專利名稱：在植物果實中生產酮類胡蘿卜素的方法
技術領域：
本發(fā)明涉及通過培養(yǎng)果實中顯示酮酶活性的遺傳修飾植物生產酮類胡蘿卜素的方法，涉及遺傳修飾的植物，涉及它們作為食物和飼料的用途及用于酮類胡蘿卜素提取物生產的用途。
背景技術：
在細菌、藻類、真菌和植物中從頭合成類胡蘿卜素。酮類胡蘿卜素，即包含至少一個酮基的類胡蘿卜素，諸如，蝦青素、角黃素、海膽酮、3-羥基海膽酮、3’-羥基海膽酮、adonirubin、金盞花黃質是一些藻類和微生物做為次生代謝物產生的天然抗氧化劑和色素。
由于它們賦予顏色的特性，酮類胡蘿卜素，特別是蝦青素用做動物營養(yǎng)，特別是鱒魚、鮭魚和蝦飼養(yǎng)中的著色助劑。
目前，主要通過化學方法合成地生產蝦青素。目前通過培養(yǎng)藻類，例如雨生紅球藻(Haematococcus pluvialis)，或者通過發(fā)酵基因工程優(yōu)化的微生物，隨后進行分離的生物技術方法，小量地獲得天然酮類胡蘿卜素，諸如，例如天然蝦青素。
因此，經濟的天然酮類胡蘿卜素的生物技術生產方法具有重要意義。
WO 98/18910描述了通過將酮酶基因引入到煙草中，煙草花蜜腺中酮類胡蘿卜素的合成。
WO 01/20011描述了利用種子特異性啟動子和來源于紅球藻屬(Haematococcus)的酮酶，在油籽植物諸如油籽油菜、向日葵、大豆和芥菜的種子中用于酮類胡蘿卜素，特別是蝦青素生產的DNA構建體。
盡管現有技術中公開的方法產生了特定組織中具有酮類胡蘿卜素含量的遺傳修飾的植物，但是它們具有下面的缺點酮類胡蘿卜素，特別是蝦青素的含量和純度還不能令人滿意。

發(fā)明內容
因此，本發(fā)明基于下述目的通過栽培植物，提供用于酮類胡蘿卜素生產的可替代方法，或者提供產生酮類胡蘿卜素的另外轉基因植物，其中所述轉基因植物具有優(yōu)化的特征，諸如，更高的酮類胡蘿卜素含量，并且其沒有上述現有技術的缺點。
因此，已經發(fā)現了通過培養(yǎng)果實中顯示酮酶活性的遺傳修飾的植物，生產酮類胡蘿卜素的方法。
酮酶活性應理解為意指酮酶的酶活性。
酮酶理解為意指具有在任選取代的類胡蘿卜素β-芷香酮環(huán)上引入酮基的酶促活性的蛋白質。
特別地，酮酶理解為意指具有轉化β-胡蘿卜素為角黃素的酶促活性的蛋白質。
因此，酮酶活性理解為意指在一定時間期內，通過酮酶蛋白質轉化的β-胡蘿卜素的量，或者形成的角黃素的量。
為了在遺傳修飾植物的果實中顯示酮酶活性，優(yōu)選的實施方式涉及使用在果實中表達酮酶的遺傳修飾植物。
因此，優(yōu)選地，本發(fā)明方法中使用果實中包含至少一個編碼酮酶的核酸的遺傳修飾植物。
已知沒有果實中顯示酮酶活性的野生型植物。特別地，下述優(yōu)選植物做為野生型在果實中不顯示酮酶活性。
在本發(fā)明中，通過起始植物的基因修飾造成遺傳修飾植物果實中的酮酶活性。因此，與遺傳未修飾的起始植物比較，本發(fā)明遺傳修飾的植物在果實中顯示酮酶活性，并因此優(yōu)選地能在果實中表達酮酶。
術語“起始植物”或“野生型”理解為意指相應的未遺傳修飾的起始植物。
術語“遺傳修飾植物”優(yōu)選地理解為意指與起始植物比較，被遺傳修飾的植物。
根據本發(fā)明上下文，術語“植物”可以理解為意指起始植物(野生型)，或者本發(fā)明遺傳修飾的植物，或者兩者。
優(yōu)選地，通過將編碼酮酶的核酸引入到起始植物中，在植物果實中實現編碼酮酶的核酸的基因表達。
因此，本發(fā)明尤其涉及上述方法，其中從起始植物開始，至少一個編碼酮酶的核酸已經引入到遺傳修飾的植物中。
為此，原則上，可以使用任何酮酶基因，即，編碼酮酶的任何核酸。
說明書中提到的所有核酸可以是例如，RNA，DNA或cDNA序列。
對于來源于真核生物來源的包含內含子的基因組酮酶序列，如果宿主植物不能或者不能被改造以致能夠表達所述酮酶，則優(yōu)選使用的核酸序列是準備好的加工后的核酸諸如相應的cDNAs。
可以在下述本發(fā)明方法中或本發(fā)明遺傳修飾植物中使用的編碼酮酶的核酸和相應酮酶的例子是例如，來源于如下生物的序列雨生紅球藻，特別是雨生紅球藻flotow em.Wille(注冊號X86782；核酸SEQ ID NO.1，蛋白質SEQ ID NO.2)，雨生紅球藻NIES-144(注冊號D45881；核酸SEQ ID NO.3，蛋白質SEQ ID NO.4)，Agrobacterium aurantiacum(注冊號D58420；核酸SEQ ID NO.5，蛋白質SEQ ID NO.6)，產堿桿菌屬種(Alcaligenes spec.)(注冊號D58422；核酸SEQ IDNO.7，蛋白質SEQ ID NO.8)，Paracoccus marcusii(注冊號Y15112；核酸SEQ ID NO.9，蛋白質SEQ ID NO.10)，集胞藻屬(Synechocystis sp.)株系PC6803(注冊號576617，NP442491；核酸SEQ ID NO.11，蛋白質SEQ ID NO.12)，
慢生根瘤菌屬(Bradyrhizobium sp.)(注冊號AF218415，BAB 74888；核酸SEQ ID NO.13，蛋白質SEQ ID NO.14)，念珠藻屬(Nostoc sp.)株系PCC7120(注冊號AP003592；核酸SEQ ID NO.15，蛋白質SEQ ID NO.16)，雨生紅球藻(注冊號AF534876，AAN03484；核酸SEQ ID NO.37，蛋白質SEQ ID NO.38)，副球菌屬(Paracoccus sp.)MBIC1143(注冊號D58420，P54972；核酸SEQ ID NO.39，蛋白質SEQ ID NO.40)，Brevundimonas aurantiaca(注冊號AY166610，AAN86030；核酸SEQ ID NO.41，蛋白質SEQ ID NO.42)，泡沫節(jié)球藻(Nodularia spumigena)NSOR10(注冊號AY210783，AAO64399；核酸SEQ ID NO.43，蛋白質SEQ ID NO.44)，點形念珠藻(Nostoc punctiforme)ATCC 29133(注冊號NZ_AABC01000195，ZP_00111258；核酸SEQ ID NO.45，蛋白質SEQ IDNO.46)，點形念珠藻ATCC 29133(注冊號NZ_AABC01000196；核酸SEQID NO.47，蛋白質SEQ ID NO.48)，耐放射異常球菌(Deinococcus radiodurans)R1(注冊號E75561，AE001872；核酸SEQ ID NO.49，蛋白質SEQ ID NO.50)。
此外，可以容易地獲得本發(fā)明中可以使用的其它天然酮酶和酮酶基因的例子，例如，通過上述序列，特別是序列SEQ ID NO.2和/或SEQ IDNO.16與數據庫中的氨基酸序列或相應反翻譯的核酸序列進行比對，可以從已知基因組序列的各種生物體獲得。
而且，通過以本身已知的方法，利用雜交技術，從上述核酸序列開始，特別是從序列SEQ ID NO.1和/或SEQ ID NO.15開始，可以從未知基因組序列的不同生物體容易地發(fā)現其它天然酮酶和酮酶基因的例子。
可以在溫和(低嚴格性)條件下或優(yōu)選地在嚴格(高嚴格性)條件下進行雜交。
例如，在Sambook，J.，Fritsch，E.F.，Maniatis，T.，《Molecular Cloning(A Laboratory Manual)》，第二版，Cold Spring Harbor Laboratory Press，1989，9.31-9.57頁，或在Current Protocols in Molecular Biology，JohnWiley & Sons，N.Y.(1989)，6.3.1-6.3.6中描述了這些雜交條件。
例如，洗滌步驟期間的條件可以從由較低嚴格性條件(在50℃，用2×SSC)和高嚴格性條件(在50℃，優(yōu)選地65℃，用0.2×SSC)(20×SSC0.3M檸檬酸鈉，3M氯化鈉，pH7.0)所限定的條件范圍內選擇。
而且，洗滌步驟期間的溫度可以從室溫下22℃的溫和條件升高到65℃的嚴格條件。
兩個參數，鹽濃度和溫度可以同時變化，或者兩個參數中的一個參數保持不變，而僅僅另一個參數變化。而且，在雜交步驟期間可以使用變性劑諸如，甲酰胺或SDS。在存在50％甲酰胺的情況下，優(yōu)選地，在42℃進行雜交。
下面示出了一些雜交和洗滌步驟條件的例子(1) 雜交條件為例如，(i)65℃，4×SSC，或(ii)45℃，6×SSC，或(iii)68℃，6×SSC，100mg/ml變性魚精DNA，或(iv)68℃，6×SSC，0.5％SDS，100mg/ml變性片斷化的鮭精DNA，或(v)42℃，6×SSC，0.5％SDS，100mg/ml變性片斷化的鮭精DNA，50％甲酰胺，或(vi)42℃，50％甲酰胺，4×SSC，或(vii)42℃，50％(體積/體積)甲酰胺，0.1％牛血清白蛋白，0.1％Ficoll，0.1％聚乙烯吡咯烷酮，50mM磷酸鈉緩沖液pH6.5，750mM NaCl，75mM檸檬酸鈉，或(viii)50℃，2×或4×SSC(溫和條件)，或(ix)42℃，30％到40％甲酰胺，2×或4×SSC(溫和條件)
(2) 每種情況下10分鐘的洗滌步驟，應用例如，(i)50℃，0.015M NaCl/0.0015M檸檬酸鈉/0.1％SDS，或(ii)65℃，0.1×SSC，或(iii)68℃，0.1×SSC，0.5％SDS，或(iv)42℃，0.1×SSC，0.5％SDS，50％甲酰胺，或者(v)42℃，0.2×SSC，0.1％SDS，或者(vi)65℃，2×SSC(溫和條件)。
在本發(fā)明方法優(yōu)選的實施方式中，引入編碼蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO.2或者通過氨基酸置換、插入或缺失衍生自該序列的序列，其中該衍生序列在氨基酸水平上與序列SEQ ID NO.2具有至少20％，優(yōu)選地至少30％，更優(yōu)選地至少40％，更優(yōu)選地至少50％，更優(yōu)選地至少60％，更優(yōu)選地至少70％，更優(yōu)選地至少80％，特別優(yōu)選地至少90％同一性，并且具有酮酶的酶特征(enzymatic characteristic)。
這可以采用通過序列比對，按上述可以從其它生物體發(fā)現的天然酮酶序列或者是通過人工改變，例如通過氨基酸置換、插入或缺失，從序列SEQID NO.2開始修飾的人工酮酶序列的形式。
在本發(fā)明方法另一個優(yōu)選的實施方式中，引入編碼蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO.16或者通過氨基酸置換、插入或缺失衍生自該序列的序列，其中所述衍生序列在氨基酸水平上與序列SEQ IDNO.16具有至少20％，優(yōu)選地至少30％，更優(yōu)選地至少40％，更優(yōu)選地至少50％，更優(yōu)選地至少60％，更優(yōu)選地至少70％，更優(yōu)選地至少80％，特別優(yōu)選地至少90％同一性，并且具有酮酶的酶特征。
這可以采用通過序列比對，按上述可以從其它生物體發(fā)現的天然酮酶序列或者通過人工改變，例如通過氨基酸置換、插入或缺失，從序列SEQID NO.16開始修飾的人工酮酶序列的形式。
在說明書中，術語“置換”理解為意指一個或多個氨基酸被一個或多個氨基酸替代。優(yōu)選進行的置換是所謂的保守性置換，其中替代的氨基酸具有與原始氨基酸相似的特征，例如Asp對Glu，Asn對Gln，Ile對Val，Ile對Leu，Thr對Ser的置換。
缺失是用直接的鍵接替代氨基酸。優(yōu)選的缺失位置是多肽末端和各單個蛋白質結構域之間的連接。
插入是向多肽鏈中引入氨基酸，其中在形式上用一個或多個氨基酸替代直接的鍵接。
兩種蛋白質之間的同一性理解為意指每種情況下在整個蛋白質長度上氨基酸的同一性，特別是通過比較計算的同一性，其中利用Clustal方法(Higgins DG，Sharp PM，Fast and sensitive multiple sequence alignmentson a microcomputer.Comput App.Biosci.1989 Apr；5(2)151-1)，借助于DNASTAR，inc.Madison，Wisconsin(USA)的Lasergene軟件幫助，設定下面參數進行該比較多序列比對參數空位罰值 10空位長度罰值 10成對比對參數k-tuple1空位罰值 3視窗 5Diagonals saved5因此，在氨基酸水平與序列SEQ ID NO.2或16具有至少20％同一性的蛋白質應理解為意指特別是利用具有上述參數設置的上述程序算法比較其序列與序列SEQ ID NO.2或16后，具有至少20％同一性的蛋白質。
例如，根據遺傳密碼，通過多肽序列的反翻譯可以獲得適宜的核酸序列。
優(yōu)選用于該目的的密碼子是根據植物特異性密碼子使用常常使用的密碼子。在對相關生物體的其它已知基因的計算機估測輔助下可以容易地確定密碼子使用。
在特別優(yōu)選的實施方式中，將包含序列SEQ ID NO.1的核酸引入到植物中。
在另一優(yōu)選的實施方式中，將包含序列SEQ ID NO.15的核酸引入到植物中。
而且，通過從核苷酸單元開始的化學合成，諸如，通過將各雙螺旋重疊互補核酸單元進行片段縮合，可以用已知的方式產生所有上述酮酶基因。例如，通過亞磷酰胺方法(Voet，Voet，第二版，Wiley Press NewYork，896-897頁)，用已知的方式，可以化學合成寡核苷酸。在Sambrook等(1989)Molecular CloningA Laboratory Manual，Cold Spring HarborLaboratory Press中描述了合成寡核苷酸的退火、通過DNA聚合酶的Klenow片段補平缺隙和連接反應及一般的克隆方法。
在本發(fā)明方法的特別優(yōu)選的實施方式中，使用果實中顯示最高酮酶表達率的遺傳修飾植物。
優(yōu)選地，這通過在果實特異性啟動子控制下進行酮酶基因表達來實現。例如，以在核酸構建體中與果實特異性啟動子功能性連接的方式，將如下所詳細描述的上述核酸引入到植物中。
根據本發(fā)明，植物優(yōu)選地理解為意指做為野生型，果實中具有色質體的植物。
其它優(yōu)選的植物做為野生型果實中還具有類胡蘿卜素，特別是β-胡蘿卜素、玉米黃質、新黃質、紫黃質，或黃體素。
進一步優(yōu)選的植物做為野生型果實中還具有羥化酶活性。
羥化酶活性理解為意指羥化酶的酶活性。
羥化酶理解為意指具有在類胡蘿卜素的任選取代的β-芷香酮環(huán)上引入羥基基團的酶促活性的蛋白質。
特別地，羥化酶理解為意指具有轉化β-胡蘿卜素為玉米黃質，或者轉化角黃素為蝦青素的酶促活性的蛋白質。
因此，羥化酶活性理解為意指在一定時間內，通過蛋白質羥化酶轉化的β-胡蘿卜素或角黃素的量，或者形成的玉米黃質或蝦青素的量。
在優(yōu)選的實施方式中，培養(yǎng)與野生型比較還顯示了增加的羥化酶活性和/或β-環(huán)化酶活性的植物。
羥化酶活性理解為意指羥化酶的酶活性。
羥化酶理解為意指具有在任選取代的類胡蘿卜素β-芷香酮環(huán)上引入羥基的酶促活性的蛋白質。
特別地，羥化酶理解為意指具有轉化β-胡蘿卜素為玉米黃質，或者轉化角黃素為蝦青素的酶促活性的蛋白質。
因此，羥化酶活性理解為意指在一定時間期間內，通過蛋白質羥化酶轉化的β-胡蘿卜素或角黃素的量，或者形成的玉米黃質或蝦青素的量。
因此，在與野生型比較羥化酶活性增加的情況下，在一定時間期間內，與野生型比較，由蛋白質羥化酶轉化的β-胡蘿卜素或角黃素的量，或者形成的玉米黃質或蝦青素的量增加。
這種羥化酶活性的增加優(yōu)選地相當于野生型羥化酶活性的至少5％，而且優(yōu)選地至少20％，而且優(yōu)選地至少50％，而且優(yōu)選地至少100％，更優(yōu)選地至少300％，甚至更優(yōu)選地至少500％，特別地至少600％。
β-環(huán)化酶活性理解為意指β-環(huán)化酶的酶活性。
β-環(huán)化酶理解為意指具有轉化番茄紅素末端線性殘基為β-芷香酮環(huán)的酶促活性的蛋白質。
特別地，β-環(huán)化酶理解為意指具有轉化γ-胡蘿卜素為β-胡蘿卜素的酶促活性的蛋白質。
因此，β-環(huán)化酶活性理解為意指在一定時間期間內，通過蛋白質β-環(huán)化酶轉化的γ-胡蘿卜素的量或形成的β-胡蘿卜素的量。
因此，在與野生型比較β-環(huán)化酶活性增加的情況下，在一定時間期間內，與野生型比較，轉化的γ-胡蘿卜素的量或形成的β-胡蘿卜素的量增加。
這種β-環(huán)化酶活性的增加優(yōu)選相當于野生型β-環(huán)化酶活性的至少5％，而且優(yōu)選地至少20％，而且優(yōu)選地至少50％，而且優(yōu)選地至少100％，更優(yōu)選地至少300％，甚至更優(yōu)選地至少500％，特別地至少600％。
根據本發(fā)明，術語“野生型”應理解為意指相應的未遺傳修飾的起始植物。
優(yōu)選地，并且特別是在不能明確地指明植物或野生型時，就羥化酶活性增加、β-環(huán)化酶活性增加和酮類胡蘿卜素含量增加而言，野生型在任何情況下均應理解為意指參照植物。
優(yōu)選地，這種參照植物是番茄(Lycopersicon esculentum)。
優(yōu)選地，在下面條件下測定本發(fā)明遺傳修飾的植物中和野生型或參照植物中羥化酶活性用Bouvier等的方法(Biochim.Biophys.Acta 1391(1998)，320-328)體外測定羥化酶活性。鐵氧還蛋白、鐵氧還蛋白-NADP氧化還原酶、過氧化氫酶、NADPH和β-胡蘿卜素與單和雙半乳糖甘油酯一起被添加到一定量植物提取物中。
特別優(yōu)選地，在下面條件下，用Bouvier，Keller，d’Harlingue和Camara的方法測定羥化酶活性(葉黃素生物合成來源于胡椒果實的類胡蘿卜素羥化酶的分子和功能特征)(Capsicum annuum L.；Biochim.Biophys.Acta 1391(1998)，320-328)在0.250ml體積中進行體外測定?；旌衔锇?0mM磷酸鉀(pH7.6)，0.025mg菠菜鐵氧還蛋白，0.5單位菠菜鐵氧還蛋白-NADP+氧化還原酶，0.25mM NADPH，0.010mgβ-胡蘿卜素(在0.1mg Tween80中乳化)，0.05mM單和雙半乳糖甘油酯混合物(1∶1)，1單位過氧化氫酶，200單和雙半乳糖甘油酯(1∶1)，0.2mg牛血清白蛋白和不同體積的植物提取物。在30℃溫育反應混合物2小時。用有機溶劑諸如丙酮或氯仿/甲醇(2∶1)提取反應產物，并且用HPLC測定。
優(yōu)選地，在下面條件下測定本發(fā)明遺傳修飾的植物中和野生型或參照植物中β-環(huán)化酶活性用Fraser和Sandmann的方法(Biochem.Biophys.Res.Comm.185(1)(1992)9-15)，體外測定β-環(huán)化酶活性。向一定量植物提取物添加下列物質做為緩沖劑的磷酸鉀(pH7.6)，做為底物的番茄紅素，來源于辣椒屬的基質蛋白質，NADP+，NADPH和ATP。
特別優(yōu)選地，在下面條件下，用Bouvier，d’Harlingue和Camara的方法(Molecular Analysis of carotenoid cylae inhibition；Arch.Biochem.Biophys.346(1)(1997)53-64)測定羥化酶活性。
在0.250ml體積中進行體外測定。混合物包含50mM磷酸鉀(pH7.6)，不同量的植物提取物，20nM番茄紅素，250μg來源于辣椒屬的色質體基質蛋白質，0.2mM NADP+，.02mM NADPH和1mM ATP。NADP/NADPH和ATP與1mg Tween 80在臨添加到溫育介質中之前一起溶解在10ml乙醇中。在30℃，60分鐘的反應時間后，通過添加氯仿/甲醇(2∶1)猝滅反應。通過HPLC方法分析氯仿中萃取的反應產物。
Fraser和Sandmann方法(Biochem.Biophys.Res.Comm.185(1)(1992)9-15)中描述了使用放射性底物的可替代測定法。
用各種方法，例如通過在表達和蛋白質水平消除抑制性調節(jié)機制，或者通過相對于野生型而言增加編碼羥化酶的核酸和/或編碼β-環(huán)化酶的核酸的基因表達，可以增加羥化酶活性和/或β-環(huán)化酶活性。
同樣可以用各種方法，例如通過用激活物誘導羥化酶基因和/或β-環(huán)化酶基因，或者通過引入一個或多個羥化酶基因拷貝和/或β-環(huán)化酶基因拷貝，即，通過向植物中引入至少一個編碼羥化酶的核酸和/或至少一個編碼β-環(huán)化酶的核酸，與野生型比較，增加編碼羥化酶的核酸的基因表達和/或編碼β-環(huán)化酶的核酸的基因表達。
根據本發(fā)明，增加編碼羥化酶和/或β-環(huán)化酶的核酸的基因表達也可以理解為意指操作植物同源的、內源的羥化酶和/或β-環(huán)化酶的表達。
例如，這可以通過修飾編碼羥化酶和/或β-環(huán)化酶的基因的啟動子DNA序列來實現。例如，可以通過DNA序列缺失或插入實現這種導致基因表達率增加的修飾。
如上所述，通過應用外源刺激物，可以修飾內源羥化酶和/或β-環(huán)化酶的表達。這可以通過特異性生理學條件，即通過外源物質施用來實現。
而且，內源羥化酶和/或β-環(huán)化酶基因的修飾的或增加的表達還可以通過未轉化植物中不存在的調節(jié)蛋白質與該基因啟動子的相互作用而獲得。
例如，如WO 96/06166中所述，這種調節(jié)物可以是由DNA結合結構域和轉錄激活結構域組成的嵌合蛋白質。
在優(yōu)選的實施方式中，通過向植物中引入至少一個編碼羥化酶的核酸和/或通過向植物中引入至少一個編碼β-環(huán)化酶的核酸實現編碼羥化酶的核酸的基因表達和/或增加編碼β-環(huán)化酶的核酸的基因表達。
原則上，任何羥化酶基因或任何β-環(huán)化酶基因，即編碼羥化酶的任何核酸和編碼β-環(huán)化酶的任何核酸都可以用于該目的。
在來源于真核生物來源的包含內含子的基因組羥化酶或β-環(huán)化酶核酸序列的情況下，如果宿主植物不能或者不能被改造以致能夠表達所述羥化酶或β-環(huán)化酶，則優(yōu)選使用準備好的加工后的核酸序列，諸如相應的cDNAs。
羥化酶基因的例子是編碼來源于雨生紅球藻的羥化酶的核酸，注冊號AX038729，WO 0061764；(核酸SEQ ID NO51，蛋白質SEQ ID NO52)和編碼下面注冊號的羥化酶的核酸|emb|CAB55626.1，CAA70427.1，CAA70888.1，CAB55625.1，AF499108_1，AF315289_1，AF296158_1，AAC49443.1，NP_194300.1，NP_200070.1，AAG10430.1，CAC06712.1，AAM88619.1，CAC95130.1，AAL80006.1，AF162276_1，AAO53295.1，AAN85601.1，CRTZ_ERWHE，CRTZ_PANAN，BAB79605.1，CRTZ_ALCSP，CRTZ_AGRAU，CAB56060.1，ZP_00094836.1，AAC44852.1，BAC77670.1，NP_745389.1，NP_344225.1，NP_849490.1，ZP_00087019.1，NP_503072.1，NP_852012.1，NP_115929.1，ZP_00013255.1而且，特別優(yōu)選的羥化酶是來源于番茄的羥化酶(核酸SEQ ID NO55，蛋白質SEQ ID NO56)。
β-環(huán)化酶基因的例子是編碼來源于番茄的β-環(huán)化酶的核酸(注冊號X86452)(核酸SEQ ID NO53，蛋白質SEQ ID NO54)，和具有下面注冊號的β-環(huán)化酶基因S66350番茄紅素β-環(huán)化酶(EC 5.5.1.-)-番茄CAA60119 番茄紅素合酶[辣椒(Capsicum annuum)]S66349番茄紅素β-環(huán)化酶(EC 5.5.1.-)-煙草(common tobacco)CAA57386 番茄紅素環(huán)化酶[煙草(Nicotiana tabacum)]AAM21152 番茄紅素β-環(huán)化酶[甜橙(Citrus sinensis)]
AAD38049 番茄紅素環(huán)化酶[葡萄柚(Citrus x paradisi)]AAN86060 番茄紅素環(huán)化酶[溫州蜜柑(Citrus unshiu)]AAF44700 番茄紅素β-環(huán)化酶[甜橙]AAK07430 番茄紅素β-環(huán)化酶[(Adonis palaestina)]AAG10429 β-環(huán)化酶[萬壽菊(Tagetes erecta)]AAA81880 番茄紅素環(huán)化酶AAB53337 番茄紅素β-環(huán)化酶AAL92175 β-番茄紅素環(huán)化酶[宮燈百合(Sandersonia aurantiaca)]CAA67331 番茄紅素環(huán)化酶[喇叭水仙(Narcissus pseudonarcissus)]AAM45381 β-環(huán)化酶[萬壽菊]AAO18661番茄紅素β-環(huán)化酶[玉米(Zea mays)]AAG21133 色質體特異性番茄紅素β-環(huán)化酶[番茄]AAF18989 番茄紅素β-環(huán)化酶[胡蘿卜(Daucus carota)]ZP_001140 推測的蛋白質[海洋原綠球藻(Prochlorococcus marinus)株系MIT9313]ZP_001050 推測的蛋白質[Prochlorococcus marinus subsp.pastoris株系CCMP1378]ZP_001046 推測的蛋白質[Prochlorococcus marinus subsp.pastoris株系CCMP1378]ZP_001134 推測的蛋白質[海洋原綠球藻株系MIT9313]ZP_001150 推測的蛋白質[聚球藻(Synechococcus sp.)WH 8102]AAF10377 番茄紅素環(huán)化酶[耐放射異常球菌]BAA29250 393氨基酸長度推測的蛋白質[Pyrococcus horikoshii]BAC77673 番茄紅素β-單環(huán)化酶[海洋細菌P99-3]AAL01999 番茄紅素環(huán)化酶[Xanthobacter sp.Py2]ZP_000190 推測的蛋白質[橙色綠屈撓菌(Chloroflexus aurantiacus)]ZP_000941 推測的蛋白質[(Novosphingobium aromaticivorans)]AAF78200 番茄紅素環(huán)化酶[慢生根瘤菌ORS278]BAB79602 crtY[成團泛菌(Pantoea agglomerans pv.milletiae)]CAA64855 番茄紅素環(huán)化酶[灰色鏈霉菌(Streptomyces griseus)]
AAA21262 Dycopene環(huán)化酶[Pantoea agglomerans]C37802crtY蛋白質-噬夏孢歐文氏菌(Erwinia uredovora)BAB79602 crtY[Pantoea agglomerans pv.milletiae]AAA64980 番茄紅素環(huán)化酶[Pantoea agglomerans]AAC44851 番茄紅素環(huán)化酶BAA09593 番茄紅素環(huán)化酶[副球菌MBIC1143]ZP_000941 推測的蛋白質[Novosphingobium aromaticivorans]CAB56061 番茄紅素β-環(huán)化酶[Paracoccus marcusii]BAA20275 番茄紅素環(huán)化酶[長赤細菌(Erythrobacter longus)]ZP_000570 推測的蛋白質[Thermobifida fusca]ZP_000190 推測的蛋白質[橙色綠屈撓菌]AAK07430 番茄紅素β-環(huán)化酶[Adonis palaestina]CAA67331 番茄紅素環(huán)化酶[喇叭水仙]AAB5337 番茄紅素β-環(huán)化酶BAC77673 番茄紅素β-單環(huán)化酶[海洋細菌(marine bacterium)P99-3]而且，特別優(yōu)選的β-環(huán)化酶是來源于番茄的色質體特異性β-環(huán)化酶(AAG21133)(核酸SEQ ID NO57，蛋白質SEQ ID NO58)。
因此在該優(yōu)選的實施方式中，與野生型比較，在本發(fā)明優(yōu)選的轉基因植物中，存在至少一個另外的羥化酶基因和/或β-環(huán)化酶基因。
在該優(yōu)選的實施方式中，例如，遺傳修飾的植物具有至少一個編碼羥化酶的外源核酸或至少兩個編碼羥化酶的內源核酸和/或至少一個編碼β-環(huán)化酶的外源核酸或至少兩個編碼β-環(huán)化酶的內源核酸。
在上述優(yōu)選實施方式中優(yōu)選地使用的羥化酶基因是編碼如下蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO52或者通過氨基酸的置換、插入或缺失衍生自該序列的序列，其中該衍生序列在氨基酸水平上與序列SEQ ID NO.52具有至少30％，優(yōu)選地至少50％，更優(yōu)選地至少70％，甚至更優(yōu)選地至少90％，最優(yōu)選地至少95％同一性，并且具有羥化酶的酶促特征。
如上述可以容易地發(fā)現其它羥化酶和羥化酶基因的例子，例如，通過SEQ ID NO.52與數據庫中的氨基酸序列或相應反翻譯核酸序列進行同源性比較，從已知基因組序列的各種生物體獲得。
而且，通過例如從序列SEQ ID NO.51開始，利用上述雜交和PCR技術，也可以從未知基因組序列的各種生物體，以本身已知的方法，容易地發(fā)現其它羥化酶和羥化酶基因的例子。
在另一特別優(yōu)選的實施方式中，將編碼包含序列SEQ ID NO.52羥化酶氨基酸序列的蛋白質的核酸引入到生物體中以增加羥化酶活性。
例如，根據遺傳密碼，通過多肽序列的反翻譯可以獲得適合的核酸序列。
優(yōu)選用于該目的的密碼子是根據植物特異性密碼子使用常常使用的密碼子。在對相關生物體其它已知基因的計算機估測輔助下可以容易地確定密碼子使用。
在特別優(yōu)選的實施方式中，將包含序列SEQ ID NO.51的核酸引入到生物體中。
在上述優(yōu)選實施方式中優(yōu)選地使用的β-環(huán)化酶基因是編碼如下蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO54或者通過氨基酸的置換、插入或缺失衍生自該序列的序列，其中該衍生序列在氨基酸水平上與序列SEQ ID NO.54具有至少30％，優(yōu)選地至少50％，更優(yōu)選地至少70％，甚至更優(yōu)選地至少90％，最優(yōu)選地至少95％同一性，并且具有β-環(huán)化酶的酶促特征。
其它β-環(huán)化酶和β-環(huán)化酶基因的例子可以如上所述容易地發(fā)現，例如，通過SEQ ID NO.54與數據庫中的氨基酸序列或相應反翻譯核酸序列進行同源性比較，從已知基因組序列的各種生物體獲得。
而且，通過例如從序列SEQ ID NO.53開始，利用雜交和PCR技術，也可以從未知基因組序列的各種生物體，以本身已知的方法，容易地發(fā)現其它β-環(huán)化酶和β-環(huán)化酶基因的例子。
在另一特別優(yōu)選的實施方式中，將編碼包含序列SEQ ID NO.54的β-環(huán)化酶氨基酸序列的蛋白質的核酸引入到生物體中以增加β-環(huán)化酶活性。
例如，根據遺傳密碼，通過多肽序列的反翻譯可以獲得核酸序列。
優(yōu)選用于該目的的密碼子是根據植物特異性密碼子使用常常使用的密碼子。在對相關生物體的其它已知基因的計算機估測輔助下可以容易地確定密碼子使用。
在特別優(yōu)選的實施方式中，將包含序列SEQ ID NO.53的核酸引入到生物體中。
而且，從核苷酸單元開始通過化學合成，諸如，通過各雙螺旋重疊互補核酸單元的片段縮合，也可以用已知的方法產生所有上述羥化酶基因或β-環(huán)化酶基因。例如，通過亞磷酰胺方法(Voet，Voet，第二版，Wiley PressNew York，896-897頁)，用已知的方式，可以化學合成寡核苷酸。在Sambrook等(1989)Molecular CloningA Laboratory Manual，ColdSpring Harbor Laboratory Press中描述了合成寡核苷酸的退火及DNA聚合酶Klenow片段對缺口的補平和連接反應和一般的克隆方法。
特別優(yōu)選的植物是選自皺子棕屬(Actinophloeus)、亮絲草屬(Aglaeonema)、風梨屬(Ananas)、草莓樹屬(Arbutus)、假檳榔屬(Archontophoenix)、Area、Aronia、天門冬屬(Asparagus)、亞塔棕屬(Attalea)、小檗屬(Berberis)、Bixia、Brachychilum、Bryonia、Caliptocalix、辣椒屬(Capsicum)、番木瓜屬(Carica)、南蛇藤屬(Celastrus)、西瓜屬(Citrullus)、柑橘屬(Citrus)、鈴蘭屬(Convallaria)、栒子屬(Cotoneaster)、山楂屬(Crataegus)、香瓜屬(Cucumis)、南瓜屬(Cucurbita)、菟絲子屬(Cuscuta)、蘇鐵屬(Cycas)、樹番茄屬(Cyphomandra)、薯蕷屬(Dioscorea)、柿屬(Diospyrus)、Dura、胡頹子屬(Elaeagnus)、油棕屬(Elaeis)、古柯屬(Erythroxylon)、衛(wèi)矛屬(Euonymus)、榕屬(Ficus)、金桔屬(Fortunella)、草莓屬(Fragaria)、梔子屬(Gardinia)、瓊欖屬(Gonocaryum)、棉屬(Gossypium)、番石榴屬(Guava)、刺棒棕屬(Guilielma)、木槿屬(Hibiscus)、沙棘屬(Hippophaea)、鳶尾屬(Iris)、山黧豆屬(Lathyrus)、忍冬屬(Lonicera)、絲瓜屬(Luffa)、枸杞屬(Lycium)、番茄屬(Lycopersicum)、金虎尾屬(Malpighia)、
芒果屬(Mangifera)、Mormodica、九里香屬(Murraya)、芭蕉屬(Musa)、能加棕屬(Nenga)、Palisota、露兜樹屬(Pandanus)、西番蓮屬(Passiflora)、鱷梨屬(Persea)、酸漿屬(Physalis)、李屬(Prunus)、Ptychandra、石榴屬(Punica)、火棘屬(Pyracantha)、梨屬(Pyrus)、茶藨子屬(Ribes)、薔薇屬(Rosa)、懸鉤子屬(Rubus)、薩巴棕屬(Sabal)、接骨木屬(Sambucus)、Seaforita、水牛果屬(Shepherdia)、茄屬(Solanum)、花楸屬(Sorbus)、Synaspadix、Tabernae、Tamus、紅豆杉屬(Taxus)、栝樓屬(Trichosanthes)、Triphasia、越桔屬(Vaccinium)、莢蒾屬(Viburnum)、Vignia或葡萄屬(Vitis)的植物。
用與Frazer等(J.Biol.Chem.272(10)6128-6135，1997)方法相似的方法測定本發(fā)明遺傳修飾植物中酮酶活性。在脂質(大豆卵磷脂)和去污劑(膽酸鈉)存在的情況下，用底物β-胡蘿卜素和角黃素測定植物提取物中酮酶活性。通過HPLC測定來源于該酮酶試驗的底物/產物比率。
在生產酮類胡蘿卜素的本發(fā)明方法中，優(yōu)選地，在遺傳修飾植物(此后也稱為轉基因植物)栽培步驟后，收獲植物和從植物果實分離酮類胡蘿卜素。
以本身已知的方法，在基質上培養(yǎng)轉基因植物，并且用適合的方法收獲該轉基因植物。
用本身已知的方法，例如干燥，接著提取，并且如果適當的地，進一步進行化學或物理純化方法，諸如，沉淀方法，結晶方法，熱分離方法諸如精餾方法，或物理分離方法諸如，層析法，從收獲的果實分離酮類胡蘿卜素。優(yōu)選地，例如，用有機溶劑諸如丙酮、己烷、乙醚或叔丁基甲基醚從果實分離酮類胡蘿卜素。
酮類胡蘿卜素的其它分離方法在例如，Egger和Kleinig(Phytochemistry(1967)6，437-440)和Egger(Phytochemistry(1965)4，609-618)中描述。
優(yōu)選地，酮類胡蘿卜素選自蝦青素、角黃素、海膽酮、3-羥基海膽酮、3’-羥基海膽酮、adonirubin和金盞花黃質。
特別優(yōu)選的酮類胡蘿卜素是蝦青素。
優(yōu)選地，通過用核酸構建體轉化起始植物產生轉基因植物，所述核酸構建體包含至少一個(也優(yōu)選地多個)功能性地連接一個或多個調節(jié)信號的上述核酸，所述調節(jié)信號確保植物中的轉錄和翻譯。
此后，將這些核酸構建體稱為表達盒，在該核酸構建體中編碼核酸序列功能性地連接一個或多個調節(jié)信號，所述調節(jié)信號確保植物中的轉錄和翻譯。
優(yōu)選地，調節(jié)信號包含一個或多個確保植物中轉錄和翻譯的啟動子。
表達盒包含調節(jié)宿主細胞中編碼序列表達的調節(jié)信號，即調節(jié)性核酸序列。根據優(yōu)選的實施方式，表達盒包含上游，即在編碼序列5’端的啟動子和下游，即在3’端的聚腺苷酸化信號，并且如果適當，還包含其它調節(jié)元件，該調節(jié)元件與居間的至少一種上述基因的編碼序列可操作地連接?？刹僮鞯剡B接理解為意指啟動子、編碼序列、終止子，及如果適當，其它調節(jié)元件順序地排列，以便當編碼序列表達時，每種調節(jié)元件能完成其預期的功能。
此后通過舉例的方式描述用于植物的優(yōu)選核酸構建體、表達盒和載體，及產生轉基因植物的方法和轉基因植物本身。
優(yōu)選可操作連接的序列是，但不限于，用于確保質外體中、液泡中、質體中、線粒體中、內質網(ER)中、核中、油體中或其它區(qū)室中亞細胞定位的導向序列，和翻譯增強子諸如煙草花葉病毒5’前導序列(Gallie等，Nucl.Acids Res.15(1987)，8693-8711)。
原則上，能控制外源基因在植物中表達的任何啟動子都適于用做表達盒的啟動子。
“組成型”啟動子意指確保大量，優(yōu)選地所有組織在植物發(fā)育的相當大部分時期，優(yōu)選地在植物發(fā)育的所有時間點表達的那些啟動子。
優(yōu)選使用的啟動子特別是植物啟動子或來源于植物病毒的啟動子。特別優(yōu)選的是CaMV花椰菜花葉病毒35S轉錄物啟動子(Franck等，(1980)Cell 21285-294；Odell等，(1985)Nature 313810-812；Shewmaker等，(1985)Virology 140281-288；Gardner等，(1986)Plant Mol Biol 6221-228)或19S CaMV啟動子(US 5,352,605；WO 84/02913；Benfey等(1989)EMBOJ 82195-2202)。
其它適合的組成型啟動子是pds啟動子(Pecker等.(1992)Proc.Natl.Acad.Sci USA 894962-4966)或“核酮糖二磷酸羧化酶-加氧酶亞基(SSU)”啟動子(US 4,962,028)，豆球蛋白B啟動子(GenBank注冊號X03677)，農桿菌屬(Agrobacterium)胭脂氨酸合酶啟動子，TR雙啟動子，來源于農桿菌屬(Agrobacterium)的OCS(章魚氨酸合酶)啟動子，泛素啟動子(Holtorf S等，(1995)Plant Mol Biol 29637-649)，泛素1啟動子(Christensen等，(1992)Plant Mol Biol 18675-689；Bruce等，(1989)Proc Natl Acad SciUSA 869692-9696)，Smas啟動子，肉桂醇脫氫酶啟動子(US 5,683,439)，液泡ATPase亞基啟動子或來源于小麥的富含脯氨酸的蛋白質的啟動子(WO 91/13991)，Pnit啟動子(Y07648.L，Hillebrand等，(1998)，Plant.Mol.Biol.36，89-99，Hillebrand等.(1996)，Gene，170，197-200)，鐵氧還蛋白-NADPH氧化還原酶啟動子(數據庫登記AB011474，位置70127到69493)，TPT啟動子(WO 03006660)，“超啟動子”(US專利5955646)，34S啟動子(US專利6051753)和本領域技術人員已知的植物中組成型表達的其它基因啟動子。
表達盒也可以包含化學誘導型啟動子(綜述論文Gatz等，(1997)AnnuRev Plant Physiol Plant Mol Biol 4889-108)，利用該啟動子，可以在特定時間點控制植物中酮酶基因的表達。同樣可以使用這種啟動子，諸如，PRP1啟動子(Ward等，(1993)Plant Mol Biol 22361-366)，水楊酸誘導型啟動子(WO 95/19443)，苯磺酰胺誘導型啟動子(EP 0 388 186)，四環(huán)素誘導型啟動子(Gatz等.(1992)Plant J 2397-404)，脫落酸誘導型啟動子(EP 0335 528)或乙醇或環(huán)己酮誘導型啟動子(WO 93/21334)。
其它優(yōu)選的啟動子是由生物或非生物脅迫誘導的啟動子，諸如，PRP1基因的病原體誘導型啟動子(Ward等，(1993)Plant Mol Biol 22361-366)，來源于番茄的熱誘導型hsp70或hsp80啟動子(US 5,187,267)，來源于馬鈴薯的冷誘導型α-淀粉酶啟動子(WO 96/12814)，光誘導型PPDK啟動子或創(chuàng)傷誘導型pinII啟動子(EP375091)。
病原體誘導型啟動子包括由于病原體攻擊而誘導的基因，諸如，PR蛋白質、SAR蛋白質、β-1，3-葡聚糖酶，幾丁質酶基因的啟動子(例如Redolfi等，(1983)Neth J Plant Pathol 89245-254；Uknes等，(1992)The Plant Cell4645-656；Van Loon(1985)Plant Mol Viral 4111-116；Marineau等，(1987)Plant Mol Biol 9335-342；Matton等，(1987)Molecular Plant-MicrobeInteractions 2325-342；Somssich等，(1986)Proc Natl Acad Sci USA832427-2430；Somssich等，(1988)Mol Gen Genetics 293-98；Chen等，(1996)Plant J 10955-966；Zhang和Sing(1994)Proc Natl Acad Sci USA912507-2511；Warner等，(1993)Plant J 3191-201；Siebertz等，(1989)Plant Cell 1961-968(1989))。
也可以包含創(chuàng)傷誘導型啟動子，諸如pinII基因(Ryan(1990)Ann RevPhytopath 28425-449；Duan等，(1996)Nat Biotech 14494-498)，wun1和wun2基因(US 5,428,148)，win1和win2基因(Stanford等，(1989)Mol GenGenet 215200-208)，系統(tǒng)素(McGurl等，(1992)Science 2251570-1573)，WIP1基因(Rohmeier等，(1993)Plant Mol Biol 22783-792；Ekelkamp等，(1993)FEBS Letters 32373-76)，MPI基因(Corderok等，(1994)ThePlant J 6(2)141-150)等等的啟動子。
其它適合的啟動子是例如果實成熟特異性啟動子，例如來源于番茄的果實成熟特異性啟動子(WO 94/21794，EP 409 625)。因為天然地，由于發(fā)育作用形成各個組織，所以發(fā)育啟動子包括的一些啟動子是組織特異性啟動子。
而且，特別優(yōu)選的啟動子是確保在發(fā)生例如，酮類胡蘿卜素或其前體的生物合成的組織或植物部分中表達的啟動子。優(yōu)選的啟動子例子是對花藥、子房、花瓣、萼片、花、葉、莖、根、果實和它們的組合具有特異性的啟動子。
例如，塊莖特異性、貯藏根特異性或根特異性啟動子是patatin啟動子I型(B33)或來源于馬鈴薯的組織蛋白酶D抑制劑啟動子。
葉特異性啟動子的例子是例如，來源于馬鈴薯的胞質FBPase啟動子(WO 97/05900)，核酮糖二磷酸羧化酶-加氧酶(核酮糖-1，5-二磷酸羧化酶)SSU啟動子(小亞基)或來源于馬鈴薯的ST-LSI啟動子(Stockhaus等，(1989)EMBO J 82445-2451).
花特異性啟動子的例子是八氫番茄紅素合酶啟動子(WO 92/16635)或P-rr基因啟動子(WO 98/22593)。
花藥特異性啟動子的例子是5126啟動子(US 5,689,049，US 5,689,051)、glob-1啟動子或g-玉米醇溶蛋白啟動子。
果實特異性啟動子是例如，來源于番茄的Pds啟動子(Genbank注冊號U46919；Corona，V.，Aracri.B.，Kosturkova，G.，Bartley，G.E.，Pitto，L.，Giorgetti，L.，Scolink，P.A.和Giuliano，G.，Regulation of a carotenoid biosynthesis gene promoterduring plant development Plant J.9(4)，505-512(1996))，SEQ ID NO.17，來源于番茄的2A11啟動子(Pear，J.R.，Ridge，N.，Rasmussen，R.，Rose，R.E.和Houck，C.M.Isolation and characterization of a fruit-specific cDNAand the corresponding genomic clone from tomato Plant Mol.Biol.13(6)，639-651(1998)，SEQ ID NO.18)，cucumisin啟動子(Yamagata，H.，Yonseu，K.，Hirata，A和Aizono，Y.，TGTCACA Motif Is a Novel cis-Regulatory Enhancer Element Involved inFruit-specific Expression of the cucumisin Gene J.Biol.Chem.277(13)，11582-11590(2002)，SEQ ID NO.19，內切多聚半乳糖醛酸酶基因啟動子(Redondo-Nevado.J.，Medina-Escobar，N.，Caballero-Repullo，J.L.和Muonz-Blanco，J.A fruitspecific and developmentally regulated endopolygalacturonase gene fromstrawberry(Fragaria x ananassa c.v.chandler)，J Experimental Botany 52(362)1941-1945(2001)，SEQ ID NO.20，來源于番茄的多聚半乳糖醛酸酶啟動子(Nicholass，F.J.，Smith，C.J.，Schuch，W.，Bird，C.R.和Grierson，D.，High levels of ripening-specificreporter gene expression directed by tomato fruit polygalacturonasegene-flanking regions，Plant Mol.Biol.28(3)，423-435(1995))，SEQ IDNO.21，TMF7和TMF9啟動子(US 5608150)，E4啟動子(Cordes A.Deikman J.Margossian LJ.Fischer RL.Interaction of a developmentally regulated DNA-binding factor with sitesflanking two different fruit-ripening genes from tomato(1989)，Plant Cell 1，1025-1034)和E8啟動子(Deikman和Fisher，Interaction of a DNA binding factorwith the 5’-flanking region of an ethylene-responsive fruit ripening genefrom tomato(1988)，EMBO J.7，3315-3320)。
現有技術中還描述了適于在植物中表達的其它啟動子(Rogers等，(1987)Meth，in Enzymol 153253-277；Schardl等，(1987)Gene 611-11；Berger等，(1989)Proc Natl Acad Sci USA 868402-8406)。
通常，本申請中描述的所有啟動子都可以使酮酶在本發(fā)明植物果實中表達。
本發(fā)明方法中特別優(yōu)選的是組成型，并且特別是果實特異性啟動子。
因此，本發(fā)明特別地涉及這樣的核酸構建體，該核酸構建體包含功能性連接的果實特異性啟動子，特別優(yōu)選上述果實特異性啟動子和編碼酮酶的核酸。
優(yōu)選地，使用例如，T.Maniatis，E.F.Fritsch和J.Sambrook，Molecular CloningA Laboratory Manual，Cold Spring HarborLaboratory，Cold Spring Harbor，NY(1989)中和T.J.Silhavy，M.L.Berman和L.W.Enquist，Experiments with Gene Fusions，Cold SpringHarbor Laboratory，Cold Spring Harbor，NY(1984)中和Ausubel，F.M.等，Current Protocols in Molecular Biology，Greene Publishing Assoc.andWiley-Interscience(1987)中所述的常規(guī)重組和克隆技術，通過融合適宜的啟動子和上述編碼酮酶的核酸和優(yōu)選地插在啟動子和核酸序列之間并且編碼質體特異性轉運肽的核酸以及聚腺苷酸化信號，制備表達盒。
編碼質體轉運肽并優(yōu)選插入的核酸將確保在質體中，特別是在色質體中定位。
也可以利用其核酸序列編碼酮酶融合蛋白的表達盒，其中融合蛋白的一部分是控制多肽轉運的轉運肽。優(yōu)選的是在酮酶轉運到色質體中后從酮酶部分上酶促切除的色質體特異性轉運肽。
特別優(yōu)選的是來源于煙草(Nicotiana tabacum)質體轉酮酶或來源于另一種轉運肽(例如，核酮糖二磷酸羧化酶-加氧酶小亞基(rbcS)轉運肽或鐵氧還蛋白-NADP氧化還原酶和異戊烯焦磷酸異構酶-2的轉運肽)的轉運肽，或者其功能等同物。
特別優(yōu)選的是以KpnI/BamHI片段形式處于3個讀框中的3個煙草質體轉酮酶質體轉運肽盒的核酸序列，其中ATG密碼子在NcoI切割位點中pTP09KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAGACTGCGGGATCC_BamHIpTP10KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAGACTGCGCTGGATCC_BamHIpTP11KpnI_GGTACCATGGCGTCTTCTTCTTCTCTCACTCTCTCTCAAGCTATCCTCTCTCGTTCTGTCCCTCGCCATGGCTCTGCCTCTTCTTCTCAACTTTCCCCTTCTTCTCTCACTTTTTCCGGCCTTAAATCCAATCCCAATATCACCACCTCCCGCCGCCGTACTCCTTCCTCCGCCGCCGCCGCCGCCGTCGTAAGGTCACCGGCGATTCGTGCCTCAGCTGCAACCGAAACCATAGAGAAAACTGAGACTGCGGGGATCC_BamHI質體轉運肽的其它例子是來源于擬南芥(Arabidopsis thaliana)的質體異戊烯焦磷酸異構酶-2(IPP-2)的轉運肽，和來源于豌豆的核酮糖二磷酸羧化酶小亞基(rbcS)轉運肽(Guerineau，F，Woolston，S，Brooks，L，Mullineaux，P(1988)An expression cassette for targeting foreign proteinsinto the chloroplasts.Nucl.Acids Res.1611380)。
本發(fā)明核酸可以合成產生，或者天然獲得，或者包含合成和天然核酸組分的混合物，由來源于各種生物體的各種異源基因片段組成。
優(yōu)選的是如上所述具有植物偏愛使用的密碼子的合成的核苷酸序列。從在大多數目的植物物種中表達的具有最高蛋白質頻率的密碼子可以確定植物偏愛的這些密碼子。
當制備表達盒時，可以操作處理各種DNA片段以獲得這樣的核苷酸序列，該核苷酸序列方便地以正確方向閱讀，并且裝配有正確的讀框。為了使DNA片段互相連接，可以給片段添加銜接頭或接頭。
方便地，可以以轉錄方向提供具有接頭或多接頭的啟動子和終止子區(qū)，其中該接頭或多接頭包含一個或多個限制位點用于該序列的插入。通常，接頭有1到10個，大多數情況下1到8個，優(yōu)選地2到6個限制位點。一般地，在調節(jié)區(qū)內，接頭有小于100bp，常常地小于60bp，但是至少是5bp的大小。啟動子相對于宿主植物而言可以是天然的、或同源的、或外源或異源的。優(yōu)選地，表達盒按5’-3’轉錄方向包含啟動子、編碼核酸序列或核酸構建體和轉錄終止區(qū)。可以按需要交換各種終止區(qū)。
終止子的例子是35S終止子(Guerineau等，(1988)Nucl Acids Res.1611380)，nos終止子(Depicker A，Stachel S，Dhases P，Zambryski P，Goodman HM.Nopaline synthasetranscript mapping and DNA sequence.J Mol Appl Genet.1982；1(6)561-73)或ocs終止子(Gielen，J，de Beuckeleer，M，Seurinck，J，Debroek，H，de Greve，H，Lemmers，M，Van Montagu，M，Schell，J(1984)The complete sequence of the TL-DNA of theAgrobacterium tumefaciens plasmid pTiAch5.EMBO J.3835-846)。
而且，可以利用操作提供適合的限制切割位點或移除多余的DNA或限制切割位點。當適宜進行插入、缺失或置換，諸如，轉換和顛換時，可以利用體外誘變、引物修補、限制切割或連接。
利用適宜的操作，諸如，限制、反嚼(chewing-back)或填平突出端獲得平端，可以提供用于連接的片段互補末端。
優(yōu)選的聚腺苷酸化信號是植物聚腺苷酸化信號，優(yōu)選基本對應于根瘤農桿菌(Agrobacterium tumefaciens)，特別是Ti質粒pTiACH5 T-DNA基因3(章魚氨酸合酶)的T-DNA聚腺苷酸化信號的聚腺苷酸化信號(Gielen等，EMBO J.3(1984)，835以及以下頁)，或功能性等同物。
外源基因向植物基因組中的轉移被稱為轉化。
為此，可以利用本身已知的用于轉化和從植物組織或植物細胞再生植物的方法，以實現瞬時或穩(wěn)定轉化。
植物轉化的適合方法是利用聚乙二醇誘導DNA攝取的原生質體轉化，利用基因槍的生物彈擊方法(其也稱為微粒轟擊方法)，電穿孔、在含有DNA的溶液中溫育干燥胚，顯微注射和上述農桿菌介導的基因轉移。例如，在B.Jenes等，Techniques for Gene Transfer，inTransgenic Plants，1卷，Engineering and Utilization，S.D.Kung和R.Wu編輯，AcademicPress(1993)，128-143中和在Potrykus，Annu.Rev.Plant Physiol.PlantMolec.Biol.42(1991)，205-225中描述了上述方法。
優(yōu)選地，將要表達的構建體克隆到適于根瘤農桿菌(Agrobacteriumtumefaciens)轉化的載體，例如，pBin19(Bevan等，Nucl.Acids Res.12(1984)，8711)或者特別優(yōu)選地pSUN2，pSUN3，pSUN4或pSUN5(WO02/00900)中。
可以用已知的方法使用已經被表達質粒轉化的農桿菌轉化植物，例如，通過在農桿菌溶液中浸浴劃破的葉或葉片段，隨后在適合的培養(yǎng)基中培養(yǎng)它們。
為了優(yōu)選地產生遺傳修飾植物，此后也稱為轉基因植物，將表達酮酶的融合表達盒克隆到載體，例如pBin19，或者特別是pSUN2中，所述載體適于被轉化到根瘤農桿菌中。
然后，可以用已知的方法使用已經被這種載體轉化的農桿菌，用于植物，特別是作物的轉化，例如，通過在農桿菌溶液中浸浴劃破的葉或葉片段，隨后在適合的培養(yǎng)基中培養(yǎng)它們。
尤其是從F.F.White，Vectors for Gene Transfer in Higher Plants；inTransgenic Plants，1卷，Engineering and Utilization，S.D.Kung和R.Wu編輯，Academic Press(1993)，15-38頁，可知如何用農桿菌轉化植物。從劃破的葉片或葉片片段的轉化細胞，用已知的方法可以再生轉基因植物，并且這種植物包含整合到表達盒中用于表達編碼酮酶的核酸的基因。
為了用編碼酮酶的核酸轉化宿主植物，表達盒做為插入物引入到重組載體中，該重組載體的載體DNA包含另外的功能性調節(jié)信號，例如用于復制或整合的序列。尤其在”Method in Plant Molecular Biology andBiotechnology”(CRC Press)，6/7章，71-119頁(1993)中描述了適合的載體。
利用上面引用的重組和克隆技術，可以將表達盒克隆到適合的載體中，這些載體將使得表達盒可以在例如大腸桿菌(E.coli)中操作。適合的克隆載體尤其是pJIT117(Guerineau等，(1988)Nucl Acids Res.1611380)，pBR332，pUC系列，M13mp系列，pACYC184，pMC1210，pMc1210和pCL1920。特別適合的是二元載體，它們在大腸桿菌(E.coli)中和農桿菌屬(Agrobacteria)中都能復制。
在本發(fā)明上下文中，根據啟動子的選擇，可以組成型地，或者優(yōu)選地特異性地在果實中發(fā)生表達。
因此，本發(fā)明還涉及遺傳修飾植物的生產方法，其中將核酸構建體引入起始植物的基因組中，該核酸構建體包含功能性連接的果實特異性啟動子和編碼酮酶的核酸。
本發(fā)明還涉及與起始植物比較，果實中具有酮酶活性的遺傳修飾植物。
在優(yōu)選的實施方式中，通過遺傳修飾植物在果實中表達酮酶的事實獲得酮酶活性。
因此，優(yōu)選的遺傳修飾植物果實中包含至少一個編碼酮酶的核酸。
在進一步優(yōu)選的實施方式中，如上所述，通過向起始植物中引入編碼酮酶的核酸來造成編碼酮酶的核酸的基因表達。
因此，特別優(yōu)選地，本發(fā)明涉及上述遺傳修飾植物，其中從起始植物開始，至少一個編碼酮酶的核酸已經被引入植物中。
特別地，本發(fā)明涉及選自下列植物的包含至少一個編碼酮酶的核酸的遺傳修飾植物皺子棕屬、亮絲草屬、風梨屬、草莓樹屬、假檳榔屬、Area、Aronia、天門冬屬、亞塔棕屬、小檗屬、Bixia、Brachychilum、Bryonia、Cliptocalix、辣椒屬、番木瓜屬、南蛇藤屬、西瓜屬、柑橘屬、鈴蘭屬、栒子屬、山楂屬、香瓜屬、南瓜屬、菟絲子屬、蘇鐵屬、樹番茄屬、薯蕷屬、柿屬、Dura、胡頹子屬、油棕屬、古柯屬、衛(wèi)矛屬、榕屬、金桔屬、草莓屬、梔子屬、瓊欖屬、棉屬、番石榴屬、刺棒棕屬、木槿屬、沙棘屬、鳶尾屬、山黧豆屬、忍冬屬、絲瓜屬、枸杞屬、番茄屬、金虎尾屬、芒果屬、Mormodica、九里香屬、芭蕉屬、能加棕屬、Palisota、露兜樹屬、西番蓮屬、鱷梨屬、酸漿屬、李屬、Ptychandra、石榴屬、火棘屬、梨屬、茶藨子屬、薔薇屬、懸鉤子屬、薩巴棕屬、接骨木屬、Seaforita、水牛果屬、茄屬、花楸屬、Synaspadix、Tabernae、Tamus、紅豆杉屬、栝樓屬、Triphasia、越桔屬、莢蒾屬、Vignia或葡萄屬。
非常特別優(yōu)選的植物屬是包含至少一種編碼酮酶的轉基因核酸的風梨屬、天門冬屬、辣椒屬、柑橘屬、香瓜屬、南瓜屬、西瓜屬、番茄屬、西番蓮屬、李屬、酸漿屬、茄屬、越桔屬和葡萄屬。
在優(yōu)選的轉基因植物中，如上所述，在果實中表達酮酶；特別優(yōu)選地，酮酶在果實中表達最高。
特別優(yōu)選的遺傳修飾植物與野生植物比較，如上所述還具有增加的羥化酶活性和/或β-環(huán)化酶活性。在上面本發(fā)明方法中描述了其它優(yōu)選的實施方式。
本發(fā)明還涉及轉基因植物，它們的繁殖材料和它們的植物細胞、組織或部分，特別是它們的果實。
如上所述，遺傳修飾植物可以用于酮類胡蘿卜素，特別是蝦青素的生產。
也可以例如直接或者在本身已知的加工后將人類和動物可以消耗的并且具有增加的酮類胡蘿卜素含量的本發(fā)明遺傳修飾植物用做食品或飼料，或者用做食品或飼料增補劑。而且，遺傳修飾的植物可以用于生產包含酮類胡蘿卜素的植物提取物，和/或用于生產飼料和食品增補劑。
遺傳修飾的植物與野生型比較具有增加的酮類胡蘿卜素含量。
通常，增加的酮類胡蘿卜素含量理解為意指增加的總酮類胡蘿卜素含量。
然而，增加的酮類胡蘿卜素含量也可以理解為特別意指優(yōu)選的酮類胡蘿卜素的含量改變，而總類胡蘿卜素含量不必增加。
在特別優(yōu)選的實施方式中，本發(fā)明遺傳修飾的植物與野生型比較，具有增加的蝦青素含量。
在這種情況下，增加的含量特別地理解為意指產生的酮類胡蘿卜素或蝦青素的含量。
具體實施例方式
現在通過下面的實施例示例本發(fā)明，但是本發(fā)明不限于這些實施例。
一般的實驗條件重組DNA的序列分析按照Sanger方法(Sanger等，Proc.Natl.Acad.Sci.USA 74(1977)，5463-5467)，利用Licor的激光熒光DNA測序儀(從MWG Biotech，Ebersbach可以購得)測序重組DNA分子。
實施例1從雨生紅球藻flotow em.Wille擴增編碼酮酶完整一級序列的cDNA用PCR方法，從雨生紅球藻(“Sammlung von Algenkulturen derUniversit_t G_ttingen”(G_ttingen大學的藻類培養(yǎng)物保藏中心)的株系192.80)懸浮培養(yǎng)物擴增編碼來源于雨生紅球藻的酮酶的cDNA。
為了從已經在室溫下紅球藻培養(yǎng)基(1.2g/l醋酸鈉，2g/l酵母提取物，0.2g/l MgCl2×6H2O，0.02 CaCl2×2H2O；pH6.8；高壓滅菌后，添加400mg/lL-天冬酰胺，10mg/l FeSO4×H2O)中用間接日照(indirect daylight)培養(yǎng)2周的雨生紅球藻(株系192.80)懸浮培養(yǎng)物制備總RNA，收獲細胞，在液氮中冷凍，并且在研缽中研磨成粉末。此后，將100mg冷凍粉碎的藻細胞轉移到反應容器中，加入0.8ml Trizol緩沖液(LifeTechnologies)。用0.2ml氯仿萃取懸浮液。在12000g離心15分鐘后，取出水性上清液，轉移到新的反應容器中，并且用1體積乙醇萃取。用1體積異丙醇沉淀RNA，用75％乙醇洗滌，并且將沉淀溶解在DEPC水(在室溫下，用1/1000體積焦碳酸二乙酯過夜溫育水，然后高壓滅菌)中。光度計地測定RNA濃度。
對于cDNA合成，60℃變性2.5μg總RNA 10分鐘，在冰上冷卻2分鐘，之后利用cDNA試劑盒(Ready-to-go-you-prime-beam，PharmaciaBiotech)，按照制造商的說明書，并且利用反義特異性引物(PR1 SEQ IDNO.29)轉錄為cDNA。
利用有義特異性引物(PR2 SEQ ID NO.30)和反義特異性引物(PR1SEQ ID NO.29)，用聚合酶鏈式反應(PCR)從雨生紅球藻擴增編碼來源于雨生紅球藻(株系192.80)的酮酶的核酸。
PCR條件如下在50μl反應混合物中進行擴增cDNA的PCR，該cDNA編碼由完整一級序列組成的酮酶蛋白質，該反應混合物包含-(如上所述制備的)4μl雨生紅球藻cDNA-0.25mM dNTPs-0.2mM PR1(SEQ ID NO.29)-0.2mM PR2(SEQ ID NO.30)-5μl10×PCR緩沖液(TAKARA)-0.25μlR Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘53℃2分鐘72℃3分鐘
1× 72℃10分鐘。
用SEQ ID NO.29和SEQ ID NO.30進行的PCR擴增產生了1155bp片段，其編碼由完整一級序列(SEQ ID NO.22)組成的蛋白質。利用標準方法，將擴增產物克隆到PCR克隆載體pGEM-Teasy(Promega)中，產生克隆pGKETO2。
用T7和SP6引物測序克隆pGKETO2，證實了只有3個密碼子73，114和119中各有一個堿基不同于公開的序列X86782的序列。在獨立擴增試驗中再現了這些核苷酸置換，因此代表在所用雨生紅球藻株系192.80中的核苷酸序列(圖3和4，序列比對)。
因此，該克隆用于克隆到表達載體pJIT117中(Guerineau等，(1988)Nucl Acids Res.1611380)。通過從pGKETO2分離1027bp SpHI片段，并且連接到SpHI切割的載體pJIT117中實現克隆。包含正確方向的雨生紅球藻酮酶基因的克隆稱為pJKETO2，其中該酮酶基因在N末端翻譯融合rbcs轉運肽序列。
實施例2擴增編碼來源于雨生紅球藻(Haematococcus pluvialis Flotowem.Wille)的酮酶的cDNA，其中該酮酶在N末端被截短了14個氨基酸通過PCR，從雨生紅球藻懸浮液培養(yǎng)物(“Sammlung vonAlgenkulturen der Universit_t G_ttingen”的株系192.80)擴增編碼來源于雨生紅球藻(株系192.80)的酮酶的cDNA，該酮酶在N末端被截短了14個氨基酸。
如實施例1中所述，從雨生紅球藻(株系192.80)懸浮液培養(yǎng)物進行總RNA制備。
如實施例1中所述進行cDNA合成。
用聚合酶鏈式反應(PCR)，利用有義特異性引物(PR3 SEQ ID NO.31)和反義特異性引物(PR1 SEQ ID NO.29)，從雨生紅球藻擴增編碼來源于雨生紅球藻(株系192.80)的酮酶的核酸，其中該酮酶在N末端被截短了14個氨基酸。
PCR反應條件如下在50μl反應混合物中進行擴增cDNA的PCR，該cDNA編碼在N末端被截短14個氨基酸的酮酶蛋白質，該反應混合物包含-(如上所述制備的)4μl雨生紅球藻cDNA-0.25mM dNTPs-0.2mM PR1(SEQ ID NO.29)-0.2mM PR2(SEQ ID NO.31)-5μl10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘53℃2分鐘72℃3分鐘1× 72℃10分鐘。
用SEQ ID NO.29和SEQ ID NO.31進行PCR擴增產生了編碼酮酶蛋白質的1111bp片段，在該酮酶蛋白質中，用單一氨基酸(亮氨酸)置換了N末端氨基酸(位置2-16)。
利用標準方法，將擴增產物克隆到PCR克隆載體pGEM-Teasy(Promega)中，并且獲得克隆pGKETO3。利用引物T7和SP6進行的測序反應證實了與序列SEQ ID NO.22相同的序列，在擴增產物SEQ IDNO.24中SEQ ID NO.22的5’區(qū)(位置1-53)已經被一個序列不同的九聚體序列置換。因此，該克隆用于克隆到表達載體pJIT117中(Guerineau等，(1988)Nucl Acids Res.1611380)。
通過從pGKETO3分離985bp SpHI片段，并且連接SpHI切割的載體pJIT117進行克隆。以正確方向包含在N末端截短了14個氨基酸的雨生紅球藻酮酶的克隆稱為pJKETO3，該酮酶在N末端翻譯融合rbcs轉運肽序列。
實施例3擴增編碼來源于雨生紅球藻(Haematococcus pluvialis Flotowem.Wille)(“Sammlung von Algenkulturen der Universit_t G_ttingen”的株系192.80)的酮酶的cDNA，該cDNA由完整的一級序列組成并融合了C末端myc標簽。
利用質粒pGKETO2(如實施例1中所述)和引物PR15(SEQ IDNO.32)，通過PCR方法制備編碼來源于雨生紅球藻的酮酶的cDNA，該cDNA由完整的一級序列組成并融合了C末端myc標簽。引物PR15由反義特異性3’區(qū)(核苷酸40-59)和編碼myc標簽的5’區(qū)(核苷酸1-39)構成。
在11.5μl反應混合物中進行pGKETO2和PR15的變性(95℃，5分鐘)和退火(在室溫下慢慢冷卻到40℃)，該反應混合物包含-1μg pGKETO2質粒DNA-0.1μg PR15(SEQ ID NO.32)。
在20μl反應混合物中補平3’末端(30℃，30分鐘)，該反應混合物包含-(如上所述制備的)11.5μl pGKETO2/PR15退火反應物-50μM dNTPs-2μl 1×Klenow緩沖液-2U Klenow酶。
利用有義特異性引物(PR2 SEQ ID NO.30)和反義特異性引物(PR15SEQ ID NO.32)，通過聚合酶鏈式反應(PCR)方法，從雨生紅球藻擴增編碼來源于雨生紅球藻(株系192.80)的酮酶的核酸，該核酸由完整的一級序列組成并融合了C末端myc標簽。
PCR條件如下在50μl反應混合物中進行擴增cDNA的PCR，該cDNA編碼具有融合的C-末端myc標簽的酮酶蛋白質，該反應混合物包含
-(如上所述制備的)1μl退火的反應物-0.25mM dNTPs-0.2μM PR15(SEQ ID NO.32)-0.2μM PR2(SEQ ID NO.30)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘53℃1分鐘72℃1分鐘1× 72℃10分鐘。
用SEQ ID NO.32和SEQ ID NO.30進行PCR擴增產生了編碼蛋白質的1032bp片段，該蛋白質由來源于雨生紅球藻的酮酶的完整一級序列組成，具有在N末端與rbcs轉運肽和在C末端與myc標簽的雙翻譯融合。
利用標準方法，將擴增產物克隆到PCR克隆載體pGEM-Teasy(Promega)中，并且獲得克隆pGKETO4。利用引物T7和SP6進行的測序反應證實了與序列SEQ ID NO.22相同的序列，在擴增產物SEQ IDNO.26中SEQ ID NO.22的3’區(qū)(位置993-1155)已經被不同的39bp序列置換。因此，該克隆用于克隆到表達載體pJIT117中(Guerineau等，(1988)Nucl Acids Res.1611380)。
通過從pGKETO4分離1038bp EcoRI/SpHI片段，并且連接到EcoRI-SpHI切割的載體pJIT117中實現克隆。這種連接在rbcS轉運肽序列C末端和酮酶序列N末端之間產生了翻譯融合。以正確方向包含具有融合的C末端myc標簽的雨生紅球藻酮酶的克隆稱為pJKET4，其中該酮酶在N末端翻譯融合rbcs轉運肽。
實施例4
用于番茄中雨生紅球藻酮酶組成型表達的表達載體的制備番茄(L.esculentum)中和萬壽菊(Tagetes erecta)中來源于雨生紅球藻的酮酶的表達受來源于CaMV的組成型啟動子d35S(Franck等，1980，Cell 21285-294)控制。用來源于豌豆的轉運肽rbcs(Anderson等，1986，Biochem J.240709-715)進行表達。
利用二元載體pSUN3(WO 02/00900)制備表達質粒，該表達質粒用于在番茄(L.esculentum)中實現農桿菌介導的雨生紅球藻酮酶的轉化。
為了制備表達載體pS3KETO2，連接來自于pJKETO2的2.8kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(圖5，構建體圖譜)。在圖5中，片段d35S包含加倍的35S啟動子(747bp)，片段rbcS包含來源于豌豆的rbcS轉運肽(204bp)，片段KETO2編碼雨生紅球藻酮酶的完整一級序列(1027bp)，片段term(761bp)包含CaMV聚腺苷酸化信號。
為了制備表達載體pS3KETO3，連接來自于pJKETO3的2.7kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(圖6，構建體圖譜)。在圖6中，片段d35S包含加倍的35S啟動子(747bp)，片段rbcS包含來源于豌豆的rbcS轉運肽(204bp)，片段KETO3(985bp)編碼截短了14個氨基酸的雨生紅球藻酮酶的一級序列，片段term(761bp)包含CaMV聚腺苷酸化信號。
為了制備表達載體pS3KETO4，連接來自于pJKETO4的2.8kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(圖7，構建體圖譜)。在圖7中，片段d35S包含加倍的35S啟動子(747bp)，片段rbcS包含來源于豌豆的rbcS轉運肽(204bp)，片段KETO4(1038bp)編碼具有C末端myc標簽的雨生紅球藻酮酶的完整一級序列，片段term(761bp)包含CaMV聚腺苷酸化信號。
實施例5制備用于番茄中雨生紅球藻酮酶表達的表達載體利用來源于豌豆的轉運肽rbcS(Anderson等，1986，Biochem J.
240709-715)，在番茄(L.esculentum)中表達來源于雨生紅球藻的酮酶。表達受擬南芥(Arabidopsis thaliana)啟動子AP3的修飾形式AP3P控制(AL132971核苷酸區(qū)9298-10200；Hill等，(1998)Development1251711-1721)。
利用(用標準方法從擬南芥分離的)基因組DNA和引物PR7(SEQ IDNO.33)和PR10(SEQ ID NO.36)，通過PCR方法制備包含來源于擬南芥(Arabidopsis thaliana)的AP3啟動子區(qū)-902到+15的DNA片段。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA包含AP3啟動子片段(-902到+15)，該反應混合物包含-100ng來源于擬南芥(Arabidopsis thaliana)的基因組DNA-0.25mM dNTPs-0.2mM PR7(SEQ ID NO.33)-0.2mM PR10(SEQ ID NO.36)-5μl 10×PCR緩沖液(Stratagene)-0.25μl Pfu聚合酶(Stratagene)-28.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘50℃1分鐘72℃1分鐘1× 72℃10分鐘。
利用標準方法，將922bp擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中，產生質粒pTAP3。
測序克隆pTAP3證實了不同于公開的AP3序列(AL132971，核苷酸區(qū)9298-10200)的序列，不同之處只在于一個插入(在序列AL132971位置9765中一個G)和一個堿基置換(在序列AL132971位置9726中一個G代替了一個A)。在獨立擴增試驗中再現了這些核苷酸差異，因此這些核苷酸差異代表了所用擬南芥(Arabidopsis thaliana)植物中實際的核苷酸序列。
利用質粒pTAP3，通過重組PCR方法制備修飾形式的AP3P。利用引物PR7(SEQ ID NO.33)和PR9(SEQ ID NO.35)擴增區(qū)域10200-9771(擴增產物A7/9)，利用引物PR8(SEQ ID NO.34)和PR10(SEQ ID NO.36)擴增區(qū)域9526-9285(擴增產物A8/10)。
PCR條件如下在50μl成批反應混合物中進行擴增DNA片段的PCR反應，該DNA片段包含AP3啟動子的區(qū)域10200-9771和區(qū)域9526-9285，該反應混合物包含-(上述)100ng AP3擴增產物-0.25mM dNTPs-0.2mM有義引物(分別是PR7 SEQ ID NO.33和PR8 SEQ ID NO.35)-0.2mM反義引物(分別是PR9 SEQ ID NO.35和PR10 SEQ ID NO.36)-5μl 10×PCR緩沖液(Stratagene)-0.25μl Pfu Taq聚合酶(Stratagene)-28.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘50℃1分鐘72℃1分鐘1× 72℃10分鐘。
重組PCR包括擴增產物A7/9和A8/10的退火(它們重疊25個核苷酸序列，互補產生雙鏈)，及隨后的擴增。這產生了修飾形式的AP3啟動子，即，AP3P，其中缺失位置9670-9526。在17.6μl反應混合物中進行兩種擴增產物A7/9和A8/10的變性(95℃，5分鐘)和退火(在室溫下慢慢地冷卻到40℃)，該反應混合物包含-0.5μg A7/9擴增產物-0.25μg A8/10擴增產物在20μl反應混合物中補平3’末端(30℃，30分鐘)，該反應混合物包含-(如上所述制備的)17.6μl A7/9和A8/10退火反應物-50μM dNTPs-2μl 1×Klenow緩沖液-2U Klenow酶。
利用有義特異性引物(PR7 SEQ ID NO.28)和反義特異性引物(PR10SEQ ID NO.36)，通過PCR方法擴增編碼修飾啟動子AP3P的核酸。
PCR條件如下在50μl反應混合物中進行擴增AP3P片段的PCR，該反應混合物包含-(如上所述制備的)1μl退火反應物-0.25mM dNTPs-0.2mM PR7(SEQ ID NO.33)-0.2mM PR10(SEQ ID NO.36)-5μl 10×PCR緩沖液(Stratagene)-0.25μl Pfu Taq聚合酶(Stratagene)-28.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘50℃1分鐘72℃1分鐘1× 72℃10分鐘。
用SEQ ID NO.33和SEQ ID NO.36進行PCR擴增產生了編碼修飾啟動子AP3P的778bp片段。將擴增產物克隆到克隆載體pCR2.1(Invitrogen)中，并且獲得了克隆pTAP3P。用引物T7和M13進行的測序反應證實了與序列AL132971，區(qū)域10200-9298具有同一性并同時缺失了內部區(qū)域9285-9526的序列。因此，該克隆用于克隆到表達載體pJIT117中(Guerineau等，(1988)Nucl Acids Res.1611380)。
通過從pTAP3P分離771bp SacI/HindIII片段并且連接到SacI/HindIII切割的載體pJIT117中進行克隆。包含啟動子AP3P而不是原始啟動子d35S的克隆稱為pJAP3P。
為了制備表達盒pJAP3PKETO2，將1027bp SpHI片段KETO2(實施例1中所述)克隆到SpHI切割的載體pJAP3P中。以正確方向包含片段KETO2的克隆稱為pJAP3PKETO2，其中該片段KETO2在N末端融合rbcS轉運肽。
為了制備表達盒pJAP3PKETO4，將1032bp SpHI/EcoRI片段KETO4(實施例3中所述)克隆到SpHI/EcoRI切割的載體pJAP3P中。以正確方向包含片段KETO4的克隆稱為pJAP3PKETO4，該片段KETO4在N末端融合rbcS轉運肽上。
利用二元載體pSUN3(WO 02/00900)制備表達載體，該表達載體用于在番茄(L.esculentum)中進行農桿菌介導的來源于雨生紅球藻的受AP3P控制的酮酶的轉化。
為了制備表達載體pS3AP3PKETO2，連接來自于pJAP3KETO2的2.8kb SacI/XhoI片段和SacI-XhoI切割的載體pSUN3(圖8，構建體圖譜)。在圖8中，片段AP3P包含修飾的AP3P啟動子(771bp)，片段rbcS包含來源于豌豆的rbcS轉運肽(204bp)，片段KETO2(1027bp)編碼雨生紅球藻酮酶的完整一級序列，片段term(761bp)包含CaMV聚腺苷酸化信號。
為了制備表達載體pS3AP3PKETO4，連接來自于pJAP3KETO4的2.8kb SacI/XhoI片段和SacI-XhoI切割的載體pSUN3(圖9，構建體圖譜)。在圖9中，片段AP3P包含修飾的AP3P啟動子(771bp)，片段rbcS包含來源于豌豆的rbcS轉運肽(204bp)，片段KETO4(1038bp)編碼具有C-末端myc標簽的雨生紅球藻酮酶的完整一級序列，片段term(761bp)包含CaMV聚腺苷酸化信號。
實施例6轉基因番茄植物的產生用Ling及其同事(Plant Cell Reports(1998)，17843-847)公開的方法轉化和再生番茄植物。較高的卡那霉素濃度(100mg/l)用于品種Microtom的篩選。
用于轉化的起始外植體是品系Microtom 7到10天齡幼苗的子葉和下胚軸。補加有2％蔗糖的Murashige和Skoog培養(yǎng)基(1962Murashige和Skoog，1962，Physiol.Plant 15，473-)，pH6.1，用于萌發(fā)。在21℃，在低光水平(20到100μE)萌芽。7到10天后，子葉被水平地分開，下胚軸被切割成5到10mm長的片段，并且置放在培養(yǎng)基MSBN(MS，pH6.1，3％蔗糖+1mg/l BAP，0.1mg/l NAA)上，在前一天該培養(yǎng)基已經加載了懸浮培養(yǎng)基中培養(yǎng)的煙草細胞。用無菌濾紙覆蓋煙草細胞，不讓氣泡產生。在上述培養(yǎng)基上預培養(yǎng)外植體3到5天。分別用質粒pS3KETO2，pS3KETO3和pS3AP3KETO2單獨轉化根瘤農桿菌株系LBA4404的細胞。在每種情況下，在28攝氏度，在添加有卡那霉素(20mg/l)的YEP培養(yǎng)基中，培養(yǎng)已經分別被二元載體pS3KETO2和pS3KETO3轉化的各農桿菌株系的過夜培養(yǎng)物，并且離心細胞。在液體MS培養(yǎng)基(3％蔗糖，pH6.1)中重懸浮細菌沉淀，并且產生0.3的光密度(在600nm)。將預培養(yǎng)的外植體轉移到懸浮液中，并且在室溫下溫育30分鐘，同時輕輕振蕩。此后，用無菌濾紙干燥外植體，并且將它們放回預培養(yǎng)培養(yǎng)基進行3天共培養(yǎng)(21℃)。
共培養(yǎng)后，將外植體轉移到MSZ2培養(yǎng)基(MS pH6.1+3％蔗糖，2mg/l玉米素，100mg/l卡那霉素，160mg/l特美汀(Timentin))，并且在21℃，保藏在低光條件下(20到100μE，16h/8h光周期)進行選擇性再生。在形成芽之前，每2到3周轉移外植體一次。從外植體分離小芽，并且在MS(pH6.1，3％蔗糖)，160mg/l特美汀，30mg/l卡那霉素，0.1mg/l IAA上生根。將生根的植物轉移到溫室。
根據上述轉化方法，用下面的表達構建體獲得了下面的株系
用pS3KETO2獲得了cs13-24，cs13-30，cs13-40。
用pS3KETO3獲得了cs14-2，cs14-3，cs14-9，cs14-19。
用pS3AP3PKETO2獲得了cs16-15，cs16-34，cs16-35，cs16-40。
實施例8轉基因果實的表征在液氮中粉碎轉基因植物的果實材料，用100％丙酮(3部分，每部分500μl)提取粉末(約250-500mg)。蒸發(fā)溶劑，并且在100μl丙酮中重懸浮類胡蘿卜素。
利用C30反相柱子可以區(qū)分類胡蘿卜素的單和雙酯。按照公開的方法(Frazer等，(2000)，Plant Journal 24(4)551-558)，修改了HPLC的條件。建立了下面HPLC條件分離柱Prontosil C30柱子，250×4.6mm(Bischoff，Leonberg，德國)流速1.0ml/分鐘洗脫液洗脫液A-100％甲醇洗脫液B-80％甲醇，0.2％醋酸銨洗脫液C-100％叔丁基甲基醚梯度曲線

檢測300-530nm。
利用光電二極管陣列檢測器讀取光譜。通過與標準樣品比較，利用類胡蘿卜素的吸收光譜和保留時間鑒定類胡蘿卜素。
表1顯示根據上述實施例產生的轉基因番茄和對照番茄植物的番茄果實中類胡蘿卜素的分布情況。與遺傳未修飾的對照植物比較，遺傳修飾的植物顯示了酮類胡蘿卜素含量，特別是蝦青素含量。
表1

+表示可檢測到類胡蘿卜素；-表示沒有檢測到類胡蘿卜素；(+)表示類胡蘿卜素濃度在檢測限值。
表2a顯示轉基因番茄和對照植物成熟果實中類胡蘿卜素的量。該數據為不同品系的平均值，并且表示為總類胡蘿卜素含量的百分數。

表2b顯示轉基因番茄和對照植物成熟果實中類胡蘿卜素的量。該數據為不同品系的平均值，并且表示為總類胡蘿卜素含量的百分數。

實施例9擴增編碼來源于點形念珠藻ATCC 29133之NP196酮酶完整一級序列的DNA用PCR方法，從點形念珠藻ATCC 29133(美國典型培養(yǎng)物保藏中心(ATCC)的株系)擴增編碼來源于點形念珠藻ATCC 29133的NP196酮酶的DNA。
為了從在25℃下BG11培養(yǎng)基(1.5g/l NaNO3，0.04g/l K2PO4×3H2O，0.075g/l MgSO4×H2O，0.036g/l CaCl2×2H2O，0.006g/l檸檬酸，0.006g/l檸檬酸鐵銨，0.001g/l EDTA二鈉鎂，0.04g/l Na2CO3，1ml痕量金屬混合物“A5+Co”，2.86g/l H3BO3，1.81g/l MnCl2×4H2O，0.222g/l ZnSO4×7H2O，0.39g/l NaMoO4×2H2O，0.079g/l CuSO4×5H2O，0.0494g/lCo(NO3)2×6H2O)中在連續(xù)振蕩(150rpm)及連續(xù)光照條件下已經培養(yǎng)1周的點形念珠藻ATCC 29133懸浮培養(yǎng)物制備基因組DNA，通過離心收集細胞，在液氮中冷凍并且在研缽中研磨成粉末。
從點形念珠藻ATCC 29133分離DNA的方法通過在8000rpm離心10分鐘從10ml液體培養(yǎng)物沉淀細菌細胞。此后，利用研杵和研缽，在液氮中弄碎和研磨細菌細胞。在1ml 10mM Tris-HCl(pH7.5)中重懸浮細胞材料，并且轉移到Eppendorf反應容器(體積2ml)中。添加100μl蛋白酶K(濃度20mg/ml)后，在37℃溫育細胞懸浮液3小時。此后，用500μl苯酚提取細胞懸浮液。在13000rpm離心5分鐘后，將水性上層相轉移到新的2ml Eppendorf反應容器中。重復苯酚提取3次。通過添加1/10體積3M醋酸鈉(pH5.2)和0.6體積異丙醇沉淀DNA，并且隨后用70％乙醇進行洗滌。在室溫下干燥DNA沉淀，加入25μl水，并且在65℃加熱溶解。
利用有義特異性引物(NP196-1，SEQ ID NO.59)和反義特異性引物(NP196-2，SEQ ID NO.60)，通過聚合酶鏈式反應(PCR)，從點形念珠藻ATCC 29133擴增編碼來源于點形念珠藻ATCC 29133的酮酶的核酸。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA編碼由完整一級序列組成的酮酶蛋白質，該反應混合物包含-(如上所述制備的)1μl點形念珠藻ATCC 29133 DNA-0.25mM dNTPs-0.2mM NP196-1(SEQ ID NO.59)-0.2mM NP196-2(SEQ ID NO.60)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘55℃1分鐘72℃3分鐘1× 72℃10分鐘。
用SEQ ID NO.59和SEQ ID NO.60進行PCR擴增產生了編碼蛋白質的792bp片段，該蛋白質由完整一級序列(NP196，SEQ ID NO.61)組成。利用標準方法，將擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中，產生克隆pNP196。
用M13F和M13R引物測序克隆pNP196證實了除了以下不同之處外與數據庫登記NZ_AABC01000196的140.571-139.810 DNA序列相同的序列(反向于此公開的數據庫登記序列)，不同之處在于用A置換了位置140.571中的G以產生標準的ATG起始密碼子。在獨立擴增試驗中再現了該核苷酸序列，由此代表所用的點形念珠藻ATCC 29133中的核苷酸序列。
因此，該克隆pNP196用于克隆到如實施例5中所述的表達載體pJAP3P中。
通過用根瘤農桿菌Ti質粒pTi15955的(章魚氨酸合酶)OCS終止子(數據庫登記X00493，位置12.541-12.350，Gielen等，(1984)EMBO J.3835-846)置換35S終止子修飾pJAP3P。
利用質粒pHELLSGATE(數據庫登記AJ311874，Wesley等，(2001)Plant J.27 581-590，用標準方法從大腸桿菌分離的)和引物OCS-1(SEQID NO.63)和OCS-2(SEQ ID NO.64)，通過PCR方法制備包含OCS終止子區(qū)的DNA片段。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA包含章魚氨酸合酶(OCS)終止子區(qū)(SEQ ID NO.65)，該反應混合物包含-100ng pHELLSGATE質粒DNA-0.25mM dNTPs-0.2mM OCS-1(SEQ ID NO.63)-0.2mM OCS-2(SEQ ID NO.64)-5μl10×PCR緩沖液(Stratagene)-0.25μl Pfu聚合酶(Stratagene)-28.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘50℃1分鐘
72℃1分鐘1× 72℃10分鐘。
利用標準方法，將210bp擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中，產生質粒pOCS。
克隆pOCS的測序證實了與根瘤農桿菌Ti質粒pTi15955上序列片段(數據庫登記X00493)位置12.541到12.350一致的序列。
通過從pOCS分離210bp SalI/XhoI片段，并且連接到SalI/XhoI切割的載體pJAP3P中進行克隆。
該克隆稱為pJOAP，因此用于克隆到表達載體pJOAPNP196中。
通過從pNP196分離782bp SphI片段，并且連接到SphI切割的載體pJOAP中實現克隆。以正確方向包含點形念珠藻NP196酮酶的克隆稱為pJOAPNP196，其中該點形念珠藻NP196酮酶在N末端翻譯融合rbcS轉運肽。
實施例10用于在番茄中果實特異性超表達NP196酮酶的表達載體的制備，該NP196酮酶來源于點形念珠藻ATCC 29133(美國典型培養(yǎng)物保藏中心(ATCC)的株系)利用來源于豌豆的轉運肽rbcS(Anderson等，1986，Biochem J.240709-715)，在番茄(L.esculentum)中表達來源于點形念珠藻的NP196酮酶。在如實施例5中所述來源于擬南介的啟動子AP3P控制下實現表達。
利用二元載體pSUN3(WO 02/00900)制備表達載體，該表達載體用于農桿菌屬介導的AP3P控制的來源于點形念珠藻ATCC 29133的NP196酮酶向番茄(L.esculentum)中的轉化。
為了制備表達載體MSP20，連接來自于pJOAPNP196的1.958kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(


圖10，構建體圖譜)。在
圖10中，片段AP3P PROM包含AP3P啟動子(765bp)，片段rbcS TPFragment包含來源于豌豆的rbcS轉運肽(194bp)，片段NP196 KETO CDS(761bp)編碼點形念珠藻NP196酮酶，片段OCS終止子(192bp)包含章魚氨酸合酶聚腺苷酸化信號。
實施例11擴增編碼來源于念珠藻PCC 7120的酮酶完整一級序列的DNA通過PCR方法，從念珠藻PCC 7120(巴斯德藍細菌培養(yǎng)物保藏中心的株系)擴增編碼來源于點形念珠藻PCC 7120的NOST酮酶的DNA。
為了從在25℃下BG11培養(yǎng)基(1.5g/l NaNO3，0.04g/l K2PO4×3H2O，0.075g/l MgSO4×H2O，0.036g/l CaCl2×2H2O，0.006g/l檸檬酸，0.006g/l檸檬酸鐵銨，0.001g/l EDTA二鈉鎂，0.04g/l Na2CO3，1ml痕量金屬混合物“A5+Co”(2.86g/l H3BO3，1.81g/l MnCl2×4H2O，0.222g/l ZnSO4×7H2O，0.39g/l NaMoO4×2H2O，0.079g/l CuSO4×5H2O，0.0494g/lCo(NO3)2×6H2O)中，在連續(xù)振蕩(150rpm)及連續(xù)光照條件下已經培養(yǎng)1周的念珠藻PCC 7120懸浮培養(yǎng)物制備基因組DNA，通過離心收集細胞，在液氮中冷凍并且在研缽中研磨成粉末。
從念珠藻PCC 7120分離DNA的方法通過在8000rpm離心10分鐘從10ml液體培養(yǎng)物沉淀細菌細胞。此后，利用研缽，在液氮中粉碎和研磨細菌細胞。在1ml 10mM Tris-HCl(pH7.5)中重懸浮細胞材料，并且轉移到Eppendorf反應容器(體積2ml)中。添加100μl蛋白酶K后(濃度20mg/ml)，在37℃溫育細胞懸浮液3小時。以后，用500μl苯酚提取懸浮液。在13000rpm離心5分鐘后，將含水的上層相轉移到新的2ml Eppendorf反應容器。重復苯酚提取3次。通過添加1/10體積3M醋酸鈉(pH5.2)和0.6體積異丙醇沉淀DNA，并且隨后用70％乙醇進行洗滌。在室溫下干燥DNA沉淀，加入25μl水，并且在65℃加熱溶解。
利用有義特異性引物(NOST-1，SEQ ID NO.66)和反義特異性引物(NOST-2，SEQ ID NO.67)，通過聚合酶鏈式反應(PCR)，從念珠藻PCC7120擴增編碼來源于念珠藻PCC 7120的酮酶的核酸。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA編碼由完整一級序列組成的酮酶蛋白質，該反應混合物包含-(如實施例9中所述制備的)1μl念珠藻PCC 7120 DNA-0.25mM dNTPs-0.2mM NOST-1(SEQ ID NO.66)-0.2mM NOST-2(SEQ ID NO.67)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘55℃1分鐘72℃3分鐘1× 72℃10分鐘。
用SEQ ID NO.66和SEQ ID NO.67進行PCR擴增產生了編碼由完整一級序列(SEQ ID NO.68)組成的蛋白質的809bp片段。利用標準方法，將擴增產物克隆到PCR克隆載體pGEM-T(Promega)中，產生克隆pNOST。
用M13F和M13R引物測序克隆pNOST證實了與數據庫登記AP003592 DNA序列相同的序列。在獨立擴增試驗中再現了該核苷酸序列，因此代表了所用的念珠藻PCC 7120中的核苷酸序列。
因此，該克隆pNOST用于克隆到表達載體pJOAP(如實施例9中所述)中。
通過從pNOST分離799bp SphI片段，并且連接到SphI切割的載體pJOAP中實現克隆。包含正確方向的來源于念珠藻PCC 7120的NOST酮酶的克隆稱為pJOAPNOST，該酮酶在N末端翻譯融合rbcS轉運肽。
實施例12用于在番茄中果實特異性超表達來源于念珠藻PCC 7120的NOST酮酶的表達載體的制備用來源于豌豆的轉運肽rbcS(Anderson等，1986，Biochem J.240709-715)，在番茄(L.esculentum)中表達來源于念珠藻PCC 7120的NOST酮酶。在來源于擬南介的啟動子AP3P(如實施例5中所述)控制下實現該表達。
利用二元載體pSUN3(WO 02/00900)產生表達載體，該表達載體用于農桿菌屬介導的AP3P控制的來源于念珠藻PCC 7120的NOST酮酶向番茄(L.esculentum)的轉化。
為了產生表達載體MSP121，連接來自于pJOAPNOST的1.982kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(
圖11，構建體圖譜)。在
圖11中，片段AP3P PROM包含AP3P啟動子(765bp)，片段rbcS TPFrag ment包含來源于豌豆的rbcS轉運肽(194bp)，片段NOST KETO CDS(774bp)編碼念珠藻PCC 7120 NOST酮酶，片段OCS終止子(192bp)包含章魚氨酸合酶聚腺苷酸化信號。
實施例13擴增編碼來源于點形念珠藻ATCC 29133的NP195酮酶完整一級序列的DNA通過PCR方法，從點形念珠藻ATCC 29133(美國典型培養(yǎng)物保藏中心(ATCC)株系)擴增編碼來源于點形念珠藻ATCC 29133的NP195酮酶的DNA。如實施例9中所述從點形念珠藻ATCC 29133懸浮液培養(yǎng)物制備基因組DNA。
通過聚合酶鏈式反應(PCR)方法，利用有義特異性引物(NP195-1 SEQID NO.70)和反義特異性引物(NP195-2，SEQ ID NO.71)，從點形念珠藻ATCC 29133擴增編碼來源于點形念珠藻ATCC 29133的酮酶的核酸。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA編碼由完整一級序列組成的酮酶蛋白質，該反應混合物包含-(如實施例9中所述制備的)1μl點形念珠藻ATCC 29133 DNA
-0.25mM dNTPs-0.2mM NP195-1(SEQ ID NO.70)-0.2mM NP195-2(SEQ ID NO.71)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘55℃1分鐘72℃3分鐘1× 72℃10分鐘。
用SEQ ID NO.70和SEQ ID NO.71進行PCR擴增產生了編碼蛋白質的819bp片段，該蛋白質由完整一級序列(SEQ ID NO.72)組成。利用標準方法，將擴增產物克隆到PCR克隆載體pGEM-T(Promega)中，獲得克隆pNP195。
用M13F和M13R引物測序克隆pNP195證實了除以下不同之處外與數據庫登記NZ_AABC010001965的55，604-56，392 DNA序列相同的序列，不同之處在于位置55 604中A被置換成了T以產生標準的ATG起始密碼子。在獨立擴增試驗中再現了該核苷酸序列，因此代表所用的點形念珠藻ATCC 29133中的核苷酸序列。
因此，該克隆pNP195用于克隆到表達載體pJOAP(如實施例9中所述)中。
通過從pNP195分離709bp SphI片段，并且連接到SphI切割的載體pJOAP中實現克隆。以正確方向包含來源于點形念珠藻ATCC 29133的NP195酮酶的克隆稱為pJOAPNP195，該酮酶在N末端翻譯融合rbcS轉運肽。
實施例14
用于在番茄中果實特異性超表達來源于點形念珠藻ATCC 29133的NP195酮酶的表達載體的制備用來源于豌豆的轉運肽rbcS(Anderson等，1986，Biochem J.240709-715)，在番茄(L.esculentum)中表達來源于點形念珠藻ATCC29133(美國典型培養(yǎng)物保藏中心(ATCC)株系)的NP195酮酶。在來源于擬南介的啟動子AP3P(如實施例5中所述)控制下實現該表達。
利用二元載體pSUN3(WO 02/00900)制備表達載體，該表達載體用于農桿菌介導的AP3P控制的來源于點形念珠藻ATCC 29133的NP195酮酶向番茄(L.esculentum)的轉化。
為了制備表達載體MPS122，連接來自于pJOAPNP195的1.992kbSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(
圖12，構建體圖譜)。在
圖12中，片段AP3P PROM包含AP3P啟動子(765bp)，片段rbcS TPFragment包含來源于豌豆的rbcS轉運肽(194bp)，片段NP195 KETOCDS(789bp)編碼點形念珠藻ATCC 29133 NP195酮酶，片段OCS終止子(192bp)包含章魚氨酸合酶聚腺苷酸化信號。
實施例15擴增編碼來源于泡沫節(jié)球藻(Nodularia spumignea)NSOR10的NODK酮酶完整一級序列的DNA用PCR方法，從泡沫節(jié)球藻NSOR10擴增編碼來源于泡沫節(jié)球藻NSOR10的酮酶的DNA。
為了從在25℃下BG11培養(yǎng)基(1.5g/l NaNO3，0.04g/l K2PO4×3H2O，0.075g/l MgSO4×H2O，0.036g/l CaCl2×2H2O，0.006g/l檸檬酸，0.006g/l檸檬酸鐵銨，0.001g/l EDTA二鈉鎂，0.04g/l Na2CO3，1ml痕量金屬混合物“A5+Co”，2.86g/l H3BO3，1.81g/l MnCl2×4H2O，0.222g/lZnSO4×7H2O，0.39g/l NaMoO4×2H2O，0.079g/l CuSO4×5H2O，0.0494g/lCo(NO3)2×6H2O)中，在連續(xù)振蕩(150rpm)及連續(xù)光照條件下已經培養(yǎng)1周的泡沫節(jié)球藻NSOR10懸浮培養(yǎng)物制備基因組DNA，通過離心收集細胞，在液氮中冷凍并且在研缽中研磨成粉末。
從泡沫節(jié)球藻NSOR10分離DNA的方法通過在8000rpm離心10分鐘從10ml液體培養(yǎng)物沉淀細菌細胞。此后，利用研杵和研缽，在液氮中弄碎和研磨細菌細胞。在1ml 10mM Tris-HCl(pH7.5)中重懸浮細胞材料，并且轉移到Eppendorf反應容器(體積2ml)中。添加100μl蛋白酶K后(濃度20mg/ml)，在37℃溫育細胞懸浮液3小時。以后，用500μl苯酚提取懸浮液。在13000rpm離心5分鐘后，將含水的上層相轉移到新的2ml Eppendorf反應容器。重復苯酚提取3次。通過添加1/10體積3M醋酸鈉(pH5.2)和0.6體積異丙醇沉淀DNA，并且隨后用70％乙醇進行洗滌。在室溫下干燥DNA沉淀，加入25μl水，并且在65℃加熱溶解。
利用有義特異性引物(NODK-1，SEQ ID NO.74)和反義特異性引物(NODK-2，SEQ ID NO.75)，通過聚合酶鏈式反應(PCR)，從泡沫節(jié)球藻NSOR10擴增編碼來源于泡沫節(jié)球藻NSOR10的酮酶的核酸。
PCR條件如下在50μl反應混合物中進行擴增DNA的PCR，該DNA編碼由完整一級序列組成的酮酶蛋白質，該反應混合物包含-(如上所述制備的)1μl泡沫節(jié)球藻NSOR10 DNA-0.25mM dNTPs-0.2mM NODK-1(SEQ ID NO.74)-0.2mM NODK-2(SEQ ID NO.75)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-25.8μl蒸餾水。
在下面循環(huán)條件下進行PCR1× 94℃2分鐘35×94℃1分鐘55℃1分鐘72℃3分鐘
1×72℃10分鐘。
用SEQ ID NO.74和SEQ ID NO.75進行PCR擴增產生了編碼蛋白質的720bp片段，該蛋白質由完整一級序列(NODK，SEQ ID NO.76)組成。利用標準方法，將擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中，并且獲得克隆pNODK。
用M13F和M13R引物測序克隆pNODK證實了與數據庫登記AY210783的2130-2819 DNA序列相同的序列(反向于此公開的數據庫登記序列)。在獨立擴增試驗中再現了該核苷酸序列，因此代表了所用泡沫節(jié)球藻NSOR10中的核苷酸序列。
因此，該克隆pNODK用于克隆到表達載體pJOAP(實施例9中所述)中。
通過從pNODK分離710bp SphI片段，并且連接到SphI切割的載體pJOAP中實現克隆。以正確方向包含來源于泡沫節(jié)球藻NSOR10的NODK酮酶的克隆稱為pJOAPNODK，該酮酶在N末端翻譯融合rbcS轉運肽。
實施例16用于在番茄中果實特異性超表達來源于泡沫節(jié)球藻NSOR10的NODK酮酶的表達載體的制備用來源于豌豆的轉運肽rbcS(Anderson等，1986，Biochem J.240709-715)，在番茄(L.esculentum)中表達來源于泡沫節(jié)球藻NSOR10的NODK酮酶。該表達受來源于擬南介的啟動子AP3P(如實施例5中所述)控制。
利用二元載體pSUN3(WO 02/00900)制備表達載體，該表達載體用于農桿菌屬介導的AP3P控制的來源于泡沫節(jié)球藻NSOR10的NP195-酮酶向番茄的轉化。
為了制備表達載體MSP123，連接來自于pJOAPNODK的1893bpSacI/XhoI片段和SacI-XhoI切割的載體pSUN3(
圖13，構建體圖譜)。在
圖13中，片段AP3P PROM包含AP3P啟動子(765bp)，片段rbcS TPFrag ment包含來源于豌豆的rbcS轉運肽(194bp)片段NODK KETO CDS(690bp)編碼泡沫節(jié)球藻NSOR10 NODK酮酶，片段OCS終止子(192bp)包含章魚氨酸合酶聚腺苷酸化信號。
實施例17用于來源于番茄的色質體特異性β-羥化酶的果實特異性超表達的表達盒的制備在來源于擬南芥的果實特異性啟動子AP3P控制下(實施例2)，在番茄中表達來源于番茄的色質體特異性β-羥化酶。所用終止子元件是來源于蠶豆(Vicia faba)的LB3(數據庫登記AX696005)。通過RNA分離，逆轉錄和PCR制備色質體特異性β-羥化酶序列(數據庫登記Y14810&BE354440)。
通過PCR方法分離包含LB3終止子區(qū)的DNA片段。
通過標準方法，從蠶豆(Vicia faba)組織分離基因組DNA，并且利用引物PR206(SEQ ID NO.78)和PR207(SEQ ID NO.79)，通過基因組PCR使用該基因組DNA。在50μl反應混合物中進行該LB3 DNA片段的PCR擴增，該反應混合物包含-(如上所述制備的)1μl基因組DNA-0.25mM dNTPs-0.2μM PR206(SEQ ID NO.78)-0.2μM PR207(SEQ ID NO.79)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-28.8μl蒸餾水。
用SEQ ID NO.78和SEQ ID NO.79進行PCR擴增產生了包含LB終止子的307bp片段(SEQ ID NO.80)。利用標準方法，將擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中，并且獲得克隆pLB3。用M13F和M13R引物測序克隆pLB3證實了與數據庫登記AX696005的3-298DNA序列相同的序列。該克隆稱為pLB3，因此用于克隆到載體pJAP3P(參見實施例5)中。
通過用蠶豆豆球蛋白LB3終止子(數據庫登記AX696005；WO03/008596)置換35S終止子修飾表達盒pJAP3P(參加下文)。
為了制備β-羥化酶序列，從番茄制備總RNA。為此，100mg冷凍粉碎的花被轉移到反應容器中，并且加入0.8ml Trizol緩沖液(Lifetechnologies)，用0.2ml氯仿提取懸浮液。在12000g離心15分鐘后，取出含水的上清液，轉移到新的反應容器中，并且用1體積乙醇萃取。用1體積異丙醇沉淀RNA，用75％乙醇洗滌，并且將沉淀溶解在DEPC水(在室溫下，用1/1000體積焦碳酸二乙酯過夜溫育水，然后高壓滅菌)中。光度計測定RNA濃度。對于cDNA合成，在60℃變性2.5μg總RNA10分鐘，在冰上冷卻2分鐘，之后利用cDNA試劑盒(Ready-to-go-you-prime-beads，Pharmacia Biotech)，按照制造商的說明書，并且利用反義特異性引物(PR17 SEQ ID NO.56)，轉錄為cDNA。
隨后PCR反應條件如下通過聚合酶鏈式反應(PCR)，利用有義特異性引物(VPR204，SEQID NO.81)和反義特異性引物(PR215，SEQ ID NO.82)從番茄擴增編碼β-羥化酶的核酸。
在50μl反應混合物中進行DNA擴增PCR，該DNA編碼由完整一級序列組成的β-羥化酶蛋白質，該反應混合物包含-(如上所述制備的)1μl cDNA-0.25mM dNTPs-0.2μM VPR204(SEQ ID NO.81)-0.2μM PR215(SEQ ID NO.82)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-28.8μl蒸餾水。
用VPR204和PR215進行PCR擴增產生了編碼β-羥化酶的1040bp片段(SEQ ID NO.83)。將擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中。該克隆稱為pCrtR-b2。
用引物M13-R和M13-R對克隆pCrtR-b2進行測序反應，證實了與數據庫登記BE354440的33-558 DNA序列相同并與數據庫登記Y14810的1-1009 DNA序列相同的序列。因此，克隆pCrtR-b2用于克隆到載體pCSP02中(參見下文)。
通過從來源于克隆載體pCR2.1(Invitrogen)的pCrtR-b2分離1034bpHindIII/EcoRI片段，并且連接HindIII/EcoRI切割的載體pJPA3P(參見實施例5)中進行第一步克隆。包含β-羥化酶片段CrtR-b2的克隆稱為pCSP02。
通過從來源于克隆載體pCR-2.1(Invitrogen)的pLB3分離301 bpEcoRI/XhoI片段，并且連接EcoRI/XhoI切割的載體pCSP02，進行第二步克隆。包含296bp終止子LB3的克隆稱為pCSP03。此連接在終止子LB3和β-羥化酶片段CrtR-b2之間造成了轉錄融合。而且，它還在AP3P啟動子和β-羥化酶片段之間造成了轉錄融合。
實施例18用于果實特異性超表達來源于番茄的B基因的表達盒的制備在來源于番茄的果實特異性啟動子PDS(八氫番茄紅素去飽和酶；數據庫登記U46919)控制下在番茄中表達來源于番茄的B基因(番茄紅素β-羥化酶；數據庫登記AF254793)。所用的終止子元件是來源于CaMV的35S。通過PCR方法，從番茄的基因組DNA制備B基因序列。
用來源于番茄的基因組DNA，通過PCR方法，以寡核苷酸引物BGEN-1(SEQ ID NO.85)和BGEN-2(SEQ ID NO.86)分離B基因。
如所述(Galbiati M等，Funct.Integr.Genomics 2000，20125-34)從番茄分離基因組DNA。
如下進行PCR擴增在25μl終體積中包含80ng基因組DNA1×Expand Long Template PCR緩沖液2.5mM MgCl2
dATP，dCTP，dGTP，dTTP各350μM0.3μM BGEN-1(SEQ ID NO.85)0.3μM BGEN-2(SEQ ID NO.86)2.5單位Expand Long Template聚合酶。
使用下面的溫度程序94℃，120秒，1個循環(huán)；94℃，10秒；48℃，30秒和68℃，3分鐘；35個循環(huán)；68℃，10分鐘，1個循環(huán)。
用BGEN-1和BGEN-2進行PCR擴增產生了編碼β-羥化酶的1505bp片段(SEQ ID NO.87)。將擴增產物克隆到PCR克隆載體pCR2.1(Invitrogen)中。該克隆稱為pBGEN。
用引物M13-R和M13-F對克隆pBGEN進行的測序反應證實了與數據庫登記AF25493的1-1497 DNA序列相同的序列。因此，該克隆pCrtR-b2用于克隆到載體pCSP02(參見下文)中。
為了從番茄制備PDS啟動子序列，用標準方法，從番茄組織分離基因組DNA，并且利用引物PDS-1和PDS-2，通過基因組PCR使用該基因組DNA。在50μl反應混合物中進行該PDS啟動子片段的PCR擴增，該反應混合物包含-(如上所述制備的)1μl基因組DNA-0.3mM dNTPs-0.2μM PDS-1(SEQ ID NO.89)-0.2μM PDS-2(SEQ ID NO.90)-5μl 10×Pfu-Turbo聚合酶(Stratagene)-1μl Pfu-Turbo聚合酶(Stratagene)-28.8μl蒸餾水。
使用下面的溫度程序94℃，120秒，1個循環(huán)；94℃，60秒；55℃，120秒和72℃，4分鐘；36個循環(huán)；
72℃，10分鐘，1個循環(huán)。
用PDS-1和PDS-2進行PCR擴增產生了包含PDS啟動子序列的片段。將擴增產物克隆到pCR4-BLUNT(Invitrogen)中。該克隆稱為pPDS。
用引物M13-R和M13-F進行測序反應，證實了與序列SEQ ID NO.91相同的序列。該克隆稱為pPDS，因此用于克隆到載體pJBGEN中(參見下文)。
通過從來源于克隆載體pCR2.1(Invitrogen)的pBGEN分離1499bpNcoI/EcoRI片段進行第一步克隆。首先，用BamHI切割pBGEN，并且用標準方法(30℃，30分鐘)補平3’末端(Klenow補平)，然后用NcoI部分消化，從中分離得到的1499bp片段。隨后，將該片段克隆到pCSP02中，pCSP02先用EcoRI切割，用標準方法補平3’末端(30℃，30分鐘)(Klenow補平)，然后用NcoI切割。包含1497bp B基因片段BGEN的克隆稱為pJAPBGEN。該連接產生了35S終止子和B基因之間的轉錄融合。
通過從pPDS分離2078bp PDS PROM片段進行第二步克隆。首先，用SmaI切割pPDS，然后用SacI部分消化，分離得到的2088bp片段。隨后，將該片段克隆到pJAPBGEN中，pJAPBGEN先用BamHI切割，用標準方法(30℃，30分鐘)補平3’末端(Klenow補平)，然后用SacI切割。該連接產生了啟動子PDS和B基因之間的轉錄融合。包含2078bp PDS啟動子BGEN的克隆稱為pJPDSBGEN。
實施例19用于在番茄中以果實特異性方式超表達B基因、表達點形念珠藻酮酶NP196和超表達來源于番茄的色質體特異性β-羥化酶的三重表達載體的制備首先，制備包含用于超表達點形念珠藻ATCC 29133 NP196酮酶和超表達β-羥化酶的表達盒的雙重構建體。
首先，以2104bp Ec1136II/XhoI片段(如實施例18中所述)從pCSP03分離包含β-羥化酶表達盒的片段AP3Pβ-羥化酶LB3。用標準方法補平3’末端(Klenow補平；30℃，30分鐘)。此后，在載體MSP120(如實施例10中所述)中，用Ec1136II和EcoRI切割該片段，并且用標準方法補平3’末端(30℃，30分鐘；Klenow補平)。連接產生包含2個表達盒的T-DNA首先是，用于色質體特異性番茄β-羥化酶超表達的盒子，其次是用于來源于點形念珠藻的酮酶NP196超表達的盒子。β-羥化酶負調節(jié)盒可以以2個方向連接到載體中。優(yōu)選地使用2個表達盒具有相同方向的形式(參見
圖14)?？梢酝ㄟ^下面所述PCR鑒定該形式在50μl反應混合物中進行擴增PR206-PR010質粒片段的PCR，該質粒片段包含β-羥化酶盒子LB3終止子和酮酶盒子AP3P啟動子的連接，該反應混合物包含-1μl質粒DNA(用標準方法制備的)-0.25mM dNTPs-0.2μM PR010(SEQ ID NO.92)-0.2μM PR206(SEQ ID NO.93)-5μl 10×PCR緩沖液(TAKARA)-0.25μl R Taq聚合酶(TAKARA)-28.8μl蒸餾水。
用PR010和PR206進行的PCR擴增產生了1080bp片段，該片段表明存在上述LB3終止子和AP3P啟動子連接及由此2個表達盒采用優(yōu)選的方向。該克隆稱為pBHYXNP196。
通過從pJPDSBGEN(參見實施例19)分離4362bp EcoRV/XhoI片段，并且連接到SmaI/XhoI切割的(上述)載體pBHYXNP196中，將該B基因超表達盒克隆到用于農桿菌介導的番茄轉化的表達載體中。該連接產生了包含3個表達盒的T-DNA首先是用于超表達B基因的盒子，其次是用于超表達來源于點形念珠藻的NP196-1酮酶的盒子，第三是用于色質體特異性超表達來源于番茄的β-羥化酶的盒子(
圖14，構建體圖譜)。該克隆稱為MSP124。在
圖14中，片段AP3P PROM(765bp)包含AP3P啟動子，片段BHYX b2 CDS(2bp)包含β-羥化酶CrtRb2，片段LB3 TERM(296bp)包含LB3終止子。
而且，片段AP3P PROM(765bp)包含AP3P啟動子，rbcS TP Fragment(194bp)包含來源于豌豆的rbcS基因的轉運肽，NP196 KETO CDS(761bp)包含來源于點形念珠藻ATCC 29133的酮酶，OCS TERM(192bp)包含章魚氨酸合酶基因的聚腺苷酸化信號。
而且，片段PDS PROM(2078bp)包含PDS啟動子、片段BGEN CDS(1497bp)包含B基因序列，和片段35S TERM(746bp)包含35S終止子。
實施例20轉基因番茄植物的產生如實施例6中所述轉化和再生番茄植物。
根據所述的轉化方法，用下面的表達構建體獲得了下面的品系用MSP120獲得了MSP120-1，MSP120-2，MSP120-3；用MSP121獲得了MSP121-1，MSP121-2，MSP121-3；用MSP122獲得了MSP122-1，MSP122-2，MSP133-3；用MSP123獲得了MSP123-1，MSP123-2，MSP123-3；用MSP124獲得了MSP124-1，MSP124-2，MSP124-3。
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<221>CDS<222>(166)..(1155)<223>
<400>1ggcacgagct tgcacgcaag tcagcgcgcg caagtcaaca cctgccggtc cacagcctca 60aataataaag agctcaagcg tttgtgcgcc tcgacgtggc cagtctgcac tgccttgaac 120ccgcgagtct cccgccgcac tgactgccat agcacagcta gacga atg cag cta gca 177Met Gln Leu Ala1gcg aca gta atg ttg gag cag ctt acc gga agc gct gag gca ctc aag 225Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser Ala Glu Ala Leu Lys5 10 15 20gag aag gag aag gag gtt gca ggc agc tct gac gtg ttg cgt aca tgg 273Glu Lys Glu Lys Glu Val Ala Gly Ser Ser Asp Val Leu Arg Thr Trp25 30 35gcg acc cag tac tcg ctt ccg tca gaa gag tca gac gcg gcc cgc ccg 321Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser Asp Ala Ala Arg Pro40 45 50gga ctg aag aat gcc tac aag cca cca cct tcc gac aca aag ggc atc 369Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser Asp Thr Lys Gly Ile55 60 65aca atg gcg cta cgt gtc atc ggc tcc tgg gcc gca gtg ttc ctc cac 417Thr Met Ala Leu Arg Val Ile Gly Ser Trp Ala Ala Val Phe Leu His70 75 80gcc att ttt caa atc aag ctt ccg acc tcc ttg gac cag ctg cac tgg 465Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu Asp Gln Leu His Trp85 90 95 100ctg ccc gtg tca gat gcc aca gct cag ctg gtt agc ggc acg agc agc 513Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val Ser Gly Thr Ser Ser105 110 115ctg ctc gac atc gtc gta gta ttc ttt gtc ctg gag ttc ctg tac aca 561
Leu Leu Asp Ile Val Val Val Phe Phe Val Leu Glu Phe Leu Tyr Thr120 125 130ggc ctt ttt atc acc acg cat gat gct atg cat ggc acc atc gcc atg 609Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile Ala Met135 140 145aga aac agg cag ctt aat gac ttc ttg ggc aga gta tgc atc tcc ttg 657Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg Val Cys Ile Ser Leu150 155 160tac gcc tgg ttt gat tac aac atg ctg cac cgc aag cat tgg gag cac 705Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg Lys His Trp Glu His165 170 175 180cac aac cac act ggc gag gtg ggc aag gac cct gac ttc cac agg gga 753His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp Phe His Arg Gly185 190 195aac cct ggc att gtg ccc tgg ttt gcc agc ttc atg tcc agc tac atg 801Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met200 205 210tcg atg tgg cag ttt gcg cgc ctc gca tgg tgg acg gtg gtc atg cag 849Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp Thr Val Val Met Gln215 220 225ctg ctg ggt gcg cca atg gcg aac ctg ctg gtg ttc atg gcg gcc gcg 897Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala230 235 240ccc atc ctg tcc gcc ttc cgc ttg ttc tac ttt ggc acg tac atg ccc 945Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Met Pro245 250 255 260cac aag cct gag cct ggc gcc gcg tca ggc tct tca cca gcc gtc atg 993His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser Ser Pro Ala Val Met265 270 275aac tgg tgg aag tcg cgc act agc cag gcg tcc gac ctg gtc agc ttt 1041Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser Asp Leu Val Ser Phe280 285 290ctg acc tgc tac cac ttc gac ctg cac tgg gag cac cac cgc tgg ccc 1089Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro295 300 305ttc gcc ccc tgg tgg gag ctg ccc aac tgc cgc cgc ctg tct ggc cga 1137Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg Arg Leu Ser Gly Arg310 315 320ggt ctg gtt cct gcc tag ctggacacac tgcagtgggc cctgctgcca 1185Gly Leu Val Pro Ala325gctgggcatg caggttgtgg caggactggg tgaggtgaaa agctgcaggc gctgctgccg1245gacacgctgc atgggctacc ctgtgtagct gccgccacta ggggaggggg tttgtagctg1305tcgagcttgc cccatggatg aagctgtgta gtggtgcagg gagtacaccc acaggccaac1365acccttgcag gagatgtctt gcgtcgggag gagtgttggg cagtgtagat gctatgattg1425
tatcttaatg ctgaagcctt taggggagcg acacttagtg ctgggcaggc aacgccctgc1485aaggtgcagg cacaagctag gctggacgag gactcggtgg caggcaggtg aagaggtgcg1545ggagggtggt gccacaccca ctgggcaaga ccatgctgca atgctggcgg tgtggcagtg1605agagctgcgt gattaactgg gctatggatt gtttgagcag tctcacttat tctttgatat1665agatactggt caggcaggtc aggagagtga gtatgaacaa gttgagaggt ggtgcgctgc1725ccctgcgctt atgaagctgt aacaataaag tggttcaaaa aaaaaa 1771<210>2<211>329<212>PRT<213>雨生紅球藻<400>2Met Gln Leu Ala Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser Ala1 5 10 15Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser Ser Asp Val20 25 30Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser Asp35 40 45Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser Asp50 55 60Thr Lys Gly Ile Thr Met Ala Leu Arg Val Ile Gly Ser Trp Ala Ala65 70 75 80Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu Asp85 90 95Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val Ser100 105 110Gly Thr Ser Ser Leu Leu Asp Ile Val Val Val Phe Phe Val Leu Glu115 120 125Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly130 135 140Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg Val145 150 155 160Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg Lys
165 170 175His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp180 185 190Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe Met195 200 205Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp Thr210 215 220Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe225 230 235 240Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly245 250 255Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser Ser260 265 270Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser Asp275 280 285Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His290 295 300His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg Arg305 310 315 320Leu Ser Gly Arg Gly Leu Val Pro Ala325<210>3<211>1662<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(168)..(1130)<223>
<400>3cggggcaact caagaaattc aacagctgca agcgcgcccc agcctcacag cgccaagtga 60gctatcgacg tggttgtgag cgctcgacgt ggtccactga cgggcctgtg agcctctgcg 120ctccgtcctc tgccaaatct cgcgtcgggg cctgcctaag tcgaaga atg cac gtc 176Met His Val1
gca tcg gca cta atg gtc gag cag aaa ggc agt gag gca gct gct tcc 224Ala Ser Ala Leu Met Val Glu Gln Lys Gly Ser Glu Ala Ala Ala Ser5 10 15agc cca gac gtc ttg aga gcg tgg gcg aca cag tat cac atg cca tcc 272Ser Pro Asp Val Leu Arg Ala Trp Ala Thr Gln Tyr His Met Pro Ser20 25 30 35gag tcg tca gac gca gct cgt cct gcg cta aag cac gcc tac aaa cct 320Glu Ser Ser Asp Ala Ala Arg Pro Ala Leu Lys His Ala Tyr Lys Pro40 45 50cca gca tct gac gcc aag ggc atc acg atg gcg ctg acc atc att ggc 368Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr Ile Ile Gly55 60 65acc tgg acc gca gtg ttt tta cac gca ata ttt caa atc agg cta ccg 416Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile Arg Leu Pro70 75 80aca tcc atg gac cag ctt cac tgg ttg cct gtg tcc gaa gcc aca gcc 464Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu Ala Thr Ala85 90 95cag ctt ttg ggc gga agc agc agc cta ctg cac atc gct gca gtc ttc 512Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala Ala Val Phe100 105 110 115att gta ctt gag ttc ctg tac act ggt cta ttc atc acc aca cat gac 560Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp120 125 130gca atg cat ggc acc ata gct ttg agg cac agg cag ctc aat gat ctc 608Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu Asn Asp Leu135 140 145ctt ggc aac atc tgc ata tca ctg tac gcc tgg ttt gac tac agc atg 656Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Ser Met150 155 160ctg cat cgc aag cac tgg gag cac cac aac cat act ggc gaa gtg ggg 704Leu His Arg Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly165 170 175aaa gac cct gac ttc cac aag gga aat ccc ggc ctt gtc ccc tgg ttc 752Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro Trp Phe180 185 190 195gcc agc ttc atg tcc agc tac atg tcc ctg tgg cag ttt gcc cgg ctg 800Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala Arg Leu200 205 210gca tgg tgg gca gtg gtg atg caa atg ctg ggg gcg ccc atg gca aat 848Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro Met Ala Asn215 220 225ctc cta gtc ttc atg gct gca gcc cca atc ttg tca gca ttc cgc ctc 896Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu230 235 240ttc tac ttc ggc act tac ctg cca cac aag cct gag cca ggc cct gca 944
Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly Pro Ala245 250 255gca ggc tct cag gtg atg gcc tgg ttc agg gcc aag aca agt gag gca 992Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr Ser Glu Ala260 265 270 275tct gat gtg atg agt ttc ctg aca tgc tac cac ttt gac ctg cac tgg 1040Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp280 285 290gag cac cac agg tgg ccc ttt gcc ccc tgg tgg cag ctg ccc cac tgc 1088Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu Pro His Cys295 300 305cgc cgc ctg tcc ggg cgt ggc ctg gtg cct gcc ttg gca tga 1130Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala310 315 320cctggtccct ccgctggtga cccagcgtct gcacaagagt gtcatgctac agggtgctgc1190ggccagtggc agcgcagtgc actctcagcc tgtatggggc taccgctgtg ccactgagca1250ctgggcatgc cactgagcac tgggcgtgct actgagcaat gggcgtgcta ctgagcaatg1310ggcgtgctac tgacaatggg cgtgctactg gggtctggca gtggctagga tggagtttga1370tgcattcagt agcggtggcc aacgtcatgt ggatggtgga agtgctgagg ggtttaggca1430gccggcattt gagagggcta agttataaat cgcatgctgc tcatgcgcac atatctgcac1490acagccaggg aaatcccttc gagagtgatt atgggacact tgtattggtt tcgtgctatt1550gttttattca gcagcagtac ttagtgaggg tgagagcagg gtggtgagag tggagtgagt1610gagtatgaac ctggtcagcg aggtgaacag cctgtaatga atgactctgt ct1662<210>4<211>320<212>PRT<213>雨生紅球藻<400>4Met His Val Ala Ser Ala Leu Met Val Glu Gln Lys Gly Ser Glu Ala1 5 10 15Ala Ala Ser Ser Pro Asp Val Leu Arg Ala Trp Ala Thr Gln Tyr His20 25 30Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Ala Leu Lys His Ala35 40 45Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr50 55 60Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile
65 70 75 80Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu85 90 95Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala100 105 110Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr115 120 125Thr His Asp Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu130 135 140Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp145 150 155 160Tyr Ser Met Leu His Arg Lys His Trp Glu His His Asn His Thr Gly165 170 175Glu Val Gly Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val180 185 190Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe195 200 205Ala Arg Leu Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro210 215 220Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala225 230 235 240Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro245 250 255Gly Pro Ala Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr260 265 270Ser Glu Ala Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp275 280 285Leu His Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu290 295 300Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala305 310 315 320
<210>5<211>729<212>DNA<213>Agrobacterium aurantiacum<220>
<221>CDS<222>(1)..(729)<223>
<400>5atg agc gca cat gcc ctg ccc aag gca gat ctg acc gcc acc agc ctg48Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15atc gtc tcg ggc ggc atc atc gcc gct tgg ctg gcc ctg cat gtg cat96Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30gcg ctg tgg ttt ctg gac gca gcg gcg cat ccc atc ctg gcg atc gca 144Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Ile Ala35 40 45aat ttc ctg ggg ctg acc tgg ctg tcg gtc gga ttg ttc atc atc gcg 192Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60cat gac gcg atg cac ggg tcg gtg gtg ccg ggg cgt ccg cgc gcc aat 240His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80gcg gcg atg ggc cag ctt gtc ctg tgg ctg tat gcc gga ttt tcg tgg 288Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95cgc aag atg atc gtc aag cac atg gcc cat cac cgc cat gcc gga acc 336Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110gac gac gac ccc gat ttc gac cat ggc ggc ccg gtc cgc tgg tac gcc 384Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125cgc ttc atc ggc acc tat ttc ggc tgg cgc gag ggg ctg ctg ctg ccc 432Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140gtc atc gtg acg gtc tat gcg ctg atc ctt ggg gat cgc tgg atg tac 480Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160gtg gtc ttc tgg ccg ctg ccg tcg atc ctg gcg tcg atc cag ctg ttc 528Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175gtg ttc ggc acc tgg ctg ccg cac cgc ccc ggc cac gac gcg ttc ccg 576Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190gac cgc cac aat gcg cgg tcg tcg cgg atc agc gac ccc gtg tcg ctg 624
Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205ctg acc tgc ttt cac ttt ggc ggt tat cat cac gaa cac cac ctg cac 672Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220ccg acg gtg ccg tgg tgg cgc ctg ccc agc acc cgc acc aag ggg gac 720Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240acc gca tga 729Thr Ala<210>6<211>242<212>PRT<213>Agrobacterium aurantiacum<400>6Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Ile Ala35 40 45Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160
Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240Thr Ala<210>7<211>1631<212>DNA<213>產堿菌屬種(Alcaligenes sp.)<220>
<221>CDS<222>(99)..(827)<223>
<400>7ctgcaggccg ggcccggtgg ccaatggtcg caaccggcag gactggaaca ggacggcggg 60ccggtctagg ctgtcgccct acgcagcagg agtttcgg atg tcc gga cgg aag cct 116Met Ser Gly Arg Lys Pro1 5ggc aca act ggc gac acg atc gtc aat ctc ggt ctg acc gcc gcg atc 164Gly Thr Thr Gly Asp Thr Ile Val Asn Leu Gly Leu Thr Ala Ala Ile10 15 20ctg ctg tgc tgg ctg gtc ctg cac gcc ttt acg cta tgg ttg cta gat 212Leu Leu Cys Trp Leu Val Leu His Ala Phe Thr Leu Trp Leu Leu Asp25 30 35gcg gcc gcg cat ccg ctg ctt gcc gtg ctg tgc ctg gct ggg ctg acc 260Ala Ala Ala His Pro Leu Leu Ala Val Leu Cys Leu Ala Gly Leu Thr40 45 50tgg ctg tcg gtc ggg ctg ttc atc atc gcg cat gac gca atg cac ggg 308Trp Leu Ser Val Gly Leu Phe Ile Ile Ala His Asp Ala Met His Gly55 60 65 70tcc gtg gtg ccg ggg cgg ccg cgc gcc aat gcg gcg atc ggg caa ctg 356Ser Val Val Pro Gly Arg Pro Arg Ala Asn Ala Ala Ile Gly Gln Leu75 80 85
gcg ctg tgg ctc tat gcg ggg ttc tcg tgg ccc aag ctg atc gcc aag 404Ala Leu Trp Leu Tyr Ala Gly Phe Ser Trp Pro Lys Leu Ile Ala Lys90 95 100cac atg acg cat cac cgg cac gcc ggc acc gac aac gat ccc gat ttc 452His Met Thr His His Arg His Ala Gly Thr Asp Asn Asp Pro Asp Phe105 110 115ggt cac gga ggg ccc gtg cgc tgg tac ggc agc ttc gtc tcc acc tat 500Gly His Gly Gly Pro Val Arg Trp Tyr Gly Ser Phe Val Ser Thr Tyr120 125 130ttc ggc tgg cga gag gga ctg ctg cta ccg gtg atc gtc acc acc tat 548Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro Val Ile Val Thr Thr Tyr135 140 145 150gcg ctg atc ctg ggc gat cgc tgg atg tat gtc atc ttc tgg ccg gtc 596Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr Val Ile Phe Trp Pro Val155 160 165ccg gcc gtt ctg gcg tcg atc cag att ttc gtc ttc gga act tgg ctg 644Pro Ala Val Leu Ala Ser Ile Gln Ile Phe Val Phe Gly Thr Trp Leu170 175 180ccc cac cgc ccg gga cat gac gat ttt ccc gac cgg cac aac gcg agg 692Pro His Arg Pro Gly His Asp Asp Phe Pro Asp Arg His Asn Ala Arg185 190 195tcg acc ggc atc ggc gac ccg ttg tca cta ctg acc tgc ttc cat ttc 740Ser Thr Gly Ile Gly Asp Pro Leu Ser Leu Leu Thr Cys Phe His Phe200 205 210ggc ggc tat cac cac gaa cat cac ctg cat ccg cat gtg ccg tgg tgg 788Gly Gly Tyr His His Glu His His Leu His Pro His Val Pro Trp Trp215 220 225 230cgc ctg cct cgt aca cgc aag acc gga ggc cgc gca tga cgcaattcct837Arg Leu Pro Arg Thr Arg Lys Thr Gly Gly Arg Ala235 240cattgtcgtg gcgacagtcc tcgtgatgga gctgaccgcc tattccgtcc accgctggat 897tatgcacggc cccctaggct ggggctggca caagtcccat cacgaagagc acgaccacgc 957gttggagaag aacgacctct acggcgtcgt cttcgcggtg ctggcgacga tcctcttcac1017cgtgggcgcc tattggtggc cggtgctgtg gtggatcgcc ctgggcatga cggtctatgg1077gttgatctat ttcatcctgc acgacgggct tgtgcatcaa cgctggccgt ttcggtatat1137tccgcggcgg ggctatttcc gcaggctcta ccaagctcat cgcctgcacc acgcggtcga1197ggggcgggac cactgcgtca gcttcggctt catctatgcc ccacccgtgg acaagctgaa1257gcaggatctg aagcggtcgg gtgtcctgcg cccccaggac gagcgtccgt cgtgatctct1317gatcccggcg tggccgcatg aaatccgacg tgctgctggc aggggccggc cttgccaacg1377gactgatcgc gctggcgatc cgcaaggcgc ggcccgacct tcgcgtgctg ctgctggacc1437gtgcggcggg cgcctcggac gggcatactt ggtcctgcca cgacaccgat ttggcgccgc1497
actggctgga ccgcctgaag ccgatcaggc gtggcgactg gcccgatcag gaggtgcggt1557tcccagacca ttcgcgaagg ctccgggccg gatatggctc gatcgacggg cgggggctga1617tgcgtgcggt gacc 1631<210>8<211>242<212>PRT<213>產堿菌屬種<400>8Met Ser Gly Arg Lys Pro Gly Thr Thr Gly Asp Thr Ile Val Asn Leu1 5 10 15Gly Leu Thr Ala Ala Ile Leu Leu Cys Trp Leu Val Leu His Ala Phe20 25 30Thr Leu Trp Leu Leu Asp Ala Ala Ala His Pro Leu Leu Ala Val Leu35 40 45Cys Leu Ala Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80Ala Ala Ile Gly Gln Leu Ala Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95Pro Lys Leu Ile Ala Lys His Met Thr His His Arg His Ala Gly Thr100 105 110Asp Asn Asp Pro Asp Phe Gly His Gly Gly Pro Val Arg Trp Tyr Gly115 120 125Ser Phe Val Ser Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140Val Ile Val Thr Thr Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160Val Ile Phe Trp Pro Val Pro Ala Val Leu Ala Ser Ile Gln Ile Phe165 170 175Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Asp Phe Pro180 185 190
Asp Arg His Asn Ala Arg Ser Thr Gly Ile Gly Asp Pro Leu Ser Leu195 200 205Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220Pro His Val Pro Trp Trp Arg Leu Pro Arg Thr Arg Lys Thr Gly Gly225 230 235 240Arg Ala<210>9<211>729<212>DNA<213>Paracoccus marcusii<220>
<221>CDS<222>(1)..(729)<223>
<400>9atg agc gca cat gcc ctg ccc aag gca gat ctg acc gcc aca agc ctg 48Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15atc gtc tcg ggc ggc atc atc gcc gca tgg ctg gcc ctg cat gtg cat 96Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30gcg ctg tgg ttt ctg gac gcg gcg gcc cat ccc atc ctg gcg gtc gcg144Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Val Ala35 40 45aat ttc ctg ggg ctg acc tgg ctg tcg gtc gga ttg ttc atc atc gcg192Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60cat gac gcg atg cac ggg tcg gtc gtg ccg ggg cgt ccg cgc gcc aat240His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80gcg gcg atg ggc cag ctt gtc ctg tgg ctg tat gcc gga ttt tcg tgg288Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95cgc aag atg atc gtc aag cac atg gcc cat cac cgc cat gcc gga acc336Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110gac gac gac cca gat ttc gac cat ggc ggc ccg gtc cgc tgg tac gcc384Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125cgc ttc atc ggc acc tat ttc ggc tgg cgc gag ggg ctg ctg ctg ccc432
Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140gtc atc gtg acg gtc tat gcg ctg atc ctg ggg gat cgc tgg atg tac 480Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160gtg gtc ttc tgg ccg ttg ccg tcg atc ctg gcg tcg atc cag ctg ttc 528Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175gtg ttc ggc act tgg ctg ccg cac cgc ccc ggc cac gac gcg ttc ccg 576Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190gac cgc cat aat gcg cgg tcg tcg cgg atc agc gac cct gtg tcg ctg 624Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205ctg acc tgc ttt cat ttt ggc ggt tat cat cac gaa cac cac ctg cac 672Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220ccg acg gtg ccg tgg tgg cgc ctg ccc agc acc cgc acc aag ggg gac 720Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240acc gca tga 729Thr Ala<210>10<211>242<212>PRT<213>Paracoccus marcusii<400>10Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Val Ala35 40 45Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95
Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240Thr Ala<210>11<211>1629<212>DNA<213>聚球藻屬種(Synechococcus sp.)<220>
<221>CDS<222>(1)..(1629)<223>
<400>11atg atc acc acc gat gtt gtc att att ggg gcg ggg cac aat ggc tta 48Met Ile Thr Thr Asp Val Val Ile Ile Gly Ala Gly His Asn Gly Leu1 5 10 15gtc tgt gca gcc tat ttg ctc caa cgg ggc ttg ggg gtg acg tta cta 96Val Cys Ala Ala Tyr Leu Leu Gln Arg Gly Leu Gly Val Thr Leu Leu20 25 30gaa aag cgg gaa gta cca ggg ggg gcg gcc acc aca gaa gct ctc atg144
Glu Lys Arg Glu Val Pro Gly Gly Ala Ala Thr Thr Glu Ala Leu Met35 40 45ccg gag cta tcc ccc cag ttt cgc ttt aac cgc tgt gcc att gac cac 192Pro Glu Leu Ser Pro Gln Phe Arg Phe Asn Arg Cys Ala Ile Asp His50 55 60gaa ttt atc ttt ctg ggg ccg gtg ttg cag gag cta aat tta gcc cag 240Glu Phe Ile Phe Leu Gly Pro Val Leu Gln Glu Leu Asn Leu Ala Gln65 70 75 80tat ggt ttg gaa tat tta ttt tgt gac ccc agt gtt ttt tgt ccg ggg 288Tyr Gly Leu Glu Tyr Leu Phe Cys Asp Pro Ser Val Phe Cys Pro Gly85 90 95ctg gat ggc caa gct ttt atg agc tac cgt tcc cta gaa aaa acc tgt 336Leu Asp Gly Gln Ala Phe Met Ser Tyr Arg Ser Leu Glu Lys Thr Cys100 105 110gcc cac att gcc acc tat agc ccc cga gat gcg gaa aaa tat cgg caa 384Ala His Ile Ala Thr Tyr Ser Pro Arg Asp Ala Glu Lys Tyr Arg Gln115 120 125ttt gtc aat tat tgg acg gat ttg ctc aac gct gtc cag cct gct ttt 432Phe Val Asn Tyr Trp Thr Asp Leu Leu Asn Ala Val Gln Pro Ala Phe130 135 140aat gct ccg ccc cag gct tta cta gat tta gcc ctg aac tat ggt tgg 480Asn Ala Pro Pro Gln Ala Leu Leu Asp Leu Ala Leu Asn Tyr Gly Trp145 150 155 160gaa aac tta aaa tcc gtg ctg gcg atc gcc ggg tcg aaa acc aag gcg 528Glu Asn Leu Lys Ser Val Leu Ala Ile Ala Gly Ser Lys Thr Lys Ala165 170 175ttg gat ttt atc cgc act atg atc ggc tcc ccg gaa gat gtg ctc aat 576Leu Asp Phe Ile Arg Thr Met Ile Gly Ser Pro Glu Asp Val Leu Asn180 185 190gaa tgg ttc gac agc gaa cgg gtt aaa gct cct tta gct aga cta tgt 624Glu Trp Phe Asp Ser Glu Arg Val Lys Ala Pro Leu Ala Arg Leu Cys195 200 205tcg gaa att ggc gct ccc cca tcc caa aag ggt agt agc tcc ggc atg 672Ser Glu Ile Gly Ala Pro Pro Ser Gln Lys Gly Ser Ser Ser Gly Met210 215 220atg atg gtg gcc atg cgg cat ttg gag gga att gcc aga cca aaa gga 720Met Met Val Ala Met Arg His Leu Glu Gly Ile Ala Arg Pro Lys Gly225 230 235 240ggc act gga gcc ctc aca gaa gcc ttg gtg aag tta gtg caa gcc caa 768Gly Thr Gly Ala Leu Thr Glu Ala Leu Val Lys Leu Val Gln Ala Gln245 250 255ggg gga aaa atc ctc act gac caa acc gtc aaa cgg gta ttg gtg gaa 816Gly Gly Lys Ile Leu Thr Asp Gln Thr Val Lys Arg Val Leu Val Glu260 265 270aac aac cag gcg atc ggg gtg gag gta gct aac gga gaa cag tac cgg 864Asn Asn Gln Ala Ile Gly Val Glu Val Ala Asn Gly Glu Gln Tyr Arg275 280 285
gcc aaa aaa ggc gtg att tct aac atc gat gcc cgc cgt tta ttt ttg 912Ala Lys Lys Gly Val Ile Ser Asn Ile Asp Ala Arg Arg Leu Phe Leu290 295 300caa ttg gtg gaa ccg ggg gcc cta gcc aag gtg aat caa aac cta ggg 960Gln Leu Val Glu Pro Gly Ala Leu Ala Lys Val Asn Gln Asn Leu Gly305 310 315 320gaa cga ctg gaa cgg cgc act gtg aac aat aac gaa gcc att tta aaa 1008Glu Arg Leu Glu Arg Arg Thr Val Asn Asn Asn Glu Ala Ile Leu Lys325 330 335atc gat tgt gcc ctc tcc ggt tta ccc cac ttc act gcc atg gcc ggg 1056Ile Asp Cys Ala Leu Ser Gly Leu Pro His Phe Thr Ala Met Ala Gly340 345 350ccg gag gat cta acg gga act att ttg att gcc gac tcg gta cgc cat 1104Pro Glu Asp Leu Thr Gly Thr Ile Leu Ile Ala Asp Ser Val Arg His355 360 365gtc gag gaa gcc cac gcc ctc att gcc ttg ggg caa att ccc gat gct 1152Val Glu Glu Ala His Ala Leu Ile Ala Leu Gly Gln Ile Pro Asp Ala370 375 380aat ccg tct tta tat ttg gat att ccc act gta ttg gac ccc acc atg 1200Asn Pro Ser Leu Tyr Leu Asp Ile Pro Thr Val Leu Asp Pro Thr Met385 390 395 400gcc ccc cct ggg cag cac acc ctc tgg atc gaa ttt ttt gcc ccc tac 1248Ala Pro Pro Gly Gln His Thr Leu Trp Ile Glu Phe Phe Ala Pro Tyr405 410 415cgc atc gcc ggg ttg gaa ggg aca ggg tta atg ggc aca ggt tgg acc 1296Arg Ile Ala Gly Leu Glu Gly Thr Gly Leu Met Gly Thr Gly Trp Thr420 425 430gat gag tta aag gaa aaa gtg gcg gat cgg gtg att gat aaa tta acg 1344Asp Glu Leu Lys Glu Lys Val Ala Asp Arg Val Ile Asp Lys Leu Thr435 440 445gac tat gcc cct aac cta aaa tct ctg atc att ggt cgc cga gtg gaa 1392Asp Tyr Ala Pro Asn Leu Lys Ser Leu Ile Ile Gly Arg Arg Val Glu450 455 460agt ccc gcc gaa ctg gcc caa cgg ctg gga agt tac aac ggc aat gtc 1440Ser Pro Ala Glu Leu Ala Gln Arg Leu Gly Ser Tyr Asn Gly Asn Val465 470 475 480tat cat ctg gat atg agt ttg gac caa atg atg ttc ctc cgg cct cta 1488Tyr His Leu Asp Met Ser Leu Asp Gln Met Met Phe Leu Arg Pro Leu485 490 495ccg gaa att gcc aac tac caa acc ccc atc aaa aat ctt tac tta aca 1536Pro Glu Ile Ala Asn Tyr Gln Thr Pro Ile Lys Asn Leu Tyr Leu Thr500 505 510ggg gcg ggt acc cat ccc ggt ggc tcc ata tca ggt atg ccc ggt aga 1584Gly Ala Gly Thr His Pro Gly Gly Ser Ile Ser Gly Met Pro Gly Arg515 520 525aat tgc gct cgg gtc ttt tta aaa caa caa cgt cgt ttt tgg taa 1629
Asn Cys Ala Arg Val Phe Leu Lys Gln Gln Arg Arg Phe Trp530 535 540<210>12<211>542<212>PRT<213>聚球藻屬種<400>12Met Ile Thr Thr Asp Val Val Ile Ile Gly Ala Gly His Asn Gly Leu1 5 10 15Val Cys Ala Ala Tyr Leu Leu Gln Arg Gly Leu Gly Val Thr Leu Leu20 25 30Glu Lys Arg Glu Val Pro Gly Gly Ala Ala Thr Thr Glu Ala Leu Met35 40 45Pro Glu Leu Ser Pro Gln Phe Arg Phe Asn Arg Cys Ala Ile Asp His50 55 60Glu Phe Ile Phe Leu Gly Pro Val Leu Gln Glu Leu Asn Leu Ala Gln65 70 75 80Tyr Gly Leu Glu Tyr Leu Phe Cys Asp Pro Ser Val Phe Cys Pro Gly85 90 95Leu Asp Gly Gln Ala Phe Met Ser Tyr Arg Ser Leu Glu Lys Thr Cys100 105 110Ala His Ile Ala Thr Tyr Ser Pro Arg Asp Ala Glu Lys Tyr Arg Gln115 120 125Phe Val Asn Tyr Trp Thr Asp Leu Leu Asn Ala Val Gln Pro Ala Phe130 135 140Asn Ala Pro Pro Gln Ala Leu Leu Asp Leu Ala Leu Asn Tyr Gly Trp145 150 155 160Glu Asn Leu Lys Ser Val Leu Ala Ile Ala Gly Ser Lys Thr Lys Ala165 170 175Leu Asp Phe Ile Arg Thr Met Ile Gly Ser Pro Glu Asp Val Leu Asn180 185 190Glu Trp Phe Asp Ser Glu Arg Val Lys Ala Pro Leu Ala Arg Leu Cys195 200 205
Ser Glu Ile Gly Ala Pro Pro Ser Gln Lys Gly Ser Ser Ser Gly Met210 215 220Met Met Val Ala Met Arg His Leu Glu Gly Ile Ala Arg Pro Lys Gly225 230 235 240Gly Thr Gly Ala Leu Thr Glu Ala Leu Val Lys Leu Val Gln Ala Gln245 250 255Gly Gly Lys Ile Leu Thr Asp Gln Thr Val Lys Arg Val Leu Val Glu260 265 270Asn Asn Gln Ala Ile Gly Val Glu Val Ala Asn Gly Glu Gln Tyr Arg275 280 285Ala Lys Lys Gly Val Ile Ser Asn Ile Asp Ala Arg Arg Leu Phe Leu290 295 300Gln Leu Val Glu Pro Gly Ala Leu Ala Lys Val Asn Gln Asn Leu Gly305 310 315 320Glu Arg Leu Glu Arg Arg Thr Val Asn Asn Asn Glu Ala Ile Leu Lys325 330 335Ile Asp Cys Ala Leu Ser Gly Leu Pro His Phe Thr Ala Met Ala Gly340 345 350Pro Glu Asp Leu Thr Gly Thr Ile Leu Ile Ala Asp Ser Val Arg His355 360 365Val Glu Glu Ala His Ala Leu Ile Ala Leu Gly Gln Ile Pro Asp Ala370 375 380Asn Pro Ser Leu Tyr Leu Asp Ile Pro Thr Val Leu Asp Pro Thr Met385 390 395 400Ala Pro Pro Gly Gln His Thr Leu Trp Ile Glu Phe Phe Ala Pro Tyr405 410 415Arg Ile Ala Gly Leu Glu Gly Thr Gly Leu Met Gly Thr Gly Trp Thr420 425 430Asp Glu Leu Lys Glu Lys Val Ala Asp Arg Val Ile Asp Lys Leu Thr435 440 445Asp Tyr Ala Pro Asn Leu Lys Ser Leu Ile Ile Gly Arg Arg Val Glu
450 455 460Ser Pro Ala Glu Leu Ala Gln Arg Leu Gly Ser Tyr Asn Gly Asn Val465 470 475 480Tyr His Leu Asp Met Ser Leu Asp Gln Met Met Phe Leu Arg Pro Leu485 490 495Pro Glu Ile Ala Asn Tyr Gln Thr Pro Ile Lys Asn Leu Tyr Leu Thr500 505 510Gly Ala Gly Thr His Pro Gly Gly Ser Ile Ser Gly Met Pro Gly Arg515 520 525Asn Cys Ala Arg Val Phe Leu Lys Gln Gln Arg Arg Phe Trp530 535 540<210>13<211>776<212>DNA<213>慢生根瘤菌屬種(Bradyrhizobium sp.)<220>
<221>CDS<222>(1)..(774)<223>
<400>13atg cat gca gca acc gcc aag gct act gag ttc ggg gcc tct cgg cgc 48Met His Ala Ala Thr Ala Lys Ala Thr Glu Phe Gly Ala Ser Arg Arg1 5 10 15gac gat gcg agg cag cgc cgc gtc ggt ctc acg ctg gcc gcg gtc atc 96Asp Asp Ala Arg Gln Arg Arg Val Gly Leu Thr Leu Ala Ala Val Ile20 25 30atc gcc gcc tgg ctg gtg ctg cat gtc ggt ctg atg ttc ttc tgg ccg144Ile Ala Ala Trp Leu Val Leu His Val Gly Leu Met Phe Phe Trp Pro35 40 45ctg acc ctt cac agc ctg ctg ccg gct ttg cct ctg gtg gtg ctg cag192Leu Thr Leu His Ser Leu Leu Pro Ala Leu Pro Leu Val Val Leu Gln50 55 60acc tgg ctc tat gta ggc ctg ttc atc atc gcg cat gac tgc atg cac240Thr Trp Leu Tyr Val Gly Leu Phe Ile Ile Ala His Asp Cys Met His65 70 75 80ggc tcg ctg gtg ccg ttc aag ccg cag gtc aac cgc cgt atc gga cag288Gly Ser Leu Val Pro Phe Lys Pro Gln Val Asn Arg Arg Ile Gly Gln85 90 95ctc tgc ctg ttc ctc tat gcc ggg ttc tcc ttc gac gct ctc aat gtc336Leu Cys Leu Phe Leu Tyr Ala Gly Phe Ser Phe Asp Ala Leu Asn Val100 105 110
gag cac cac aag cat cac cgc cat ccc ggc acg gcc gag gat ccc gat 384Glu His His Lys His His Arg His Pro Gly Thr Ala Glu Asp Pro Asp115 120 125ttc gac gag gtg ccg ccg cac ggc ttc tgg cac tgg ttc gcc agc ttt 432Phe Asp Glu Val Pro Pro His Gly Phe Trp His Trp Phe Ala Ser Phe130 135 140ttc ctg cac tat ttc ggc tgg aag cag gtc gcg atc atc gca gcc gtc 480Phe Leu His Tyr Phe Gly Trp Lys Gln Val Ala Ile Ile Ala Ala Val145 150 155 160tcg ctg gtt tat cag ctc gtc ttc gcc gtt ccc ttg cag aac atc ctg 528Ser Leu Val Tyr Gln Leu Val Phe Ala Val Pro Leu Gln Asn Ile Leu165 170 175ctg ttc tgg gcg ctg ccc ggg ctg ctg tcg gcg ctg cag ctg ttc acc 576Leu Phe Trp Ala Leu Pro Gly Leu Leu Ser Ala Leu Gln Leu Phe Thr180 185 190ttc ggc acc tat ctg ccg cac aag ccg gcc acg cag ccc ttc gcc gat 624Phe Gly Thr Tyr Leu Pro His Lys Pro Ala Thr Gln Pro Phe Ala Asp195 200 205cgc cac aac gcg cgg acg agc gaa ttt ccc gcg tgg ctg tcg ctg ctg 672Arg His Asn Ala Arg Thr Ser Glu Phe Pro Ala Trp Leu Ser Leu Leu210 215 220acc tgc ttc cac ttc ggc ttt cat cac gag cat cat ctg cat ccc gat 720Thr Cys Phe His Phe Gly Phe His His Glu His His Leu His Pro Asp225 230 235 240gcg ccg tgg tgg cgg ctg ccg gag atc aag cgg cgg gcc ctg gaa agg 768Ala Pro Trp Trp Arg Leu Pro Glu Ile Lys Arg Arg Ala Leu Glu Arg245 250 255cgt gac ta776Arg Asp<210>14<211>258<212>PRT<213>慢生根瘤菌屬種<400>14Met His Ala Ala Thr Ala Lys Ala Thr Glu Phe Gly Ala Ser Arg Arg1 5 10 15Asp Asp Ala Arg Gln Arg Arg Val Gly Leu Thr Leu Ala Ala Val Ile20 25 30Ile Ala Ala Trp Leu Val Leu His Val Gly Leu Met Phe Phe Trp Pro35 40 45Leu Thr Leu His Ser Leu Leu Pro Ala Leu Pro Leu Val Val Leu Gln
50 55 60Thr Trp Leu Tyr Val Gly Leu Phe Ile Ile Ala His Asp Cys Met His65 70 75 80Gly Ser Leu Val Pro Phe Lys Pro Gln Val Asn Arg Arg Ile Gly Gln85 90 95Leu Cys Leu Phe Leu Tyr Ala Gly Phe Ser Phe Asp Ala Leu Asn Val100 105 110Glu His His Lys His His Arg His Pro Gly Thr Ala Glu Asp Pro Asp115 120 125Phe Asp Glu Val Pro Pro His Gly Phe Trp His Trp Phe Ala Ser Phe130 135 140Phe Leu His Tyr Phe Gly Trp Lys Gln Val Ala Ile Ile Ala Ala Val145 150 155 160Ser Leu Val Tyr Gln Leu Val Phe Ala Val Pro Leu Gln Asn Ile Leu165 170 175Leu Phe Trp Ala Leu Pro Gly Leu Leu Ser Ala Leu Gln Leu Phe Thr180 185 190Phe Gly Thr Tyr Leu Pro His Lys Pro Ala Thr Gln Pro Phe Ala Asp195 200 205Arg His Asn Ala Arg Thr Ser Glu Phe Pro Ala Trp Leu Ser Leu Leu210 215 220Thr Cys Phe His Phe Gly Phe His His Glu His His Leu His Pro Asp225 230 235 240Ala Pro Trp Trp Arg Leu Pro Glu Ile Lys Arg Arg Ala Leu Glu Arg245 250 255Arg Asp<210>15<211>777<212>DNA<213>念珠藻屬種(Nostoc sp.)<220>
<221>CDS
<222>(1)..(777)<223>
<400>15atg gtt cag tgt caa cca tca tct ctg cat tca gaa aaa ctg gtg tta 48Met Val Gln Cys Gln Pro Ser Ser Leu His Ser Glu Lys Leu Val Leu1 5 10 15ttg tca tcg aca atc aga gat gat aaa aat att aat aag ggt ata ttt 96Leu Ser Ser Thr Ile Arg Asp Asp Lys Asn Ile Asn Lys Gly Ile Phe20 25 30att gcc tgc ttt atc tta ttt tta tgg gca att agt tta atc tta tta144Ile Ala Cys Phe Ile Leu Phe Leu Trp Ala Ile Ser Leu Ile Leu Leu35 40 45ctc tca ata gat aca tcc ata att cat aag agc tta tta ggt ata gcc192Leu Ser Ile Asp Thr Ser Ile Ile His Lys Ser Leu Leu Gly Ile Ala50 55 60atg ctt tgg cag acc ttc tta tat aca ggt tta ttt att act gct cat240Met Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His65 70 75 80gat gcc atg cac ggc gta gtt tat ccc aaa aat ccc aga ata aat aat288Asp Ala Met His Gly Val Val Tyr Pro Lys Asn Pro Arg Ile Asn Asn85 90 95ttt ata ggt aag ctc act cta atc ttg tat gga cta ctc cct tat aaa336Phe Ile Gly Lys Leu Thr Leu Ile Leu Tyr Gly Leu Leu Pro Tyr Lys100 105 110gat tta ttg aaa aaa cat tgg tta cac cac gga cat cct ggt act gat384Asp Leu Leu Lys Lys His Trp Leu His His Gly His Pro Gly Thr Asp115 120 125tta gac cct gat tat tac aat ggt cat ccc caa aac ttc ttt ctt tgg432Leu Asp Pro Asp Tyr Tyr Asn Gly His Pro Gln Asn Phe Phe Leu Trp130 135 140tat cta cat ttt atg aag tct tat tgg cga tgg acg caa att ttc gga480Tyr Leu His Phe Met Lys Ser Tyr Trp Arg Trp Thr Gln Ile Phe Gly145 150 155 160tta gtg atg att ttt cat gga ctt aaa aat ctg gtg cat ata cca gaa528Leu Val Met Ile Phe His Gly Leu Lys Asn Leu Val His Ile Pro Glu165 170 175aat aat tta att ata ttt tgg atg ata cct tct att tta agt tca gta576Asn Asn Leu Ile Ile Phe Trp Met Ile Pro Ser Ile Leu Ser Ser Val180 185 190caa cta ttt tat ttt ggt aca ttt ttg cct cat aaa aag cta gaa ggt624Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Lys Lys Leu Glu Gly195 200 205ggt tat act aac ccc cat tgt gcg cgc agt atc cca tta cct ctt ttt672Gly Tyr Thr Asn Pro His Cys Ala Arg Ser Ile Pro Leu Pro Leu Phe210 215 220tgg tct ttt gtt act tgt tat cac ttc ggc tac cac aag gaa cat cac720
Trp Ser Phe Val Thr Cys Tyr His Phe Gly Tyr His Lys Glu His His225 230 235 240gaa tac cct caa ctt cct tgg tgg aaa tta cct gaa gct cac aaa ata768Glu Tyr Pro Gln Leu Pro Trp Trp Lys Leu Pro Glu Ala His Lys Ile245 250 255tct tta taa777Ser Leu<210>16<211>258<212>PRT<213>念珠藻屬種<400>16Met Val Gln Cys Gln Pro Ser Ser Leu His Ser Glu Lys Leu Val Leu1 5 10 15Leu Ser Ser Thr Ile Arg Asp Asp Lys Asn Ile Asn Lys Gly Ile Phe20 25 30Ile Ala Cys Phe Ile Leu Phe Leu Trp Ala Ile Ser Leu Ile Leu Leu35 40 45Leu Ser Ile Asp Thr Ser Ile Ile His Lys Ser Leu Leu Gly Ile Ala50 55 60Met Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His65 70 75 80Asp Ala Met His Gly Val Val Tyr Pro Lys Asn Pro Arg Ile Asn Asn85 90 95Phe Ile Gly Lys Leu Thr Leu Ile Leu Tyr Gly Leu Leu Pro Tyr Lys100 105 110Asp Leu Leu Lys Lys His Trp Leu His His Gly His Pro Gly Thr Asp115 120 125Leu Asp Pro Asp Tyr Tyr Asn Gly His Pro Gln Asn Phe Phe Leu Trp130 135 140Tyr Leu His Phe Met Lys Ser Tyr Trp Arg Trp Thr Gln Ile Phe Gly145 150 155 160Leu Val Met Ile Phe His Gly Leu Lys Asn Leu Val His Ile Pro Glu165 170 175
Asn Asn Leu Ile Ile Phe Trp Met Ile Pro Ser Ile Leu Ser Ser Val180 185 190Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Lys Lys Leu Glu Gly195 200 205Gly Tyr Thr Asn Pro His Cys Ala Arg Ser Ile Pro Leu Pro Leu Phe210 215 220Trp Ser Phe Val Thr Cys Tyr His Phe Gly Tyr His Lys Glu His His225 230 235 240Glu Tyr Pro Gln Leu Pro Trp Trp Lys Leu Pro Glu Ala His Lys Ile245 250 255Ser Leu<210>17<211>2093<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(2093)<223>
<400>17tttgccagta ttacaacagc ttatatgttg agcaggtaaa agcttcaatg ccctattctt 60tctacagtta tcaatgttgc tcgtctaata tctggtgttc ttctcgaaat gtcaattggc 120ttgcagcaca ttgtcctcta atatccattc aagcttctta gatgatgaaa catttgtcaa 180atttattaat ttcatagtgt tcagtctcaa ttctttagct ggttcctcat agtaaagttg 240tctaatatga aatgaaaatg ttctgtgtgt tgtactaata ccttttcatg gttgtctata 300gaacgtcgat gaagagccaa acagaaacta ttttgggctg cgatttctga taccattgta 360tctgaatgct gggtgggagc tcatcagaag ctttacaatg ggtcacatat atggagccgg 420tatgaggaat gctgggaatc agttgcgttt cgcgtgctag gacttttcct tcctggtatt 480tctgcccaca gcccagttga ttacgtgaac tccgtcagac ttggaaagga gagaagtacc 540caaatgtcgt ctttttagaa atacttttgt cacaaaatag cggggtttac agctacagaa 600gatcatgcag aaggcgtcca gtttagtttt tgaaggttgt ttggagttta tttatctaaa 660gtaaacttaa atcagctttt tgtttatgag ttcagtgaac tatatgttca aataagactt 720
ccctttgtag atatgtgttt tttttgttgt tgagcacttt gtgtgcattg gataaacccc 780caacgtgtaa tagctaccat acaagagaag taactcgcac tgtccatgtc ttatgtggct 840cgactcagaa agcattcagg gggattgata accaccctcc aaaccaactg aaccattgtg 900aataaccacc cttcaaatca accgagtcct cgtgaaggac aaatatgtgg ttttatatac 960attaaatttt gtttttacat gcttcctctt acttctttag ttttcttgac catatcttgc1020gtttttccct tctgtaattg acacttttct tcaaaccatc cagcaatgtg gaagcttgac1080gattttcctt cagagtagaa attgaaaaga atcaactaaa aaggatagtc cttcgatttg1140atttccggct taaaaataaa ctaataagaa tgagagagcg aataatagaa tattttgaaa1200ttttaaagat attcaactat gttaaattgc gttataaatt tcttaaatta gtagcaccta1260atagtttagt tctcaaaagt caaaactact acataatgtg ctcatttttc acattaaaat1320gcctacatga tgtaaaagta aaactcgtag cattctacgt gttttactca actcaaacat1380cctgttcatt ttaataaacg tacgatgagc ttctctctcc aattttcttt tctttttttt1440ttttaaaaaa atattttttt ttatatcaat ccaaatgggc tccaatttat cataaattag1500gtagaaactt agatattaaa gaaagaaaag ggtttatctc gcaagtgtgg ctatggtggg1560acgtgtcaaa ttttggattg tagccaaaca tgagatttga tttaaaggga attggccaaa1620tcaccgaaag caggcatctt catcataaat tagtttgttt atttatacag aattatacgc1680ttttactagt tatagcattc ggtatctttt tctgggtaac tgccaaacca ccacaaattt1740caagtttcca tttaactctt caacttcaac ccaaccaaat ttatttgctt aattgtgcag1800aaccactccc tatatcttct aggtgctttc attcgttccg aggtaagaaa agatttttgt1860ttctttgaat gctttatgcc actcgtttaa cttctgaggt ttgtggatct tttaggcgac1920tttttttttt tttgtatgta aaatttgttt cataaatgct tctcaacata aatcttgaca1980aagagaagga attttaccaa gtatttaggt tcagaaatgg ataattttct tactgtgaaa2040tatccttatg gcaggtttta ctgttatttt tcagtaaaat gcctcaaatt gga 2093<210>18<211>4760<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(4760)<223>
<400>18tctagattga aataaacctt attgcattta gtatatgaga atgcatctat aaaataatgt 60
ctatttttgg tggaaaatat ttgtgcgcca aagcacggtt tgtattttat attttacaat 120atttttgcac ggtaatatag ttgcaaggtt ttacaaacga attatctctt gaactttaaa 180ttaagttcac agtttattcc aaaaataatg ttcaacttct aatcatatct ccccctattg 240ctagaaaaat ataacattta cgcccaactt catttaggat ccatttttat gcatggtgga 300gcaattggat catatactac atattttttt aaaaaaaata gatagaaatt atttaatctt 360gattccgaat caattgtgat gggaaaacct tattagtttg atgtgtacat ataatgtttt 420atgtcaaata aatttatttt atactaaatt ttatttgaaa gtatttttct cataacaaat 480aatttaacta tattggagac atgaaaattc tacaaaacca acttgcatta tcaacataat 540tttatagttt gaaattgtgc tcttaattaa acaattcaag ataacaatct ggtaaaatta 600aaattacaag ttgataacaa acatatacat atgtacatct catagatgca ttcattaaat 660catataatag taaatgcttc acaatagaag ggtctatatt catttttttt ttatgtgtca 720aacaattttg aggaattcaa tttcatcttt aactggtaca ataatcattt tatcatgaaa 780ataagcagct caagagaatt tttgaagaat cttttatttc tttaacattt aaccacatga 840atttttaatt tttttttgca atacatttaa accgaaatgg tcaaacgatc aaccaactga 900tctttattct aataaacttc tagtttacat ttgcatgtga gtgcatcatc attatcatat 960ttgtacacaa caaacaagaa aaaaatataa acaatatttt atttaaatat ttatattcca1020ctttgactgt agatattaaa tcttgtcatc atttatagtc tcaatattat aattttttta1080ttttttcaaa attcaaaagt ttacaattat ttttttgaac tataatatta tccaagatga1140acatctcaag aagaaaatta ttaatattgt tatggttaaa attttacata caatacttgt1200tttttgcttt acttttatct taccgtagat acacaatcga cgataactta gtgatcacac1260aataataatt attttgttca tgacacaata tttataagaa atacttattt ctttctttta1320tccttcagta gttcataata aaaacatacc ataatatttg tgatgcattc atagtacgta1380atgaaatgac aatttatgtc aaattatttt cttttatact ctcaaacctc ccgtaaaggt1440gagatgagtc atttatccaa ttatacataa atatgtcttt attcatgctc tttatcacat1500tctgacacat tcacttaatt tcaagagtaa gcaagcatga taactgaaac tatttatgcg1560tatcttacct tgatatttga cacattacat gacacacctc aacatcactt tcaaagatta1620agcgcaccac catattatct ttcttttttt ttttatgaag gttttataaa attattaaat1680taggtccaaa aaattgtttg tcaaataacc ttttatacta gattgatgac aaaaattacc1740tttacgtttt gaaagaccat tttaagacct aatctatcag tgactcctta aagttggcac1800aatatttcac ttagacaccc taattgaatg atgttcattt taaacaccca atgtagggtt1860ccgctatatc attttgacac atttcttaac atcaacaaaa atatataatg agtatgtgat1920
atactcgcga atgacgtgaa aaatgaagac atttgttatt tgtatcaaag tagttactaa1980ataattaatt ttgaataaaa ataaaagctg accagtaaat caataacaca taatattttc2040cacctaataa ttaaaatata aaataaaaaa gagccatctc agggtcatct gcccaccatt2100gctatttcaa agaaatttgt acgttagttt atagaaattg atgttaaaat tctttcaaga2160aaaatttatg aatgaattta ttctctaatt taaaaatatt ttctgttatt tttgttgaaa2220gaaatttaac ttggataaaa tggtggttaa aactggaaag aagaaaagag aaaaaataat2280taaaaatcat ttcacgctct aatcaatgag cgtatcacat tcattatgtt atataagcaa2340aagtgacaaa acgaaaataa tatattacat gaaatgtcta aaataaatat cgtctaatta2400aaatatctaa gtaacatatt gtgcctaact ttagagggat catcaataag ttaaacccca2460ttttaataac tcataattgt cctttttatt taatattgtc acaaatcaca atgataatta2520acattaattt gtcctttgtg acgtccatat tcatgcattt aaccaatcat cttcatttgg2580acttattatc acaattatcc cactttcctc acaaaatgga gcattcaagt ggaatagact2640acacgatttt taatttcatc aaaaacatct ttttgcttta ttcattatta tattgtcgct2700attgttgaat tttatttgcc ctaaatttct taccataaat agatttttct tttagaaaaa2760ggagattgac taattctttt cttgtaggaa aaggtttagg actctataaa tagagacata2820ttccttctaa cttaatcaac atttacaatg tagtcttaaa gactttgaaa gtttttggtt2880agggggagaa attgtgggtc acaagcttga tacgttatca attgtgtaaa cctcccatgt2940attctgagtg aatttggttg aggttgtttc cctctgtatt ttgtactctc atatttatag3000tggattgttc atctctttcg tggacgtagg tcgattgacc gtcgattgac cgaaccacgt3060taaatctttg tattttttga tatatttctc attatcttct tactcgtgat ctttcaaggt3120ttgcattgct atcttccgcg ttacaccaac ttatttacga tcctaacagc tatggtgtgg3180aaacataaat caaacatttt actgatataa acacatcttt gattataaca tgatagaaat3240ttgagcccaa ctttttatca tcattatata caaaaagttc taaatttttt ttttgatgta3300gtaaaactta aatccatagt cttgccccta aaccaatgac ataatatata acccaaaata3360tactagtttt cgccctcgag ccctttaaaa agtatagtca atatttacgg tgaccgtgaa3420tttcttaatt atgatatata atttaaaaga aatcatgatc acattctact gatgagaaca3480tgtgctaatc aagggaaaac atggatgtga aaaatacttt ttgttaaaag taaaaaaaaa3540tgtgaaattt tgttagttat ttactaccta tacattattt gagcatgtgc aaactttaca3600aatacctaat agaagatttt cacctgcctg tatatatgta aattaattat aatgaacact3660ctcacataaa ataattatca gtatatacat taatacttgc cctccacaat gaattaaata3720aaatgtagaa catgatctac acttcaataa aactaagacc ataaagaata atttcaaaat3780
atacacatgt caacaataaa ttatttgcat attatattaa cttactaaac aatctttact3840tttgaaatat aaaaataatc aagttataag tctgctcaaa gtaaagcact tgttagactc3900atctgatttt gagaaggtaa gcaaattgat ggtgcataat agtcacaagt aaaatataaa3960atagatttca ttagtaaaat tgttttttac tttctttata tataattatc aatatccttc4020aatggtaggt taattatatt gttaacttct tgttgaatta aagcaataag acaagaatat4080taaagataaa agaacaataa aaatagaaag actaagagat aagagttttc ttattcttct4140ttcaataagt atcatcaagt gtatacaata taaatttttg tatttttgat ctatctattt4200ataatgttat atataagcat acaaaagatc agtcataaat atgactttaa tcatgaaaat4260aatgaaagag attatgaagg cgtaaggtta ctagaataat agtcattaaa aaaaggggtt4320atctttataa ttgaataatt gatgaagtaa tggagataat tagtgagcat aaattttttt4380aaaaaaatgg acatttacac tataatattt tataacactt tcccttaaac atctaggtat4440aaataatgag tcttgtcaaa atcttagtag gaaaaattct gtgaaatttt tttagtgaaa4500acaaatgata taaatatctt gaatactcat tatttgttgt ctcattaaaa atcttatctg4560acctataaaa taaattattt gctcaactca aaatagtttt tcattctaaa attagtataa4620ttattagtga atatttaatt aacataattg tatactaagg ggcctataaa ttggattctt4680ctcaaagaaa aataaaatca ccacacaact ttcttcttct gctcatcaat tagcaattaa4740tccaaaacca ttatggctgc4760<210>19<211>1229<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(1229)<223>
<400>19gatcttactt taccataatg gtgaaaagga tagagaccca catggttttt acttcgttat 60agagacaaga tgaaaacaaa tctaaaattt aatattatag atggatagat gatggacaac 120aaaaagagaa aagaagatac tggtcattgg tccaaaacag ccacccgaat caatatatga 180ccgaaaaaca aaagctacag aatcatatct gtgcaacggt gccacagtgc tataggatag 240cacaaccaca ctgtcacata aaaaagagga ttttgcactc gttttagatg gagtttcgta 300attttcgggt ctttcaagct taaatatata cttcattaaa gcttcgaatt ttgtaatgtt 360caattctacc tctttgatgt tcgataccta taaaataatt aaataaacgt atagacgtag 420
gaacaattaa gcggagttag atagtgcatt tatgattcta cctgtgagtg caatggtaaa 480atggacatta taaaagagta ggggcaaaga gggaagtgaa aaattctccc cacttagcca 540tgtttaatat agtagggata ggaatatgta ataagtagtg ttttttctat ttaattttct 600gtatacttct tccatctcct ttaattatta aaaggttttc ctctctttac tctttctctc 660taaattacta ttctgaagta tattttcttt tataaaaaga gtaataaact ttatttccat 720taaaagaaca aacaacaaga aatgataatc aaatacacat tcatattttt aaaaaaaaag 780ttaaacaaga tatagaaata gttatcaaat atatttatgt tgtcattcct tgtatacaat 840ggcattcctt tagctttgtt tatgtatttc ctgagcttct cttagtgtac tatatccttt 900aatattaatg catctttcga tcttgctaag atatgataaa aatagacgac acgtgtcaca 960acctaattga gatatttcga tgtactttct atccgtctta gcttgtaatt aattattgtt1020aaaaaagaat actcaattaa ctagaaacaa gaaataagaa acgaaaacat tacaaaacgg1080agttgaagcg tgcaaatttg tggaaatgat tgttatcatg aaccagaaaa cattaaataa1140ctcttcctat aaaaggccct tattcttcac tttctcaaat cacgtcctaa agatatcaaa1200gatttcaact gatagcaaaa agcactact 1229<210>20<211>845<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(845)<223>
<400>20ctgttattga atttctataa aatgttataa tattgatttc ttaatgatca gttaactacg 60tgattatttg atatgttttt aatctaaaat gtgatatgta aaatatagaa gaaaaaaaat 120taaaaagaac tttaagaaaa aaatttcaac ccaccccaac ctaaaatcct aggtccgcca 180tggtaattat agatatatga tgatgaaggg caaatattgg tctatgagaa tttcggtgat 240actaccgctt gaagagcaat aatggttttg ggactccgat gagggaaaca ttcaaatatg 300atggattttg gtgatactat gtttacccga gctagctatc acagaataat ctacatccca 360caaatgaaat atgttatagg ctaccaatta ggaagtagtg gaattatgaa gaagtaggga 420tgtgcaaata taagagaaaa tttgaaaatt atgattgaaa caagttatgt ttttttaact 480agatgaatta aatggtttaa agatttgtag atttataatc aaacaattac cgctactcta 540tcggtgacta ccaattccat cattgtaaat aacaaataac agattcgttg ctggatgtct 600
tagtgccgtg aagcctacaa atcacactat aaactgctta gctctcgagc gttactaatt 660tggtgattac caattccaac attgcgactt cttctactag tagtactaaa atagcaagta 720atatgcattt gtggtaagat gtttggtgtt aacctttcct aaccagacta taaatgacct 780caacactata gtggagtttc atcgatcatc attctaaacg aaaaacttga agtgaaagca 840tcaag 845<210>21<211>3417<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(3417)<223>
<400>21aagcttggct gcaggtcgac ctgcaggtca acggatcaat gccttgttaa taatatgaaa 60ataagacgta aaagaagtct tgcatatgca ccataatatt agacttatgg acaaaagtaa 120gttggttcaa attacgcttt tatttatcca catagcaaga aaataatact caaaatccaa 180cggtatcggt tattttatat tttactctac atgtatatat gtagtataat ggacataaat 240tctgtcgtaa ttatacatat attaataatg aggattgtaa aataatatgc aaaaacgtcg 300tatttgacat actaatagct aaaatactac ctactatcat atataattag ttaactatgt 360gccttttaag aaaaattacg tgaaataaca aatatttaga gcatattatg taatatagct 420gtagttttat tattttttgt taatggctac aatttcgcaa aattttccta ttttgtttct 480taatcgtata aatccaaatt ttgtataatt atgaccttaa ttgtttaatt cagatttcgt 540ataaaattcg atttttgatt ttataaatta aaatttatac ttactttagc tacttgttta 600tgatttatca aaaaattcat attaatctat ttgtatatgg acaagcaaaa tatacaaatg 660gagttctgaa aatttctaaa tgcatatact taatatcttt gatggtcact caactatcaa 720ctttttccat aaaaagtcac ttaacattga ttttcaactc gaaaatcact caactatgaa 780atctttgtat agaaagtcac tcaacctatt taattatttt tttccattat atctgttgtc 840acgaaatatt atttctaact aatattctaa gaataaacat acatccattt aaatcattta 900ataaacccgc ccacttgacc taacccacat aatattaaca cttttgtttt acttttattc 960tccaaaatta ttttcttggt ttcccattct ttctcctttg cttttttttt cttcttctca1020atttcagcct ttttcttcct ttttttagta aacctcagtc aaataggaat tagattgtga1080ttaaaatatt attagaagga tgcagggttg tacaaagaga gtttattaag agataatcta1140
taaaaaaaaa aaagtcagat aatgcatatt cagattcaga gatcattaaa tgatgacttt1200tttcgtaata ggttttcttt aaatcctttc gccttcatac gacgactctc gataataaca1260tcgtttaaag ctaataatgc taatgaacaa taatcaaaat aaaaaagaat tcggatacaa1320gagaaaatga tttagtgaga gaaaaaattg agatattcct tattcctaac taaacgaagg1380aagaagaggc taaaattgag attcagttaa aaaaaaaaaa caaagaaaaa cgcaatggag1440atgagagaaa gtaattttga aaaataaaaa taaattaaga gggtaaatat tttattttta1500gcgagttggg ttaagtggtg ccggtcatta aatggatata tgtttatttc ttaaaatttt1560agttagaaat acaaatttca aatcaacaaa ttttaatgaa aaaataatta aataggttga1620gtggctttct atgcaaagat ctcatagttg agtgattttt gagtagaaaa tcatagttaa1680gtgagtttct gtgaaaaaaa attgatagtt gagtgactat caaagatatt aactctagac1740ttgtcatatt cgtatactta catacgaaat atacaaacct ctgcctccat gacaagcaaa1800aaactataac tatgaaacaa tattttcgaa atcatagcta taaagtctta ttatatctaa1860tatctttact atttttaaaa atttcacata attttaatac ataaataatt tacttttaac1920taacgaaaaa ggacattttt atgtcacctg agagcccatc ggtagattca tcacattttt1980tcgtttcttg taataaactg tacacatata aggagaaatt aaattagaga ttatttttcc2040attttgagga gattaataaa tttaaaatgt aacttaacat gtaaactgct ataaaggtaa2100caaaacacgt aaactgctat aaaggtaatt ctatttaaaa gataaataaa tgcttaaaag2160aagtgccaaa aaaacacaaa caaacaaatg aaactaaacc tacttcaagg gaagttcttg2220tagtataaaa ataaataaag tcaacttatt cacgacattt ctttttggtt ttcttttggc2280tacgtattca tatttaagtc tgactaattt agattctcgc tatatataaa agattcaggg2340gtggctcaac gcaattggag gcctagagca aaatttcaat tcgcggccta atatattata2400tactttatat acctatttat tcaaaattta ttttttttac actatttaga tggaaattat2460tagtacttaa tattgttttt tcagttatta gttttaggta aaattttatt aatacaacat2520tgaaaaacat cctttaagtg agacaattat tatatgtatt gttaacatag tgctataagt2580aataagtaaa taaatattaa ataaaaataa gagtaagaac catagaattt gacacaagaa2640gttgatgact tggtatacct cattttaaca tgcttgtact ttagtaatgc ttgaatctaa2700aatttaaaaa gaaataaaaa agaatttgta atccactttt tccaacactt ttcactgtta2760attcttattt ttaacatagt acaaaaaata ttaaaatgga taaaataatt tattttataa2820aagattatat atatattttt ttatcatata taactaattt ttctataaaa atttaaacac2880ataatttaat tttaaaaaaa atttggggct ttggggccta agacaaaggc cttaaaggac2940aaaacataga gccgcccctg aaaagatctc attcgaaaga aaatatgcat taccaatgat3000
ttttcgtacc cagagctcaa aatcaaaatt gtactgttat ttttttaaaa aatttcatct3060cagactaaat ggaatttttt tctttggtta acctgtttga tcaatctttt ggaatcagtt3120aattttgaaa aataaattaa tgagaaataa tttgtatttg tccagcttat ttaagaatta3180tttttgagca acaatttata tttagtcacg cttttaagtg tattttttaa aataaaatta3240aggtattatt tgaaaaaatt acttttaaaa aaattgaatt aaattctgtt actcttatta3300tatactccta tataatttga ttgccaaaaa tatcaaacgt ttaatatttg aagttgatgt3360gagggattac ttcttgatta aattgtacta caatgtaata ttatcaaatt aaagctt 3417<210>22<211>1155<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(6)..(995)<223>
<400>22gaagc atg cag cta gca gcg aca gta atg ttg gag cag ctt acc gga agc 50Met Gln Leu Ala Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser1 5 10 15gct gag gca ctc aag gag aag gag aag gag gtt gca ggc agc tct gac98Ala Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Set Ser Asp20 25 30gtg ttg cgt aca tgg gcg acc cag tac tcg ctt ccg tca gag gag tca 146Val Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser35 40 45gac gcg gcc cgc ccg gga ctg aag aat gcc tac aag cca cca cct tcc 194Asp Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser50 55 60gac aca aag ggc atc aca atg gcg cta gct gtc atc ggc tcc tgg gcc 242Asp Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser Trp Ala65 70 75gca gtg ttc ctc cac gcc att ttt caa atc aag ctt ccg acc tcc ttg 290Ala Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu80 85 90 95gac cag ctg cac tgg ctg ccc gtg tca gat gcc aca gct cag ctg gtt 338Asp Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val100 105 110agc ggc agc agc agc ctg ctg cac atc gtc gta gta ttc ttt gtc ctg 386Ser Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe Val Leu115 120 125gag ttc ctg tac aca ggc ctt ttt atc acc acg cat gat gct atg cat 434Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His130 135 140
ggc acc atc gcc atg aga aac agg cag ctt aat gac ttc ttg ggc aga482Gly Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg145 150 155gta tgc atc tcc ttg tac gcc tgg ttt gat tac aac atg ctg cac cgc530Val Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg160 165 170 175aag cat tgg gag cac cac aac cac act ggc gag gtg ggc aag gac cct578Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro180 185 190gac ttc cac agg gga aac cct ggc att gtg ccc tgg ttt gcc agc ttc626Asp Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe195 200 205atg tcc agc tac atg tcg atg tgg cag ttt gcg cgc ctc gca tgg tgg674Met Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp210 215 220acg gtg gtc atg cag ctg ctg ggt gcg cca atg gcg aac ctg ctg gtg722Thr Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val225 230 235ttc atg gcg gcc gcg ccc atc ctg tcc gcc ttc cgc ttg ttc tac ttt770Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe240 245 250 255ggc acg tac atg ccc cac aag cct gag cct ggc gcc gcg tca ggc tct818Gly Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser260 265 270tca cca gcc gtc atg aac tgg tgg aag tcg cgc act agc cag gcg tcc866Ser Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser275 280 285gac ctg gtc agc ttt ctg acc tgc tac cac ttc gac ctg cac tgg gag914Asp Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu290 295 300cac cac cgc tgg ccc ttt gcc ccc tgg tgg gag ctg ccc aac tgc cgc962His His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg305 310 315cgc ctg tct ggc cga ggt ctg gtt cct gcc tag ctggacacac tgcagtgggc 1015Arg Leu Ser Gly Arg Gly Leu Val Pro Ala320 325cctgctgcca gctgggcatg caggttgtgg caggactggg tgaggtgaaa agctgcaggc 1075gctgctgccg gacacgctgc atgggctacc ctgtgtagct gccgccacta ggggaggggg 1135tttgtagctg tcgagcttgc 1155<210>23<211>329<212>PRT<213>雨生紅球藻<400>23
Met Gln Leu Ala Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser Ala1 5 10 15Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser Ser Asp Val20 25 30Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser Asp35 40 45Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser Asp50 55 60Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser Trp Ala Ala65 70 75 80Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu Asp85 90 95Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val Ser100 105 110Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe Val Leu Glu115 120 125Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly130 135 140Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg Val145 150 155 160Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg Lys165 170 175His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp180 185 190Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe Met195 200 205Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp Thr210 215 220Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe225 230 235 240Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly
245 250 255Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser Ser260 265 270Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser Asp275 280 285Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His290 295 300His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg Arg305 310 315 320Leu Ser Gly Arg Gly Leu Val Pro Ala325<210>24<211>1111<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(4)..(951)<223>
<400>24tgc atg cta gag gca ctc aag gag aag gag aag gag gtt gca ggc agc48Met Leu Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser1 5 10 15tct gac gtg ttg cgt aca tgg gcg acc cag tac tcg ctt ccg tca gaa96Ser Asp Val Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu20 25 30gag tca gac gcg gcc cgc ccg gga ctg aag aat gcc tac aag cca cca 144Glu Ser Asp Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro35 40 45cct tcc gac aca aag ggc atc aca atg gcg cta gct gtc atc ggc tcc 192Pro Ser Asp Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser50 55 60tgg gcc gca gtg ttc ctc cac gcc att ttt caa atc aag ctt ccg acc 240Trp Ala Ala Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr65 70 75tcc ttg gac cag ctg cac tgg ctg ccc gtg tca gat gcc aca gct cag 288Ser Leu Asp Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln80 85 90 95ctg gtt agc ggc agc agc agc ctg ctg cac atc gtc gta gta ttc ttt 336Leu Val Ser Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe100 105 110
gtc ctg gag ttc ctg tac aca ggc ctt ttt atc acc acg cat gat gct 384Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala115 120 125atg cat ggc acc atc gcc atg aga aac agg cag ctt aat gac ttc ttg 432Met His Gly Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu130 135 140ggc aga gta tgc atc tcc ttg tac gcc tgg ttt gat tac aac atg ctg 480Gly Arg Val Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu145 150 155cac cgc aag cat tgg gag cac cac aac cac act ggc gag gtg ggc aag 528His Arg Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys160 165 170 175gac cct gac ttc cac agg gga aac cct ggc att gtg ccc tgg ttt gcc 576Asp Pro Asp Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala180 185 190agc ttc atg tcc agc tac atg tcg atg tgg cag ttt gcg cgc ctc gca 624Ser Phe Met Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala195 200 205tgg tgg acg gtg gtc atg cag ctg ctg ggt gcg cca atg gcg aac ctg 672Trp Trp Thr Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu210 215 220ctg gtg ttc atg gcg gcc gcg ccc atc ctg tcc gcc ttc cgc ttg ttc 720Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe225 230 235tac ttt ggc acg tac atg ccc cac aag cct gag cct ggc gcc gcg tca 768Tyr Phe Gly Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser240 245 250 255ggc tct tca cca gcc gtc atg aac tgg tgg aag tcg cgc act agc cag 816Gly Ser Ser Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln260 265 270gcg tcc gac ctg gtc agc ttt ctg acc tgc tac cac ttc gac ctg cac 864Ala Ser Asp Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His275 280 285tgg gag cac cac cgc tgg ccc ttc gcc ccc tgg tgg gag ctg ccc aac 912Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn290 295 300tgc cgc cgc ctg tct ggc cga ggt ctg gtt cct gcc tag ctggacacac961Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala305 310 315tgcagtgggc cctgctgcca gctgggcatg caggttgtgg caggactggg tgaggtgaaa1021agctgcaggc gctgctgccg gacacgttgc atgggctacc ctgtgtagct gccgccacta1081ggggaggggg tttgtagctg tcgagcttgc 1111<210>25<211>315
<212>PRT<213>雨生紅球藻<400>25Met Leu Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser Ser1 5 10 15Asp Val Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu20 25 30Ser Asp Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro35 40 45Ser Asp Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser Trp50 55 60Ala Ala Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser65 70 75 80Leu Asp Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu85 90 95Val Ser Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe Val100 105 110Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met115 120 125His Gly Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly130 135 140Arg Val Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His145 150 155 160Arg Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp165 170 175Pro Asp Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser180 185 190Phe Met Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp195 200 205Trp Thr Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu210 215 220Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr
225 230 235 240Phe Gly Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly245 250 255Ser Ser Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala260 265 270Ser Asp Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp275 280 285Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys290 295 300Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala305 310 315<210>26<211>1031<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(6)..(1031)<223>
<400>26gaagc atg cag cta gca gcg aca gta atg ttg gag cag ctt acc gga agc 50Met Gln Leu Ala Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser1 5 10 15gct gag gca ctc aag gag aag gag aag gag gtt gca ggc agc tct gac98Ala Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser Ser Asp20 25 30gtg ttg cgt aca tgg gcg acc cag tac tcg ctt ccg tca gag gag tca 146Val Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser35 40 45gac gcg gcc cgc ccg gga ctg aag aat gcc tac aag cca cca cct tcc 194Asp Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser50 55 60gac aca aag ggc atc aca atg gcg cta gct gtc atc ggc tcc tgg gct 242Asp Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser Trp Ala65 70 75gca gtg ttc ctc cac gcc att ttt caa atc aag ctt ccg acc tcc ttg 290Ala Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu80 85 90 95gac cag ctg cac tgg ctg ccc gtg tca gat gcc aca gct cag ctg gtt 338Asp Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val100 105 110
agc ggc agc agc agc ctg ctg cac atc gtc gta gta ttc ttt gtc ctg 386Ser Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe Val Leu115 120 125gag ttc ctg tac aca ggc ctt ttt atc acc acg cat gat gct atg cat 434Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His130 135 140ggc acc atc gcc atg aga aac agg cag ctt aat gac ttc ttg ggc aga 482Gly Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg145 150 155gta tgc atc tcc ttg tac gcc tgg ttt gat tac aac atg ctg cac cgc 530Val Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg160 165 170 175aag cat tgg gag cac cac aac cac act ggc gag gtg ggc aag gac cct 578Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro180 185 190gac ttc cac agg gga aac cct ggc att gtg ccc tgg ttt gcc agc ttc 626Asp Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe195 200 205atg tcc agc tac atg tcg atg tgg cag ttt gcg cgc ctc gca tgg tgg 674Met Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp210 215 220acg gtg gtc atg cag ctg ctg ggt gcg cca atg gcg aac ctg ctg gtg 722Thr Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val225 230 235ttc atg gcg gcc gcg ccc atc ctg tcc gcc ttc cgc ttg ttc tac ttt 770Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe240 245 250 255ggc acg tac atg ccc cac aag cct gag cct ggc gcc gcg tca ggc tct 818Gly Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser260 265 270tca cca gcc gtc atg aac tgg tgg aag tcg cgc act agc cag gcg tcc 866Ser Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser275 280 285gac ctg gtc agc ttt ctg acc tgc tac cac ttc gac ctg cac tgg gag 914Asp Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu290 295 300cac cac cgc tgg ccc ttt gcc ccc tgg tgg gag ctg ccc aac tgc cgc 962His His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg305 310 315cgc ctg tct ggc cga ggt ctg gtt cct gcc gag caa aaa ctc atc tca 1010Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Glu Gln Lys Leu Ile Ser320 325 330 335gaa gag gat ctg aat agc tag 1031Glu Glu Asp Leu Asn Ser340
<210>27<211>341<212>PRT<213>雨生紅球藻<400>27Met Gln Leu Ala Ala Thr Val Met Leu Glu Gln Leu Thr Gly Ser Ala1 5 10 15Glu Ala Leu Lys Glu Lys Glu Lys Glu Val Ala Gly Ser Ser Asp Val20 25 30Leu Arg Thr Trp Ala Thr Gln Tyr Ser Leu Pro Ser Glu Glu Ser Asp35 40 45Ala Ala Arg Pro Gly Leu Lys Asn Ala Tyr Lys Pro Pro Pro Ser Asp50 55 60Thr Lys Gly Ile Thr Met Ala Leu Ala Val Ile Gly Ser Trp Ala Ala65 70 75 80Val Phe Leu His Ala Ile Phe Gln Ile Lys Leu Pro Thr Ser Leu Asp85 90 95Gln Leu His Trp Leu Pro Val Ser Asp Ala Thr Ala Gln Leu Val Ser100 105 110Gly Ser Ser Ser Leu Leu His Ile Val Val Val Phe Phe Val Leu Glu115 120 125Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly130 135 140Thr Ile Ala Met Arg Asn Arg Gln Leu Asn Asp Phe Leu Gly Arg Val145 150 155 160Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Asn Met Leu His Arg Lys165 170 175His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp180 185 190Phe His Arg Gly Asn Pro Gly Ile Val Pro Trp Phe Ala Ser Phe Met195 200 205Ser Ser Tyr Met Ser Met Trp Gln Phe Ala Arg Leu Ala Trp Trp Thr210 215 220
Val Val Met Gln Leu Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe225 230 235 240Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly245 250 255Thr Tyr Met Pro His Lys Pro Glu Pro Gly Ala Ala Ser Gly Ser Ser260 265 270Pro Ala Val Met Asn Trp Trp Lys Ser Arg Thr Ser Gln Ala Ser Asp275 280 285Leu Val Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His290 295 300His Arg Trp Pro Phe Ala Pro Trp Trp Glu Leu Pro Asn Cys Arg Arg305 310 315 320Leu Ser Gly Arg Gly Leu Val Pro Ala Glu Gln Lys Leu Ile Ser Glu325 330 335Glu Asp Leu Asn Ser340<210>28<211>777<212>DNA<213>擬南芥(Arabidopsis thaliana)<220>
<221>啟動子<222>(1)..(777)<223>
<400>28gagctcactc actgatttcc attgcttgaa aattgatgat gaactaagat caatccatgt 60tagtttcaaa acaacagtaa ctgtggccaa cttagttttg aaacaacact aactggtcga 120agcaaaaaga aaaaagagtt tcatcatata tctgatttga tggactgttt ggagttagga 180ccaaacatta tctacaaaca aagacttttc tcctaacttg tgattccttc ttaaacccta 240ggggtaatat tctattttcc aaggatcttt agttaaaggc aaatccggga aattattgta 300atcatttggg gaaacatata aaagatttga gttagatgga agtgacgatt aatccaaaca 360tatatatctc tttcttctta tttcccaaat taacagacaa aagtagaata ttggctttta 420acaccaatat aaaaacttgc ttcacaccta aacacttttg tttactttag ggtaagtgca 480
aaaagccaac caaatccacc tgcactgatt tgacgtttac aaacgccgtt aagtcgatgt 540ccgttgattt aaacagtgtc ttgtaattaa aaaaatcagt ttacataaat ggaaaattta 600tcacttagtt ttcatcaact tctgaactta cctttcatgg attaggcaat actttccatt 660tttagtaact caagtggacc ctttacttct tcaactccat ctctctcttt ctatttcact 720tctttcttct cattatatct cttgtcctct ccaccaaatc tcttcaacaa aaagctt777<210>29<211>22<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(22)<223>
<400>29gcaagctcga cagctacaaa cc 22<210>30<211>24<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(24)<223>
<400>30gaagcatgca gctagcagcg acag 24<210>31<211>30<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(30)<223>
<400>31tgcatgctag aggcactcaa ggagaaggag 30<210>32<211>59<212>DNA<213>人工序列
<220>
<221>primer_bind<222>(1)..(59)<223>
<400>32ctagctattc agatcctctt ctgagatgag tttttgctcg gcaggaacca gacctcggc 59<210>33<211>28<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(28)<223>
<400>33gagctcactc actgatttcc attgcttg28<210>34<211>37<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(37)<223>
<400>34cgccgttaag tcgatgtccg ttgatttaaa cagtgtc 37<210>35<211>34<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(34)<223>
<400>35atcaacggac atcgacttaa cggcgtttgt aaac 34<210>36<211>25<212>DNA<213>人工序列<220>
<221>primer_bind<222>(1)..(25)<223>
<400>36taagcttttt gttgaagaga tttgg 25<210>37<211>831<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(1)..(831)<223>
<400>37atg cca tcc gag tcg tca gac gca gct cgt cct gtg ttg aag cac gcc 48Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Val Leu Lys His Ala1 5 10 15tat aaa cct cca gca tct gac gcc aag ggc atc act atg gcg ctg acc 96Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr20 25 30atc att ggc acc tgg acc gca gtg ttt tta cac gca ata ttc caa atc144Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile35 40 45agg cta ccg aca tcc atg gac cag ctt cac tgg ttg cct gtg tcc gaa192Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu50 55 60gcc aca gcc cag ctg ttg ggc gga agc agc agc cta ttg cac atc gcc240Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala65 70 75 80gca gtc ttc att gta ctt gag ttt ctg tac act ggt cta ttc atc acc288Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr85 90 95acg cat gat gca atg cat ggc acc ata gct ttg agg aac agg cag ctc336Thr His Asp Ala Met His Gly Thr Ile Ala Leu Arg Asn Arg Gln Leu100 105 110aat gat ctc ctt ggc aac atc tgc ata tca ctg tac gcc tgg ttt gac384Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp115 120 125tac agc atg cac tgg gag cac cac aac cat act ggc gaa gtg ggg aaa432Tyr Ser Met His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys130 135 140gac cct gac ttc cac aaa gga aat cct ggc ctt gtc ccc tgg ttc gcc480Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro Trp Phe Ala145 150 155 160agc ttc atg tcc agc tac atg tcc ctg tgg cag ttt gcc cgg ctg gca528
Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala Arg Leu Ala165 170 175tgg tgg gca gtg gtg atg caa acg ttg ggg gcc ccc atg gcg aat ctc 576Trp Trp Ala Val Val Met Gln Thr Leu Gly Ala Pro Met Ala Asn Leu180 185 190cta gtc ttc atg gct gca gcc cca atc ttg tca gca ttc cgc ctc ttc 624Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe195 200 205tacttc ggc act tac ctg cca cac aag cct gag cca ggc cct gca gca672Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly Pro Ala Ala210 215 220ggc tct cag gtc atg tct tgg ttc agg gcc aag aca agt gag gca tct 720Gly Ser Gln Val Met Ser Trp Phe Arg Ala Lys Thr Ser Glu Ala Ser225 230 235 240gat gtg atg agc ttc ctg aca tgc tac cac ttt gac ctg ttt gcc ccc 768Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu Phe Ala Pro245 250 255tgg tgg cag ctg ccc cac tgc cgc cgc ctg tct ggg cgt ggc ctg gtg 816Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val260 265 270cct gcc ttg gca tga 831Pro Ala Leu Ala275<210>38<211>276<212>PRT<213>雨生紅球藻<400>38Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Val Leu Lys His Ala1 5 10 15Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr20 25 30Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile35 40 45Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu50 55 60Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala65 70 75 80Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr85 90 95
Thr His Asp Ala Met His Gly Thr Ile Ala Leu Arg Asn Arg Gln Leu100 105 110Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp115 120 125Tyr Ser Met His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys130 135 140Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro Trp Phe Ala145 150 155 160Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala Arg Leu Ala165 170 175Trp Trp Ala Val Val Met Gln Thr Leu Gly Ala Pro Met Ala Asn Leu180 185 190Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe195 200 205Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly Pro Ala Ala210 215 220Gly Ser Gln Val Met Ser Trp Phe Arg Ala Lys Thr Ser Glu Ala Ser225 230 235 240Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu Phe Ala Pro245 250 255Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val260 265 270Pro Ala Leu Ala275<210>39<211>729<212>DNA<213>副球菌屬種(Paracoccus sp.)MBIC1143<220>
<221>CDS<222>(1)..(729)<223>
<400>39atg agc gca cat gcc ctg ccc aag gca gat ctg acc gcc acc agc ctg 48
Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15atc gtc tcg ggc ggc atc atc gcc gct tgg ctg gcc ctg cat gtg cat 96Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30gcg ctg tgg ttt ctg gac gca gcg gcg cat ccc atc ctg gcg atc gca144Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Ile Ala35 40 45aat ttc ctg ggg ctg acc tgg ctg tcg gtc gga ttg ttc atc atc gcg192Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60cat gac gcg atg cac ggg tcg gtg gtg ccg ggg cgt ccg cgc gcc aat240His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80gcg gcg atg ggc cag ctt gtc ctg tgg ctg tat gcc gga ttt tcg tgg288Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95cgc aag atg atc gtc aag cac atg gcc cat cac cgc cat gcc gga acc336Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110gac gac gac ccc gat ttc gac cat ggc ggc ccg gtc cgc tgg tac gcc384Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125cgc ttc atc ggc acc tat ttc ggc tgg cgc gag ggg ctg ctg ctg ccc432Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140gtc atc gtg acg gtc tat gcg ctg atc ctt ggg gat cgc tgg atg tac480Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160gtg gtc ttc tgg ccg ctg ccg tcg atc ctg gcg tcg atc cag ctg ttc528Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175gtg ttc ggc acc tgg ctg ccg cac cgc ccc ggc cac gac gcg ttc ccg576Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190gac cgc cac aat gcg cgg tcg tcg cgg atc agc gac ccc gtg tcg ctg624Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205ctg acc tgc ttt cac ttt ggc ggt tat cat cac gaa cac cac ctg cac672Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His210 215 220ccg acg gtg ccg tgg tgg cgc ctg ccc agc acc cgc acc aag ggg gac720Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240acc gca tga729Thr Ala
<210>40<211>242<212>PRT<213>副球菌MBIC1143<400>40Met Ser Ala His Ala Leu Pro Lys Ala Asp Leu Thr Ala Thr Ser Leu1 5 10 15Ile Val Ser Gly Gly Ile Ile Ala Ala Trp Leu Ala Leu His Val His20 25 30Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala Ile Ala35 40 45Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala50 55 60His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn65 70 75 80Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe Ser Trp85 90 95Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala Gly Thr100 105 110Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala115 120 125Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro130 135 140Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr145 150 155 160Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln Leu Phe165 170 175Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala Phe Pro180 185 190Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val Ser Leu195 200 205Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His
210 215 220Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp225 230 235 240Thr Ala<210>41<211>735<212>DNA<213>Brevundimonas aurantiaca<220>
<221>CDS<222>(1)..(735)<223>
<400>41atg acc gcc gcc gtc gcc gag cca cgc acc gtc ccg cgc cag acc tgg 48Met Thr Ala Ala Val Ala Glu Pro Arg Thr Val Pro Arg Gln Thr Trp1 5 10 15atc ggt ctg acc ctg gcg gga atg atc gtg gcg gga tgg gcg gtt ctg 96Ile Gly Leu Thr Leu Ala Gly Met Ile Val Ala Gly Trp Ala Val Leu20 25 30cat gtc tac ggc gtc tat ttt cac cga tgg ggg ccg ttg acc ctg gtg144His Val Tyr Gly Val Tyr Phe His Arg Trp Gly Pro Leu Thr Leu Val35 40 45atc gcc ccg gcg atc gtg gcg gtc cag acc tgg ttg tcg gtc ggc ctt192Ile Ala Pro Ala Ile Val Ala Val Gln Thr Trp Leu Ser Val Gly Leu50 55 60ttc atc gtc gcc cat gac gcc atg tac ggc tcc ctg gcg ccg gga cgg240Phe Ile Val Ala His Asp Ala Met Tyr Gly Ser Leu Ala Pro Gly Arg65 70 75 80ccg cgg ctg aac gcc gca gtc ggc cgg ctg acc ctg ggg ctc tat gcg288Pro Arg Leu Asn Ala Ala Val Gly Arg Leu Thr Leu Gly Leu Tyr Ala85 90 95ggc ttc cgc ttc gat cgg ctg aag acg gcg cac cac gcc cac cac gcc336Gly Phe Arg Phe Asp Arg Leu Lys Thr Ala His His Ala His His Ala100 105 110gcg ccc ggc acg gcc gac gac ccg gat ttt cac gcc ccg gcg ccc cgc384Ala Pro Gly Thr Ala Asp Asp Pro Asp Phe His Ala Pro Ala Pro Arg115 120 125gcc ttc ctt ccc tgg ttc ctg aac ttc ttt cgc acc tat ttc ggc tgg432Ala Phe Leu Pro Trp Phe Leu Asn Phe Phe Arg Thr Tyr Phe Gly Trp130 135 140cgc gag atg gcg gtc ctg acc gcc ctg gtc ctg atc gcc ctc ttc ggc480Arg Glu Met Ala Val Leu Thr Ala Leu Val Leu Ile Ala Leu Phe Gly145 150 155 160
ctg ggg gcg cgg ccg gcc aat ctc ctg acc ttc tgg gcc gcg ccg gcc 528Leu Gly Ala Arg Pro Ala Asn Leu Leu Thr Phe Trp Ala Ala Pro Ala165 170 175ctg ctt tca gcg ctt cag ctc ttc acc ttc ggc acc tgg ctg ccg cac 576Leu Leu Ser Ala Leu Gln Leu Phe Thr Phe Gly Thr Trp Leu Pro His180 185 190cgc cac acc gac cag ccg ttc gcc gac gcg cac cac gcc cgc agc agc 624Arg His Thr Asp Gln Pro Phe Ala Asp Ala His His Ala Arg Ser Ser195 200 205ggc tac ggc ccc gtg ctt tcc ctg ctc acc tgt ttc cac ttc ggc cgc 672Gly Tyr Gly Pro Val Leu Ser Leu Leu Thr Cys Phe His Phe Gly Arg210 215 220cac cac gaa cac cat ctg agc ccc tgg cgg ccc tgg tgg cgt ctg tgg 720His His Glu His His Leu Ser Pro Trp Arg Pro Trp Trp Arg Leu Trp225 230 235 240cgc ggc gag tct tga 735Arg Gly Glu Ser<210>42<211>244<212>PRT<213>Brevundimonas aurantiaca<400>42Met Thr Ala Ala Val Ala Glu Pro Arg Thr Val Pro Arg Gln Thr Trp1 5 10 15Ile Gly Leu Thr Leu Ala Gly Met Ile Val Ala Gly Trp Ala Val Leu20 25 30His Val Tyr Gly Val Tyr Phe His Arg Trp Gly Pro Leu Thr Leu Val35 40 45Ile Ala Pro Ala Ile Val Ala Val Gln Thr Trp Leu Ser Val Gly Leu50 55 60Phe Ile Val Ala His Asp Ala Met Tyr Gly Ser Leu Ala Pro Gly Arg65 70 75 80Pro Arg Leu Asn Ala Ala Val Gly Arg Leu Thr Leu Gly Leu Tyr Ala85 90 95Gly Phe Arg Phe Asp Arg Leu Lys Thr Ala His His Ala His His Ala100 105 110Ala Pro Gly Thr Ala Asp Asp Pro Asp Phe His Ala Pro Ala Pro Arg
115 120 125Ala Phe Leu Pro Trp Phe Leu Asn Phe Phe Arg Thr Tyr Phe Gly Trp130 135 140Arg Glu Met Ala Val Leu Thr Ala Leu Val Leu Ile Ala Leu Phe Gly145 150 155 160Leu Gly Ala Arg Pro Ala Asn Leu Leu Thr Phe Trp Ala Ala Pro Ala165 170 175Leu Leu Ser Ala Leu Gln Leu Phe Thr Phe Gly Thr Trp Leu Pro His180 185 190Arg His Thr Asp Gln Pro Phe Ala Asp Ala His His Ala Arg Ser Ser195 200 205Gly Tyr Gly Pro Val Leu Ser Leu Leu Thr Cys Phe His Phe Gly Arg210 215 220His His Glu His His Leu Ser Pro Trp Arg Pro Trp Trp Arg Leu Trp225 230 235 240Arg Gly Glu Ser<210>43<211>690<212>DNA<213>泡沫節(jié)球藻(Nodularia spumigena)NSOR10<220>
<221>CDS<222>(1)..(690)<223>
<400>43atg gcg atc gcc att att agt ata tgg gct atc agc cta ggt ttg tta 48Met Ala Ile Ala Ile Ile Ser Ile Trp Ala Ile Ser Leu Gly Leu Leu1 5 10 15ctt tat att gat ata tcc caa ttc aag ttt tgg atg ttg tta ccg ctc 96Leu Tyr Ile Asp Ile Ser Gln Phe Lys Phe Trp Met Leu Leu Pro Leu20 25 30ata ttt tgg caa aca ttt tta tat acg gga tta ttt att aca gct cat144Ile Phe Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His35 40 45gat gcc atg cat ggg gta gtt ttt ccc aaa aat ccc aaa atc aac cat192Asp Ala Met His Gly Val Val Phe Pro Lys Asn Pro Lys Ile Asn His50 55 60
ttc att ggc tca ttg tgc ctg ttt ctt tat ggt ctt tta cct tat caa 240Phe Ile Gly Ser Leu Cys Leu Phe Leu Tyr Gly Leu Leu Pro Tyr Gln65 70 75 80aaa ctt tta aaa aag cat tgg cta cat cac cat aat cca gcc agt gaa 288Lys Leu Leu Lys Lys His Trp Leu His His His Asn Pro Ala Ser Glu85 90 95aca gat cca gat ttt cac aac ggg aag cag aaa aac ttt ttt gct tgg 336Thr Asp Pro Asp Phe His Asn Gly Lys Gln Lys Asn Phe Phe Ala Trp100 105 110tat tta tat ttt atg aag cgt tac tgg agt tgg tta caa att atc aca 384Tyr Leu Tyr Phe Met Lys Arg Tyr Trp Ser Trp Leu Gln Ile Ile Thr115 120 125tta atg att att tat aac tta cta aaa tat ata tgg cat ttt cca gag 432Leu Met Ile Ile Tyr Asn Leu Leu Lys Tyr Ile Trp His Phe Pro Glu130 135 140gat aat atg act tat ttt tgg gta gtt ccc tca att tta agt tct tta 480Asp Asn Met Thr Tyr Phe Trp Val Val Pro Ser Ile Leu Ser Ser Leu145 150 155 160caa tta ttt tat ttt gga act ttt cta ccc cac agt gag cct gta gaa 528Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Ser Glu Pro Val Glu165 170 175ggt tat aaa gag cct cat cgt tcc caa act att agc cgt ccc att tgg 576Gly Tyr Lys Glu Pro His Arg Ser Gln Thr Ile Ser Arg Pro Ile Trp180 185 190tgg tca ttt ata act tgt tac cat ttt ggt tat cat tac gaa cat cat 624Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr His Tyr Glu His His195 200 205gaa tac ccc cat gtt cct tgg tgg caa tta cca gaa att tat aaa atg 672Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro Glu Ile Tyr Lys Met210 215 220tct aaa tca aat ttg tga 690Ser Lys Ser Asn Leu225<210>44<211>229<212>PRT<213>泡沫節(jié)球藻NSOR10<400>44Met Ala Ile Ala Ile Ile Ser Ile Trp Ala Ile Ser Leu Gly Leu Leu1 5 10 15Leu Tyr Ile Asp Ile Ser Gln Phe Lys Phe Trp Met Leu Leu Pro Leu20 25 30Ile Phe Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His
35 40 45Asp Ala Met His Gly Val Val Phe Pro Lys Asn Pro Lys Ile Asn His50 55 60Phe Ile Gly Ser Leu Cys Leu Phe Leu Tyr Gly Leu Leu Pro Tyr Gln65 70 75 80Lys Leu Leu Lys Lys His Trp Leu His His His Asn Pro Ala Ser Glu85 90 95Thr Asp Pro Asp Phe His Asn Gly Lys Gln Lys Asn Phe Phe Ala Trp100 105 110Tyr Leu Tyr Phe Met Lys Arg Tyr Trp Ser Trp Leu Gln Ile Ile Thr115 120 125Leu Met Ile Ile Tyr Asn Leu Leu Lys Tyr Ile Trp His Phe Pro Glu130 135 140Asp Asn Met Thr Tyr Phe Trp Val Val Pro Ser Ile Leu Ser Ser Leu145 150 155 160Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Ser Glu Pro Val Glu165 170 175Gly Tyr Lys Glu Pro His Arg Ser Gln Thr Ile Ser Arg Pro Ile Trp180 185 190Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr His Tyr Glu His His195 200 205Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro Glu Ile Tyr Lys Met210 215 220Ser Lys Ser Asn Leu225<210>45<211>789<212>DNA<213>點形念珠藻(Nostoc punctiforme)ATCC 29133<220>
<221>CDS<222>(1)..(789)<223>
<400>45ttg aat ttt tgt gat aaa cca gtt agc tat tat gtt gca ata gag caa 48Leu Asn Phe Cys Asp Lys Pro Val Ser Tyr Tyr Val Ala Ile Glu Gln1 5 10 15tta agt gct aaa gaa gat act gtt tgg ggg ctg gtg att gtc ata gta 96Leu Ser Ala Lys Glu Asp Thr Val Trp Gly Leu Val Ile Val Ile Val20 25 30att att agt ctt tgg gta gct agt ttg gct ttt tta cta gct att aat144Ile Ile Ser Leu Trp Val Ala Ser Leu Ala Phe Leu Leu Ala Ile Asn35 40 45tat gcc aaa gtc cca att tgg ttg ata cct att gca ata gtt tgg caa192Tyr Ala Lys Val Pro Ile Trp Leu Ile Pro Ile Ala Ile Val Trp Gln50 55 60atg ttc ctt tat aca ggg cta ttt att act gca cat gat gct atg cat240Met Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His Asp Ala Met His65 70 75 80ggg tca gtt tat cgt aaa aat ccc aaa att aat aat ttt atc ggt tca288Gly Ser Val Tyr Arg Lys Asn Pro Lys Ile Asn Asn Phe Ile Gly Ser85 90 95cta gct gta gcg ctt tac gct gtg ttt cca tat caa cag atg tta aag336Leu Ala Val Ala Leu Tyr Ala Val Phe Pro Tyr Gln Gln Met Leu Lys100 105 110aat cat tgc tta cat cat cgt cat cct gct agc gaa gtt gac cca gat384Asn His Cys Leu His His Arg His Pro Ala Ser Glu Val Asp Pro Asp115 120 125ttt cat gat ggt aag aga aca aac gct att ttc tgg tat ctc cat ttc432Phe His Asp Gly Lys Arg Thr Asn Ala Ile Phe Trp Tyr Leu His Phe130 135 140atg ata gaa tac tcc agt tgg caa cag tta ata gta cta act atc cta480Met Ile Glu Tyr Ser Ser Trp Gln Gln Leu Ile Val Leu Thr Ile Leu145 150 155 160ttt aat tta gct aaa tac gtt ttg cac atc cat caa ata aat ctc atc528Phe Asn Leu Ala Lys Tyr Val Leu His Ile His Gln Ile Asn Leu Ile165 170 175tta ttt tgg agt att cct cca att tta agt tcc att caa ctg ttt tat576Leu Phe Trp Ser Ile Pro Pro Ile Leu Ser Ser Ile Gln Leu Phe Tyr180 185 190ttc gga aca ttt ttg cct cat cga gaa ccc aag aaa gga tat gtt tat624Phe Gly Thr Phe Leu Pro His Arg Glu Pro Lys Lys Gly Tyr Val Tyr195 200 205ccc cat tgc agc caa aca ata aaa ttg cca act ttt ttg tca ttt atc672Pro His Cys Ser Gln Thr Ile Lys Leu Pro Thr Phe Leu Ser Phe Ile210 215 220gct tgc tac cac ttt ggt tat cat gaa gaa cat cat gag tat ccc cat720Ala Cys Tyr His Phe Gly Tyr His Glu Glu His His Glu Tyr Pro His225 230 235 240gta cct tgg tgg caa ctt cca tct gta tat aag cag aga gta ttc aac768
Val Pro Trp Trp Gln Leu Pro Ser Val Tyr Lys Gln Arg Val Phe Asn245 250 255aat tca gta acc aat tcg taa 789Asn Ser Val Thr Asn Ser260<210>46<211>262<212>PRT<213>點形念珠藻ATCC 29133<400>46Leu Asn Phe Cys Asp Lys Pro Val Ser Tyr Tyr Val Ala Ile Glu Gln1 5 10 15Leu Ser Ala Lys Glu Asp Thr Val Trp Gly Leu Val Ile Val Ile Val20 25 30Ile Ile Ser Leu Trp Val Ala Ser Leu Ala Phe Leu Leu Ala Ile Asn35 40 45Tyr Ala Lys Val Pro Ile Trp Leu Ile Pro Ile Ala Ile Val Trp Gln50 55 60Met Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His Asp Ala Met His65 70 75 80Gly Ser Val Tyr Arg Lys Asn Pro Lys Ile Asn Asn Phe Ile Gly Ser85 90 95Leu Ala Val Ala Leu Tyr Ala Val Phe Pro Tyr Gln Gln Met Leu Lys100 105 110Asn His Cys Leu His His Arg His Pro Ala Ser Glu Val Asp Pro Asp115 120 125Phe His Asp Gly Lys Arg Thr Asn Ala Ile Phe Trp Tyr Leu His Phe130 135 140Met Ile Glu Tyr Ser Ser Trp Gln Gln Leu Ile Val Leu Thr Ile Leu145 150 155 160Phe Asn Leu Ala Lys Tyr Val Leu His Ile His Gln Ile Asn Leu Ile165 170 175Leu Phe Trp Ser Ile Pro Pro Ile Leu Ser Ser Ile Gln Leu Phe Tyr180 185 190
Phe Gly Thr Phe Leu Pro His Arg Glu Pro Lys Lys Gly Tyr Val Tyr195 200 205Pro His Cys Ser Gln Thr Ile Lys Leu Pro Thr Phe Leu Ser Phe Ile210 215 220Ala Cys Tyr His Phe Gly Tyr His Glu Glu His His Glu Tyr Pro His225 230 235 240Val Pro Trp Trp Gln Leu Pro Ser Val Tyr Lys Gln Arg Val Phe Asn245 250 255Asn Ser Val Thr Asn Ser260<210>47<211>762<212>DNA<213>點形念珠藻ATCC 29133<220>
<221>CDS<222>(1)..(762)<223>
<400>47gtg atc cag tta gaa caa cca ctc agt cat caa gca aaa ctg act cca 48Val Ile Gln Leu Glu Gln Pro Leu Ser His Gln Ala Lys Leu Thr Pro1 5 10 15gta ctg aga agt aaa tct cag ttt aag ggg ctt ttc att gct att gtc 96Val Leu Arg Ser Lys Ser Gln Phe Lys Gly Leu Phe Ile Ala Ile Val20 25 30att gtt agc gca tgg gtc att agc ctg agt tta tta ctt tcc ctt gac144Ile Val Ser Ala Trp Val Ile Ser Leu Ser Leu Leu Leu Ser Leu Asp35 40 45atc tca aag cta aaa ttt tgg atg tta ttg cct gtt ata cta tgg caa192Ile Ser Lys Leu Lys Phe Trp Met Leu Leu Pro Val Ile Leu Trp Gln50 55 60aca ttt tta tat acg gga tta ttt att aca tct cat gat gcc atg cat240Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ser His Asp Ala Met His65 70 75 80ggc gta gta ttt ccc caa aac acc aag att aat cat ttg att gga aca288Gly Val Val Phe Pro Gln Asn Thr Lys Ile Asn His Leu Ile Gly Thr85 90 95ttg acc cta tcc ctt tat ggt ctt tta cca tat caa aaa cta ttg aaa336Leu Thr Leu Ser Leu Tyr Gly Leu Leu Pro Tyr Gln Lys Leu Leu Lys100 105 110aaa cat tgg tta cac cac cac aat cca gca agc tca ata gac ccg gat384
Lys His Trp Leu His His His Asn Pro Ala Ser Ser Ile Asp Pro Asp115 120 125ttt cac aat ggt aaa cac caa agt ttc ttt gct tgg tat ttt cat ttt 432Phe His Asn Gly Lys His Gln Ser Phe Phe Ala Trp Tyr Phe His Phe130 135 140atg aaa ggt tac tgg agt tgg ggg caa ata att gcg ttg act att att 480Met Lys Gly Tyr Trp Ser Trp Gly Gln Ile Ile Ala Leu Thr Ile Ile145 150 155 160tat aac ttt gct aaa tac ata ctc cat atc cca agt gat aat cta act 528Tyr Asn Phe Ala Lys Tyr Ile Leu His Ile Pro Ser Asp Asn Leu Thr165 170 175tac ttt tgg gtg cta ccc tcg ctt tta agt tca tta caa tta ttc tat 576Tyr Phe Trp Val Leu Pro Ser Leu Leu Ser Ser Leu Gln Leu Phe Tyr180 185 190ttt ggt act ttt tta ccc cat agt gaa cca ata ggg ggt tat gtt cag 624Phe Gly Thr Phe Leu Pro His Ser Glu Pro Ile Gly Gly Tyr Val Gln195 200 205cct cat tgt gcc caa aca att agc cgt cct att tgg tgg tca ttt atc 672Pro His Cys Ala Gln Thr Ile Ser Arg Pro Ile Trp Trp Ser Phe Ile210 215 220acg tgc tat cat ttt ggc tac cac gag gaa cat cac gaa tat cct cat 720Thr Cys Tyr His Phe Gly Tyr His Glu Glu His His Glu Tyr Pro His225 230 235 240att tct tgg tgg cag tta cca gaa att tac aaa gca aaa tag 762Ile Ser Trp Trp Gln Leu Pro Glu Ile Tyr Lys Ala Lys245 250<210>48<211>253<212>PRT<213>點形念珠藻ATCC 29133<400>48Val Ile Gln Leu Glu Gln Pro Leu Ser His Gln Ala Lys Leu Thr Pro1 5 10 15Val Leu Arg Ser Lys Ser Gln Phe Lys Gly Leu Phe Ile Ala Ile Val20 25 30Ile Val Ser Ala Trp Val Ile Ser Leu Ser Leu Leu Leu Ser Leu Asp35 40 45Ile Ser Lys Leu Lys Phe Trp Met Leu Leu Pro Val Ile Leu Trp Gln50 55 60Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ser His Asp Ala Met His65 70 75 80
Gly Val Val Phe Pro Gln Asn Thr Lys Ile Asn His Leu Ile Gly Thr85 90 95Leu Thr Leu Ser Leu Tyr Gly Leu Leu Pro Tyr Gln Lys Leu Leu Lys100 105 110Lys His Trp Leu His His His Asn Pro Ala Ser Ser Ile Asp Pro Asp115 120 125Phe His Asn Gly Lys His Gln Ser Phe Phe Ala Trp Tyr Phe His Phe130 135 140Met Lys Gly Tyr Trp Ser Trp Gly Gln Ile Ile Ala Leu Thr Ile Ile145 150 155 160Tyr Asn Phe Ala Lys Tyr Ile Leu His Ile Pro Ser Asp Asn Leu Thr165 170 175Tyr Phe Trp Val Leu Pro Ser Leu Leu Ser Ser Leu Gln Leu Phe Tyr180 185 190Phe Gly Thr Phe Leu Pro His Ser Glu Pro Ile Gly Gly Tyr Val Gln195 200 205Pro His Cys Ala Gln Thr Ile Ser Arg Pro Ile Trp Trp Ser Phe Ile210 215 220Thr Cys Tyr His Phe Gly Tyr His Glu Glu His His Glu Tyr Pro His225 230 235 240Ile Ser Trp Trp Gln Leu Pro Glu Ile Tyr Lys Ala Lys245 250<210>49<211>1536<212>DNA<213>耐放射異常球菌(Deinococcus radiodurans)R1<220>
<221>CDS<222>(1)..(1536)<223>
<400>49atg ccg gat tac gac ctg atc gtc atg ggc gcg ggc cac aac gcg ctg 48Met Pro Asp Tyr Asp Leu Ile Val Met Gly Ala Gly His Asn Ala Leu1 5 10 15gtg act gct gcc tac gcc gcc cgg gcg ggc ctg aaa gtc ggc gtg ttc 96
Val Thr Ala Ala Tyr Ala Ala Arg Ala Gly Leu Lys Val Gly Val Phe20 25 30gag cgg cgg cac ctc gtc ggc ggg gcg gtc agc acc gag gag gtc gtg 144Glu Arg Arg His Leu Val Gly Gly Ala Val Ser Thr Glu Glu Val Val35 40 45ccc ggt tac cgc ttc gac tac ggc ggc agc gcc cac atc ctg att cgg 192Pro Gly Tyr Arg Phe Asp Tyr Gly Gly Ser Ala His Ile Leu Ile Arg50 55 60atg acg ccc atc gtg cgc gaa ctc gaa ctc acg cgg cac ggg ctg cat 240Met Thr Pro Ile Val Arg Glu Leu Glu Leu Thr Arg His Gly Leu His65 70 75 80tac ctc gaa gtg gac cct atg ttt cac gct tcc gac ggt gaa acg ccc 288Tyr Leu Glu Val Asp Pro Met Phe His Ala Ser Asp Gly Glu Thr Pro85 90 95tgg ttc att cac cgc gac gcc ggg cgg acc atc cgc gaa ctg gac gaa 336Trp Phe Ile His Arg Asp Ala Gly Arg Thr Ile Arg Glu Leu Asp Glu100 105 110aag ttt ccc ggg cag ggc gac gcc tac ggg cgc ttt ctc gac gat tgg 384Lys Phe Pro Gly Gln Gly Asp Ala Tyr Gly Arg Phe Leu Asp Asp Trp115 120 125aca ccc ttc gcg cgc gcc gtg gcc gac ctg ttc aac tcg gcg ccg ggg 432Thr Pro Phe Ala Arg Ala Val Ala Asp Leu Phe Asn Ser Ala Pro Gly130 135 140ccg ctc gac ctg ggc aaa atg gtg atg cgc agc ggc cag ggc aag gac 480Pro Leu Asp Leu Gly Lys Met Val Met Arg Ser Gly Gln Gly Lys Asp145 150 155 160tgg aac gag cag ctc ccg cgc atc ctg cgg ccc tac ggc gac gtg gcg 528Trp Asn Glu Gln Leu Pro Arg Ile Leu Arg Pro Tyr Gly Asp Val Ala165 170 175cgc gag tac ttc agc gag gag cgc gtg cgg gct ccc ctg acc tgg atg 576Arg Glu Tyr Phe Ser Glu Glu Arg Val Arg Ala Pro Leu Thr Trp Met180 185 190gcg gcc cag agc ggc ccc cca ccc tcg gac ccg ctg agc gcg ccc ttt 624Ala Ala Gln Ser Gly Pro Pro Pro Ser Asp Pro Leu Ser Ala Pro Phe195 200 205ttg ctg tgg cac ccg ctc tac cac gaa ggc ggc gtg gcg cgg ccc aaa 672Leu Leu Trp His Pro Leu Tyr His Glu Gly Gly Val Ala Arg Pro Lys210 215 220ggc ggc agc ggc ggc ctg acc aaa gcc ctg cgc cgg gcc acc gag gcc 720Gly Gly Ser Gly Gly Leu Thr Lys Ala Leu Arg Arg Ala Thr Glu Ala225 230 235 240gaa ggc ggc gag gtc ttc acc gac gcg ccg gtc aag gaa att ctg gtc 768Glu Gly Gly Glu Val Phe Thr Asp Ala Pro Val Lys Glu Ile Leu Val245 250 255aag gac ggc aag gcg cag ggc atc cgg ctg gaa agc ggc gag acg tac 816Lys Asp Gly Lys Ala Gln Gly Ile Arg Leu Glu Ser Gly Glu Thr Tyr260 265 270
acc gcc cgc gcc gtc gtg tcg ggc gtc cac atc ctg acc act gcg aat864Thr Ala Arg Ala Val Val Ser Gly Val His Ile Leu Thr Thr Ala Asn275 280 285gcc ctg ccc gcc gaa tat gtc cct agc gcc gcc agg aat gtg cgc gtg912Ala Leu Pro Ala Glu Tyr Val Pro Ser Ala Ala Arg Asn Val Arg Val290 295 300ggc aac ggc ttc ggc atg att ttg cgc ctc gcc ctc agt gaa aaa gtc960Gly Asn Gly Phe Gly Met Ile Leu Arg Leu Ala Leu Ser Glu Lys Val305 310 315 320aaa tac cgt cac cac acc gag ccc gac tca cgc atc ggc ctg gga ttg 1008Lys Tyr Arg His His Thr Glu Pro Asp Ser Arg Ile Gly Leu Gly Leu325 330 335ctg atc aaa aac gag cgg caa atc atg cag ggc tac ggc gaa tac ctc 1056Leu Ile Lys Asn Glu Arg Gln Ile Met Gln Gly Tyr Gly Glu Tyr Leu340 345 350gcc ggg cag ccc acc acc gac ccg ccc ctc gtc gcc atg agc ttc agc 1104Ala Gly Gln Pro Thr Thr Asp Pro Pro Leu Val Ala Met Ser Phe Ser355 360 365gcg gtg gac gac tcg ctc gcc cca ccg aac ggc gac gtg ttg tgg ctg 1152Ala Val Asp Asp Ser Leu Ala Pro Pro Asn Gly Asp Val Leu Trp Leu370 375 380tgg gcg cag tac tac ccc ttc gag ctc gcc acc ggg agc tgg gaa acg 1200Trp Ala Gln Tyr Tyr Pro Phe Glu Leu Ala Thr Gly Ser Trp Glu Thr385 390 395 400cgc acc gcc gaa gcg cgg gag aac atc ctg cgg gcc ttt gag cac tac 1248Arg Thr Ala Glu Ala Arg Glu Asn Ile Leu Arg Ala Phe Glu His Tyr405 410 415gcg ccg ggc acc cgc gac acg att gtg ggc gaa ctc gtg cag acg ccg 1296Ala Pro Gly Thr Arg Asp Thr Ile Val Gly Glu Leu Val Gln Thr Pro420 425 430cag tgg ctg gaa acc aac ctc ggc ctg cac cgg ggc aac gtg atg cac 1344Gln Trp Leu Glu Thr Asn Leu Gly Leu His Arg Gly Asn Val Met His435 440 445ctg gaa atg tcc ttc gac cag atg ttc tcc ttc cgc ccc tgg ctg aaa 1392Leu Glu Met Ser Phe Asp Gln Met Phe Ser Phe Arg Pro Trp Leu Lys450 455 460gcg agc cag tac cgc tgg ccg ggc gtg cag ggg ctg tac ctc acc ggc 1440Ala Ser Gln Tyr Arg Trp Pro Gly Val Gln Gly Leu Tyr Leu Thr Gly465 470 475 480gcc agc acc cac ccc ggc gga ggc atc atg ggc gcc tcg gga cgc aac 1488Ala Ser Thr His Pro Gly Gly Gly Ile Met Gly Ala Ser Gly Arg Asn485 490 495gcg gcg cgg gtc atc gtg aag gac ctg acg cgg agg cgc tgg aaa tga 1536Ala Ala Arg Val Ile Val Lys Asp Leu Thr Arg Arg Arg Trp Lys500 505 510
<210>50<211>511<212>PRT<213>耐放射異常球菌R1<400>50Met Pro Asp Tyr Asp Leu Ile Val Met Gly Ala Gly His Asn Ala Leu1 5 10 15Val Thr Ala Ala Tyr Ala Ala Arg Ala Gly Leu Lys Val Gly Val Phe20 25 30Glu Arg Arg His Leu Val Gly Gly Ala Val Ser Thr Glu Glu Val Val35 40 45Pro Gly Tyr Arg Phe Asp Tyr Gly Gly Ser Ala His Ile Leu Ile Arg50 55 60Met Thr Pro Ile Val Arg Glu Leu Glu Leu Thr Arg His Gly Leu His65 70 75 80Tyr Leu Glu Val Asp Pro Met Phe His Ala Ser Asp Gly Glu Thr Pro85 90 95Trp Phe Ile His Arg Asp Ala Gly Arg Thr Ile Arg Glu Leu Asp Glu100 105 110Lys Phe Pro Gly Gln Gly Asp Ala Tyr Gly Arg Phe Leu Asp Asp Trp115 120 125Thr Pro Phe Ala Arg Ala Val Ala Asp Leu Phe Asn Ser Ala Pro Gly130 135 140Pro Leu Asp Leu Gly Lys Met Val Met Arg Ser Gly Gln Gly Lys Asp145 150 155 160Trp Asn Glu Gln Leu Pro Arg Ile Leu Arg Pro Tyr Gly Asp Val Ala165 170 175Arg Glu Tyr Phe Ser Glu Glu Arg Val Arg Ala Pro Leu Thr Trp Met180 185 190Ala Ala Gln Ser Gly Pro Pro Pro Ser Asp Pro Leu Ser Ala Pro Phe195 200 205Leu Leu Trp His Pro Leu Tyr His Glu Gly Gly Val Ala Arg Pro Lys210 215 220
Gly Gly Ser Gly Gly Leu Thr Lys Ala Leu Arg Arg Ala Thr Glu Ala225 230 235 240Glu Gly Gly Glu Val Phe Thr Asp Ala Pro Val Lys Glu Ile Leu Val245 250 255Lys Asp Gly Lys Ala Gln Gly Ile Arg Leu Glu Ser Gly Glu Thr Tyr260 265 270Thr Ala Arg Ala Val Val Ser Gly Val His Ile Leu Thr Thr Ala Asn275 280 285Ala Leu Pro Ala Glu Tyr Val Pro Ser Ala Ala Arg Asn Val Arg Val290 295 300Gly Asn Gly Phe Gly Met Ile Leu Arg Leu Ala Leu Ser Glu Lys Val305 310 315 320Lys Tyr Arg His His Thr Glu Pro Asp Ser Arg Ile Gly Leu Gly Leu325 330 335Leu Ile Lys Asn Glu Arg Gln Ile Met Gln Gly Tyr Gly Glu Tyr Leu340 345 350Ala Gly Gln Pro Thr Thr Asp Pro Pro Leu Val Ala Met Ser Phe Ser355 360 365Ala Val Asp Asp Ser Leu Ala Pro Pro Asn Gly Asp Val Leu Trp Leu370 375 380Trp Ala Gln Tyr Tyr Pro Phe Glu Leu Ala Thr Gly Ser Trp Glu Thr385 390 395 400Arg Thr Ala Glu Ala Arg Glu Asn Ile Leu Arg Ala Phe Glu His Tyr405 410 415Ala Pro Gly Thr Arg Asp Thr Ile Val Gly Glu Leu Val Gln Thr Pro420 425 430Gln Trp Leu Glu Thr Asn Leu Gly Leu His Arg Gly Asn Val Met His435 440 445Leu Glu Met Ser Phe Asp Gln Met Phe Ser Phe Arg Pro Trp Leu Lys450 455 460Ala Ser Gln Tyr Arg Trp Pro Gly Val Gln Gly Leu Tyr Leu Thr Gly
465 470 475 480Ala Ser Thr His Pro Gly Gly Gly Ile Met Gly Ala Ser Gly Arg Asn485 490 495Ala Ala Arg Val Ile Val Lys Asp Leu Thr Arg Arg Arg Trp Lys500 505 510<210>51<211>1608<212>DNA<213>雨生紅球藻<220>
<221>CDS<222>(3)..(971)<223>
<400>51ct aca ttt cac aag ccc gtg agc ggt gca agc gct ctg ccc cac atc 47Thr Phe His Lys Pro Val Ser Gly Ala Ser Ala Leu Pro His Ile1 5 10 15ggc cca cct cct cat ctc cat cgg tca ttt gct gct acc acg atg ctg 95Gly Pro Pro Pro His Leu His Arg Ser Phe Ala Ala Thr Thr Met Leu20 25 30tcg aag ctg cag tca atc agc gtc aag gcc cgc cgc gtt gaa cta gcc143Ser Lys Leu Gln Ser Ile Ser Val Lys Ala Arg Arg Val Glu Leu Ala35 40 45cgc gac atc acg cgg ccc aaa gtc tgc ctg cat gct cag cgg tgc tcg191Arg Asp Ile Thr Arg Pro Lys Val Cys Leu His Ala Gln Arg Cys Ser50 55 60tta gtt cgg ctg cga gtg gca gca cca cag aca gag gag gcg ctg gga239Leu Val Arg Leu Arg Val Ala Ala Pro Gln Thr Glu Glu Ala Leu Gly65 70 75acc gtg cag gct gcc ggc gcg ggc gat gag cac agc gcc gat gta gca287Thr Val Gln Ala Ala Gly Ala Gly Asp Glu His Ser Ala Asp Val Ala80 85 90 95ctc cag cag ctt gac cgg gct atc gca gag cgt cgt gcc cgg cgc aaa335Leu Gln Gln Leu Asp Arg Ala Ile Ala Glu Arg Arg Ala Arg Arg Lys100 105 110cgg gag cag ctg tca tac cag gct gcc gcc att gca gca tca att ggc383Arg Glu Gln Leu Ser Tyr Gln Ala Ala Ala Ile Ala Ala Ser Ile Gly115 120 125gtg tca ggc att gcc atc ttc gcc acc tac ctg aga ttt gcc atg cac431Val Ser Gly Ile Ala Ile Phe Ala Thr Tyr Leu Arg Phe Ala Met His130 135 140atg acc gtg ggc ggc gca gtg cca tgg ggt gaa gtg gct ggc act ctc479Met Thr Val Gly Gly Ala Val Pro Trp Gly Glu Val Ala Gly Thr Leu145 150 155
ctc ttg gtg gtt ggt ggc gcg ctc ggc atg gag atg tat gcc cgc tat 527Leu Leu Val Val Gly Gly Ala Leu Gly Met Glu Met Tyr Ala Arg Tyr160 165 170 175gca cac aaa gcc atc tgg cat gag tcg cct ctg ggc tgg ctg ctg cac 575Ala His Lys Ala Ile Trp His Glu Ser Pro Leu Gly Trp Leu Leu His180 185 190aag agc cac cac aca cct cgc act gga ccc ttt gaa gcc aac gac ttg 623Lys Ser His His Thr Pro Arg Thr Gly Pro Phe Glu Ala Asn Asp Leu195 200 205ttt gca atc atc aat gga ctg ccc gcc atg ctc ctg tgt acc ttt ggc 671Phe Ala Ile Ile Asn Gly Leu Pro Ala Met Leu Leu Cys Thr Phe Gly210 215 220ttc tgg ctg ccc aac gtc ctg ggg gcg gcc tgc ttt gga gcg ggg ctg 719Phe Trp Leu Pro Asn Val Leu Gly Ala Ala Cys Phe Gly Ala Gly Leu225 230 235ggc atc acg cta tac ggc atg gca tat atg ttt gta cac gat ggc ctg 767Gly Ile Thr Leu Tyr Gly Met Ala Tyr Met Phe Val His Asp Gly Leu240 245 250 255gtg cac agg cgc ttt ccc acc ggg ccc atc gct ggc ctg ccc tac atg 815Val His Arg Arg Phe Pro Thr Gly Pro Ile Ala Gly Leu Pro Tyr Met260 265 270aag cgc ctg aca gtg gcc cac cag cta cac cac agc ggc aag tac ggt 863Lys Arg Leu Thr Val Ala His Gln Leu His His Ser Gly Lys Tyr Gly275 280 285ggc gcg ccc tgg ggt atg ttc ttg ggt cca cag gag ctg cag cac att 911Gly Ala Pro Trp Gly Met Phe Leu Gly Pro Gln Glu Leu Gln His Ile290 295 300cca ggt gcg gcg gag gag gtg gag cga ctg gtc ctg gaa ctg gac tgg 959Pro Gly Ala Ala Glu Glu Val Glu Arg Leu Val Leu Glu Leu Asp Trp305 310 315tcc aag cgg tag ggtgcggaac caggcacgct ggtttcacac ctcatgcctg 1011Ser Lys Arg320tgataaggtg tggctagagc gatgcgtgtg agacgggtat gtcacggtcg actggtctga1071tggccaatgg catcggccat gtctggtcat cacgggctgg ttgcctgggt gaaggtgatg1131cacatcatca tgtgcggttg gaggggctgg cacagtgtgg gctgaactgg agcagttgtc1191caggctggcg ttgaatcagt gagggtttgt gattggcggt tgtgaagcaa tgactccgcc1251catattctat ttgtgggagc tgagatgatg gcatgcttgg gatgtgcatg gatcatggta1311gtgcagcaaa ctatattcac ctagggctgt tggtaggatc aggtgaggcc ttgcacattg1371catgatgtac tcgtcatggt gtgttggtga gaggatggat gtggatggat gtgtattctc1431agacgtagac cttgactgga ggcttgatcg agagagtggg ccgtattctt tgagagggga1491ggctcgtgcc agaaatggtg agtggatgac tgtgacgctg tacattgcag gcaggtgaga1551
tgcactgtct cgattgtaaa atacattcag atgcaaaaaa aaaaaaaaaa aaaaaaa 1608<210>52<211>322<212>PRT<213>雨生紅球藻<400>52Thr Phe His Lys Pro Val Ser Gly Ala Ser Ala Leu Pro His Ile Gly1 5 10 15Pro Pro Pro His Leu His Arg Ser Phe Ala Ala Thr Thr Met Leu Ser20 25 30Lys Leu Gln Ser Ile Ser Val Lys Ala Arg Arg Val Glu Leu Ala Arg35 40 45Asp Ile Thr Arg Pro Lys Val Cys Leu His Ala Gln Arg Cys Ser Leu50 55 60Val Arg Leu Arg Val Ala Ala Pro Gln Thr Glu Glu Ala Leu Gly Thr65 70 75 80Val Gln Ala Ala Gly Ala Gly Asp Glu His Ser Ala Asp Val Ala Leu85 90 95Gln Gln Leu Asp Arg Ala Ile Ala Glu Arg Arg Ala Arg Arg Lys Arg100 105 110Glu Gln Leu Ser Tyr Gln Ala Ala Ala Ile Ala Ala Ser Ile Gly Val115 120 125Ser Gly Ile Ala Ile Phe Ala Thr Tyr Leu Arg Phe Ala Met His Met130 135 140Thr Val Gly Gly Ala Val Pro Trp Gly Glu Val Ala Gly Thr Leu Leu145 150 155 160Leu Val Val Gly Gly Ala Leu Gly Met Glu Met Tyr Ala Arg Tyr Ala165 170 175His Lys Ala Ile Trp His Glu Ser Pro Leu Gly Trp Leu Leu His Lys180 185 190Ser His His Thr Pro Arg Thr Gly Pro Phe Glu Ala Asn Asp Leu Phe195 200 205
Ala Ile Ile Asn Gly Leu Pro Ala Met Leu Leu Cys Thr Phe Gly Phe210 215 220Trp Leu Pro Asn Val Leu Gly Ala Ala Cys Phe Gly Ala Gly Leu Gly225 230 235 240Ile Thr Leu Tyr Gly Met Ala Tyr Met Phe Val His Asp Gly Leu Val245 250 255His Arg Arg Phe Pro Thr Gly Pro Ile Ala Gly Leu Pro Tyr Met Lys260 265 270Arg Leu Thr Val Ala His Gln Leu His His Ser Gly Lys Tyr Gly Gly275 280 285Ala Pro Trp Gly Met Phe Leu Gly Pro Gln Glu Leu Gln His Ile Pro290 295 300Gly Ala Ala Glu Glu Val Glu Arg Leu Val Leu Glu Leu Asp Trp Ser305 310 315 320Lys Arg<210>53<211>1503<212>DNA<213>番茄<220>
<221>CDS<222>(1)..(1503)<223>
<400>53atg gat act ttg ttg aaa acc cca aat aac ctt gaa ttt ctg aac cca 48Met Asp Thr Leu Leu Lys Thr Pro Asn Asn Leu Glu Phe Leu Asn Pro1 5 10 15cat cat ggt ttt gct gtt aaa gct agt acc ttt aga tct gag aag cat 96His His Gly Phe Ala Val Lys Ala Ser Thr Phe Arg Ser Glu Lys His20 25 30cat aat ttt ggt tct agg aag ttt tgt gaa act ttg ggt aga agt gtt144His Asn Phe Gly Ser Arg Lys Phe Cys Glu Thr Leu Gly Arg Ser Val35 40 45tgt gtt aag ggt agt agt agt gct ctt tta gag ctt gta cct gag acc192Cys Val Lys Gly Ser Ser Ser Ala Leu Leu Glu Leu Val Pro Glu Thr50 55 60aaa aag gag aat ctt gat ttt gag ctt cct atg tat gac cct tca aaa240
Lys Lys Glu Asn Leu Asp Phe Glu Leu Pro Met Tyr Asp Pro Ser Lys65 70 75 80ggg gtt gtt gtg gat ctt gct gtg gtt ggt ggt ggc cct gca gga ctt 288Gly Val Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu85 90 95gct gtt gca cag caa gtt tct gaa gca gga ctc tct gtt tgt tca att 336Ala Val Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Cys Ser Ile100 105 110gat ccg aat cct aaa ttg ata tgg cct aat aac tat ggt gtt tgg gtg 384Asp Pro Asn Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val115 120 125gat gaa ttt gag gct atg gac ttg tta gat tgt cta gat gct acc tgg 432Asp Glu Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp130 135 140tct ggt gca gca gtg tac att gat gat aat acg gct aaa gat ctt cat 480Ser Gly Ala Ala Val Tyr Ile Asp Asp Asn Thr Ala Lys Asp Leu His145 150 155 160aga cct tat gga agg gtt aac cgg aaa cag ctg aaa tcg aaa atg atg 528Arg Pro Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Met165 170 175cag aaa tgt ata atg aat ggt gtt aaa ttc cac caa gcc aaa gtt ata 576Gln Lys Cys Ile Met Asn Gly Val Lys Phe His Gln Ala Lys Val Ile180 185 190aag gtg att cat gag gaa tcg aaa tcc atg ttg ata tgc aat gat ggt 624Lys Val Ile His Glu Glu Ser Lys Ser Met Leu Ile Cys Asn Asp Gly195 200 205att act att cag gca acg gtg gtg ctc gat gca act ggc ttc tct aga 672Ile Thr Ile Gln Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg210 215 220tct ctt gtt cag tat gat aag cct tat aac ccc ggg tat caa gtt gct 720Ser Leu Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala225 230 235 240tat ggc att ttg gct gaa gtg gaa gag cac ccc ttt gat gta aac aag 768Tyr Gly Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asn Lys245 250 255atg gtt ttc atg gat tgg cga gat tct cat ttg aag aac aat act gat 816Met Val Phe Met Asp Trp Arg Asp Ser His Leu Lys Asn Asn Thr Asp260 265 270ctc aag gag aga aat agt aga ata cca act ttt ctt tat gca atg cca 864Leu Lys Glu Arg Asn Ser Arg Ile Pro Thr Phe Leu Tyr Ala Met Pro275 280 285ttt tca tcc aac agg ata ttt ctt gaa gaa aca tca ctc gta gct cgt 912Phe Ser Ser Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg290 295 300cct ggc ttg cgt ata gat gat att caa gaa cga atg gtg gct cgt tta 960Pro Gly Leu Arg Ile Asp Asp Ile Gln Glu Arg Met Val Ala Arg Leu305 310 315 320
aac cat ttg ggg ata aaa gtg aag agc att gaa gaa gat gaa cat tgt 1008Asn His Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu His Cys325 330 335cta ata cca atg ggt ggt cca ctt cca gta tta cct cag aga gtc gtt 1056Leu Ile Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val340 345 350gga atc ggt ggt aca gct ggc atg gtt cat cca tcc acc ggt tat atg 1104Gly Ile Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met355 360 365gtg gca agg aca cta gct gcg gct cct gtt gtt gcc aat gcc ata att 1152Val Ala Arg Thr Leu Ala Ala Ala Pro Val Val Ala Asn Ala Ile Ile370 375 380caa tac ctc ggt tct gaa aga agt cat tcg ggt aat gaa tta tcc aca 1200Gln Tyr Leu Gly Ser Glu Arg Ser His Ser Gly Asn Glu Leu Ser Thr385 390 395 400gct gtt tgg aaa gat ttg tgg cct ata gag agg aga cgt caa aga gag 1248Ala Val Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu405 410 415ttc ttc tgc ttc ggt atg gat att ctt ctg aag ctt gat tta cct gct 1296Phe Phe Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Pro Ala420 425 430aca aga agg ttc ttt gat gca ttc ttt gac tta gaa cct cgt tat tgg 1344Thr Arg Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp435 440 445cat ggc ttc tta tcg tct cga ttg ttt cta cct gaa ctc ata gtt ttt 1392His Gly Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Ile Val Phe450 455 460ggg ctg tct cta ttc tct cat gct tca aat act tct aga ttt gag ata 1440Gly Leu Ser Leu Phe Ser His Ala Ser Asn Thr Ser Arg Phe Glu Ile465 470 475 480atg aca aag gga act gtt cca tta gta aat atg atc aac aat ttg tta 1488Met Thr Lys Gly Thr Val Pro Leu Val Asn Met Ile Asn Asn Leu Leu485 490 495cag gat aaa gaa tga 1503Gln Asp Lys Glu500<210>54<211>500<212>PRT<213>番茄<400>54Met Asp Thr Leu Leu Lys Thr Pro Asn Asn Leu Glu Phe Leu Asn Pro1 5 10 15His His Gly Phe Ala Val Lys Ala Ser Thr Phe Arg Ser Glu Lys His
20 25 30His Asn Phe Gly Ser Arg Lys Phe Cys Glu Thr Leu Gly Arg Ser Val35 40 45Cys Val Lys Gly Ser Ser Ser Ala Leu Leu Glu Leu Val Pro Glu Thr50 55 60Lys Lys Glu Asn Leu Asp Phe Glu Leu Pro Met Tyr Asp Pro Ser Lys65 70 75 80Gly Val Val Val Asp Leu Ala Val Val Gly Gly Gly Pro Ala Gly Leu85 90 95Ala Val Ala Gln Gln Val Ser Glu Ala Gly Leu Ser Val Cys Ser Ile100 105 110Asp Pro Asn Pro Lys Leu Ile Trp Pro Asn Asn Tyr Gly Val Trp Val115 120 125Asp Glu Phe Glu Ala Met Asp Leu Leu Asp Cys Leu Asp Ala Thr Trp130 135 140Ser Gly Ala Ala Val Tyr Ile Asp Asp Asn Thr Ala Lys Asp Leu His145 150 155 160Arg Pro Tyr Gly Arg Val Asn Arg Lys Gln Leu Lys Ser Lys Met Met165 170 175Gln Lys Cys Ile Met Asn Gly Val Lys Phe His Gln Ala Lys Val Ile180 185 190Lys Val Ile His Glu Glu Ser Lys Ser Met Leu Ile Cys Asn Asp Gly195 200 205Ile Thr Ile Gln Ala Thr Val Val Leu Asp Ala Thr Gly Phe Ser Arg210 215 220Ser Leu Val Gln Tyr Asp Lys Pro Tyr Asn Pro Gly Tyr Gln Val Ala225 230 235 240Tyr Gly Ile Leu Ala Glu Val Glu Glu His Pro Phe Asp Val Asn Lys245 250 255Met Val Phe Met Asp Trp Arg Asp Ser His Leu Lys Asn Asn Thr Asp260 265 270
Leu Lys Glu Arg Asn Ser Arg Ile Pro Thr Phe Leu Tyr Ala Met Pro275 280 285Phe Ser Ser Asn Arg Ile Phe Leu Glu Glu Thr Ser Leu Val Ala Arg290 295 300Pro Gly Leu Arg Ile Asp Asp Ile Gln Glu Arg Met Val Ala Arg Leu305 310 315 320Asn His Leu Gly Ile Lys Val Lys Ser Ile Glu Glu Asp Glu His Cys325 330 335Leu Ile Pro Met Gly Gly Pro Leu Pro Val Leu Pro Gln Arg Val Val340 345 350Gly Ile Gly Gly Thr Ala Gly Met Val His Pro Ser Thr Gly Tyr Met355 360 365Val Ala Arg Thr Leu Ala Ala Ala Pro Val Val Ala Asn Ala Ile Ile370 375 380Gln Tyr Leu Gly Ser Glu Arg Ser His Ser Gly Asn Glu Leu Ser Thr385 390 395 400Ala Val Trp Lys Asp Leu Trp Pro Ile Glu Arg Arg Arg Gln Arg Glu405 410 415Phe Phe Cys Phe Gly Met Asp Ile Leu Leu Lys Leu Asp Leu Pro Ala420 425 430Thr Arg Arg Phe Phe Asp Ala Phe Phe Asp Leu Glu Pro Arg Tyr Trp435 440 445His Gly Phe Leu Ser Ser Arg Leu Phe Leu Pro Glu Leu Ile Val Phe450 455 460Gly Leu Ser Leu Phe Ser His Ala Ser Asn Thr Ser Arg Phe Glu Ile465 470 475 480Met Thr Lys Gly Thr Val Pro Leu Val Asn Met Ile Asn Asn Leu Leu485 490 495Gln Asp Lys Glu500<210>55
<211>1125<212>DNA<213>番茄(Lycopersicon esculentum)<220>
<221>CDS<222>(20)..(946)<223>
<400>55ttggtcatct ccacaatca atg gct gcc gcc gcc aga atc tcc gcc tcc tct 52Met Ala Ala Ala Ala Arg Ile Ser Ala Ser Ser1 5 10acc tca cga act ttt tat ttc cgt cat tca ccg ttt ctt ggc cca aaa 100Thr Ser Arg Thr Phe Tyr Phe Arg His Ser Pro Phe Leu Gly Pro Lys15 20 25cct act tcg aca acc tca cat gtt tct cca atc tct cct ttt tct ctt 148Pro Thr Ser Thr Thr Ser His Val Ser Pro Ile Ser Pro Phe Ser Leu30 35 40aat cta ggc cca att ttg agg tct aga aga aaa ccc agt ttc act gtt 196Asn Leu Gly Pro Ile Leu Arg Ser Arg Arg Lys Pro Ser Phe Thr Val45 50 55tgc ttt gtt ctc gag gat gag aag ctg aaa cct caa ttt gac gat gag 244Cys Phe Val Leu Glu Asp Glu Lys Leu Lys Pro Gln Phe Asp Asp Glu60 65 70 75gct gag gat ttt gaa aag aag att gag gaa cag atc tta gct act cgc 292Ala Glu Asp Phe Glu Lys Lys Ile Glu Glu Gln Ile Leu Ala Thr Arg80 85 90ttg gcg gag aaa ctg gct agg aag aaa tcg gag agg ttt act tat ctt 340Leu Ala Glu Lys Leu Ala Arg Lys Lys Ser Glu Arg Phe Thr Tyr Leu95 100 105gtg gct gct ata atg tct agt ttt ggg att act tct atg gct gtt atg 388Val Ala Ala Ile Met Ser Ser Phe Gly Ile Thr Ser Met Ala Val Met110 115 120gct gtt tat tac aga ttt tcg tgg caa atg gag gga gga gaa gtt cct 436Ala Val Tyr Tyr Arg Phe Ser Trp Gln Met Glu Gly Gly Glu Val Pro125 130 135gta acc gaa atg ttg ggt aca ttt gct ctc tct gtt ggt gct gct gta 484Val Thr Glu Met Leu Gly Thr Phe Ala Leu Ser Val Gly Ala Ala Val140 145 150 155gga atg gag ttt tgg gcg aga tgg gca cac aaa gca ctg tgg cat gct 532Gly Met Glu Phe Trp Ala Arg Trp Ala His Lys Ala Leu Trp His Ala160 165 170tca cta tgg cac atg cat gag tca cac cac aaa cca aga gaa gga cct 580Ser Leu Trp His Met His Glu Ser His His Lys Pro Arg Glu Gly Pro175 180 185ttt gag ctg aac gac gtt ttc gcc ata aca aac gct gtt cca gca ata 628Phe Glu Leu Asn Asp Val Phe Ala Ile Thr Asn Ala Val Pro Ala Ile190 195 200
gcc ctc ctc aac tat ggt ttc ttc cat aaa ggc ctc att gcc gga cta 676Ala Leu Leu Asn Tyr Gly Phe Phe His Lys Gly Leu Ile Ala Gly Leu205 210 215tgc ttc ggt gct ggg cta ggg atc aca gta ttt gga atg gca tac atg 724Cys Phe Gly Ala Gly Leu Gly Ile Thr Val Phe Gly Met Ala Tyr Met220 225 230 235ttt gtt cac gat ggt ttg gtt cac aag aga ttc cca gtt gga cct gta 772Phe Val His Asp Gly Leu Val His Lys Arg Phe Pro Val Gly Pro Val240 245 250gcc aat gta cct tat ctt agg aag gtg gct gct gct cat tcg ctt cat 820Ala Asn Val Pro Tyr Leu Arg Lys Val Ala Ala Ala His Ser Leu His255 260 265cac tca gag aag ttc aat ggt gtc cca tat ggc ttg ttc ttc gga cct 868His Ser Glu Lys Phe Asn Gly Val Pro Tyr Gly Leu Phe Phe Gly Pro270 275 280aag gaa ctg gaa gaa gta gga ggg acg gaa gag ttg gaa aag gaa gtg 916Lys Glu Leu Glu Glu Val Gly Gly Thr Glu Glu Leu Glu Lys Glu Val285 290 295ata cga agg acg aga ctt tcg aaa gga tca tgaacgattg ttcataaaca 966Ile Arg Arg Thr Arg Leu Ser Lys Gly Ser300 305tagaatgtca ttttacactt cttatcaatg aggaagggtg atttttgatg tatttgatag1026tagagaaaaa tgtagctctc ttgatgaaat gaatttgtat ttatgtaggc tcttcttatt1086cagtaagatt ttttcttttt tttgatctcg tgccgaatt 1125<210>56<211>309<212>PRT<213>番茄<400>56Met Ala Ala Ala Ala Arg Ile Ser Ala Ser Ser Thr Ser Arg Thr Phe1 5 10 15Tyr Phe Arg His Ser Pro Phe Leu Gly Pro Lys Pro Thr Ser Thr Thr20 25 30Ser His Val Ser Pro Ile Ser Pro Phe Ser Leu Asn Leu Gly Pro Ile35 40 45Leu Arg Ser Arg Arg Lys Pro Ser Phe Thr Val Cys Phe Val Leu Glu50 55 60Asp Glu Lys Leu Lys Pro Gln Phe Asp Asp Glu Ala Glu Asp Phe Glu65 70 75 80
Lys Lys Ile Glu Glu Gln Ile Leu Ala Thr Arg Leu Ala Glu Lys Leu85 90 95Ala Arg Lys Lys Ser Glu Arg Phe Thr Tyr Leu Val Ala Ala Ile Met100 105 110Ser Ser Phe Gly Ile Thr Ser Met Ala Val Met Ala Val Tyr Tyr Arg115 120 125Phe Ser Trp Gln Met Glu Gly Gly Glu Val Pro Val Thr Glu Met Leu130 135 140Gly Thr Phe Ala Leu Ser Val Gly Ala Ala Val Gly Met Glu Phe Trp145 150 155 160Ala Arg Trp Ala His Lys Ala Leu Trp His Ala Ser Leu Trp His Met165 170 175His Glu Ser His His Lys Pro Arg Glu Gly Pro Phe Glu Leu Asn Asp180 185 190Val Phe Ala Ile Thr Asn Ala Val Pro Ala Ile Ala Leu Leu Asn Tyr195 200 205Gly Phe Phe His Lys Gly Leu Ile Ala Gly Leu Cys Phe Gly Ala Gly210 215 220Leu Gly Ile Thr Val Phe Gly Met Ala Tyr Met Phe Val His Asp Gly225 230 235 240Leu Val His Lys Arg Phe Pro Val Gly Pro Val Ala Asn Val Pro Tyr245 250 255Leu Arg Lys Val Ala Ala Ala His Ser Leu His His Ser Glu Lys Phe260 265 270Asn Gly Val Pro Tyr Gly Leu Phe Phe Gly Pro Lys Glu Leu Glu Glu275 280 285Val Gly Gly Thr Glu Glu Leu Glu Lys Glu Val Ile Arg Arg Thr Arg290 295 300Leu Ser Lys Gly Ser305<210>57
<211>1666<212>DNA<213>番茄<220>
<221>CDS<222>(1)..(1494)<223>
<400>57atg gaa gct ctt ctc aag cct ttt cca tct ctt tta ctt tcc tct cct 48Met Glu Ala Leu Leu Lys Pro Phe Pro Ser Leu Leu Leu Ser Ser Pro1 5 10 15aca ccc cat agg tct att ttc caa caa aat ccc tct ttt cta agt ccc 96Thr Pro His Arg Ser Ile Phe Gln Gln Asn Pro Ser Phe Leu Ser Pro20 25 30acc acc aaa aaa aaa tca aga aaa tgt ctt ctt aga aac aaa agt agt144Thr Thr Lys Lys Lys Ser Arg Lys Cys Leu Leu Arg Asn Lys Ser Ser35 40 45aaa ctt ttt tgt agc ttt ctt gat tta gca ccc aca tca aag cca gag192Lys Leu Phe Cys Ser Phe Leu Asp Leu Ala Pro Thr Ser Lys Pro Glu50 55 60tct tta gat gtt aac atc tca tgg gtt gat cct aat tcg aat cgg gct240Ser Leu Asp Val Asn Ile Ser Trp Val Asp Pro Asn Ser Asn Arg Ala65 70 75 80caa ttc gac gtg atc att atc gga gct ggc cct gct ggg ctc agg cta288Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg Leu85 90 95gct gaa caa gtt tct aaa tat ggt att aag gta tgt tgt gtt gac cct336Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro100 105 110tca cca ctc tcc atg tgg cca aat aat tat ggt gtt tgg gtt gat gag384Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu115 120 125ttt gag aat tta gga ctg gaa aat tgt tta gat cat aaa tgg cct atg432Phe Glu Asn Leu Gly Leu Glu Asn Cys Leu Asp His Lys Trp Pro Met130 135 140act tgt gtg cat ata aat gat aac aaa act aag tat ttg gga aga cca480Thr Cys Val His Ile Asn Asp Asn Lys Thr Lys Tyr Leu Gly Arg Pro145 150 155 160tat ggt aga gtt agt aga aag aag ctg aag ttg aaa ttg ttg aat agt528Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Lys Leu Leu Asn Ser165 170 175tgt gtt gag aac aga gtg aag ttt tat aaa gct aag gtt tgg aaa gtg576Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys Val180 185 190gaa cat gaa gaa ttt gag tct tca att gtt tgt gat gat ggt aag aag624Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys Lys195 200 205
ata aga ggt agt ttg gtt gtg gat gca agt ggt ttt gct agt gat ttt672Ile Arg Gly Ser Leu Val Val Asp Ala Ser Gly Phe Ala Ser Asp Phe210 215 220ata gag tat gac agg cca aga aac cat ggt tat caa att gct cat ggg720Ile Glu Tyr Asp Arg Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly225 230 235 240gtt tta gta gaa gtt gat aat cat cca ttt gat ttg gat aaa atg gtg768Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met Val245 250 255ctt atg gat tgg agg gat tct cat ttg ggt aat gag cca tat tta agg816Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg260 265 270gtg aat aat gct aaa gaa cca aca ttc ttg tat gca atg cca ttt gat864Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp275 280 285aga gat ttg gtt ttc ttg gaa gag act tct ttg gtg agt cgt cct gtt912Arg Asp Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val290 295 300tta tcg tat atg gaa gta aaa aga agg atg gtg gca aga tta agg cat960Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg His305 310 315 320ttg ggg atc aaa gtg aaa agt gtt att gag gaa gag aaa tgt gtg atc 1008Leu Gly Ile Lys Val Lys Ser Val Ile Glu Glu Glu Lys Cys Val Ile325 330 335cct atg gga gga cca ctt ccg cgg att cct caa aat gtt atg gct att 1056Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala Ile340 345 350ggt ggg aat tca ggg ata gtt cat cca tca aca ggg tac atg gtg gct 1104Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val Ala355 360 365agg agc atg gct tta gca cca gta cta gct gaa gcc atc gtc gag ggg 1152Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Glu Gly370 375 380ctt ggc tca aca aga atg ata aga ggg tct caa ctt tac cat aga gtt 1200Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg Val385 390 395 400tgg aat ggt ttg tgg cct ttg gat aga aga tgt gtt aga gaa tgt tat 1248Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Val Arg Glu Cys Tyr405 410 415tca ttt ggg atg gag aca ttg ttg aag ctt gat ttg aaa ggg act agg 1296Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg420 425 430aga ttg ttt gac gct ttc ttt gat ctt gat cct aaa tac tgg caa ggg 1344Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln Gly435 440 445ttc ctt tct tca aga ttg tct gtc aaa gaa ctt ggt tta ctc agc ttg 1392
Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Gly Leu Leu Ser Leu450 455 460tgt ctt ttc gga cat ggc tca aac atg act agg ttg gat att gtt aca 1440Cys Leu Phe Gly His Gly Ser Asn Met Thr Arg Leu Asp Ile Val Thr465 470 475 480aaa tgt cct ctt cct ttg gtt aga ctg att ggc aat cta gca ata gag 1488Lys Cys Pro Leu Pro Leu Val Arg Leu Ile Gly Asn Leu Ala Ile Glu485 490 495agc ctt tgaatgtgaa aagtttgaat cattttcttc attttaattt ctttgattat 1544Ser Leutttcatattt tctcaattgc aaaagtgaga taagagctac atactgtcaa caaataaact1604actattggaa agttaaaata tgtgtttgtt gtatgttatt ctaatggaat ggattttgta1664aa 1666<210>58<211>498<212>PRT<213>番茄<400>58Met Glu Ala Leu Leu Lys Pro Phe Pro Ser Leu Leu Leu Ser Ser Pro1 5 10 15Thr Pro His Arg Ser Ile Phe Gln Gln Asn Pro Ser Phe Leu Ser Pro20 25 30Thr Thr Lys Lys Lys Ser Arg Lys Cys Leu Leu Arg Asn Lys Ser Ser35 40 45Lys Leu Phe Cys Ser Phe Leu Asp Leu Ala Pro Thr Ser Lys Pro Glu50 55 60Ser Leu Asp Val Asn Ile Ser Trp Val Asp Pro Asn Ser Asn Arg Ala65 70 75 80Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg Leu85 90 95Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro100 105 110Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu115 120 125Phe Glu Asn Leu Gly Leu Glu Asn Cys Leu Asp His Lys Trp Pro Met
130 135140Thr Cys Val His Ile Asn Asp Asn Lys Thr Lys Tyr Leu Gly Arg Pro145 150 155 160Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Lys Leu Leu Asn Ser165 170 175Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys Val180 185 190Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys Lys195 200 205Ile Arg Gly Ser Leu Val Val Asp Ala Ser Gly Phe Ala Ser Asp Phe210 215 220Ile Glu Tyr Asp Arg Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly225 230 235 240Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met Val245 250 255Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg260 265 270Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp275 280 285Arg Asp Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val290 295 300Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg His305 310 315 320Leu Gly Ile Lys Val Lys Ser Val Ile Glu Glu Glu Lys Cys Val Ile325 330 335Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala Ile340 345 350Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val Ala355 360 365Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Glu Gly370 375 380
Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg Val385 390 395 400Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Val Arg Glu Cys Tyr405 410 415Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg420 425 430Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln Gly435 440 445Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Gly Leu Leu Ser Leu450 455 460Cys Leu Phe Gly His Gly Ser Asn Met Thr Arg Leu Asp Ile Val Thr465 470 475 480Lys Cys Pro Leu Pro Leu Val Arg Leu Ile Gly Asn Leu Ala Ile Glu485 490 495Ser Leu<210>59<211>37<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(37)<223>
<400>59gcgcatgcat ctagaaatga tccagttaga acaacca 37<210>60<211>37<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(37)<223>
<400>60gcgcatgctc tagactattt tgctttgtaa atttctg 37
<210>61<211>792<212>DNA<213>點形念珠藻ATCC 29133<220>
<221>CDS<222>(5)..(775)<223>
<400>61gcgc atg cat cta gaa atg atc cag tta gaa caa cca ctc agt cat caa 49Met His Leu Glu Met Ile Gln Leu Glu Gln Pro Leu Ser His Gln1 5 10 15gca aaa ctg act cca gta ctg aga agt aaa tct cag ttt aag ggg ctt97Ala Lys Leu Thr Pro Val Leu Arg Ser Lys Ser Gln Phe Lys Gly Leu20 25 30ttc att gct att gtc att gtt agc gca tgg gtc att agc ctg agt tta 145Phe Ile Ala Ile Val Ile Val Ser Ala Trp Val Ile Ser Leu Ser Leu35 40 45tta ctt tcc ctt gac atc tca aag cta aaa ttt tgg atg tta ttg cct 193Leu Leu Ser Leu Asp Ile Ser Lys Leu Lys Phe Trp Met Leu Leu Pro50 55 60gtt ata cta tgg caa aca ttt tta tat acg gga tta ttt att aca tct 241Val Ile Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ser65 70 75cat gat gcc atg cat ggc gta gta ttt ccc caa aac acc aag att aat 289His Asp Ala Met His Gly Val Val Phe Pro Gln Asn Thr Lys Ile Asn80 85 90 95cat ttg att gga aca ttg acc cta tcc ctt tat ggt ctt tta cca tat 337His Leu Ile Gly Thr Leu Thr Leu Ser Leu Tyr Gly Leu Leu Pro Tyr100 105 110caa aaa cta ttg aaa aaa cat tgg tta cac cac cac aat cca gca agc 385Gln Lys Leu Leu Lys Lys His Trp Leu His His His Asn Pro Ala Ser115 120 125tca ata gac ccg gat ttt cac aat ggt aaa cac caa agt ttc ttt gct 433Ser Ile Asp Pro Asp Phe His Asn Gly Lys His Gln Ser Phe Phe Ala130 135 140tgg tat ttt cat ttt atg aaa ggt tac tgg agt tgg ggg caa ata att 481Trp Tyr Phe His Phe Met Lys Gly Tyr Trp Ser Trp Gly Gln Ile Ile145 150 155gcg ttg act att att tat aac ttt gct aaa tac ata ctc cat atc cca 529Ala Leu Thr Ile Ile Tyr Asn Phe Ala Lys Tyr Ile Leu His Ile Pro160 165 170 175agt gat aat cta act tac ttt tgg gtg cta ccc tcg ctt tta agt tca 577Ser Asp Asn Leu Thr Tyr Phe Trp Val Leu Pro Ser Leu Leu Ser Ser180 185 190
tta caa tta ttc tat ttt ggt act ttt tta ccc cat agt gaa cca ata 625Leu Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Ser Glu Pro Ile195 200 205ggg ggt tat gtt cag cct cat tgt gcc caa aca att agc cgt cct att 673Gly Gly Tyr Val Gln Pro His Cys Ala Gln Thr Ile Ser Arg Pro Ile210 215 220tgg tgg tca ttt atc acg tgc tat cat ttt ggc tac cac gag gaa cat 721Trp Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr His Glu Glu His225 230 235cac gaa tat cct cat att tct tgg tgg cag tta cca gaa att tac aaa 769His Glu Tyr Pro His Ile Ser Trp Trp Gln Leu Pro Glu Ile Tyr Lys240 245 250 255gca aaa tagtctagag catgcgc792Ala Lys<210>62<211>257<212>PRT<213>點形念珠藻ATCC 29133<400>62Met His Leu Glu Met Ile Gln Leu Glu Gln Pro Leu Ser His Gln Ala1 5 10 15Lys Leu Thr Pro Val Leu Arg Ser Lys Ser Gln Phe Lys Gly Leu Phe20 25 30Ile Ala Ile Val Ile Val Ser Ala Trp Val Ile Ser Leu Ser Leu Leu35 40 45Leu Ser Leu Asp Ile Ser Lys Leu Lys Phe Trp Met Leu Leu Pro Val50 55 60Ile Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ser His65 70 75 80Asp Ala Met His Gly Val Val Phe Pro Gln Asn Thr Lys Ile Asn His85 90 95Leu Ile Gly Thr Leu Thr Leu Ser Leu Tyr Gly Leu Leu Pro Tyr Gln100 105 110Lys Leu Leu Lys Lys His Trp Leu His His His Asn Pro Ala Ser Ser115 120 125Ile Asp Pro Asp Phe His Asn Gly Lys His Gln Ser Phe Phe Ala Trp130 135 140
Tyr Phe His Phe Met Lys Gly Tyr Trp Ser Trp Gly Gln Ile Ile Ala145 150 155 160Leu Thr Ile Ile Tyr Asn Phe Ala Lys Tyr Ile Leu His Ile Pro Ser165 170 175Asp Asn Leu Thr Tyr Phe Trp Val Leu Pro Ser Leu Leu Ser Ser Leu180 185 190Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Ser Glu Pro Ile Gly195 200 205Gly Tyr Val Gln Pro His Cys Ala Gln Thr Ile Ser Arg Pro Ile Trp210 215 220Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr His Glu Glu His His225 230 235 240Glu Tyr Pro His Ile Ser Trp Trp Gln Leu Pro Glu Ile Tyr Lys Ala245 250 255Lys<210>63<211>26<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(26)<223>
<400>63gtcgaccctg ctttaatgag atatgc 26<210>64<211>27<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(27)<223>
<400>64
ctcgagcttg gacaatcagt aaattga 27<210>65<211>210<212>DNA<213>根癌農桿菌(Agrobacterium tumefaciens)<220>
<221>終止子<222>(1)..(210)<223>
<400>65gtcgaccctg ctttaatgag atatgcgaga cgcctatgat cgcatgatat ttgctttcaa 60ttctgttgtg cacgttgtaa aaaacctgag catgtgtagc tcagatcctt accgccggtt 120tcggttcatt ctaatgaata tatcacccgt tactatcgta tttttatgaa taatattctc 180cgttcaattt actgattgtc caagctcgag 210<210>66<211>35<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(35)<223>
<400>66gcgcatgcat ctagaaatgg ttcagtgtca accat35<210>67<211>35<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(35)<223>
<400>67gcgcatgctc tagaccttat aaagatattt tgtga35<210>68<211>809<212>DNA<213>念珠藻PCC 7120<220>
<221>CDS<222>(5)..(790)<223>
<400>68gcgc atg cat cta gaa atg gtt cag tgt caa cca tca tct ctg cat tca 49Met His Leu Glu Met Val Gln Cys Gln Pro Ser Ser Leu His Ser1 5 10 15gaa aaa ctg gtg tta ttg tca tcg aca atc aga gat gat aaa aat att97Glu Lys Leu Val Leu Leu Ser Ser Thr Ile Arg Asp Asp Lys Asn Ile20 25 30aat aag ggt ata ttt att gcc tgc ttt atc tta ttt tta tgg gca att 145Asn Lys Gly Ile Phe Ile Ala Cys Phe Ile Leu Phe Leu Trp Ala Ile35 40 45agt tta atc tta tta ctc tca ata gat aca tcc ata att cat aag agc 193Ser Leu Ile Leu Leu Leu Ser Ile Asp Thr Ser Ile Ile His Lys Ser50 55 60tta tta ggt ata gcc atg ctt tgg cag acc ttc tta tat aca ggt tta 241Leu Leu Gly Ile Ala Met Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu65 70 75ttt att act gct cat gat gcc atg cac ggc gta gtt tat ccc aaa aat 289Phe Ile Thr Ala His Asp Ala Met His Gly Val Val Tyr Pro Lys Asn80 85 90 95ccc aga ata aat aat ttt ata ggt aag ctc act cta atc ttg tat gga 337Pro Arg Ile Asn Asn Phe Ile Gly Lys Leu Thr Leu Ile Leu Tyr Gly100 105 110cta ctc cct tat aaa gat tta ttg aaa aaa cat tgg tta cac cac gga 385Leu Leu Pro Tyr Lys Asp Leu Leu Lys Lys His Trp Leu His His Gly115 120 125cat cct ggt act gat tta gac cct gat tat tac aat ggt cat ccc caa 433His Pro Gly Thr Asp Leu Asp Pro Asp Tyr Tyr Asn Gly His Pro Gln130 135 140aac ttc ttt ctt tgg tat cta cat ttt atg aag tct tat tgg cga tgg 481Asn Phe Phe Leu Trp Tyr Leu His Phe Met Lys Ser Tyr Trp Arg Trp145 150 155acg caa att ttc gga tta gtg atg att ttt cat gga ctt aaa aat ctg 529Thr Gln Ile Phe Gly Leu Val Met Ile Phe His Gly Leu Lys Asn Leu160 165 170 175gtg cat ata cca gaa aat aat tta att ata ttt tgg atg ata cct tct 577Val His Ile Pro Glu Asn Asn Leu Ile Ile Phe Trp Met Ile Pro Ser180 185 190att tta agt tca gta caa cta ttt tat ttt ggt aca ttt ttg cct cat 625Ile Leu Ser Ser Val Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His195 200 205aaa aag cta gaa ggt ggt tat act aac ccc cat tgt gcg cgc agt atc 673Lys Lys Leu Glu Gly Gly Tyr Thr Asn Pro His Cys Ala Arg Ser Ile210 215 220
cca tta cct ctt ttt tgg tct ttt gtt act tgt tat cac ttc ggc tac721Pro Leu Pro Leu Phe Trp Ser Phe Val Thr Cys Tyr His Phe Gly Tyr225 230 235cac aag gaa cat cac gaa tac cct caa ctt cct tgg tgg aaa tta cct769His Lys Glu His His Glu Tyr Pro Gln Leu Pro Trp Trp Lys Leu Pro240 245 250 255gaa gct cac aaa ata tct tta taaggtctag agcatgcgc 809Glu Ala His Lys Ile Ser Leu260<210>69<211>262<212>PRT<213>念珠藻PCC 7120<400>69Met His Leu Glu Met Val Gln Cys Gln Pro Ser Ser Leu His Ser Glu1 5 10 15Lys Leu Val Leu Leu Ser Ser Thr Ile Arg Asp Asp Lys Asn Ile Asn20 25 30Lys Gly Ile Phe Ile Ala Cys Phe Ile Leu Phe Leu Trp Ala Ile Ser35 40 45Leu Ile Leu Leu Leu Ser Ile Asp Thr Ser Ile Ile His Lys Ser Leu50 55 60Leu Gly Ile Ala Met Leu Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe65 70 75 80Ile Thr Ala His Asp Ala Met His Gly Val Val Tyr Pro Lys Asn Pro85 90 95Arg Ile Asn Asn Phe Ile Gly Lys Leu Thr Leu Ile Leu Tyr Gly Leu100 105 110Leu Pro Tyr Lys Asp Leu Leu Lys Lys His Trp Leu His His Gly His115 120 125Pro Gly Thr Asp Leu Asp Pro Asp Tyr Tyr Asn Gly His Pro Gln Asn130 135 140Phe Phe Leu Trp Tyr Leu His Phe Met Lys Ser Tyr Trp Arg Trp Thr145 150 155 160Gln Ile Phe Gly Leu Val Met Ile Phe His Gly Leu Lys Asn Leu Val165 170 175
His Ile Pro Glu Asn Asn Leu Ile Ile Phe Trp Met Ile Pro Ser Ile180 185 190Leu Ser Ser Val Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Lys195 200 205Lys Leu Glu Gly Gly Tyr Thr Asn Pro His Cys Ala Arg Ser Ile Pro210 215 220Leu Pro Leu Phe Trp Ser Phe Val Thr Cys Tyr His Phe Gly Tyr His225 230 235 240Lys Glu His His Glu Tyr Pro Gln Leu Pro Trp Trp Lys Leu Pro Glu245 250 255Ala His Lys Ile Ser Leu260<210>70<211>39<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(39)<223>
<400>70gcgcatgcat ctagaaatga atttttgtga taaaccagt39<210>71<211>37<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(37)<223>
<400>71gcgcatgctc tagattacga attggttact gaattgt 37<210>72<211>819<212>DNA<213>點形念珠藻ATCC 29133
<220>
<221>CDS<222>(5)..(802)<223>
<400>72gcgc atg cat cta gaa atg aat ttt tgt gat aaa cca gtt agc tat tat49Met His Leu Glu Met Asn Phe Cys Asp Lys Pro Val Ser Tyr Tyr1 5 10 15gtt gca ata gag caa tta agt gct aaa gaa gat act gtt tgg ggg ctg 97Val Ala Ile Glu Gln Leu Ser Ala Lys Glu Asp Thr Val Trp Gly Leu20 25 30gtg att gtc ata gta att att agt ctt tgg gta gct agt ttg gct ttt145Val Ile Val Ile Val Ile Ile Ser Leu Trp Val Ala Ser Leu Ala Phe35 40 45tta cta gct att aat tat gcc aaa gtc cca att tgg ttg ata cct att193Leu Leu Ala Ile Asn Tyr Ala Lys Val Pro Ile Trp Leu Ile Pro Ile50 55 60gca ata gtt tgg caa atg ttc ctt tat aca ggg cta ttt att act gca241Ala Ile Val Trp Gln Met Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala65 70 75cat gat gct atg cat ggg tca gtt tat cgt aaa aat ccc aaa att aat289His Asp Ala Met His Gly Ser Val Tyr Arg Lys Asn Pro Lys Ile Asn80 85 90 95aat ttt atc ggt tca cta gct gta gcg ctt tac gct gtg ttt cca tat337Asn Phe Ile Gly Ser Leu Ala Val Ala Leu Tyr Ala Val Phe Pro Tyr100 105 110caa cag atg tta aag aat cat tgc tta cat cat cgt cat cct gct agc385Gln Gln Met Leu Lys Asn His Cys Leu His His Arg His Pro Ala Ser115 120 125gaa gtt gac cca gat ttt cat gat ggt aag aga aca aac gct att ttc433Glu Val Asp Pro Asp Phe His Asp Gly Lys Arg Thr Asn Ala Ile Phe130 135 140tgg tat ctc cat ttc atg ata gaa tac tcc agt tgg caa cag tta ata481Trp Tyr Leu His Phe Met Ile Glu Tyr Ser Ser Trp Gln Gln Leu Ile145 150 155gta cta act atc cta ttt aat tta gct aaa tac gtt ttg cac atc cat529Val Leu Thr Ile Leu Phe Asn Leu Ala Lys Tyr Val Leu His Ile His160 165 170 175caa ata aat ctc atc tta ttt tgg agt att cct cca att tta agt tcc577Gln Ile Asn Leu Ile Leu Phe Trp Ser Ile Pro Pro Ile Leu Ser Ser180 185 190att caa ctg ttt tat ttc gga aca ttt ttg cct cat cga gaa ccc aag625Ile Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Arg Glu Pro Lys195 200 205aaa gga tat gtt tat ccc cat tgc agc caa aca ata aaa ttg cca act673Lys Gly Tyr Val Tyr Pro His Cys Ser Gln Thr Ile Lys Leu Pro Thr210 215 220
ttt ttg tca ttt atc gct tgc tac cac ttt ggt tat cat gaa gaa cat 721Phe Leu Ser Phe Ile Ala Cys Tyr His Phe Gly Tyr His Glu Glu His225 230 235cat gag tat ccc cat gta cct tgg tgg caa ctt cca tct gta tat aag 769His Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro Ser Val Tyr Lys240 245 250 255cag aga gta ttc aac aat tca gta acc aat tcg taatctagag catgcgc819Gln Arg Val Phe Asn Asn Ser Val Thr Asn Ser260 265<210>73<211>266<212>PRT<213>點形念珠藻ATCC 29133<400>73Met His Leu Glu Met Asn Phe Cys Asp Lys Pro Val Ser Tyr Tyr Val1 5 10 15Ala Ile Glu Gln Leu Ser Ala Lys Glu Asp Thr Val Trp Gly Leu Val20 25 30Ile Val Ile Val Ile Ile Ser Leu Trp Val Ala Ser Leu Ala Phe Leu35 40 45Leu Ala Ile Asn Tyr Ala Lys Val Pro Ile Trp Leu Ile Pro Ile Ala50 55 60Ile Val Trp Gln Met Phe Leu Tyr Thr Gly Leu Phe Ile Thr Ala His65 70 75 80Asp Ala Met His Gly Ser Val Tyr Arg Lys Asn Pro Lys Ile Asn Asn85 90 95Phe Ile Gly Ser Leu Ala Val Ala Leu Tyr Ala Val Phe Pro Tyr Gln100 105 110Gln Met Leu Lys Asn His Cys Leu His His Arg His Pro Ala Ser Glu115 120 125Val Asp Pro Asp Phe His Asp Gly Lys Arg Thr Asn Ala Ile Phe Trp130 135 140Tyr Leu His Phe Met Ile Glu Tyr Ser Ser Trp Gln Gln Leu Ile Val145 150 155 160Leu Thr Ile Leu Phe Asn Leu Ala Lys Tyr Val Leu His Ile His Gln
165 170 175Ile Asn Leu Ile Leu Phe Trp Ser Ile Pro Pro Ile Leu Ser Ser Ile180 185 190Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Arg Glu Pro Lys Lys195 200 205Gly Tyr Val Tyr Pro His Cys Ser Gln Thr Ile Lys Leu Pro Thr Phe210 215 220Leu Ser Phe Ile Ala Cys Tyr His Phe Gly Tyr His Glu Glu His His225 230 235 240Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro Ser Val Tyr Lys Gln245 250 255Arg Val Phe Asn Asn Ser Val Thr Asn Ser260 265<210>74<211>33<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(33)<223>
<400>74gcgcatgcat ctagaaatgg cgatcgccat tat 33<210>75<211>32<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(32)<223>
<400>75gcgcatgctc tagatcacaa atttgattta ga 32<210>76<211>720<212>DNA<213>泡沫節(jié)球藻NSOR10
<220>
<221>CDS<222>(5)..(703)<223>
<400>76gcgc atg cat cta gaa atg gcg atc gcc att att agt ata tgg gct atc 49Met His Leu Glu Met Ala Ile Ala Ile Ile Ser Ile Trp Ala Ile1 5 10 15agc cta ggt ttg tta ctt tat att gat ata tcc caa ttc aag ttt tgg97Ser Leu Gly Leu Leu Leu Tyr Ile Asp Ile Ser Gln Phe Lys Phe Trp20 25 30atg ttg tta ccg ctc ata ttt tgg caa aca ttt tta tat acg gga tta 145Met Leu Leu Pro Leu Ile Phe Trp Gln Thr Phe Leu Tyr Thr Gly Leu35 40 45ttt att aca gct cat gat gcc atg cat ggg gta gtt ttt ccc aaa aat 193Phe Ile Thr Ala His Asp Ala Met His Gly Val Val Phe Pro Lys Asn50 55 60ccc aaa atc aac cat ttc att ggc tca ttg tgc ctg ttt ctt tat ggt 241Pro Lys Ile Asn His Phe Ile Gly Ser Leu Cys Leu Phe Leu Tyr Gly65 70 75ctt tta cct tat caa aaa ctt tta aaa aag cat tgg cta cat cac cat 289Leu Leu Pro Tyr Gln Lys Leu Leu Lys Lys His Trp Leu His His His80 85 90 95aat cca gcc agt gaa aca gat cca gat ttt cac aac ggg aag cag aaa 337Asn Pro Ala Ser Glu Thr Asp Pro Asp Phe His Asn Gly Lys Gln Lys100 105 110aac ttt ttt gct tgg tat tta tat ttt atg aag cgt tac tgg agt tgg 385Asn Phe Phe Ala Trp Tyr Leu Tyr Phe Met Lys Arg Tyr Trp Ser Trp115 120 125tta caa att atc aca tta atg att att tat aac tta cta aaa tat ata 433Leu Gln Ile Ile Thr Leu Met Ile Ile Tyr Asn Leu Leu Lys Tyr Ile130 135 140tgg cat ttt cca gag gat aat atg act tat ttt tgg gta gtt ccc tca 481Trp His Phe Pro Glu Asp Asn Met Thr Tyr Phe Trp Val Val Pro Ser145 150 155att tta agt tct tta caa tta ttt tat ttt gga act ttt cta ccc cac 529Ile Leu Ser Ser Leu Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His160 165 170 175agt gag cct gta gaa ggt tat aaa gag cct cat cgt tcc caa act att 577Ser Glu Pro Val Glu Gly Tyr Lys Glu Pro His Arg Ser Gln Thr Ile180 185 190agc cgt ccc att tgg tgg tca ttt ata act tgt tac cat ttt ggt tat 625Ser Arg Pro Ile Trp Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr195 200 205cat tac gaa cat cat gaa tac ccc cat gtt cct tgg tgg caa tta cca 673His Tyr Glu His His Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro
210 215 220gaa att tat aaa atg tct aaa tca aat ttg tgatctagag catgcgc720Glu Ile Tyr Lys Met Ser Lys Ser Asn Leu225 230<210>77<211>233<212>PRT<213>泡沫節(jié)球藻NSOR10<400>77Met His Leu Glu Met Ala Ile Ala Ile Ile Ser Ile Trp Ala Ile Ser1 5 10 15Leu Gly Leu Leu Leu Tyr Ile Asp Ile Ser Gln Phe Lys Phe Trp Met20 25 30Leu Leu Pro Leu Ile Phe Trp Gln Thr Phe Leu Tyr Thr Gly Leu Phe35 40 45Ile Thr Ala His Asp Ala Met His Gly Val Val Phe Pro Lys Asn Pro50 55 60Lys Ile Asn His Phe Ile Gly Ser Leu Cys Leu Phe Leu Tyr Gly Leu65 70 75 80Leu Pro Tyr Gln Lys Leu Leu Lys Lys His Trp Leu His His His Asn85 90 95Pro Ala Ser Glu Thr Asp Pro Asp Phe His Asn Gly Lys Gln Lys Asn100 105 110Phe Phe Ala Trp Tyr Leu Tyr Phe Met Lys Arg Tyr Trp Ser Trp Leu115 120 125Gln Ile Ile Thr Leu Met Ile Ile Tyr Asn Leu Leu Lys Tyr Ile Trp130 135 140His Phe Pro Glu Asp Asn Met Thr Tyr Phe Trp Val Val Pro Ser Ile145 150 155 160Leu Ser Ser Leu Gln Leu Phe Tyr Phe Gly Thr Phe Leu Pro His Ser165 170 175Glu Pro Val Glu Gly Tyr Lys Glu Pro His Arg Ser Gln Thr Ile Ser180 185 190
Arg Pro Ile Trp Trp Ser Phe Ile Thr Cys Tyr His Phe Gly Tyr His195 200 205Tyr Glu His His Glu Tyr Pro His Val Pro Trp Trp Gln Leu Pro Glu210 215 220Ile Tyr Lys Met Ser Lys Ser Asn Leu225 230<210>78<211>24<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(24)<223>
<400>78gaattcctgc aatagaatgt tgag 24<210>79<211>25<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(25)<223>
<400>79ctcgagctta cgagcatttt ctaag 25<210>80<211>307<212>DNA<213>蠶豆(Vicia faba)<220>
<221>終止子<222>(1)..(307)<223>
<400>80gaattcctgc aatagaatgt tgaggtgacc actttctgta ataaaataat tataaaataa 60atttagaatt gctgtagtca agaacatcag ttctaaaata ttaataaagt tatggccttt 120tgacatatgt gtttcgataa aaaaatcaaa ataaattgag atttattcga aatacaatga 180aagtttgcag atatgagata tgtttctaca aaataataac ttaaaactca actatatgct 240
aatgtttttc ttggtgtgtt tcatagaaaa ttgtatccgt ttcttagaaa atgctcgtaa 300gctcgag 307<210>81<211>26<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(26)<223>
<400>81aagcttgaat ttggatccgc caccgt 26<210>82<211>25<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(25)<223>
<400>82gaattcccaa taataatcta cagcc25<210>83<211>1040<212>DNA<213>番茄<220>
<221>CDS<222>(29)..(970)<223>
<400>83aagcttgaat ttggatccgc caccgtcc atg gcg gcc gga att tca gcc tcc 52Met Ala Ala Gly Ile Ser Ala Ser1 5gct agt tcc cga acc att cgc ctc cgt cat aac ccg ttt ctc agt cca 100Ala Ser Ser Arg Thr Ile Arg Leu Arg His Asn Pro Phe Leu Ser Pro10 15 20aaa tcc gcc tca acc gcc ccg ccg gtt ctg ttc ttc tct ccg tta act 148Lys Ser Ala Ser Thr Ala Pro Pro Val Leu Phe Phe Ser Pro Leu Thr25 30 35 40cgc aat ttt ggc gca att ttg ctg tct aga aga aag ccg aga ttg gcg 196
Arg Asn Phe Gly Ala Ile Leu Leu Ser Arg Arg Lys Pro Arg Leu Ala45 50 55gtt tgt ttt gtg ctg gag aat gag aaa ttg aat agt act atc gaa agt244Val Cys Phe Val Leu Glu Asn Glu Lys Leu Asn Ser Thr Ile Glu Ser60 65 70gag agt gaa gta ata gag gat cgg ata caa gta gag att aat gag gag292Glu Ser Glu Val Ile Glu Asp Arg Ile Gln Val Glu Ile Asn Glu Glu75 80 85aag agt tta gct gcc agt tgg ctg gcg gag aaa ttg gcg agg aag aaa340Lys Ser Leu Ala Ala Ser Trp Leu Ala Glu Lys Leu Ala Arg Lys Lys90 95 100tcg gag agg ttt act tat ctt gtg gca gct gtg atg tct agt ttg ggg388Ser Glu Arg Phe Thr Tyr Leu Val Ala Ala Val Met Ser Ser Leu Gly105 110 115 120att act tct atg gcg att ttg gcg gtt tat tac aga ttt tca tgg caa436Ile Thr Ser Met Ala Ile Leu Ala Val Tyr Tyr Arg Phe Ser Trp Gln125 130 135atg gag ggt gga gaa gtg cct ttt tct gaa atg tta gct aca ttc act484Met Glu Gly Gly Glu Val Pro Phe Ser Glu Met Leu Ala Thr Phe Thr140 145 150ctc tcg ttt ggc gct gcc gta gga atg gag tac tgg gcg aga tgg gct532Leu Ser Phe Gly Ala Ala Val Gly Met Glu Tyr Trp Ala Arg Trp Ala155 160 165cat aga gca cta tgg cat gct tct tta tgg cac atg cac gag tcg cac580His Arg Ala Leu Trp His Ala Ser Leu Trp His Met His Glu Ser His170 175 180cat aga cca aga gaa gga cct ttt gag atg aac gac gtt ttc gcc ata628His Arg Pro Arg Glu Gly Pro Phe Glu Met Asn Asp Val Phe Ala Ile185 190 195 200aca aat gct gtt cca gct ata ggt ctt ctt tcc tac ggt ttc ttc cat676Thr Asn Ala Val Pro Ala Ile Gly Leu Leu Ser Tyr Gly Phe Phe His205 210 215aaa ggg atc gtc cct ggc ctc tgt ttc ggc gct gga ttg ggg atc aca724Lys Gly Ile Val Pro Gly Leu Cys Phe Gly Ala Gly Leu Gly Ile Thr220 225 230gta ttt ggg atg gct tac atg ttc gtt cac gat gga ctg gtt cat aag772Val Phe Gly Met Ala Tyr Met Phe Val His Asp Gly Leu Val His Lys235 240 245aga ttt ccc gta ggg cct att gcc aac gtg cct tac ttt cgg agg gta820Arg Phe Pro Val Gly Pro Ile Ala Asn Val Pro Tyr Phe Arg Arg Val250 255 260gct gca gca cat cag ctt cat cac tcg gac aaa ttt gat ggt gtc cca868Ala Ala Ala His Gln Leu His His Ser Asp Lys Phe Asp Gly Val Pro265 270 275 280tat ggc ttg ttt cta gga cct aag gaa ttg gaa gaa gta gga gga ctt916Tyr Gly Leu Phe Leu Gly Pro Lys Glu Leu Glu Glu Val Gly Gly Leu285 290 295
gaa gag tta gaa aag gaa gtc aac cga agg att aaa att tct aag gga964Glu Glu Leu Glu Lys Glu Val Asn Arg Arg Ile Lys Ile Ser Lys Gly300 305 310tta tta tgatcaaaag atacgtctga taataataaa atgcgattgt atttaggctg1020Leu Leutagattatta ttgggaattc 1040<210>84<211>314<212>PRT<213>番茄<400>84Met Ala Ala Gly Ile Ser Ala Ser Ala Ser Ser Arg Thr Ile Arg Leu1 5 10 15Arg His Asn Pro Phe Leu Ser Pro Lys Ser Ala Ser Thr Ala Pro Pro20 25 30Val Leu Phe Phe Ser Pro Leu Thr Arg Asn Phe Gly Ala Ile Leu Leu35 40 45Ser Arg Arg Lys Pro Arg Leu Ala Val Cys Phe Val Leu Glu Asn Glu50 55 60Lys Leu Asn Ser Thr Ile Glu Ser Glu Ser Glu Val Ile Glu Asp Arg65 70 75 80Ile Gln Val Glu Ile Asn Glu Glu Lys Ser Leu Ala Ala Ser Trp Leu85 90 95Ala Glu Lys Leu Ala Arg Lys Lys Ser Glu Arg Phe Thr Tyr Leu Val100 105 110Ala Ala Val Met Ser Ser Leu Gly Ile Thr Ser Met Ala Ile Leu Ala115 120 125Val Tyr Tyr Arg Phe Ser Trp Gln Met Glu Gly Gly Glu Val Pro Phe130 135 140Ser Glu Met Leu Ala Thr Phe Thr Leu Ser Phe Gly Ala Ala Val Gly145 150 155 160Met Glu Tyr Trp Ala Arg Trp Ala His Arg Ala Leu Trp His Ala Ser165 170 175
Leu Trp His Met His Glu Ser His His Arg Pro Arg Glu Gly Pro Phe180 185 190Glu Met Asn Asp Val Phe Ala Ile Thr Asn Ala Val Pro Ala Ile Gly195 200 205Leu Leu Ser Tyr Gly Phe Phe His Lys Gly Ile Val Pro Gly Leu Cys210 215 220Phe Gly Ala Gly Leu Gly Ile Thr Val Phe Gly Met Ala Tyr Met Phe225 230 235 240Val His Asp Gly Leu Val His Lys Arg Phe Pro Val Gly Pro Ile Ala245 250 255Asn Val Pro Tyr Phe Arg Arg Val Ala Ala Ala His Gln Leu His His260 265 270Ser Asp Lys Phe Asp Gly Val Pro Tyr Gly Leu Phe Leu Gly Pro Lys275 280 285Glu Leu Glu Glu Val Gly Gly Leu Glu Glu Leu Glu Lys Glu Val Asn290 295 300Arg Arg Ile Lys Ile Ser Lys Gly Leu Leu305 310<210>85<211>34<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(34)<223>
<400>85ccatggaagc tcttctcaag ccttttccat ctct 34<210>86<211>34<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(34)<223>
<400>86ggatcctcaa aggctctcta ttgctagatt gcca 34<210>87<211>1505<212>DNA<213>番茄<220>
<221>CDS<222>(3)..(1505)<223>
<400>87cc atg gaa gct ctt ctc aag cct ttt cca tct ctt tta ctt tcc tct 47Met Glu Ala Leu Leu Lys Pro Phe Pro Ser Leu Leu Leu Ser Ser1 5 10 15cct aca ccc cat agg tct att ttc caa caa aat ccc tct ttt cta agt 95Pro Thr Pro His Arg Ser Ile Phe Gln Gln Asn Pro Ser Phe Leu Ser20 25 30ccc acc acc aaa aaa aaa tca aga aaa tgt ctt ctt aga aac aaa agt143Pro Thr Thr Lys Lys Lys Ser Arg Lys Cys Leu Leu Arg Asn Lys Ser35 40 45agt aaa ctt ttt tgt agc ttt ctt gat tta gca ccc aca tca aag cca191Ser Lys Leu Phe Cys Ser Phe Leu Asp Leu Ala Pro Thr Ser Lys Pro50 55 60gag tct tta gat gtt aac atc tca tgg gtt gat cct aat tcg aat cgg239Glu Ser Leu Asp Val Asn Ile Ser Trp Val Asp Pro Asn Ser Asn Arg65 70 75gct caa ttc gac gtg atc att atc gga gct ggc cct gct ggg ctc agg287Ala Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg80 85 90 95cta gct gaa caa gtt tct aaa tat ggt att aag gta tgt tgt gtt gac335Leu Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp100 105 110cct tca cca ctc tcc atg tgg cca aat aat tat ggt gtt tgg gtt gat383Pro Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp115 120 125gag ttt gag aat tta gga ctg gaa aat tgt tta gat cat aaa tgg cct431Glu Phe Glu Asn Leu Gly Leu Glu Asn Cys Leu Asp His Lys Trp Pro130 135 140atg act tgt gtg cat ata aat gat aac aaa act aag tat ttg gga aga479Met Thr Cys Val His Ile Asn Asp Asn Lys Thr Lys Tyr Leu Gly Arg145 150 155cca tat ggt aga gtt agt aga aag aag ctg aag ttg aaa ttg ttg aat527Pro Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Lys Leu Leu Asn160 165 170 175
agt tgt gtt gag aac aga gtg aag ttt tat aaa gct aag gtt tgg aaa 575Ser Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys180 185 190gtg gaa cat gaa gaa ttt gag tct tca att gtt tgt gat gat ggt aag 623Val Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys195 200 205aag ata aga ggt agt ttg gtt gtg gat gca agt ggt ttt gct agt gat 671Lys Ile Arg Gly Ser Leu Val Val Asp Ala Ser Gly Phe Ala Ser Asp210 215 220ttt ata gag tat gac agg cca aga aac cat ggt tat caa att gct cat 719Phe Ile Glu Tyr Asp Arg Pro Arg Asn His Gly Tyr Gln Ile Ala His225 230 235ggg gtt tta gta gaa gtt gat aat cat cca ttt gat ttg gat aaa atg 767Gly Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met240 245 250 255gtg ctt atg gat tgg agg gat tct cat ttg ggt aat gag cca tat tta 815Val Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu260 265 270agg gtg aat aat gct aaa gaa cca aca ttc ttg tat gca atg cca ttt 863Arg Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe275 280 285gat aga gat ttg gtt ttc ttg gaa gag act tct ttg gtg agt cgt cct 911Asp Arg Asp Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro290 295 300gtt tta tcg tat atg gaa gta aaa aga agg atg gtg gca aga tta agg 959Val Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg305 310 315cat ttg ggg atc aaa gtg aaa agt gtt att gag gaa gag aaa tgt gtg 1007His Leu Gly Ile Lys Val Lys Ser Val Ile Glu Glu Glu Lys Cys Val320 325 330 335atc cct atg gga gga cca ctt ccg cgg att cct caa aat gtt atg gct 1055Ile Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala340 345 350att ggt ggg aat tca ggg ata gtt cat cca tca aca ggg tac atg gtg 1103Ile Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val355 360 365gct agg agc atg gct tta gca cca gta cta gct gaa gcc atc gtc gag 1151Ala Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Glu370 375 380ggg ctt ggc tca aca aga atg ata aga ggg tct caa ctt tac cat aga 1199Gly Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg385 390 395gtt tgg aat ggt ttg tgg cct ttg gat aga aga tgt gtt aga gaa tgt 1247Val Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Val Arg Glu Cys400 405 410 415tat tca ttt ggg atg gag aca ttg ttg aag ctt gat ttg aaa ggg act 1295Tyr Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr
420 425 430agg aga ttg ttt gac gct ttc ttt gat ctt gat cct aaa tac tgg caa 1343Arg Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln435 440 445ggg ttc ctt tct tca aga ttg tct gtc aaa gaa ctt ggt tta ctc agc 1391Gly Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Gly Leu Leu Ser450 455 460ttg tgt ctt ttc gga cat ggc tca aac atg act agg ttg gat att gtt 1439Leu Cys Leu Phe Gly His Gly Ser Asn Met Thr Arg Leu Asp Ile Val465 470 475aca aaa tgt cct ctt cct ttg gtt aga ctg att ggc aat cta gca ata 1487Thr Lys Cys Pro Leu Pro Leu Val Arg Leu Ile Gly Asn Leu Ala Ile480 485 490 495gag agc ctt tga gga tcc 1505Glu Ser Leu Gly Ser500<210>88<211>498<212>PRT<213>番茄<400>88Met Glu Ala Leu Leu Lys Pro Phe Pro Ser Leu Leu Leu Ser Ser Pro1 5 10 15Thr Pro His Arg Ser Ile Phe Gln Gln Asn Pro Ser Phe Leu Ser Pro20 25 30Thr Thr Lys Lys Lys Ser Arg Lys Cys Leu Leu Arg Asn Lys Ser Ser35 40 45Lys Leu Phe Cys Ser Phe Leu Asp Leu Ala Pro Thr Ser Lys Pro Glu50 55 60Ser Leu Asp Val Asn Ile Ser Trp Val Asp Pro Asn Ser Asn Arg Ala65 70 75 80Gln Phe Asp Val Ile Ile Ile Gly Ala Gly Pro Ala Gly Leu Arg Leu85 90 95Ala Glu Gln Val Ser Lys Tyr Gly Ile Lys Val Cys Cys Val Asp Pro100 105 110Ser Pro Leu Ser Met Trp Pro Asn Asn Tyr Gly Val Trp Val Asp Glu115 120 125
Phe Glu Asn Leu Gly Leu Glu Asn Cys Leu Asp His Lys Trp Pro Met130 135 140Thr Cys Val His Ile Asn Asp Asn Lys Thr Lys Tyr Leu Gly Arg Pro145 150 155 160Tyr Gly Arg Val Ser Arg Lys Lys Leu Lys Leu Lys Leu Leu Asn Ser165 170 175Cys Val Glu Asn Arg Val Lys Phe Tyr Lys Ala Lys Val Trp Lys Val180 185 190Glu His Glu Glu Phe Glu Ser Ser Ile Val Cys Asp Asp Gly Lys Lys195 200 205Ile Arg Gly Ser Leu Val Val Asp Ala Ser Gly Phe Ala Ser Asp Phe210 215 220Ile Glu Tyr Asp Arg Pro Arg Asn His Gly Tyr Gln Ile Ala His Gly225 230 235 240Val Leu Val Glu Val Asp Asn His Pro Phe Asp Leu Asp Lys Met Val245 250 255Leu Met Asp Trp Arg Asp Ser His Leu Gly Asn Glu Pro Tyr Leu Arg260 265 270Val Asn Asn Ala Lys Glu Pro Thr Phe Leu Tyr Ala Met Pro Phe Asp275 280 285Arg Asp Leu Val Phe Leu Glu Glu Thr Ser Leu Val Ser Arg Pro Val290 295 300Leu Ser Tyr Met Glu Val Lys Arg Arg Met Val Ala Arg Leu Arg His305 310 315 320Leu Gly Ile Lys Val Lys Ser Val Ile Glu Glu Glu Lys Cys Val Ile325 330 335Pro Met Gly Gly Pro Leu Pro Arg Ile Pro Gln Asn Val Met Ala Ile340 345 350Gly Gly Asn Ser Gly Ile Val His Pro Ser Thr Gly Tyr Met Val Ala355 360 365Arg Ser Met Ala Leu Ala Pro Val Leu Ala Glu Ala Ile Val Glu Gly370 375 380
Leu Gly Ser Thr Arg Met Ile Arg Gly Ser Gln Leu Tyr His Arg Val385 390 395 400Trp Asn Gly Leu Trp Pro Leu Asp Arg Arg Cys Val Arg Glu Cys Tyr405 410 415Ser Phe Gly Met Glu Thr Leu Leu Lys Leu Asp Leu Lys Gly Thr Arg420 425 430Arg Leu Phe Asp Ala Phe Phe Asp Leu Asp Pro Lys Tyr Trp Gln Gly435 440 445Phe Leu Ser Ser Arg Leu Ser Val Lys Glu Leu Gly Leu Leu Ser Leu450 455 460Cys Leu Phe Gly His Gly Ser Asn Met Thr Arg Leu Asp Ile Val Thr465 470 475 480Lys Cys Pro Leu Pro Leu Val Arg Leu Ile Gly Asn Leu Ala Ile Glu485 490 495Ser Leu<210>89<211>37<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(37)<223>
<400>89gagctcgata tctttgccag tattacaaca gcttata 37<210>90<211>31<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(31)<223>
<400>90
cccgggttta ctgaaaaata acagtaaaac c 31<210>91<211>2096<212>DNA<213>番茄<220>
<221>啟動子<222>(1)..(2096)<223>
<400>91gagctcgata tctttgccag tattacaaca gcttatatgt tgagcaggta aaagcttcaa 60tgccctattc tttctacagt tatcaatgtt gctcgtctaa tatctggtgt tcttctcgaa 120atgtcaattg gcttgcagca cattgtcctc taatatccat tcaagcttct tagatgatga 180aacatttgtc aaatttatta atttcatagt gttcagtctc aattctttag ctgtttcctc 240atagtaaagt tgtctaatat gaaatgaaaa tgttctgtgt gttgtactaa taccttttca 300tggttgtcta tagaacgtcg atgaagagcc aaacagaaac tattttgggc tgcgatttct 360gataccattg tatctgaatg ctgggtggga gctcatcaga agctttacaa tgggtcacat 420atatggagcc gagtatgagg aatgctggga atcagttgtg cttcgcgtgc taggactttt 480ccttcctggt atttctgccc acagcccagt tgattacgtg aactccgtca gacttggaaa 540ggagagaagt acccaaatgt cgtcttttta gaaatacttt tgtcacaaaa tagcggggtt 600tacagctaca gaagatcatg cagaaggcgt ccagtttagt ttttgaaggt tgtttggagt 660ttatttatct aaagtaaact taaatcagct ttttgtttat gagttcagtg aactatatgt 720tcaaataaga cttccctttg tagaatatgt gttttttttt gttgttgagc actttgtgtg 780cattggataa acccccaacg tgtaatagct accatacaag agaagtaact cgcactgtcc 840atgtcttatg tggctcgact cagaaagcat tcagggggat tgataaccac cctccaaacc 900aactgaacca ttgtgaataa ccacccttca aatcaaccga gtcctcgtga aggacaaata 960tgtggtttta tatacattaa attttgtttt tacatgcttc ctcttacttc tttagttttc1020ttgaccatat cttctttttc ccttctgtaa ttgacatttt cttcaaacca tccagcaatg1080tggaagcttg acgattttcc ttcagagtag aaattgaaaa gaatcaacta aaaaggatag1140tccttcgatt tgatttccgg cttaaaaata aactaataag aatgagagag cgaataatag1200aatattttga aattttaaag atattcaact atgttaaatt gcgttataaa tttcttaaat1260tagtagcacc taatagttta gttctcaaaa gtcaaaacta ctacataatg tgctcatttt1320tcacattaaa atgcctacat gatgtaaaag taaaactcgt agcattctac gtgttttact1380
caactcaaac atcctgttca ttttaataaa cgtacgatga gcttctctct ccaattttct1440tttctttttt ttttttaaaa aaatattttt ttttatatca atccaaatgg gctccaattt1500atcataaatt aggtagaaac ttagatatta aagaaagaaa agggtttatc tcgcaagtgt1560ggctatggtg ggacgtgtca aattttggat tgtagccaaa catgagattt gatttaaagg1620gaattggcca aatcaccgaa agcaggcatc ttcatcataa attagtttgt ttatttatac1680agaattatac gcttttacta gttatagcat tcggtatctt tttctgggta actgccaaac1740caccacaaat ttcaagtttc catttaactc ttcaacttca acccaaccaa atttatttgc1800ttaattgtgc agaaccactc cctatatctt ctaggtgctt tcattcgttc cgaggtaaga1860aaagattttt gtttctttga atgctttatg ccactcgttt aacttctgag gtttgtggat1920cttttaggcg actttttttt tttttgtatg taaaatttgt ttcataaatg cttctcaaca1980taaatcttga caaagagaag gaattttacc aagtatttag gttcagaaat ggataatttt2040cttactgtga aatatcctta tggcaggttt tactgttatt tttcagtaaa cccggg2096<210>92<211>25<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(25)<223>
<400>92taagcttttt gttgaagaga tttgg25<210>93<211>24<212>DNA<213>人工序列<220>
<221>引物<222>(1)..(24)<223>
<400>93gaattcctgc aatagaatgt tgag 2權利要求
1.一種生產酮類胡蘿卜素的方法，該方法包括培養(yǎng)果實中顯示酮酶活性的遺傳修飾植物。
2.如權利要求1所述的方法，其中使用果實中表達酮酶的遺傳修飾植物。
3.如權利要求1或2所述的方法，其中使用在果實中包含至少一個編碼酮酶的核酸的遺傳修飾植物。
4.如權利要求3所述的方法，其中使用從起始植物開始已經引入至少一個編碼酮酶的核酸的遺傳修飾植物。
5.如權利要求4所述的方法，其中引入編碼蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO.2或者通過氨基酸置換、插入或缺失而衍生自該序列的序列，其中該衍生序列在氨基酸水平上與序列SEQ ID NO.2具有至少20％同一性，并且具有酮酶的酶特征。
6.如權利要求4所述的方法，其中引入包含序列SEQ ID NO.1的核酸。
7.如權利要求4所述的方法，其中引入編碼蛋白質的核酸，該蛋白質包含氨基酸序列SEQ ID NO.16或者通過氨基酸置換、插入或缺失而衍生自該序列的序列，其中該衍生序列在氨基酸水平上與序列SEQ ID NO.16具有至少20％同一性，并且具有酮酶的酶特征。
8.如權利要求6所述的方法，其中引入包含序列SEQ ID NO.15的核酸。
9.如權利要求1至8任一所述的方法，其中使用果實中顯示最高酮酶表達率的遺傳修飾植物。
10.如權利要求9所述的方法，其中在果實特異性啟動子控制下實現酮酶的基因表達。
11.如權利要求1至10任一所述的方法，其中該植物與野生型比較還顯示出具有增加的選自羥化酶活性和β-環(huán)化酶活性的至少一種活性。
12.如權利要求11所述的方法，其中為了另外增加至少一種所述活性，與野生型比較，增加了選自編碼羥化酶的核酸和編碼β-環(huán)化酶的核酸中的至少一種核酸的基因表達。
13.如權利要求12所述的方法，其中為了增加至少一種所述核酸的基因表達，將選自編碼羥化酶的核酸和編碼β-環(huán)化酶的核酸中的至少一種核酸引入植物中。
14.如權利要求13所述的方法，其中編碼下述羥化酶的核酸作為編碼羥化酶的核酸被引入，所述羥化酶包含氨基酸序列SEQ ID NO52或者通過氨基酸的置換、插入或缺失而衍生自該序列的序列，其中所述衍生序列在氨基酸水平上與序列SEQ ID NO.52具有至少20％同一性。
15.如權利要求14所述的方法，其中引入包含序列SEQ ID NO.51的核酸。
16.如權利要求13所述的方法，其中編碼下述β-環(huán)化酶的核酸作為編碼β-環(huán)化酶的核酸被引入，所述β-環(huán)化酶包含氨基酸序列SEQ ID NO54或者通過氨基酸的置換、插入或缺失而衍生自該序列的序列，其中所述衍生序列在氨基酸水平上與序列SEQ ID NO.54具有至少20％同一性。
17.如權利要求16所述的方法，其中引入包含序列SEQ ID NO.53的核酸。
18.如權利要求11至17任一所述的方法，其中使用在花中具有最高羥化酶和/或β-環(huán)化酶表達率的遺傳修飾植物。
19.如權利要求18所述的方法，其中在花特異性啟動子控制下實現羥化酶和/或β-環(huán)化酶的基因表達。
20.如權利要求1至10任一所述的方法，其中所用植物是果實中有色質體的植物。
21.如權利要求1至20任一所述的方法，其中所用植物是選自皺子棕屬、亮絲草屬、風梨屬、草莓樹屬、假檳榔屬、Area、Aronia、天門冬屬、亞塔棕屬、小檗屬、Bixia、Brachychilum、Bryonia、Caliptocalix、辣椒屬、番木瓜屬、南蛇藤屬、西瓜屬、柑橘屬、鈴蘭屬、栒子屬、山楂屬、香瓜屬、南瓜屬、菟絲子屬、蘇鐵屬、樹番茄屬、薯蕷屬、柿屬、Dura、胡頹子屬、油棕屬、古柯屬、衛(wèi)矛屬、榕屬、金桔屬、草莓屬、梔子屬、瓊欖屬、棉屬、番石榴屬、刺棒棕屬、木槿屬、沙棘屬、鳶尾屬、山黧豆屬、忍冬屬、絲瓜屬、枸杞屬、番茄屬、金虎尾屬、芒果屬、Mormodica、九里香屬、芭蕉屬、能加棕屬、Palisota、露兜樹屬、西番蓮屬、鱷梨屬、酸漿屬、李屬、Ptychandra、石榴屬、火棘屬、梨屬、茶藨子屬、薔薇屬、懸鉤子屬、薩巴棕屬、接骨木屬、Seaforita、水牛果屬、茄屬、花楸屬、Synaspadix、Tabernae、Tamus、紅豆杉屬、栝樓屬、Triphasia、越桔屬、莢蒾屬、Vignia和葡萄屬的植物。
22.如權利要求1至21任一所述的方法，其中，栽培后，收獲遺傳修飾植物，隨后從植物果實分離酮類胡蘿卜素。
23.如權利要求1至22任一所述的方法，其中酮類胡蘿卜素選自蝦青素、角黃素、海膽酮、3-羥基海膽酮、3’-羥基海膽酮、adonirubin和金盞花黃質。
24.一種核酸構建體，包含功能性連接的果實特異性啟動子和編碼酮酶的核酸。
25.一種遺傳修飾植物，其在果實中顯示了酮酶活性。
26.如權利要求25所述的遺傳修飾植物，其中該遺傳修飾植物在果實中表達酮酶。
27.如權利要求25或26所述的遺傳修飾植物，其果實中包含至少一個編碼酮酶的核酸。
28.如權利要求25至27任一所述的遺傳修飾植物，其中從起始植物開始，至少一個編碼酮酶的核酸已經被引入到植物中。
29.如權利要求25至28任一所述的遺傳修飾植物，其中與野生型植物比較，該遺傳修飾還使選自羥化酶活性和β-環(huán)化酶活性中的至少一種活性增加。
30.一種選自皺子棕屬、亮絲草屬、風梨屬、草莓樹屬、假檳榔屬、Area、Aronia、天門冬屬、亞塔棕屬、小檗屬、Bixia、Brachychilum、Bryonia、Caliptocalix、辣椒屬、番木瓜屬、南蛇藤屬、西瓜屬、柑橘屬、鈴蘭屬、栒子屬、山楂屬、香瓜屬、南瓜屬、菟絲于屬、蘇鐵屬、樹番茄屬、薯蕷屬、柿屬、Dura、胡頹子屬、油棕屬、古柯屬、衛(wèi)矛屬、榕屬、金桔屬、草莓屬、梔子屬、瓊欖屬、棉屬、番石榴屬、刺棒棕屬、木槿屬、沙棘屬、鳶尾屬、山黧豆屬、忍冬屬、絲瓜屬、枸杞屬、番茄屬、金虎尾屬、芒果屬、Mormodica、九里香屬、芭蕉屬、能加棕屬、Palisota、露兜樹屬、西番蓮屬、鱷梨屬、酸漿屬、李屬、Ptychandra、石榴屬、火棘屬、梨屬、茶藨子屬、薔薇屬、懸鉤子屬、薩巴棕屬、接骨木屬、Seaforita、水牛果屬、茄屬、花楸屬、Synaspadix、Tabernae、Tamus、紅豆杉屬、栝樓屬、Triphasia、越桔屬、莢蒾屬、Vignia和葡萄屬植物的遺傳修飾植物，其包含至少一個編碼酮酶的核酸。
31.如權利要求30所述的遺傳修飾植物，其中在果實中表達酮酶。
32.如權利要求25至31任一所述的遺傳修飾植物，其中果實中酮酶表達率最高。
33.權利要求25至32任一所述遺傳修飾植物作為飼料或食品的用途。
34.權利要求25至32任一所述遺傳修飾植物的果實用于生產包含酮類胡蘿卜素的提取物或用于生產飼料添加劑或食品添加劑的用途。
35.一種產生權利要求32所述遺傳修飾植物的方法，其中包含功能性連接的果實特異性啟動子和編碼酮酶的核酸的核酸構建體被引入到起始植物基因組中。
全文摘要
本發(fā)明涉及通過栽培果實中顯示酮酶活性的遺傳修飾植物，生產酮類胡蘿卜素的方法。
文檔編號A23K1/16GK1688713SQ03824350
公開日2005年10月26日 申請日期2003年8月18日 優(yōu)先權日2002年8月20日
發(fā)明者C·R·朔普費爾, R·弗拉赫曼, K·赫伯斯, I·孔策, M·紹爾, M·克勒布薩特爾 申請人:太陽基因兩合公司