CN1860211A - 用于在转基因生物中产生多不饱和脂肪酸的方法 - Google Patents
用于在转基因生物中产生多不饱和脂肪酸的方法 Download PDFInfo
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Abstract
本发明涉及通过向生物导入编码具有Δ5-延伸酶活性的多肽的核酸用于在这种生物中产生多不饱和脂肪酸的方法。有利地,这些核酸序列可在这种生物中表达,根据需要这些核酸序列可与编码脂肪酸或脂类代谢中的其它编码多肽的核酸序列共同在这种生物中表达。编码Δ6-去饱和酶、Δ5-去饱和酶、Δ4-去饱和酶和/或Δ6-延伸酶活性的核酸序列尤其有利。这些去饱和酶和延伸酶有利地源自海链藻属、裸藻属或Ostreococcus。本发明还涉及用于产生具有长链多不饱和脂肪酸含量升高的油和/或三酰甘油酯的方法。在优选的实施方案中,本发明进一步涉及用于产生不饱和ω3-脂肪酸的方法和涉及用于产生具有不饱和脂肪酸,尤其具备超过3个双键的ω3-脂肪酸含量升高的三酰甘油的方法。本发明涉及转基因生物的产生,优选地涉及转基因植物或转基因微生物的产生,其中所述转基因生物由于本发明方法中所用的延伸酶和去饱和酶的表达,有利地与ω3-去饱和酶例如来源于腐霉科(Pythiaceae)例如疫霉属(Phytophthora)比如致病疫霉属或种的真菌的ω3-去饱和酶或源于如草绿藻科例如Ostreococcus属尤其Ostreococcus tauri属和种的藻类或海链藻属尤其假矮海链藻(Thalassiosira pseudonana)属和种的硅藻的ω3-去饱和酶共同表达,具有含量升高的具备ω3-双键的不饱和ω3-脂肪酸、油或脂类。本发明进一步涉及核酸序列、包含本发明核酸序列的核酸构建体、载体和生物、包含所述核酸序列和/或核酸构建体的载体并且涉及包含以上提到的核酸序列、核酸构建体和/或载体的转基因生物。本发明的其它部分涉及由本发明方法产生的油、脂类和/或脂肪酸并涉及它们的用途。本发明进一步涉及不饱和脂肪酸和涉及具有不饱和脂肪酸含量升高的三酰甘油并且涉及它们的用途。
Description
本发明涉及通过将编码具有Δ5延伸酶活性的多肽的核酸导入生物体,在生物中产生多不饱和脂肪酸的方法。这些核酸序列,根据需要与编码脂肪酸生物合成或脂代谢的多肽的其它核酸序列一起可有利地在生物中表达。特别有利的核酸序列是编码Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ12去饱和酶和/或Δ6延伸酶活性的核酸序列。这些去饱和酶和延伸酶有利地来源于海链藻属(Thalassiosira)、裸藻属(Euglena)或Ostreococcus。本发明还涉及产生具有含量升高的长链多不饱和脂肪酸的油类和/或三酰甘油酯的方法。
在优选的实施方案中,本发明进一步涉及产生不饱和ω3脂肪酸的方法和产生具有含量升高的不饱和脂肪酸尤其具有超过三个双键的ω3脂肪酸的甘油三酯的方法。本发明涉及转基因生物的产生,优选地为转基因植物或转基因微生物的产生,所述的转基因生物由于本发明方法中使用的延伸酶和去饱和酶,有利地与ω3去饱和酶类共同的表达,具有含量升高的不饱和ω3脂肪酸、具有ω3双键的油类或脂类,其中所述的ω3去饱和酶例如来自真菌腐霉科(Pythiaceae)如疫霉属(Phytophthora),例如致病疫霉(Phytophthora infestans)属和种的真菌,或来自藻类如草绿藻科(Prasinophyceae),例如Ostreococcus属,特别是Ostreococcus tauri属和种的藻类,或来自硅藻如海链藻属,特别是假矮海链藻(Thalassiosirapseudonana)属和种的硅藻。
本发明进一步涉及核酸序列、包含本发明核酸序列的核酸构建体、载体和生物、包含核酸序列和/或核酸构建体的载体并且涉及含有以上提及的核酸序列、核酸构建体和/或载体的转基因生物。
本发明的一部分还涉及根据本发明方法产生的油、脂类和/或脂肪酸和它们的用途。本发明还涉及不饱和脂肪酸和具有含量升高的不饱和脂肪酸的甘油三酯以及它们的用途。
脂肪酸和三酰甘油酯在食品工业、动物营养、化妆品和药学领域内具有多种应用。根据它们是否为游离饱和或不饱和脂肪酸或为具有含量升高的饱和或不饱和脂肪酸的三酰甘油酯,这些脂肪酸和三酰甘油酯适合迥然不同的应用。因为哺乳动物不能产生多不饱和脂肪酸,所以多不饱和脂肪酸例如亚油酸和亚麻酸为哺乳动物必需。故多不饱和ω3脂肪酸和ω6脂肪酸是动物和人类营养的重要构成要素。
鉴于多不饱和长链ω3脂肪酸在健康领域包括儿童脑发育、眼的功能性、激素和其它信号物质的合成以及心血管疾病、癌症和糖尿病的预防的多种作用,多不饱和长链ω3脂肪酸例如二十碳五烯酸(EPA,C20:5Δ5,8,11,14,17)或二十二碳六烯酸(DHA,C22:6Δ4,7,10,13,16,19)是人类营养的重要成分(Poulos,A,Lipids 30:1-14,1995;Horrocks,LA和YeoYK,Pharmacol Res 40:211-225,1999)。这是为什么需要产生多不饱和长链脂肪酸的的原因。
鉴于目前人类食物的组成,向食物中添加优先地发现于鱼油中的多不饱和ω3脂肪酸极为重要。因此例如将多不饱和脂肪酸如二十二碳六烯酸(DHA,C22:6Δ4,7,10,13,16,19)或二十碳五烯酸(EPA,C20:5Δ5,8,11,14,17)添加至婴儿配制方中以改善营养价值。据称不饱和脂肪酸DHA对脑功能的发育和维持有积极影响。
下文中多不饱和脂肪酸称作PUFA、PUFAs、LCPUFA或LCPUFAs(
poly
unsaturated
fatty
acids,
PUFA,
long
chain
polyunsaturated
fatty
acids,
LCPUFA)。
多种脂肪酸和甘油三酯主要从微生物如被孢霉属(Mortierella)和Schizochytrium中获得或从产油植物如大豆、油菜籽、藻类如隐甲藻属(Crypthecodinium)或褐指藻属(Phaeodactylum)和其它生物中获得,其中所述多种脂肪酸和甘油三酯通常以其三酰甘油酯(=甘油三酯=三酰甘油)形式获得。然而这些脂肪酸和甘油三酯也能从动物如鱼中获得。游离脂肪酸有利地通过水解制备。极长链多不饱和脂肪酸例如DHA、EPA、花生四烯酸(ARA,C20:4Δ5,8,11,14)、二高γ-亚麻酸(C20:3Δ8,11,14)或二十二碳五烯酸(DPA,C22:5Δ7,10,13,16,19)不能在油料作物例如油菜籽、大豆、向日葵或红花中合成。这些脂肪酸的常规天然来源是鱼如鲱鱼、鲑鱼、沙丁鱼、雄鲑、鳗鲡、鲤鱼、鳟鱼、大比目鱼、鲭鱼、梭鲈或金枪鱼或藻类。
根据目的用途,具有饱和或不饱和脂肪酸的油为优选。例如在人类营养中,具有不饱和脂肪酸尤其具有多不饱和脂肪酸的脂类为优选。据称多不饱和ω3脂肪酸对血液中胆固醇水平具有正面影响并且因此正面地影响心脏病的预防可能性。通过向食物中添加这些ω3脂肪酸可显著降低心脏病、中风或高血压的风险。ω3脂肪酸也正面影响与免疫性疾病如类风湿性关节炎有关的炎性过程,尤其是慢性炎性过程。因此将它们添加至食品,尤其食用性食品中或应用于药品。由于我们日常的饮食摄入,ω6脂肪酸如花生四烯酸往往不利地影响与这些风湿性疾病相关的这类功能紊乱。
ω3和ω6脂肪酸是称作类二十烷酸的组织激素如前列腺素的前体,其中这类组织激素自二高γ亚麻酸、花生四烯酸和二十碳五烯酸衍生,同时ω3和ω6脂肪酸也是凝血烷和白三烯的前体,而凝血烷和白三烯衍生自花生四烯酸和二十碳五烯酸。形成自ω6脂肪酸的类二十烷酸(称作PG2系列)通常促进了炎性反应,而ω3脂肪酸来源的类二十烷酸(称作PG3系列)具有很少或没有促炎效应。
鉴于多不饱和脂肪酸的积极特征,过去从未停止尝试获得参与合成这些脂肪酸或甘油三酯的基因以便在不饱和脂肪酸的含量经改变的多种生物中产生油。因而WO 91/13972和它的美国等同公开描述了Δ9去饱和酶。WO 93/11245要求了Δ15去饱和酶的权利并且WO 94/11516要求了Δ12去饱和酶的权利。而且其它去饱和酶在例如EP-A-0550162、WO 94/18337、WO 97/30582、WO 97/21340、WO 95/18222、EP-A-0794250、Stukey等,J.Biol.Chem.,265,1990:20144-20149、Wada等,Nature 347,1990:200-203或Huang等,Lipids 34,1999:649-659中得到了描述。然而迄今多种去饱和酶的生物化学特征描述得仍不充分,因为这些酶作为膜结合蛋白严重阻碍了对它们的分离和特征描述(McKeon等,Methods in Enzymol.71,1981:12141-12147,Wang等,Plant Physiol.Biochem.,26,1988:777-792)。原则上将膜结合的去饱和酶通过导入合适的生物并随后通过分析起始物质和产物而分析酶活性来表征。Δ6去饱和酶在WO 93/06712、US 5614393、US5614393、WO 96/21022、WO 00/21557和WO 99/27111中描述并且这种酶用于转基因生物中产生脂肪酸的申请在WO 98/46763、WO 98/46764和WO98/46765中描述。在本上下文中,多种去饱和酶的表达和多不饱和脂肪酸的形成也在WO 99/64616或WO 98/46776中进行了描述和要求权利。就去饱和酶的表达效率和这种表达效率对多不饱和脂肪酸形成的影响而言,必须指出单一去饱和酶的表达如迄今所述仅仅获得了低含量的不饱和脂肪酸/脂类如γ-亚麻酸和硬脂艾杜糖酸(stearidonic acid)。此外通常获得的是ω3和ω6脂肪酸的混合物。
尤其适合用于产生PUFA的微生物是微藻如三角褐指藻(Phaeodactylum tricornutum)、Porphiridium某些种、破囊壶菌属(Thraustochytrium)某些种、Schizochytrium种或隐甲藻属某些种、纤毛虫例如棘尾虫属(Stylonychia)或豆形虫属(Colpidium)、真菌如被孢霉、虫霉菌属(Entomophthora)和毛霉属(Mucor)和/或藓类如剑叶藓属(Physcomitrella)、角齿藓属(Ceratodon)和地钱属(Marchantia)(R.Vazhappilly和F.Chen(1998)Botanica Marina 41:553-558;K.Totani和K.Oba(1987)Lipids 22:1060-1062;M.Akimoto等,(1998)Appl.Biochemistry and Biotechnology 73:269-278)。株系的选择已经导致了所讨论的多种微生物突变株系的开发,这些突变株系产生了包括PUFA在内的一系列目标化合物。然而对株系进行突变并选择具有改善特定分子如多不饱和脂肪酸产生的株系是一个费时且困难的过程。这就是为什么只要可能就优选如上所述的重组方法的原因。
然而目的多不饱和脂肪酸如DPA、EPA或ARA在以上提到的微生物的协助下仅仅可有限量地产生并且通常根据所用的微生物,这些脂肪酸作为如EPA、DPA或ARA的脂肪酸混合物获得。
讨论了花生四烯酸、二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)(图1)合成的多种合成途径。因此,EPA或DHA通过聚酮化合物途径在海生细菌如弧菌(Vibrio sp.)或希瓦菌(Shewanella sp.)中产生(Yu,R.等,Lipids 35:1061-1064,2000;Takeyama,H.等,Microbiology 143:2725-2731,1997)。
另一个备选策略是改变去饱和酶和延伸酶的活性(Zank,T.K.等,PlantJournal 31:255-268,2002;Sakuradani,E.等,Gene 238:445-453,1999)。经Δ6去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和Δ4去饱和酶修饰的上述途径是哺乳动物中的Sprecher途径(Sprecher 2000,Biochim.Biophys.Acta 1486:219-231)。实施进一步的延伸步骤而非Δ4去饱和过程以产生C24,随后通过进一步Δ6-去饱和作用和最终的β-氧化作用产生了C22的链长度。由于调节机制未知,故称作Sprecher的途径(见图1)并不适于植物和微生物中的生产。
这类多不饱和脂肪酸可根据它们的去饱和形式分为两个大类,即ω6或ω3脂肪酸,其中这些脂肪酸在代谢和功能性活性方面有差异(图1)。
ω6代谢途径的起始物质是脂肪酸亚油酸(18:2Δ9,12)而ω3途径经亚麻酸(18:3Δ9,12,15)进行。亚麻酸通过ω3去饱和酶的活性形成(Tocher等,1998,Prog.Lipid Res.37,73-117;Domergue等,2002,Eur.J.Biochem.269,4105-4113)。
哺乳动物及人类没有相应的去饱和酶活性(Δ12和ω3去饱和酶)并且必须通过食物摄入这类脂肪酸(必需脂肪酸)。生理上重要的多不饱和脂肪酸花生四烯酸(ARA,20:4Δ5,8,11,14),一种ω6脂肪酸及两种ω3脂肪酸二十碳五烯酸(EPA,20:5Δ5,8,11,14,17)和二十二碳六烯酸(DHA,22:6Δ4,7,10,13,17,19)从这类前体开始通过依次的去饱和酶以及延伸酶反应合成。ω3脂肪酸在心血管疾病(Shimikawa 2001,World Rev.Nutr.Diet.88,100-108)、Entzündungen(Calder 2002,Proc.Nutr.Soc.61,345-358)和关节炎(Cleland和James 2000,J.Rheumatol.27,2305-2307)的治疗应用中表现了如上所述的治疗活性、
因此就营养生理而言在合成多不饱和脂肪酸时,成功实现ω6合成途径和ω3合成途径间的转化(见图1)以产生更多ω3脂肪酸是重要的。多种使C18:2、C22:4或C22:5脂肪酸去饱和的ω3去饱和酶的酶活性已在文献中描述(见图1)。然而已经由生物化学所描述的去饱和酶无一能使ω6合成途径中广泛类型的底物转化为ω3合成途径中相应的脂肪酸。
因而对适于产生ω3多不饱和脂肪酸的ω3去饱和酶的需求一直十分迫切。所有已知的植物和蓝细菌ω3去饱和酶可用亚油酸为底物使C18脂肪酸去饱和,但是不能使任何C20或C22脂肪酸去饱和。
已知真菌Saprolegnia dicilina具有使C20多不饱和脂肪酸去饱和的ω3去饱和酶[Pereira等2004,Biochem.J.378(Pt2):665-71]。然而,这种ω3去饱和酶的缺点是不能使任何C18或C22 PUFA如ω6合成途径中重要脂肪酸C18:2、C22:4或C22:5脂肪酸去饱和。更为不利的是这种酶不能使任何结合至磷脂的脂肪酸去饱和。这种酶仅仅转化辅酶A-脂肪酸酯。
脂肪酸按照2或4个C原子通过延伸酶加以延伸对于分别产生C20和C22 PUFA极其重要。这个过程经历4个步骤。第一步是通过酮酰基辅酶A合成酶(KCS,此处以下称为延伸酶)使丙二酸单酰辅酶A和脂肪酸酰基辅酶A缩合。随后是一个还原步骤(酮酰基辅酶A还原酶,KCR)、一个脱水步骤(脱水酶)和一个最后的还原步骤(烯酰辅酶A还原酶)。据推测延伸酶的活性影响了这个过程整体的特异性和速率(Millar和Kunst,1997 Plant Journal 12:121-131)。
过去已进行了获取延伸酶基因的多次尝试。Millar和Kunst,1997(Plant Journal 12:121-131)并且Millar等1999,(Plant Cell 11:825-838)描述了用于合成单不饱和长链脂肪酸(C22:1)的植物延伸酶的特征和用于合成在植物中形成蜡的极长链脂肪酸(C28~C32)的植物延伸酶的特征。关于花生四烯酸和EPA合成的描述可在例如WO0159128、WO0012720、WO02077213和WO0208401中找到。多不饱和C24脂肪酸的合成在例如Tvrdik等,2000,JCB 149:707-717或WO0244320中得到描述。
迄今仍未描述在天然不产生DHA(C22:6 n-3)的生物中用于产生DHA的特异性延伸酶。目前只描述了可提供C20或C24脂肪酸的延伸酶。迄今没有Δ5延伸酶活性的描述。
高等植物包含多不饱和脂肪酸如亚油酸(C18:2)和亚麻酸(C18:3)。但ARA、EPA和DHA在所有高等植物的种子油中完全找不到或仅少量发现(E.Ucciani:Nouveau Dictionnaire des Huiles Végétales[NewDictionary of Vegetable oils].Technique & Documentation-Lavoisier,1995.ISBN:2-7430-0009-0)。然而在高等植物,优选在油料作物例如油菜籽、亚麻子、向日葵和大豆产生LCPUFA将是有利的因为有可能经济地获得大量高质量的LCPUFA用于食品工业、动物营养和药用目的。为实现此目标,编码LCPUFA生物合成酶基因通过重组方法向油料作物中导入并且在其中表达这些基因是有利的。例如这些基因编码例如Δ6去饱和酶、Δ6延伸酶、Δ5去饱和酶或Δ4去饱和酶。这些基因可有利地从产生LCPUFA并且使LCPUFA掺入膜或三酰甘油酯的微生物和低等植物中分离。因此已有可能从藓类植物展叶剑叶藓(Physcomitrella patens)中分离Δ6去饱和酶基因并且从展叶剑叶藓和秀丽线虫(C.elegans)中分离Δ6延伸酶基因。
包含并表达编码LCPUFA生物合成酶的基因并且由此产生LCPUFA的第一个转基因植物例如在DE-A-10219203(用于在植物中产生多不饱和脂肪酸的方法)中得到首次描述。然而这些植物仅以需要进一步优化而用于目前工厂中油料加工的量产生了LCPUFA。
为实现以这类多不饱和脂肪酸强化食物和/或饲料,用于产生尤其是在真核细胞系统内产生这类多不饱和脂肪酸的简单、廉价的方法极为需要。
因此本发明的一个目标是提供适于合成LCPUFA的其他基因或酶,尤其提供具有Δ5-延伸酶、Δ5去饱和酶、Δ4去饱和酶、Δ12去饱和酶或Δ6去饱和酶活性的基因以产生多不饱和脂肪酸。本发明的又一个目标是提供有可能自ω6脂肪酸转化为ω3脂肪酸的基因或酶。本发明的另一个目标是开发在生物中,有利地在真核生物中,优选地在植物或微生物中产生多不饱和脂肪酸的方法。这个目标通过本发明的用于转基因生物中产生式I化合物的方法实现,其中以所述转基因生物的总脂类含量为基础,这种转基因生物具有至少1%重量含量的式I化合物,其中式I化合物为:
所述的方法包括如下步骤:
a)将至少一个编码Δ9延伸酶和/或Δ6去饱和酶活性的核酸序列导入生物体,以及
b)将至少一个编码Δ8去饱和酶和/或Δ6延伸酶活性的核酸序列导入生物体,以及
c)将至少一个编码Δ5去饱和酶活性的核酸序列导入生物体,以及
d)将至少一个编码Δ5延伸酶活性的核酸序列导入生物体,以及
e)将至少一个编码Δ4去饱和酶活性的核酸序列导入生物体,并且在此方法中式I的变量和取代基的含义如下:
R1=羟基、辅酶A(硫酯)、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇、鞘碱(sphingo base)或式II的基团,
其中在所述的式II中
R2=氢、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇或饱和或不饱和的C2~C24烷基羰基,
R3=氢、饱和或不饱和的C2~C24烷基羰基,或R2和R3彼此独立地是式Ia的基团:
其中所述的式Ia中
n=2、3、4、5、6、7或9,m=2、3、4、5或6并且p=0或3。
式I中的R1为羟基、辅酶A(硫酯)、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇、鞘碱或式II的基团。
以上提及的基团R1总是与式I的硫酯形式化合物结合。
式II中的R2为氢、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇或饱和或不饱和C2~C24烷基羰基。
可提及的烷基是包含一个或多个双键的取代或未取代、饱和或不饱和的C2~C24烷基羰基链如乙基羰基、正丙基羰基、正丁基羰基、正戊基羰基、正己基羰基、正庚基羰基、正辛基羰基、正壬基羰基、正癸基羰基、正十一烷基羰基、正十二烷基羰基、正十三烷基羰基、正十四烷基羰基、正十五烷基羰基、正十六烷基羰基、正十七烷基羰基、正十八烷基羰基、正十九烷基羰基、正二十烷基羰基、正二十二烷基羰基或正二十四烷基羰基。包含一个或多个双键的饱和或不饱和C10~C22烷基羰基如正癸基羰基、正十一烷基羰基、正十二烷基羰基、正十三烷基羰基、正十四烷基羰基、正十五烷基羰基、正十六烷基羰基、正十七烷基羰基、正十八烷基羰基、正十九烷基羰基、正二十烷基羰基、正二十二烷基羰基或正二十四烷基羰基为优选的。尤其优选包含一个或多个双键的饱和和/或不饱和C10~C22烷基羰基如C10烷基羰基、C11烷基羰基、C12烷基羰基、C13烷基羰基、C14烷基羰基、C16烷基羰基、C18烷基羰基、C20烷基羰基或C22烷基羰基。非常尤其优选包含一个或多个双键的饱和或不饱和C16~C22烷基羰基如C16烷基羰基、C18烷基羰基、C20烷基羰基或C22烷基羰基。这些有利的基团可以包含二、三、四、五或六个双键。脂肪酸链中具有20或22个碳原子的尤其优选的基团包含多达六个双键,有利地包含三、四、五或六个双键,尤其优选地包含五或六个双键。以上提到的所有基团均衍生自相应的脂肪酸。
式II中的R3为氢、饱和或不饱和的C2~C24烷基羰基。
可提及的烷基是包含一个或多个双键的取代或未取代、饱和或不饱和的C2~C24烷基羰基链例如乙基羰基、正丙基羰基、正丁基羰基、正戊基羰基、正己基羰基、正庚基羰基、正辛基羰基、正壬基羰基、正癸基羰基、正十一烷基羰基、正十二烷基羰基、正十三烷基羰基、正十四烷基羰基、正十五烷基羰基、正十六烷基羰基、正十七烷基羰基、正十八烷基羰基、正十九烷基羰基、正二十烷基羰基、正二十二烷基羰基或正二十四烷基羰基。优选包含一个或多个双键的饱和或不饱和C10~C22烷基羰基例如正癸基羰基、正十一烷基羰基、正十二烷基羰基、正十三烷基羰基、正十四烷基羰基、正十五烷基羰基、正十六烷基羰基、正十七烷基羰基、正十八烷基羰基、正十九烷基羰基、正二十烷基羰基、正二十二烷基羰基或正二十四烷基羰基。尤其优选包含一个或多个双键的饱和和/或不饱和C10~C22烷基羰基例如C10烷基羰基、C11烷基羰基、C12烷基羰基、C13烷基羰基、C14烷基羰基、C16烷基羰基、C18烷基羰基、C20烷基羰基或C22烷基羰基。非常尤其优选包含一个或多个双键的饱和或不饱和C16~C22烷基羰基如C16烷基羰基、C18烷基羰基、C20烷基羰基或C22烷基羰基。这些有利的基团可包含二、三、四、五或六个双键。在脂肪酸链中具有20或22个碳原子的尤其优选的基团包含多达六个双键,有利地包含三、四、五或六个双键,尤其优选地包含五或六个双键。以上提到的所有基团均衍生自相应的脂肪酸。
以上提及的R1、R2和R3基团可以被羟基和/或环氧基取代和/或可以包含三键。
本发明的方法中所产生的多不饱和脂肪酸有利地包含至少两个,有利地包含三、四、五或六个双键。这些脂肪酸尤其有利地包含四、五或六个双键。方法中所产生的脂肪酸在脂肪酸链中有利地具有18、20或22个C原子;这些脂肪酸在脂肪酸链中优选地包含20或22个碳原子。饱和的脂肪酸通过方法中所用核酸有利地得以小程度地反应至或者完全未得到反应。小程度应当理解为与多不饱和脂肪酸相比,饱和脂肪酸以低于5%的活性、有利地以低于3%、尤其有利地以低于2%、非常尤其有利地以低于1、0.5、0.25或0.125%的活性反应。这些已经产生的脂肪酸可以在方法中作为单个产物产生或在脂肪酸混合物中出现。
本发明方法中所用的核酸序列是编码具有Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶活性的多肽的经分离的核酸序列。
本发明方法中有利使用的核酸序列是那些编码具有Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶活性的多肽的核酸序列,这些核酸序列选自:
a)具有SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示序列的核酸序列,或
b)作为遗传密码简并结果可由SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132或SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示核酸序列的衍生物,其编码与SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132 or SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184在氨基酸水平具有至少40%同一性的多肽或者/并且具有Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶活性。
式I和II中的取代基R2或R3有利地并彼此独立地是饱和或不饱和的C18~C22烷基羰基,这些取代基尤其有利地彼此独立地是具有至少两个双键的不饱和的C18、C20或C22烷基羰基。
方法中一个优选实施方案的特征在于将这样的核酸序列额外地导入生物体,其中所述核酸序列编码具有ω3去饱和酶活性的多肽,选自:
a)具有SEQ ID NO:87或SEQ ID NO:105中所示序列的核酸序列,或
b)作为遗传密码简并结果可由SEQ ID NO:88或SEQ ID NO:106中所示的氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:87或SEQ ID NO:105中所示核酸序列的衍生物,其编码与SEQ ID NO:88或SEQ ID NO:106在氨基酸水平具有至少60%同一性的多肽并且具有ω3去饱和酶活性。
在另一个优选实施方案中,方法包括向生物体中额外地导入这样的核酸序列,其中所述核酸序列编码具有Δ12去饱和酶活性的多肽,选自:
a)具有SEQ ID NO:107或SEQ ID NO:109中所示序列的核酸序列,或
b)作为遗传密码简并结果可由SEQ ID NO:108或SEQ ID NO:110中所示的氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:107或SEQ ID NO:109中所示核酸序列的衍生物,其编码与SEQ ID NO:108或SEQ ID NO:110在氨基酸水平具有至少60%同一性的多肽并且具有Δ12去饱和酶活性。
以上提及的Δ12去饱和酶序列可与方法中所用的并且编码Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶的核酸序列共同使用,单独或与ω3去饱和酶序列联合使用。
表1显示了核酸序列、来源生物和序列ID号
No. | 生物体 | 活性 | 序列号 |
1. | 细小裸藻(Euglena gracilis) | Δ8-去饱和酶 | SEQ ID NO:1 |
2. | 绿光等鞭金藻(Isochrysis galbana) | Δ9-延伸酶 | SEQ ID NO:3 |
3. | 三角褐指藻(Phaedodactylum tricornutum) | Δ5-去饱和酶 | SEQ ID NO:5 |
4. | 角齿藓(Ceratodon pupureus) | Δ5-去饱和酶 | SEQ ID NO:7 |
5. | 展叶剑叶藓(Physcomitrella patens) | Δ5-去饱和酶 | SEQ ID NO:9 |
6. | 破囊壶菌属中的种(Thraustrochytrium sp.) | Δ5-去饱和酶 | SEQ ID NO:11 |
7. | 高山被孢霉(Mortierella alpina) | Δ5-去饱和酶 | SEQ ID NO:13 |
8. | 秀丽隐杆线虫(Caenorhabditis elegans) | Δ5-去饱和酶 | SEQ ID NO:15 |
9. | 琉璃苣(Borago officinalis) | Δ6-去饱和酶 | SEQ ID NO:17 |
10. | 角齿藓 | Δ6-去饱和酶 | SEQ ID NO:19 |
11. | 三角褐指藻 | Δ6-去饱和酶 | SEQ ID NO:21 |
12. | 展叶剑叶藓 | Δ6-去饱和酶 | SEQ ID NO:23 |
13. | 秀丽隐杆线虫 | Δ6-去饱和酶 | SEQ ID NO:25 |
14. | 展叶剑叶藓 | Δ6-延伸酶 | SEQ ID NO:27 |
15. | 破囊壶菌属中的种 | Δ6-延伸酶 | SEQ ID NO:29 |
16. | 致病疫霉(Phytopthera infestans) | Δ6-延伸酶 | SEQ ID NO:31 |
17. | 高山被孢霉 | Δ6-延伸酶 | SEQ ID NO:33 |
18. | 高山被孢霉 | Δ6-延伸酶 | SEQ ID NO:35 |
19. | 秀丽隐杆线虫 | Δ6-延伸酶 | SEQ ID NO:37 |
20. | 细小裸藻 | Δ4-去饱和酶 | SEQ ID NO:39 |
21. | 破囊壶菌属中的种 | Δ4-去饱和酶 | SEQ ID NO:41 |
22. | 假矮海链藻(Thalassiosira pseudonana) | Δ5-延伸酶 | SEQ ID NO:43 |
23. | 假矮海链藻 | Δ6-延伸酶 | SEQ ID NO:45 |
24. | 寇氏隐甲藻(Crypthecodinium cohnii) | Δ5-延伸酶 | SEQ ID NO:47 |
25. | 寇氏隐甲藻 | Δ5-延伸酶 | SEQ ID NO:49 |
26. | 虹鳟(Oncorhynchus mykiss) | Δ5-延伸酶 | SEQ ID NO:51 |
27. | 虹鳟 | Δ5-延伸酶 | SEQ ID NO:53 |
28. | 假矮海链藻 | Δ5-延伸酶 | SEQ ID NO:59 |
29. | 假矮海链藻 | Δ5-延伸酶 | SEQ ID NO:61 |
30. | 假矮海链藻 | Δ5-延伸酶 | SEQ ID NO:63 |
31. | 鲜黄破囊壶菌(Thraustrochytrium aureum) | Δ5-延伸酶 | SEQ ID NO:65 |
32. | Ostreococcus tauri | Δ5-延伸酶 | SEQ ID NO:67 |
33. | Ostreococcus tauri | Δ6-延伸酶 | SEQ ID NO:69 |
34. | 有粉报春(Primula farinosa) | Δ6-去饱和酶 | SEQ ID NO:71 |
35. | 高穗花报春(Primula vialii) | Δ6-去饱和酶 | SEQ ID NO:73 |
36. | Ostreococcus tauri | Δ5-延伸酶 | SEQ ID NO:75 |
37. | Ostreococcus tauri | Δ5-延伸酶 | SEQ ID NO:77 |
38. | Ostreococcus tauri | Δ5-延伸酶 | SEQ ID NO:79 |
39. | Ostreococcus tauri | Δ6-延伸酶 | SEQ ID NO:81 |
40. | 破囊壶菌属中的种 | Δ5-延伸酶 | SEQ ID NO:83 |
41. | 假矮海链藻 | Δ5-延伸酶 | SEQ ID NO:85 |
42. | 致病疫霉 | ω3-去饱和酶 | SEQ ID NO:87 |
43. | Ostreococcus tauri | Δ6-去饱和酶 | SEQ ID NO:89 |
44. | Ostreococcus tauri | Δ5-去饱和酶 | SEQ ID NO:91 |
45. | Ostreococcus tauri | Δ5-去饱和酶 | SEQ ID NO:93 |
46. | Ostreococcus tauri | Δ4-去饱和酶 | SEQ ID NO:95 |
47. | 假矮海链藻 | Δ6-去饱和酶 | SEQ ID NO:97 |
48. | 假矮海链藻 | Δ5-去饱和酶 | SEQ ID NO:99 |
49. | 假矮海链藻 | Δ5-去饱和酶 | SEQ ID NO:101 |
50. | 假矮海链藻 | Δ4-去饱和酶 | SEQ ID NO:103 |
51. | 假矮海链藻 | ω3-去饱和酶 | SEQ ID NO:105 |
52. | Ostreococcus tauri | Δ12-去饱和酶 | SEQ ID NO:107 |
53. | 假矮海链藻 | Δ12-去饱和酶 | SEQ ID NO:109 |
54. | Ostreococcus tauri | Δ6-延伸酶 | SEQ ID NO:111 |
55. | Ostreococcus tauri | Δ5-延伸酶 | SEQ ID NO:113 |
56. | 非洲爪蟾(Xenopus laevis)(BC044967) | Δ5-延伸酶 | SEQ ID NO:117 |
57. | 玻璃海鞘(Ciona intestinalis)(AK112719) | Δ5-延伸酶 | SEQ ID NO:119 |
58. | 细小裸藻 | Δ5-延伸酶 | SEQ ID NO:131 |
59. | 细小裸藻 | Δ5-延伸酶 | SEQ ID NO:133 |
60. | 拟南芥菜(Arabidopsis thaliana) | Δ5-延伸酶 | SEQ ID NO:135 |
61. | 拟南芥菜 | Δ5-延伸酶 | SEQ ID NO:137 |
62. | 三角褐指藻 | Δ6-延伸酶 | SEQ ID NO:183 |
方法中所产生的多不饱和脂肪酸有利地结合膜脂类和/或三酰甘油酯,但是也可以在生物中以游离脂肪酸或与其它脂肪酸酯的结合形式出现。在本上下文中,它们可作为“纯的产物”或者有利地以多种脂肪酸的混合物或不同甘油酯的混合物的形式出现。三酰甘油酯中所结合的多种脂肪酸可由具有4至6个C原子的短链脂肪酸、具有8至12个C原子的中等链脂肪酸或14至24个C原子的长链脂肪酸衍生,优选从长链脂肪酸衍生,更为优选地从具有18、20和/或22个C原子的长链多不饱和脂肪酸衍生。
本发明的方法有利地产生了在脂肪酸酯中具有至少两个双键,有利地在脂肪酸酯中具有至少三个、四个、五个或六个双键,尤其有利地在脂肪酸酯中具有至少五个或六个双键的多不饱和C18、C20和/或C22脂肪酸分子的脂肪酸酯并且有利地导致亚油酸(LA,C18:2Δ9,12)、γ亚麻酸(GLA,C18:3Δ6,9,12)、硬脂艾杜糖酸(SDA,C18:4Δ6,9,12,15)、二高γ亚麻酸(DGLA,20:3Δ8,11,14)、ω3花生四烯酸(ETA,C20:4Δ5,8,11,14)、花生四烯酸(ARA,C20:4Δ5,8,11,14)、二十碳五烯酸(EPA,C20:5Δ5,8,11,14,17)、ω6二十二碳五烯酸(C22:5Δ4,7,10,13,16)、ω6二十二碳四烯酸(C22:4Δ7,10,13,16)、ω3二十二碳五烯酸(DPA,C22:5Δ7,10,13,16,19)、二十二碳六烯酸(DHA,C22:6Δ4,7,10,13,16,19)或这些脂肪酸混合物的合成,优选导致ARA、EPA和/或DHA的合成。非常尤其优选产生了ω3脂肪酸如EPA和/或DHA。
具有多不饱和的C18、C20和/或C22脂肪酸分子的脂肪酸酯可以油或脂的形式,例如以化合物如鞘脂类、磷酸甘油酯、脂、糖脂例如糖鞘脂类、磷脂例如磷脂酰乙醇胺、磷脂酰胆碱、磷脂酰丝氨酸、磷脂酰甘油、磷脂酰肌醇或二磷脂酰甘油、单酰甘油酯、二酰甘油酯、三酰甘油酯或者其它脂肪酸酯例如乙酰基辅酶A酯的形式分离,其中所述的脂肪酸酯包含源于已用来制备脂肪酸酯的生物体的具有至少两个、三个、四个、五个或六个双键,优选地具有五个或六个双键的多不饱和脂肪酸;这些脂肪酸酯优选地以其二酰甘油酯、三酰甘油酯和/或以磷脂酰胆碱的形式,尤其优选地以三酰甘油酯的形式分离。除了这些酯以外,多不饱和脂肪酸也可以作为游离的或结合于其它化合物中的脂肪酸在生物,有利地在植物中存在。通常以上提到的多种化合物(脂肪酸酯和游离脂肪酸)在生物体中以如下的大致分布出现,即以多种化合物的总体计为100%重量,甘油三酯占80至90%重量、二酰甘油占2至5%重量、单酰甘油占5至10%重量、游离脂肪酸占1至5%重量、磷脂占2至8%重量。
本发明的方法产生了LCPUFA,其中所产生的LCPUFA的含量以转基因生物,优选地以转基因植物中的总脂肪酸为基础占至少3%重量、有利地占至少5%重量,优选地占至少8%重量,尤其优选地占至少10%重量、最为优选地占至少15%重量。在本上下文中仅仅提到两个例子,将宿主生物中的C18和/或C20脂肪酸转化了至少10%、优选地至少20%、尤其优选地至少30%、最优选地至少40%以产生相应的产物如DPA或DHA是有利的。这些脂肪酸有利地以结合的形式产生。在本发明方法中所用的核酸的辅助下,这些不饱和脂肪酸可以在已经有利地产生的甘油三酯的sn1、sn2和/或sn3位置处定位。由于多个反应步骤在本发明的方法中通过起始化合物亚油酸(C18:2)和亚麻酸(C18:3)实施,故方法的终产物例如花生四烯酸(ARA),二十碳五烯酸(EPA)、ω6二十二碳五烯酸或DHA不能作为绝对纯的产物获得;在终产物中总是出现微小痕量的前体。例如假设亚油酸和亚麻酸两者均在起始生物和起始植物中出现,则终产物如ARA、EPA或DHA作为混合物出现。以所讨论终产物的量为基础,前体应当有利地不超过20%重量、优选地不超过15%重量、尤其优选地不超过10%重量,最优选地不超过5%重量。有利地,根据本发明的方法,仅仅结合的或作为游离酸的ARA、EPA或仅DHA作为转基因植物中的终产物得到产生。如果同时产生化合物ARA、EPA和DHA,它们有利地以至少1∶1∶2(EPA∶ARA∶DHA)、有利地以至少1∶1∶3、优选地以至少1∶1∶4、尤其优选地以1∶1∶5的比率得到产生。
由本发明的方法所产生的脂肪酸酯或脂肪酸混合物有利地包含6至15%的棕榈酸、1至6%的硬脂酸、7-85%的油酸、0.5至8%的11-十八碳烯酸、0.1至1%的花生酸、7至25%的饱和脂肪酸、8至85%的单不饱和脂肪酸和60至85%的多不饱和脂肪酸,每种情况均以100%为基础并且以生物中总脂肪酸含量为基础。以总脂肪酸含量为基础,在脂肪酸酯或脂肪酸混合物出现的有利多不饱和脂肪酸优选地是至少0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9或1%的花生四烯酸。而且通过本发明方法所产生的脂肪酸酯或脂肪酸混合物有利地包含选自芥酸(13-二十二碳烯酸)、苹婆酸(9,10-亚甲基十八碳-9-烯酸)、锦葵酸(8,9-亚甲基十七碳-8-烯酸)、大风子油酸(环戊烯十二烷酸)、呋喃脂肪酸(9,12-环氧十八碳-9,11-二烯酸)、斑鸠菊酸(9,10-环氧十八碳-12-烯酸)、果脂酸(6-十八碳炔酸)、6-十九碳炔酸、santalbic acid(反11-十八碳烯-9-炔酸)、6,9-十八碳烯炔酸、pyrulic acid(反10-十七烯-8-炔酸)、crepenyninic acid(9-十八烯-12-炔酸)、13,14-二氢oropheic acid、十八碳-13-烯-9,11-二炔酸、伞形花子油酸(顺-6-十八烯酸)、9顺,12反-十八碳二烯酸、金盏花素酸(calendulicacid)(8反10反12顺-十八碳三烯酸)、梓甙酸(catalpic acid)(9反11反13顺-十八碳三烯酸)、桐酸(9顺11反13反-十八碳三烯酸)、iacaric acid(8顺10反12顺-十八碳三烯酸)、石榴酸(9顺11反13顺-十八碳三烯酸)、麦饼树酸(9顺11反13反15顺-十八碳四烯酸)、皮诺敛酸(pinolenicacid)(全-顺-5,9,12-十八碳三烯酸)、laballenic acid(5,6-十八碳二烯丙二烯酸)、蓖麻油酸(12-羟油酸)和/或革盖菌素酸(coriolic acid)(13-羟-9顺,11反-十八碳二烯酸)的脂肪酸。上述脂肪酸通常有利地仅在本发明方法所产生的脂肪酸酯或脂肪酸的混合物中痕量地存在,这就是说,以总脂肪酸为基础,它们的存在小于30%、优选地小于25%、24%、23%、22%或21%、尤其优选地低于20%、15%、10%、9%、8%、7%、6%或5%,非常尤其优选地小于4%、3%、2%或1%。在本发明更为优选的形式中,以总脂肪酸为基础,这些上述脂肪酸的存在小于0.9%、0.8%、0.7%、0.6%或0.5%,尤其优选地小于0.4%、0.3%、0.2%、0.1%。以总脂肪酸为基础,由本发明方法所产生的脂肪酸酯或脂肪酸混合物有利地包含小于0.1%和/或无丁酸、无胆固醇、无鰶鱼酸(二十二碳五烯酸,C22:5Δ4,8,12,15,21)以及无尼生酸(二十四碳六烯酸,C23:6Δ3,8,12,15,18,21)。
当脂肪酸以GC分析检测时,与非转基因的起始生物如酵母、藻类、真菌或植物如拟南芥(arabidopsis)或亚麻子相比较,由于核酸序列或在方法中所用的核酸序列,多不饱和脂肪酸的产量可获得至少50%、有利地为至少80%、尤其有利地为至少100%、极其有利地为至少150%的提高(见实施例)。
化学纯的多不饱和脂肪酸或脂肪酸组合物也能通过以上描述的方法合成。为达此目的,脂肪酸或脂肪酸组合物按照已知的方法如通过提取、蒸馏、结晶、层析或这些方法的组合从生物如微生物或植物,或从其中生物已经在其内或其上生长的培养基,或从生物和培养基中分离。这些化学纯的脂肪酸或脂肪酸组合物有利地在食品工业、化妆品应用并且尤其在药学领域内应用。
原则上在本发明方法中用于产生的合适生物可以是任何生物如微生物、非人类动物或植物。
合适的植物原则上是能合成脂肪酸的所有植物如所有双子叶或单子叶植物、藻类或藓类。有利的植物选自Adelotheciaceae、漆树科(Anacardiaceae)、菊科(Asteraceae)、伞形科(Apiaceae)、桦木科(Betulaceae)、紫草科(Boraginaceae)、十字花科(Brassicaceae)、凤梨科(Bromeliaceae)、番木瓜科(Caricaceae)、大麻科(Cannabaceae)、旋花科(Convolvulaceae)、藜科(Chenopodiaceae)、隐甲藻科(Crypthecodiniaceae)、葫芦科(Cucurbitaceae)、牛毛藓科(Ditrichaceae)、胡颓子科(Elaeagnaceae)、杜鹃花科(Ericaceae)、大戟科(Euphorbiaceae)、豆科(Fabaceae)、牻牛儿苗科(Geraniaceae)、禾本科(Gramineae)、核桃科(Juglandaceae)、樟科(Lauraceae)、豆科(Leguminosae)、亚麻科(Linaceae)、裸藻科(Euglenaceae)、草绿藻科或蔬菜植物或装饰性植物如万寿菊属(Tagetes)。
可提到的例子是选自以下的植物:Adelotheciaceae如剑叶藓属,例如展叶剑叶藓的属和种、漆树科如黄连木属(Pistacia)、芒果属(Mangifera)、腰果属(Anacardium)例如阿月混子(Pistacia vera[阿月混子实])、芒果(Mangifer indica[芒果])或腰果(Anacardiumoceidentale[腰果])的属和种、菊科如金盏花属(Calendula)、红蓝花属(Carthamus)、矢车菊属(Centaurea)、菊苣属(Cichorium)、菜蓟属(Cynara)、向日葵属(Helianthus)、莴苣属(Lactuca)、Locusta、万寿菊属、缬草属(Valeriana)例如金盏花(Calendula officinalis[普通金盏花])、红花(Carthamus tinctorius[红花])、矢车菊(Centaurea cyanus[矢车菊])、菊苣(Cichorium intybus[菊苣])、洋蓟(Cynara scolymus[朝鲜蓟])、向日葵(Helianthus annus [向日葵])、莴苣(Lactuca sativa)、皱叶莴苣(Lactuca crispa)、芋(Lactuca esculenta)、毒莴苣的某些种(Lactuca scariola L.ssp.Sativa)、Lactuca scariola L.var.integrata、Lactuca scariola L.var.integrifolia、莴苣romana亚种(Lactuca sativasubsp.Romana)、Locusta communis、莴苣缬草(Valeriana locusta[沙拉蔬菜])、香叶万寿菊(Tagetes lucida)、万寿菊(Tagetes erecta)或细叶万寿菊(Tagetes tenuifolia)[非洲或法国金盏花]的属和种、伞形科如胡萝卜属(Daucus)例如胡萝卜(Daucus carota[胡萝卜])的属和种、桦木科如榛属(Corylus)例如欧洲榛(Corylus avellana)或榛子(Coryluscolurna[榛子])的属和种、紫草科如琉璃苣属(Borago)例如琉璃苣(Borago officinalis[琉璃苣])的属和种、十字花科如芸苔属(Brassica)、亚麻荠属(Camelina)、Melanosinapis、白芥属(Sinapis)、拟南芥属(Arabadopsis)例如欧洲油菜(Brassica napus)、芜青属某些种(Brassicarapa ssp.[油菜籽])、野生欧白芥(Sinapis arvensis)、芥菜(Brassicajuncea)、Brassica juncea var.juncea、皱叶芥菜(Brassica juncea var.crispifolia)、大叶芥菜(Brassica juncea var.foliosa)、黑芥(Brassicanigra)、Brassica sinapioides、亚麻荠(Camelina sativa)、芥菜(Melanosinapis communis[芥菜])、甘蓝(Brassica oleracea[饲用甜菜])或拟南芥菜(Arabidopsis thaliana)的属和种、凤梨科如凤梨属(Anana)、Bromelia(菠萝)例如菠萝(Anana comosus)、Ananas ananas或Bromeliacomosa[菠萝]的属和种、番木瓜科如番木瓜属(Carica)例如番木瓜(Carica papaya[番木瓜])的属和种、大麻科如大麻属(Cannabis)例如大麻(Cannabis sativa[大麻])的属和种、旋花科如番薯属(Ipomea)、旋花属(Convolvulus)例如甘薯(Ipomoea batatus)、提琴叶牵牛花(Ipomoea pandurata)、Convolvulus batatas、Convolvulus tiliaceus、Ipomoea fastigiata、Ipomoea tiliacea、三裂叶薯(Ipomoea triloba)或Convolvulus panduratus[甜薯、番薯]的属和种、藜科如甜菜属(Betavulgaris)例如甜菜(Beta vulgaris)、Beta vulgaris var.altissima、甜菜普通变种(Beta vulgaris var.vulgaris)、Beta maritima、甜菜宿根变种Beta vulgaris var.perennis、Beta vulgaris var.Conditiva或Beta vulgarisvar.esculenta[甜菜]的属和种、隐甲藻科如隐甲藻属例如寇氏隐甲藻(Cryptecodinium cohnii)的属和种、葫芦科如南瓜属(Cucurbita)例如笋瓜(Cucurbita maxima)、灰子南瓜(Cucurbita mixta)、西葫芦(Cucurbita pepo)或南瓜(Cucurbita moschata[西葫芦/南瓜])的属和种、桥弯藻科(Cymbellaceae)如Amphora、桥弯藻属(Cymbella)、Okedenia、褐指藻属(Phaeodactylum)、仰叶藓属(Reimeria)例如三角褐指藻的属和种、牛毛藓科(Ditrichaceae)如牛毛藓属、高地藓属(Astomiopsis)、角齿藓属(Ceratodon)、Chrysoblastella、牛毛藓属(Ditrichum)、对叶藓属(Distichium)、Eccremidium、Lophidion、Philibertiella、丛毛藓属(Pleuridium)、石缝藓属(Saelania)、毛齿藓属(Trichodon)、Skottsbergia,例如以下的属和种:Ceratodonantarcticus、Ceratodon columbiae、Ceratodon heterophyllus、角齿藓(Ceratodon purpurascens)、Ceratodon purpureus、Ceratodonpurpureus ssp.convolutus、Ceratodon purpureus ssp.Stenocarpus、角齿藓圆叶变种(Ceratodon purpureus var.Rotundifolius)、Ceratodonratodon、疣蒴角齿藓(Ceratodon stenocarpus)、Chrysoblastellachilensis、Ditrichum ambiguum、Ditrichum brevisetum、皱波牛毛藓(Ditrichum crispatissimum)、卷叶牛毛藓(Ditrichum difficile)、Ditrichum falcifolium、扭叶牛毛藓(Ditrichum flexicaule)、Ditrichumgiganteum、牛毛藓(Ditrichum heteromallum)、Ditrichum lineare、Ditrichum lineare、Ditrichum montanum、Ditrichum montanum、黄牛毛藓(Ditrichum pallidum)、Ditrichum punctulatum、细叶牛毛藓(Ditrichum pusillum)、细叶牛毛藓扭叶变种(Ditrichum pusillum var.tortile、Ditrichum rhynchostegium)、Ditrichum schimperi、Ditrichumtortile、对叶藓(Distichium capillaceum)、小对叶藓(Distichiumhagenii)、斜蒴对叶藓(Distichium inclinatum)、Distichium macounii、Eccremidium floridanum、Eccremidium whiteleggei、Lophidionstrictus、尖叶丛毛藓(Pleuridium acuminatum)、Pleuridiumalternifolium、Pleuridium holdridgei、Pleuridium mexicanum、Pleuridium ravenelii、丛毛藓(Pleuridium subulatum)、石缝藓(Saelaniaglaucescens)、Trichodon borealis、毛齿藓(Trichodon cylindricus)或Trichodon cylindricus var.oblongus、胡颓子科如胡颓子属(Elaeagnus)例如油橄榄(Olea europaea[橄榄])的属和种、杜鹃花科如山月桂属(Kalmia)例如阔叶山月桂(Kalmia latifolia)、狭叶山月桂(Kalmiaangustifolia)、小叶山月桂(Kalmia microphylla)、沼泽山月桂(Kalmiapolifolia)、西洋山月桂(Kalmia occidentalis)、Cistus chamaerhodendros或山月桂(Kalmia lucida[山月桂])的属和种、裸藻科如囊胞藻属(Ascoglena)、变胞藻属(Astasia)、胶柄藻属(Colacium)、Cyclidiopsis、裸藻属、拟裸藻属(Euglenopsis)、Hyalaphacus、克氏藻属(Khawkinea)、鳞孔藻属(Lepocinclis)、扁裸藻属(Phacus)、陀螺藻属(Strombomonas)、囊裸藻属(Trachelomonas)例如细小裸藻的属和种;大戟科如木薯属(Manihot)、Janipha、麻风树属(Jatropha)、蓖麻属(Ricinus)例如木薯(Manihot utilissima)、Janipha manihot、Jatropha manihot、Manihot aipil、甜木薯(Manihot dulcis)、Manihotmanihot、Manihot melanobasis、木薯(Manihot esculenta[木薯])或蓖麻(Ricinus communis[蓖麻])的属和种、豆科如豌豆属(Pisum)、合欢属(Albizia)、Cathormion、Feuillea、Inga、Pithecolobium、金合欢属(Acacia)、含羞草属(Mimosa)、Medicajo、大豆属(Glycine)、扁豆属(Dolichos)、菜豆属Phaseolus、大豆属(Soybean)例如豌豆(Pisumsativum)、饲料豌豆(Pisum arvense)、Pisum humile[豌豆]、Albiziaberteriana、合欢(Albizia julibrissin)、阔荚合欢(Albizia lebbeck)、Acacia berteriana、Acacia littoralis、Albizia berteriana、Albizziaberteriana、Cathormion berteriana、Feuillea berteriana、Ingafragrans、Pithecellobium berterianum、Pithecellobium fragrans、Pithecolobium berterianum、Pseudalbizzia berteriana、Acaciajulibrissin、Acacia nemu、Albizia nemu、Feuilleea julibrissin、Mimosajulibrissin、Mimosa speciosa、Sericanrda julibrissin、阔荚金合欢(Acacia lebbeck)、Acacia macrophylla、大叶合欢(Albizia lebbeck)、Feuilleea lebbeck、大叶含羞草(Mimosa lebbeck)、Mimosa speciosa、紫苜蓿(Medicago sativa)、野苜蓿(Medicago falcata)、杂交苜蓿(Medicagovaria)[紫花苜蓿]、大豆(Glycine max)、镰扁豆(Dolichos soja)、宽叶蔓豆(Glycine gracilis)、大豆(Glycine hispida)、大菜豆(Phaseolusmax)、Soja hispida或Soja max[大豆]的属和种、葫芦藓科(Funariaceae)如Aphanorrhegma、Entosthodon、葫芦藓属(Funaria)、剑叶藓属(Physcomitrella)、立碗藓属(Physcomitrium)例如Aphanorrhegma serratum、Entosthodon attenuatus、Entosthodonbolanderi、Entosthodon bonplandii、Entosthodon californicus、Entosthodon drummondii、Entosthodon jamesonii、Entosthodonleibergii、Entosthodon neoscoticus、Entosthodon rubrisetus、Entosthodon spathulifolius、Entosthodon tucsoni、美国葫芦藓(Funariaamericana)、Funaria bolanderi、齿叶葫芦藓(Funaria calcarea)、加利福尼亚葫芦藓(Funaria californica)、Funaria calvescens、皱叶葫芦藓(Funaria convoluta)、Funaria flavicans、Funaria groutiana、葫芦藓(Funaria hygrometrica)、葫芦藓北极变种(Funaria hygrometrica var.Arctica)、葫芦藓暖地变种(Funaria hygrometrica var.Calvescens)、葫芦藓皱叶变种(Funaria hygrometrica var.Convoluta)、Funariahygrometrica var.muralis、Funaria hygrometrica var.utahensis、小口葫芦藓(Funaria microstoma)、Funaria microstoma var.obtusifolia、刺边葫芦藓(Funaria muhlenbergii)、Funaria orcuttii、Funariaplano-convexa、Funaria polaris、Funaria ravenelii、Funaria rubriseta、Funaria serrata、Funaria sonorae、Funaria sublimbatus、Funariatucsoni、加利福尼亚剑叶藓(Physcomitrella californica)、展叶剑叶藓、Physcomitrella readeri、Physcomitrium australe、加利福尼亚立碗藓(Physcomitrium californicum)、Physcomitrium collenchymatum、Physcomitrium coloradense、Physcomitrium cupuliferum、Physcomitrium drummondii、红蒴立碗藓(Physcomitriumeurystomum)、Physcomitrium flexifolium、Physcomitrium hookeri、Physcomitrium hookeri var.serratum、隐蒴立碗藓(Physcomitriumimmersum)、Physcomitrium kellermanii、Physcomitriummegalocarpum、梨蒴立碗藓(Physcomitrium pyriforme)、Physcomitriumpyriforme var.serratum、Physcomitrium rufipes、Physcomitriumsandbergii、Physcomitrium subsp haericum、华盛顿立碗藓(Physcomitrium washingtoniense)的属和种、牻牛儿苗科(Geraniaceae)如天竺葵属(Pelargonium)、椰子属(Cocos)、Oleum例如椰子(Cocosnucifera)、茶麋子天竺葵(Pelargonium grossularioides)或椰子油(Oleumcocois[椰子])的属和种、禾本科如甘蔗属(Saccharum)例如甘蔗(Saccharum officinarum)的属和种、胡桃科如胡桃属(Juglans)、Wallia例如胡桃(Juglans regia)、Juglans ailanthifolia、山核桃(Juglanssieboldiana)、灰核桃(Juglans cinerea)、Wallia cinerea、Juglansbixbyi、加州黑核桃(Juglans californica)、印度黑核桃(Juglanshindsii)、Juglans intermedia、Juglans jamaicensis、大核桃(Juglansmajor)、Juglans microcarpa、黑核桃(Juglans nigra)或胡桃(Wallianigra)[胡桃]的属和种、樟科如鳄梨属(Persea)、月桂属(Laurus)例如月桂(Laurus nobilis[bay])、鳄梨(Persea americana)、鳄梨油(Perseagratissima)或Persea persea[avocado]的属和种、豆科如落花生属(Arachis)例如落花生(Arachis hypogaea[花生])的属和种、亚麻科如Adenolinum属例如亚麻(Linum usitatissimum)、Linum humile、奥地利亚麻(Linum austriacum)、Linum bienne、窄叶亚麻(Linumangustifolium)、泻亚麻(Linum catharticum)、金黄亚麻(Linumflavum)、大花亚麻(Linum grandiflorum)、Adenolinum grandiflorum、刘易斯亚麻(Linum lewisii)、那旁亚麻(Linum narbonense)、宿根亚麻(Linum perenne)、宿根亚麻刘易斯变种(Linum perenne var.lewisii)、Linum pratense或亚麻子(Linum trigynum[亚麻子])的属和种、Lythrarieae如石榴属(Punica)例如石榴(Punica granatum[pomegranate])的属和种、锦葵科(Malvaceae)如棉属(Gossypium)例如陆地棉(Gossypium hirsutum)、树棉(Gossypium arboreum)、海岛棉(Gossypium barbadense)、草棉(Gossypium herbaceum)或瑟伯棉(Gossypium thurberi[棉])的属和种、地钱科(Marchantiaceae)如地钱属(Marchantia)例如Marchantia berteroana、Marchantia foliacea、大孢地钱(Marchantia macropora)的属和种、芭蕉科(Musaceae)如芭蕉属(Musa)例如香蕉(Musa nana)、小果野蕉(Musa acuminata)、大蕉(Musa paradisiaca)、芭蕉属的某些种(Musa spp.[banana])的属和种、柳叶菜科(Onagraceae)如Camissonia属、月见草属(Oenothera)例如月见草(Oenothera biennis)或夜来香(Camissonia brevipes[月见草])的属和种、棕榈科(Palmae)如油棕属(Elaeis),例如以下的属和种:油棕(Elaeis guineensis[油棕榈])、罂粟科(Papaveraceae)如罂粟属(Papaver),例如以下的属和种:东方罂粟(Papaver orientale)、虞美人(Papaver rhoeas)、长荚罂粟(Papaver dubium[罂粟])、胡麻科(Pedaliaceae)如胡麻属(Sesamum)例如胡麻(Sesamum indicum[sesame])的属和种、胡椒科(Piperaceae)如胡椒属(Piper)、Artanthe、草胡椒属(Peperomia)、Steffensia例如树胡椒(Piper aduncum)、Piperamalago、狭叶胡椒(Piper angustifolium)、大胡椒(Piper auritum)、萎叶(Piper betel)、荜澄茄(Piper cubeba)、荜菝(Piper longum)、胡椒(Piper nigrum)、假荜拔(Piper retrofractum)、Artanthe adunca、Artanthe elongata、长胡椒(Peperomia elongata)、Piper elongatum、Steffensia elongata[cayenne pepper]的属和种、禾本科(Poaceae)如大麦属(Hordeum)、黑麦属(Secale)、燕麦属(Avena)、高梁属(Sorghum)、须芒草属(Andropogon)、绒毛草属(Holcus)、黍属(Panicum)、稻属(Oryza)、玉蜀黍属(Zea(maize))、小麦属(Triticum)例如大麦(Hordeum vulgare)、芒颖大麦草(Hordeum jubatum)、鼠大麦草(Hordeum murinum)、短芒大麦草(Hordeum secalinum)、栽培二棱大麦(Hordeum distichon)、Hordeum aegiceras、六列大麦(Hordeumhexastichon)、六棱大麦(Hordeum hexastichum)、不规则大麦(Hordeumirregulare)、大麦(Hordeum sativum)、短芒大麦草(Hordeumsecalinum)[大麦]、黑麦(Secale cereale[黑麦])、燕麦(Avena sativa)、野燕麦(Avena fatua)、地中海红燕麦(Avena byzantina)、Avena fatuavar.sativa、杂种燕麦(Avena hybrida[燕麦])、二色高梁(Sorghumbicolor)、石茅高梁(Sorghum halepense)、甜高梁(Sorghumsaccharatum)、高梁(Sorghum vulgare)、Andropogon drummondii、二色绒毛草(Holcus bicolor)、Holcus sorghum、Sorghum aethiopicum、Sorghum arundinaceum、Sorghum caffrorum、垂穗高梁草(Sorghumcernuum)、工艺高梁(Sorghum dochna)、Sorghum drummondii、硬高粱草(Sorghum durra)、Sorghum guineense、Sorghum lanceolatum、多脉高粱草(Sorghum nervosum)、甜高梁(Sorghum saccharatum)、Sorghum subglabrescens、垂叶高梁草(Sorghum verticilliflorum)、高梁(Sorghum vulgare)、石茅高梁(Holcus halepensis)、Sorghummiliaceum、黍粟(Panicum militaceum[millet])、稻(Oryza sativa)、Oryza latifolia[稻]、玉米(Zea mays[maize])、小麦(Triticum aestivum)、硬粒小麦(Triticum durum)、圆柱小麦(Triticum turgidum)、Triticumhybernum、马卡小麦(Triticum macha)、普通小麦(Triticum sativum或Triticum vulgare)[小麦]、紫球藻科(Porphyridiaceae)如Chroothece属、Flintiella属、Petrovanella属、紫球藻属(Porphyridium)、Rhodella、Rhodosorus、Vanhoeffenia例如紫球藻(Porphyridiumcruentum)的属和种、山龙眼科(Proteaceae)如澳洲坚果属(Macadamia)例如全缘叶澳洲坚果(Macadamia intergrifolia[macadamia])的属和种、草绿藻科如肾藻属(Nephroselmis)、Prasinococcus、Scherffelia、扁藻属(Tetraselmis)、Mantoniella、Ostreococcus例如绿肾藻(Nephroselmisolivacea)、Prasinococcus capsulatus、Scherffelia dubia、周氏扁藻(Tetraselmis chui)、四鞭毛藻(Tetraselmis suecica)、Mantoniellasquamata、Ostreococcus tauri属和种、茜草科(Rubiaceae)如咖啡属(Coffea)例如咖啡属(Coffea)某些种、小果咖啡(Coffea arabica)、中果咖啡(Coffea canephora)或大果咖啡(Coffea liberica[coffee])、玄参科(Scrophulariaceae)如毛蕊花属(Verbascum)例如以下的属和种:毛瓣毛蕊花(Verbascum blattaria)、东方毛蕊花(Verbascum chaixii)、密叶毛蕊花(Verbascum densiflorum)、Verbascum lagurus、长叶毛蕊花(Verbascum longifolium)、Verbascum lychnitis、Verbascum nigrum、奥林匹克毛蕊花(Verbascum olympicum)、Verbascum phlomoides、紫毛蕊花(Verbascum phoenicum)、Verbascum pulverulentum或毛蕊花(Verbascum thapsus[毛蕊花])、茄科(Solanaceae)如辣椒属(Capsicum)、烟草属(Nicotiana)、茄属(Solanum)、番茄属(Lycopersicon)例如以下的属和种:辣椒(Capsicum annuum)、Capsicumannuum var.glabriusculum、五色椒(Capsicum frutescens [pepper])、红椒(Capsicum annuum[paprika])、烟草(Nicotiana tabacum)、花烟草(Nicotiana alata)、长头烟草(Nicotiana attenuata)、光烟草(Nicotianaglauca)、Nicotiana langsdorffii、Nicotiana obtusifolia、Nicotianaquadrivalvis、Nicotiana repanda、黄花烟草(Nicotiana rustica)、Nicotiana sylvestris[烟草]、马铃薯(Solanum tuberosum[番茄])、茄(Solanum melongena[eggplant])、番茄(Lycopersicon esculentum)、Lycopersicon lycopersicum、梨形番茄(Lycopersicon pyriforme)、红茄(Solanum integrifolium)或番茄(Solanum lycopersicum)[番茄]、梧桐科(Sterculiaceae)如可可树属(Theobroma)例如可可树(Theobroma cacao[可可])的属和种或山茶科(Theaceae)如山茶属(Camellia)例如大叶茶(Camellia sinensis[茶])的属和种。
有利的微生物例如选自毛壳菌科(Chaetomiaceae)、笄霉科(Choanephoraceae)、隐球酵母科(Cryptococcaceae)、小克银汉霉科(Cunninghamellaceae)、暗梗胞科(Demetiaceae)、从梗胞科(Moniliaceae)、被孢霉科、毛霉科(Mucoraceae)、腐霉科(Pythiaceae)、酵母菌科(Saccharomycetaceae)、水霉科(Saprolegniaceae)、裂殖酵母科(Schizosacharomycetaceae)、粪壳科(Sodariaceae)或瘤座孢科(Tuberculariaceae)科的真菌。
可提到的微生物的例子为选自如下的微生物组:笄霉科如布拉霉属(Blakeslea)、笄霉属(Choanephora)例如三孢布拉霉(Blakesleatrispora)、瓜笄霉(Choanephora cucurbitarum)、漏斗笄霉瓜变种(Choanephora infundibulifera var.Cucurbitarum)的属和种、被孢霉科如被孢霉属例如深黄被孢霉(Mortierella isabellina)、多头被孢霉(Mortierella polycephala)、拉曼被孢霉(Mortierella ramanniana)、葡酒色被孢霉(Mortierella vinacea)、环带被孢霉(Mortierella zonata)的属和种、腐霉科如腐霉属(Phytium)、疫霉属(Phytophthora)例如德巴利腐霉(Pythium debaryanum)、间型腐霉(Pythium intermedium)、畸雌腐霉(Pythium irregulare)、大雄腐霉(Pythium megalacanthum)、侧雄腐霉(Pythium paroecandrum)、Pythium sylvaticum、终极腐霉(Pythium ultimum)、恶疫霉(Phytophthora cactorum)、樟疫霉(Phytophthora cinnamomi)、柑桔生疫霉(Phytophthora citricola)、柑桔褐腐疫霉(Phytophthora citrophthora)、隐地疫霉(Phytophthoracryptogea)、德雷疫霉(Phytophthora drechsleri)、红瘤疫霉(Phytophthora erythroseptica)、侧生疫霉(Phytophthora lateralis)、大雄疫霉(Phytophthora megasperma)、烟草疫霉(Phytophthoranicotianae)、烟草疫霉寄生变种(Phytophthora nicotianae var.parasitica)、棕榈疫霉(Phytophthora palmivora)、寄生疫霉(Phytophthora parasitica)、丁香疫霉(Phytophthora syringae)的属和种、酵母菌科如汉逊属(Hansenula)、毕赤属(Pichia)、酵母属(Saccharomyces)、类酵母属(Saccharomycodes)、亚罗酵母属(Yarrowia)例如异常汉逊酵母(Hansenula anomala)、加利福尼亚汉逊酵母(Hansenula californica)、加拿大汉逊酵母(Hansenula canadensis)、碎囊汉逊酵母(Hansenula capsulata)、西弗汉逊酵母(Hansenulaciferrii)、葡糖酶汉逊酵母(Hansenula glucozyma)、亨利汉逊酵母(Hansenula henricii)、Hansenula holstii、小汉逊酵母(Hansenulaminuta)、不发酵汉逊酵母(Hansenula nonfermentans)、Hansenulaphilodendri、多形汉逊酵母(Hansenula polymorpha)、土星汉逊酵母(Hansenula saturnus)、亚膜汉逊酵母(Hansenula subpelliculosa)、魏氏汉逊酵母(Hansenula wickerhamii)、温奇汉逊酵母(Hansenulawingei)、嗜酒毕赤酵母(Pichia alcoholophila)、Pichia angusta、异常毕赤酵母(Pichia anomala)、二孢毕赤酵母(Pichia bispora)、伯顿毕赤酵母(Pichia burtonii)、加拿大毕赤酵母(Pichia canadensis)、荚膜毕赤酵母(Pichia capsulata)、卡氏毕赤酵母(Pichia carsonii)、聚纤维二糖毕赤酵母(Pichia cellobiosa)、西弗毕赤酵母(Pichia ciferrii)、粉状毕赤酵母(Pichia farinosa)、发酵毕赤酵母(Pichia fermentans)、芬兰毕赤酵母(Pichia finlandica)、葡糖酶毕赤酵母(Pichia glucozyma)、季氏毕赤酵母(Pichia guilliermondii)、甲虫毕赤酵母(Pichiahaplophila)、Pichia henricii、Pichia holstii、Pichia jadinii、Pichialindnerii、膜醭毕赤酵母(Pichia membranaefaciens)、甲醇毕赤酵母(Pichia methanolica)、微小毕赤酵母微小变种(Pichia minuta var.minuta)、微小毕赤酵母不发酵变种(Pichia minuta var.nonfermentans)、挪威毕赤酵母(Pichia norvegensis)、奥默毕赤酵母(Pichia ohmer)、巴斯德毕赤酵母(Pichia pastoris)、Pichiaphilodendri、Pichia pini、多形毕赤酵母(Pichia polymorpha)、栎毕赤酵母(Pichia quercuum)、罗丹毕赤酵母(Pichia rhodanensis)、Pichiasargentensis、树干毕赤酵母(Pichia stipitis)、斯地毕赤酶母(Pichiastrasburgensis)、亚膜毕赤酵母(Pichia subpelliculosa)、帽孢毕赤酵母(Pichia toletana)、喜海藻糖毕赤酵母(Pichia trehalophila)、Pichiavini、木糖毕赤酵母(Pichia xylosa)、酸酒酵母(Saccharomyces aceti)、拜耳酵母(Saccharomyces bailii)、贝酵母(Saccharomyces bayanus)、二孢酵母(Saccharomyces bisporus)、好望角酵母(Saccharomycescapensis)、卡尔酵母(Saccharomyces carlsbergensis)、酿酒酵母(Saccharomyces cerevisiae)、椭圆酿酒酵母(Saccharomyces cerevisiaevar.ellipsoideus)、薛瓦酵母(Saccharomyces chevalieri)、德布尔酵母(Saccharomyces delbrueckii)、糖化酵母(Saccharomyces diastaticus)、Saccharomyces drosophilarum、雅致酵母(Saccharomyces elegans)、椭圆酵母(Saccharomyces ellipsoideus)、发酵性酵母(Saccharomycesfermentati)、Saccharomyces florentinus、脆壁酵母(Saccharomycesfragilis)、异质酵母(Saccharomyces heterogenicus)、橄榄油酵母(Saccharomyces hienipiensis)、荷兰啤酒酵母(Saccharomycesinusitatus)、意大利酵母(Saccharomyces italicus)、克鲁弗酵母(Saccharomyces kluyveri)、克鲁斯酵母(Saccharomyces krusei)、乳酸酵母(Saccharomyces lactis)、马克斯酵母(Saccharomycesmarxianus)、小椭圆酵母(Saccharomyces microellipsoides)、山地酵母(Saccharomyces montanus)、诺地酵母(Saccharomyces norbensis)、橄榄酵母(Saccharomyces oleaceus)、奇异酵母(Saccharomycesparadoxus)、Saccharomyces pastorianus、普地酵母(Saccharomycespretoriensis)、罗斯酵母(Saccharomyces rosei)、鲁氏酵母(Saccharomyces rouxii)、葡萄汁酵母(Saccharomyces uvarum)、路德类酵母(Saccharomycodes ludwigii)、解脂亚罗酵母(Yarrowialipolytica)、裂殖酵母科(Schizosacharomycetaceae)如裂殖酵母属(Schizosaccharomyces)例如日本裂殖酵母日本变种(Schizosaccharomyces japonicus var.japonicus)、日本裂殖酵母多变变种(Schizosaccharomyces japonicus var.versatilis)、解苹果酸裂殖酵母(Schizosaccharomyces malidevorans)、八孢裂殖酵母(Schizosaccharomyces octosporus)、粟酒裂殖酵母解苹果酸变种(Schizosaccharomyces pombe var.malidevorans)、粟酒裂殖酵母粟酒变种(Schizosaccharomyces pombe var.pombe)、破囊壶菌科(Thraustochytriaceae)如Althornia、Aplanochytrium、Japonochytrium、裂殖壶菌(Schizochytrium)、破囊壶菌属(破囊壶菌)例如聚集裂殖壶菌(Schizochytrium aggregatum),Schizochytriumlimacinum,红树林裂殖壶菌(Schizochytrium mangrovei),小裂殖壶菌(Schizochytrium minutum),八孢裂殖壶菌(Schizochytriumoctosporum),聚集破囊壶菌(Thraustochytrium aggregatum),Thraustochytrium amoeboideum,南极破囊壶菌(Thraustochytriumantacticum),Thraustochytrium arudimentale,鲜黄破囊壶菌(Thraustochytrium aureum),Thraustochytrium benthicola,球形破囊壶菌(Thraustochytrium globosum),Thraustochytrium indicum,Thraustochytrium kerguelense,Thraustochytrium kinnei,运动破囊壶菌(Thraustochytrium motivum),多初级破囊壶菌(Thraustochytriummultirudimentale),厚皮破囊壶菌(Thraustochytrium pachydermum),增殖破囊壶菌(Thraustochytrium proliferum),粉红破囊壶菌(Thraustochytrium roseum),罗氏破囊壶菌(Thraustochytrium rossii),Thraustochytrium striatum或Thraustochytrium visurgense的属和种。
更有利的微生物是例如选自芽孢杆菌科(Bacillaceae)、肠杆菌科(Enterobacteriacae)或根瘤菌科(Rhizobiaceae)的细菌。
可能提到的例子为选自如下的微生物:芽孢杆菌科如芽孢杆菌属例如酸热芽孢杆菌(bacillus acidocaldarius)、酸土环脂芽孢杆菌(bacillusacidoterrestris)、嗜碱芽孢杆菌(bacillus alcalophilus)、解淀粉芽孢杆菌(bacillus amyloliquefaciens)、溶淀粉芽孢杆菌(bacillus amylolyticus)、短芽孢杆菌(bacillus brevis)、蜡样芽孢杆菌(bacillus cereus)、环状芽孢杆菌(bacillus circulans)、凝结芽孢杆菌(bacillus coagulans)、球形芽孢杆菌梭形亚种(bacillus sphaericus subsp.fusiformis)、嗜乳糖芽孢杆菌(bacillus galactophilus)、园孢芽孢杆菌(bacillus globisporus)、园孢芽孢杆菌海生亚种(bacillus globisporus subsp.marinus)、嗜盐芽孢杆菌(bacillus halophilus)、缓病芽孢杆菌(bacillus lentimorbus)、迟缓芽孢杆菌(bacillus lentus)、地衣芽孢杆菌(bacillus licheniformis)、巨大芽孢杆菌(bacillus megaterium)、多粘芽孢杆菌(bacillus polymyxa)、冷解糖芽孢杆菌(bacillus psychrosaccharolyticus)、短小芽孢杆菌(bacilluspumilus)、球形芽孢杆菌(bacillus sphaericus)、枯草芽孢杆菌斯氏亚种(bacillus subtilis subsp.spizizenii)、枯草芽孢杆菌枯草亚种(bacillussubtilis subsp.subtilis)或苏云金芽孢杆菌(bacillus thuringiensis)的属和种;肠杆菌科例如柠檬酸细菌属(Citrobacter)、爱德华氏菌属(Edwardsiella)、肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、埃希氏菌属(Escherichia)、克雷伯氏菌属(Klebsiella)、沙门氏菌属(Salmonella)或沙雷氏菌属(Serratia)例如无丙二酸柠檬酸菌(Citrobacter amalonaticus)、异型柠檬酸菌(Citrobacter diversus)、弗氏柠檬酸菌(Citrobacter freundii)、Citrobacter genomospecies、Citrobacter gillenii、中间型柠檬酸菌(Citrobacter intermedium)、柯氏柠檬酸菌(Citrobacter koseri)、Citrobacter murliniae、柠檬酸菌种(Citrobacter sp.)、保科爱德华菌(Edwardsiella hoshinae)、Edwardsiellaictaluri、迟缓爱德华菌(Edwardsiella tarda)、Erwinia alni、解淀粉欧文氏菌(Erwinia amylovora)、Erwinia ananatis、Erwinia aphidicola、Erwinia billingiae、灭仙人掌欧文氏菌(Erwinia cacticida)、生癌欧文氏菌(Erwinia cancerogena)、大仙人掌欧文氏菌(Erwinia carnegieana)、胡萝卜软腐欧文氏菌黑腐亚种(Erwinia carotovora subsp.atroseptica)、胡萝卜软腐欧文氏菌亚种(Erwinia carotovora subsp.betavasculorum)、胡萝卜软腐欧文氏菌气味亚种(Erwinia carotovora subsp.odorifera)、胡萝卜软腐欧文氏菌wasabiae亚种(Erwinia carotovora subsp.wasabiae)、菊欧文氏菌(Erwinia chrysanthemi)、杓兰欧文氏菌(Erwinia cypripedii)、融解欧文氏菌(Erwinia dissolvens)、草生欧文氏菌(Erwinia herbicola)、野梧桐欧文氏菌(Erwinia mallotivora)、鸡血藤欧文氏菌(Erwiniamilletiae)、流黑欧文氏菌(Erwinia nigrifluens)、超压欧文氏菌(Erwinianimipressuralis)、粉红欧文氏菌(Erwinia persicina)、Erwinia psidii、Erwinia pyrifoliae、栎欧文氏菌(Erwinia quercina)、大黄欧文氏菌(Erwinia rhapontici)、生红欧文氏菌(Erwinia rubrifaciens)、柳欧文氏菌(Erwinia salicis)、斯氏欧文氏菌(Erwinia stewartii)、嗜管欧文氏菌(Erwinia tracheiphila)、噬夏孢欧文氏菌(Erwinia uredovora)、非脱羧埃希氏菌(Escherichia adecarboxylata)、Escherichia anindolica、金色埃希氏菌(Escherichia aurescens)、螳螂埃希氏菌(Escherichiablattae)、大肠埃希氏菌(Escherichia coli)、大肠埃希氏菌共生变种(Escherichia coli var.Communior)、大肠可变埃希氏菌(Escherichiacoli-mutabile)、福氏埃希氏菌(Escherichia fergusonii)、黑氏埃希氏菌(Escherichia hermannii)、埃希氏菌种(Escherichia sp.)、伤口埃希氏菌(Escherichia vulneris)、产气克雷伯氏菌(Klebsiella aerogenes)、贾氏克雷伯氏菌亚特兰大亚种(Klebsiella edwardsii subsp.Atlantae)、解鸟氨酸克雷伯氏菌(Klebsiella ornithinolytica)、催产克雷伯氏菌(Klebsiellaoxytoca)、植物克雷伯氏菌(Klebsiella planticola)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、肺炎克雷伯氏菌肺炎亚种(Klebsiellapneumoniae subsp.pneumoniae)、克雷伯氏菌种(Klebsiella sp.)、土生克雷伯氏菌(Klebsiella terrigena)、特氏克雷伯氏菌(Klebsiella trevisanii)、奥博尼沙门氏菌(Salmonella abony)、亚利桑那沙门氏菌(Salmonellaarizonae)、邦戈尔沙门氏菌(Salmonella bongori)、猪霍乱沙门氏菌亚利桑那亚种(Salmonella choleraesuis subsp.arizonae)、猪霍乱沙门氏菌邦戈尔亚种(Salmonella choleraesuis subsp.bongori)、猪霍乱沙门氏菌猪霍乱亚种(Salmonella choleraesuis subsp.cholereasuis)、猪霍乱沙门氏菌双亚利桑那亚种(Salmonella choleraesuis subsp.diarizonae)、猪霍乱沙门氏菌豪斯顿亚种(Salmonella choleraesuis subsp.houtenae)、猪霍乱沙门氏菌英迪加亚种(Salmonella choleraesuis subsp.indica)、猪霍乱沙门氏菌萨拉姆亚种(Salmonella choleraesuis subsp.salamae)、达累斯萨拉姆沙门氏菌(Salmonella daressalaam)、肠道沙门氏菌豪斯顿亚种(Salmonella enterica subsp.houtenae)、肠道沙门氏菌萨拉姆亚种(Salmonella enterica subsp.salamae)、肠炎沙门氏菌(Salmonellaenteritidis)、鸡沙门氏菌(Salmonella gallinarum)、海德堡沙门氏菌(Salmonella heidelberg)、巴拿马沙门氏菌(Salmonella panama)、森夫顿堡沙门氏菌(Salmonella senftenberg)、鼠伤寒沙门氏菌(Salmonellatyphimurium)、嗜虫沙雷菌(Serratia entomophila)、无花果沙雷菌(Serratia ficaria)、居泉沙雷菌(Serratia fonticola)、格式沙雷菌(Serratiagrimesii)、液化沙雷菌(Serratia liquefaciens)、粘质沙雷菌(Serratiamarcescens)、粘质沙雷菌粘质亚种(Serratia marcescens subsp.marcescens)、海红氏沙雷菌(Serratia marinorubra)、气味沙雷菌(Serratiaodorifera)、普利茅斯沙雷菌(Serratia plymouthensis)、普城沙雷菌(Serratia plymuthica)、变形斑沙雷菌(Serratia proteamaculans)、变形斑沙雷菌解奎宁亚种(Serratia proteamaculans subsp.quinovora)、解奎宁沙雷菌(Serratia quinivorans)或悬钩子沙雷菌(Serratia rubidaea)的属和种;根瘤菌科(Rhizobiaceae)例如农杆菌属(Agrobacterium)、嗜碳杆菌属(Carbophilus)、Chelatobacter、剑菌属(Ensifer)、根瘤菌属(Rhizobium)、中华根瘤菌属(Sinorhizobium)例如亚特兰农杆菌属(Agrobacterium atlanticum)、Agrobacterium ferrugineum、解明胶农杆菌属(Agrobacterium gelatinovorum)、Agrobacterium larrymoorei、Agrobacterium meteori、放射形农杆菌属(Agrobacterium radiobacter)、发根农杆菌属(Agrobacterium rhizogenes)、悬钩子农杆菌(Agrobacteriumrubi)、星斑农杆菌(Agrobacterium stellulatum)、根癌农杆菌(Agrobacterium tumefaciens)、Agrobacterium vitis、Carbophiluscarboxidus、Chelatobacter heintzii、剑菌(Ensifer adhaerens)、Ensiferarboris、费氏剑菌属(Ensifer fredii)、Ensifer kostiensis、鸡眼草剑菌属(Ensifer kummerowiae)、Ensifer medicae、苜蓿剑菌属(Ensifer meliloti)、萨赫尔剑菌属(Ensifer saheli)、Ensifer terangae、新疆剑菌属(Ensiferxinjiangensis)、鹰嘴豆根瘤菌(Rhizobium ciceri)、埃特里根瘤菌(Rhizobium etli)、费氏根瘤菌(Rhizobium fredii)、山羊豆根瘤菌(Rhizobium galegae)、Rhizobium gallicum、Rhizobium giardinii、海南根瘤菌(Rhizobium hainanense)、华癸根瘤菌(Rhizobium huakuii)、胡特根瘤菌(Rhizobium huautlense)、木兰根瘤菌(Rhizobiumindigoferae)、日本根瘤菌属(Rhizobium japonicum)、豌豆根瘤菌(Rhizobium leguminosarum)、黄土根瘤菌(Rhizobium loessense)、百脉根根瘤菌(Rhizobium loti)、羽扇豆根瘤菌(Rhizobium lupini)、地中海根瘤菌属(Rhizobium mediterraneum)、苜蓿根瘤菌(Rhizobiummeliloti)、蒙古根瘤菌(Rhizobium mongolense)、菜豆根瘤菌(Rhizobiumphaseoli)、放射形根瘤菌(Rhizobium radiobacter)、发根根瘤菌(Rhizobium rhizogenes)、Rhizobium rubi、Rhizobium sullae、天山根瘤菌(Rhizobium tianshanense)、三叶草根瘤菌(Rhizobium trifolii)、热带根瘤菌(Rhizobium tropici)、Rhizobium undicola、Rhizobium vitis、粘附中华根瘤菌(Sinorhizobium adhaerens)、Sinorhizobium arboris、费氏中华根瘤菌(Sinorhizobium fredii)、Sinorhizobium kostiense、鸡眼草中华根瘤菌(Sinorhizobium kummerowiae)、Sinorhizobium medicae、苜蓿中华根瘤菌(Sinorhizobium meliloti)、莫雷兰中华根瘤菌(Sinorhizobium morelense)、萨赫尔中华根瘤菌(Sinorhizobium saheli)或新疆中华根瘤菌(Sinorhizobium xinjiangense)的微生物。
用于本发明方法的有利微生物的其它例子是原生动物或硅藻,选自:甲藻科(Dinophyceae)、Turaniellidae或Oxytrichidae科,如寇氏隐甲藻(Crypthecodinium cohnii)如三角褐指藻、贻贝棘尾虫(Stylonychiaamytilus)、鬃棘尾虫(Stylonychia pustulata)、Stylonychiaputrina、背状棘尾虫(Stylonychia notophora)、棘尾虫种(Stylonychia sp.)、弯豆形虫(Colpidium campylum)或豆形虫种(Colpidium sp.)的属和种的。
那些有利地在本发明方法中应用的生物是转基因生物如真菌类如被孢霉属或破囊壶菌属、酵母属如酵母或裂殖酵母、藓类如剑叶藓属或角齿藓属、非人动物如隐杆线虫(Caenorhabditis)、藻类如肾藻属、Pseudoscourfielda、Prasinococcus、Scherffelia、扁藻属、Mantoniella、Ostreococcus、隐甲藻属或褐指藻属或植物类如双子叶植物或单子叶植物。尤其有利地在本发明方法中应用的生物属于产油的生物,也即用于产生油的生物、如真菌例如被孢霉属或破囊壶菌属、藻类例如肾藻属、Pseudoscourfielda、Prasinococcus、Scherffelia、扁藻属、Mantoniella、Ostreococcus、隐甲藻属、褐指藻属或者植物,尤其优选包含大量脂类化合物的油籽或油料作物植物,如花生、油菜籽、芸苔、向日葵、红花(Carthamus tinctoria)、罂粟、芥菜、大麻、蓖麻、橄榄、芝麻、金盏花、石榴、月见草、毛蕊花、大蓟、野玫瑰、榛子、杏仁、澳洲坚果、鳄梨、月桂、西葫芦/南瓜、亚麻籽、大豆、阿月混子、琉璃苣、树(油棕榈树、椰子树或胡桃树)或可耕种的作物例如玉米、小麦、黑麦、燕麦、小黑麦、稻、大麦、棉、木薯、胡椒、万寿菊、茄科植物例如马铃薯、烟草、茄子和番茄、蚕豆属的种、豌豆、紫花苜蓿或灌木(咖啡、可可、茶)、柳属的种和宿生牧草及饲料作物。优选的植物根据本发明是油料作物植物如花生、油菜籽、芸苔、向日葵、红花、罂粟、芥菜、大麻、蓖麻、橄榄、金盏花、石榴、月见草、西葫芦/南瓜、亚麻子、大豆、琉璃苣、树(油棕榈树、可可树)。尤其优选的植物是含有高C18:2和/或C18:3脂肪酸的植物,如向日葵、红花、烟草、毛蕊花、芝麻、棉、西葫芦/南瓜、罂粟、月见草、胡桃、亚麻子、大麻或大蓟。非常尤其优选的植物是例如红花、向日葵、罂粟、月见草、胡桃、亚麻子或大麻。
因此对于本发明的上述方法而言,除了在方法步骤(a)至(d)中所导入的核酸以及任选地所导入的编码ω3去饱和酶的核酸序列以外,额外地向生物中导入其它编码脂肪酸或脂类代谢的酶的核酸是有利的。
脂肪酸或脂类代谢中的所有基因原则上均可在产生多不饱和脂肪酸的方法中使用,有利地与Δ5延伸酶,Δ6延伸酶和/或ω3去饱和酶组合使用[为了本发明目的,将复数形式理解为包含单数,并且反之亦然]。选自酰基辅酶A脱氢酶、酰基ACP[酰基载体蛋白]去饱和酶、酰基ACP硫酯酶、脂肪酸酰基转移酶、酰基-辅酶A:溶血磷脂酰基转移酶、脂肪酸合成酶、脂肪酸羟化酶、乙酰基-辅酶A羧化酶、酰辅酶A氧化酶、脂肪酸去饱和酶、脂肪酸炔属酶(acetylenase)、脂加氧酶、三酰甘油酯脂肪酶、环丙烯合成酶、氢过氧化物裂合酶或脂肪酸延伸酶的脂肪酸和脂类代谢中的基因有利地与Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶组合使用。选自Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、Δ6延伸酶或Δ9延伸酶的基因尤其优选地与上述Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶的基因组合使用,可与单种基因或多种基因组合使用。
与人类延伸酶或来自非人类动物的延伸酶如那些来自大马哈鱼属(Oncorhynchus)、爪蟾(Xenopus)或海鞘(Ciona)的延伸酶比较,本发明的Δ5延伸酶具备有利特性,即它们不使C22脂肪酸延伸为相应的C24脂肪酸。而且它们有利地不转化具有Δ6位置双键的脂肪酸,而上述脂肪酸则由人类延伸酶或来自非人类动物的延伸酶所转化。尤其有利的Δ5延伸酶优选地只转化不饱和C20脂肪酸。这些有利的Δ5延伸酶具有数个推定的跨膜螺旋(5-7个)。有利地,所述Δ5延伸酶仅转化在Δ5位置上具有一个双键的C20脂肪酸,其中优选转化ω3-C20脂肪酸(EPA)。在本发明的一个优选实施方案中,上述Δ5延伸酶具有如此特性即除了Δ5延伸酶活性外,这类酶还有利地没有或仅有较小的Δ6延伸酶活性。相反,人类的延伸酶或来自非人类动物的延伸酶对具有Δ6或Δ5双键的脂肪酸表现了大致相同的活性。将这类有利的延伸酶称作公知的单功能延伸酶。相反,将人类的延伸酶或来自非人类动物的延伸酶称作多功能延伸酶,所述多功能延伸酶除了以上提到的底物之外,还可使例如具有Δ9或Δ11双键的单不饱和C16和C18脂肪酸转化。在酵母喂饲实验中,其中已向酵母中喂饲EPA作为底物,单功能延伸酶有利地使所喂饲的EPA至少15%重量,有利地至少20%重量,尤其有利地至少25%重量转化为二十二碳五烯酸(DPA,C22:5Δ7,10,13,16,19)。若喂饲作为底物的γ-亚麻酸(GLA,C18:3Δ6,9,12),γ-亚麻酸有利地根本未得到延伸。同样C18:3Δ5,9,12也未得到延伸。在另一个有利的实施方案中,单功能延伸酶将所喂饲GLA以小于60%重量、有利地小于55%重量、尤其优选地小于50%重量,尤其有利地小于45%重量、非常尤其有利地小于40%重量转化为二高γ亚麻酸(C20:3Δ8,11,14)。根据本发明在又一个非常尤其优选的Δ5延伸酶活性实施方案中,GLA未得到转化。
图27和28显示了不同延伸酶经测定的底物特异性、图27显示了非洲爪蛙(Xenopus laevis)(图27A)、玻璃海鞘(Ciona intestinalis)(图27B)和虹鳟(Oncorhynchus mykiss)(图27C)的多功能延伸酶的特异性。所有这些延伸酶均转化了广泛类型的底物。在本发明的方法中,这可能产生了必须由其它酶活性来转化的副产物。这就是为什么这些酶在本发明的方法中较次优选的原因。优选的单功能延伸酶和它们的底物特异性在图28中显示。图28A显示了Ostreococcus tauri Δ5延伸酶特异性。这种酶仅转化在Δ5位置上具有双键的脂肪酸。有利地仅使C20-脂肪酸转化。假矮海链藻Δ5延伸酶显示了相似的高底物特异性(图28C)。OstreococcustauriΔ6延伸酶(图28B)和假矮海链藻Δ6延伸酶(图28D)均有利地仅转化在Δ6位置上具有双键的脂肪酸。有利地仅使C18脂肪酸转化。来自拟南芥菜和细小裸藻的Δ5延伸酶也根据它们的特异性进行了区分。
本发明有利的Δ6延伸酶,也即优选地延伸C18脂肪酸的延伸酶同样根据高特异性进行区分。这类Δ6延伸酶有利地转化在Δ6-位置上具有双键的脂肪酸。尤其有利的Δ6延伸酶有利地转化在分子中具有三个或四个双键的C18脂肪酸,其中所述的脂肪酸必须在Δ6位置上包含一个双键。在本发明的优选实施方案中,这些Δ6延伸酶还具有如下特征:即除了Δ6延伸酶活性外,这些Δ6延伸酶还有利地没有或仅有相对较小的Δ5延伸酶活性。相反,人类的延伸酶或来自非人类动物的延伸酶对具有Δ6或Δ5双键的脂肪酸表现了大致相同的活性。将所述有利的延伸酶称作公知的单功能延伸酶。相反,如以上描述,将人类延伸酶或来自非人类动物的延伸酶称作多功能延伸酶,其中这类酶除了以上提到的底物以外,还可使具有例如Δ9或Δ11双键的单不饱和的C16和C18脂肪酸转化。在酵母喂饲实验中,其中已经向酵母中喂饲EPA作为底物,所述单功能延伸酶有利地转化了所喂饲的α-亚麻酸(ALA,C18:3Δ9,12,15)至少10%重量或转化了所喂饲的γ-亚麻酸(GLA,C18:3Δ6,9,12)至少40%重量,有利地至少20%重量或50%重量,尤其有利地至少25%重量或60%重量。C18:4Δ6,9,12,15(硬脂艾杜糖酸)也尤其有利地得到延伸。在本文中,SDA至少40%重量,有利地至少50%重量,尤其有利地至少60%重量、非常尤其有利地至少70%重量得到转化。尤其有利的Δ6延伸酶对如下底物:C18:1Δ6、C18:1Δ9、C18:1Δ11、C20:2Δ11,14、C20:3Δ11,14,17、C20:3Δ8,11,14、C20:4Δ5,8,11,14、C20:5Δ5,8,11,14,17或C22:4Δ7,10,13,16表现出没有活性或仅表现出极小的活性(转化率小于0.1%重量)
图29和30及表18列出了多种延伸酶经测量的底物特异性。
与已知的ω3去饱和酶相反,本发明的ω3去饱和酶具有有利的特征即它能够使广泛类型的ω6脂肪酸去饱和;可优选地使C20和C22脂肪酸如C20:2、C20:3、C20:4、C22:4或C22:5脂肪酸去饱和。然而也能有利地使更短的C18脂肪酸如C18:2或C18:3脂肪酸去饱和。根据ω3去饱和酶的这些特点,将在生物中有利地将在植物或真菌中的脂肪酸类型由ω6脂肪酸转化至ω3脂肪酸有利地成为可能。本发明的ω3去饱和酶优选地使C20脂肪酸去饱和。这些来自当前脂肪酸库中的脂肪酸至少10%、15%、20%、25%或30%于生物内转化为对应的ω3脂肪酸。ω3去饱和酶对C18-脂肪酸的活性小10倍,即仅仅约1.5至3%的存在于脂肪酸库中的C18-脂肪酸转化为相应的ω3脂肪酸。本发明ω3去饱和酶的优选底物是结合于磷脂中的ω6脂肪酸。以去饱和二高γ-亚麻酸[C20:4Δ8,11,14]为参考,图19清晰显示在去饱和期间,ω3去饱和酶有利地不区别在sn1位置结合或在sn2位置结合的脂肪酸。在磷脂sn1位置结合的脂肪酸和sn2位置结合的脂肪酸均得以去饱和。而且ω3去饱和酶对广泛类型的磷脂如磷脂酰胆碱(PC)、磷脂酰肌醇(PIS)或磷脂酰乙醇胺(PE)的转化是有利的。最后,去饱和产物也可在中性脂类(NL)中,也即在甘油三酯中找到。
与已知的Δ4去饱和酶、Δ5去饱和酶和Δ6去饱和酶比较,本发明的Δ4去饱和酶、Δ5去饱和酶和Δ6去饱和酶的优点是它们能够转化结合至磷脂或辅酶A脂肪酸酯的脂肪酸,有利地转化结合至辅酶A脂肪酸酯的脂肪酸。
本发明方法中所用的Δ12去饱和酶有利地将油酸(C18:1Δ9)转化为亚油酸(C18:2Δ9,12)或有利地将C18:2Δ6,9转化为C18:3Δ6,9,12(GLA)。所用Δ12去饱和酶有利地转化结合至磷脂或辅酶A脂肪酸酯的脂肪酸,有利地转化结合至辅酶A脂肪酸酯的那些脂肪酸。
由于本发明方法中所用的核酸的酶活性。其中所述核酸编码具有Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶活性的多肽,有利地与编码脂肪酸或脂类代谢的多肽如具有Δ4、Δ5、Δ6、Δ8、Δ12去饱和酶或者Δ5、Δ6或Δ9延伸酶活性的其它多肽的核酸序列联合使用,种类广泛的多不饱和脂肪酸可在本发明的方法中产生。根据所选择的生物如本发明方法中所用的有利的植物,多种的多不饱和脂肪酸混合物或单种的多不饱和脂肪酸如EPA或ARA可以以游离或结合形式产生。根据起始植物中优势的脂肪酸组成(C18:2或C18:3脂肪酸),由C18:2脂肪酸衍生的脂肪酸如GLA、DGLA或ARA;或由C18:3脂肪酸衍生的脂肪酸如SDA、ETA或EPA因此获得。若仅仅亚油酸(LA,C18:2Δ9,12)作为不饱和脂肪酸在方法所用的植物中出现,方法仅能提供作为产物的GLA、DGLA和ARA,所有产物均能以游离的脂肪酸或以结合的形式出现。若仅仅α-亚麻酸(ALA,C18:3Δ9,12,15)作为不饱和脂肪酸在方法所用的植物中如在亚麻籽中存在,方法仅能提供作为产物的SDA、ETA或EPA和/或DHA,如上所述的所有产物均能以游离脂肪酸或以结合形式存在。由于对Δ5延伸酶酶活性的修饰,其有利地与在合成中起作用的Δ4、Δ5、Δ6、Δ12去饱和酶和/或Δ6延伸酶、或Δ4、Δ5、Δ8、Δ12去饱和酶和/或Δ9延伸酶联合使用,可以在以上提及的生物,有利地在以上提及的植物中以定向的方式只产生单个的产物。由于Δ6去饱和酶和Δ6延伸酶的活性,依据起始的植物和不饱和脂肪酸,形成了例如GLA和DGLA或SDA和ETA。DGLA或ETA或这些脂肪酸的混合物优选地形成。若向生物,有利地向植物中额外地导入Δ5去饱和酶、Δ5延伸酶和Δ4去饱和酶,ARA、EPA和/或DHA额外地形成。这也适用于事先已导入Δ8去饱和酶和Δ9延伸酶的生物。根据在生物或在植物中作为合成起始物质而存在的脂肪酸,仅ARA、EPA或DHA或这些脂肪酸的混合物有利地得到合成。因为涉及生物合成的级联,所讨论的终产物未能在生物中以纯的形态出现。小量的前体化合物总是额外地在终产物中存在。基于终产物DGLA、ETA或其混合物,或者ARA、EPA、DHA或其混合物,有利地以EPA或DHA或其混合物,这些前体化合物小量占小于20%重量、有利地占小于15%重量、尤其有利地占小于10%重量、最有利地占小于5、4、3、2或1%重量。
本发明的核酸所编码的蛋白质对用于DHA合成的两种前体C18:4Δ6,9,12,15和C20:5Δ5,8,11,14,17脂肪酸表现了高度的特异性(前体和DHA的合成见图1)。因此SEQ NO:53编码的蛋白质对带有一个额外ω3双键的Δ6和Δ5脂肪酸表现了特异性(图2)。Δ5延伸酶具备有利地使酰基辅酶A酯的脂肪酸残基延伸2个碳原子的酮酰基辅酶A合成酶活性。
在Δ5延伸酶基因、褐指藻Δ5去饱和酶和裸藻Δ4去饱和酶的辅助下,DHA的合成有可能在酵母(酿酒酵母)中展示(图3)。
用于本发明的Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶的起始脂肪酸除了直接在生物中产生以外,所述脂肪酸还能自外部添加。因为经济原因在生物中生产为优选。ω3去饱和酶的优选底物是亚油酸(C18:2Δ9,12)、γ-亚麻酸(C18:3Δ6,9,12)、二十碳二烯酸(C20:2Δ11,14)、二高γ-亚麻酸(C20:3Δ8,11,14)、花生四烯酸(C20:4Δ5,8,11,14)、二十二碳四烯酸(C22:4Δ7,10,13,16)和二十二碳五烯酸(C22:5Δ4,7,10,13,15)。
为了在上述用于产生多不饱和脂肪酸含量有利地得到升高的油和/或甘油三酯的方法中提高产量,增加合成脂肪酸的起始产物的量是有利的;这可以例如通过向生物中导入编码Δ12去饱和酶多肽的核酸实现。在产油生物如那些来自十字花科如芸苔属,例如油菜籽;胡颓子科如胡颓子属,例如油橄榄的属和种或豆科如大豆属,例如具有较高油酸的大豆的属和种中这种方法尤其有利。因为这些生物仅有较低的亚油酸(Mikoklajczak等,Journal of the American oils Chemical Society,38,1961,678-681),以上提到的Δ12去饱和酶用于产生起始物质亚油酸是有利的。
本发明方法中所用的核酸有利地来自植物如藻类,例如草绿藻科的藻类如异毛藻属(Heteromastix)、Mammella、Mantoniella、微单藻属(Micromonas)、肾藻属、Ostreococcus、绿枝藻属(Prasinocladus)、Prasinococcus、Pseudoscourfielda、Pycnococcus、塔胞藻属(Pyramimonas)、Scherffelia、扁藻属例如Heteromastix longifillis、Mamiella gilva、Mantoniella squamata、Micromonas pusilla、绿肾藻(Nephroselmis olivacea)、Nephroselmis pyriformis、圆形肾藻(Nephroselmis rotunda)、Ostreococcus tauri、Ostreococcus sp.、Prasinocladus ascus、Prasinocladus lubricus、Pycnococcus provasolii、Pyramimonas amylifera、Pyramimonas disomata、Pyramimonasobovata、Pyramimonas orientalis、Pyramimonas parkeae、Pyramimonas spinifera、Pyramimonas sp.、Tetraselmis apiculata、Tetraselmis carteriaformis、周氏扁藻(Tetraselmis chui)、皱叶扁藻(Tetraselmis convolutae)、Tetraselmis desikacharyi、纤细扁藻(Tetraselmis gracilis)、Tetraselmis hazeni、Tetraselmis impellucida、Tetraselmis inconspicua、Tetraselmis levis、有斑扁藻(Tetraselmismaculata)、Tetraselmis marina、Tetraselmis striata、Tetraselmissubcordiformis、Tetraselmis suecica、Tetraselmis tetrabrachia、Tetraselmis tetrathele、Tetraselmis verrucosa、Tetraselmis verrucosa fo.Rubens或Tetraselmis sp.的属和种或选自裸藻科的藻类如囊胞藻属(Ascoglena)、变胞藻属(Astasia)、胶柄藻属、Cyclidiopsis、裸藻属、拟裸藻属(Euglenopsis)、Hyalophacus、克氏藻属(Khawkinea)、鳞孔藻属、扁裸藻属、陀螺藻属(Strombomonas)或囊裸藻属例如梭形裸藻(Euglena acus)、膝曲裸藻(Euglena geniculata)、细小裸藻、Euglenamixocylindracea、喙状裸藻(Euglena rostrifera)、绿色裸藻(Euglenaviridis)、Colacium stentorium、圆柱囊裸藻(Trachelomonas cylindrica)或旋转囊裸藻(Trachelomonas volvocina)的属和种。所用核酸有利地衍生自裸藻属、Mantoniella或Ostreococcus属的藻类。
更有利的植物是藻类如等鞭金藻属(Isochrysis)或隐甲藻属、藻类/硅藻如海链藻属或褐指藻属、藓类如剑叶藓属或角齿藓属、或者高等植物如报春花科(Primulaceae)例如Aleuritia、Calendula stellata、刺状骨籽菊(Osteospermum spinescens)或Osteospermum hyoseroides、微生物如真菌例如曲霉属(Aspergillus)、破囊壶菌属、疫霉属、虫霉属、毛霉属或被孢霉属,细菌如希瓦氏菌,酵母或动物如线虫例如隐杆线虫、昆虫、蛙、鲍鱼或鱼。本发明经分离的核酸序列有利地源自脊椎动物目的动物。所述核酸序列优选地源自脊椎动物纲;Euteleostomi,辐鳍鱼纲(Actinopterygii);新鳍鱼亚纲(Neopterygii);硬骨鱼类(Teleostei);Euteleostei、原鳍鱼总目(Protacanthopterygii)、鲑形目(Salmoniformes);鲑科(Salmonidae)或大马哈鱼属(Oncorhynchus)或脊椎动物(Vertebrata)、两栖纲(Amphibia)、无尾两栖目(Anura)、负子蟾科(Pipidae)、非洲爪蟾属或无脊椎动物(Evertebrata)例如原索动物门(Protochordata)、被囊亚门(Tunicata)、海参纲(Holothuroidea)、海鞘科(Cionidae)例如Amaroucium constellatum、Botryllus schlosseri、玻璃海鞘、Molgula citrina、Molgula manhattensis、Perophora viridis或Styela partita。所述核酸尤其有利地源自真菌、动物、或源自植物如藻类或藓类,优选地源自鲑形目如鲑科如鲑属例如来自虹鳟、Trutta trutta或亚东鲑(Salmo trutta fario)的属和种,源自藻类如Mantoniella或Ostreococcus,或源自硅藻如海链藻属或褐指藻属或者源自藻类如隐甲藻属。
本发明的方法有利地包含以上提及的核酸序列或它们的衍生物或同系物,其编码这样的多肽,所述多肽保留了由以上提及的核酸序列所编码的蛋白质的酶活性。这些序列单独地或与编码Δ12去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶的核酸序列组合而克隆于表达构建体内并用于向生物中导入和在生物中表达。由于它们的构建,这些表达构建体使得可有利地优化合成本发明方法中所产生的多不饱和脂肪酸。
在一个优选的实施方案中,所述方法进一步包括获得细胞或完整生物的步骤,其中所述的细胞或完整生物包含方法中所用的核酸序列,这些细胞和/或生物在所述的方法中由本发明编码Δ12去饱和酶、Δ4去饱和酶,Δ5去饱和酶、Δ6去饱和酶、Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶的核酸序列、如上所述的基因构建体或载体,单独地或与编码脂肪酸或脂类代谢的蛋白质的核酸序列组合而转化。在一个更优选的实施方案中,所述方法进一步包括从生物或培养物中获得油、脂类或游离脂肪酸的步骤。培养物可采用例如发酵培养物的形式例如在培养微生物如被孢霉、海链藻、Mantoniella、Ostreococcus、酵母或破囊壶菌的情况下或采用植物的温室培养物或田间生长培养物形式。由此所产生的细胞或微生物有利地是产油生物的细胞如油料作物例如花生、油菜籽、卡诺拉油菜(Canola)、亚麻籽、大麻、花生、大豆、红花、大麻、向日葵或琉璃苣的细胞。
在为植物细胞、植物组织或植物器官的情况下,“生长”理解为意指例如在营养培养基内或培养基上培养、或完整植物在基底物上或基底物内的培养例如在水培、盆载培养料内或在可耕地上的培养。
为了本发明目的,“转基因的”或“重组的”意指例如核酸序列、表达盒(基因构建体)或包含核酸序列的载体或由本发明的核酸序列、表达盒或载体所转化的生物,所有通过重组方法产生的构建体中的
a)本发明的核酸序列,或
b)有效地连接于本发明核酸序列的遗传控制序列,例如启动子,或
c)a)和b)
均不处于它们天然的遗传环境中或均已经通过重组方法修饰,对这种修饰而言可采取例如一个或多个核苷酸残基的替代、添加、删除、倒位或插入的形式。将天然遗传环境理解为意指原始生物中的天然基因组位点或染色体位点或在基因组文库中的存在。在为基因组文库的情况下,优选地保留,至少部分保留所述核酸序列的天然遗传环境。所述环境位于所述核酸序列的至少一侧并且具有至少50bp、优选地至少500bp、尤其优选地至少1000bp、最为优选地至少5000bp的序列长度。天然存在的表达盒-例如所述核酸序列的天然启动子与相应的Δ12去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6延伸酶和/或Δ5延伸酶基因的天然存在的组合-当经过非天然、合成的(“人工的”)方法例如通过诱变处理修饰后变成了转基因的表达盒。合适的方法在美国5.565.350或WO001/15815中进行了描述。
用于本发明目的的转基因生物或转基因植物因此理解为意指上述方法中所用的核酸不处于它们在生物的基因组中的天然基因座处,对这类核酸而言可同源或异源性地表达。然而如所提及,转基因也意指即便本发明的核酸处于生物基因组中它们的天然位置上,这种序列相对于天然序列已经过了修饰,和/或天然序列的调节序列已经过了修饰。转基因优选地理解为意指本发明的核酸在基因组内非天然基因座处的表达,即发生了这种核酸的同源表达,或优选地异源表达。优选的转基因生物是真菌如被孢霉或疫霉、藓类如剑叶藓、藻类如Mantoniella、裸藻、隐甲藻或Ostreococcus、硅藻如海链藻或褐指藻或者植物如油料作物。
生物或宿主生物对于本发明方法中所用的核酸、表达盒或载体而言原则上是能够有利地合成脂肪酸尤其不饱和脂肪酸和/或适用于表达重组基因的所有生物。可提到例子是植物如拟南芥、菊科植物例如金盏花或作物植物如大豆、花生、蓖麻油植物、向日葵、玉米、棉、亚麻、油菜籽、椰子、油棕榈、红花(Carthamus tinctorius)或可可豆;微生物如真菌例如被孢霉属、破囊壶菌属、水霉属、疫霉属或腐霉属;细菌如埃希氏菌属或希瓦氏菌属;酵母如酵母属、蓝细菌属、纤毛虫属,藻类如Mantoniella、裸藻属、海链藻属如Ostreococcus;或原生动物如甲藻例如隐甲藻。优选的生物是那些能够天然合成大量油的生物如真菌如高山被孢霉(Mortierella alpina)、Pythium insidiosum、致病疫霉或植物如大豆、油菜籽、椰子、油棕榈、红花、亚麻、大麻、金盏花、蓖麻油植物、花生、可可豆或向日葵,或者酵母如酿酒酵母,并且尤其优选大豆、亚麻、油菜籽、红花、向日葵、金盏花、被孢霉或酿酒酵母。除了以上提到的转基因生物以外,宿主生物原则上还可以是转基因动物,有利地为非人类的动物例如秀丽线虫、玻璃海鞘或非洲爪蟾。
可利用的宿主细胞进一步在Goeddel,Gene Expression Technology:Methods in Enzymology 185,Academic Press,San Diego,CA(1990)中详述。
可使用的表达株系如那些具有较低蛋白酶活性的株系在Gottesman,S.,Gene Expression Technology:Methods in Enzymology 185,Academic Press,San Diego,California(1990)119-128中描述。
这些株系包括植物细胞和某些组织、器官和植物的所有表型形式中的部分如花药、纤维、根毛、茎、胚、愈伤组织、cotelydon、叶柄、所收获的材料、植物组织、可繁殖的组织和来自实际的转基因植物的和/或可用于产生转基因植物的细胞培养物。
包含本发明方法中所合成的多不饱和脂肪酸的转基因植物能够有利地直接销售而无需分离所合成的油、脂类或脂肪酸。本发明方法的植物罗列如下:完整植物和植物的所有部分、植物器官或植物部分如叶、茎、种子、根、块茎、花药、纤维、根毛、茎、胚、愈伤组织、cotelydons、叶柄、所收获的材料、植物组织、可繁殖的组织和来自实际转基因植物的细胞培养物和/或可用于产生转基因植物的细胞培养物。在本上下文中,种子包含种子的所有部分如种皮、表皮细胞、种子细胞、胚乳或胚组织。本发明方法中所产生的化合物也可以油、脂肪、脂类和/或游离脂肪酸的形式从生物,有利地从植物中分离。由所述方法产生的多不饱和脂肪酸可通过从所生长的作物中或从田野中收获这类生物获得。这可通过对植物部分优选对植物种子的压榨或提取实现。在本上下文中,油、脂肪、脂类和/或游离脂肪酸通过公知的冷敲打(cold-beating)或冷榨可获得而无需加热。为了更容易地破裂植物部分尤其种子,它们事先经过粉碎、蒸热或烘烤。随后经如此方式预处理的种子可用溶剂如温己烷压榨或提取。然后去除溶剂。在为微生物时,微生物在收获后例如可直接提取而无需进一步的加工步骤或在破裂后经技术人员所熟悉的多种方法提取。按照这种方式,可分离所述方法产生的超过96%的化合物。此后所得到的产物经过进一步加工即精制。在该方法中,例如植物黏液和悬浮物首先去除。众知的所谓除去矿泥可通过酶或例如通过加入酸如磷酸而化学-物理性地完成。此后游离脂肪酸用碱例如氢氧化钠溶液处理去除。得到的产物用水彻底洗涤以去除产物中残留的碱,然后干燥。为了除去产物中残留的色素,利用滤土或活性碳漂白产物。最后例如利用蒸汽使产物脱嗅。
通过该方法所产生的PUFA或LCPUFA有利地是C18、C20或C22脂肪酸分子,有利地是C20或C22脂肪酸分子,这些脂肪酸分子具有至少两个双键,优选地具有三、四、五或六个双键。这些C18、C20或C22脂肪酸分子可以以油、脂类或游离脂肪酸的形式从生物中分离。合适的生物是例如以上提到的那些生物。优选的生物是转基因植物。
因此本发明的一个实施方案是通过以上描述的方法产生的油、脂类或脂肪酸或它们的级分,尤其优选含有PUFA并且来自转基因植物的油、脂类或脂肪酸组合物。
如以上所描述,在每一情况下,以100%为基础并且以生物的总脂肪酸含量为基础,这些油、脂类或脂肪酸有利地包含6至15%的棕榈酸、1至6%的硬脂酸、7-85%的油酸、0.5至8%的11-十八碳烯酸、0.1至1%的花生酸、7至25%的饱和脂肪酸、8至85%的单不饱和脂肪酸和60至85%的多不饱和脂肪酸。以总脂肪酸含量为基础,这些脂肪酸酯或脂肪酸混合物中存在的有利的多不饱和脂肪酸优选地为至少0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9或1%的花生四烯酸。而且通过本发明方法产生的脂肪酸酯或脂肪酸混合物有利地包含选自如下的脂肪酸:芥酸(二十二碳烯-13酸)、苹婆酸(9,10-亚甲基十八碳-9-烯酸)、锦葵酸(8,9-亚甲基十七碳-8-烯酸)、大风子油酸(环戊烯十二烷酸)、呋喃脂肪酸(9,12-环氧十八碳-9,11-二烯酸)、斑鸠菊酸(9,10-环氧十八碳-12-烯酸)、果脂酸(6-十八碳炔酸)、6-十九碳炔酸、santalbic acid(反11-十八碳烯-9-炔酸)、6,9-十八碳烯炔酸、pyrulic acid(反10-十七烯-8-炔酸)、crepenyninic acid(9-十八烯-12-炔酸)、13,14-二氢oropheic acid、十八碳-13-烯-9,11-二炔酸、伞形花子油酸(顺-6-十八烯酸)、9顺,12反-十八碳二烯酸、金盏花素酸(8反10反12顺-十八碳三烯酸)、梓甙酸(9反11反13顺-十八碳三烯酸)、桐酸(9顺11反13反-十八碳三烯酸)、iacaric acid(8顺10反12顺-十八碳三烯酸),石榴酸(9顺11反13顺-十八碳三烯酸)、麦饼树酸(9顺11反13反15顺-十八碳四烯酸)、皮诺敛酸(全-顺-5,9,12-十八碳三烯酸)、laballenic acid(5,6-十八碳二烯丙二烯酸)、蓖麻油酸(12-羟油酸)和/或革盖菌素酸(13-羟-9顺,11反-十八碳二烯酸)。通常上面提到的脂肪酸有利地仅在本发明方法产生的脂肪酸酯或脂肪酸的混合物中痕量地存在,也就是说以总脂肪酸为基础,它们的含量小于30%,优选地小于25%、24%、23%、22%或21%,尤其优选地小于20%、15%、10%、9%、8%、7%、6%或5%、非常尤其优选地小于4%、3%、2%或1%。在本发明更优选的实施方案中,以总脂肪酸为基础,以上这些提到的脂肪酸的含量小于0-9%、0.8%、0.7%、0.6%或0.5%,尤其优选地小于小于0.4%、0.3%、0.2%、0.1%。以总脂肪酸为基础,由本发明方法所产生的脂肪酸酯或脂肪酸混合物有利地包含小于0.1%和/或无丁酸、无胆固醇、无鰶鱼酸(二十二碳五烯酸,C22:5Δ4,8,12,15,21)以及无尼生酸(二十四碳六烯酸,C23:6Δ3,8,12,15,18,21)。
以生产生物,有利地是植物,尤其优选地是油料作物植物如大豆、油菜籽、椰子、油棕榈、红花、亚麻、大麻、蓖麻、金盏花、花生、可可豆、向日葵或以上提到的其它单子叶或双子叶油料作物植物的总脂肪酸含量为基础,本发明的油、脂类或脂肪酸包含至少0.5%、1%、2%、3%、4%或5%,有利地至少6%、7%、8%、9%或10%,尤其有利地至少11%、12%、13%、14%或15%的ARA或至少0.5%、1%、2%、3%、4%或5%,有利地至少6%或7%,尤其有利地至少8%、9%或10%的EPA和/或DHA。
本发明的又一个实施方案是油、脂、脂肪酸和/或脂肪酸组合物在饲料、食品、化妆品或药品中的用途。本发明的油、脂、脂肪酸或脂肪酸混合物可按照技术人员熟悉的方式与动物来源的其它油、脂类、脂肪酸或脂肪酸混合物例如鱼油混合使用。这些由植物或动物组分所组成的油、脂类、脂肪酸或脂肪酸混合物也可用于制备饲料、食品、化妆品或药品。
术语“油”、“脂类”或“脂肪”理解为意指包含不饱和的、饱和的,优选地已酯化的脂肪酸的脂肪酸混合物。油、脂类或脂肪优选地具有较高多不饱和的游离脂肪酸或有利地具有较高酯化的脂肪酸,特别是亚油酸、γ-亚麻酸、二高γ-亚麻酸、花生四烯酸、α-亚麻酸、硬脂艾杜糖酸、二十二碳四烯酸、二十碳五烯酸、二十二碳五烯酸或二十二碳六烯酸。
酯化的不饱和脂肪酸的量优选地占约30%含量,50%的含量更为优选,60%、70%、80%或更高的含量尤其优选。为了分析,脂肪酸的含量例如可在脂肪酸经酯转换转化为甲酯后通过气相层析测定。油、脂类或脂肪可包含多种其它饱和或不饱和的脂肪酸例如金盏花素酸、棕榈酸、棕榈油酸、硬脂酸、油酸等。在油或脂肪中的多种脂肪酸含量可变化,这尤其取决于起始生物。
如上所述,在方法中所产生的有利地具有至少两个双键的多不饱和脂肪酸如上所述是例如鞘脂类、磷酸甘油酯、脂类、糖脂、磷脂、单酰甘油酯、二酰甘油酯、三酰甘油酯或其它脂肪酸酯。
从通过本发明方法制备的有利地具有至少五个或六个双键的多不饱和脂肪酸开始,所存在的多不饱和脂肪酸经碱例如KOH或NaOH水溶液处理,或者有利地于存在醇例如甲醇或乙醇时经酸水解,或者经酶切割释放并且通过例如相分离及随后例如H2SO4的酸化作用而分离。这类脂肪酸也可无需以上描述的加工步骤直接释放。
方法中所用的核酸在导入生物体,有利地导入植物细胞或植物后,可以在单独的质粒中存在或者有利地可以整合入宿主细胞的基因组内。在整合入基因组内的情况下,整合可以是随机的或可通过重组实现,从而所导入的拷贝替代了原来的基因,由此细胞产生的目的化合物是被调节的,或整合可通过运用基因反式而实现,从而这种基因有效地与功能性表达单元连接,其中这个表达单元包含至少一段确保基因表达的序列和至少一段确保功能性转录的基因多聚腺苷酸化的序列。这类核酸通过多重表达盒或构建体有利地向生物中导入以便多重平行表达,有利地向植物中导入以便基因的多重平行种子特异性的表达。
藓类和藻类是唯一已知的产生大量多不饱和脂肪酸如花生四烯酸(ARA)和/或二十碳五烯酸(EPA)和/或二十二碳六烯酸(DHA)的植物系统。藓类在膜脂中包含PUFA;而藻类、与藻类有关的生物及少数真菌也在三酰甘油酯的级分中聚集了大量的PUFA。这是为什么从三酰甘油酯级分中聚集PUFA的株系所分离的核酸分子尤其有利地用于本发明方法并且因此用于修饰宿主中脂类和PUFA产生系统的原因,特别是修饰植物中如油料作物例如油菜籽、卡诺拉油菜、亚麻籽、大麻、大豆、向日葵和琉璃苣中的脂类和PUFA产生系统。因此这类核酸分子可有利地用于本发明的方法。
有利地适于本发明方法中所用的核酸的底物,其中所述核酸编码Δ12去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9延伸酶、Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶活性和/或适于所用的其它核酸的底物有利地是C16、C18、或C20脂肪酸,其中所述其它的核酸是编码选自酰基-辅酶A脱氢酶、酰基ACP[酰基载体蛋白]去饱和酶、酰基ACP硫酯酶、脂肪酸酰基转移酶、酰基-辅酶A:溶血磷脂酰基转移酶(酶)、脂肪酸合成酶、脂肪酸羟化酶、乙酰基辅酶A羧化酶、酰基辅酶A氧化酶、脂肪酸去饱和酶、脂肪酸炔属酶、脂加氧酶、三酰甘油酯脂肪酶、环丙烯合酶、氢过氧化物裂合酶或脂肪酸延伸酶的核酸。方法中作为底物转化的脂肪酸优选地以它们酰基辅酶A酯形式和/或它们的磷脂形式的酯转化的脂肪酸或脂代谢中的多肽。
为产生本发明的长链PUFA,多不饱和C18脂肪酸首先必须通过去饱和酶的酶活性去饱和并且接下来通过延伸酶延伸至少两个碳原子。在一次延伸循环后,延伸酶的酶活性产生了C20脂肪酸并且经过两轮延伸循环后,产生了C22脂肪酸。本发明方法中所用的去饱和酶和延伸酶的活性优选地产生C18、C20和/或C22脂肪酸,在脂肪酸分子中有利地具有至少两个双键,优选地具有三、四、五或六个双键,尤其优选地产生C20和/或C22脂肪酸,在脂肪酸分子中具有至少两个双键,优选地具有三、四、五或六个双键,非常尤其优选地在分子中具有五或六个双键。在第一次去饱和和延伸发生后,可发生进一步的去饱和和延伸步骤例如在Δ5和Δ4位置处的去饱和。尤其优选的本发明方法的产物是二高γ-亚麻酸、花生四烯酸、二十碳五烯酸、二十二碳五烯酸和/或二十二碳六烯酸。在脂肪酸分子中具有至少两个双键的C20脂肪酸可以以游离脂肪酸的形式或以酯的形式如磷脂类、糖脂类、鞘脂类、磷酸甘油酯、单酰甘油酯、二酰甘油酯或三酰甘油酯通过本发明的酶活性加以延伸。
脂肪酸、油、脂类或脂肪在有利使用的植物中优选的生物合成部位例如通常是种子或种子的细胞层,因此对方法中所用的核酸进行种子特异性表达是有意义的。然而显然脂肪酸、油或脂类的生物合成并非限定于种子组织中,同时也可在植物的所有其它部分例如在表皮细胞或在块茎中以组织特异性方式进行。
若微生物如酵母例如酵母属或裂殖酵母属、真菌如被孢霉属、曲霉属、疫霉属、虫霉属、毛霉属或破囊壶菌属、藻类如等鞭金藻属、Mantoniella、裸藻属、Ostreococcus、褐指藻属或隐甲藻属作为本发明方法中的生物使用,这些生物有利地在发酵培养物内生长。
由于本发明编码Δ5延伸酶的核酸的利用,与未重组的不含有这种核酸的生物的野生型比较,方法中产生的多不饱和脂肪酸可增加至少5%,优选地至少10%,尤其优选地至少20%,非常尤其优选地至少50%。
原则上,可以两种不同方式增加在方法所用的生物中由本发明方法产生的多不饱和脂肪酸。游离的多不饱和脂肪酸库和/或由方法产生的酯化多不饱和脂肪酸含量可有利地扩大。转基因生物中酯化的多不饱和脂肪酸库通过本发明方法有利地扩大。
若微生物作为本发明方法中的生物使用,根据宿主生物,将它们按技术人员熟悉的方式生长或培养。微生物一般在液体培养基中生长,其中所述培养基通常含有糖形式的碳源、有机氮源形式的氮源如酵母提取物或盐如硫酸铵、微量元素如铁、锰和镁盐和根据需要含有维生素,该培养基的温度在0℃和100℃之间,优选地在10℃和60℃之间,同时通氧。液体培养基的pH可保持恒定即在培养期间受到调节,或不保持恒定。培养物可以分批式、半分批式或连续地生长。营养物可在发酵开始时提供或半连续或连续性地输入。所产生的多不饱和脂肪酸可从如上描述的生物中通过技术人员所知的方法例如提取、蒸馏、结晶、根据需要以盐沉淀和/或层析分离。为此目的,可有利地事先破裂生物体。
若宿主生物为微生物,本发明的方法有利地在0℃和95℃之间,优选地在10℃和85℃间,尤其优选地在15℃和75℃间,非常尤其优选地在15℃和45℃间实施。
将本发明方法中pH值优选地维持在pH 4和12之间、优选地维持在pH 6和9之间、尤其优选地维持在pH 7和8之间。
本发明的方法可分批式、半分批式或连续地操作。对已知培养方法的综述可在Chmiel的教材(Bioprozeβtechnik 1.Einführung in dieBioverfahrenstechnik[Bioprocess technology 1.Introduction to Bioprocesstechnology](Gustav Fischer Verlag,Stuttgart,1991))中或在Storhas的教材(Bioreaktoren und periphere Einrichtungen[Bioreactors andperipheral equipment](Vieweg Verlag,Braunschweig/Wiesbaden,1994))中找到。
预备使用的培养基必须符合所讨论的株系的需要。用于各种微生物的培养基的描述可在教材″Manual of Methods für Genaeral Bacteriology″ofthe American Society for Bacteriology(Washington D.C.,USA,1981)中找到。
如以上描述,根据本发明的可使用的这些培养基通常包含一种或多种碳源、氮源、无机盐、维生素和/或微量元素。
优选的碳源是糖如单糖、双糖或多糖。极好碳源的例子是葡萄糖、果糖、甘露糖、半乳糖、核糖、山梨糖、核酮糖、乳糖、麦芽糖、蔗糖、棉子糖、淀粉或纤维素。糖还可以复杂的化合物如糖蜜或其它来自糖蜜三糖化(raffination)的副产物加入培养基。添加多种碳源的混合物也是有利的。其它可能的碳源是油和脂肪例如大豆油、向日葵油、花生油和/或可可脂、脂肪酸如棕榈酸、硬脂酸和/或亚油酸、醇和/或多元醇例如甘油、甲醇和/或乙醇和/或有机酸例如乙酸和/或乳酸。
氮源通常是有机或无机氮化合物或含有这些化合物的物质。氮源的例子包括液体或气体形式的氨或者铵盐如硫酸铵、氯化铵、磷酸铵、碳酸铵或硝酸铵、硝酸盐、尿素、氨基酸或者复杂氮源例如玉米浆、大豆粕、大豆蛋白质、酵母提取物、肉膏和其它等。氮源可单独地或作为混合物使用。
可在培养基中存在的无机盐化合物包括钙、镁、钠、钴、钼、钾、锰、锌、铜和铁的氯化物、磷酸盐和硫酸盐。
含无机硫的化合物例如硫酸盐、亚硫酸盐、连二亚硫酸盐、连四硫酸盐、硫代硫酸盐、硫化物或有机硫化合物如硫醇和硫醇类可以用作硫源以产生含硫的精细化学品,特别是甲硫氨酸。
磷酸、磷酸二氢钾或磷酸氢二钾或相应的钠盐可用作磷源。
鳌合剂可加入培养基以维持溶液中的金属离子。尤其合适的鳌合剂包括二羟基酚如邻苯二酚或原儿茶酸盐和有机酸如柠檬酸。
本发明使用的用于培养微生物的发酵培养基通常也含有其它生长因子如维生素或生长促进剂,包括例如生物素、核黄素、维生素B1、叶酸、烟酸、泛酸盐和吡哆醇。生长因子和盐经常来自复杂的培养基成分如酵母提取物、糖蜜、玉米浆等。更有可能向培养基中加入合适的前体。培养基化合物的明确组分很大程度取决于特定的实验并且单独取决于每个特定的情况。优化培养基的信息可在教材″Applied Microbiol.Physiology,APractical Approach″(Editors P.M.Rhodes,P.F.Stanbury,IRL Press(1997)pp.53-73,ISBN 0 19 963577 3)中找到。生长培养基例如标准1(Merck)或BHI(脑心浸液,DIFCO)等也可从商业供应商处获得。
所有培养基的成分均经加热(1.5巴,121℃20分钟)或过滤灭菌法灭菌。各成分可共同或根据需要分别灭菌。培养基的所有成分可在培养开始时存在或根据需要连续或分批式加入。
培养温度通常在15℃和45℃之间,优选地从25℃至40℃,并且可维持恒定或可在实验期间变动。培养基的pH应该处于5至8.5之间,优选地在7.0附近。用于培养的pH在培养期间可通过加入碱性化合物如氢氧化钠、氢氧化钾、氨和液氨或酸性化合物如磷酸或硫酸控制。起泡可使用消泡剂例如脂肪酸聚乙二醇脂控制。为维持质粒的稳定性,可向培养基中加入合适的具有选择效应的物质如抗生素。需氧条件通过向培养基中导入氧或含氧的气体混合物例如环境空气维持。培养基的温度通常为20℃至40℃并且优选地为25℃至40℃。持续培养直至最大限度地形成目的产物。这个目标通常在10至160小时内达到。
以如此方式所获的发酵培养液,尤其那些含有多不饱和脂肪酸的培养液通常含有7.5至25%重量的干物质。
这种发酵培养液随后可经过进一步加工。生物质根据需要可通过分离法例如离心、过滤、倾析或这些方法的组合从发酵培养基中完全或部分地移出或完全滞留在所述的培养基中。生物质有利地在分离后进行加工。
然而,发酵液也可使用已知的方法例如在旋转蒸发器、薄膜蒸发器、降膜蒸发器的辅助下通过反向渗透或通过纳米过滤加以增稠或浓缩而无需分离细胞。最终这种浓缩的发酵液可经加工以获得存在其中的脂肪酸。
所述方法中所获的脂肪酸也适用于化学合成其它目的的产物。例如它们可单独或彼此联合地用于制备药物、食品、动物饲料或化妆品。
本发明还涉及经分离的编码具有Δ5延伸酶的多肽的核酸序列,其中由所述核酸序列编码的Δ5延伸酶转化在脂肪酸分子中具有至少四个双键的C20脂肪酸,这种C20脂肪酸有利地最终掺入二酰甘油酯和/或三酰甘油酯内。
有利的经分离的核酸序列是编码具有Δ5延伸酶活性的多肽并且编码包含选自具有SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141或SEQ ID NO:142中所示氨基酸序列的氨基酸的核酸序列。
进一步有利的经分离的核酸序列是编码具有Δ5延伸酶活性的多肽并且编码包含选自以下氨基酸序列的联合的核酸序列:
a)SEQ ID NO:115和SEQ ID NO:139、SEQ ID NO:115和SEQ IDNO:140或SEQ ID NO:139和SEQ ID NO:140;或
b)SEQ ID NO:116和SEQ ID NO:141、SEQ ID NO:116和SEQ IDNO:142或SEQ ID NO:141和SEQ ID NO:142;或
c)SEQ ID NO:115、SEQ ID NO:139和SEQ ID NO:140或SEQ IDNO:116、SEQ ID NO:141和SEQ ID NO:142。
序列SEQ ID NO:115(NXXXHXXMYXYYX)、SEQ ID NO:116(HHXXXXWAWW)、SEQ ID NO:139(LHXXHH)、SEQ ID NO:140(TXXQXXQF)、SEQ ID NO:141(DTXFMV)和SEQ ID NO:142(TQAQXXQF)中所示的序列组成了多种延伸酶的保守性区域。上述核酸序列中出现的标注为X的氨基酸的含义在表2中显示(列3)。在多个位置处所优选的氨基酸也可以在该表中(列3)找到。列1指示SEQ ID NO,列2指示序列中的位置。
表2 共有序列中标注为X的氨基酸的含义
SEQ ID NO: | 在序列中X的位置 | 氨基酸 | 优选的氨基酸 |
115(NXXXHXXMYXYYX) | 2 | Ser,Cys,Leu,Gly | Cys,Leu |
115 | 3 | Thr,Phe,Ile,Ser,Val,Trp,Gly | Phe,Trp |
115 | 4 | Val,Ile | Val,Ile |
115 | 6 | Val,Ile,Thr | Val,Ile |
115 | 7 | Ile,Phe,Val,Leu,Cys | Cys,Val |
115 | 10 | Ser,Gly,Tyr,Thr,Ala | Thr,Ser |
115 | 13 | Phe,Met,Thr,Leu,Ala,Gly | Leu |
116(HHXXXXWAWW) | 3 | Ala,Ser,Thr | Ala,Ser,尤其优选Ala |
116 | 4 | Thr,Met,Val,Leu,Ile,Ser | Leu,Thr,尤其优选Leu |
116 | 5 | Val,Thr,Met,Leu,Ile | Ile,Ser,尤其优选Ile |
SEQ ID NO: | 在序列中X的位置 | 氨基酸 | 优选的氨基酸 |
116 | 6 | Val,Met,Leu,Ile,Ala,Pro,Ser,Phe | Ile,Ser,尤其优选Ile |
139LHXXHH | 3 | Val,Tyr,Ile | Val,Thr |
139 | 4 | Tyr,Phe | Tyr |
140TXXQXXQF | 2 | Asn,Asp,Thr,Gln,Met,Ser,Ala | Gln |
140 | 3 | Thr,Cys,Leu,Met,Ala,Ile,Val,Phe | Ala,Met |
140 | 5 | Met,Ile,Leu | Met |
140 | 6 | Val,Ile,Leu,Thr,Phe | Leu |
141DTXFMV | 3 | Leu,Ile,Val,Tyr,Phe,Ala | Phe |
142TQAQXXQF | 5 | Met,Ile,Leu | Met,Leu,尤其优选Met |
142 | 6 | Val,Ile,Leu,Thr,Phe | Leu |
尤其有利的Δ5延伸酶包含序列SEQ ID NO:116、SEQ ID NO:141和/或SEQ ID NO:142中的至少一个序列。
尤其有利的经分离的核酸序列选自以下序列:
a)具有SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ IDNO:113、SEQ ID NO:131或SEQ ID NO:133中所示序列的核酸序列;
b)作为遗传密码简并性的结果,可从SEQ ID NO:44,SEQ ID NO:46,SEQ ID NO:48,SEQ ID NO:50,SEQ ID NO:60,SEQ IDNO:62,SEQ ID NO:64,SEQ ID NO:66,SEQ ID NO:68,SEQID NO:76,SEQ ID NO:78,SEQ ID NO:80,SEQ ID NO:84,SEQ ID NO:86,SEQ ID NO:114,SEQ ID NO:132或SEQ ID NO:134中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ IDNO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:113、SEQ ID NO:131或SEQ ID NO:133中所示核酸序列的衍生物,其编码与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ IDNO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQID NO:66、SEQ ID NO:68、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:113、SEQ ID NO:131或SEQ ID NO:133在氨基酸水平具有至少40%同源性的多肽并且具有Δ5延伸酶活性。
本发明还涉及编码具有Δ6延伸酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ ID NO:184中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示核酸序列的衍生物,其编码与SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ ID NO:184在氨基酸水平具有至少40%同源性的多肽并且具有Δ6延伸酶活性。
本发明还涉及编码具有ω3去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:87或SEQ ID NO:105中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:88或SEQ ID NO:106中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:87或SEQ ID NO:105中所示核酸序列的衍生物,其编码与SEQ ID NO:88或SEQ ID NO:106在氨基酸水平具有至少60%同一性的多肽并且具有ω3去饱和酶活性。
本发明还涉及编码具有Δ6去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:89或SEQ ID NO:97中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:90或SEQ ID NO:98中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:89或SEQ ID NO:97中所示核酸序列的衍生物,其编码与SEQ ID NO:90或SEQ ID NO:98在氨基酸水平具有至少40%同源性的多肽并且具有Δ6去饱和酶活性。
本发明还涉及编码具有Δ5去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQ ID NO:101中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:100或SEQ ID NO:102中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQ ID NO:101中所示核酸序列的衍生物,其编码与SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:100或SEQ ID NO:102在氨基酸水平具有至少40%同源性的多肽并且具有Δ5去饱和酶活性。
本发明还涉及编码具有Δ4去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:95或SEQ ID NO:103中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:96或SEQ ID NO:104中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:95或SEQ ID NO:103中所示核酸序列的衍生物,其编码与SEQ ID NO:96或SEQ ID NO:104在氨基酸水平具有至少40%同源性的多肽并且具有Δ4去饱和酶活性。
本发明还涉及编码具有Δ12去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:107或SEQ ID NO:109中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:108或SEQ ID NO:110中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:107或SEQ ID NO:109中所示核酸序列的衍生物,其编码与SEQ ID NO:108或SEQ ID NO:110在氨基酸水平具有至少50%同源性的多肽并且具有Δ12去饱和酶活性。
本发明还涉及包含SEQ ID NO:43、SEQ ID NO:45、SEQ IDNO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ IDNO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ IDNO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的核酸序列的基因构建体,其中根据本发明,所述核酸有效地与一个或多个调节信号连接。此外,脂肪酸或脂类代谢的其它生物合成基因可在基因构建体中存在,其中所述其他生物合成基因选自酰基辅酶A脱氢酶、酰基ACP[酰基载体蛋白]去饱和酶、酰基ACP硫酯酶、脂肪酸酰基转移酶、酰基辅酶A:溶血磷脂酰基转移酶、脂肪酸合成酶、脂肪酸羟化酶、乙酰辅酶A羧化酶、酰基辅酶A氧化酶、脂肪酸去饱和酶、脂肪酸炔属酶、脂加氧酶、三酰甘油酯脂肪酶、环丙烯合酶、氢过氧化物裂合酶或脂肪酸延伸酶。选自Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、Δ6延伸酶、Δ9延伸酶或ω3去饱和酶的脂肪酸或脂类代谢中的生物合成基因有利地额外存在。
本发明方法中所用的全部核酸序列有利地衍生自真核生物如植物、微生物或动物。核酸序列优选地衍生自鳟形目、藻类如Mantoniella,隐甲藻属,裸藻属或Ostreococcus、真菌如疫霉属或者硅藻如海链藻属或褐指藻属。
方法中所用的编码具有ω3去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、Δ5延伸酶、Δ6延伸酶或Δ9延伸酶活性的蛋白的核酸序列有利地单独导入,或优选地与有可能使所述核酸在生物中,有利地在植物或微生物中表达的表达盒(核酸构建体)组合导入。所述核酸构建体可以包含具有酶活性如Δ12去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ5延伸酶、Δ6延伸酶和/或ω3去饱和酶活性的多于一种的核酸序列。
为了导入方法中所用的核酸,所述核酸有利地通过众知的方式扩增和连接。优选地遵循用于Pfu DNA聚合酶或Pfu/TaqDNA聚合酶混合物的符合规程的程序。引物在考虑所要扩增的序列后选择。引物应当以如此方式有利地选择以便扩增物包含从起始密码子至终止密码子的完整编码序列。扩增物在扩增后便利地加以分析。例如凝胶电泳分离可以实施,然后进行定量和定性分析。此后扩增物可按照标准规程纯化(如Qiagen)。而后一份经纯化的扩增物在随后的克隆步骤中使用。合适的克隆载体通常为技术人员所知。这类载体特别包括那些能够在微生物系统中复制的载体,即那些确保在酵母或真菌中有效克隆和那些有可能稳定转化植物的主要载体。那些特别可提及的载体是适用于T-DNA介导的转化的多种二元和共整合的载体系统。这类载体系统通常以其包含至少为农杆菌介导的转染所需的vir基因和T-DNA分界序列(T-DNA边界)为特征。这类载体系统还有利地包含其它顺式调节区域,如启动子和终止子序列和/或选择标记,其中已合适转化的生物通过选择标记可以鉴定。在共整合载体系统的情况下,vir基因和T-DNA序列在相同载体中排列,而二元系统至少以两个载体为基础,其中一个载体携带了vir基因但没有T-DNA,同时第二个载体携带了T-DNA而无vir基因。因此所提及的后一种载体相对较小,容易在大肠杆菌(E.coli)中和农杆菌中操作和复制。这类二元载体包括来自pBIB-HYG、pPZP、pBecks、pGreen系列的载体。根据本发明,Bin19、pBI101、pBinAR、pGPTV和pCAMBIA优选使用。二元载体和它们的用途的综述可在Hellens等,Trends in Plant Science(2000)5,446-451中找到。为了制备载体,载体首先以限制性核酸内切酶线性化并且然后按照合适的方式进行酶修饰。此后纯化载体并且将一份纯化的载体用于克隆步骤。在克隆步骤中,使用连接酶将已经酶切割并且根据需要已纯化的扩增物与已按类似方式制备的载体片段克隆。在本上下文中,具体的核酸构建体、或载体或质粒构建体可具有一个或超过一个的编码性基因片段。将编码性基因片段在这些构建体中优选地与调节性序列有效连接。调节性序列尤其包括植物序列,如以上提到的启动子和终止子序列。这类构建体可在微生物,特别在大肠杆菌和根癌农杆菌中于选择性条件下稳定增殖并且有可能向植物或微生物中转移异源DNA。
方法中所用的核酸、发明性核酸和核酸构建体可有利地使用克隆载体向生物如微生物中或有利地向植物中导入,并且因此用于转化植物,如那些在《Plant Molecular Biology and Biotechnology》(1993年佛罗里达CRCPress出版社Boca Raton)第6/7章第71-119页;1993年Academic Press出版社Kung和R.Wu编辑的《Transgenic plants》第1卷《Engineering andUtilization》第15-38页F.F.White的Vectors for Gene Transfer inHigher Plants;1993年Academic Press出版社Kung和R.Wu编辑的《Transgenic plants》第1卷《Engineering and Utilization》第128-143中B.Jen es等,Techniques for Gene Transfer;Potrykus,Annu.Rev.Plant Physiol.Plant Molec.Biol.42(1991),205-225中发表和引用的植物。因此方法中所用的核酸、创新的核酸和核酸构建体和/或载体可用于重组性修饰广泛类型的生物,有利地修饰植物,因此经重组性修饰的植物成为更好和/或更高效的PUFA生产者。
存在一系列可修饰Δ12去饱和酶、Δ5延伸酶、Δ6延伸酶、Δ5去饱和酶、Δ4去饱和酶、Δ6去饱和酶和/或ω3去饱和酶蛋白质和方法中所用的其它蛋白质如Δ12去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶或Δ4去饱和酶蛋白质的机制,因此有利的多不饱和脂肪酸在植物、优选在油料作物植物或微生物中的产量、产生和/或产生效率由于经修饰的蛋白质可直接受到影响。Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶蛋白质或基因的量或活性可以得到增加,因此产生了更大量的基因产物并且最终产生了更大量的通式I化合物。在向缺乏该化合物生物合成活性和能力的生物中导入相应基因之前,从头(de novo)合成也为可能。这类似地适用于脂肪酸和脂类代谢中的其它去饱和酶或延伸酶或其它酶的组合。利用多种趋异的序列即在DNA水平不同的序列在本上下文中也是有利的,或者利用基因表达的启动子也是有利的,其中所述的启动子可使不同基因的表达随时间过程不同,例如与种子或贮油组织成熟程度相关。
由于向生物中单独导入Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶基因或与细胞中其它基因组合导入,不但有可能增加指向终产物的生物合成流,而且还有可能增加或产生全新的相应三酰甘油酯的组合物。
同样,可增加其它基因的数量或活性,其中所述的其它基因参与为一种或多种脂肪酸、油、极性和/或中性脂类的生物合成所需的营养素的输入,由此增加这些前体、辅因子或中间体在细胞中或贮存区室中的浓度,从而如以下描述可进一步增强细胞产生PUFA的能力。通过优化参与这类化合物生物合成的一种或多种Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的活性或增加其量,或通过破坏参与这些化合物降解的一种或多种基因的活性,有可能在生物中强化脂肪酸和脂类分子的产量、生产和/或生产效率。
本发明方法中所用的经分离的核酸分子编码这样的蛋白质或其部分,其中所述蛋白质或单个蛋白质或其部分包含这样的氨基酸序列,所述氨基酸序列与序列SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中所示的氨基酸序列具有足够的同源性,因此所述蛋白质或其部分保留了Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的活性。由所述核酸分子编码的蛋白质或其部分优选地在生物中,有利地在植物中保留了它们的基本酶活性以及参与代谢细胞膜或脂肪体合成所需的化合物的能力,或参与跨越这类膜的分子运输的能力。所述核酸分子编码的蛋白质有利地具有与SEQ ID NO:2、SEQ ID NO:4、SEQ IDNO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ IDNO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ lD NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中所示氨基酸序列至少约50%、优选地至少约60%并且更优选地至少约70%、80%和90%并且最为优选地至少约85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的同一性。为了本发明目的,将同源性或同源的分别理解为意指同一性或同一的。
可计算涵盖整个氨基酸或核酸序列区域的同源性。技术人员可获得基于多种序列比较的多种算法的一系列程序。这里Needleman和Wunsch或Smith和Waterman的算法产生了尤其可靠的结果。作为GCG软件包[Genetics Computer Group,575 Science Drive,Madison,Wisconsin,USA 5371l(1991)]的部分的PileUp程序(J.Mol.Evolution.,25,351-360,1987,Higgins等,CABIOS,5 1989:151-153)或Gap和BestFit程序[Needleman和Wunsch(J.Mol.Biol.48;443-453(1970)和Smith及Waterman(Adv.Appl.Math.2;482-489(1981))用于序列比对。使用GAP程序和如下设置:缺口权重:50,长度权重:3,平均匹配:10.000和平均错配:0.000确定如以上以百分率所示的涵盖全长序列的序列同源性值。除非说明,这些设置总是作为标准设置用于序列比对。
本发明方法中所用的Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的基本酶活性理解为意指与序列SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183和它们的衍生物所编码的蛋白质/酶比较,它们保留了至少10%,优选地为20%,尤其优选地为30%和非常尤其地为40%的酶活性并且因此可参与在生物中,有利地为在植物或在植物细胞中,或在分子跨膜运输中合成脂肪酸、脂肪酸酯如二酰甘油酯和/或三酰甘油酯所需的化合物的代谢,这意味着脂肪酸分子中C18,C20或C22碳链在至少两处,有利地在三、四、五或六处位置具有双键。
有利地用于方法的核酸可源自细菌、真菌、硅藻、动物如隐杆线虫或大马哈鱼属或者植物如藻类或者藓类如希瓦氏菌属、剑叶藓属、破囊壶菌属、镰孢属(Fusarium)、疫霉属、角齿藓属、Mantoniella、Ostreococcus、等鞭金藻属、Aleurita、Muscarioides、被孢霉属、琉璃苣属、褐指藻属、隐甲藻属,尤其来自虹鳟、非洲爪蟾、玻璃海鞘、假矮海链藻、Mantoniella squamata、Ostreococcus sp.、Ostreococcus tauri、细小裸藻、展叶剑叶藓、致病疫霉、Fusarium graminaeum、寇氏隐甲藻、角齿藓(Ceratodon purpureus)、球等鞭金藻(Isocrydid galbana)、Aleurita farinosa、破囊壶菌属的种、Muscarioides viallii、高山被孢霉、玻璃莴苣、三角褐指藻、秀丽线虫的属和种或尤其有利地来自虹鳟、细小裸藻、假矮海链藻或寇氏隐甲藻。
可在本发明方法中备选地使用编码Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶并且与SEQ ID NO:1,SEQ ID NO:3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11,SEQ ID NO:13,SEQ ID NO:15,SEQ ID NO:17,SEQ ID NO:19,SEQ ID NO:21,SEQ ID NO:23,SEQ ID NO:25,SEQ ID NO:27,SEQ ID NO:29,SEQ ID NO:31,SEQ ID NO:33,SEQ ID NO:35,SEQ ID NO:37,SEQ ID NO:39,SEQ ID NO:41,SEQ ID NO:43,SEQ ID NO:45,SEQ ID NO:47,SEQ ID NO:49,SEQ ID NO:51,SEQ ID NO:53,SEQ ID NO:59,SEQ ID NO:61,SEQ ID NO:63,SEQ ID NO:65,SEQ ID NO:67,SEQ ID NO:69,SEQ ID NO:71,SEQ ID NO:73,SEQ ID NO:75,SEQ ID NO:77,SEQ ID NO:79,SEQ ID NO:81,SEQ ID NO:83,SEQ ID NO:85,SEQ ID NO:87,SEQ ID NO:89,SEQ ID NO:91,SEQ ID NO:93,SEQ ID NO:95,SEQ ID NO:97,SEQ ID NO:99,SEQ ID NO:101,SEQ ID NO:103,SEQ ID NO:105,SEQ ID NO:107,SEQ ID NO:109,SEQ ID NO:111,SEQ ID NO:113,SEQ ID NO:117,SEQ ID NO:119,SEQ ID NO:131,SEQ ID NO:133,SEQ ID NO:135,SEQ ID NO:137或SEQ ID NO:183中所示核酸序列在严格条件下有利杂交的核酸序列。
方法中所用的核酸序列有利地向表达盒导入,其中所述的表达盒有可能在生物如微生物或植物中表达这种核酸。
为达到此目的,将编码Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的核酸序列与有利地用于增强基因表达的一个或多个调节信号有效地连接。这类调节序列用于使基因和蛋白质特异性表达。根据宿主生物,这可以意味例如仅在导入已经发生后才表达和/或过表达这种基因,或立即表达和/或过表达这种基因。例如这些调节序列采取了诱导物和阻抑物结合的序列形式,因此控制这种核酸的表达。除了这些新的调节序列以外或作为这些序列的替代,这些序列的天然调节元件仍可在实际的结构基因之前存在并且根据需要可以如此方式加以遗传性修饰从而消除了它们的天然调节能力而且增强这些基因的表达。然而这种表达盒(表达构建体=基因构建体)也可以更为简单地构建,也就是说在核酸序列或其衍生物的前面未插入额外的调节信号而且并不除去天然启动子和其调节功能。作为替代,天然调节序列已经以如此方式突变从而调节不再发生和/或增强了基因的表达。这些经修饰的启动子也可本身以部分序列的形式位于天然基因前(用于本发明核酸序列的一部分的启动子)以增强活性。而且这种基因构建体也可有利地包含与启动子有效连接的一个或多个称作增强子的序列,而这有可能增强核酸序列的表达。额外有利的序列如其它调节元件或终止子序列也可在DNA序列的3′端插入。Δ12去饱和酶、ω3去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ5延伸酶、Δ6延伸酶和/或Δ9延伸酶基因可在表达盒(基因构建体)中存在单个或多个拷贝。优选地,仅仅这些基因的单个拷贝存在于每种表达盒中。这种基因构建体或这类基因构建体可在宿主生物中共同表达。在本上下文中,基因构建体可插入在一种或多种载体中并且在细胞中以游离的形式存在,或插入基因组中。当待表达的基因共同存在于一个基因构建体中时,其他基因在基因组中的插入有利的。
在本上下文中,调节序列或调节因子如上所述可优选地积极影响所导入基因的基因表达,从而增强基因表达。因此调节元件的增强过程可通过使用强转录信号如启动子和/或增强子有利地在转录水平上发生。此外例如通过改善mRNA的稳定性,经增强的翻译同样是可能的。
本发明的另一个实施方案是一种或多种这样的基因构建体,其中所述基因构建体包含一个或多个如SEQ ID NO:1、SEQ ID NO:3、SEQ IDNO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ IDNO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183所示的序列或其衍生物并且编码如SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中所示的多肽。以上提到的Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶蛋白质有利地使脂肪酸分子中有利地具有一个、二个、三个、四个、五个或六个双键并且有利地具有18、20或22个碳原子的底物去饱和或延伸。这同样适用于它们的同源物、衍生物或类似物,其与有利地增强基因表达的一个或多个调节信号有效连接。
用于新方法的有利的调节序列在例如cos、tac、trp、tet、trp-tet、lpp、lac、lpp-lac、lacIq、T7、T5、T3、gal、trc、ara、SP6、λ-PR或λ-PL的启动子中存在并且有利地在革兰氏阴性菌中应用。其它有利的调节序列是例如革兰氏阳性启动子amy和SPO2、酵母或真菌启动子ADC1、MFα、AC、P-60、CYC1、GAPDH、TEF、rp28、ADH或者植物启动子CaMV/35S[Franck等,Cell 21(1980)285-294]、PRP1[ward等,Plant.Mol.Biol.22(1993)]、SSU、OCS、lib4、usp、STLS1、B33、nos或者泛蛋白或菜豆蛋白启动子中存在。可诱导启动子在本上下文中同样是有利的如在EP-A-0 388 186(苯磺酰胺可诱导)、Plant J.2,1992:397-404(Gatz等,四环素可诱导)、EP-A-0 335 528(脱落酸可诱导)或WO 93/21334(乙醇或环己醇可诱导)中的启动子。其它合适的植物启动子是马铃薯胞质FBP酶启动子或ST-LSI启动子(Stockhaus等,EMBO J.8,1989,2445)、大豆磷酸核糖焦磷酸酰氨基转移酶启动子(GenbankAccession No.U87999)或在EP-A-0 249 676中所描述的结节特异性启动子。
尤其有利的启动子是有可能使得在参与脂肪酸生物合成的组织中表达的启动子。种子特异性的启动子如所描述的USP启动子非常尤其有利并且其它启动子如LeB4、DC3、菜豆蛋白或油菜籽蛋白启动子同样如此。进一步更有利的启动子是种子特异性启动子,这种启动子能用于单子叶或双子叶植物并且在US 5,608,152(油菜籽的油菜籽蛋白启动子)、WO 98/45461(拟南芥的油质蛋白启动子)、US 5,504,200(菜豆的菜豆蛋白启动子)WO 91/13980(芸苔的Bce4启动子)、Baeumlein等,Plant J.,2,2,1992:233-239(来自豆科植物的LeB4启动子)中描述。适于单子叶植物的启动子是例如大麦lpt-2或lpt-1启动子(WO 95/15389和WO 95/23230)、大麦醇溶蛋白启动子和WO 99/16890中所描述的其它合适启动子。
原则上,对于新方法可使用所有天然启动子及其调节序列,如那些如上提及的天然启动子及其调节序列。也可有利地使用合成启动子,可组合或者单独地使用,尤其当这类合成启动子介导了种子特异性表达时,如那些WO 99/16890中所描述的启动子。
为了取得特别高的PUFA含量,尤其在转基因植物中,PUFA生物合成基因应当有利地在油料作物中以种子特异性的方式表达。为实现此目的,可使用种子特异性启动子或在胚和/或在胚乳中活跃的那些启动子。原则上,种子特异性启动子既可从双子叶植物也可从单子叶植物中分离。如下列出了优选的启动子:USP(未知种子蛋白质)和豌豆球蛋白(豌豆((Viciafaba))[Bumlein等,Mol.Gen Genet.,1991,225(3)],油菜籽蛋白(油菜籽)[US 5,608,152]、酰基载体蛋白(油菜籽)[US 5,315,001和WO92/18634]、油质蛋白(拟南芥)[WO 98/45461和WO 93/20216]、菜豆蛋白(菜豆)[US 5,504,200]、Bce4[WO 91/13980]、豆球蛋白B4(LegB4启动子)[Bumlein等,Plant J.,2,2,1992],Lpt2和lpt1(大麦)[WO95/15389和WO95/23230]、来自稻、玉米和小麦的种子特异性启动子[WO99/16890 ]、Amy32b、Amy 6-6和aleurain[US 5,677,474]、Bce4(油菜籽)[US 5,530,149]、大豆球蛋白(大豆)[EP 571 741]、磷酸烯醇丙酮酸羧化酶(大豆)[JP 06/62870]、ADR12-2(大豆)[WO 98/08962]、异柠檬酸裂合酶(油菜籽)[US 5,689,040]或α淀粉酶(大麦)[EP 781 849]。
植物的基因表达也可通过化学可诱导启动子促进(见于综述Gatz1997,Annu.Rev.Plant Physiol.Plant Mol.Biol.,48:89-108)。在需要以时间特异性方式进行基因表达时,这种化学可诱导启动子尤其合适。此类启动子的例子是水杨酸可诱导启动子(WO 95/19443)、四环素可诱导启动子(Gatz等(1992)Plant J.2,397-404)和乙醇可诱导启动子。
为确保这类生物合成基因经多个世代后仍然稳定地在转基因植物中整合,编码Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶并且用于方法中的每种核酸应当在一个独立的启动子,优选地在不同于其它启动子的启动子控制下表达,因为重复序列基序可导致T-DNA不稳定或导致重组事件。在本上下文中,表达盒有利地按如此方式构建从而启动子后是适宜的切割位点,这个切割位点有利地位于多位点接头中以便插入将要表达的核酸,并且根据需要使终止子序列位于这个多位点接头的后面。上述的这种序列重复多次,优选地重复三、四或五次,由此多达五个基因可在一个构建体中组合并导入转基因植物中以便表达。这种序列有利地重复了多至三次。为了表达核酸序列,核酸序列通过例如多位点接头中的合适切割位点在启动子后面插入。每种核酸序列有利地具有自身的启动子并且根据需要具有自身的终止子序列。这类有利的构建体在例如DE 101 02 337或DE 101 02 338中公开。然而仍然可在启动子后面并且根据需要在终止子序列前插入多个核酸序列。这里表达盒中所插入核酸的插入位点或其序列并不特别重要,也就是说核酸序列可在表达盒中最先的位置或最末的位置插入而不显著地影响这种核酸序列的表达。不同的启动子例如USP、LegB4或DC3启动子和不同的终止子序列可有利地在表达盒中使用。然而也可在表达盒中只使用一种类型的启动子。但是这可能导致不为所需的重组事件。
如以上所述,已导入基因的转录应当通过位于已导入的生物合成基因3’端的合适终止子序列有利地终止(在终止密码子之后)。在本上下文中,可使用序列的例子是OCS1终止子序列。如同启动子的情况,对每个基因应该使用不同的终止子序列。
如以上所述,基因构建体还可包含欲导入生物体的其它基因。向宿主生物中导入并且因此表达调节基因如诱导物、阻抑物或酶基因是可能并且有利的,其中这些诱导物、阻抑物或酶因其活性参与了生物合成途径中一种或多种基因的调节。这类调节基因可为异源或同源。而且脂肪酸或脂类代谢中的其它生物合成基因可在核酸构建体或基因构建体中有利地存在;然而这些基因还可以位于一种或多种其它核酸构建体中。优选使用的脂肪酸或脂类代谢中的生物合成基因是选自酰基辅酶A脱氢酶、酰基ACP[酰基载体蛋白]去饱和酶、酰基ACP硫酯酶、脂肪酸酰基转移酶、酰基辅酶A:溶血磷脂酰基转移酶、脂肪酸合成酶、脂肪酸羟化酶、乙酰基辅酶A羧化酶、酰基辅酶A氧化酶、脂肪酸去饱和酶、脂肪酸炔属酶、脂加氧酶、三酰甘油酯脂肪酶、环丙烯合酶、氢过氧化物裂合酶或脂肪酸延伸酶或它们组合的基因。尤其有利的核酸序列是脂肪酸或脂类代谢中的生物合成基因,这些生物合成基因选自酰基辅酶A:溶血磷脂酰基转移酶、ω3去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、Δ5延伸酶、Δ6延伸酶和/或Δ9延伸酶。
在本上下文中,以上提到的核酸或基因可与其它延伸酶和去饱和酶组合在以上提及的表达盒中克隆并在农杆菌属协助下转化植物。
这里调节序列或调节因子可如以上所述优选地积极影响已导入基因的表达,并且因此增强了已导入基因的表达。因此,调节元件的增强可通过使用强转录信号例如启动子和/或增强子有利地在转录水平发生。然而例如通过改善mRNA稳定性同样可增强翻译。原则上,这类表达盒可直接用于导入植物或导入载体。
这些有利的载体优选地表达载体包含编码Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶并且用于方法中的核酸,或包含核酸构建体,其中所述核酸单独使用或者与脂肪酸或脂类代谢中的其它生物合成基因如酰基辅酶A:溶血磷脂酰基转移酶、ω3去饱和酶、Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6延伸酶和/或Δ9延伸酶组合。如在本上下文中所用,术语“载体”指这样的核酸分子,其能够运输与这种核酸分子所结合的其它核酸。载体的一个类型是“质粒”,即额外的DNA片段可加以连入的环状双链DNA环。载体的另一个类型是病毒载体,对额外的DNA片段而言有可能向这种病毒的基因组内连接。某些载体能够在已导入这些载体的宿主细胞中自主复制(例如具有细菌复制原点的细菌载体)。其它载体当向宿主细胞导入时有利地在宿主细胞的基因组内整合并且因此随宿主基因组一起复制。而且某些载体能够控制与这些载体有效连接的基因的表达。这些载体在本上下文中称为“表达载体”。通常适用于DNA重组技术的表达载体采用了质粒的形式。在本说明书中由于质粒是最常用的载体形式,故“质粒”和“载体”可交互使用。然而本发明还将覆盖具有类似功能的其它形式的表达载体如病毒载体。而且术语“载体”还将包含技术人员熟知的其它载体如噬菌体、病毒如SV40、CMV、TMV、转座子、IS元件、噬粒、噬菌粒、粘粒、线形或环形DNA。
有利地在方法中使用的重组表达载体包含具有宿主细胞中适于所用核酸表达的形式的如下所述核酸或以上提及的基因构建体,即所述重组表达载体包含基于用于表达的宿主细胞所选择的一种或多种调节序列,其中这种(类)调节序列有效地与将要表达的核酸序列连接。在重组表达载体中,“有效连接的”意指将目的核苷酸序列与调节序列以如此方式连接即有可能使这种核苷酸序列表达并且它们彼此连接而使这两类序列(例如在体外转录/翻译系统中,或如果载体导入了宿主细胞,在宿主细胞中)都执行了属于各序列的预期功能。术语“调节序列”旨在包含启动子、增强子和其它表达控制元件(例如多聚腺苷酸化信号)。这些调节序列在例如Goeddel:Gene Expression Technology:Methods in Enzymology 185,AcademicPress,San Diego,CA(1990)一书中描述或参见:Gruber和Crosby,Methods in Plant Molecular Biology and Biotechnolgy,CRC Press,BocaRaton,Florida,Glick和Thompson编著,Chapter 7,89-108,包括其中引用的参考文献。调节序列包含那些在多种类型的宿主细胞中控制核苷酸序列组成型表达的调节序列和那些控制核苷酸序列仅仅在特定的宿主细胞中于特定条件下直接表达的调节序列。技术人员知道表达载体的设计可依赖多种因素如待转化的宿主细胞的选择、蛋白质的目的表达水平及其它因素。
所用的重组表达载体可设计用于在原核或真核细胞中表达Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶。为方便起见,载体构建的中间步骤常在微生物中实施,因此这样设计是有利的。例如Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶基因可使用载体按照如WO 98/01572中所描述的转化方法在细菌细胞、昆虫细胞(使用杆状病毒表达载体)、酵母和其它真菌细胞(见Romanos,M.A.等(1992)“Foreign geneexpression in yeast:a review”,Yeast 8:423-488:van den Hondel、C.A.M.J.J.等.(1991)“Heterologous gene expression in filamentous fungi”,in:More Gene Manipulations in Fungi,J.W.Bennet和L.L.Lasure编辑,第396-428页:Academic Press:San Diego:和van den Hondel、C.A.M.J.J.& Punt,P.J.(1991)”Gene transfer systems and vectordevelopment for filamentous fungi,in:Applied Molecular Genetics ofFungi,Peberdy,J.F.等编辑,第1-28页Cambridge University Press:Cambridge)、藻类(Falciatore等,1999,Marine Biotechnology.1,3:239-251)、多种类型的纤毛虫:Holotrichia,缘毛亚纲(Peritrichia),旋毛亚纲(Spirotrichia),吸管亚纲(Suctoria),四膜虫(Tetrahymena),草履虫(Paramecium),豆形虫(colpidium),瞬目虫(Glaucoma),匙口虫(Platyophrya),Potomacus,Desaturaseudocohnilembus,游仆虫(Euplotes),Engelmaniella和stylonychia(Stylonychia),尤其是Stylonychia lemnae并且优选地在多细胞植物的细胞中(见Schmidt,R.和Willmitzer,L.(1988)“High efficiency Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana leaf and cotyledonexplants”Plant Cell Rep.:583-586;Plant Molecular Biology andBiotechnology,C Press,Boca Raton,Florida,Chapter 6/7,pp.71-119(1993);F.F.White,B.Jenes等,Techniques for Gene Transfer,in:Transgenic Plants,第一卷,Engineering and Utilization,Ed.:Kung和R.Wu,Academic Press(1993),128-43;Potrykus,Annu.Rev.Plant Physiol.Plant Molec.Biol.42(1991),205-225(并且其中所应用的参考文献))表达。合适的宿主细胞在Goeddel,Gene Expression Technology:Methods inEnzymology 185,Academic Press,San Diego,CA(1990)中详细讨论。作为备选,重组表达载体可例如使用T7-启动子调节序列和T7-聚合酶体外转录和翻译。
在大多数情况下,原核生物中蛋白质的表达涉及使用包含控制融合或非融合蛋白质表达的组成型或可诱导启动子的载体。典型的融合表达载体尤其为pGEX(Pharmacia Biotech Inc;Smith,D.B.和Johnson,K.S.(1988)Gene 67:31-40)、pMAL(New England Biolabs,Beverly,MA)和pRIT5(Pharmacia,Piscataway,NJ),其中谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白和蛋白A分别与重组靶蛋白融合。
合适的可诱导的非融合大肠杆菌表达载体的例子尤其为pTrc(Amann等(1988)Gene 69:301-315)和pET 11d(1990年加利福尼亚圣迭哥Academic Press出版社《Methods in Enzymology》185的Studier等《GeneExpression Technology》第60-89页)。靶基因自pTrc载体的表达是基于宿主RNA聚合酶自杂合trp-lac融合启动子的转录。靶基因自pET 11d载体的表达是基于经共表达的病毒RNA聚合酶(T7-gn1)所介导的T7-gn10-lac融合启动子的转录,这种病毒RNA聚合酶由宿主菌株BL21(DE3)或HMS174(DE3)的定居λ-原噬菌体提供,其中所述定居λ-原噬菌体携带了受lacUV 5启动子转录控制的T7 gn1基因。
其它适用于原核生物的载体为技术人员所知,这类载体是例如大肠杆菌的pLG338、pACYC184、pBR系列如pBR322、pUC系列如pUC18或pUC19、M113mp系列、pKC30、pRep4、pHS1、pHS2、pPLc236、pMBL24、pLG200、pUR290、pIN-III113-B1、λgt11或pBdCI,链霉菌中的pIJ101、pIJ364、pIJ702或pIJ361,芽孢杆菌属中的pUB110、pC194或pBD214,棒杆菌属中的pSA77或pAJ667。
在另一个实施方案中,表达载体是酵母表达载体。用于酿酒酵母中表达的载体的例子包含pYeDesaturasec1(Baldari等.(1987)Embo J.6:229-234)、pMFa(Kurjan和Herskowitz(1982)Cell 30:933-943)、pJRY88(Schultz等(1987)Gene 54:113-123)和pYES2(InvitrogenCorporation,San Diego,CA)。用于构建适用于其它真菌如丝状真菌的载体和构建方法包含在1991年剑桥大学的剑桥大学出版社J.F.Peberdy等编辑的《Applied Molecular Genetics of fungi》第1-28页,van den Hondel、C.A.M.J.J;和Pun,P.J.的“Gene transfer systems and vector developmentfor filamentous fungi”中或在《More Gene Manipulations in Fungi》[圣迭哥Academic Press出版社J.W.Bennet和L.L.Lasure编辑,第396-428页]中所详细描述的那些载体和方法。其它合适的酵母载体为例如pAG-1、YEp6、YEp13和pEMBLYe23。
作为备选,Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶可在昆虫细胞中用杆状病毒载体表达。可用于在培养的昆虫细胞(例如Sf9细胞)中表达蛋白质的杆状病毒表达载体包含pAc系列(Smith等(1983)Mol.Cell Biol.3:2156-2165)和pVL系列(Lucklow和Summers(1989)Virology 170:31-39)。
以上提到的载体仅概略回顾了可能适合的载体。其它的质粒为技术人员所知并且在例如《Cloning vector》(1985年阿姆斯特旦-纽约-牛津Elsevier出版社,Pouwels,P.H.等编辑,ISBN 0 444 904018)中描述。对于其它合适用于原核及真核细胞的表达系统,见1989年纽约冷泉港ColdSpring Harbor Laboratory出版社,Sambrook,J.、Fritsch,E.F.和Maniatis,T.的《Molecular Cloning:A Laboratory Manual》第二版第16和第17章中的描述。
在方法的又一个实施方案中,Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶可在单细胞植物细胞(如藻类)中表达,参见Falciatore等,1999,Marine Biotechnology 1(3):239-251及其中引用的参考文献,并且可在高等植物的植物细胞中表达(如种子植物例如耕作作物)。植物表达载体的例子包括在1992年Becker,D.、Kemper,E.、Schell,J.和Masterson,R.“New plant binary vectors with selectable markers locatedproximal to the left border”,Plant Mol.Biol.20:1195-1197;和1984年Bevan,M.W.“Binary Agrobacterium vectors for plant transformation”,Nucl.Acids Res.12:8711-8721;在1993年Academic Press出版社Kung和R.Wu编辑的《Transgenic Plants:第1卷Engineering and Utilization》第15-38页的Vectors for Gene Transfer in Higher Plants中所详细描述的那些植物表达载体。
植物表达盒优选地包含能够在植物细胞中控制基因表达并且有效连接的调节序列,如此使每一种序列能够实现它的功能,例如多聚腺苷酸化信号的转录终止功能。优选的多聚腺苷酸化信号是衍生自根癌农杆菌T-DNA如Ti质粒pTiACH5(1984年Gielen等,EMBO J.3期,835等)的基因3的那些多聚腺苷酸化信号,其中已知基因3是章鱼氨酸合酶,或者是那些多聚腺苷酸化信号的等同物,但是所有其它在植物中具有功能性活性的终止子序列同样合适。
由于植物基因表达通常不限于在转录水平,植物表达盒优选地包含其它有效连接的序列如翻译增强子例如超驱动序列,所述的超驱动序列增强烟草花叶病毒5’-非翻译前导序列,这种前导序列提高了蛋白质/RNA比(Gallie等,1987,Nucl.Acids Research 15:8693-8711)。
如以上所述,植物基因表达必须有效地与合适启动子连接,其中这种启动子引发基因以正确的时间过程或以细胞或组织特异性方式表达。可使用的启动子是组成型启动子(Benfey等,EMBO J.8(1989)2195-2202),例如那些衍生自植物病毒如35S CaMV(Franck等,Cell 21(1980)285-294)、19S CaMV(也见US 5352605和WO 84/02913)的启动子或植物启动子如在US 4,962,028中所描述Rubisco亚单位的启动子。
用于在植物基因表达盒中有效连接的其它优选的序列是靶向序列,其中这种靶向序列为基因产物向该基因产物对应的细胞区室中(见Kermode,Crit.Rev.Plant Sci.15,4(1996)285-423的综述及其引用的参考文献)例如向液泡、细胞核、所有类型质体如造粉体、叶绿体、色质体、胞外空间、线粒体、内质网、油质体、过氧化物酶体和植物细胞的其它区室中导入所需。
如以上所述,植物基因表达还可以通过化学可诱导启动子(见Gatz1997,Annu.Rev.Plant Physiol.Plant Mol.Biol.,48:89-108的综述)实现。当希望基因表达需以时间特异性发生时,化学可诱导启动子尤其适合。这类启动子的例子是水杨酸可诱导启动子(WO 95/19443)、四环素可诱导启动子(Gatz等(1992)Plant J.2,397-404)和乙醇可诱导启动子。
对生物性或非生物应激条件反应的启动子如病原诱导的PRP1基因启动子(Ward等,Plant.Mol.Biol.22(1993)361-366)、热诱导的番茄hsp80启动子(US 5,187,267)、冷冻诱导的马铃薯α淀粉酶启动子(WO96/12814)或外伤诱导的pinII启动子(EP-A-0 375 091)同样适合。
那些在进行脂肪酸、脂类和油生物合成的组织和器官中,在种子细胞如胚乳细胞和发育胚的细胞中引起基因表达的启动子尤其有利。这类合适的启动子是油菜籽的油菜籽蛋白启动子(US 5,608,152)、蚕豆的USP启动子(Baeumlein等,Mol Gengenet,1991,225(3):459-67)、拟南芥的油质蛋白启动子(WO 98/45461)、菜豆的菜豆蛋白启动子(US 5,504,200)、芸苔属的Bce4启动子(WO 91/13980)或豆球蛋白B4启动子(LeB4;Baeumlein等,1992,Plant Journal,2(2):233-9)及在单子叶植物如玉米、大麦、小麦、黑麦、稻等中引起种子特异性表达的启动子。重要的合适启动子是大麦的lpt2或lpt1基因启动子(WO 95/15389和WO 95/23230)或WO 99/16890中所描述来自大麦的大麦醇溶蛋白基因、稻的谷蛋白基因、稻的水稻素基因、稻的谷醇溶蛋白基因、小麦的麦醇溶蛋白基因、小麦的谷蛋白基因、玉米的玉米醇溶蛋白基因、燕麦的谷蛋白基因、高梁的kasirin基因或黑麦的裸麦醇溶蛋白基因的启动子。
引起方法中所用的Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶的多重平行表达可能尤为所需。这类表达盒可通过同时转化多种单独的表达构建体,或优选地通过在一个载体构建体上组合多种表达盒而产生。同样在每一情况下多种载体可以用多重表达盒转化然后向宿主细胞转移。
因为质体是合成脂类生物合成的前体和某些终产物的区室,同样尤其适合的其它启动子是那些引起质体特异性表达的启动子。这类合适的启动子如病毒RNA聚合酶启动子在WO 95/16783和WO 97/06250中描述以及来自拟南芥的clpP启动子在WO 99/46394中描述。
载体DNA可通过常规的转化或转染技术向原核和真核细胞内导入。术语“转化”和“转染”、接合和转导如在本上下文中所用,旨在包含本领域众知的用于向宿主细胞内导入外源核酸(如DNA)的多种方法,包括磷酸钙或氯化钙共沉淀、DEAE葡聚糖介导的转染、脂质体转染、天然感受态、化学介导的转移、电穿孔或粒子轰击。适用于转化或转染包括植物细胞在内的宿主细胞的方法可在Sambrook等(1989年纽约冷泉港ColdSpring Harbor Laboratory出版社《Molecular Cloning:A LaboratoryManual》第二版)和其它的实验室教材如1995年新泽西Totowa的HumanaPress出版社《Methods in Molecular Biology》第44卷Gartland和Davey编辑的《Agrobacterium protocols》中找到。
适于摄入本发明的核酸、本发明的基因产物或本发明的载体的宿主细胞是所有原核或真核生物。有利使用的宿主生物是微生物如真菌或酵母或者植物细胞,优选植物或其部分。优选使用真菌、酵母或植物,尤其优选使用植物,非常尤其优选使用具有高脂类化合物的植物如油料作物,例如油菜籽、月见草、大麻、大蓟、花生、卡诺拉油菜、亚麻子、大豆、红花、向日葵、琉璃苣,或植物如玉米、小麦、黑麦、燕麦、小黑麦、稻、大麦、棉、木薯、胡椒、万寿菊,茄科植物如马铃薯、烟草、茄和番茄、豌豆属的种、豌豆、紫花苜蓿,灌木植物(咖啡、可可、茶),柳属的种、树(油棕榈,椰子)和宿生牧草及饲料作物。本发明尤其优选的植物是油料作物如大豆、花生、油菜籽、卡诺拉油菜、亚麻子、大麻、月见草、向日葵、红花、树(油棕榈、椰子)。
本发明还涉及编码具有Δ5延伸酶活性的多肽的上述经分离核酸序列,其中由这种核酸序列所编码的延伸酶转化具有一个双键的C16和C18脂肪酸并且有利地转化具有一个Δ6双键的多不饱和C18脂肪酸以及具有一个Δ5双键的多不饱和C20脂肪酸。这种延伸酶不延伸C22脂肪酸。
有利的经分离的核酸序列是编码具有Δ5延伸酶活性并且包含选自具有序列SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:139、SEQ IDNO:140、SEQ ID NO:141或SEQ ID NO:142中所示氨基酸序列的多肽的核酸序列。
其它有利的经分离的核酸序列是编码具有Δ5延伸酶活性并且包含选自如下的氨基酸序列组合的多肽的核酸序列:
a)SEQ ID NO:115和SEQ ID NO:139、SEQ ID NO:115和SEQ IDNO:140或者SEQ ID NO:139和SEQ ID NO:140;或
b)SEQ ID NO:116和SEQ ID NO:141、SEQ ID NO:116和SEQ IDNO:142或者SEQ ID NO:141和SEQ ID NO:142;或
c)SEQ ID NO:115、SEQ ID NO:139和SEQ ID NO:140或者SEQID NO:116、SEQ ID NO:141和SEQ ID NO:142。
编码具有Δ5延伸酶活性的多肽的优选核酸序列有利地包含以上提及的氨基酸序列。以上提及的氨基酸序列在表2中较详细地描述。
尤其有利的经分离的核酸序列是选自如下的核酸序列:
a)具有SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ IDNO:113、SEQ ID NO:131或SEQ ID NO:133中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:60、SEQ IDNO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:114、SEQ ID NO:132或SEQ ID NO:134中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ IDNO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:113、SEQ ID NO:131或SEQ ID NO:133中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ IDNO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:84、SEQID NO:86、SEQ ID NO:114、SEQ ID NO:132或SEQ ID NO:134在氨基酸水平具有至少40%同源性的多肽并且具有Δ5延伸酶活性。
本发明还涉及如以下所编号并且编码Δ6延伸酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶或Δ12去饱和酶的核酸序列。
其它有利的经分离的核酸序列是选自如下的核酸序列:
a)具有SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ ID NO:184中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ IDNO:184在氨基酸水平具有至少40%同源性的多肽并且具有Δ6延伸酶活性。
编码具有ω3去饱和酶活性的经分离的核酸序列,其选自:
a)具有SEQ ID NO:87或SEQ ID NO:105中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:88或SEQ ID NO:106中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:87或SEQ ID NO:105中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ ID NO:88或SEQ ID NO:106在氨基酸水平具有至少60%同源性的多肽并且具有ω3去饱和酶活性.
编码具有Δ6去饱和酶活性的经分离的核酸序列,其选自:
a)具有SEQ ID NO:89或SEQ ID NO:97中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:90或SEQ ID NO:98中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:89或SEQ ID NO:97中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ ID NO:90或SEQ ID NO:98在氨基酸水平具有至少40%同源性的多肽并且具有Δ6去饱和酶活性。
编码具有Δ5去饱和酶活性的经分离的核酸序列,其选自:
a)具有SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQID NO:101中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:100或自SEQ ID NO:102中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQ ID NO:101中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ IDNO:92、SEQ ID NO:94、SEQ ID NO:100或SEQ ID NO:102在氨基酸水平具有至少40%同源性的多肽并且具有Δ5去饱和酶活性。
编码具有Δ12去饱和酶活性的经分离的核酸序列,其选自:
a)具有SEQ ID NO:95或SEQ ID NO:103中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:96或SEQ ID NO:104中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:95或SEQ ID NO:103中所示核酸序列的衍生物,其中这种核酸序列的衍生物编码与SEQ ID NO:96或SEQ ID NO:104在氨基酸水平具有至少40%同源性的多肽并且具有Δ6去饱和酶活性。
编码具有Δ12去饱和酶活性的经分离的核酸序列,其选自:
a)具有SEQ ID NO:107或SEQ ID NO:109中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可从SEQ ID NO:108或SEQ ID NO:110中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:107或SEQ ID NO:109中所示核酸序列的衍生物,其中所述的衍生物编码与SEQ ID NO:108或SEQ ID NO:110在氨基酸水平具有至少50%同源性的多肽并且具有Δ12去饱和酶活性。
本发明以上提及的核酸序源自能合成PUFA的生物如非人类动物、纤毛虫、真菌、植物如藻类或腰鞭毛虫(dinofalgellates)。
以上提及的经分离的核酸序列有利地源自鲑形目、爪蟾科或海鞘科、硅藻的海链藻属或隐甲藻属,或者源自草绿藻科例如Ostreococcus属、或裸藻科如裸藻属、或疫霉科例如疫霉属。
本发明还涉及如以上所述编码具有ω3去饱和酶活性的多肽的经分离核酸序列,其中这种核酸序列所编码的ω3去饱和酶转化具有二、三、四或五个双键的C18、C20和C22脂肪酸并且有利地转化具有二或三个双键的多不饱和C18-脂肪酸和具有二、三或四个双键的C20-脂肪酸。这种ω3去饱和酶也可使具有四个或五个双键的C22-脂肪酸去饱和。
如以上所述,本发明还涉及编码具有Δ12去饱和酶、Δ4去饱和酶、Δ5去饱和酶和Δ6去饱和酶活性的多肽的经分离核酸序列,其中由这些核酸序列所编码的Δ12去饱和酶、Δ4去饱和酶、Δ5去饱和酶和Δ6去饱和酶转化具有一、二、三、四或五个双键的C18、C20和C22脂肪酸并且有利地转化具有一、二或三个双键的多不饱和C18脂肪酸如C18:1Δ9、C18:2Δ9,12或C18:3Δ9,12,15,有利地转化具有三或四个双键的多不饱和C20脂肪酸如C20:3Δ8,11,14或C20:4Δ8,11,14,17或有利地转化具有四或五个双键的多不饱和C22脂肪酸如C22:4Δ7,10,13,16或C22:5Δ7,10,13,16,19。这些脂肪酸有利地在磷脂或者辅酶A脂肪酸酯中去饱和,有利地在辅酶A脂肪酸酯中去饱和。
在一个有利的实施方案中,术语“核酸(分子)”如在本上下文中所用还包含位于编码区的3’端和5’端的非翻译序列:位于编码区5’端上游序列的至少500个,优选200个,尤其优选100个核苷酸序列和位于编码区的3’端下游序列的至少100个,优选50个,特别优选20个核苷酸的序列。“经分离的”核酸分子独立于在这种核酸的天然来源中存在的其它核酸分子。“经分离的”核酸优选地不具备这种核酸所来源的生物的基因组DNA中天然分布于这种核酸两侧的序列(例如位于这种核酸序列的5’端和3’端的序列)。在不同的实施方案中,经分离的Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的核酸分子可包含例如小于约5kb、4kb、3kb、2kb、1kb、0.5kb或0.1kb的核苷酸序列,而这些核苷酸序列在来源于这类核酸分子所来源的细胞的基因组DNA中天然地分布在这类核酸分子的两侧。
方法中所用的核酸分子,例如具有SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的核苷酸序列或者其部分的核酸分子可使用分子生物学标准技术和这里提供的序列信息分离。DNA或氨基酸水平的同源序列或同源的保守序列区域例如还可在比较性算法的协助下鉴定。它们可用作杂交探针和用于标准杂交技术(例如在1989年纽约冷泉港Cold Spring Harbor Laboratory出版社《Molecular Cloning:A Laboratory Manual》第二版所描述的那些探针和标准杂交技术)以分离可用于方法的其它核酸序列。而且包含SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的完整序列或其部分的核酸分子可通过聚合酶链反应分离,其中寡核苷酸引物以这种序列或其部分为基础使用(例如包含完整序列或其部分的核酸分子通过以相同的序列为基础所产生的寡核苷酸引物进行的聚合酶链反应分离)。例如mRNA可从细胞中分离(如通过Chirgwin等(1979)Biochemistry 18:5294-5299的硫氰酸胍提取方法)和通过逆转录酶获得cDNA(例如可以用从Gibco/BRL、Bethesda、MD获得的莫洛尼MLV逆转录酶或可以用从Seikagaku America,Inc.,St.Petersburg,FL获得的AMV逆转录酶)。用于聚合酶链反应扩增的合成寡核苷酸引物可基于SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示的一种序列产生或在SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中详述的氨基酸序列协助下产生。本发明的核酸可使用cDNA或备选地使用基因组DNA作为模板并且使用合适的寡核苷酸引物通过标准PCR扩增技术扩增。所扩增的核酸因此可向合适的载体克隆并且通过DNA序列分析描述特征。与去饱和酶核苷酸序列对应的寡核苷酸可通过标准合成方法如使用自动DNA合成仪产生。
与具有序列SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的核酸序列的同源物意指例如与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示的核苷酸序列或其同源物、衍生物或类似物或者它们的部分具有至少约50或60%,优选地具有至少约60或70%,更为优选地具有至少约70或80%、90%或95%并且甚至更为优选地具有至少约85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高同一性或同源性的等位基因变体。具有与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示诸核苷酸序列之一或其部分杂交的核苷酸序列的经分离核酸分子可在例如严格条件下进行杂交。根据本发明,将其部分理解为意指至少25个碱基对(bp)、50bp、75bp、100bp、125bp或150bp,优选至少175bp、200bp、225bp、250bp、275bp或300bp,尤其优选350bp、400bp、450bp、500bp或更多的碱基对为杂交所用。全长序列的使用同样是可能并且有利的。等位基因变体尤其包含功能性变体,这种功能性变体可自/向SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所详述序列通过核苷酸的删除、插入或替代而获得,然而即便由于插入了一个或多个核苷酸,预计所合成的最终蛋白质的酶活性仍然有利地得以保留。保留了Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶的酶活性的蛋白质,即蛋白质的酶活性基本上未减少意指与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183所编码的蛋白质比较,这类蛋白质具有至少10%,优选地具有20%,尤其优选地具有30%,非常尤其优选地具有40%的原初酶活性。计算了涵盖整个氨基酸或核酸序列区域的同源性。技术人员可获得基于多种序列比较的多种算法的一系列程序。这里Needleman和Wunsch或Smith和Waterman的算法产生了尤其可靠的结果。这里作为GCG软件包[Genetics Computer Group,575 ScienceDrive,Madison,Wisconsin,USA 53711(1991)]的部分的PileUp程序(J.Mol.Evolution.,25,351-360,1987,Higgins等,CABIOS,51989:151-153)或Gap和BestFit程序[Needleman和Wunsch(J.Mol.Biol.48;443-453(1970)和Smith及Waterman(Adv.Appl.Math.2;482-489(1981))用于序列比对。使用GAP程序并如下设置:缺口权重:50,长度权重:3,平均匹配:10.000和平均错配:0.000测定了如以上以百分率所示的涵盖全长序列的序列同源性值。除非另有说明,这些设置总是作为标准设置用于序列比对。
SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的同源物同样意指例如细菌、真菌和植物的同源物、经截短的序列、编码和非编码的DNA序列的单链DNA或单链RNA。
SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183的同源物还意指衍生物例如启动子变体。通过交换一个或多个核苷酸、通过(多次)插入和/或(多次)删除可修饰位于所详述的核苷酸序列上游的启动子同时并未不利地影响这种启动子的功能或活性。进一步有可能通过修饰启动子序列增强启动子的活性或者有可能以包括来自异源生物的启动子在内的更为活跃的启动子完全取代这类启动子。
将具有Δ12去饱和酶、ω3去饱和酶、Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶和/或Δ4去饱和酶活性并且参与脂类和脂肪酸、PUFA辅因子和酶的代谢或参与亲脂化合物跨膜运输的以上提及的核酸和蛋白质分子在本发明的方法中使用,以便在转基因植物有利地在植物如玉米、小麦、黑麦、燕麦、小黑麦、稻、大麦、大豆、花生、棉花、亚麻属的种例如亚麻子或亚麻,芸苔属的种例如油菜籽、卡诺拉油菜和芜箐、胡椒、向日葵、琉璃苣、月见草和万寿菊,茄科的植物例如马铃薯、烟草、茄和番茄,豌豆属的种,豌豆,木薯,紫花苜蓿,灌木植物(咖啡、可可、茶),柳属的种,树(油棕榈、椰子)和宿生牧草和饲料作物中调节PUFA的产生,所述的调节为直接调节(例如当脂肪酸生物合成中的蛋白质过表达或优化表达对经修饰的生物中脂肪酸的产量、生产和/或生产效率有直接影响时)和/或具有间接影响,然而这种间接效果无论如何总是引起了PUFA产量、生产和/或生产效率的增强或所不需的化合物的减少(例如当调节细胞中脂类和脂肪酸、辅因子和酶的代谢引起了所需化合物的产量、生产和/或生产效率的改变或细胞中目的化合物组分的改变,而这反过来又可影响一种或多种脂肪酸时)。
多种前体分子和生物合成酶的组合导致了多种脂肪酸分子的产生,这决定性地影响了脂类组成,因为多不饱和脂肪酸(PUFA)不仅掺入三酰甘油酯而且也掺入膜脂类。
芸苔科、紫草科、报春花科或亚麻科特别适于产生PUFA如硬脂艾杜糖酸、二十碳五烯酸和二十二碳六烯酸。亚麻籽(linum usitatissimum)尤其有利地适合以本发明所述的核酸序列组合其它去饱和酶和延伸酶用于产生PUFA。
脂类合成可划分为两个阶段:脂肪酸的合成和它们与sn-甘油-3-磷酸的结合及极性首基的添加或修饰。通常用于膜中的脂类包含磷脂类、糖脂类、鞘脂类和磷酸甘油酯。脂肪酸的合成始于通过乙酰基辅酶A羧化酶将乙酰基辅酶A转化为丙二酸单酰辅酶A或通过乙酰基转酰基酶将乙酰基辅酶A转化为乙酰基ACP。这两种产物分子在缩合反应后共同形成了乙酰乙酰基ACP,这种分子经一系列缩合、还原和脱水反应转化,因而获得了具有所需链长度的饱和脂肪酸分子。从这些分子产生不饱和脂肪酸的过程由特异性去饱和酶通过分子氧有氧性地催化,或者无氧性地催化(关于在微生物中合成脂肪酸,见F.C.Neidhardt等(1996)E.coli and Salmonella.ASM Press:Washington,D.C.第612-636页和其中所引用的参考文献;Lengeler等(Ed.)(1999)Biology of Procaryotes.Thieme:Stuttgart,New York和其中的参考文献及Magnuson,K.,等(1993)MicrobiologicalReviews 57:522-542和其中的参考文献)。为进一步实施延伸步骤,所得的与磷脂结合的脂肪酸必须返回至脂肪酸辅酶A酯库。酰基辅酶A:溶血磷脂酰基转移酶实施此步骤。而且这些酶能够将已延伸的脂肪酸从辅酶A酯再次转移至磷脂。根据需要可以重复这种反应顺序。
用于生物合成PUFA的前体的例子是油酸、亚油酸和亚麻酸。必须将C18碳的脂肪酸延伸至C20和C22以获得二十和二十二链型的脂肪酸。在方法中所用的去饱和酶如Δ12、ω3、Δ4、Δ5、Δ6和Δ8去饱和酶和/或Δ5、Δ6、Δ9延伸酶的协助下,可以获得花生四烯酸、二十碳五烯酸、二十二碳五烯酸或二十二碳六烯酸,有利地获得二十碳五烯酸和/或二十二碳六烯酸并且可在涉及食品、饲料、化妆品或药品方面的多种用途中应用。以上提到的酶可用于制备脂肪酸分子中具有至少两个双键,有利地具有至少三、四、五或六个双键的C20和/或C22脂肪酸,优选地制备在脂肪酸分子中有利地具有四个、五个或六个双键的C20或C22脂肪酸。去饱和可在延伸所讨论的脂肪酸之前或之后发生。这是为什么去饱和酶活性的产物和其它可能的去饱和延伸步骤的产物产生了具有更高去饱和程度的优选PUFA的原因,包括将C20至C22脂肪酸进一步延伸为如γ-亚麻酸、二高γ-亚麻酸、花生四烯酸、硬脂艾杜糖酸、二十碳四烯酸或二十碳五烯酸的脂肪酸。本发明方法中所用的去饱和酶和延伸酶的底物是C16、C18或C20脂肪酸例如亚油酸、γ-亚麻酸、α-亚麻酸、二高γ-亚麻酸、二十碳四烯酸或硬脂艾杜糖酸。优选的底物是亚油酸、γ-亚麻酸和/或α-亚麻酸、二高γ-亚麻酸、花生四烯酸、二十碳四烯酸或二十碳五烯酸。脂肪酸中具有至少两、三、四、五或六个双键的所合成的C20或C22脂肪酸在本发明的方法中以游离脂肪酸形式或以其酯形式如以其甘油酯形式获得。
将术语“甘油酯”理解为意指用一、二或三个羧基所酯化的甘油(单酰甘油、二酰甘油或三酰甘油)。将“甘油酯”理解为意指多种甘油酯的混合物。甘油酯或甘油酯的混合物可以包含其它添加物例如游离脂肪酸、抗氧化剂、蛋白质、糖类、维生素和/或其它物质。
为了本发明的目的,也将“甘油酯”理解为意指甘油衍生物。除以上所描述的脂肪酸甘油酯以外,这些甘油衍生物还包括甘油磷脂和甘油糖脂。可在本上下文中提到的优选的例子为甘油磷脂如卵磷脂(磷脂酰胆碱)、心磷脂、磷脂酰甘油、磷脂酰丝氨酸和烷基酰基甘油磷脂。
而且随后脂肪酸必须易位至不同的修饰位点并且掺入三酰甘油酯贮藏脂。脂类合成的又一个重要步骤是例如通过甘油脂肪酸酰基转移酶将脂肪酸转移至极性首基(见Frentzen,1998,Lipid,100(4-5):161-166)。
关于植物脂肪酸生物合成、去饱和、脂类新陈代谢和脂类化合物的膜运输、β氧化、脂肪酸修饰和辅因子、三酰甘油酯贮藏和三酰甘油酯组装的发表物,包括其中的参考文献参见如下文章:1997年JK Setlow编辑的《Genetic Engeneering》19第149-166页Kinney;Ohlrogge和Browse,1995,Plant Cell 7:957-970;Shanklin和Cahoon,1998,Annu.Rev.Plant Physiol.Plant Mol.Biol.49:611-641;1996年JK Setlow编辑的《Genetic Engeneering》18第111-13页Voelker;Gerhardt,1992,Prog.Lipid R.31:397-417;Gühnemann-Schfer和Kindl,1995,Biochim.Biophys Acta 1256:181-186;Kunau等,1995,Prog.Lipid Res.34:267-342;Rockville美国植物生理学学会1993年Murata和Somerville编辑的《Biochemistry and Molecular Biology of Membrane and StorageLipids of Plants》第150-158页Stymne等;Murphy和Ross 1998,PlantJournal.13(1):1-16。
方法中所产生的PUFA包含高等动物不再能合成并且因此必须摄入的一组分子或者高等动物不再能自行足量地合成并且因此必须摄入额外量的一组分子,虽然这类PUFA易于由其它生物例如细菌合成,但是猫不再能合成花生四烯酸。
为了本发明目的将磷脂理解为意指磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、磷脂酰甘油和/或磷脂酰肌醇,有利地为磷脂酰胆碱。术语“产生”或“生产量(productivity)”在本领域内为公知并且包含在特定时间段内和在特定发酵体积内形成的发酵产物(式I化合物)的浓度(例如kg产物每小时每升)。这个术语还包含在植物细胞或植物中的生产量,也即相对于这种细胞或植物中所有脂肪酸含量,方法中所产生的目的脂肪酸的含量。术语“产生效率”包含为获得特定产量所需的时间(如细胞需要用于建立精细化学品的某种流通量速率的时间)。术语产量或产物/碳产量为本领域公知并且包含将碳源转化为产物(即精细化学品)的效率。例如通常将产量表述为每公斤碳源kg产物。通过增加化合物的产量或产生,增加了在特定培养基中经特定时间段所获得这种化合物的分子的量或所获得的该化合物的合适分子的量。术语“生物合成”或“生物合成途径”为本领域所知并且包含例如在多步骤且受强烈调控的过程中通过细胞从中间体合成化合物,优选地合成有机化合物。术语“分解代谢或分解代谢的途径”为本领域所知并且包含例如在多步骤且受强烈调控的过程中由细胞切割化合物,优选地切割有机化合物以产生分解代谢产物(以更为通俗的术语描述,较小的或复杂度较低的分子)。术语代谢为本领域所知并且包含生物中发生的全部生物化学反应。某种化合物的代谢(例如脂肪酸的代谢)因此包含与这种化合物有关的细胞中该化合物全部的生物合成途径、修饰途径和分解途径。
在又一个实施方案中,SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183中所代表的本发明的核酸分子衍生物编码与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:132、SEQ ID NO:134或SEQ ID NO:184的全长氨基酸序列具有至少40%,有利地具有约50或60%,有利地具有至少约60或70%和更优选地具有至少约70或80%、80至90%、90至95%以及最为优选地具有至少约96%、97%、98%、99%或更高同源性(同一性)的蛋白质。计算了涵盖整个氨基酸或核酸序列区域的同源性。作为GCG软件包[Genetics Computer Group,575 Science Drive,Madison,Wisconsin,USA 53711(1991)]的部分的PileUp程序(J.Mol.Evolution.,25,351-360,1987,Higgins等,CABIOS,5 1989:151-153)或Gap和BestFit程序[Needleman和Wunsch(J.Mol.Biol.48;443-453(1970)和Smith及Waterman(Adv.Appl.Math.2;482-489(1981))用于序列比对。使用BestFit程序和如下设置:缺口权重:50,长度权重:3,平均匹配:10.000和平均错配:0.000确定了如以上以百分率所显示的覆盖全序列的序列同源性值。除非另外说明,这些设置总是用作标准设置用于序列比对。
本发明进一步包含由于遗传密码子简并而不同于SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133 或SEQ ID NO:183中所示诸核苷酸序列之一并且因此编码与SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183中所示那些核苷酸序列编码相同的Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶或Δ6延伸酶的核酸分子(和其部分)。
除了在SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183中所示的Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶或Δ6延伸酶以外,技术人员将认识到引起Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶和/或Δ6延伸酶的氨基酸序列改变的DNA序列多态性可在群体中存在。Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶和/或Δ6延伸酶基因中的这类遗传多态性可因为天然变异在群体中的个体之间存在。这些天然变体通常在Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶和/或Δ6延伸酶的核苷酸序列中产生1至5%的变异。本发明将包含这些Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶和/或Δ6延伸酶中的每一种和每一个核苷酸变异以及所导致的氨基酸多态性,它们是天然变异的结果并且不改变这些酶的功能活性。
有利于本发明方法的核酸分子由于与Δ12去饱和酶、ω3去饱和酶、Δ5延伸酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶和/或Δ6延伸酶核酸序列的同源性可以遵循标准杂交技术,使用这些酶的核酸序列或其部分为探针在严格杂交条件下分离。例如本上下文中可使用这样的经分离的核酸分子,其中所述核酸分子长至少15个核苷酸并且与包含SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183的核苷酸序列的核酸分子在严格条件下杂交。具有至少25、50、100、200或更多个核苷酸的核酸也可以使用。如本上下文中所用的“在严格条件下杂交”用以描述杂交和洗涤条件,通常彼此具有至少60%同源性的核苷酸序列在这样的条件下可保持相互杂交。优选的杂交条件是这样:彼此具有至少约65%,优选至少约70%和尤其优选至少75%或更高同源性的序列间通常保持相互杂交。这些严格条件为技术人员所知并且在例如1989年N.Y.的John Wiley& Sons出版社的《Current Protocols in Molecular Biology》第6.3.1-6.3.6中描述。一个优选的非限制性严格杂交条件的例子是在6×氯化钠/柠檬酸钠(SSC)中约45℃时杂交,然后是一个或多个于0.2x SSC、0.1%SDS中50至65℃的洗涤步骤。技术人员知道根据核酸类型和例如当存在有机溶剂时,温度和缓冲剂浓度在杂交条件中是不同的。在“标准杂交条件”下,例如根据核酸类型,杂交温度在0.1至5×SSC(pH 7.2)浓度的缓冲水溶液中处于42℃和58℃之间。如果在以上提到的缓冲液中存在有机溶剂例如50%甲酰胺,标准条件下的杂交温度是大约42℃。对于DNA:DNA杂交体的杂交条件例如是0.1×SSC和20℃至45℃,优选地为30℃至45℃。例如DNA:RNA杂交体的杂交条件是0.1×SSC和30℃至55℃,优选地为45℃至55℃杂交。以上提到的杂交条件通过长约100bp(碱基对)并且G+C含量为50%的核酸在无甲酰胺的情况下测定。技术人员以上面提到的教材或基于如1989年Cold Spring Harbor Laboratory出版社Sambrook等的《Molecular Cloning》;1985年牛津的牛津大学出版社中IRL Press出版社Hames和Higgins编辑《Nucliec AcidHybridization:A Practical Approach》;1991年牛津的牛津大学出版社中IRL Press出版社Brown编辑的《Essential Molecular Biology:A PracticalApproach》所述知道如何确定所需的杂交条件。
为了确定两个氨基酸序列(如SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:132、SEQ ID NO:134或SEQ ID NO:184序列中的一个序列)或两种核酸(例如SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183)的同源性(同源性)百分数,将序列书写为使一个序列处于另一个序列下以便优化比较(例如可向一个蛋白质序列或一个核酸序列中导入缺口以便产生与另一个蛋白质或另一个核酸的优化比对)。然后比较相应氨基酸位置上或相应核苷酸位置上的氨基酸残基或核苷酸。如果一个序列的某个位置由另一个序列相应位置的相同氨基酸残基或相同核苷酸占据,则在此位置上这些分子同源(即氨基酸或核酸“同源性”如在本发明中所用对应于氨基酸或核酸“同源性”)。两个序列间的同源性百分数是两个序列间所共有的位置数目的函数(即同源性%=相同的位置数目/总位置数目×100)。因此将术语“同源性”和“同源性”视为同义。
编码与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:106、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:114、SEQ ID NO:132、SEQ ID NO:134或SEQ ID NO:184的蛋白质序列同源的Δ12去饱和酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ5延伸酶和/或Δ6延伸酶的经分离核酸分子可通过在/向SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183的核苷酸序列中导入一个或多个核苷酸的替代、添加或删除而产生,从而将一个或多个氨基酸的替代、添加或删除导入/到所编码的蛋白质中。序列SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183之一中的突变可通过标准技术如定点诱变和PCR介导的诱变导入。优选对所预测的一个或多个非关键氨基酸残基进行保守性氨基酸替代。在“保守性氨基酸替代”中,氨基酸残基由具有类似侧链的另一种氨基酸残基替代。本领域中具有类似侧链的氨基酸残基家族已经得到定义。这些家族包含具有碱性侧链(如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此Δ12去饱和酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ5延伸酶或Δ6延伸酶中所预测的非关键氨基酸残基由来自相同侧链家族的另一种氨基酸残基优选地替换。在另一个实施方案中,这种突变可备选地在编码Δ12去饱和酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ5延伸酶或Δ6延伸酶的完整序列或其部分中随机导入,如通过饱和诱变导入,并且将由此所得的突变体可通过重组表达此处所描述的Δ12去饱和酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ5延伸酶或Δ6延伸酶活性而筛选以鉴定保持了Δ12去饱和酶、ω3去饱和酶、Δ6去饱和酶、Δ5去饱和酶、Δ4去饱和酶、Δ5延伸酶或Δ6延伸酶活性的突变体。在SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183中的序列之一经过诱变后,这种诱变后序列所编码的蛋白质可重组表达,并且所编码蛋白质的活性可经例如本文中所描述的试验测定。
本发明还涉及这样的非人类的转基因生物,其包含本发明的核酸SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:87、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:105、SEQ ID NO:107、SEQ ID NO:109、SEQ ID NO:113、SEQ ID NO:131、SEQ ID NO:133或SEQ ID NO:183或者包含含有本发明这些核酸序列的基因构建体或载体。有利的非人类生物是微生物、非人类动物或植物,尤其优选为植物。
本发明通过以下实施例更详细地说明,这不得解释为作为限制性的说明。本专利申请中所引用的参考文献、专利申请、专利和已公开的专利申请的全部内容此处引用作为参考。
实施例
实施例1:通用的克隆方法
克隆方法例如限制性切割、琼脂糖凝胶电泳、纯化DNA片段、核酸转移至硝酸纤维素膜和尼龙膜、连接DNA片段、转化大肠杆菌细胞、细菌培养和重组DNA的序列分析按照Sambrook等(1989)(Cold SpringHarbor Laboratory Press:ISBN 0-87969-309-6)所描述实施。
实施例2:重组DNA的序列分析
重组DNA分子以ABI激光荧光DNA测序仪按照Sanger(Sanger等(1977)Proc.Natl.Acad.Sci.USA74,5463-5467)的方法测序。通过聚合酶链反应获得的片段经过测序和核对以避免待表达的构建体中的聚合酶偏差。
实施例3:虹鳟基因的克隆
在对应于本申请所详述的延伸酶基因的蛋白质序列中对保守区域的搜索鉴定了两个在Genebank序列数据库中具有相应基序的序列。
基因名称 | Genbank No. | 氨基酸 |
OmELO2 | CA385234,CA364848,CA366480 | 264 |
OmELO3 | CA360014,CA350786 | 295 |
虹鳟的总RNA用来自Qiagen的RNAeasy试剂盒(Valencia,CA,美国)分离。聚腺苷酸化RNA(mRNA)以寡聚-dT-纤维素从这种总RNA中分离(Sambrook等,1989)。这种RNA使用来自Promega的逆转录系统试剂盒进行逆转录并且将所合成的cDNA克隆至载体λZAP(lambdaZAP Gold,Stratagene)。然后根据制造商的说明将所述cDNA去包装以产生质粒DNA。然后将这种cDNA质粒文库用于克隆表达质粒的PCR。
实施例4:用于酵母中异源表达的表达载体的克隆
以下寡核苷酸用于克隆两个在酵母中异源表达的序列的PCR反应:
引物 核苷酸序列
5’f*OmELO2 5’aagcttacataatggcttcaacatggcaa(SEQ ID NO:179)
3’r*OmELO2 5’ggatccttatgtcttcttgctcttcctgtt(SEQ ID NO:180)
5’fOmELO3 5’aagcttacataatggagacttttaat(SEQ ID NO:181)
3’rOmELO3 5’ggatccttcagtcccccctcactttcc(SEQ ID NO:182)
f:正向引物,r:反向引物
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10×缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶HindIII和BamHI 37℃温育2小时。酵母表达载体pYES3(Invitrogen)以同样方式温育。此后经琼脂糖凝胶电泳分离812bp的PCR产物、905bp的PCR产物和载体并且切下相应的DNA片段。然后这些DNA以Qiagen凝胶纯化试剂盒遵循制造商的说明纯化。此后连接载体和延伸酶的cDNA。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pYES3-OmELO2和pYES3-OmELO3经测序证实并且通过电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。pYES3作为对照进行平行转化。此后将酵母在补加了2%葡萄糖的撤除色氨酸的基本培养基上铺板。能在无色氨酸的培养基内生长的细胞包含相应的质粒pYES3、pYES3-OmELO2(SEQ ID NO:51)和pYES3-OmELO3(SEQID NO:53)。在选择后,对每种情况挑选了两个转化体用于进一步的功能性表达。
实施例5:植物中种子特异性表达的表达载体的克隆
生成基于pSUN-USP的另一个转化载体以转化植物。使用以下引物对将NotI切割位点导入编码序列的5′和3′端以实现此目的:
PSUN-OmELO2
正向引物:5′-GCGGCCGCATAATGGCTTCAACATGGCAA(SEQID NO:175)
反向引物:3′-GCGGCCGCTTATGTCTTCTTGCTCTTCCTGTT(SEQ ID NO:176)
PSUN-OMELO3
正向引物:5′-GCGGCCGCataatggagacttttaat(SEQ ID NO:177)
反向引物:3′-GCGGCCGCtcagtcccccctcactttcc(SEQ ID NO:178)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以向样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pSUN-OmELO2和pSUN-OmELO3经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:
5′-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-3′,SEQ ID NO:174)通过遵循标准方法的PCR反应扩增。将PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例6:从酵母和种子中提取脂类
遗传性修饰对植物、真菌、藻类、纤毛虫的影响或对产生目的产物(例如脂肪酸)的影响可通过将经修饰的微生物或经修饰的植物在合适条件下(例如那些以上所描述的条件下)生长并且分析用于产生升高的目的产物(即脂类或脂肪酸)的培养基和/或细胞性成分而测定。这些分析的技术为技术人员所知并包含光谱学、薄层层析、多种染色方法、酶和微生物学方法以及分析性层析如高效液相层析(例如参见:Ullman,Encyclopedia ofIndustrial Chemistry,Vol.A2,p.89-90和p.443-613,VCH:Weinheim(1985);FaHon,A.等人,(1987)“Applications of HPLC in Biochemistry“,在:Laboratory Techniques in Biochemistry and Molecular Biology,Vol.17一书中;Rehm等人(1 993)Biotechnology,Vol.3,Chapter III:“Productrecovery and purification“,p.469-714,VCH:Weinheim;Belter,P.A.等人(1988)Bioseparations:downstream processing for Biotechnology,JohnWiley and Sons;Kennedy,J.F.和Cabral,J.M.S.(1992)Recovery processesfor biological Materials,John Wiley and Sons;Shaeiwitz,J.A.和Henry,J.D.(1988)Biochemical Separations,在:Ullmann’s Encyclopedia ofIndustrial Chemistry,Vol.B3;Chapter 11,p.1-27,VCH:Weinheim一书中;以及Dechow,F.J.(1989)Separation and purification techniques inbiotechnology,Noyes Publications)。
除以上提到的方法,植物脂类可如Cahoon等(1999)Proc.Natl.Acad.Sci.USA 96(22):12935-12940和Browse等(1986)Analytic Biochemistry152:141-145所描述从植物材料中提取。脂类或脂肪酸的定性和定量分析由Christie,William W.,Advances in Lipid Methodology,Ayr/Scotland:Oily Press(Oily Press Lipid Library;2);Christie,William W.,GasChromatography and Lipids.A Practical Guide-Ayr,Scotland:Oily Press,1989,Repr.1992,IX,307 pp.(Oily Press Lipid Library;1);“Progress inLipid Research,Oxford:Pergamon Press,1(1952)-16(1977)在题为Progress in the Chemistry of Fats and Other Lipids CODEN的文章中描述。
除了测量发酵的终产物以外,还可分析用于产生目的化合物的代谢途径中的其它成分例如中间体和副产物以测定该化合物的总生产效率。分析方法包括测量培养基中营养物的量(例如糖、碳水化合物、氮源、磷酸盐和其它离子),测量生物量的组成和增长,分析生物合成途径中的常规代谢物的产生并且测量发酵期间所产生的气体。用于这些测量的标准方法在IRL Press出版社P.M.Rhodes和P.F.Stanbury编辑的《Applied MicrobialPhysiology;A Practical Approach》(ISBN:0199635773)的第103-129页、第131-163页和第165-192页以及其中所引用的参考文献中描述。
一个分析的例子是脂肪酸的分析(缩略语:FAME,脂肪酸甲酯;GC-MS,气液色谱/质谱;TAG,三酰甘油酯;TLC,薄层层析)。
可如Christie及其中的参考文献(1997年Dundee的Oily Press出版社Christie的《Advances on Lipid Methodology》第四版中第119-169页;1998年Gaschromatographie-Massenspektrometrie-Verfahren[GasChromatography/mass spectrometric methodsl,Lipide 33:343-353)所数次描述的那样使用分析性标准方法:GC、GC-MS或TLC清楚无误地检测脂肪酸产物的存在。
欲分析的材料可通过超声处理、玻璃磨中研磨、液氮和研磨或者通过其它可利用的方法加以破碎。这种材料在破碎后必须离心。在蒸馏水中重悬沉淀,100℃加热10分钟,冰上冷却并且再次离心,然后在含0.5M硫酸和2%二甲氧丙烷的甲醇溶液中90℃提取1小时,由此产生了已水解的油和脂类化合物,产生了已转甲基化的脂类。这些脂肪酸甲酯在石油醚中提取并且最后通过使用毛细管柱(Chrompack,WCOT Fused Silica,CP-Wax-52 CB,25μm,0.32mm)经20分钟的170℃至240℃之间的温度梯度并且在240℃维持5分钟加以GC分析。得到的脂肪酸甲酯的身份必须使用可从商业来源获得的标准品(即Sigma)确定。
首先通过往返于研杵和研钵之间机械地均化植物材料以使其更便于提取。随后100℃加热10分钟并且在冰上冷却后再次离心。细胞沉淀以1M甲醇硫酸和2%二甲氧丙烷90℃水解一小时并且将脂类转甲基化。得到的脂肪酸甲酯(FAME)在石油醚中抽提。所提取的FAME通过使用毛细管柱(Chrompack,WCOT Fused Silica,CP-Wax-52 CB,25μm,0.32mm)的气液色谱经20分钟的170℃至240℃之间的温度梯度并且在240℃维持5分钟加以分析。脂肪酸甲酯的身份通过与相应FAME标准品(Sigma)的比较确认。双键的身份和位置通过使FAME混合物适当地化学性衍生以产生例如4,4-二甲氧唑啉衍生物(Christie,1998)经GC-MS进一步分析。
对如实施例4中所描述的质粒pYES3、pYES3-OmELO2和pYES3-OmELO3转化了的酵母进行如下分析:
来自主培养的酵母细胞经离心(100×g、10分钟、20℃)收获并用pH 8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)由酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME通过石油醚(PE)提取两次。为了除去未衍生的脂肪酸,在每种情况下,有机相以2ml pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。此后PE相用Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在Hewlett-Packard 6850气相色谱火焰电离探测器内的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)内分离。GLC分析的条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250℃并且最后250℃维持10分钟(保持)。
通过与相应脂肪酸标准品(Sigma)的保留时间比较鉴定信号。
例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585;Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例7:OmELO2和OmELO3的功能特征
OmELO2未表现延伸酶活性,而OmELO3表现了对不同底物的明显活性。OmElo3的底物特异性在表达后并且喂饲了多种脂肪酸(图2)后加以测定。所喂饲的底物可大量在所有转基因酵母中检测到。所有转基因酵母均显示合成了作为OmElo3反应产物的新脂肪酸。这意味基因OmElo3的功能性表达已为可能。
图2显示OmElo3具有高度特异地导致带有一个ω3双键的Δ5和Δ6脂肪酸延伸的底物特异性。而且ω6脂肪酸(C18和C20)也可以较低的特异性延伸。OmElo3的最佳底物(高达66%的延伸)是硬脂艾杜糖酸(C18:4ω3)和二十碳五烯酸(C20:5ω3)。
实施例8:酵母中DHA合成的重建。
通过共表达OmElo3与细小裸藻Δ4去饱和酶或通过共表达OmElo3与三角褐指藻Δ5去饱和酶和细小裸藻Δ4去饱和酶由EPA(20:5ω3)或硬脂艾杜糖酸(18:4ω3)开始实施DHA(22:6ω3)生物合成的重建。为达此目的,还构建了表达载体pYes2-EgD4和pESCLeu-PtD5。上述已由pYes3-OtElo3(SEQ ID NO:55)转化的酵母株以pYes2-EgD4或同时以pYes2-EgD4和pESCLeu-PtD5进一步转化。在pYes3-OmELO/pYes2-EgD4株的情况下,经转化的酵母以补加了2%葡萄糖的无色氨酸和尿嘧啶的完全基本培养基琼脂平板选择;在pYes3-OmELO/pYes2-EgD4+pESCLeu-PtD5株的情况下,经转化的酵母以无色氨酸、尿嘧啶和亮氨酸的完全基本培养基琼脂平板选择。如上所示,酶的表达通过加入2%(w/v)半乳糖诱导。培养物进一步15℃温育120小时。
图3显示了已经喂饲了20:5ω3的转基因酵母中的脂肪酸曲线。在已由pYes3-OmElo3载体和空白载体pYes2转化的对照酵母(A)中,20:5ω3经过极高效地延伸以产生22:5ω3(65%延伸)。额外导入的EgΔ4去饱和酶导致22:5ω3转化为22:6ω3(DHA)。转基因酵母中的脂肪酸组成在图5中显示。在共表达OmElo3和EgD4后,酵母中检测到了高达3%的DHA。
在又一个共表达实验中,将OmElo3、EgD4和三角褐指藻Δ5去饱和酶(PtD5)共表达。所述转基因酵母喂饲了硬脂艾杜糖酸(18:4ω3)并且进行了分析(图4)。这些酵母的脂肪酸组成在图5中显示。所喂饲的脂肪酸18:4ω3通过OmElo3延伸以产生20:4ω3(60%延伸)。20:4ω3经过PtD5去饱和以产生20:5ω3。PtD5的活性是15%。而且有可能通过OmElo3将20:5ω3延伸以产生22:5ω3。此后新近合成的22:5ω3经去饱和成为22:6ω3(DHA)。高达0.7%的DHA在这些实验中获得。
从这些实验中可看出本发明中所用的序列OmElo3、EgD4和PtD5适于真核细胞中DHA的产生。
实施例9:转基因植物的产生
a)转基因油菜籽植物的产生(Moloney等,1992,Plant CellReports,8:238-242的改良方法)
根癌农杆菌C58C1:pGV2260或大肠杆菌中的二元载体(Deblaere等,1984,Nucl.Acids.Res.13,4777-4788)可用于产生转基因油菜籽植物。为了转化油菜籽植物(Var.Drakkar,NPZ NordeutschePflanzenzucht,Hohenlieth,Germany),将阳性转化的农杆菌菌落在补加3%蔗糖(3MS培养基)的Murashige-Skoog培养基(Murashige和Skoog 1962 Physiol.Plant.15,473)中的过夜培养物1∶50稀释而使用。将新近发芽的无菌油菜籽植物的叶柄或下胚轴(在每一情况下约1cm2)与1∶50的农杆菌稀释物在培养皿中共同温育5-10分钟。然后这些叶柄或下胚轴在补加了0.8%Bacto琼脂的3MS培养基上黑暗中25℃共温育3天。然后将这些培养物在16小时光照/8小时黑暗中生长3天并且这种培养按照每周周期继续在补加了1.6g/L葡萄糖的500mg/L Claforan(头孢噻肟钠)、50mg/L卡那霉素、20μM苄氨基嘌呤(BAP)的MS培养基上生长。生长的芽转移至补加了2%蔗糖、250mg/L Claforan和0.8%Bacto琼脂的MS培养基中。若三周后根未发育,向培养基中加入作为生长激素用于生根的2-吲哚基丁酸。
已再生的苗在补加了卡那霉素和Claforan的2 MS培养基上获得;生根之后,将这些苗转移至培养料并且在受控的环境箱或温室中生长两周后,以便开花,并且收获成熟的种子及通过脂类分析法分析延伸酶的表达如Δ5延伸酶或Δ6延伸酶活性或ω3去饱和酶活性。具有含量已升高的多不饱和C20和C22脂肪酸的株系可经如此方式鉴定。
b)转基因亚麻子植物的产生
转基因亚麻子植物可通过例如Bell等,1999,In Vitro Cell.Dev.Biol.Plant.35(6):456-465的方法经粒子轰击产生。亚麻籽通常以例如Mlynarova等(1994),Plant Cell Report 13:282-285的方法经农杆菌介导的转化作用而转化。
实施例10:从鲜黄破囊壶菌ATCC34304和破囊壶菌属某些种中克隆Δ5延伸酶基因
通过比较本发明中所发现的多种延伸酶的蛋白质序列,有可能确定保守的核酸区域(组氨酸盒:His-Val-X-His-His、色氨酸盒:Met-Tyr-X-Tyr-Tyr)。在这些序列的辅助下筛选了鲜黄破囊壶菌ATCC34304和破囊壶菌属某些种的EST数据库以发现其它的Δ5延伸酶。发现了如下所列的新序列:
基因名称 | 核苷酸 | 氨基酸 |
BioTaurELO1 | 828bp | 275 |
TL16y2 | 831 | 276 |
用来自Qiagen的RNAeasy试剂盒(Valencia,CA,美国)分离了鲜黄破囊壶菌ATCC34304和破囊壶菌属某些种的总RNA。以PolyATract分离系统(Promega)从这些总RNA中分离mRNA。这些RNA使用Marathon cDNA扩增试剂盒(BD Biosciences)遵循制造商的说明逆转录并且连接了人工接头。然后将这种cDNA文库用于通过5’和3’RACE(cDNA末端快速扩增)方法PCR克隆表达质粒。
实施例11:用于酵母中异源表达的表达质粒的克隆
以下的寡核苷酸用于PCR反应以克隆酵母中异源表达的序列:
引物 核苷酸序列
5’f*BioTaurELO1 5’gacataatgacgagcaacatgag(SEQ ID NO:170)
3’r*BioTaurELO1 5’cggcttaggccgacttggccttggg(SEQ ID NO:171)
5’f*TL16y2 5’agacataatggacgtcgtcgagcagcaatg(SEQ ID NO:172)
3’r*TL16y2 5’ttagatggtcttctgcttcttgggcgcc(SEQ ID NO:173)
*f:正向引物,r:反向引物
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10×缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR混合物的组成(50μl):
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物BioTaurELO1(见SEQ ID NO:65)和TL16y2(见SEQ IDNO:83)与酵母表达载体pYES2.1-TOPO(Invitrogen)根据制造商说明21℃温育30分钟。这里通过T突出端和拓扑异构酶(Invitrogen)的活性向载体中连接PCR产物。在温育后转化大肠杆菌DH5α细胞。合适的克隆通过PCR鉴定,质粒DNA通过Qiagen DNAeasy试剂盒分离并且经测序证实。正确的序列随后经电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。以空白载体pYES2.1平行转化作为对照。此后将酵母在补加了2%葡萄糖的无尿嘧啶基本培养基上铺板。能在无尿嘧啶的培养基内生长的细胞包含相应的质粒pYES2.1、pYES2.1-BioTaurELO1和pYES2.1-TL16y2。在选择后,对每种情况挑选两个转化体用于进一步的功能性表达。
实施例12:植物中种子特异性表达的表达载体的克隆
产生了另一个基于pSUN-USP的转化载体以转化植物。为达此目的,利用以下引物对将NotI切割位点导入编码序列的5′和3′端:
PSUN-BioTaurELO1
正向引物:5‘-GCGGCCGCATAATGACGAGCAACATGAGC(SEQID NO:166)
反向引物:3‘-GCGGCCGCTTAGGCCGACTTGGCCTTGGG(SEQID NO:167)
PSUN-TL16y2
正向引物:
5’-GCGGCCGCACCATGGACGTCGTCGAGCAGCAATG(SEQ IDNO:168)
反向引物:
5’-GCGGCCGCTTAGATGGTCTTCTGCTTCTTGGGCGCC(SEQID NO:169)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后通过Qiagen凝胶纯化试剂盒遵循制造商说明将DNA纯化。此后将载体和PCR产物连接。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pSUN-BioTaurELO1和pSUN-TL16Y2经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。DerUSP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-3’,SEQ ID NO:165)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
如实施例6中所描述从酵母和种子中提取脂类。
实施例13:BioTaurELO1和TL16y2的功能特征
BioTaurELO1的底物特异性在表达和喂饲多种脂肪酸后加以测定(图6)。图6显示了以包含pYes2.1载体(对照)或包含Δ5延伸酶的载体pYes2.1-BioTaurELO1(BioTaur)的酵母株测定功能性和底物特异性的喂饲实验。在两个批量中,将200μM的γ-亚麻酸和二十碳五烯酸喂饲酵母温育培养基并且温育24小时。脂肪酸从酵母中提取之后经转甲基化并且以气相层析分离。从所喂饲的两种脂肪酸获得的延伸产物如箭头所示。
所喂饲的底物可大量地在所有转基因酵母中检测到。全部转基因酵母均显示合成了作为BioTaurELO1反应产物的新脂肪酸。这意味基因BioTaurELO1的功能性表达已为可能。
图6表明BioTaurELO1具有高度特异性地引起带有一个ω3双键的Δ5和Δ6脂肪酸延伸的底物特异性。而且ω6脂肪酸(C18和C20)也进一步地加以延伸。γ-亚麻酸(C18:3ω6)以65.28%的速率转化,硬脂艾杜糖酸(C18:4ω3)以65.66%的速率转化并且二十碳五烯酸(C20:5ω3)以22.01%的速率转化。多个饲喂实验的底物特异性在表3中显示(见本描述的末尾)。
当喂饲GLA和EPA时,GLA的转化率为65.28%。当喂饲等量的GLA和EPA时,EPA的转化率为9.99%。如果仅喂饲EPA,EPA的转化率为22.01%。当喂饲花生四烯酸(ARA)时,花生四烯酸也被转化。花生四烯酸的转化率为14.47%。硬脂艾杜糖酸(SDA)也被转化。在此情况下,硬脂艾杜糖酸转化率为65.66%。
TL16y2的功能性和底物特异性在表达后和喂饲多种脂肪酸后进行了测定了。表4显示了喂饲实验。这个喂饲实验按照例对BioTaurELO1描述的同样方式进行。喂饲的底物可在所有转基因酵母大量地检测到。全部转基因酵母均显示合成了作为TL16y2反应产物的新脂肪酸(图11)。这意味基因TL16y2的功能性表达已为可能。
表4:TL16y2在酵母中的表达
气相色谱分析的面积(以%表示) | |||||||||
质粒 | 脂肪酸 | C18:3(n-6) | C18:4(n-3) | C20:3(n-6) | C20:4(n-6) | C20:4(n-3) | C20:5(n-3) | C22:4(n-6) | C22:5(n-3) |
pYES | 250uM EPA | 13.79 | |||||||
TL16y2 | 250uM EPA | 25.81 | 2.25 | ||||||
pYES | 50uM EPA | 5.07 | |||||||
TL16y2 | 50uM EPA | 2.48 | 1.73 |
pYES | 250uM GLA | 8.31 | |||||||
TL16y2 | 250uM GLA | 3.59 | 10.71 | ||||||
pYES | 250uM ARA | 16.03 | |||||||
TL16y2 | 250uM ARA | 15.2 | 3.87 | ||||||
pYES | 250uM SDA | 26.79 | 0.35 | ||||||
TL16y2 | 250uM SDA | 7.74 | 29.17 |
将TL16y2的结果与对照比较,如表4所示,显示出以百分率表示的以下转化率:a)%转化率EPA(250μM):8%,b)%转化率EPA(50μM):41%,c)%转化率ARA:20.3%,d)%转化率SDA:79.4%,以及e)%转化率GLA:74.9%。
因此,TL16y2显示Δ5-、Δ6-和Δ8-延伸酶活性。其中对具有Δ6-双键的C18-脂肪酸具有最高活性。根据所喂饲的脂肪酸浓度,接下来延伸具有一个Δ5-或Δ8-双键的C20-脂肪酸。
实施例14:从Ostreococcus tauri中克隆基因
在本申请中所列具有Δ5延伸酶活性或Δ6延伸酶活性的延伸酶基因的辅助下通过搜索蛋白质序列中的保守序列,在Ostreococcus tauri序列数据库(基因组序列)中鉴定了两个具有相应基序的序列。如下是这两个序列:
基因名称 | SEQ ID | 氨基酸 |
OtELO1,(Δ5-延伸酶) | SEQ ID NO:67 | 300 |
OtELO2,(Δ6-延伸酶) | SEQ ID NO:69 | 292 |
OtElo1与斑马鱼(Danio rerio)延伸酶(GenBank AAN77156;约26%同源性)具有最高的相似性,同时OtElo与剑叶藓Elo(PSE)[约36%同源性]具有最大的相似性(用tBLASTn算法进行比较(Altschul等,J.Mol.Biol.1990,215:403-410)。
克隆程序按如下方式进行:
将稳定期的40ml Ostreococcus tauri培养物离心并且将沉淀在100μl双蒸水中重悬并且-20℃保存。基于PCR法扩增了相关的基因组DNA。相应的引物对以这样的方式选择即引物包含紧接起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。在每种情况下,使用1μl解冻的细胞、200μM dNTP、2.5U Taq聚合酶和每种引物100pmol于总计50μl体积中进行OtElo-DNA的扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃ 30秒、55℃ 1分钟和72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
实施例15:用于酵母中异源表达的表达质粒的克隆
为描述Ostreococcus tauri延伸酶的功能特征,将所讨论的诸DNA的可读框克隆至pYE2.1/V5-His-TOPO(Invitrogen)的半乳糖可诱导GAL1启动子的下游,产生了pOTE1和pOTE2。
酿酒酵母334株分别以载体pOTE1和pOTE2经电穿孔(1500V)转化。空白载体pYES2转化的酵母用作对照。在补加了2%葡萄糖的无尿嘧啶完全基本培养基(CMdum)琼脂平板上选择已转化的酵母。选择后,在每种情况下挑选三个转化体用于进一步的功能性表达。
在每种情况下,为表达Ot延伸酶,以所选择的转化体首先接种由5ml补加了2%(w/v)棉子糖但缺乏尿嘧啶的CMdum培养基组成的预培养基并且以每分钟200转30℃温育2天。然后以预培养物接种5ml补加了2%棉子糖和300μM多种脂肪酸的CMdum(尿嘧啶撤除)液体培养基至OD6000.05。酶的表达通过加入2%(w/v)的半乳糖诱导。培养物进一步20℃温育96小时。
实施例16:用于植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用以下引物对向编码序列的5′和3′端导入NotI切割位点。对应的引物序列从OtElo1和OtElo2的5′和3′区域衍生。
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pSUN-OtELO1和pSUN-OtELO2经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant trahsformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-3’,SEQ ID NO:164)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例17:OtELO1和OtELO2在酵母中的表达
用如实施例15中所述质粒pYES3、pYES3-OtELO1和pYES3-OtELO2转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
通过与相应的脂肪酸标准品(Sigma)的保留时间比较而鉴定信号。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例18:OtELO1和OtELO2的功能特征
OtElo1底物特异性在表达后和在喂饲多种脂肪酸后测定(表5)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为OtElo1反应产物的新脂肪酸。这意味基因OtElo1的功能性表达已为可能。
表4表明OtElo1具有窄范围的底物特异性。OtElo1只能延伸C20脂肪酸二十碳五烯酸(图7)和花生四烯酸(图8),但优选地延伸ω3去饱和的二十碳五烯酸。
表5:
脂肪酸底物 | 转化率(%) |
16:0 | - |
16:1Δ9 | - |
18:0 | - |
18:1Δ9 | - |
18:1Δ11 | - |
18:2Δ9,12 | - |
18:3Δ6,9,12 | - |
18:3Δ5,9,12 | - |
20:3Δ8,11,14 | - |
20:4Δ5,8,11,14 | 10.8±0.6 |
20:5Δ5,8,11,14,17 | 46.8±3.6 |
22:4Δ7,10,13,16 | - |
22:6Δ4,7,10,13,16,19 | - |
表5显示了与多种脂肪酸比较,延伸酶OtElo1对于在Δ5位置具有双键的C20-多不饱和脂肪酸的底物特异性。
经载体pOTE1转化的酵母在所述脂肪酸存在的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。每个值显示为平均值(n=3)±标准差。
OtElo2(SEQ IN NO:81)的底物特异性在表达后和喂饲多种脂肪酸后测定(表6)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为OtElo2反应产物的新脂肪酸。这意味基因OtElo2的功能性表达已为可能。
表6:
脂肪酸底物 | 转化率(%) |
16:0 | - |
16:1Δ9 | - |
16:3Δ7,10,13 | - |
18:0 | - |
18:1Δ6 | - |
18:1Δ9 | - |
18:1Δ11 | - |
18:2Δ9,12 | - |
18:3Δ6,9,12 | 15.3± |
18:3Δ5,9,12 | - |
18:4Δ6,9,12,15 | 21.1± |
20:2Δ11,14 | - |
20:3Δ8,11,14 | - |
20:4Δ5,8,11,14 | - |
20:5Δ5,8,11,14,17 | - |
22:4Δ7,10,13,16 | - |
22:5Δ7,10,13,16,19 | - |
22:6Δ4,7,10,13,16,19 | - |
表6显示了延伸酶OtElo2相对于多种脂肪酸的底物特异性。
经载体pOTE2转化的酵母在所述脂肪酸存在的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。每个值显示为平均值(n=3)±标准差。
表3中所示的酶活性清楚表明OtElo2为Δ6-延伸酶。
实施例19:从假矮海链藻中克隆基因
在如本申请中所详述的具有Δ5-延伸酶活性或Δ6-延伸酶活性的延伸酶基因的辅助下通过搜索蛋白质序列中的保守区域,可在假矮海链藻序列数据库(基因组序列)中鉴定到两个具有相应基序的序列。这两个序列如下:
基因名称 | SEQ ID | 氨基酸 |
TpELO1(Δ5-延伸酶) | 43 | 358 |
TpELO2(Δ5-延伸酶) | 59 | 358 |
TpELO3(Δ6-延伸酶) | 45 | 272 |
2L假矮海链藻培养物在f/2培养基中(《Culture of MarineInvertebrate Animals》中(纽约Plenum Press出版Smith,W.L.和ChanleyM.H编辑)中第29-60页Guillard,R.R.L.1975.Culture of phytoplanktonfor feeding marine invertebrates)于80E/cm2光密度下经14天长成。在细胞离心后,RNA用来自Qiagen的RNAeasy试剂盒(Valencia,CA,美国)遵循制造商说明分离。mRNA遵循制造商的说明使用Marathon cDNA扩增试剂盒(BD Biosciences)逆转录并且连接了人工接头。然后此cDNA文库用于通过5’和3’RACE(cDNA末端快速扩增)方法PCR克隆表达质粒。
实施例20:用于酵母中异源表达的表达质粒的克隆
所讨论的引物对以这样方式选择即所述引物具有毗邻起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。在每种情况下使用1μl cDNA、200μM dNTP、2.5U Advantage聚合酶和每种引物100pmol于总计50μl体积中实施TpElo DNA的扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃30秒、55℃ 1分钟和72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
用于PCR反应的以下寡核苷酸用于克隆在酵母中异源表达的序列
基因名称和SEQ ID NO: | 引物序列 |
TpELO1(Δ5-延伸酶),SEQID NO:59 | F:5’-accatgtgctcaccaccgccgtc(SEQ ID NO:158)R:5’-ctacatggcaccagtaac(SEQ ID NO:159) |
TpELO2(Δ5-延伸酶),SEQID NO:85 | F:5’-accatgtgctcatcaccgccgtc(SEQ ID NO:160)R:5’-ctacatggcaccagtaac(SEQ ID NO:161) |
TpELO3(Δ6-延伸酶),SEQID NO:45 | F:5’-accatggacgcctacaacgctgc(SEQ ID NO:162)R:5’-ctaagcactcttcttcttt(SEQ ID NO:163) |
*F=正向引物,R=反向引物
PCR产物与酵母表达载体pYES2.1-TOPO(Invitrogen)根据制造商说明21℃温育30分钟。这里通过T突出端和拓扑异构酶(Invitrogen)活性向载体连接PCR产物。温育后转化大肠杆菌DH5α细胞。合适的克隆通过PCR鉴定,质粒DNA通过Qiagen DNAeasy试剂盒分离并且经测序证实。然后将正确的序列经电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。平行转化空白载体pYES2.1作为对照。此后酵母在补加了2%葡萄糖的无尿嘧啶的基本培养基上铺板。因此能够在无尿嘧啶的培养基中生长的细胞包含相应的质粒pYES2.1、pYES2.1-TpELO1、pYES2.1-ELO2和pYES2.1-TpELO3。选择后,在每种情况下挑选了两个转化体用于进一步的功能性表达。
实施例21:克隆用于植物中种子特异性表达的表达质粒
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用以下引物对向编码序列的5′和3′端导入NotI切割位点:
PSUN-TPELO1
正向引物:5‘-GCGGCCGCACCATGTGCTCACCACCGCCGTC(SEQ ID NO:152)
反向引物:3‘-GCGGCCGCCTACATGGCACCAGTAAC(SEQ IDNO:153)
PSUN-TPELO2
正向引物:5‘-GCGGCCGCACCATGTGCTCATCACCGCCGTC(SEQ ID NO:154)
反向引物:3‘-GCGGCCGCCTACATGGCACCAGTAAC(SEQ IDNO:155)
PSUN-TPELO3
正向引物:5‘-GCGGCCGCaccatggacgcctacaacgctgc(SEQ ID NO:156)
反向:3‘-GCGGCCGCCTAAGCACTCTTCTTCTTT(SEQ ID NO:157)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pSUN-TPELO1、pSUN-TPELO2和pSUN-TPELO3经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCCGGATCTGCTGGCTATGAA-3’;SEQ ID NO:151)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
如实施例6中所描述从酵母和种子中提取脂类。
实施例22:TpELO1、TpELO2和TpELO3在酵母中的表达
用如实施例4中所描述的质粒pYES2、pYES2-TpELO1、pYES2-TpELO2和pYES2-TpELO3转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg、5分钟、20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例23:TpELO1和TpELO3的功能特征
TpElo1的底物特异性在表达后和喂饲多种脂肪酸之后测定(图9)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为TpElo1反应产物的新脂肪酸。这意味基因TpElo1的功能性表达已为可能。
表7表明TpElo1具有窄范围的底物特异性。TpElo1只能延伸C20脂肪酸二十碳五烯酸(图7)和花生四烯酸,但优选地延伸ω3去饱和的二十碳五烯酸。
已由载体pYES2-TpElo1转化的酵母于所述脂肪酸存在的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
表7:TpElo1在酵母中的表达。列1和列3是列2(喂饲250μM20:4Δ5,8,11,14)和列4(喂饲250μM 20:5Δ5,8,11,14,17)的对照反应。
表达 | 表达 | 表达 | 表达 | |
脂肪酸 | 1 | 2 | 3 | 4 |
16:0 | 18.8 | 17.8 | 25.4 | 25.2 |
16:1Δ9 | 28.0 | 29.8 | 36.6 | 36.6 |
18:0 | 5.2 | 5.0 | 6.8 | 6.9 |
18:1Δ9 | 25.5 | 23.6 | 24.6 | 23.9 |
20:4Δ5,8,11,14 | 22.5 | 23.4 | - | - |
22:4Δ7,10,13,16 | - | 0.4 | - | - |
20:5Δ5,8,11,14,17 | - | - | 6.6 | 6.5 |
22:5Δ7,10,13,16,19 | - | - | - | 0.9 |
%转化率 | 0 | 1.7 | 0 | 12.2 |
TpElo3的底物特异性在表达后和在喂饲多种脂肪酸之后测定(图10)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为TpElo3反应产物的新脂肪酸。这意味基因TpElo3的功能性表达已为可能。
表8表明TpElo3具有较窄范围的底物特异性。TpElo3只能延伸C18脂肪酸γ-亚麻酸和硬脂艾杜糖酸,但优选地延伸ω3去饱和的硬脂艾杜糖酸。
已由载体pYES2-TpElo3转化的酵母于所述脂肪酸存在的基本培养基中生长。然后,脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
表8:TpElo3在酵母中的表达。列1显示了未饲入的酵母的脂肪酸曲线。列2显示了对照反应。在列3至列6饲入了γ-亚麻酸、硬脂艾杜糖酸、花生四烯酸和二十碳五烯酸(每种脂肪酸各250μM)。
脂肪酸 | 1 | 2 | 3 | 4 | 5 | 6 |
16:0 | 17.9 | 20.6 | 17.8 | 16.7 | 18.8 | 18.8 |
16:1Δ9 | 41.7 | 18.7 | 27.0 | 33.2 | 24.0 | 31.3 |
18:0 | 7.0 | 7.7 | 6.4 | 6.6 | 5.2 | 6.0 |
18:1Δ9 | 33.3 | 16.8 | 24.2 | 31.8 | 25.5 | 26.4 |
18:2Δ9,12 | - | 36.1 | - | - | - | - |
18:3Δ6,9,12 | - | - | 6.1 | - | - | |
18:4Δ6,9,12,15 | - | - | - | 1.7 | - | |
20:2Δ11,14 | - | 0 | - | - | - | |
20:3Δ8,11,14 | - | - | 18.5 | - | - | |
20:4Δ8,11,14,17 | - | - | - | 10.0 | - | |
20:4Δ5,8,11,14 | - | - | - | - | 22.5 |
22:4Δ7,10,13,16 | - | - | - | - | 0 | |
20:5Δ5,8,11,14,17 | - | - | - | - | - | 17.4 |
22:5Δ7,10,13,16,19 | - | - | - | - | - | 0 |
%转化率 | 0 | 0 | 75 | 85 | 0 | 0 |
实施例24:用于酵母中Pi-ω3Des异源表达的表达质粒的克隆
为了在酵母中异源表达,将Pi-ω3Des的克隆以合适的特异性Pi-ω3Des引物通过PCR克隆至酵母表达载体pYES3。仅仅扩增了这个编码Pi-ω3Des蛋白质的基因的可读框;提供了具备两个向表达载体pYES3中克隆的切割位点的Pi-ω3Des基因可读框。
正向引物:5’-TAAGCTTACATGGCGACGAAGGAGG(SEQ IDNO:149)
反向引物:5’-TGGATCCACTTACGTGGACTTGGT(SEQ ID NO:150)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10×缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶HindIII和BamHI 37℃温育2小时。酵母表达载体pYES3(Invitrogen)以同样方式温育。此后经琼脂糖凝胶电泳分离了1104bp的PCR产物和载体并且切下了相应的DNA片段。然后DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和去饱和酶的cDNA。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pYES3-Pi-ω3Des经测序证实并且通过电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。平行转化pYES3作为对照。此后酵母在补加了2%葡萄糖的无色氨酸基本培养基上铺板。能够在无色氨酸的培养基内生长的细胞包含相应的质粒pYES3、pYES3-Pi-ω3Des。选择后,在每种情况下挑选了两个转化体用于进一步的功能表达。
实施例25:用于植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用以下引物对向编码序列的5′和3′端导入NotI切割位点:
PSUN-Pi-ω3Des
反向引物:3‘-GCGGCCGCTTACGTGGACTTGGTC(SEQ ID NO:147)
正向引物:5‘-GCGGCCGCatGGCGACGAAGGAGG(SEQ ID NO:148)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育4小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒pSUN-Piω3Des经测序证实。
实施例26:Pi-ω3Des在酵母中表达
将如实施例24中所描述的质粒pYES3或pYES3-Pi-ω3Des转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每种情况下为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例27:Pi-ω3Des的功能特征
在表达和喂饲多种脂肪酸后测定Pi-ω3Des的底物特异性(图12至18)。所喂饲的底物在全部转基因酵母中可大量地检测到,证明酵母摄取了这些脂肪酸。转基因酵母显示合成了作为Pi-ω3Des反应产物的新脂肪酸。这意味基因Pi-ω3Des的功能性表达已为可能。
图12表明Pi-ω3Des将亚油酸(18:2ω-6脂肪酸)去饱和为α亚麻酸(18:3ω-3脂肪酸)。脂肪酸甲酯通过对已由空白载体pYES2(图12A)或载体pYes3-Pi-ω3Des(图12 B)转化的完整细胞经酸甲醇分解作用合成。酵母于存在C18:2Δ9,12-脂肪酸(300μM)的基本培养基中生长。随后这些FAME通过GLC分析。
图13表明Pi-ω3Des使γ-亚麻酸(18:3ω-6脂肪酸)去饱和为硬脂艾杜糖酸(18:4ω-3脂肪酸)。脂肪酸甲酯通过对已由空白载体pYES2(图13A)或载体pYes3-Pi-ω3De(图13B)转化的完整细胞经酸甲醇分解作用合成。酵母于存在γ-C18:3Δ6,9,12-脂肪酸(300μM)的基本培养基中生长。随后这些FAME通过GLC分析。
图14表明Pi-ω3Des使C20:2-ω-6脂肪酸去饱和为C20:3-ω-3脂肪酸。脂肪酸甲酯通过对已由空白载体pYES2(图14A)或载体pYes3-Pi-ω3De(图14B)转化的完整细胞经酸甲醇分解作用合成。酵母于存在C20:2Δ11,14-脂肪酸(300μM)的基本培养基中生长。随后这些FAME通过GLC分析。
图15表明Pi-ω3Des使C20:3-ω-6脂肪酸去饱和为C20:4-ω-3脂肪酸。脂肪酸甲酯通过对已由空白载体pYES2(图15A)或载体pYes3-Pi-ω3De(图15B)转化的完整细胞经酸甲醇分解作用合成。酵母于存在C20:3Δ8,11,14-脂肪酸(300μM)的基本培养基中生长。随后这些FAME通过GLC分析。
图16表明Pi-ω3Des将花生四烯酸(C20:4-ω-6脂肪酸)去饱和为二十碳五烯酸(C20:4-ω-3脂肪酸)。
脂肪酸甲酯通过对已由空白载体pYES2(图16A)或载体pYes3-Pi-ω3De(图16B)转化的完整细胞经酸甲醇分解作用合成这些酵母于存在C20:4Δ5,8,11,14脂肪酸(300μM)的基本培养基中生长。随后这些FAME通过GLC分析。
图17表明Pi-ω3Des使二十二碳四烯酸(C22:4-ω-6脂肪酸)去饱和为二十二碳五烯酸(C22:5-ω-3脂肪酸)。脂肪酸甲酯通过对已由空白载体pYES2(图17A)或载体pYes3-Pi-ω3De(图17B)转化的完整细胞经酸甲醇分解作用合成。将酵母在C22:4Δ7,10,13,16脂肪酸(300μM)存在时于基本培养基中生长。随后这些FAME通过GLC分析。
Pi-ω3Des对多种脂肪酸的底物特异性可从图18中发现。已由载体pYes3-Pi-ω3Des转化的酵母于存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。每个值代表为三个测量的平均值。使用下式:[产物]/[产物]+[底物]×100计算转化率(%去饱和)。
如实施例9中所描述,Pi-ω3Des还可用于产生转基因植物。然后可从这些植物的种子中如实施例6中所描述提取脂类。
实施例28:从Ostreococcus tauri克隆去饱和酶基因
在保守基序(His盒,Domergue等人,2002,Eur.J.Biochem.269,4105-4113)的辅助下搜索蛋白质序列中的保守区域可在Ostreococcus tauri序列数据库(基因组序列)中鉴定五个具有相应基序的序列。这些序列如下显示:
基因名称 | SEQ ID | 氨基酸 | 同源性 |
OtD4 | SEQ ID NO:95 | 536 | Δ4-去饱和酶 |
OtD5.1 | SEQ ID NO:91 | 201 | Δ5-去饱和酶 |
OtD5.2 | SEQ ID NO:93 | 237 | Δ5-去饱和酶 |
OtD6.1 | SEQ ID NO:89 | 457 | Δ6-去饱和酶 |
OtFad2 | SEQ ID NO:107 | 361 | Δ12-去饱和酶 |
使用tBLASTn算法(Altschul等,J.Mol.Biol.1990,215:403-410)实施了发现单个基因间同源性的比对。
按如下方式进行克隆:
将稳定期的40ml Ostreococcus tauri培养物经离心并且沉淀在100μl双蒸水中重悬并-20℃保存。相关的基因组DNA基于PCR法扩增。相应的引物对以如此方式选择即引物包含紧接起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。在每一情况下使用1μl解冻的细胞、200μM dNTP、2.5 U Taq聚合酶和每种引物100pmol于总计50μl体积中实施了OtDes-DNA的扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃ 30秒、55℃ 1分钟和72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
以下的引物用于PCR:
OtDes6.1正向引物:5’ggtaccacataatgtgcgtggagacggaaaataacg3’(SEQ ID NO:145)
OtDes6.1反向引物:5’ctcgagttacgccgtctttccggagtgttggcc3’(SEQ ID NO:146)
实施例29:酵母中异源表达的表达质粒的克隆
为描述来自Ostreococcus tauri的去饱和酶OtDes6.1(=Δ6-去饱和酶)的功能特征,将位于pYES2.1/V5-His-TOPO(Invitrogen)的半乳糖可诱导启动子GAL1上游的DNA可读框克隆,产生了相应的pYES2.1-OtDes6.1克隆,以类似方式可克隆其它Ostreococcus去饱和酶基因。
酿酒酵母334株以载体pYES2.1-OtDes6.1经电穿孔(1500V)转化。由空白载体pYES2转化的酵母用作对照。已转化的酵母在补加了2%葡萄糖的无尿嘧啶的完全基本培养基(CMdum)琼脂平板上选择。选择后,在每一情况下挑选三个转化体用于进一步的功能性表达。
在每一情况下,为表达OtDes6.1去饱和酶,由5ml补加了2%(w/v)棉子糖的尿嘧啶撤除的CMdum培养基组成的预培养基首先以所选择的转化体接种并且每分钟200转30℃温育2天。然后对5ml补加了2%棉子糖和300μM多种脂肪酸的CMdum(尿嘧啶撤除)液体培养基接种预培养物至OD600 0.05。酶的表达通过加入2%(w/v)半乳糖诱导。培养物进一步20℃温育96小时。
实施例30:克隆用于植物中种子特异性表达的表达质粒
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用PCR向编码序列的5′和3′端导入NotI切割位点。对应的引物序列从去饱和酶的5′和3′区域衍生。
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离PCR产物和载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。为此目的,使用了来自Roche的快速连接试剂盒。得到的质粒经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3’,SEQ ID NO:144)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例31:OtDes6.1在酵母中的表达
经质粒pYES2和pYES2-OtDes6.1如实施例4中所描述转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每种情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例32:Ostreococcus去饱和酶的功能特征
去饱和酶的底物特异性可在酵母中表达酶后通过喂饲、使用多种酵母(见克隆去饱和酶基因、酵母表达的实施例)测定。对单种活性测定的描述可在WO 93/11245中对Δ15去饱和酶的描述;WO 94/11516中对Δ12去饱和酶的描述;WO 93/06712、US 5,614,393、US 5,614,393、WO 96/21022、WO 0021557和WO 99/27111中对Δ6去饱和酶的描述;Qiu等,2001,J.Biol.Chem.276,31561-31566对Δ4去饱和酶的描述;Hong等,2002,Lipids 37,863-868对Δ5去饱和酶的描述中找到。
表9显示了OtDes6.1去饱和酶对多种脂肪酸的底物特异性。OtDes6.1的底物特异性在表达后和喂饲多种脂肪酸后测定。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为OtDes6.1反应产物的新脂肪酸(图20)。这意味着功能性表达了基因OtDes6.1。
用载体pYes2-OtDes6.1转化的酵母于所述脂肪酸存在的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。每个值表示为平均值(n=3)±标准差。活性是使用公式底物/(底物+产物)*100]计算的转化率。
表9表明OtDes6.1去饱和酶对油酸和亚油酸(18:2和18:3)具有底物特异性,原因在于这些脂肪酸引起了最高的活性。相反,对油酸(18:1)和棕榈油酸(16:1)的活性基本不显著。优选转化亚油酸和亚麻酸表明这种去饱和酶适合于产生多不饱和脂肪酸。
表9
底物 | 活性(%) |
16:1Δ9 | 5.6 |
18:1Δ9 | 13.1 |
18:2Δ9,12 | 68.7 |
18:3Δ9,12,15 | 64.6 |
图20表明OtDes6.1转化了亚油酸。FAME通过气相色谱分析。所喂饲的底物(C18:2)转化为γ-C18:3。箭头指示起始材料和形成的产物。
图21表明当存在OtDes6.1时分别使亚油酸(LA)和α-亚麻酸(ALA)转化以产生γ-亚麻酸(GLA)和硬脂艾杜糖酸(STA)(图21A和C)。图21还显示当Δ6去饱和酶OtDes6.1与来自展叶剑叶藓的Δ6延伸酶PSE1(Zank等2002,Plant J.31:255-268)和来自三角褐指藻的Δ5去饱和酶PtD5(Domergue等2002,Eur.J.Biochem.269,4105-4113)共同存在时分别使亚油酸(LA)和α-亚麻酸(ALA)转化以产生二高γ-亚麻酸(DHGLA)和花生四烯酸(ARA,图21B)以及产生二高硬脂艾杜糖酸(DHSTA)和二十碳五烯酸(EPA,图21D)。图21清晰地表明当存在Δ6延伸酶PSE1时几乎定量地使Δ6去饱和酶OtDes6.1的反应产物GLA和STA分别延伸以产生DHGLA和DHSTA。接下来由Δ5去饱和酶PtD5进行去饱和以分别产生ARA和EPA同样是毫无困难的。大约25%-30%的延伸酶产物被去饱和(图21B和D)。
此处的表10为已克隆的Ostreococcus去饱和酶的概览。
Ostreococcus tauri去饱和酶 | |||||||
名称 | bp | aa | 同源性 | Cyt.B5 | His盒1 | His盒2 | His盒3 |
OtD4 | 1611 | 536 | Δ4-去饱和酶 | HPGG | HCANH | WRYHHQVSHH | QVEHHLFP |
OtD5.1 | 606 | 201 | Δ5-去饱和酶 | - | - | - | QVVHHLFP |
OtD5.2 | 714 | 237 | Δ5-去饱和酶 | - | - | WRYHHMVSHH | QIEHHLPF |
OtD6.1 | 1443 | 480 | Δ6-去饱和酶 | HPGG | HEGGH | WNSMHNKHH | QVIHHLFP |
OtFAD2 | 1086 | 361 | Δ12-去饱和酶 | - | HECGH | WQRSHAVHH | HVAHH |
实施例33:从假矮海链藻中克隆去饱和酶基因
在保守基序(His盒,参见基序)的辅助下搜索蛋白质序列中的保守区域可在假矮海链藻序列数据库(基因组序列)中鉴定六个具有相应基序的序列。序列如下:
基因名称 | SEQ ID | 氨基酸 | 同源性 |
TpD4 | SEQ ID NO:103 | 503 | Δ-4-去饱和酶 |
TpD5-1 | SEQ ID NO:99 | 476 | Δ-5-去饱和酶 |
TpD5-2 | SEQ ID NO:101 | 482 | Δ-5-去饱和酶 |
TpD6 | SEQ ID NO:97 | 484 | Δ-6-去饱和酶 |
TpFAD2 | SEQ ID NO:109 | 434 | Δ-12-去饱和酶 |
TpO3 | SEQ ID NO:105 | 418 | ω-3-去饱和酶 |
克隆程序按如下方式进行:
稳定期的40ml假矮海链藻培养物经过离心并且沉淀在100μl双蒸水中重悬并且-20℃保存。相关的基因组DNA基于PCR法扩增。以如此方式选择相应的引物对即引物包含紧接起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。在每一情况下使用1μl解冻的细胞、200μM dNTP、2.5U Taq聚合酶和每种引物100pmol于总计50μl体积中实施了TpDes-DNA的扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃30秒、55℃ 1分钟和在72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
实施例34:用于酵母中异源表达的表达质粒的克隆
为描述假矮海链藻去饱和酶的功能特征,将所讨论的DNA的可读框克隆于pYES2.1/V5-His-TOPO(Invitrogen)的半乳糖可诱导启动子GAL1的下游,产生了相应的pYES2.1克隆。
酿酒酵母334株以载体pYES2.1-TpDesatauresen经电穿孔(1500V)转化。经空白载体pYES2转化的酵母作为对照使用。已转化的酵母在补加了2%葡萄糖但缺乏尿嘧啶的完全基本培养基(CMdum)琼脂平板上选择。选择后,每种情况挑选三个转化体用于进一步的功能性表达。
在每一情况下,为了表达Tp去饱和酶,5ml由补加了2%(w/v)棉子糖但缺乏尿嘧啶的CMdum培养基组成的预培养基首先以所选择的转化体接种并且每分钟200转30℃温育2天。然后将5ml补加了2%棉子糖和300μM多种脂肪酸的CMdum(尿嘧啶撤除)液体培养基接种预培养物至OD600 0.05。酶的表达通过加入2%(w/v)半乳糖诱导。培养物在20℃下进一步温育96小时。
实施例35:植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用PCR向编码序列的5′和3′端导入NotI切割位点。对应的引物序列从去饱和酶的5′和3′区域衍生。
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离PCR产物和载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。使用了来自Roche的快速连接试剂盒以便连接。得到的质粒经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3’;SEQ ID NO:143)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例36:Tp去饱和酶在酵母中的表达
如实施例4中所描述由质粒pYES2和pYES2-Tp转化的酵母以如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例37:假矮海链藻去饱和酶的功能特征
去饱和酶的底物特异性可在酵母中表达后通过使用多种酵母的喂饲(见克隆去饱和酶基因、酵母表达的实施例)测定。对单种活性测定的描述可在WO 93/11245中对Δ15去饱和酶的描述;WO 94/11516中对Δ12去饱和酶的描述;WO 93/06712、US 5,614,393、US 5,614,393、WO 96/21022、WO 0021557和WO 99/27111中对Δ6去饱和酶的描述;Qiu等,2001,J.Biol.Chem.276,31561-31566对Δ4去饱和酶的描述;Hong等,2002,Lipids 37,863-868对Δ5去饱和酶的描述中找到。
单种去饱和酶的活性使用公式:底物/(底物+产物)*100]从转化率计算。
以下表11和12为经克隆的假矮海链藻去饱和酶的概览。
表11:经克隆的海链藻去饱和酶的长度和特征
去饱和酶 | cDNA(bp) | 蛋白质(aa) | Cyt.B5 | His盒1 | His盒2 | His盒3 |
TpD4 | 1512 | 503 | HPGG | HDGNH | WELQHMLGHH | QIEHHLFP |
TpD5-1 | 1431 | 476 | HPGG | HDANH | WMAQHWTHH | QVEHHLFP |
TpD5-2 | 1443 | 482 | HPGG | HDANH | WLAQHWTHH | QVEHHLFP |
TpD6 | 1449 | 484 | HPGG | HDFLH | WKNKHNGHH | QVDHHLFP |
TpFAD2(d12) | 1305 | 434 | - | HECGH | HAKHH | HVAHHLFH |
TpO3 | 1257 | 419 | - | HDAGH | WLFMVTYLQHH | HVVHHLF |
表12:经克隆的去饱和酶的长度、外显子、同源性和同源性
Des. | GDNA(bp) | 外显子1 | 外显子2 | 第一BlastHit | Hom./Iden. |
TpD4 | 2633 | 496-1314 | 1571-2260 | 破囊壶菌D4-des | 56%/43% |
TpD5-1 | 2630 | 490-800 | 900-2019 | 褐指藻D5-des | 74%/62% |
TpD5-2 | 2643 | 532-765 | 854-2068 | 褐指藻D5-des | 72%/61% |
TpD6 | 2371 | 379-480 | 630-1982 | 褐指藻D6-des | 83%/69% |
TpFAD2 | 2667 | 728-2032 | - | 褐指藻FAD2 | 76%/61% |
TpO3 | 2402 | 403-988 | 1073-1743 | 隐杆线虫Fad2 | 49%/28% |
来自Ostreococcus和海链藻的Δ12去饱和酶基因也可按以上提及的实施例类似地克隆。
实施例38:从非洲爪蟾和玻璃海鞘中克隆延伸酶基因
在本申请中所详述的具有Δ5-延伸酶活性或Δ6-延伸酶活性的延伸酶基因的辅助下,通过在基因数据库(Genebank)中搜索蛋白质序列内的保守区域(见共有序列SEQ ID NO:115和SEQ ID NO:116),可鉴定和分离其它生物来源的其它延伸酶。可使用合适的基序分别鉴定非洲爪蟾和玻璃海鞘来源的其它序列。这些序列如下显示:
基因名称 | 生物 | Genbank No. | SEQ IDNO: | 氨基酸 |
ELO(XI) | 非洲爪蟾 | BC044967 | 117 | 303 |
ELO(Ci) | 玻璃海鞘 | AK112719 | 119 | 290 |
非洲爪蟾cDNA克隆从NIH(国立健康研究所)[genetic and genomictools for Xenopus research:The NIH Xenopus initiative,Dev.Dyn.225(4),384-391(2002)]获得。
玻璃海鞘cDNA克隆从Kyoto大学[Satou,Y.,Yamada,L.,Mochizuki,Y.,Takatori,N.,Kawashima,T.,Sasaki,A.,Hamaguchi,M.,Awazu,S.,Yagi,K.,Sasakura,Y.,Nakayama,A.,Ishikawa,H.,Inaba,K.and Satoh,N.“A cDNA resource fromthe basal chordate Ciona intestinalis”JOURNAL Genesis 33(4),153-154(2002)]获得。
实施例39:用于酵母中异源表达的表达质粒的克隆
延伸酶DNA在每一情况下以1μl cDNA、200μM dNTP、2.5UAdvantage聚合酶和每种引物100pmol于总计50μl体积中扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃ 30秒、55℃ 1分钟和72℃2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
以下寡核苷酸用于克隆酵母中异源表达的序列的PCR反应:
基因名称和SEQ ID NO: | 引物序列 |
ELO(XI)SEQ ID NO:121SEQ ID NO:122 | F:5’-AGGATCC ATGGCCTTCAAGGAGCTCACATCR:5’-CCTCGAGTCAATGGTTTTTGCTTTTCAATGCACCG |
ELO(Ci),SEQ ID NO:123SEQ ID NO:124 | F:5’-TAAGCTT ATGGACGTACTTCATCGTR:5’-TCAGATCT TTAATCGGTTTTACCATT |
*F=正向引物,R=反向引物
PCR产物与酵母表达载体pYES2.1-TOPO(Invitrogen)根据制造商说明21℃温育30分钟。这里通过T突出端和拓扑异构酶(Invitrogen)活性向载体连入PCR产物。在温育后转化大肠杆菌DH5α细胞。合适的克隆通过PCR鉴定,质粒DNA通过Qiagen DNAeasy试剂盒分离并且经测序证实。然后将正确的序列经电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。平行转化空白载体pYES2.1作为对照。此后将酵母在补加了2%葡萄糖的尿嘧啶撤除基本培养基上铺板。能够在无尿嘧啶的培养基中生长的细胞包含相应的质粒pYES2.1、pYES2.1-ELO(XI)和pYES2.1-ELO(Ci)。选择后,在每种情况下挑选了两个转化体用于进一步的功能性表达。
实施例40:植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用以下引物对向编码序列的5′和3′端导入NotI切割位点:
pSUN-ELO(XI)
正向引物:
5‘-GCGGCCGCACC
ATGGCCTTCAAGGAGCTCACATC
(SEQ ID NO:125)
反向引物:
3‘-GCGGCCGCCT
TCAATGGTTTTTGCTTTTCAATGCACCG
(SEQ ID NO:126)
pSUN-ELO(Ci)
正向引物:5‘-GCGGCCGCACC
ATGGACGTACTTCATCGT
(SEQ ID NO:127)
反向引物:3‘-GCGGCCGCT
TTAATCGGTTTTACCATT
(SEQ ID NO:128)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。使用了来自Roche的快速连接试剂盒以便连接。得到的质粒pSUN-ELO(XI)和pSUN-ELO(Ci)经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacterium binaryvectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3’;SEQ ID NO:129))通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
如实施例6所述自酵母和种子提取脂类。
实施例41:ELO(XI)和ELO(Ci)在酵母中的表达
如实施例4中所描述的由质粒pYES2,pYES2-ELO(XI)和pYES2-ELO(Ci)转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例42:ELO(XI)和ELO(Ci)的功能特征
ELO(XI)的底物特异性在表达和在喂饲多种脂肪酸后测定(图22)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为ELO(XI)反应产物的新脂肪酸。这意味基因ELO(XI)功能性地得以表达。
表13表明ELO(XI)具有宽泛的底物特异性。C18脂肪酸和C20脂肪酸均得到延伸,优选地观察到了经Δ5和Δ6去饱和的脂肪酸。
经载体pYES2-ELO(XI)所转化的酵母于存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
表13:ELO(XI)在酵母中的表达。显示了不同起始物质(在每一情况下以250μM喂饲)的转化率。
起始物质 | ELO(XI)对起始物质的转化(%) |
16:0 | 3 |
16:1Δ9 | 0 |
18:0 | 2 |
18:1Δ9 | 0 |
18:2Δ9,12 | 3 |
18:3Δ6,9,12 | 12 |
18:3Δ5,9,12 | 13 |
18:3Δ9,12,15 | 3 |
起始物质 | ELO(XI)对起始物质的转化(%) |
18:4Δ6,9,12,15 | 20 |
20:3Δ8,11,14 | 5 |
20:3Δ11,14,17 | 13 |
20:4Δ5,8,11,14 | 15 |
20:5Δ5,8,11,14,17 | 10 |
22:4Δ7,10,13,16 | 0 |
22:6Δ4,7,10,13,16,19 | 0 |
ELO(Ci)的底物特异性在表达和在喂饲多种脂肪酸后测定(图23)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为ELO(Ci)反应产物的新脂肪酸。这意味基因ELO(Ci)得到功能性表达。
表14:ELO(Ci)在酵母中的表达。显示了不同起始物质(在每一情况下以250μM喂饲)的转化率。
起始物质 | ELO(Ci)对起始物质的转化(%) |
16:0 | 0 |
16:1Δ9 | 0 |
18:0 | 0 |
18:1Δ9 | 0 |
18:2Δ9,12 | 23 |
18:3Δ6,9,12 | 10 |
起始物质 | ELO(Ci)对起始物质的转化(%) |
18:3Δ5,9,12 | 38 |
18:3Δ9,12,15 | 25 |
18:4Δ6,9,12,15 | 3 |
20:3Δ8,11,14 | 10 |
20:3Δ11,14,17 | 8 |
20:4Δ5,8,11,14 | 10 |
20:5Δ5,8,11,14,17 | 15 |
22:4Δ7,10,13,16 | 0 |
22:6Δ4,7,10,13,16,19 | 0 |
表14表明ELO(Ci)具有宽泛的底物特异性。C18脂肪酸和C20脂肪酸均得到了延伸,优选地观察到了经Δ5和Δ6去饱和的脂肪酸。
经载体pYES2-ELO(Ci)所转化的酵母于存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
实施例43:从Ostreococcus tauri克隆基因
在此处所描述的具有Δ5-延伸酶活性或Δ6-延伸酶活性的延伸酶基因的辅助下,通过搜索蛋白质序列中的保守区域,在每种情况下可在Ostreococcus tauri序列数据库(基因组序列)中鉴定2个具有相应基序的序列。序列如下:
基因名称 | SEQ ID | 氨基酸 |
OtELO1,(Δ5-延伸酶) | SEQ ID NO:67 | 300 |
OtELO1.2,(Δ5-延伸酶) | SEQ ID NO:113 | 300 |
OtELO2,(Δ6-延伸酶) | SEQ ID NO:69 | 292 |
OtELO2.1,(Δ6-延伸酶) | SEQ ID NO:111 | 292 |
OtElo1和OtElo1.2表现了与斑马鱼来源(GenBank AAN77156;约26%同源性)的延伸酶最高程度的相似性,而OtElo2和OtElo2.1表现了与剑叶藓Elo(PSE)最高程度的相似性[约36%同源性](用tBLASTn算法进行比对(Altschul等,J.Mol.Biol.1990,215:403-410))。
延伸酶按如下方式克隆:
稳定期的40ml Ostreococcus tauri培养物经过离心并且沉淀在100μl双蒸水中重悬并-20℃保存。相关的基因组DNA基于PCR法扩增。以如此方式选择了相应的引物对即引物携有紧邻起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。在每一情况下使用1μl解冻的细胞、200μM dNTP、2.5U Taq聚合酶和每种引物100pmol于总计50μl体积中实施了OtElo-DNA的扩增。PCR的条件如下:首先95℃变性5分钟,其后为94℃ 30秒、55℃ 1分钟和72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
实施例44:酵母中异源表达的表达质粒的克隆
为描述Ostreococcus tauri延伸酶的功能特征,将所讨论的DNA的可读框克隆于pYES2.1/V5-His-TOPO(Invitrogen)的半乳糖可诱导启动子GAL1下游,产生了相应的pOTE1、pOTE1.2、pOTE2和pOTE2.1克隆。
酿酒酵母334株分别以载体pOTE1,pOTE1.2,pOTE2和pOTE2.1经电穿孔(1500V)转化。经空白载体pYES2转化的酵母用作对照。已转化的酵母在补加了2%葡萄糖但缺乏尿嘧啶的完全基本培养基(CMdum)琼脂平板上选择。选择后,在每种情况下挑选三个转化体用于进一步的功能性表达。
为表达Ot延伸酶,在每种情况下对5ml由补加了2%(w/v)棉子糖但缺乏尿嘧啶的CMdum培养基组成的预培养基首先以经选择的转化体接种并且每分钟200转30℃温育2天。然后将5ml补加了2%棉子糖和300μM多种脂肪酸的CMdum(尿嘧啶撤除)液体培养基以预培养物接种至OD6000.05。酶的表达通过加入2%(w/v)半乳糖诱导。培养物进一步20℃温育96小时。
实施例45:植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用PCR向编码序列的5′和3′端导入NotI切割位点。对应的引物序列从OtElo1、OtElo1.2、OtElo2和OtElo2.1的5′和3′区域衍生。
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离PCR产物和载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。使用了来自Roche的快速连接试剂盒以便连接。得到的质粒pSUN-OtElIO1、pSUN-OtELO1.2、pSUN-OtELO2和pSUN-OtELO2.2经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacteriumbinary vectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。USP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3’.SEQ ID NO:130)通过遵循标准方法的PCR反应扩增。这个PCR片段再用EcoRI/SalI切割并且插入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例46:OtElo1、OtElo1.2、OtElo2和OtELO2.2在酵母中的表达
如实施例15中所描述的由质粒pYES3,pYES3-OtELO1,pYES3-OtELO1.2,pYES3-OtELO2和pYES3-OtELO2.2转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例47:OtElo1、OtElo1.2、OtElo2和OtELO2.1的功能特征
OtElo1的底物特异性在表达和在喂饲多种脂肪酸后测定(表15)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为OtElo1反应产物的新脂肪酸。这意味基因OtElo1得到功能性地表达。
表15表明OtElo1和OtElo1.2具有窄的底物特异性。OtElo1和OtElo1.2只能延伸C20脂肪酸二十碳五烯酸(图24A、24B)和花生四烯酸(图25A、25B),但是优选延伸ω-3去饱和的二十碳五烯酸。
表15表明同多种脂肪酸比较,延伸酶OtElo1和OtElo1.2对Δ5位置具备双键的C20多不饱和脂肪酸具有底物特异性。
分别由载体pOtElo1和pOtElo1.2转化的酵母在存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
OtElo1(SEQ ID NO:81)和OtElo2.1(SEQ ID NO:111)的底物特异性在表达和在喂饲多种脂肪酸后测定(表16)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为OtElo2反应产物的新脂肪酸。这意味着功能性表达了基因OtElo2和OtElo2.1。
表15
脂肪酸底物 | 转化率(%)OtElo1 | 转化率(%)OtElo1.2 |
16:0 | - | - |
脂肪酸底物 | 转化率(%)OtElo1 | 转化率(%)OtElo1.2 |
16:1Δ9 | - | - |
18:0 | - | - |
18:1Δ9 | - | - |
18:1Δ11 | - | - |
18:2Δ9,12 | - | - |
18:3Δ6,9,12 | - | - |
18:3Δ5,9,12 | - | - |
20:3Δ8,11,14 | - | - |
20:4Δ5,8,11,14 | 10.8±0.6 | 38.0 |
20:5Δ5,8,11,14,17 | 46.8±3.6 | 68.6 |
22:4Δ7,10,13,16 | - | - |
22:6Δ4,7,10,13,16,19 | - | - |
表16显示了延伸酶OtElo2和OtElo2.1对多种脂肪酸的底物特异性。OtElo2.1的活性明显较高。
已分别由载体pOtElo2和pOtElo2.1转化的酵母在存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。
在表16中显示的酶活性清楚地表明OtElo2或OtElo2.1是Δ6延伸酶。
表16:
脂肪酸底物 | 转化率(%)OtElo2 | 转化率(%)OtELO2.2 |
16:0 | - | - |
16:1Δ9 | - | - |
16:3Δ7,10,13 | - | - |
18:0 | - | - |
18:1Δ6 | - | - |
18:1Δ9 | - | - |
18:1Δ11 | - | - |
18:2Δ9,12 | - | - |
18:3Δ6,9,12 | 15.3 | 55.7 |
18:3Δ5,9,12 | - | - |
18:4Δ6,9,12,15 | 21.1 | 70.4 |
20:2Δ11,14 | - | - |
20:3Δ8,11,14 | - | - |
20:4Δ5,8,11,14 | - | - |
20:5Δ5,8,11,14,17 | - | - |
22:4Δ7,10,13,16 | - | - |
22:5Δ7,10,13,16,19 | - | - |
22:6Δ4,7,10,13,16,19 | - | - |
图24A-D分别显示了OtElo1(B)和OtElo1.2(D)对二十碳五烯酸的延伸。对照(A、C)未显示延伸产物(22:5ω3)。
图25A-D分别显示了OtElo1(B)和OtElo1.2(D)对花生四烯酸的延伸。对照(A、C)未显示延伸产物(22:4ω6)。
实施例48:从细小裸藻和拟南芥菜中克隆基因
在本申请中所详述的具有Δ5延伸酶活性或Δ6延伸酶活性的延伸酶基因的辅助下,通过搜索蛋白质序列中的保守区域,可在序列数据库(Genbank,裸藻EST文库)中鉴定分别来自拟南芥菜和细小裸藻的具有相应基序的序列。序列如下:
基因名称 | SEQ ID | 氨基酸 |
EGY1019(细小裸藻) | SEQ ID NO:131 | 262 |
EGY2019(细小裸藻) | SEQ ID NO:133 | 262 |
At3g06460(拟南芥菜) | SEQ ID NO:135 | 298 |
At3g06470(拟南芥菜) | SEQ ID NO:137 | 278 |
将细小裸藻延伸酶按如下方式克隆:
细小裸藻1224-5/25株从Sammlung für Algenkulturen Gttingen[Gttingen collection of algal cultures](SAG)获得。该株在培养基II(Calvayrac R和Douce R,FEBS Letters 7:259-262,1970)中于23℃以8小时/16小时光照/黑暗(光照密度35mol s-1 m-2)周期生长4天。
用来自Qiagen的RNAeasy试剂盒(Valencia,CA,美国)分离了四日龄的裸藻培养物的总RNA。聚腺苷酸化RNA(mRNA)以寡聚-dT-纤维素从这种总RNA中分离(Sambrook等,1989)。这种RNA使用来自Promega的逆转录系统试剂盒进行逆转录并且将已合成的cDNA克隆入载体λZAP(lambda ZAP Gold,Stratagene)。然后这种cDNA根据制造商的说明去包装以产生质粒DNA,并且对克隆以随机测序方式进行部分测序。mRNA以PolyATract分离系统(Promega)从总RNA中分离。这种mRNA使用Marathon cDNA扩增试剂盒(BD Biosciences)遵循制造商的说明进行逆转录并且连接了人工接头。然后这种cDNA文库用于通过5’和3’RACE(cDNA末端快速扩增)方法的PCR克隆表达质粒。
将拟南芥菜延伸酶按如下方式克隆:
从基因组DNA开始,在每种情况下用于这两个基因的引物衍生自可读框5’和3’端。
将Chrigwin等(1979)的方法用于从拟南芥菜中分离总RNA。21日龄的植物的叶片于液氮中经研杵和研钵捣碎、以破碎缓冲液处理并于37℃温育15分钟。离心后(10分钟、4℃,12000×g)将上清中的RNA以0.02体积3M pH 5.0的乙酸钠和0.75体积乙醇-20℃沉淀5小时。然后经再次离心后,将RNA置于每克起始材料1ml的TES中,并且以1倍体积的酚/氯仿提取一次并且以1倍体积的氯仿再提取一次,以及RNA以2.5M LiCI沉淀。RNA经再次离心并且经80%乙醇洗涤后重悬于水中。cDNA如Sambrook等所描述合成,并且RT-PCR以衍生的引物实施。将PCR产物遵循制造商说明克隆入载体pYES2.1-TOPO(Invitrogen)。
实施例49:酵母中异源表达的表达质粒的克隆
为描述来自拟南芥菜去饱和酶的功能特征,将所讨论的DNA的可读框克隆至pYES2.1/V5-His-TOPO(Invitrogen)的半乳糖可诱导启动子GAL1下游,产生了相应的pAt60和pAt70克隆。
酿酒酵母334株分别以载体pAt60和pAt70经电穿孔(1500V)转化。经空白载体pYES2.1转化的酵母用作对照。已转化的酵母在补加了2%葡萄糖但缺乏尿嘧啶的完全基本培养基(CMdum)琼脂平板上选择。选择后,在每种情况下挑选三个转化体用于进一步的功能性表达。
为表达At延伸酶,在每种情况下对5ml由补加了2%(w/v)棉子糖但缺乏尿嘧啶的CMdum液体培养基组成的预培养基首先以经选择的转化体接种并且每分钟200转30℃温育2天。
然后5ml补加了2%棉子糖和300μM多种脂肪酸的CMdum(尿嘧啶撤除)液体培养基以预培养物接种至OD600 0.05。酶的表达通过加入2%(w/v)半乳糖诱导。培养物进一步20℃温育96小时。
实施例50:pAt60和pAt70在酵母中的表达
如实施例5中所描述的由质粒pYES2.1、pAt60和pAt70分别转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下,为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例51:pAt60和pAt70的功能特征
延伸酶At3g06460和At3g06470的底物特异性在表达和在喂饲多种脂肪酸后分别测定(表17、图26)。所喂饲的底物在全部转基因酵母中可检测到,转基因酵母显示分别合成了作为At3g06460和At3g06470基因产物的新脂肪酸。这意味这些基因得到了功能性表达。
表17:EPA分别经延伸酶At3g06460和At3g06470延伸。在喂饲250μM EPA之后,分析酵母提取物。
基因 | 喂饲的脂肪酸 | C20:5n-3含量 | C22:5n-3含量 | ||
At3g06460 | EPA(C20:5n-3) | 20.8 | 0.6 | ||
At3g06460 | EPA(C20:5n-3) | 25.4 | 1.1 | ||
EPA的转化率 | At3g06460:3.0% | At3g06470:4.1% |
图26显示20:5n-3经延伸酶At3g06470得到了延伸。
实施例52:从三角褐指藻中克隆延伸酶
在本申请中所详述的具有Δ6-延伸酶活性的延伸酶基因的辅助下从蛋白质序列中的保守区域开始,产生了简并引物并且这些引物通过PCR方法用于筛选褐指藻属的cDNA文库:
引物名 | 序列5’-3’方向 | 相应的氨基酸 |
Phaelo正向引物1 | AA(C/T)CTUCTUTGGCTUTT(C/T)TA(SEQ ID NO.185) | NLLWLFY |
Phaelo反向引物1 | GA(C/T)TGUAC(A/G)AA(A/G) | FAQFFVQS |
AA(C/T)TGUG C(A/G)AA(SEQ ID NO.186) |
括号中的核苷酸碱基意指在每种情况下存在的一种或另一种核苷酸碱基的寡核苷酸混合物。
褐指藻属cDNA文库的制备:
2L三角褐指藻UTEX 646培养物在f/2培养基中(1975年纽约PlenumPress出版社(Smith,W.L.和Chanley,M.H.编辑)《Culture of MarineInvertebrate Animals》中第29-60页的Guillard,R.R.L.Culture ofphytoplankton for feeding marine invertebrates)经14天长成35E/cm2光密度。离心后,冷冻的细胞在液氮存在时研成细粉并且在2ml均化作用缓冲液(在0.2M Tris-Cl pH 8.5中的0.33M山梨糖醇、0.3M NaCl、10mMEDTA、10mM EGTA、2%SDS、2%巯基乙醇)中重悬。在加入4ml酚和2ml氯仿后,将混合物45-50℃剧烈振摇15分钟。随后离心该混合物(10分钟×10000g),并且使用氯仿逐步抽提水相。然后通过加入1/20体积的4M碳酸氢钠沉淀核酸并且加以离心。将沉淀置于80mM Tris硼酸pH 7.0和1mM EDTA中,以8M氯化锂沉淀RNA。RNA沉淀经离心和用70%乙醇洗涤后置于无RNA酶的水中。Poly(A)-RNA以Dynabeads(Dynal,Oslo,Norway)遵循制造商的说明分离,并且第一链cDNA的合成使用来自Roche(Mannheim)的MLV逆转录酶实施。第二链的合成随后以DNA聚合酶I和Klenow片段实施,然后以RNA酶H消化。所述cDNA经过T4 DNA聚合酶处理并且随后通过T4连接酶增加了EcoRI/XhoI接头(Pharmacia,Freiburg)。在经过XhoI消化、磷酸化作用和凝胶分离之后,大于300bp的片段遵循制造商说明连接进入噬菌体λZAP Express(荷兰阿姆斯特丹Stratagene)内。在cDNA文库的大量切割和质粒回收后,将质粒文库转化入大肠杆菌DH10B细胞并且用于PCR筛选。
可使用以上提到的简并引物产生具有SEQ ID NO:187的PCR片段。
所述片段以洋地黄毒苷(Roche,Mannheim)标记并且用作筛选噬菌体文库的探针。
使用序列SEQ ID NO:187可获得基因序列SEQ ID NO:183,其构成褐指藻属Δ6-延伸酶的全长RNA分子。
实施例53:酵母中异源表达的表达质粒的克隆
所讨论的引物对以如此方式选择即它们具有紧邻起始密码子的用于高效翻译的酵母共有序列(Kozak,Cell 1986,44:283-292)。PtELO6 DNA的扩增在每一情况下使用1μl cDNA、200μM dNTP、2.5U Advantage聚合酶和每种引物100pmol于总计50μl体积中实施。PCR的条件如下:首先95℃变性5分钟,其后为94℃ 30秒、55℃ 1分钟和72℃ 2分钟的30个循环,并且终末延伸步骤为72℃ 10分钟。
以下寡核苷酸用于克隆在酵母中异源表达的序列的PCR反应
基因名称和SEQ ID NO: | 引物序列 |
PtELO6(SEQ ID NO:183) | F:5’-GCGGCCGCACATAATGATGGTACCTTCAAG(SEQ ID NO:188)R:3’-GAAGACAGCTTAATAGACTAGT(SEQ ID NO:189) |
*F=正向引物,R=反向引物
PCR产物与酵母表达载体pYES2.1-TOPO(Invitrogen)根据制造商说明21℃温育30分钟。通过T突出端和拓扑异构酶(Invitrogen)活性向载体连入PCR产物(见SEQ ID NO:192)。在温育后转化大肠杆菌DH5α细胞。合适的克隆通过PCR鉴定,质粒DNA通过Qiagen DNAeasy试剂盒分离并且经测序证实。然后正确的序列经电穿孔(1500V)转化入酵母株INVSc1(Invitrogen)。平行转化空白载体pYES2.1作为对照。此后酵母在补加了2%葡萄糖的无尿嘧啶基本培养基上铺板。能够在无尿嘧啶的培养基中生长的细胞包含相应的质粒pYES2.1和pYES2.1-PtELO6。选择后,在每种情况下挑选了两个转化体用于进一步的功能表达。
实施例54:植物中种子特异性表达的表达质粒的克隆
产生了基于pSUN-USP的另一个转化载体用于转化植物。为达此目的,使用以下引物对向编码序列的5′和3′端导入NotI切割位点:
PSUN-PtELO6
正向引物:5‘-GCGGCCGCACCATGATGGTACCTTCAAGTTA(SEQ ID NO:190)
反向引物:3‘-GAAGACAGCTTAATAGGCGGCCGC(SEQ IDNO:191)
PCR混合物的组成(50μl):
5.00μl模板cDNA
5.00μl 10x缓冲液(Advantage聚合酶)+25mM MgCl2
5.00μl 2mM的dNTP
1.25μl每种引物(10pmol/μl)
0.50μl Advantage聚合酶
使用了来自Clontech的Advantage聚合酶。
PCR反应条件:
复性温度:55℃ 1分钟
变性温度:94℃ 1分钟
延伸温度:72℃ 2分钟
循环次数:35
PCR产物与限制性酶NotI 37℃温育16小时。植物表达载体pSUN300-USP以同样方式温育。此后通过琼脂糖凝胶电泳分离了PCR产物和7624bp的载体并且切下相应的DNA片段。此后这些DNA通过Qiagen凝胶纯化试剂盒遵循制造商说明纯化。此后连接载体和PCR产物。使用了来自Roche的快速连接试剂盒以便连接。得到的质粒pSUN-PtELO经测序证实。
pSUN300是质粒pPZP的衍生物(Hajdukiewicz,P、Svab,Z、Maliga,P.,(1994)The small versatile pPZP family of Agrobacteriumbinary vectors for plant transformation.Plant Mol Biol 25:989-994)。通过向pSUN300中插入作为EcoRI片段的USP启动子从pSUN300衍生了pSUN-USP。加A信号是来自根癌农杆菌Ti质粒的章鱼氨酸合酶基因的加A信号(ocs终止子,Genbank Accession V00088)(De Greve,H、Dhaese,P.、Seurinck,J.、Lemmers,M、Van Montagu,M.和Schell,J.Nucleotide sequence and transcript map of the Agrobacteriumtumefaciens Ti plasmid-encoded octopine synthase geneJ.Mol.Appl.Genet.1(6),499-511(1982)。DerUSP启动子对应于第1-684位的核苷酸(Genbank Accession X56240),其中USP基因非编码区的一部分存在于USP启动子中。684碱基对大小的USP启动子片段以商业上可获得的T7标准引物(Stratagene)和经合成的引物(引物序列:5’-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3’;SEQ ID NO:151)通过遵循标准方法的PCR反应扩增。所述PCR片段再用EcoRI/SalI切割并且导入带OCS终止子的pSUN300载体。由此产生了名为pSUN-USP的质粒。这种构建体用于转化拟南芥菜、油菜籽、烟草和亚麻子。
实施例55:PtElo在酵母中的表达
如实施例4中所描述的由质粒pYES2和pYES2-PtELO6转化的酵母按如下方式分析:
来自主培养的酵母细胞经离心(100xg,5分钟,20℃)收获并以pH8.0的100mM NaHCO3洗涤以除去残留的培养基和脂肪酸。脂肪酸甲酯(FAME)从酵母细胞沉淀经酸甲醇分解作用制备。为达此目的,细胞沉淀与2ml的1N甲醇硫酸和2%(v/v)二甲氧丙烷80℃温育1小时。FAME以石油醚(PE)提取两次。在每一情况下为除去未衍生的脂肪酸,有机相以2ml的pH 8.0的100mM NaHCO3和2ml蒸馏水洗涤。随后PE相以Na2SO4干燥,在氩气下蒸发并置于100μl PE中。样品在装备有火焰电离探测器的Hewlett-Packard 6850气相色谱仪的DB-23毛细管柱(30m,0.25mm,0.25μm,Agilent)中分离。GLC分析条件如下:将炉腔温度设置为以每分钟5℃的速率从50℃升至250°并且最后在250℃维持10分钟(保持)。
信号通过比较相应的脂肪酸标准品(Sigma)的保留时间加以鉴定。例如在Napier和Michaelson,2001,Lipids.36(8):761-766;Sayanova等,2001,Journal of Experimental Botany.52(360):1581-1585,Sperling等,2001,Arch.Biochem.Biophys.388(2):293-298和Michaelson等,1998,FEBS Letters.439(3):215-218中描述了这种方法。
实施例56:PtELO6的功能特征
图29显示了C18:3Δ6,9,12和C18:4Δ6,9,12,15的转化。在每一情况下底物延伸了两个碳原子,分别形成了脂肪酸C20:3Δ8,11,14和C20:4Δ8,11,14,17。PtELO6的底物特异性在表达和在喂饲多种脂肪酸后测定(图30)。所喂饲的底物在全部转基因酵母中可大量地检测到。转基因酵母显示合成了作为PtELO6反应产物的新脂肪酸。这意味PtELO6基因得到功能性的表达。
表18表明PtELO6具有窄的底物特异性。PtELO6只能延伸C18脂肪酸亚油酸、亚麻酸、γ-亚麻酸和硬脂艾杜糖酸,但是优选地延伸ω3去饱和的硬脂艾杜糖酸(见图30)。
喂饲实验:每一情况下喂饲250μM的脂肪酸(粗体)。下划线的脂肪酸为新形成。
表18:PtELO6的底物特异性
喂饲的脂肪酸: | +18:2 | +18:3 | +18:3 | +18:4 | |
16:0 | 16.2 | 18.2 | 15.2 | 20 | 04:48 |
16:1 | 50.6 | 20.5 | 22.8 | 33.5 | 34.2 |
18:0 | 5.4 | 6.3 | 6.2 | 5.2 | 12.4 |
18:1 | 27.7 | 14.6 | 19.6 | 19.3 | 16.7 |
18:2 | 40 | ||||
18:3 | 32.9 | ||||
18:3 | 12.3 | ||||
18:4 | 4.5 | ||||
20:2 | 0.4 | ||||
20:3 | 3.4 | ||||
20:3 | 9.7 |
20:4 | 14.5 | ||||
%延伸 | 0.0 | 0.99 | 9.37 | 44.09 | 76.32 |
喂饲了以下脂肪酸,但是未得到转化。
●18:1Δ6,18:1Δ9,18:1Δ11
●20:2Δ11,14,20:3Δ11,14,17,20:3Δ8,11,14,20:4Δ5,8,11,14,20:5Δ5,8,11,14,17
●22:4Δ7,10,13,16
经载体pYES2-PtELO6转化的酵母在存在所述脂肪酸的基本培养基中生长。脂肪酸甲酯通过对完整细胞的酸甲醇分解作用合成。随后这些FAME通过GLC分析。通过这种方法,获得了在图29和30及表16中所示的结果。
等同物:
文中描述的本发明的特定实施方案的许多等同物可由技术人员仅借助常规实验确定或找到。这些等同物落入本专利权利要求的范围内。
<PN 053637>
表3 喂饲的脂肪酸的转化率。使用下式计算转化率:[转化率]=[产物]/[底物]+[产物]*100.
GC分析中以%表示的BioTaur克隆面积 | ||||||||||||||
克隆 | 脂肪酸 | C16:0 | C16:1(n-7) | C18:0 | C18:1(n-9) | C18:3(n-6) | C18:4(n-3) | C20:3(n-6) | C20:4(n-6) | C20:4(n-3) | C20:5(n-3) | C22:4(n-6) | C22:4(n-3) | C22:5(n-3) |
载体 | 无 | 21.261 | 41.576 | 4.670 | 25.330 | |||||||||
BioTaur | 无 | 20.831 | 37.374 | 4.215 | 26.475 | |||||||||
载体 | GLA+EPA | 22.053 | 23.632 | 5.487 | 17.289 | 11.574 | 13.792 | |||||||
BioTaur | GLA+EPA | 20.439 | 25.554 | 6.129 | 19.587 | 3.521 | 6.620 | 10.149 | 1.127 | |||||
载体 | EPA | 20.669 | 28.985 | 6.292 | 21.712 | 16.225 | ||||||||
BioTaur | EPA | 20.472 | 26.913 | 6.570 | 23.131 | 11.519 | 3.251 | |||||||
载体 | ARA | 23.169 | 23.332 | 6.587 | 12.735 | 27.069 | ||||||||
BioTaur | ARA | 20.969 | 31.281 | 5.367 | 21.351 | 9.648 | 1.632 | |||||||
载体 | SDA | 18.519 | 12.626 | 6.642 | 6.344 | 47.911 | ||||||||
BioTaur | SDA | 19.683 | 15.878 | 7.246 | 8.403 | 13.569 | 25.946 | 0.876 |
序列表
<110>巴斯福植物科学有限公司
<120>用于在转基因生物中产生多不饱和脂肪酸的方法
<130>PF54756
<140>20030601
<141>2003-08-01
<160>192
<170>PatentIn版本3.1
<210>1
<211>1266
<212>DNA
<213>细小裸藻(Euglena gracilis)
<220>
<221>CDS
<222>(1)..(1266)
<223>Δ8-去饱和酶
<400>1
atg aag tca aag cgc caa gcg ctt ccc ctt aca att gat gga aca aca 48
Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr
1 5 10 15
tat gat gtg tct gcc tgg gtc aat ttc cac cct ggt ggt gcg gaa att 96
Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile
20 25 30
ata gag aat tac caa gga agg gat gcc act gat gcc ttc atg gtt atg 144
lle Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met
35 40 45
cac tct caa gaa gcc ttc gac aag ctc aag cgc atg ccc aaa atc aat 192
His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn
50 55 60
ccc agt tct gag ttg cca ccc cag gct gca gtg aat gaa gct caa gag 240
Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu
65 70 75 80
gat ttc cgg aag ctc cga gaa gag ttg atc gca act ggc atg ttt gat 288
Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp
85 90 95
gcc tcc ccc ctc tgg tac tca tac aaa atc agc acc aca ctg ggc ctt 336
Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu
100 105 110
gga gtg ctg ggt tat ttc ctg atg gtt cag tat cag atg tat ttc att 384
Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile
115 120 125
ggg gca gtg ttg ctt ggg atg cac tat caa cag atg ggc tgg ctt tct 432
Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser
130 135 140
cat gac att tgc cac cac cag act ttc aag aac cgg aac tgg aac aac 480
His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn
145 150 155 160
ctc gtg gga ctg gta ttt ggc aat ggt ctg caa ggt ttt tcc gtg aca 528
Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr
165 170 175
tgc tgg aag gac aga cac aat gca cat cat tcg gca acc aat gtt caa 576
Cys Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln
180 185 190
ggg cac gac cct gat att gac aac ctc ccc ctc tta gcc tgg tct gag 624
Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Scr Glu
195 200 205
gat gac gtc aca cgg gcg tca ccg att tcc cgc aag ctc att cag ttc 672
Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe
210 215 220
cag cag tat tat ttc ttg gtc atc tgt atc ttg ttg cgg ttc att tgg 720
Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp
225 230 235 240
tgt ttc cag agc gtg ttg acc gtg cgc agt ctg aag gac aga gat aac 768
Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn
245 250 255
caa ttc tat cgc tct cag tat aag aag gag gcc att ggc ctc gcc ctg 816
Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu
260 265 270
cat tgg aca ttg aag gcc ctg ttc cac tta ttc ttt atg ccc agc atc 864
His Trp Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile
275 280 285
ctc aca tcg ctg ttg gta ttt ttc gtt tcg gag ctg gtt ggc ggc ttc 912
Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe
290 295 300
ggc att gcg atc gtg gtg ttc atg aac cac tac cca ctg gag aag atc 960
Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile
305 310 315 320
ggg gac tcg gtc tgg gat ggc cat gga ttc tcg gtt ggc cag atc cat 1008
Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His
325 330 335
gag acc atg aac att cgg cga ggg att atc aca gat tgg ttt ttc gga 1056
Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly
340 345 350
ggc ttg aac tac cag atc gag cac cat ttg tgg ccg acc ctc cct cgc 1104
Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg
355 360 365
cac aac ctg aca gcg gtt agc tac cag gtg gaa cag ctg tgc cag aag 1152
His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys
370 375 380
cac aac ctg ccg tat cgg aac ccg ctg ccc cat gaa ggg ttg gtc atc 1200
His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile
385 390 395 400
ctg ctg cgc tat ctg gcg gtg ttc gcc cgg atg gcg gag aag caa ccc 1248
Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro
405 410 415
gcg ggg aag gct cta taa 1266
Ala Gly Lys Ala Leu
420
<210>2
<21l>42l
<212>PRT
<213>细小裸藻
<400>2
Met Lys Ser Lys Arg Gln Ala Leu Pro Leu Thr Ile Asp Gly Thr Thr
1 5 10 15
Tyr Asp Val Ser Ala Trp Val Asn Phe His Pro Gly Gly Ala Glu Ile
20 25 30
Ile Glu Asn Tyr Gln Gly Arg Asp Ala Thr Asp Ala Phe Met Val Met
35 40 45
His Ser Gln Glu Ala Phe Asp Lys Leu Lys Arg Met Pro Lys Ile Asn
50 55 60
Pro Ser Ser Glu Leu Pro Pro Gln Ala Ala Val Asn Glu Ala Gln Glu
65 70 75 80
Asp Phe Arg Lys Leu Arg Glu Glu Leu Ile Ala Thr Gly Met Phe Asp
85 90 95
Ala Ser Pro Leu Trp Tyr Ser Tyr Lys Ile Ser Thr Thr Leu Gly Leu
100 105 110
Gly Val Leu Gly Tyr Phe Leu Met Val Gln Tyr Gln Met Tyr Phe Ile
115 120 125
Gly Ala Val Leu Leu Gly Met His Tyr Gln Gln Met Gly Trp Leu Ser
130 135 140
His Asp Ile Cys His His Gln Thr Phe Lys Asn Arg Asn Trp Asn Asn
145 150 155 160
Leu Val Gly Leu Val Phe Gly Asn Gly Leu Gln Gly Phe Ser Val Thr
165 170 175
Cys Trp Lys Asp Arg His Asn Ala His His Ser Ala Thr Asn Val Gln
180 185 190
Gly His Asp Pro Asp Ile Asp Asn Leu Pro Leu Leu Ala Trp Ser Glu
195 200 205
Asp Asp Val Thr Arg Ala Ser Pro Ile Ser Arg Lys Leu Ile Gln Phe
210 215 220
Gln Gln Tyr Tyr Phe Leu Val Ile Cys Ile Leu Leu Arg Phe Ile Trp
225 230 235 240
Cys Phe Gln Ser Val Leu Thr Val Arg Ser Leu Lys Asp Arg Asp Asn
245 250 255
Gln Phe Tyr Arg Ser Gln Tyr Lys Lys Glu Ala Ile Gly Leu Ala Leu
260 265 270
His Trp Thr Leu Lys Ala Leu Phe His Leu Phe Phe Met Pro Ser Ile
275 280 285
Leu Thr Ser Leu Leu Val Phe Phe Val Ser Glu Leu Val Gly Gly Phe
290 295 300
Gly Ile Ala Ile Val Val Phe Met Asn His Tyr Pro Leu Glu Lys Ile
305 310 315 320
Gly Asp Ser Val Trp Asp Gly His Gly Phe Ser Val Gly Gln Ile His
325 330 335
Glu Thr Met Asn Ile Arg Arg Gly Ile Ile Thr Asp Trp Phe Phe Gly
340 345 350
Gly Leu Asn Tyr Gln Ile Glu His His Leu Trp Pro Thr Leu Pro Arg
355 360 365
His Asn Leu Thr Ala Val Ser Tyr Gln Val Glu Gln Leu Cys Gln Lys
370 375 380
His Asn Leu Pro Tyr Arg Asn Pro Leu Pro His Glu Gly Leu Val Ile
385 390 395 400
Leu Leu Arg Tyr Leu Ala Val Phe Ala Arg Met Ala Glu Lys Gln Pro
405 410 415
Ala Gly Lys Ala Leu
420
<210>3
<211>777
<212>DNA
<213>绿光等鞭金藻(Isochrysis galbana)
<220>
<221>CDS
<222>(1)..(777)
<223>Δ9-延伸酶
<400>3
atg gcc ctc gca aac gac gcg gga gag cgc atc tgg gcg gct gtg acc 48
Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr
1 5 10 15
gac ccg gaa atc ctc att ggc acc ttc tcg tac ttg cta ctc aaa ccg 96
Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro
20 25 30
ctg ctc cgc aat tcc ggg ctg gtg gat gag aag aag ggc gca tac agg 144
Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg
35 40 45
acg tcc atg atc tgg tac aac gtt ctg ctg gcg ctc ttc tct gcg ctg 192
Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu
50 55 60
agc ttc tac gtg acg gcg acc gcc ctc ggc tgg gac tat ggt acg ggc 240
Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly
65 70 75 80
gcg tgg ctg cgc agg caa acc ggc gac aca ccg cag ccg ctc ttc cag 288
Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln
85 90 95
tgc ccg tcc ccg gtt tgg gac tcg aag ctc ttc aca tgg acc gcc aag 336
Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr Trp Thr Ala Lys
100 105 110
gca ttc tat tac tcc aag tac gtg gag tac ctc gac acg gcc tgg ctg 384
Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu
115 120 125
agg gtc tcc ttt ctc cag gcc ttc cac cac ttt ggc gcg ccg tgg gat 432
Arg Val Ser Phe Leu Gln Ala Phe His His Phe Gly Ala Pro Trp Asp
130 135 140
gtg tac ctc ggc att cgg ctg cac aac gag ggc gta tgg atc ttc atg 480
Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly Val Trp Ile Phe Met
145 150 155 160
ttt ttc aac tcg ttc att cac acc atc atg tac acc tac tac ggc ctc 528
Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr Thr Tyr Tyr Gly Leu
165 170 175
acc gcc gcc ggg tat aag ttc aag gcc aag ccg ctc atc acc gcg atg 576
Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro Leu Ile Thr Ala Met
180 l85 190
cag atc tgc cag ttc gtg ggc ggc ttc ctg ttg gtc tgg gac tac atc 624
Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu Val Trp Asp Tyr Ile
195 200 205
aac gtc ccc tgc ttc aac tcg gac aaa ggg aag ttg ttc agc tgg gct 672
Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys Leu Phe Ser Trp Ala
210 215 220
ttc aac tat gca tac gtc ggc tcg gtc ttc ttg ctc ttc tgc cac ttt 720
Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu Leu Phe Cys His Phe
225 230 235 240
ttc tac cag gac aac ttg gca acg aag aaa tcg gcc aag gcg ggc aag 768
Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser Ala Lys Ala Gly Lys
245 250 255
cag ctc tag 777
Gln Leu
<210>4
<211>258
<212>PRT
<213>绿光等鞭金藻
<400>4
Met Ala Leu Ala Asn Asp Ala Gly Glu Arg Ile Trp Ala Ala Val Thr
1 5 10 15
Asp Pro Glu Ile Leu Ile Gly Thr Phe Ser Tyr Leu Leu Leu Lys Pro
20 25 30
Leu Leu Arg Asn Ser Gly Leu Val Asp Glu Lys Lys Gly Ala Tyr Arg
35 40 45
Thr Ser Met Ile Trp Tyr Asn Val Leu Leu Ala Leu Phe Ser Ala Leu
50 55 60
Ser Phe Tyr Val Thr Ala Thr Ala Leu Gly Trp Asp Tyr Gly Thr Gly
65 70 75 80
Ala Trp Leu Arg Arg Gln Thr Gly Asp Thr Pro Gln Pro Leu Phe Gln
85 90 95
Cys Pro Ser Pro Val Trp Asp Ser Lys Leu Phe Thr Trp Thr Ala Lys
100 105 110
Ala Phe Tyr Tyr Ser Lys Tyr Val Glu Tyr Leu Asp Thr Ala Trp Leu
115 120 125
Arg Val Ser Phe Leu Gln Ala Phe His His Phe Gly Ala Pro Trp Asp
130 135 140
Val Tyr Leu Gly Ile Arg Leu His Asn Glu Gly Val Trp Ile Phe Met
145 150 155 160
Phe Phe Asn Ser Phe Ile His Thr Ile Met Tyr Thr Tyr Tyr Gly Leu
165 170 175
Thr Ala Ala Gly Tyr Lys Phe Lys Ala Lys Pro Leu Ile Thr Ala Met
180 185 190
Gln Ile Cys Gln Phe Val Gly Gly Phe Leu Leu Val Trp Asp Tyr Ile
195 200 205
Asn Val Pro Cys Phe Asn Ser Asp Lys Gly Lys Leu Phe Ser Trp Ala
210 215 220
Phe Asn Tyr Ala Tyr Val Gly Ser Val Phe Leu Leu Phe Cys His Phe
225 230 235 240
Phe Tyr Gln Asp Asn Leu Ala Thr Lys Lys Ser Ala Lys Ala Gly Lys
245 250 255
Gln Leu
<210>5
<211>1410
<212>DNA
<213>三角褐指藻(Phaeodactylum tricornutum)
<220>
<221>CDS
<222>(1)..(1410)
<223>Δ5-去饱和酶
<400>5
atg gct ccg gat gcg gat aag ctt cga caa cgc cag acg act gcg gta 48
Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val
1 5 10 15
gcg aag cac aat gct gct acc ata tcg acg cag gaa cgc ctt tgc agt 96
Ala Lys H1s Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser
20 25 30
ctg tct tcg ctc aaa ggc gaa gaa gtc tgc atc gac gga atc atc tat 144
Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr
35 40 45
gac ctc caa tca ttc gat cat ccc ggg ggt gaa acg atc aaa atg ttt 192
Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe
50 55 60
ggt ggc aac gat gtc act gta cag tac aag atg att cac ccg tac cat 240
Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr His
65 70 75 80
acc gag aag cat ttg gaa aag atg aag cgt gtc ggc aag gtg acg gat 288
Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp
85 90 95
ttc gtc tgc gag tac aag ttc gat acc gaa ttt gaa cgc gaa atc aaa 336
Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys
100 105 110
cga gaa gtc ttc aag att gtg cga cga ggc aag gat ttc ggt act ttg 384
Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu
115 120 125
gga tgg ttc ttc cgt gcg ttt tgc tac att gcc att ttc ttc tac ctg 432
Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu
130 135 140
cag tac cat tgg gtc acc acg gga acc tct tgg ctg ctg gcc gtg gcc 480
Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala
145 150 155 160
tac gga atc tcc caa gcg atg att ggc atg aat gtc cag cac gat gcc 528
Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala
165 170 175
aac cac ggg gcc acc tcc aag cgt ccc tgg gtc aac gac atg cta ggc 576
Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly
180 185 190
ctc ggt gcg gat ttt att ggt ggt tcc aag tgg ctc tgg cag gaa caa 624
Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln
195 200 205
cac tgg acc cac cac gct tac acc aat cac gcc gag atg gat ccc gat 672
His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp
210 215 220
agc ttt ggt gcc gaa cca atg ctc cta ttc aac gac tat ccc ttg gat 720
Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp
225 230 235 240
cat ccc gct cgt acc tgg cta cat cgc ttt caa gca ttc ttt tac atg 768
His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met
245 250 255
ccc gtc ttg gct gga tac tgg ttg tcc gct gtc ttc aat cca caa att 816
Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile
260 265 270
ctt gac ctc cag caa cgc ggc gca ctt tcc gtc ggt atc cgt ctc gac 864
Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp
275 280 285
aac gct ttc att cac tcg cga cgc aag tat gcg gtt ttc tgg cgg gct 912
Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala
290 295 300
gtg tac att gcg gtg aac gtg att gct ccg ttt tac aca aac tcc ggc 960
Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly
305 310 315 320
ctc gaa tgg tcc tgg cgt gtc ttt gga aac atc atg ctc atg ggt gtg 1008
Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val
325 330 335
gcg gaa tcg ctc gcg ctg gcg gtc ctg ttt tcg ttg tcg cac aat ttc 1056
Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe
340 345 350
gaa tcc gcg gat cgc gat ccg acc gcc cca ctg aaa aag acg gga gaa 1104
Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu
355 360 365
cca gtc gac tgg ttc aag aca cag gtc gaa act tcc tgc act tac ggt 1152
Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly
370 375 380
gga ttc ctt tcc ggt tgc ttc acg gga ggt ctc aac ttt cag gtt gaa 1200
Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu
385 390 395 400
cac cac ttg ttc cca cgc atg agc agc gct tgg tat ccc tac att gcc 1248
His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala
405 410 415
ccc aag gtc cgc gaa att tgc gcc aaa cac ggc gtc cac tac gcc tac 1296
Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr
420 425 430
tac ccg tgg atc cac caa aac ttt ctc tcc acc gtc cgc tac atg cac 1344
Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His
435 440 445
gcg gcc ggg acc ggt gcc aac tgg cgc cag atg gcc aga gaa aat ccc 1392
Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro
450 455 460
ttg acc gga cgg gcg taa 1410
Leu Thr Gly Arg Ala
465
<210>6
<211>469
<212>PRT
<213>三角褐指藻
<400>6
Met Ala Pro Asp Ala Asp Lys Leu Arg Gln Arg Gln Thr Thr Ala Val
1 5 10 15
Ala Lys His Asn Ala Ala Thr Ile Ser Thr Gln Glu Arg Leu Cys Ser
20 25 30
Leu Ser Ser Leu Lys Gly Glu Glu Val Cys Ile Asp Gly Ile Ile Tyr
35 40 45
Asp Leu Gln Ser Phe Asp His Pro Gly Gly Glu Thr Ile Lys Met Phe
50 55 60
Gly Gly Asn Asp Val Thr Val Gln Tyr Lys Met Ile His Pro Tyr Hls
65 70 75 80
Thr Glu Lys His Leu Glu Lys Met Lys Arg Val Gly Lys Val Thr Asp
85 90 95
Phe Val Cys Glu Tyr Lys Phe Asp Thr Glu Phe Glu Arg Glu Ile Lys
100 105 110
Arg Glu Val Phe Lys Ile Val Arg Arg Gly Lys Asp Phe Gly Thr Leu
115 120 125
Gly Trp Phe Phe Arg Ala Phe Cys Tyr Ile Ala Ile Phe Phe Tyr Leu
130 135 140
Gln Tyr His Trp Val Thr Thr Gly Thr Ser Trp Leu Leu Ala Val Ala
145 150 155 160
Tyr Gly Ile Ser Gln Ala Met Ile Gly Met Asn Val Gln His Asp Ala
165 170 175
Asn His Gly Ala Thr Ser Lys Arg Pro Trp Val Asn Asp Met Leu Gly
180 185 190
Leu Gly Ala Asp Phe Ile Gly Gly Ser Lys Trp Leu Trp Gln Glu Gln
195 200 205
His Trp Thr His His Ala Tyr Thr Asn His Ala Glu Met Asp Pro Asp
210 215 220
Ser Phe Gly Ala Glu Pro Met Leu Leu Phe Asn Asp Tyr Pro Leu Asp
225 230 235 240
His Pro Ala Arg Thr Trp Leu His Arg Phe Gln Ala Phe Phe Tyr Met
245 250 255
Pro Val Leu Ala Gly Tyr Trp Leu Ser Ala Val Phe Asn Pro Gln Ile
260 265 270
Leu Asp Leu Gln Gln Arg Gly Ala Leu Ser Val Gly Ile Arg Leu Asp
275 280 285
Asn Ala Phe Ile His Ser Arg Arg Lys Tyr Ala Val Phe Trp Arg Ala
290 295 300
Val Tyr Ile Ala Val Asn Val Ile Ala Pro Phe Tyr Thr Asn Ser Gly
305 310 315 320
Leu Glu Trp Ser Trp Arg Val Phe Gly Asn Ile Met Leu Met Gly Val
325 330 335
Ala Glu Ser Leu Ala Leu Ala Val Leu Phe Ser Leu Ser His Asn Phe
340 345 350
Glu Ser Ala Asp Arg Asp Pro Thr Ala Pro Leu Lys Lys Thr Gly Glu
355 360 365
Pro Val Asp Trp Phe Lys Thr Gln Val Glu Thr Ser Cys Thr Tyr Gly
370 375 380
Gly Phe Leu Ser Gly Cys Phe Thr Gly Gly Leu Asn Phe Gln Val Glu
385 390 395 400
His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala
405 410 415
Pro Lys Val Arg Glu Ile Cys Ala Lys His Gly Val His Tyr Ala Tyr
420 425 430
Tyr Pro Trp Ile His Gln Asn Phe Leu Ser Thr Val Arg Tyr Met His
435 440 445
Ala Ala Gly Thr Gly Ala Asn Trp Arg Gln Met Ala Arg Glu Asn Pro
450 455 460
Leu Thr Gly Arg Ala
465
<210>7
<211>1344
<212>DNA
<213>角齿藓(ceratodon purpureus)
<220>
<221>CDS
<222>(1)..(1344)
<223>Δ5-去饱和酶
<400>7
atg gta tta cga gag caa gag cat gag cca ttc ttc att aaa att gat 48
Met Val Leu Arg Glu Gln Glu His Glu Pro Phe Phe Ile Lys Ile Asp
1 5 10 15
gga aaa tgg tgt caa att gac gat gct gtc ctg aga tca cat cca ggt 96
Gly Lys Trp Cys Gln Ile Asp Asp Ala Val Leu Arg Ser His Pro Gly
20 25 30
ggt agt gca att act acc tat aaa aat atg gat gcc act acc gta ttc 144
Gly Ser Ala Ile Thr Thr Tyr Lys Asn Met Asp Ala Thr Thr Val Phe
35 40 45
cac aca ttc cat act ggt tct aaa gaa gcg tat caa tgg ctg aca gaa 192
His Thr Phe His Thr Gly Ser Lys Glu Ala Tyr Gln Trp Leu Thr Glu
50 55 60
ttg aaa aaa gag tgc cct aca caa gaa cca gag atc cca gat att aag 240
Leu Lys Lys Glu Cys Pro Thr Gln Glu Pro Glu Ile Pro Asp Ile Lys
65 70 75 80
gat gac cca atc aaa gga att gat gat gtg aac atg gga act ttc aat 288
Asp Asp Pro Ile Lys Gly Ile Asp Asp Val Asn Met Gly Thr Phe Asn
85 90 95
att tct gag aaa cga tct gcc caa ata aat aaa agt ttc act gat cta 336
Ile Ser Glu Lys Arg Ser Ala Gln Ile Asn Lys Ser Phe Thr Asp Leu
100 105 110
cgt atg cga gtt cgt gca gaa gga ctt atg gat gga tct cct ttg ttc 384
Arg Met Arg Val Arg Ala Glu Gly Leu Met Asp Gly Ser Pro Leu Phe
115 120 125
tac att aga aaa att ctt gaa aca atc ttc aca att ctt ttt gca ttc 432
Tyr Ile Arg Lys Ile Leu Glu Thr Ile Phe Thr Ile Leu Phe Ala Phe
130 135 140
tac ctt caa tac cac aca tat tat ctt cca tca gct att cta atg gga 480
Tyr Leu Gln Tyr His Thr Tyr Tyr Leu Pro Ser Ala Ile Leu Met Gly
145 150 155 160
gtt gcg tgg caa caa ttg gga tgg tta atc cat gaa ttc gca cat cat 528
Val Ala Trp Gln Gln Leu Gly Trp Leu Ile His Glu Phe Ala His His
165 170 175
cag ttg ttc aaa aac aga tac tac aat gat ttg gcc agc tat ttc gtt 576
Gln Leu Phe Lys Asn Arg Tyr Tyr Asn Asp Leu Ala Ser Tyr Phe Val
180 185 190
gga aac ttt tta caa gga ttc tca tct ggt ggt tgg aaa gag cag cac 624
Gly Asn Phe Leu Gln Gly Phe Ser Ser Gly Gly Trp Lys Glu Gln His
195 200 205
aat gtg cat cac gca gcc aca aat gtt gtt gga cga gac gga gat ctt 672
Asn Val His His Ala Ala Thr Asn Val Val Gly Arg Asp Gly Asp Leu
210 215 220
gat tta gtc cca ttc tat gct aca gtg gca gaa cat ctc aac aat tat 720
Asp Leu Val Pro Phe Tyr Ala Thr Val Ala Glu His Leu Asn Asn Tyr
225 230 235 240
tct cag gat tca tgg gtt atg act cta ttc aga tgg caa cat gtt cat 768
Ser Gln Asp Ser Trp Val Met Thr Leu Phe Arg Trp Gln His Val His
245 250 255
tgg aca ttc atg tta cca ttc ctc cgt ctc tcg tgg ctt ctt cag tca 816
Trp Thr Phe Met Leu Pro Phe Leu Arg Leu Ser Trp Leu Leu Gln Ser
260 265 270
atc att ttt gtt agt cag atg cca act cat tat tat gac tat tac aga 864
Ile Ile Phe Val Ser Gln Met Pro Thr His Tyr Tyr Asp Tyr Tyr Arg
275 280 285
aat act gcg att tat gaa cag gtt ggt ctc tct ttg cac tgg gct tgg 912
Asn Thr Ala Ile Tyr Glu Gln Val Gly Leu Ser Leu His Trp Ala Trp
290 295 300
tca ttg ggt caa ttg tat ttc cta ccc gat tgg tca act aga ata atg 960
Ser Leu Gly Gln Leu Tyr Phe Leu Pro Asp Trp Ser Thr Arg Ile Met
305 310 315 320
ttc ttc ctt gtt tct cat ctt gtt gga ggt ttc ctg ctc tct cat gta 1008
Phe Phe Leu Val Ser His Leu Val Gly Gly Phe Leu Leu Ser His Val
325 330 335
gtt act ttc aat cat tat tca gtg gag aag ttt gca ttg agc tcg aac 1056
Val Thr Phe Asn His Tyr Ser Val Glu Lys Phe Ala Leu Ser Ser Asn
340 345 350
atc atg tca aat tac gct tgt ctt caa atc atg acc aca aga aat atg 1104
Ile Met Ser Asn Tyr Ala Cys Leu Gln Ile Met Thr Thr Arg Asn Met
355 360 365
aga cct gga aga ttc att gac tgg ctt tgg gga ggt ctt aac tat cag 1152
Arg Pro Gly Arg Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln
370 375 380
att gag cac cat ctt ttc cca acg atg cca cga cac aac ttg aac act 1200
Ile Glu His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Thr
385 390 395 400
gtt atg cca ctt gtt aag gag ttt gca gca gca aat ggt tta cca tac 1248
Val Met Pro Leu Val Lys Glu Phe Ala Ala Ala Asn Gly Leu Pro Tyr
405 410 415
atg gtc gac gat tat ttc aca gga ttc tgg ctt gaa att gag caa ttc 1296
Met Val Asp Asp Tyr Phe Thr Gly Phe Trp Leu Glu Ile Glu Gln Phe
420 425 430
cga aat att gca aat gtt gct gct aaa ttg act aaa aag att gcc tag 1344
Arg Asn Ile Ala Asn Val Ala Ala Lys Leu Thr Lys Lys Ile Ala
435 440 445
<210>8
<211>447
<212>PRT
<213>角齿藓
<400>8
Met Val Leu Arg Glu Gln Glu His Glu Pro Phe Phe Ile Lys Ile Asp
1 5 10 15
Gly Lys Trp Cys Gln Ile Asp Asp Ala Val Leu Arg Ser His Pro Gly
20 25 30
Gly Ser Ala Ile Thr Thr Tyr Lys Asn Met Asp Ala Thr Thr Val Phe
35 40 45
His Thr Phe His Thr Gly Ser Lys Glu Ala Tyr Gln Trp Leu Thr Glu
50 55 60
Leu Lys Lys Glu Cys Pro Thr Gln Glu Pro Glu Ile Pro Asp Ile Lys
65 70 75 80
Asp Asp Pro Ile Lys Gly Ile Asp Asp Val Asn Met Gly Thr Phe Asn
85 90 95
Ile Ser Glu Lys Arg Ser Ala Gln Ile Asn Lys Ser Phe Thr Asp Leu
100 105 110
Arg Met Arg Val Arg Ala Glu Gly Leu Met Asp Gly Ser Pro Leu Phe
115 120 125
Tyr Ile Arg Lys Ile Leu Glu Thr Ile Phe Thr Ile Leu Phe Ala Phe
130 135 140
Tyr Leu Gln Tyr His Thr Tyr Tyr Leu Pro Ser Ala Ile Leu Met Gly
145 150 155 160
Val Ala Trp Gln Gln Leu Gly Trp Leu Ile His Glu Phe Ala His His
165 170 175
Gln Leu Phe Lys Asn Arg Tyr Tyr Asn Asp Leu Ala Ser Tyr Phe Val
180 185 190
Gly Asn Phe Leu Gln Gly Phe Ser Ser Gly Gly Trp Lys Glu Gln His
195 200 205
Asn Val His His Ala Ala Thr Asn Val Val Gly Arg Asp Gly Asp Leu
210 215 220
Asp Leu Val Pro Phe Tyr Ala Thr Val Ala Glu His Leu Asn Asn Tyr
225 230 235 240
Ser Gln Asp Ser Trp Val Met Thr Leu Phe Arg Trp Gln His Val His
245 250 255
Trp Thr Phe Met Leu Pro Phe Leu Arg Leu Ser Trp Leu Leu Gln Ser
260 265 270
Ile Ile Phe Val Ser Gln Met Pro Thr His Tyr Tyr Asp Tyr Tyr Arg
275 280 285
Asn Thr Ala Ile Tyr Glu Gln Val Gly Leu Ser Leu His Trp Ala Trp
290 295 300
Ser Leu Gly Gln Leu Tyr Phe Leu Pro Asp Trp Ser Thr Arg Ile Met
305 310 315 320
Phe Phe Leu Val Ser His Leu Val Gly Gly Phe Leu Leu Ser His Val
325 330 335
Val Thr Phe Asn His Tyr Ser Val Glu Lys Phe Ala Leu Ser Ser Asn
340 345 350
Ile Met Ser Asn Tyr Ala Cys Leu Gln Ile Met Thr Thr Arg Asn Met
355 360 365
Arg Pro Gly Arg Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln
370 375 380
Ile Glu His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Thr
385 390 395 400
Val Met Pro Leu Val Lys Glu Phe Ala Ala Ala Asn Gly Leu Pro Tyr
405 410 415
Met Val Asp Asp Tyr Phe Thr Gly Phe Trp Leu Glu Ile Glu Gln Phe
420 425 430
Arg Asn Ile Ala Asn Val Ala Ala Lys Leu Thr Lys Lys Ile Ala
435 440 445
<210>9
<211>1443
<212>DNA
<213>展叶剑叶藓(Physcomitrella patens)
<220>
<221>CDS
<222>(1)..(1443)
<223>Δ5-去饱和酶
<400>9
atg gcg ccc cac tct gcg gat act gct ggg ctc gtg cct tct gac gaa 48
Met Ala Pro His Ser Ala Asp Thr Ala Gly Leu Val Pro Ser Asp Glu
1 5 10 15
ttg agg cta cga acg tcg aat tca aag ggt ccc gaa caa gag caa act 96
Leu Arg Leu Arg Thr Ser Asn Ser Lys Gly Pro Glu Gln Glu Gln Thr
20 25 30
ttg aag aag tac acc ctt gaa gat gtc agc cgc cac aac acc cca gca 144
Leu Lys Lys Tyr Thr Leu Glu Asp Val Ser Arg His Asn Thr Pro Ala
35 40 45
gat tgt tgg ttg gtg ata tgg ggc aaa gtc tac gat gtc aca agc tgg 192
Asp Cys Trp Leu Val Ile Trp Gly Lys Val Tyr Asp Val Thr Ser Trp
50 55 60
att ccc aat cat ccg ggg ggc agt ctc atc cac gta aaa gca ggg cag 240
Ile Pro Asn His Pro Gly Gly Ser Leu Ile His Val Lys Ala Gly Gln
65 70 75 80
gat tcc act cag ctt ttc gat tcc tat cac ccc ctt tat gtc agg aaa 288
Asp Ser Thr Gln Leu Phe Asp Ser Tyr His Pro Leu Tyr Val Arg Lys
85 90 95
atg ctc gcg aag tac tgt att ggg gaa tta gta ccg tct gct ggt gat 336
Met Leu Ala Lys Tyr Cys Ile Gly Glu Leu Val Pro Ser Ala Gly Asp
100 105 110
gac aag ttt aag aaa gca act ctg gag tat gca gat gcc gaa aat gaa 384
Asp Lys Phe Lys Lys Ala Thr Leu Glu Tyr Ala Asp Ala Glu Asn Glu
115 120 125
gat ttc tat ttg gtt gtg aag caa cga gtt gaa tct tat ttc aag agt 432
A5p Phe Tyr Leu Val Val Lys Gln Arg Val Glu Ser Tyr Phe Lys Ser
130 135 140
aac aag ata aac ccc caa att cat cca cat atg atc ctg aag tca ttg 480
Asn Lys Ile Asn Pro Gln Ile His Pro His Met Ile Leu Lys Ser Leu
145 150 155 160
ttc att ctt ggg gga tat ttc gcc agt tac tat tta gcg ttc ttc tgg 528
Phe Ile Leu Gly Gly Tyr Phe Ala Ser Tyr Tyr Leu Ala Phe Phe Trp
165 170 175
tct tca agt gtc ctt gtt tct ttg ttt ttc gca ttg tgg atg ggg ttc 576
Ser Ser Ser Val Leu Val Ser Leu Phe Phe Ala Leu Trp Met Gly Phe
180 185 190
ttc gca gcg gaa gtc ggc gtg tcg att caa cat gat gga aat cat ggt 624
Phe Ala Ala Glu Val Gly Val Ser Ile Gln His Asp Gly Asn His Gly
195 200 205
tca tac act aaa tgg cgt ggc ttt gga tat atc atg gga gcc tcc cta 672
Ser Tyr Thr Lys Trp Arg Gly Phe Gly Tyr Ile Met Gly Ala Ser Leu
210 215 220
gat cta gtc gga gcc agt agc ttc atg tgg aga cag caa cac gtt gtg 720
Asp Leu Val Gly Ala Ser Ser Phe Met Trp Arg Gln Gln His Val Val
225 230 235 240
gga cat cac tcg ttt aca aat gtg gac aac tac gat cct gat att cgt 768
Gly His His Ser Phe Thr Asn Val Asp Asn Tyr Asp Pro Asp Ile Arg
245 250 255
gtg aaa gat cca gat gtc agg agg gtt gcg acc aca caa cca aga caa 816
Val Lys Asp Pro Asp Val Arg Arg Val Ala Thr Thr Gln Pro Arg Gln
260 265 270
tgg tat cat gcg tat cag cat atc tac ctg gca gta tta tat gga act 864
Trp Tyr His Ala Tyr Gln His Ile Tyr Leu Ala Val Leu Tyr Gly Thr
275 280 285
cta gct ctt aag agt att ttt cta gat gat ttc ctt gcg tac ttc aca 912
Leu Ala Leu Lys Ser Ile Phe Leu Asp Asp Phe Leu Ala Tyr Phe Thr
290 295 300
gga tca att ggc cct gtc aag gtg gcg aaa atg acc ccc ctg gag ttc 960
Gly Ser Ile Gly Pro Val Lys Val Ala Lys Met Thr Pro Leu Glu Phe
305 310 315 320
aac atc ttc ttt cag gga aag ctg cta tat gcg ttc tac atg ttc gtg 1008
Asn Ile Phe Phe Gln Gly Lys Leu Leu Tyr Ala Phe Tyr Met Phe Val
325 330 335
ttg cca tct gtg tac ggt gtt cac tcc gga gga act ttc ttg gca cta 1056
Leu Pro Ser Val Tyr Gly Val His Ser Gly Gly Thr Phe Leu Ala Leu
340 345 350
tat gtg gct tct cag ctc att aca ggt tgg atg tta gct ttt ctt ttt 1104
Tyr Val Ala Ser Gln Leu Ile Thr Gly Trp Met Leu Ala Phe Leu Phe
355 360 365
caa gta gca cat gtc gtg gat gat gtt gca ttt cct aca cca gaa ggt 1152
Gln Val Ala His Val Val Asp Asp Val Ala Phe Pro Thr Pro Glu Gly
370 375 380
ggg aag gtg aag gga gga tgg gct gca atg cag gtt gca aca act acg 1200
Gly Lys Val Lys Gly Gly Trp Ala Ala Met Gln Val Ala Thr Thr Thr
385 390 395 400
gat ttc agt cca cgc tca tgg ttc tgg ggt cat gtc tct gga gga tta 1248
Asp Phe Ser Pro Arg Ser Trp Phe Trp Gly His Val Ser Gly Gly Leu
405 410 415
aac aac caa att gag cat cat ctg ttt cca gga gtg tgc cat gtt cat 1296
Asn Asn Gln Ile Glu His His Leu Phe Pro Gly Val Cys His Val His
420 425 430
tat cca gcc att cag cct att gtc gag aag acg tgc aag gaa ttc gat 1344
Tyr Pro Ala Ile Gln Pro Ile Val Glu Lys Thr Cys Lys Glu Phe Asp
435 440 445
gtg cct tat gta gcc tac cca act ttt tgg act gcg ttg aga gcc cac 1392
Val Pro Tyr Val Ala Tyr Pro Thr Phe Trp Thr Ala Leu Arg Ala His
450 455 460
ttt gcg cat ttg aaa aag gtt gga ttg aca gag ttt cgg ctc gat ggc 1440
Phe Ala His Leu Lys Lys Val Gly Leu Thr Glu Phe Arg Leu Asp Gly
465 470 475 480
tga 1443
<210>10
<211>480
<212>PRT
<213>展叶剑叶藓
<400>10
Met Ala Pro His Ser Ala Asp Thr Ala Gly Leu Val Pro Ser Asp Glu
1 5 10 15
Leu Arg Leu Arg Thr Ser Asn Ser Lys Gly Pro Glu Gln Glu Gln Thr
20 25 30
Leu Lys Lys Tyr Thr Leu Glu Asp Val Ser Arg His Asn Thr Pro Ala
35 40 45
Asp Cys Trp Leu Val Ile Trp Gly Lys Val Tyr Asp Val Thr Ser Trp
50 55 60
Ile Pro Asn His Pro Gly Gly Ser Leu Ile His Val Lys Ala Gly Gln
65 70 75 80
Asp Ser Thr Gln Leu Phe Asp Ser Tyr His Pro Leu Tyr Val Arg Lys
85 90 95
Met Leu Ala Lys Tyr Cys Ile Gly Glu Leu Val Pro Ser Ala Gly Asp
100 105 110
Asp Lys Phe Lys Lys Ala Thr Leu Glu Tyr Ala Asp Ala Glu Asn Glu
115 120 125
Asp Phe Tyr Leu Val Val Lys Gln Arg Val Glu Ser Tyr Phe Lys Ser
130 135 140
Asn Lys Ile Asn Pro Gln Ile His Pro His Met Ile Leu Lys Ser Leu
145 150 155 160
Phe Ile Leu Gly Gly Tyr Phe Ala Ser Tyr Tyr Leu Ala Phe Phe Trp
165 170 175
Ser Ser Ser Val Leu Val Ser Leu Phe Phe Ala Leu Trp Met Gly Phe
180 185 190
Phe Ala Ala Glu Val Gly Val Ser Ile Gln His Asp Gly Asn His Gly
195 200 205
Ser Tyr Thr Lys Trp Arg Gly Phe Gly Tyr Ile Met Gly Ala Ser Leu
2l0 215 220
Asp Leu Val Gly Ala Ser Ser Phe Met Trp Arg Gln Gln His Val Val
225 230 235 240
Gly His His Ser Phe Thr Asn Val Asp Asn Tyr Asp Pro Asp Ile Arg
245 250 255
Val Lys Asp Pro Asp Val Arg Arg Val Ala Thr Thr Gln Pro Arg Gln
260 265 270
Trp Tyr His Ala Tyr Gln His Ile Tyr Leu Ala Val Leu Tyr Gly Thr
275 280 285
Leu Ala Leu Lys Ser Ile Phe Leu Asp Asp Phe Leu Ala Tyr Phe Thr
290 295 300
Gly Ser Ile Gly Pro Val Lys Val Ala Lys Met Thr Pro Leu Glu Phe
305 310 315 320
Asn Ile Phe Phe Gln Gly Lys Leu Leu Tyr Ala Phe Tyr Met Phe Val
325 330 335
Leu Pro Ser Val Tyr Gly Val His Ser Gly Gly Thr Phe Leu Ala Leu
340 345 350
Tyr Val Ala Ser Gln Leu Ile Thr Gly Trp Met Leu Ala Phe Leu Phe
355 360 365
Gln Val Ala His Val Val Asp Asp Val Ala Phe Pro Thr Pro Glu Gly
370 375 380
Gly Lys Val Lys Gly Gly Trp Ala Ala Met Gln Val Ala Thr Thr Thr
385 390 395 400
Asp Phe Ser Pro Arg Ser Trp Phe Trp Gly His Val Ser Gly Gly Leu
405 410 415
Asn Asn Gln Ile Glu His His Leu Phe Pro Gly Val Cys His Val His
420 425 430
Tyr Pro Ala Ile Gln Pro Ile Val Glu Lys Thr Cys Lys Glu Phe Asp
435 440 445
Val Pro Tyr Val Ala Tyr Pro Thr Phe Trp Thr Ala Leu Arg Ala His
450 455 460
Phe Ala His Leu Lys Lys Val Gly Leu Thr Glu Phe Arg Leu Asp Gly
465 470 475 480
<210>11
<211>1320
<212>DNA
<213>破囊壶菌属(Thraustrochytrium)
<220>
<221>CDS
<222>(1)..(1320)
<223>
<400>11
atg ggc aag ggc agc gag ggc cgc agc gcg gcg cgc gag atg acg gcc 48
Met Gly Lys Gly Ser Glu Gly Arg Ser Ala Ala Arg Glu Met Thr Ala
1 5 10 15
gag gcg aac ggc gac aag cgg aaa acg att ctg atc gag ggc gtc ctg 96
Glu Ala Asn Gly Asp Lys Arg Lys Thr Ile Leu Ile Glu Gly Val Leu
20 25 30
tac gac gcg acg aac ttt aag cac ccg ggc ggt tcg atc atc aac ttc 144
Tyr Asp Ala Thr Asn Phe Lys His Pro Gly Gly Ser Ile Ile Asn Phe
35 40 45
ttg acc gag ggc gag gcc ggc gtg gac gcg acg cag gcg tac cgc gag 192
Leu Thr Glu Gly Glu Ala Gly Val Asp Ala Thr Gln Ala Tyr Arg Glu
50 55 60
ttt cat cag cgg tcc ggc aag gcc gac aag tac ctc aag tcg ctg ccg 240
Phe His Gln Arg Ser Gly Lys Ala Asp Lys Tyr Leu Lys Ser Leu Pro
65 70 75 80
aag ctg gat gcg tcc aag gtg gag tcg cgg ttc tcg gcc aaa gag cag 288
Lys Leu Asp Ala Ser Lys Val Glu Ser Arg Phe Ser Ala Lys Glu Gln
85 90 95
gcg cgg cgc gac gcc atg acg cgc gac tac gcg gcc ttt cgc gag gag 336
Ala Arg Arg Asp Ala Met Thr Arg Asp Tyr Ala Ala Phe Arg Glu Glu
100 105 110
ctc gtc gcc gag ggg tac ttt gac ccg tcg atc ccg cac atg att tac 384
Leu Val Ala Glu Gly Tyr Phe Asp Pro Ser Ile Pro His Met Ile Tyr
115 120 125
cgc gtc gtg gag atc gtg gcg ctc ttc gcg ctc tcg ttc tgg ctc atg 432
Arg Val Val Glu Ile Val Ala Leu Phe Ala Leu Ser Phe Trp Leu Met
130 135 140
tcc aag gcc tcg ccc acc tcg ctc gtg ctg ggc gtg gtg atg aac ggc 480
Ser Lys Ala Ser Pro Thr Ser Leu Val Leu Gly Val Val Met Asn Gly
145 150 155 160
att gcg cag ggc cgc tgc ggc tgg gtc atg cac gag atg ggc cac ggg 528
Ile Ala Gln Gly Arg Cys Gly Trp Val Met His Glu Met Gly His Gly
165 170 175
tcg ttc acg ggc gtc atc tgg ctc gac gac cgg atg tgc gag ttc ttc 576
Ser Phe Thr Gly Val Ile Trp Leu Asp Asp Arg Met Cys Glu Phe Phe
180 185 190
tac ggc gtc ggc tgc ggc atg agc ggg cac tac tgg aag aac cag cac 624
Tyr Gly Val Gly Cys Gly Met Ser Gly His Tyr Trp Lys Asn Gln His
195 200 205
agc aag cac cac gcc gcg ccc aac cgc ctc gag cac gat gtc gat ctc 672
Ser Lys His His Ala Ala Pro Asn Arg Leu Glu His Asp Val Asp Leu
210 215 220
aac acg ctg ccc ctg gtc gcc ttt aac gag cgc gtc gtg cgc aag gtc 720
Asn Thr Leu Pro Leu Val Ala Phe Asn Glu Arg Val Val Arg Lys Val
225 230 235 240
aag ccg gga tcg ctg ctg gcg ctc tgg ctg cgc gtg cag gcg tac ctc 768
Lys Pro Gly Ser Leu Leu Ala Leu Trp Leu Arg Val Gln Ala Tyr Leu
245 250 255
ttt gcg ccc gtc tcg tgc ctg ctc atc ggc ctt ggc tgg acg ctc tac 816
Phe Ala Pro Val Ser Cys Leu Leu Ile Gly Leu Gly Trp Thr Leu Tyr
260 265 270
ctg cac ccg cgc tac atg ctg cgc acc aag cgg cac atg gag ttc gtc 864
Leu His Pro Arg Tyr Met Leu Arg Thr Lys Arg His Met Glu Phe Val
275 280 285
tgg atc ttc gcg cgc tac att ggc tgg ttc tcg ctc atg ggc gct ctc 912
Trp Ile Phe Ala Arg Tyr Ile Gly Trp Phe Ser Leu Met Gly Ala Leu
290 295 300
ggc tac tcg ccg ggc acc tcg gtc ggg atg tac ctg tgc tcg ttc ggc 960
Gly Tyr Ser Pro Gly Thr Ser Val Gly Met Tyr Leu Cys Ser Phe Gly
305 310 315 320
ctc ggc tgc att tac att ttc ctg cag ttc gcc gtc agc cac acg cac 1008
Leu Gly Cys Ile Tyr Ile Phe Leu Gln Phe Ala Val Ser His Thr His
325 330 335
ctg ccg gtg acc aac ccg gag gac cag ctg cac tgg ctc gag tac gcg 1056
Leu Pro Val Thr Asn Pro Glu Asp Gln Leu His Trp Leu Glu Tyr Ala
340 345 350
gcc gac cac acg gtg aac att agc acc aag tcc tgg ctc gtc acg tgg 1104
Ala Asp His Thr Val Asn Ile Ser Thr Lys Ser Trp Leu Val Thr Trp
355 360 365
tgg atg tcg aac ctg aac ttt cag atc gag cac cac ctc ttc ccc acg 1152
Trp Met Ser Asn Leu Asn Phe Gln Ile Glu His His Leu Phe Pro Thr
370 375 380
gcg ccg cag ttc cgc ttc aag gaa atc agt cct cgc gtc gag gcc ctc 1200
Ala Pro Gln Phe Arg Phe Lys Glu Ile Ser Pro Arg Val Glu Ala Leu
385 390 395 400
ttc aag cgc cac aac ctc ccg tac tac gac ctg ccc tac acg agc gcg 1248
Phe Lys Arg His Asn Leu Pro Tyr Tyr Asp Leu Pro Tyr Thr Ser Ala
405 410 415
gtc tcg acc acc ttt gcc aat ctt tat tcc gtc ggc cac tcg gtc ggc 1296
Val Ser Thr Thr Phe Ala Asn Leu Tyr Ser Val Gly His Ser Val Gly
420 425 430
gcc gac acc aag aag cag gac tga 1320
Ala Asp Thr Lys Lys Gln Asp
435
<210>12
<211>439
<212>PRT
<213>破囊壶菌属
<400>12
Met Gly Lys Gly Ser Glu Gly Arg Ser Ala Ala Arg Glu Met Thr Ala
1 5 10 15
Glu Ala Asn Gly Asp Lys Arg Lys Thr Ile Leu Ile Glu Gly Val Leu
20 25 30
Tyr Asp Ala Thr Asn Phe Lys His Pro Gly Gly Ser Ile Ile Asn Phe
35 40 45
Leu Thr Glu Gly Glu Ala Gly Val Asp Ala Thr Gln Ala Tyr Arg Glu
50 55 60
Phe His Gln Arg Ser Gly Lys Ala Asp Lys Tyr Leu Lys Ser Leu Pro
65 70 75 80
Lys Leu Asp Ala Ser Lys Val Glu Ser Arg Phe Ser Ala Lys Glu Gln
85 90 95
Ala Arg Arg Asp Ala Met Thr Arg Asp Tyr Ala Ala Phe Arg Glu Glu
100 105 110
Leu Val Ala Glu Gly Tyr Phe Asp Pro Ser Ile Pro His Met Ile Tyr
115 120 125
Arg Val Val Glu Ile Val Ala Leu Phe Ala Leu Ser Phe Trp Leu Met
130 135 140
Ser Lys Ala Ser Pro Thr Ser Leu Val Leu Gly Val Val Met Asn Gly
145 150 155 160
Ile Ala Gln Gly Arg Cys Gly Trp Val Met His Glu Met Gly His Gly
165 170 175
Ser Phe Thr Gly Val Ile Trp Leu Asp Asp Arg Met Cys Glu Phe Phe
180 185 190
Tyr Gly Val Gly Cys Gly Met Ser Gly His Tyr Trp Lys Asn Gln His
195 200 205
Ser Lys His His Ala Ala Pro Asn Arg Leu Glu His Asp Val Asp Leu
210 215 220
Asn Thr Leu Pro Leu Val Ala Phe Asn Glu Arg Val Val Arg Lys Val
225 230 235 240
Lys Pro Gly Ser Leu Leu Ala Leu Trp Leu Arg Val Gln Ala Tyr Leu
245 250 255
Phe Ala Pro Val Ser Cys Leu Leu Ile Gly Leu Gly Trp Tnr Leu Tyr
260 265 270
Leu His Pro Arg Tyr Met Leu Arg Thr Lys Arg His Met Glu Phe Val
275 280 285
Trp Ile Phe Ala Arg Tyr Ile Gly Trp Phe Ser Leu Met Gly Ala Leu
290 295 300
Gly Tyr Ser Pro Gly Thr Ser Val Gly Met Tyr Leu Cys Ser Phe Gly
305 310 315 320
Leu Gly Cys Ile Tyr Ile Phe Leu Gln Phe Ala Val Ser His Thr His
325 330 335
Leu Pro Val Thr Asn Pro Glu Asp Gln Leu His Trp Leu Glu Tyr Ala
340 345 350
Ala Asp His Thr Val Asn Ile Ser Thr Lys Ser Trp Leu Val Thr Trp
355 360 365
Trp Met Ser Asn Leu Asn Phe Gln Ile Glu His His Leu Phe Pro Thr
370 375 380
Ala Pro Gln Phe Arg Phe Lys Glu Ile Ser Pro Arg Val Glu Ala Leu
385 390 395 400
Phe Lys Arg His Asn Leu Pro Tyr Tyr Asp Leu Pro Tyr Thr Ser Ala
405 410 415
Val Ser Thr Thr Phe Ala Asn Leu Tyr Ser Val Gly His Ser Val Gly
420 425 430
Ala Asp Thr Lys Lys Gln AsP
435
<210>13
<211>1341
<212>DNA
<213>高山被孢霉(Mortierella alpina)
<220>
<221>CDS
<222>(1)..(1341)
<223>Δ5-去饱和酶
<400>13
atg gga acg gac caa gga aaa acc ttc acc tgg gaa gag ctg gcg gcc 48
Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala
1 5 10 15
cat aac acc aag gac gac cta ctc ttg gcc atc cgc ggc agg gtg tac 96
His Asn Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr
20 25 30
gat gtc aca aag ttc ttg agc cgc cat cct ggt gga gtg gac act ctc 144
Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu
35 40 45
ctg ctc gga gct ggc cga gat gtt act ccg gtc ttt gag atg tat cac 192
Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His
50 55 60
gcg ttt ggg gct gca gat gcc att atg aag aag tac tat gtc ggt aca 240
Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr
65 70 75 80
ctg gtc tcg aat gag ctg ccc atc ttc ccg gag cca acg gtg ttc cac 288
Leu Val Ser Ash Glu Leu Pro Ile Phe Pro Glu Pro Thr Val Phe His
85 90 95
aaa acc atc aag acg aga gtc gag ggc tac ttt acg gat cgg aac att 336
Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn Ile
100 105 110
gat ccc aag aat aga cca gag atc tgg gga cga tac gct ctt atc ttt 384
Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe
115 120 125
gga tcc ttg atc gct tcc tac tac gcg cag ctc ttt gtg cct ttc gtt 432
Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val
130 135 140
gtc gaa cgc aca tgg ctt cag gtg gtg ttt gca atc atc atg gga ttt 480
Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe
145 150 155 160
gcg tgc gca caa gtc gga ctc aac cct ctt cat gat gcg tct cac ttt 528
Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe
165 170 175
tca gtg acc cac aac ccc act gtc tgg aag att ctg gga gcc acg cac 576
Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His
180 185 190
gac ttt ttc aac gga gca tcg tac ctg gtg tgg atg tac caa cat atg 624
Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met
195 200 205
ctc ggc cat cac ccc tac acc aac att gct gga gca gat ccc gac gtg 672
Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val
210 215 220
tcg acg tct gag ccc gat gtt cgt cgt atc aag ccc aac caa aag tgg 720
Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp
225 230 235 240
ttt gtc aac cac atc aac cag cac atg ttt gtt cct ttc ctg tac gga 768
Phe Val Asn His Ile Asn Gln His Met Phe Val Pro Phe Leu Tyr Gly
245 250 255
ctg ctg gcg ttc aag gtg cgc att cag gac atc aac att ttg tac ttt 816
Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe
260 265 270
gtc aag acc aat gac gct att cgt gtc aat ccc atc tcg aca tgg cac 864
Val Lys Thr Asn Asp Ala Ile Arg Val Asn Pro Ile Ser Thr Trp His
275 280 285
act gtg atg ttc tgg ggc ggc aag gct ttc ttt gtc tgg tat cgc ctg 912
Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu
290 295 300
att gtt ccc ctg cag tat ctg ccc ctg ggc aag gtg ctg ctc ttg ttc 960
Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe
305 310 315 320
acg gtc gcg gac atg gtg tcg tct tac tgg ctg gcg ctg acc ttc cag 1008
Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Lau Thr Phe Gln
325 330 335
gcg aac cac gtt gtt gag gaa gtt cag tgg ccg ttg cct gac gag aac 1056
Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn
340 345 350
ggg atc atc caa aag gac tgg gca gct atg cag gtc gag act acg cag 1104
Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln
355 360 365
gat tac gca cac gat tcg cac ctc tgg acc agc atc act ggc agc ttg 1152
Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu
370 375 380
aac tac cag gct gtg cac cat ctg ttc ccc aac gtg tcg cag cac cat 1200
Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His
385 390 395 400
tat ccc gat att ctg gcc atc atc aag aac acc tgc agc gag tac aag 1248
Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys
405 410 415
gtt cca tac ctt gtc aag gat acg ttt tgg caa gca ttt gct tca cat 1296
Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His
420 425 430
ttg gag cac ttg cgt gtt ctt gga ctc cgt ccc aag gaa gag tag 1341
Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu
435 440 445
<210>14
<211>446
<212>PRT
<213>高山被孢霉
<400>14
Met Gly Thr Asp Gln Gly Lys Thr Phe Thr Trp Glu Glu Leu Ala Ala
1 5 10 15
His Asn Thr Lys Asp Asp Leu Leu Leu Ala Ile Arg Gly Arg Val Tyr
20 25 30
Asp Val Thr Lys Phe Leu Ser Arg His Pro Gly Gly Val Asp Thr Leu
35 40 45
Leu Leu Gly Ala Gly Arg Asp Val Thr Pro Val Phe Glu Met Tyr His
50 55 60
Ala Phe Gly Ala Ala Asp Ala Ile Met Lys Lys Tyr Tyr Val Gly Thr
65 70 75 80
Leu Val Ser Asn Glu Leu Pro Ile Phe Pro Glu Pro Thr Val Phe His
85 90 95
Lys Thr Ile Lys Thr Arg Val Glu Gly Tyr Phe Thr Asp Arg Asn Ile
100 105 110
Asp Pro Lys Asn Arg Pro Glu Ile Trp Gly Arg Tyr Ala Leu Ile Phe
115 120 125
Gly Ser Leu Ile Ala Ser Tyr Tyr Ala Gln Leu Phe Val Pro Phe Val
130 135 140
Val Glu Arg Thr Trp Leu Gln Val Val Phe Ala Ile Ile Met Gly Phe
145 150 155 160
Ala Cys Ala Gln Val Gly Leu Asn Pro Leu His Asp Ala Ser His Phe
165 170 175
Ser Val Thr His Asn Pro Thr Val Trp Lys Ile Leu Gly Ala Thr His
180 185 190
Asp Phe Phe Asn Gly Ala Ser Tyr Leu Val Trp Met Tyr Gln His Met
195 200 205
Leu Gly His His Pro Tyr Thr Asn Ile Ala Gly Ala Asp Pro Asp Val
210 215 220
Ser Thr Ser Glu Pro Asp Val Arg Arg Ile Lys Pro Asn Gln Lys Trp
225 230 235 240
Phe Val Asn His Ile Asn Gln His Met Phe Val Pro Phe Leu Tyr Gly
245 250 255
Leu Leu Ala Phe Lys Val Arg Ile Gln Asp Ile Asn Ile Leu Tyr Phe
260 265 270
Val Lys Thr Asn Asp A1a Ile Arg Val Asn Pro Ile Ser Thr Trp His
275 280 285
Thr Val Met Phe Trp Gly Gly Lys Ala Phe Phe Val Trp Tyr Arg Leu
290 295 300
Ile Val Pro Leu Gln Tyr Leu Pro Leu Gly Lys Val Leu Leu Leu Phe
305 3l0 315 320
Thr Val Ala Asp Met Val Ser Ser Tyr Trp Leu Ala Leu Thr Phe Gln
325 330 335
Ala Asn His Val Val Glu Glu Val Gln Trp Pro Leu Pro Asp Glu Asn
340 345 350
Gly Ile Ile Gln Lys Asp Trp Ala Ala Met Gln Val Glu Thr Thr Gln
355 360 365
Asp Tyr Ala His Asp Ser His Leu Trp Thr Ser Ile Thr Gly Ser Leu
370 375 380
Asn Tyr Gln Ala Val His His Leu Phe Pro Asn Val Ser Gln His His
385 390 395 400
Tyr Pro Asp Ile Leu Ala Ile Ile Lys Asn Thr Cys Ser Glu Tyr Lys
405 410 415
Val Pro Tyr Leu Val Lys Asp Thr Phe Trp Gln Ala Phe Ala Ser His
420 425 430
Leu Glu His Leu Arg Val Leu Gly Leu Arg Pro Lys Glu Glu
435 440 445
<210>15
<211>1344
<212>DNA
<213>秀丽隐杆线虫(Caenorhabditis elegans)
<220>
<221>CDS
<222>(1)..(1344)
<223>Δ5-去饱和酶
<400>15
atg gta tta cga gag caa gag cat gag cca ttc ttc att aaa att gat 48
Met Val Leu Arg Glu Gln Glu His Glu Pro Phe Phe Ile Lys Ile Asp
1 5 10 15
gga aaa tgg tgt caa att gac gat gct gtc ctg aga tca cat cca ggt 96
Gly Lys Trp Cys Gln Ile Asp Asp Ala Val Leu Arg Ser His Pro Gly
20 25 30
ggt agt gca att act acc tat aaa aat atg gat gcc act acc gta ttc 144
Gly Ser Ala Ile Thr Thr Tyr Lys Asn Met Asp Ala Thr Thr Val Phe
35 40 45
cac aca ttc cat act ggt tct aaa gaa gcg tat caa tgg ctg aca gaa 192
His Thr Phe His Thr Gly Ser Lys Glu Ala Tyr Gln Trp Leu Thr Glu
50 55 60
ttg aaa aaa gag tgc cct aca caa gaa cca gag atc cca gat att aag 240
Leu Lys Lys Glu Cys Pro Thr Gln Glu Pro Glu Ile Pro Asp Ile Lys
65 70 75 80
gat gac cca atc aaa gga att gat gat gtg aac atg gga act ttc aat 288
Asp Asp Pro I1e Lys Gly Ile Asp Asp Val Asn Met Gly Thr Phe Asn
85 90 95
att tct gag aaa cga tct gcc caa ata aat aaa agt ttc act gat cta 336
Ile Ser Glu Lys Arg Ser Ala Gln Ile Asn Lys Ser Phe Thr Asp Leu
100 105 110
cgt atg cga gtt cgt gca gaa gga ctt atg gat gga tct cct ttg ttc 384
Arg Met Arg Val Arg Ala Glu Gly Leu Met Asp Gly Ser Pro Leu Phe
115 120 125
tac att aga aaa att ctt gaa aca atc ttc aca att ctt ttt gca ttc 432
Tyr Ile Arg Lys Ile Leu Glu Thr Ile Phe Thr Ile Leu Phe Ala Phe
130 135 140
tac ctt caa tac cac aca tat tat ctt cca tca gct att cta atg gga 480
Tyr Leu Gln Tyr His Thr Tyr Tyr Leu Pro Ser Ala Ile Leu Met Gly
l45 150 155 160
gtt gcg tgg caa caa ttg gga tgg tta atc cat gaa ttc gca cat cat 528
Val Ala Trp Gln Gln Leu Gly Trp Leu Ile His Glu Phe Ala His His
165 170 175
cag ttg ttc aaa aac aga tac tac aat gat ttg gcc agc tat ttc gtt 576
Gln Leu Phe Lys Asn Arg Tyr Tyr Asn Asp Leu Ala Ser Tyr Phe Val
180 185 190
gga aac ttt tta caa gga ttc tca tct ggt ggt tgg aaa gag cag cac 624
Gly Asn Phe Leu Gln Gly Phe Ser Ser Gly Gly Trp Lys Glu Gln His
195 200 205
aat gtg cat cac gca gcc aca aat gtt gtt gga cga gac gga gat ctt 672
Asn Val His His Ala Ala Thr Asn Val Val Gly Arg Asp Gly Asp Leu
210 215 220
gat tta gtc cca ttc tat gct aca gtg gca gaa cat ctc aac aat tat 720
Asp Leu Val Pro Phe Tyr Ala Thr Val Ala Glu His Leu Asn Asn Tyr
225 230 235 240
tct cag gat tca tgg gtt atg act cta ttc aga tgg caa cat gtt cat 768
Ser Gln Asp Ser Trp Val Met Thr Leu Phe Arg Trp Gln His Val His
245 250 255
tgg aca ttc atg tta cca ttc ctc cgt ctc tcg tgg ctt ctt cag tca 816
Trp Thr Phe Met Leu Pro Phe Leu Arg Leu Ser Trp Leu Leu Gln Ser
260 265 270
atc att ttt gtt agt cag atg cca act cat tat tat gac tat tac aga 864
Ile Ile Phe Val Ser Gln Met Pro Thr His Tyr Tyr Asp Tyr Tyr Arg
275 280 285
aat act gcg att tat gaa cag gtt ggt ctc tct ttg cac tgg gct tgg 912
Asn Thr Ala Ile Tyr Glu Gln Val Gly Leu Ser Leu His Trp Ala Trp
290 295 300
tca ttg ggt caa ttg tat ttc cta ccc gat tgg tca act aga ata atg 960
Ser Leu Gly Gln Leu Tyr Phe Leu Pro Asp Trp Ser Thr Arg Ile Met
305 3l0 315 320
ttc ttc ctt gtt tct cat ctt gtt gga ggt ttc ctg ctc tct cat gta 1008
Phe Phe Leu Val Ser His Leu Val Gly Gly Phe Leu Leu Ser His Val
325 330 335
gtt act ttc aat cat tat tca gtg gag aag ttt gca ttg agc tcg aac 1056
Val Thr Phe Asn His Tyr Ser Val Glu Lys Phe Ala Leu Ser Ser Asn
340 345 350
atc atg tca aat tac gct tgt ctt caa atc atg acc aca aga aat atg 1104
Ile Met Ser Asn Tyr Ala Cys Leu Gln Ile Met Thr Thr Arg Asn Met
355 360 365
aga cct gga aga ttc att gac tgg ctt tgg gga ggt ctt aac tat cag 1152
Arg Pro Gly Arg Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln
370 375 380
att gag cac cat ctt ttc cca acg atg cca cga cac aac ttg aac act 1200
Ile Glu His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Thr
385 390 395 400
gtt atg cca ctt gtt aag gag ttt gca gca gca aat ggt tta cca tac 1248
Val Met Pro Leu Val Lys Glu Phe Ala Ala Ala Asn Gly Leu Pro Tyr
405 410 415
atg gtc gac gat tat ttc aca gga ttc tgg ctt gaa att gag caa ttc 1296
Met Val Asp Asp Tyr Phe Thr Gly Phe Trp Leu Glu Ile Glu Gln Phe
420 425 430
cga aat att gca aat gtt gct gct aaa ttg act aaa aag att gcc tag 1344
Arg Asn Ile Ala Asn Val Ala Ala Lys Leu Thr Lys Lys Ile Ala
435 440 445
<210>16
<211>447
<212>PRT
<213>秀丽隐杆线虫
<400>16
Met Val Leu Arg Glu Gln Glu His Glu Pro Phe Phe Ile Lys Ile Asp
1 5 10 15
Gly Lys Trp Cys Gln Ile Asp Asp Ala Val Leu Arg Ser His Pro Gly
20 25 30
Gly Ser Ala Ile Thr Thr Tyr Lys Asn Met Asp Ala Thr Thr Val Phe
35 40 45
His Thr Phe His Thr Gly Ser Lys Glu Ala Tyr Gln Trp Leu Thr Glu
50 55 60
Leu Lys Lys Glu Cys Pro Thr Gln Glu Pro Glu Ile Pro Asp Ile Lys
65 70 75 80
Asp Asp Pro Ile Lys Gly Ile Asp Asp Val Asn Met Gly Thr Phe Asn
85 90 95
Ile Ser Glu Lys Arg Ser Ala Gln Ile Asn Lys Ser Phe Thr Asp Leu
100 105 110
Arg Met Arg Val Arg Ala Glu Gly Leu Met Asp Gly Ser Pro Leu Phe
115 120 125
Tyr Ile Arg Lys Ile Leu Glu Thr Ile Phe Thr Ile Leu Phe Ala Phe
130 135 140
Tyr Leu Gln Tyr His Thr Tyr Tyr Leu Pro Ser Ala Ile Leu Met Gly
145 150 155 160
Val Ala Trp Gln Gln Leu Gly Trp Leu Ile His Glu Phe Ala His His
165 170 175
Gln Leu Phe Lys Asn Arg Tyr Tyr Asn Asp Leu Ala Ser Tyr Phe Val
180 185 190
Gly Asn Phe Leu Gln Gly Phe Ser Ser Gly Gly Trp Lys Glu Gln His
195 200 205
Asn Val His His Ala Ala Thr Asn Val Val Gly Arg Asp Gly Asp Leu
210 215 220
Asp Leu Val Pro Phe Tyr Ala Thr Val Ala Glu His Leu Asn Asn Tyr
225 230 235 240
Ser Gln Asp Ser Trp Val Met Thr Leu Phe Arg Trp Gln His Val His
245 250 255
Trp Thr Phe Met Leu Pro Phe Leu Arg Leu Ser Trp Leu Leu Gln Ser
260 265 270
Ile Ile Phe Val Ser Gln Met Pro Thr His Tyr Tyr Asp Tyr Tyr Arg
275 280 285
Asn Thr Ala Ile Tyr Glu Gln Val Gly Leu Ser Leu His Trp Ala Trp
290 295 300
Ser Leu Gly Gln Leu Tyr Phe Leu Pro Asp Trp Ser Thr Arg Ile Met
305 310 315 320
Phe Phe Leu Val Ser His Leu Val Gly Gly Phe Leu Leu Ser His Val
325 330 335
Val Thr Phe Asn His Tyr Ser Val Glu Lys Phe Ala Leu Ser Ser Asn
340 345 350
Ile Met Ser Asn Tyr Ala Cys Leu Gln Ile Met Thr Thr Arg Asn Met
355 360 365
Arg Pro Gly Arg Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln
370 375 380
Ile Glu His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Thr
385 390 395 400
Val Met Pro Leu Val Lys Glu Phe Ala Ala Ala Asn Gly Leu Pro Tyr
405 410 415
Met Val Asp Asp Tyr Phe Thr Gly Phe Trp Leu Glu Ile Glu Gln Phe
420 425 430
Arg Asn Ile Ala Asn Val Ala Ala Lys Leu Thr Lys Lys Ile Ala
435 440 445
<210>17
<211>1683
<212>DNA
<213>琉璃苣(Borago officinalis)
<220>
<221>CDS
<222>(42)..(1388)
<223>Δ6-去饱和酶
<400>17
tatctgccta ccctcccaaa gagagtagtc atttttcatc a atg gct gct caa atc 56
Met Ala Ala Gln Ile
1 5
aag aaa tac att acc tca gat gaa ctc aag aac cac gat aaa ccc gga 104
Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn His Asp Lys Pro Gly
10 15 20
gat cta tgg atc tcg att caa ggg aaa gcc tat gat gtt tcg gat tgg 152
Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr Asp Val Ser Asp Trp
25 30 35
gtg aaa gac cat cca ggt ggc agc ttt ccc ttg aag agt ctt gct ggt 200
Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu Lys Ser Leu Ala Gly
40 45 50
caa gag gta act gat gca ttt gtt gca ttc cat cct gcc tct aca tgg 248
Gln Glu Val Thr Asp Ala Phe Val Ala Phe His Pro Ala Ser Thr Trp
55 60 65
aag aat ctt gat aag ttt ttc act ggg tat tat ctt aaa gat tac tct 296
Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr Leu Lys Asp Tyr Ser
70 75 80 85
gtt tct gag gtt tct aaa gat tat agg aag ctt gtg ttt gag ttt tct 344
Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu Val Phe Glu Phe Ser
90 95 100
aaa atg ggt ttg tat gac aaa aaa ggt cat att atg ttt gca act ttg 392
Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile Met Phe Ala Thr Leu
105 110 115
tgc ttt ata gca atg ctg ttt gct atg agt gtt tat ggg gtt ttg ttt 440
Cys Phe Ile Ala Met Leu Phe Ala Met Ser Val Tyr Gly Val Leu Phe
120 125 130
tgt gag ggt gtt ttg gta cat ttg ttt tct ggg tgt ttg atg ggg ttt 488
Cys Glu Gly Val Leu Val His Leu Phe Ser Gly Cys Leu Met Gly Phe
135 140 145
ctt tgg att cag agt ggt tgg att gga cat gat gct ggg cat tat atg 536
Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp Ala Gly His Tyr Met
150 155 160 165
gta gtg tct gat tca agg ctt aat aag ttt atg ggt att ttt gct gca 584
Val Val Ser Asp Ser Arg Leu Asn Lys Phe Met Gly Ile Phe Ala Ala
170 175 180
aat tgt ctt tca gga ata agt att ggt tgg tgg aaa tgg aac cat aat 632
Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp Lys Trp Asn His Asn
185 190 195
gca cat cac att gcc tgt aat agc ctt gaa tat gac cct gat tta caa 680
Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr Asp Pro Asp Leu Gln
200 205 210
tat ata cca ttc ctt gtt gtg tct tcc aag ttt ttt ggt tca ctc acc 728
Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Phe Phe Gly Ser Leu Thr
215 220 225
tct cat ttc tat gag aaa agg ttg act ttt gac tct tta tca aga ttc 776
Ser His Phe Tyr Glu Lys Arg Leu Thr Phe Asp Ser Leu Ser Arg Phe
230 235 240 245
ttt gta agt tat caa cat tgg aca ttt tac cct att atg tgt gct gct 824
Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro Ile Met Cys Ala Ala
250 255 260
agg ctc aat atg tat gta caa tct ctc ata atg ttg ttg acc aag aga 872
Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met Leu Leu Thr Lys Arg
265 270 275
aat gtg tcc tat cga gct cag gaa ctc ttg gga tgc cta gtg ttc tcg 920
Asn Val Ser Tyr Arg Ala Gln Glu Leu Leu Gly Cys Leu Val Phe Ser
280 285 290
att tgg tac ccg ttg ctt gtt tct tgt ttg cct aat tgg ggt gaa aga 968
Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro Asn Trp Gly Glu Arg
295 300 305
att atg ttt gtt att gca agt tta tca gtg act gga atg caa caa gtt 1016
Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr Gly Met Gln Gln Val
3l0 315 320 325
cag ttc tcc ttg aac cac ttc tct tca agt gtt tat gtt gga aag cct 1064
Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val Tyr Val Gly Lys Pro
330 335 340
aaa ggg aat aat tgg ttt gag aaa caa acg gat ggg aca ctt gac att 1112
Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp Gly Thr Leu Asp Ile
345 350 355
tct tgt cct cct tgg atg gat tgg ttt cat ggt gga ttg caa ttc caa 1160
Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly Gly Leu Gln Phe Gln
360 365 370
att gag cat cat ttg ttt ccc aag atg cct aga tgc aac ctt agg aaa 1208
Ile Glu His His Leu Phe Pro Lys Met Pro Arg Cys Asn Leu Arg Lys
375 380 385
atc tcg ccc tac gtg atc gag tta tgc aag aaa cat aat ttg cct tac 1256
Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys His Asn Leu Pro Tyr
390 395 400 405
aat tat gca tct ttc tcc aag gcc aat gaa atg aca ctc aga aca ttg 1304
Asn Tyr Ala Ser Phe Ser Lys Ala Asn Glu Met Thr Leu Arg Thr Leu
410 415 420
agg aac aca gca ttg cag gct agg gat ata acc aag ccg ctc ccg aag 1352
Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile Thr Lys Pro Leu Pro Lys
425 430 435
aat ttg gta tgg gaa gct ctt cac act cat ggt taa aattaccctt 1398
Asn Leu Val Trp Glu Ala Leu His Thr His Gly
440 445
agttcatgta ataatttgag attatgtatc tcctatgttt gtgtcttgtc ttggttctac 1458
ttgttggagt cattgcaact tgtcttttat ggtttattag atgtttttta atatatttta 1518
gaggttttgc tttcatctcc attattgatg aataaggagt tgcatattgt caattgttgt 1578
gctcaatatc tgatattttg gaatgtactt tgtaccactg tgttttcagt tgaagctcat 1638
gtgtacttct atagactttg tttaaatggt tatgtcatgt tattt 1683
<210>18
<211>448
<212>PRT
<213>琉璃苣
<400>18
Met Ala Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn
1 5 10 15
His Asp Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr
20 25 30
Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu
35 40 45
Lys Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His
50 55 60
Pro Ala Ser Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr
65 70 75 80
Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu
85 90 95
Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile
100 105 110
Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met Ser Val
115 120 125
Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Phe Ser Gly
130 135 140
Cys Leu Met Gly Phe Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp
145 150 155 160
Ala Gly His Tyr Met Val Val Ser Asp Ser Arg Leu Asn Lys Phe Met
165 170 175
Gly Ile Phe Ala Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp
180 185 190
Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr
195 200 205
Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Phe
210 215 220
Phe Gly Ser Leu Thr Ser His Phe Tyr Glu Lys Arg Leu Thr Phe Asp
225 230 235 240
Ser Leu Ser Arg Phe Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro
245 250 255
Ile Met Cys Ala Ala Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met
260 265 270
Leu Leu Thr Lys Arg Asn Val Ser Tyr Arg Ala Gln Glu Leu Leu Gly
275 280 285
Cys Leu Val Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro
290 295 300
Asn Trp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr
305 310 315 320
Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val
325 330 335
Tyr Val Gly Lys Pro Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp
340 345 350
Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly
355 360 365
Gly Leu Gln Phe Gln Ile Glu His His Leu Pne Pro Lys Met Pro Arg
370 375 380
Cys Asn Leu Arg Lys Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys
385 390 395 400
His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe Ser Lys Ala Asn Glu Met
405 410 415
Thr Leu Arg Thr Leu Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile Thr
420 425 430
Lys Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu His Thr His Gly
435 440 445
<210>19
<211>1563
<212>DNA
<213>角齿藓
<220>
<221>CDS
<222>(1)..(1563)
<223>Δ6-去饱和酶
<400>19
atg gtg tcc cag ggc ggc ggt ctc tcg cag ggt tcc att gaa gaa aac 48
Met Val Ser Gln Gly Gly Gly Leu Ser Gln Gly Ser Ile Glu Glu Asn
1 5 10 15
att gac gtt gag cac ttg gca acg atg ccc ctc gtc agt gac ttc cta 96
Ile Asp Val Glu His Leu Ala Thr Met Pro Leu Val Ser Asp Phe Leu
20 25 30
aat gtc ctg gga acg act ttg ggc cag tgg agt ctt tcc act aca ttc 144
Asn Val Leu Gly Thr Thr Leu Gly Gln Trp Ser Leu Ser Thr Thr Phe
35 40 45
gct ttc aag agg ctc acg act aag aaa cac agt tcg gac atc tcg gtg 192
Ala Phe Lys Arg Leu Thr Thr Lys Lys His Ser Ser Asp Ile Ser Val
50 55 60
gag gca caa aaa gaa tcg gtt gcg cgg ggg cca gtt gag aat att tct 240
Glu Ala Gln Lys Glu Ser Val Ala Arg Gly Pro Val Glu Asn Ile Ser
65 70 75 80
caa tcg gtt gcg cag ccc atc agg cgg agg tgg gtg cag gat aaa aag 288
Gln Ser Val Ala Gln Pro Ile Arg Arg Arg Trp Val Gln Asp Lys Lys
85 90 95
ccg gtt act tac agc ctg aag gat gta gct tcg cac gat atg ccc cag 336
Pro Val Thr Tyr Ser Leu Lys Asp Val Ala Ser His Asp Met Pro Gln
100 105 110
gac tgc tgg att ata atc aaa gag aag gtg tat gat gtg agc acc ttc 384
Asp Cys Trp Ile Ile Ile Lys Glu Lys Val Tyr Asp Val Ser Thr Phe
115 120 125
gct gag cag cac cct gga ggc acg gtt atc aac acc tac ttc gga cga 432
Ala Glu Gln His Pro Gly Gly Thr Val Ile Asn Thr Tyr Phe Gly Arg
130 135 140
gac gcc aca gat gtt ttc tct act ttc cac gca tcc acc tca tgg aag 480
Asp Ala Thr Asp Val Phe Ser Thr Phe His Ala Ser Thr Ser Trp Lys
145 150 155 160
att ctt cag aat ttc tac atc ggg aac ctt gtt agg gag gag ccg act 528
Ile Leu Gln Asn Phe Tyr Ile Gly Asn Leu Val Arg Glu Glu Pro Thr
165 170 175
ttg gag ctg ctg aag gag tac aga gag ttg aga gcc ctt ttc ttg aga 576
Leu Glu Leu Leu Lys Glu Tyr Arg Glu Leu Arg Ala Leu Phe Leu Arg
180 185 190
gaa cag ctt ttc aag agt tcc aaa tcc tac tac ctt ttc aag act ctc 624
Glu Gln Leu Phe Lys Ser Ser Lys Ser Tyr Tyr Leu Phe Lys Thr Leu
195 200 205
ata aat gtt tcc att gtt gcc aca agc att gcg ata atc agt ctg tac 672
Ile Asn Val Ser Ile Val Ala Thr Ser Ile Ala Ile Ile Ser Leu Tyr
210 215 220
aag tct tac cgg gcg gtt ctg tta tca gcc agt ttg atg ggc ttg ttt 720
Lys Ser Tyr Arg Ala Val Leu Leu Ser Ala Ser Leu Met Gly Leu Phe
225 230 235 240
att caa cag tgc gga tgg ttg tct cac gat ttt cta cac cat cag gta 768
Ile Gln Gln Cys Gly Trp Leu Ser His Asp Phe Leu His His Gln Val
245 250 255
ttt gag aca cgc tgg ctc aat gac gtt gtt ggc tat gtg gtc ggc aac 816
Phe Glu Thr Arg Trp Leu Asn Asp Val Val Gly Tyr Val Val Gly Asn
260 265 270
gtt gtt ctg gga ttc agt gtc tcg tgg tgg aag acc aag cac aac ctg 864
Val Val Leu Gly Phe Ser Val Ser Trp Trp Lys Thr Lys His Asn Leu
275 280 285
cat cat gct gct ccg aat gaa tgc gac caa aag tac aca ccg att gat 912
His His Ala Ala Pro Asn Glu Cys Asp Gln Lys Tyr Thr Pro Ile Asp
290 295 300
gag gat att gat act ctc ccc atc att gct tgg agt aaa gat ctc ttg 960
Glu Asp Ile Asp Thr Leu Pro Ile Ile Ala Trp Ser Lys Asp Leu Leu
305 310 315 320
gcc act gtt gag agc aag acc atg ttg cga gtt ctt cag tac cag cac 1008
Ala Thr Val Glu Set Lys Thr Met Leu Arg Val Leu Gln Tyr Gln His
325 330 335
cta ttc ttt ttg gtt ctt ttg acg ttt gcc cgg gcg agt tgg cta ttt 1056
Leu Phe Phe Leu Val Leu Leu Thr Phe Ala Arg Ala Ser Trp Leu Phe
340 345 350
tgg agc gcg gcc ttc act ctc agg ccc gag ttg acc ctt ggc gag aag 1104
Trp Ser Ala Ala Phe Thr Leu Arg Pro Glu Leu Thr Leu Gly Glu Lys
355 360 365
ctt ttg gag agg gga acg atg gct ttg cac tac att tgg ttt aat agt 1152
Leu Leu Glu Arg Gly Thr Met Ala Leu His Tyr Ile Trp Phe Asn Ser
370 375 380
gtt gcg ttt tat ctg ctc ccc gga tgg aaa cca gtt gta tgg atg gtg 1200
Val Ala Phe Tyr Leu Leu Pro Gly Trp Lys Pro Val Val Trp Met Val
385 390 395 400
gtc agc gag ctc atg tct ggt ttc ctg ctg gga tac gta ttt gta ctc 1248
Val Ser Glu Leu Met Ser Gly Phe Leu Leu Gly Tyr Val Phe Val Leu
405 410 415
agt cac aat gga atg gag gtg tac aat acg tca aag gac ttc gtg aat 1296
Ser His Asn Gly Met Glu Val Tyr Asn Thr Ser Lys Asp Phe Val Asn
420 425 430
gcc cag att gca tcg act cgc gac arc aaa gca ggg gtg ttt aat gat 1344
Ala Gln Ile Ala Ser Thr Arg Asp Ile Lys Ala Gly Val Phe Asn Asp
435 440 445
tgg ttc acc gga ggt ctc aac aga cag att gag cat cat cta ttt cca 1392
Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu His His Leu Phe Pro
450 455 460
acg atg ccc agg cac aac ctt aat aaa att tct cct cac gtg gag act 1440
Thr Met Pro Arg His Asn Leu Asn Lys Ile Ser Pro His Val Glu Thr
465 470 475 480
ttg tgc aag aag cat gga ctg gtc tac gaa gac gtg agc atg gct tcg 1488
Leu Cys Lys Lys His Gly Leu Val Tyr Glu Asp Val Ser Met Ala Ser
485 490 495
ggc act tac cgg gtt ttg aaa aca ctt aag gac gtt gcc gat gct gct 1536
Gly Thr Tyr Arg Val Leu Lys Thr Leu Lys Asp Val Ala Asp Ala Ala
500 505 510
tca cac cag cag ctt gct gcg agt tga 1563
Ser His Gln Gln Leu Ala Ala Ser
515 520
<210>20
<211>520
<212>PRT
<213>角齿藓
<400>20
Met Val Ser Gln Gly Gly Gly Leu Ser Gln Gly Ser Ile Glu Glu Asn
1 5 10 15
Ile Asp Val Glu His Leu Ala Thr Met Pro Leu Val Ser Asp Phe Leu
20 25 30
Asn Val Leu Gly Thr Thr Leu Gly Gln Trp Ser Leu Ser Thr Thr Phe
35 40 45
Ala Phe Lys Arg Leu Thr Thr Lys Lys His Ser Ser Asp Ile Ser Val
50 55 60
Glu Ala Gln Lys Glu Ser Val Ala Arg Gly Pro Val Glu Asn Ile Ser
65 70 75 80
Gln Ser Val Ala Gln Pro Ile Arg Arg Arg Trp Val Gln Asp Lys Lys
85 90 95
Pro Val Thr Tyr Ser Leu Lys Asp Val Ala Ser His Asp Met Pro Gln
100 105 110
Asp Cys Trp Ile Ile Ile Lys Glu Lys Val Tyr Asp Val Ser Thr Phe
115 120 125
Ala Glu Gln His Pro Gly Gly Thr Val Ile Asn Thr Tyr Phe Gly Arg
130 135 140
Asp Ala Thr Asp Val Phe Ser Thr Phe His Ala Ser Thr Ser Trp Lys
145 150 155 160
Ile Leu Gln Asn Phe Tyr Ile Gly Asn Leu Val Arg Glu Glu Pro Thr
165 170 175
Leu Glu Leu Leu Lys Glu Tyr Arg Glu Leu Arg Ala Leu Phe Leu Arg
180 185 190
Glu Gln Leu Phe Lys Ser Ser Lys Ser Tyr Tyr Leu Phe Lys Thr Leu
195 200 205
Ile Asn Val Ser Ile Val Ala Thr Ser Ile Ala Ile Ile Ser Leu Tyr
210 215 220
Lys Ser Tyr Arg Ala Val Leu Leu Ser Ala Ser Leu Met Gly Leu Phe
225 230 235 240
Ile Gln Gln Cys Gly Trp Leu Ser His Asp Phe Leu His His Gln Val
245 250 255
Phe Glu Thr Arg Trp Leu Asn Asp Val Val Gly Tyr Val Val Gly Asn
260 265 270
Val Val Leu Gly Phe Ser Val Ser Trp Trp Lys Thr Lys His Asn Leu
275 280 285
His His Ala Ala Pro Asn Glu Cys Asp Gln Lys Tyr Thr Pro Ile Asp
290 295 300
Glu Asp Ile Asp Thr Leu Pro Ile Ile Ala Trp Ser Lys Asp Leu Leu
305 310 315 320
Ala Thr Val Glu Ser Lys Thr Met Leu Arg Val Leu Gln Tyr Gln His
325 330 335
Leu Phe Phe Leu Val Leu Leu Thr Phe Ala Arg Ala Ser Trp Leu Phe
340 345 350
Trp Ser Ala Ala Phe Thr Leu Arg Pro Glu Leu Thr Leu Gly Glu Lys
355 360 365
Leu Leu Glu Arg Gly Thr Met Ala Leu His Tyr Ile Trp Phe Asn Ser
370 375 380
Val Ala Phe Tyr Leu Leu Pro Gly Trp Lys Pro Val Val Trp Met Val
385 390 395 400
Val Ser Glu Leu Met Ser Gly Phe Leu Leu Gly Tyr Val Phe Val Leu
405 410 415
Ser His Asn Gly Met Glu Val Tyr Asn Thr Ser Lys Asp Phe Val Asn
420 425 430
Ala Gln Ile Ala Ser Thr Arg Asp Ile Lys Ala Gly Val Phe Asn Asp
435 440 445
Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu His His Leu Phe Pro
450 455 460
Thr Met Pro Arg His Asn Leu Asn Lys Ile Ser Pro His Val Glu Thr
465 470 475 480
Leu Cys Lys Lys His Gly Leu Val Tyr Glu Asp Val Ser Met Ala Ser
485 490 495
Gly Thr Tyr Arg Val Leu Lys Thr Leu Lys Asp Val Ala Asp Ala Ala
500 505 510
Ser His Gln Gln Leu Ala Ala Ser
515 520
<210>21
<211>1434
<212>DNA
<213>三角褐指藻
<220>
<221>CDS
<222>(1)..(1434)
<223>Δ6-去饱和酶
<400>21
atg ggc aaa gga ggg gac gct cgg gcc tcg aag ggc tca acg gcg gct 48
Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr Ala Ala
1 5 10 15
cgc aag atc agt tgg cag gaa gtc aag acc cac gcg tct ccg gag gac 96
Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro Glu Asp
20 25 30
gcc tgg atc att cac tcc aat aag gtc tac gac gtg tcc aac tgg cac 144
Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser Asn Trp His
35 40 45
gaa cat ccc gga ggc gcc gtc att ttc acg cac gcc ggt gac gac atg 192
Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly Asp Asp Met
50 55 60
acg gac att ttc gct gcc ttt cac gca ccc gga tcg cag tcg ctc atg 240
Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln Ser Leu Met
65 70 75 80
aag aag ttc tac att ggc gaa ttg ctc ccg gaa acc acc ggc aag gag 288
Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr Gly Lys Glu
85 90 95
ccg cag caa atc gcc ttt gaa aag ggc tac cgc gat ctg cgc tcc aaa 336
Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu Arg Ser Lys
100 105 110
ctc atc atg atg ggc atg ttc aag tcc aac aag tgg ttc tac gtc tac 384
Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe Tyr Val Tyr
115 120 125
aag tgc ctc agc aac atg gcc att tgg gcc gcc gcc tgt gct ctc gtc 432
Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys Ala Leu Val
130 135 140
ttt tac tcg gac cgc ttc tgg gta cac ctg gcc agc gcc gtc atg ctg 480
Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala Val Met Leu
145 150 155 160
gga aca ttc ttt cag cag tcg gga tgg ttg gca cac gac ttt ctg csc 528
Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp Phe Leu His
165 170 175
cac cag gtc ttc acc aag cgc aag cac ggg gat ctc gga gga ctc ttt 576
His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly Gly Leu Phe
180 185 190
tgg ggg aac ctc atg cag ggt tac tcc gta cag tgg tgg aaa aac aag 624
Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys
195 200 205
cac aac gga cac cac gcc gtc ccc aac ctc cac tgc tcc tcc gca gtc 672
His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser Ser Ala Val
210 215 220
gcg caa gat ggg gac ccg gac atc gat acc atg ccc ctt ctc gcc tgg 720
Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp
225 230 235 240
tcc gtc cag caa gcc cag tct tac cgg gaa ctc caa gcc gac gga aag 768
Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys
245 250 255
gat tcg ggt ttg gtc aag ttc atg atc cgt aac caa tcc tac ttt tac 816
Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr
260 265 270
ttt ccc atc ttg ttg ctc gcc cgc ctg tcg tgg ttg aac gag tcc ttc 864
Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe
275 280 285
aag tgc gcc ttt ggg ctt gga gct gcg tcg gag aac gct gct ctc gaa 912
Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu
290 295 300
ctc aag gcc aag ggt ctt cag tac ccc ctt ttg gaa aag gct ggc atc 960
Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala Gly Ile
305 310 315 320
ctg ctg cac tac gct tgg atg ctt aca gtt tcg tcc ggc ttt gga cgc 1008
Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe Gly Arg
325 330 335
ttc tcg ttc gcg tac acc gca ttt tac ttt cta acc gcg acc gcg tcc 1056
Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr Ala Ser
340 345 350
tgt gga ttc ttg ctc gcc att gtc ttt ggc ctc ggc cac aac ggc atg 1104
Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His Asn Gly Met
355 360 365
gcc acc tac aat gcc gac gcc cgt ccg gac ttc tgg aag ctc caa gtc 1152
Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu Gln Val
370 375 380
acc acg act cgc aac gtc acg ggc gga cac ggt ttc ccc caa gcc ttt 1200
Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro Gln Ala Phe
385 390 395 400
gtc gac tgg ttc tgt ggt ggc ctc cag tac caa gtc gac cac cac tta 1248
Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His His Leu
405 410 415
ttc ccc agc ctg ccc cga cac aat ctg gcc aag aca cac gca ctg gtc 1296
Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His Ala Leu Val
420 425 430
gaa tcg ttc tgc aag gag tgg ggt gtc cag tac cac gaa gcc gac ctt 1344
Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala Asp Leu
435 440 445
gtg gac ggg acc atg gaa gtc ttg cac cat ttg ggc agc gtg gcc ggc 1392
Val Asp Gly Thr Met Glu Val Leu His His Leu Gly Ser Val Ala Gly
450 455 460
gaa ttc gtc gtg gat ttt gta cgc gat gga ccc gcc atg taa 1434
Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met
465 470 475
<210>22
<211>477
<212>PRT
<213>三角褐指藻
<400>22
Met Gly Lys Gly Gly Asp Ala Arg Ala Ser Lys Gly Ser Thr Ala Ala
1 5 10 15
Arg Lys Ile Ser Trp Gln Glu Val Lys Thr His Ala Ser Pro Glu Asp
20 25 30
Ala Trp Ile Ile His Ser Asn Lys Val Tyr Asp Val Ser Asn Trp His
35 40 45
Glu His Pro Gly Gly Ala Val Ile Phe Thr His Ala Gly Asp Asp Met
50 55 60
Thr Asp Ile Phe Ala Ala Phe His Ala Pro Gly Ser Gln Ser Leu Met
65 70 75 80
Lys Lys Phe Tyr Ile Gly Glu Leu Leu Pro Glu Thr Thr Gly Lys Glu
85 90 95
Pro Gln Gln Ile Ala Phe Glu Lys Gly Tyr Arg Asp Leu Arg Ser Lys
100 105 110
Leu Ile Met Met Gly Met Phe Lys Ser Asn Lys Trp Phe Tyr Val Tyr
115 120 125
Lys Cys Leu Ser Asn Met Ala Ile Trp Ala Ala Ala Cys Ala Leu Val
130 135 140
Phe Tyr Ser Asp Arg Phe Trp Val His Leu Ala Ser Ala Val Met Leu
145 150 155 160
Gly Thr Phe Phe Gln Gln Ser Gly Trp Leu Ala His Asp Phe Leu His
165 170 175
His Gln Val Phe Thr Lys Arg Lys His Gly Asp Leu Gly Gly Leu Phe
180 185 190
Trp Gly Asn Leu Met Gln Gly Tyr Ser Val Gln Trp Trp Lys Asn Lys
195 200 205
His Asn Gly His His Ala Val Pro Asn Leu His Cys Ser Ser Ala Val
210 215 220
Ala Gln Asp Gly Asp Pro Asp Ile Asp Thr Met Pro Leu Leu Ala Trp
225 230 235 240
Ser Val Gln Gln Ala Gln Ser Tyr Arg Glu Leu Gln Ala Asp Gly Lys
245 250 255
Asp Ser Gly Leu Val Lys Phe Met Ile Arg Asn Gln Ser Tyr Phe Tyr
260 265 270
Phe Pro Ile Leu Leu Leu Ala Arg Leu Ser Trp Leu Asn Glu Ser Phe
275 280 285
Lys Cys Ala Phe Gly Leu Gly Ala Ala Ser Glu Asn Ala Ala Leu Glu
290 295 300
Leu Lys Ala Lys Gly Leu Gln Tyr Pro Leu Leu Glu Lys Ala Gly Ile
305 310 315 320
Leu Leu His Tyr Ala Trp Met Leu Thr Val Ser Ser Gly Phe Gly Arg
325 330 335
Phe Ser Phe Ala Tyr Thr Ala Phe Tyr Phe Leu Thr Ala Thr Ala Ser
340 345 350
Cys Gly Phe Leu Leu Ala Ile Val Phe Gly Leu Gly His Asn Gly Met
355 360 365
Ala Thr Tyr Asn Ala Asp Ala Arg Pro Asp Phe Trp Lys Leu Gln Val
370 375 380
Thr Thr Thr Arg Asn Val Thr Gly Gly His Gly Phe Pro Gln Ala Phe
385 390 395 400
Val Asp Trp Phe Cys Gly Gly Leu Gln Tyr Gln Val Asp His His Leu
405 410 415
Phe Pro Ser Leu Pro Arg His Asn Leu Ala Lys Thr His Ala Leu Val
420 425 430
Glu Ser Phe Cys Lys Glu Trp Gly Val Gln Tyr His Glu Ala Asp Leu
435 440 445
Val Asp Gly Thr Met Glu Val Leu His His Leu Gly Ser Val Ala Gly
450 455 460
Glu Phe Val Val Asp Phe Val Arg Asp Gly Pro Ala Met
465 470 475
<210>23
<211>1578
<212>DNA
<213>展叶剑叶藓
<220>
<221>CDS
<222>(1)..(1578)
<223>Δ6-去饱和酶
<400>23
atg gta ttc gcg ggc ggt gga ctt cag cag ggc tct ctc gaa gaa aac 48
Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn
1 5 10 15
atc gac gtc gag cac att gcc agt atg tct ctc ttc agc gac ttc ttc 96
Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe
20 25 30
agt tat gtg tct tca act gtt ggt tcg tgg agc gta cac agt ata caa 144
Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln
35 40 45
cct ttg aag cgc ctg acg agt aag aag cgt gtt tcg gaa agc gct gcc 192
Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala
50 55 60
gtg caa tgt ata tca gct gaa gtt cag aga aat tcg agt acc cag gga 240
Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly
65 70 75 80
act gcg gag gca ctc gca gaa tca gtc gtg aag ccc acg aga cga agg 288
Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg
85 90 95
tca tct cag tgg aag aag tcg aca cac ccc cta tca gaa gta gca gta 336
Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val
100 105 110
cac aac aag cca agc gat tgc tgg att gtt gta aaa aac aag gtg tat 384
His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr
115 120 125
gat gtt tcc aat ttt gcg gac gag cat ccc gga gga tca gtt att agt 432
Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser
130 135 140
act tat ttt gga cga gac ggc aca gat gtt ttc tct agt ttt cat gca 480
Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala
145 150 155 160
gct tct aca tgg aaa att ctt caa gac ttt tac att ggt gac gtg gag 528
Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu
165 170 175
agg gtg gag ccg act cca gag ctg ctg aaa gat ttc cga gaa atg aga 576
Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe Arg Glu Met Arg
180 185 190
gct ctt ttc ctg agg gag caa ctt ttc aaa agt tcg aaa ttg tac tat 624
Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr
195 200 205
gtt atg aag ctg ctc acg aat gtt gct att ttt gct gcg agc att gca 672
Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe Ala Ala Ser Ile Ala
210 215 220
ata ata tgt tgg agc aag act att tca gcg gtt ttg gct tca gct tgt 720
Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val Leu Ala Ser Ala Cys
225 230 235 240
atg atg gct ctg tgt ttc caa cag tgc gga tgg cta tcc cat gat ttt 768
Met Met Ala Leu Cys Phe Gln Gln Cys Gly Trp Leu Ser His Asp Phe
245 250 255
ctc cac aat cag gtg ttt gag aca cgc tgg ctt aat gaa gtt gtc ggg 816
Leu His Asn Gln Val Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly
260 265 270
tat gtg atc ggc aac gcc gtt ctg ggg ttt agt aca ggg tgg tgg aag 864
Tyr Val Ile Gly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys
275 280 285
gag aag cat aac ctt cat cat gct gct cca aat gaa tgc gat cag act 912
Glu Lys His Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr
290 295 300
tac caa cca att gat gaa gat att gat act ctc ccc ctc att gcc tgg 960
Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp
305 310 315 320
agc aag gac ata ctg gcc aca gtt gag aat aag aca ttc ttg cga atc 1008
Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile
325 330 335
ctc caa tac cag cat ctg ttc ttc atg ggt ctg tta ttt ttc gcc cgt 1056
Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg
340 345 350
ggt agt tgg ctc ttt tgg agc tgg aga tat acc tct aca gca gtg ctc 1104
Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu
355 360 365
tca cct gtc gac agg ttg ttg gag aag gga act gtt ctg ttt cac tac 1152
Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr
370 375 380
ttt tgg ttc gtc ggg aca gcg tgc tat ctt ctc cct ggt tgg aag cca 1200
Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro
385 390 395 400
tta gta tgg atg gcg gtg act gag ctc atg tcc ggc atg ctg ctg ggc 1248
Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met Leu Leu Gly
405 410 415
ttt gta ttt gta ctt agc cac aat ggg atg gag gtt tat aat tcg tct 1296
Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val Tyr Asn Ser Ser
420 425 430
aaa gaa ttc gtg agt gca cag atc gta tcc aca cgg gat atc aaa gga 1344
Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr Arg Asp Ile Lys Gly
435 440 445
aac ata ttc aac gac tgg ttc act ggt ggc ctt aac agg caa ata gag 1392
Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly Leu Asn Asg Gln Ile Glu
450 455 460
cat cat ctt ttc cca aca atg ccc agg cat aat tta aac aaa ata gca 1440
His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Lys Ile Ala
465 470 475 480
cct aga gtg gag gtg ttc tgt aag aaa cac ggt ctg gtg tac gaa gac 1488
Pso Asg Val Glu Val Phe Cys Lys Lys His Gly Leu Val Tys Glu Asp
485 490 495
gta tct att gct acc ggc act tgc aag gtt ttg aaa gca ttg aag gaa 1536
Val Ser Ile Ala Thr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu
500 505 510
gtc gcg gag gct gcg gca gag cag cat gct acc acc agt taa 1578
Val Ala Glu Ala Ala Ala Glu Gln His Ala Thr Thr Ser
515 520 525
<210>24
<211>525
<212>PRT
<213>展叶剑叶藓
<400>24
Met Val Phe Ala Gly Gly Gly Leu Gln Gln Gly Ser Leu Glu Glu Asn
1 5 10 15
Ile Asp Val Glu His Ile Ala Ser Met Ser Leu Phe Ser Asp Phe Phe
20 25 30
Ser Tyr Val Ser Ser Thr Val Gly Ser Trp Ser Val His Ser Ile Gln
35 40 45
Pro Leu Lys Arg Leu Thr Ser Lys Lys Arg Val Ser Glu Ser Ala Ala
50 55 60
Val Gln Cys Ile Ser Ala Glu Val Gln Arg Asn Ser Ser Thr Gln Gly
65 70 75 80
Thr Ala Glu Ala Leu Ala Glu Ser Val Val Lys Pro Thr Arg Arg Arg
85 90 95
Ser Ser Gln Trp Lys Lys Ser Thr His Pro Leu Ser Glu Val Ala Val
100 105 110
His Asn Lys Pro Ser Asp Cys Trp Ile Val Val Lys Asn Lys Val Tyr
115 120 125
Asp Val Ser Asn Phe Ala Asp Glu His Pro Gly Gly Ser Val Ile Ser
130 135 140
Thr Tyr Phe Gly Arg Asp Gly Thr Asp Val Phe Ser Ser Phe His Ala
145 150 155 160
Ala Ser Thr Trp Lys Ile Leu Gln Asp Phe Tyr Ile Gly Asp Val Glu
165 170 175
Arg Val Glu Pro Thr Pro Glu Leu Leu Lys Asp Phe Arg Glu Met Arg
180 185 190
Ala Leu Phe Leu Arg Glu Gln Leu Phe Lys Ser Ser Lys Leu Tyr Tyr
195 200 205
Val Met Lys Leu Leu Thr Asn Val Ala Ile Phe Ala Ala Ser Ile Ala
210 215 220
Ile Ile Cys Trp Ser Lys Thr Ile Ser Ala Val Leu Ala Ser Ala Cys
225 230 235 240
Met Met Ala Leu Cys Phe Gln Gln Cys Gly Trp Leu Ser His Asp Phe
245 250 255
Leu His Asn Gln Val Phe Glu Thr Arg Trp Leu Asn Glu Val Val Gly
260 265 270
Tyr Val Ile Gly Asn Ala Val Leu Gly Phe Ser Thr Gly Trp Trp Lys
275 280 285
Glu Lys His Asn Leu His His Ala Ala Pro Asn Glu Cys Asp Gln Thr
290 295 300
Tyr Gln Pro Ile Asp Glu Asp Ile Asp Thr Leu Pro Leu Ile Ala Trp
305 310 315 320
Ser Lys Asp Ile Leu Ala Thr Val Glu Asn Lys Thr Phe Leu Arg Ile
325 330 335
Leu Gln Tyr Gln His Leu Phe Phe Met Gly Leu Leu Phe Phe Ala Arg
340 345 350
Gly Ser Trp Leu Phe Trp Ser Trp Arg Tyr Thr Ser Thr Ala Val Leu
355 360 365
Ser Pro Val Asp Arg Leu Leu Glu Lys Gly Thr Val Leu Phe His Tyr
370 375 380
Phe Trp Phe Val Gly Thr Ala Cys Tyr Leu Leu Pro Gly Trp Lys Pro
385 390 395 400
Leu Val Trp Met Ala Val Thr Glu Leu Met Ser Gly Met Leu Leu Gly
405 410 415
Phe Val Phe Val Leu Ser His Asn Gly Met Glu Val Tyr Asn Ser Ser
420 425 430
Lys Glu Phe Val Ser Ala Gln Ile Val Ser Thr Arg Asp Ile Lys Gly
435 440 445
Asn Ile Phe Asn Asp Trp Phe Thr Gly Gly Leu Asn Arg Gln Ile Glu
450 455 460
His His Leu Phe Pro Thr Met Pro Arg His Asn Leu Asn Lys Ile Ala
465 470 475 480
Pro Arg Val Glu Val Phe Cys Lys Lys His Gly Leu Val Tyr Glu Asp
485 490 495
Val Ser Ile Ala Thr Gly Thr Cys Lys Val Leu Lys Ala Leu Lys Glu
500 505 510
Val Ala Glu Ala Ala Ala Glu Gln His Ala Thr Thr Ser
515 520 525
<210>25
<211>1332
<212>DNA
<213>秀丽隐杆线虫
<220>
<221>CDS
<222>(1)..(1332)
<223>Δ6-去饱和酶
<400>25
atg gtc gtc gac aag aat gcc tcc ggg ctt cga atg aag gtc gat ggc 48
Met Val Val Asp Lys Asn Ala Ser Gly Leu Arg Met Lys Val Asp Gly
1 5 10 15
aaa tgg ctc tac ctt agc gag gaa ttg gtg aag aaa cat cca gga gga 96
Lys Trp Leu Tyr Leu Ser Glu Glu Leu Val Lys Lys His Pro Gly Gly
20 25 30
gct gtt att gaa caa tat aga aat tcg gat gct act cat att ttc cac 144
Ala Val Ile Glu Gln Tyr Arg Asn Ser Asp Ala Thr His Ile Phe His
35 40 45
gct ttc cac gaa gga tct tct cag gct tat aag caa ctt gac ctt ctg 192
Ala Phe His Glu Gly Ser Ser Gln Ala Tyr Lys Gln Leu Asp Leu Leu
50 55 60
aaa aag cac gga gag cac gat gaa ttc ctt gag aaa caa ttg gaa aag 240
Lys Lys His Gly Glu His Asp Glu Phe Leu Glu Lys Gln Leu Glu Lys
65 70 75 80
aga ctt gac aaa gtt gat atc aat gta tca gca tat gat gtc agt gtt 288
Arg Leu Asp Lys Val Asp Ile Asn Val Ser Ala Tyr Asp Val Ser Val
85 90 95
gca caa gaa aag aaa atg gtt gaa tca ttc gaa aaa cta cga cag aag 336
Ala Gln Glu Lys Lys Met Val Glu Ser Phe Glu Lys Leu Arg Gln Lys
100 105 110
ctt cat gat gat gga tta atg aaa gca aat gaa aca tat ttc ctg ttt 384
Leu His Asp Asp Gly Leu Met Lys Ala Asn Glu Thr Tyr Phe Leu Phe
115 120 125
aaa gcg att tca aca ctt tca att atg gca ttt gca ttt tat ctt cag 432
Lys Ala Ile Ser Thr Leu Ser Ile Met Ala Phe Ala Phe Tyr Leu Gln
130 135 140
tat ctt gga tgg tat att act tct gca tgt tta tta gca ctt gca tgg 480
Tyr Leu Gly Trp Tyr Ile Thr Ser Ala Cys Leu Leu Ala Leu Ala Trp
145 150 155 160
caa caa ttc gga tgg tta aca cat gag ttc tgc cat caa cag cca aca 528
Gln Gln Phe Gly Trp Leu Thr His Glu Phe Cys His Gln Gln Pro Thr
165 170 175
aag aac aga cct ttg aat gat act att tct ttg ttc ttt ggt aat ttc 576
Lys Asn Arg Pro Leu Asn Asp Thr Ile Ser Leu Phe Phe Gly Asn Phe
180 185 190
tta caa gga ttt tca aga gat tgg tgg aag gac aag cat aac act cat 624
Leu Gln Gly Phe Ser Arg Asp Trp Trp Lys Asp Lys His Asn Thr His
195 200 205
cac gct gcc aca aat gta att gat cat gac ggt gat atc gac ttg gca 672
Hia Ala Ala Thr Asn Val Ile Asp His Asp Gly Asp Ile Asp Leu Ala
210 215 220
cca ctt ttc gca ttt att cca gga gat ttg tgc aag tat aag gcc agc 720
Pro Leu Phe Ala Phe Ile Pro Gly Asp Leu Cys Lys Tyr Lys Ala Ser
225 230 235 240
ttt gaa aaa gca att ctc aag att gta cca tat caa cat ctc tat ttc 768
Phe Glu Lys Ala Ile Leu Lys Ile Val Pro Tyr Gln His Leu Tyr Phe
245 250 255
acc gca atg ctt cca atg ctc cgt ttc tca tgg act ggt cag tca gtt 816
Thr Ala Met Leu Pro Met Leu Arg Phe Ser Trp Thr Gly Gln Ser Val
260 265 270
caa tgg gta ttc aaa gag aat caa atg gag tac aag gtc tat caa aga 864
Gln Trp Val Phe Lys Glu Asn Gln Met Glu Tyr Lys Val Tyr Gln Arg
275 280 285
aat gca ttc tgg gag caa gca aca att gtt gga cat tgg gct tgg gta 912
Asn Ala Phe Trp Glu Gln Ala Thr Ile Val Gly His Trp Ala Trp Val
290 295 300
ttc tat caa ttg ttc tta tta cca aca tgg cca ctt cgg gtt gct tat 960
Phe Tyr Gln Leu Phe Leu Leu Pro Thr Trp Pro Leu Arg Val Ala Tyr
305 310 315 320
ttc att att tca caa atg gga gga ggc ctt ttg att gct cac gta gtc 1008
Phe Ile Ile Ser Gln Met Gly Gly Gly Leu Leu Ile Ala His Val Val
325 330 335
act ttc aac cat aac tct gtt gat aag tat cca gcc aat tct cga att 1056
Thr Phe Asn His Asn Ser Val Asp Lys Tyr Pro Ala Asn Ser Arg Ile
340 345 350
tta aac aac ttc gcc gct ctt caa att ttg acc aca cgc aac atg act 1104
Leu Asn Asn Phe Ala Ala Leu Gln Ile Leu Thr Thr Arg Asn Met Thr
355 360 365
cca tct cca ttc att gat tgg ctt tgg ggt gga ctc aat tat cag atc 1152
Pro Ser Pro Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln Ile
370 375 380
gag cac cac ttg ttc cca aca atg cca cgt tgc aat ctg aat gct tgc 1200
Glu His His Leu Phe Pro Thr Met Pro Arg Cys Asn Leu Asn Ala Cys
385 390 395 400
gtg aaa tat gtg aaa gaa tgg tgc aaa gag aat aat ctt cct tac ctc 1248
Val Lys Tyr Val Lys Glu Trp Cys Lys Glu Asn Asn Leu Pro Tyr Leu
405 410 415
gtc gat gac tac ttt gac gga tat gca atg aat ttg caa caa ttg aaa 1296
Val Asp Asp Tyr Phe Asp Gly Tyr Ala Met Asn Leu Gln Gln Leu Lys
420 425 430
aat atg gct gag cac att caa gct aaa gct gcc taa 1332
Asn Met Ala Glu His Ile Gln Ala Lys Ala Ala
435 440
<210>26
<211>443
<212>PRT
<213>秀丽隐杆线虫
<400>26
Met Val Val Asp Lys Asn Ala Ser Gly Leu Arg Met Lys Val Asp Gly
1 5 10 15
Lys Trp Leu Tyr Leu Ser Glu Glu Leu Val Lys Lys His Pro Gly Gly
20 25 30
Ala Val Ile Glu Gln Tyr Arg Asn Ser Asp Ala Thr His Ile Phe His
35 40 45
Ala Phe His Glu Gly Ser Ser Gln Ala Tyr Lys Gln Leu Asp Leu Leu
50 55 60
Lys Lys His Gly Glu His Asp Glu Phe Leu Glu Lys Gln Leu Glu Lys
65 70 75 80
Arg Leu Asp Lys Val Asp Ile Asn Val Ser Ala Tyr Asp Val Ser Val
85 90 95
Ala Gln Glu Lys Lys Met Val Glu Ser Phe Glu Lys Leu Arg Gln Lys
100 105 110
Leu His Asp Asp Gly Leu Met Lys Ala Asn Glu Thr Tyr Phe Leu Phe
115 120 125
Lys Ala Ile Ser Thr Leu Ser Ile Met Ala Phe Ala Phe Tyr Leu Gln
130 135 140
Tyr Leu Gly Trp Tyr Ile Thr Ser Ala Cys Leu Leu Ala Leu Ala Trp
145 150 155 160
Gln Gln Phe Gly Trp Leu Thr His Glu Phe Cys His Gln Gln Pro Thr
165 170 175
Lys Asn Arg Pro Leu Asn Asp Thr Ile Ser Leu Phe Phe Gly Asn Phe
180 185 190
Leu Gln Gly Phe Ser Arg Asp Trp Trp Lys Asp Lys His Asn Thr His
195 200 205
His Ala Ala Thr Asn Val Ile Asp His Asp Gly Asp Ile Asp Leu Ala
210 215 220
Pro Leu Phe Ala Phe Ile Pro Gly Asp Leu Cys Lys Tyr Lys Ala Ser
225 230 235 240
Phe Glu Lys Ala Ile Leu Lys Ile Val Pro Tyr Gln His Leu Tyr Phe
245 250 255
Thr Ala Met Leu Pro Met Leu Arg Phe Ser Trp Thr Gly Gln Ser Val
260 265 270
Gln Trp Val Phe Lys Glu Asn Gln Met Glu Tyr Lys Val Tyr Gln Arg
275 280 285
Asn Ala Phe Trp Glu Gln Ala Thr Ile Val Gly His Trp Ala Trp Val
290 295 300
Phe Tyr Gln Leu Phe Leu Leu Pro Thr Trp Pro Leu Arg Val Ala Tyr
305 310 315 320
Phe Ile Ile Ser Gln Met Gly Gly Gly Leu Leu Ile Ala His Val Val
325 330 335
Thr Phe Asn His Asn Ser Val Asp Lys Tyr Pro Ala Asn Ser Arg Ile
340 345 350
Leu Asn Asn Phe Ala Ala Leu Gln Ile Leu Thr Thr Arg Asn Met Thr
355 360 365
Pro Ser Pro Phe Ile Asp Trp Leu Trp Gly Gly Leu Asn Tyr Gln Ile
370 375 380
Glu His His Leu Phe Pro Thr Met Pro Arg Cys Asn Leu Asn Ala Cys
385 390 395 400
Val Lys Tyr Val Lys Glu Trp Cys Lys Glu Asn Asn Leu Pro Tyr Leu
405 410 415
Val Asp Asp Tyr Phe Asp Gly Tyr Ala Met Asn Leu Gln Gln Leu Lys
420 425 430
Asn Met Ala Glu His Ile Gln Ala Lys Ala Ala
435 440
<210>27
<211>873
<212>DNA
<213>展叶剑叶藓
<220>
<221>CDS
<222>(1)..(873)
<223>Δ6-延伸酶
<400>27
atg gag gtc gtg gag aga ttc tac ggt gag ttg gat ggg aag gtc tcg 48
Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser
1 5 10 15
cag ggc gtg aat gca ttg ctg ggt agt ttt ggg gtg gag ttg acg gat 96
Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp
20 25 30
acg ccc act acc aaa ggc ttg ccc ctc gtt gac agt ccc aca ccc atc 144
Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile
35 40 45
gtc ctc ggt gtt tct gta tac ttg act att gtc att gga ggg ctt ttg 192
Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu
50 55 60
tgg ata aag gcc agg gat ctg aaa ccg cgc gcc tcg gag cca ttt ttg 240
Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu
65 70 75 80
ctc caa gct ttg gtg ctt gtg cac aac ctg ttc tgt ttt gcg ctc agt 288
Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser
85 90 95
ctg tat atg tgc gtg ggc atc gct tat cag gct att acc tgg cgg tac 336
Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr
100 105 110
tct ctc tgg ggc aat gca tac aat cct aaa cat aaa gag atg gcg att 384
Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile
115 120 125
ctg gta tac ttg ttc tac atg tct aag tac gtg gaa ttc atg gat acc 432
Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr
130 135 140
gtt atc atg ata ctg aag cgc agc acc agg caa ata agc ttc ctc cac 480
Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His
145 150 155 160
gtt tat cat cat tct tca att tcc ctc att tgg tgg gct att gct cat 528
Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His
165 170 175
cac gct cct ggc ggt gaa gca tat tgg tct gcg gct ctg aac tca gga 576
His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly
180 185 190
gtg cat gtt ctc atg tat gcg tat tac ttc ttg gct gcc tgc ctt cga 624
Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg
195 200 205
agt agc cca aag tta aaa aat aag tac ctt ttt tgg ggc agg tac ttg 672
Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu
210 215 220
aca caa ttc caa atg ttc cag ttt atg ctg aac tta gtg cag gct tac 720
Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr
225 230 235 240
tac gac atg aaa acg aat gcg cca tat cca caa tgg ctg atc aag att 768
Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile
245 250 255
ttg ttc tac tac atg atc tcg ttg ctg ttt ctt ttc ggc aat ttt tac 816
Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr
260 265 270
gta caa aaa tac atc aaa ccc tct gac gga aag caa aag gga gct aaa 864
Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys
275 280 285
act gag tga 873
Thr Glu
290
<210>28
<211>290
<212>PRT
<213>展叶剑叶藓
<400>28
Met Glu Val Val Glu Arg Phe Tyr Gly Glu Leu Asp Gly Lys Val Ser
1 5 10 15
Gln Gly Val Asn Ala Leu Leu Gly Ser Phe Gly Val Glu Leu Thr Asp
20 25 30
Thr Pro Thr Thr Lys Gly Leu Pro Leu Val Asp Ser Pro Thr Pro Ile
35 40 45
Val Leu Gly Val Ser Val Tyr Leu Thr Ile Val Ile Gly Gly Leu Leu
50 55 60
Trp Ile Lys Ala Arg Asp Leu Lys Pro Arg Ala Ser Glu Pro Phe Leu
65 70 75 80
Leu Gln Ala Leu Val Leu Val His Asn Leu Phe Cys Phe Ala Leu Ser
85 90 95
Leu Tyr Met Cys Val Gly Ile Ala Tyr Gln Ala Ile Thr Trp Arg Tyr
100 105 110
Ser Leu Trp Gly Asn Ala Tyr Asn Pro Lys His Lys Glu Met Ala Ile
115 120 125
Leu Val Tyr Leu Phe Tyr Met Ser Lys Tyr Val Glu Phe Met Asp Thr
130 135 140
Val Ile Met Ile Leu Lys Arg Ser Thr Arg Gln Ile Ser Phe Leu His
145 150 155 160
Val Tyr His His Ser Ser Ile Ser Leu Ile Trp Trp Ala Ile Ala His
165 170 175
His Ala Pro Gly Gly Glu Ala Tyr Trp Ser Ala Ala Leu Asn Ser Gly
180 185 190
Val His Val Leu Met Tyr Ala Tyr Tyr Phe Leu Ala Ala Cys Leu Arg
195 200 205
Ser Ser Pro Lys Leu Lys Asn Lys Tyr Leu Phe Trp Gly Arg Tyr Leu
210 215 220
Thr Gln Phe Gln Met Phe Gln Phe Met Leu Asn Leu Val Gln Ala Tyr
225 230 235 240
Tyr Asp Met Lys Thr Asn Ala Pro Tyr Pro Gln Trp Leu Ile Lys Ile
245 250 255
Leu Phe Tyr Tyr Met Ile Ser Leu Leu Phe Leu Phe Gly Asn Phe Tyr
260 265 270
Val Gln Lys Tyr Ile Lys Pro Ser Asp Gly Lys Gln Lys Gly Ala Lys
275 280 285
Thr Glu
290
<210>29
<211>1049
<212>DNA
<213>破囊壶菌属(Thraustochytrium)
<220>
<221>CDS
<222>(43)..(858)
<223>Δ6-延伸酶
<400>29
gaattcggca cgagagcgcg cggagcggag acctcggccg cg atg atg gag ccg 54
Met Met Glu Pro
1
ctc gac agg tac agg gcg ctg gcg gag ctc gcc gcg agg tac gcc agc 102
Leu Asp Arg Tyr Arg Ala Leu Ala Glu Leu Ala Ala Arg Tyr Ala Ser
5 10 15 20
tcg gcg gcc ttc aag tgg caa gtc acg tac gac gcc aag gac agc ttc 150
Ser Ala Ala Phe Lys Trp Gln Val Thr Tyr Asp Ala Lys Asp Ser Phe
25 30 35
gtc ggg ccc ctg gga atc cgg gag ccg ctc ggg ctc ctg gtg ggc tcc 198
Val Gly Pro Leu Gly Ile Arg Glu Pro Leu Gly Leu Leu Val Gly Ser
40 45 50
gtg gtc ctc tac ctg agc ctg ctg gcc gtg gtc tac gcg ctg cgg aac 246
Val Val Leu Tyr Leu Ser Leu Leu Ala Val Val Tyr Ala Leu Arg Asn
55 60 65
tac ctt ggc ggc ctc atg gcg ctc cgc agc gtg cat aac ctc ggg ctc 294
Tyr Leu Gly Gly Leu Met Ala Leu Arg Ser Val His Asn Leu Gly Leu
70 75 80
tgc ctc ttc tcg ggc gcc gtg tgg atc tac acg agc tac ctc atg atc 342
Cys Leu Phe Ser Gly Ala Val Trp Ile Tyr Thr Ser Tyr Leu Met Ile
85 90 95 100
cag gat ggg cac ttt cgc agc ctc gag gcg gca acg tgc gag ccg ctc 390
Gln Asp Gly His Phe Arg Ser Leu Glu Ala Ala Thr Cys Glu Pro Leu
105 110 115
aag cat ccg cac ttc cag ctc atc agc ttg ctc ttt gcg ctg tcc aag 438
Lys His Pro His Phe Gln Leu Ile Ser Leu Leu Phe Ala Leu Ser Lys
120 125 130
atc tgg gag tgg ttc gac acg gtg ctc ctc atc gtc aag ggc aac aag 486
Ile Trp Glu Trp Phe Asp Thr Val Leu Leu Ile Val Lys Gly Asn Lys
135 140 145
ctc cgc ttc ctg cac gtc ttg cac cac gcc acg acc ttt tgg ctc tac 534
Leu Arg Phe Leu His Val Leu His His Ala Thr Thr Phe Trp Leu Tyr
150 155 160
gcc atc gac cac atc ttt ctc tcg tcc atc aag tac ggc gtc gcg gtc 582
Ala Ile Asp His Ile Phe Leu Ser Ser Ile Lys Tyr Gly Val Ala Val
165 170 175 180
aat gct ttc atc cac acc gtc atg tac gcg cac tac ttc cgc cca ttc 630
Asn Ala Phe Ile His Thr Val Met Tyr Ala His Tyr Phe Arg Pro Phe
185 190 195
ccg aag ggc ttg cgc ccg ctt att acg cag ttg cag atc gtc cag ttc 678
Pro Lys Gly Leu Arg Pro Leu Ile Thr Gln Leu Gln Ile Val Gln Phe
200 205 210
att ttc agc atc ggc atc cat acc gcc att tac tgg cac tac gac tgc 726
Ile Phe Ser Ile Gly Ile His Thr Ala Ile Tyr Trp His Tyr Asp Cys
215 220 225
gag ccg ctc gtg cat acc cac ttt tgg gaa tac gtc acg ccc tac ctt 774
Glu Pro Leu Val His Thr His Phe Trp Glu Tyr Val Thr Pro Tyr Leu
230 235 240
ttc gtc gtg ccc ttc ctc atc ctc ttt ttc aat ttt tac ctg cag cag 822
Phe Val Val Pro Phe Leu Ile Leu Phe Phe Asn Phe Tyr Leu Gln Gln
245 250 255 260
tac gtc ctc gcg ccc gca aaa acc aag aag gca tag ccacgtaaca 868
Tyr Val Leu Ala Pro Ala Lys Thr Lys Lys Ala
265 270
gtagaccagc agcgccgagg acgcgtgccg cgttatcgcg aagcacgaaa taaagaagat 928
catttgattc aacgaggcta cttgcggcca cgagaaaaaa aaaaaaaaaa aaaaaaaaaa 988
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1048
c 1049
<210>30
<211>271
<212>PRT
<213>破囊壶菌属
<400>30
Met Met Glu Pro Leu Asp Arg Tyr Arg Ala Leu Ala Glu Leu Ala Ala
1 5 10 15
Arg Tyr Ala Ser Ser Ala Ala Phe Lys Trp Gln Val Thr Tyr Asp Ala
20 25 30
Lys Asp Ser Phe Val Gly Pro Leu Gly Ile Arg Glu Pro Leu Gly Leu
35 40 45
Leu Val Gly Ser Val Val Leu Tyr Leu Ser Leu Leu Ala Val Val Tyr
50 55 60
Ala Leu Arg Asn Tyr Leu Gly Gly Leu Met Ala Leu Arg Ser Val His
65 70 75 80
Asn Leu Gly Leu Cys Leu Phe Ser Gly Ala Val Trp Ile Tyr Thr Ser
85 90 95
Tyr Leu Met Ile Gln Asp Gly His Phe Arg Ser Leu Glu Ala Ala Thr
100 105 110
Cys Glu Pro Leu Lys His Pro His Phe Gln Leu Ile Ser Leu Leu Phe
115 120 125
Ala Leu Ser Lys Ile Trp Glu Trp Phe Asp Thr Val Leu Leu Ile Val
130 135 140
Lys Gly Asn Lys Leu Arg Phe Leu His Val Leu His His Ala Thr Thr
145 150 155 160
Phe Trp Leu Tyr Ala Ile Asp His Ile Phe Leu Ser Ser Ile Lys Tyr
165 170 175
Gly Val Ala Val Asn Ala Phe Ile His Thr Val Met Tyr Ala His Tyr
180 185 190
Phe Arg Pro Phe Pro Lys Gly Leu Arg Pro Leu Ile Thr Gln Leu Gln
195 200 205
Ile Val Gln Phe Ile Phe Ser Ile Gly Ile His Thr Ala Ile Tyr Trp
210 215 220
His Tyr Asp Cys Glu Pro Leu Val His Thr His Phe Trp Glu Tyr Val
225 230 235 240
Thr Pro Tyr Leu Phe Val Val Pro Phe Leu Ile Leu Phe Phe Asn Phe
245 250 255
Tyr Leu Gln Gln Tyr Val Leu Ala Pro Ala Lys Thr Lys Lys Ala
260 265 270
<210>31
<211>837
<212>DNA
<213>致病疫霉(Phytophthora infestans)
<220>
<221>CDS
<222>(1)..(837)
<223>Δ6-延伸酶
<400>31
atg tcg act gag cta ctg cag agc tac tac gcg tgg gcc aac gcc acg 48
Met Ser Thr Glu Leu Leu Gln Ser Tyr Tyr Ala Trp Ala Asn Ala Thr
1 5 10 15
gag gcc aag ctg ctg gac tgg gtc gac cct gag ggc ggc tgg aag gtg 96
Glu Ala Lys Leu Leu Asp Trp Val Asp Pro Glu Gly Gly Trp Lys Val
20 25 30
cat cct atg gca gac tac ccc cta gcc aac ttc tcc agc gtc tac gcc 144
His Pro Met Ala Asp Tyr Pro Leu Ala Asn Phe Ser Ser Val Tyr Ala
35 40 45
atc tgc gtc gga tac ttg ctc ttc gta atc ttc ggc acg gcc ctg atg 192
Ile Cys Val Gly Tyr Leu Leu Phe Val Ile Phe Gly Thr Ala Leu Met
50 55 60
aaa atg gga gtc ccc gcc atc aag acc agt cca tta cag ttt gtg tac 240
Lys Met Gly Val Pro Ala Ile Lys Thr Ser Pro Leu Gln Phe Val Tyr
65 70 75 80
aac ccc atc caa gtc att gcc tgc tct tat atg tgc gtg gag gcc gcc 288
Asn Pro Ile Gln Val Ile Ala Cys Ser Tyr Met Cys Val Glu Ala Ala
85 90 95
atc cag gcc tac cgc aac ggc tac acc gcc gcc ccg tgc aac gcc ttt 336
Ile Gln Ala Tyr Arg Asn Gly Tyr Thr Ala Ala Pro Cys Asn Ala Phe
100 105 110
aag tcc gac gac ccc gtc atg ggc aac gtt ctg tac ctc ttc tat ctc 384
Lys Ser Asp Asp Pro Val Met Gly Asn Val Leu Tyr Leu Phe Tyr Leu
115 120 125
tcc aag atg ctc gac ctg tgc gac aca gtc ttc att atc cta gga aag 432
Ser Lys Met Leu Asp Leu Cys Asp Thr Val Phe Ile Ile Leu Gly Lys
130 135 140
aag tgg aaa cag ctt tcc atc ttg cac gtg tac cac cac ctt acc gtg 480
Lys Trp Lys Gln Leu Ser Ile Leu His Val Tyr His His Leu Thr Val
145 150 155 160
ctt ttc gtc tac tat gtg acg ttc cgc gcc gct cag gac ggg gac tca 528
Leu Phe Val Tyr Tyr Val Thr Phe Arg Ala Ala Gln Asp Gly Asp Ser
165 170 175
tat gct acc atc gtg ctc aac ggc ttc gtg cac acc atc atg tac act 576
Tyr Ala Thr Ile Val Leu Asn Gly Phe Val His Thr Ile Met Tyr Thr
180 185 190
tac tac ttc gtc agc gcc cac acg cgc aac att tgg tgg aag aag tac 624
Tyr Tyr Phe Val Ser Ala His Thr Arg Asn Ile Trp Trp Lys Lys Tyr
195 200 205
ctc acg cgc att cag ctt atc cag ttc gtg acc atg aac gtg cag ggc 672
Leu Thr Arg Ile Gln Leu Ile Gln Phe Val Thr Met Asn Val Gln Gly
210 215 220
tac ctg acc tac tct cga cag tgc cca ggc atg cct cct aag gtg ccg 720
Tyr Leu Thr Tyr Ser Arg Gln Cys Pro Gly Met Pro Pro Lys Val Pro
225 230 235 240
ctc atg tac ctt gtg tac gtg cag tca ctc ttc tgg ctc ttc atg aat 768
Leu Met Tyr Leu Val Tyr Val Gln Ser Leu Phe Trp Leu Phe Met Asn
245 250 255
ttc tac att cgc gcg tac gtg ttc ggc ccc aag aaa ccg gcc gtg gag 816
Phe Tyr Ile Arg Ala Tyr Val Phe Gly Pro Lys Lys Pro Ala Val Glu
260 265 270
gaa tcg aag aag aag ttg taa 837
Glu Ser Lys Lys Lys Leu
275
<210>32
<211>278
<212>PRT
<213>致病疫霉
<400>32
Met Ser Thr Glu Leu Leu Gln Ser Tyr Tyr Ala Trp Ala Asn Ala Thr
1 5 10 15
Glu Ala Lys Leu Leu Asp Trp Val Asp Pro Glu Gly Gly Trp Lys Val
20 25 30
His Pro Met Ala Asp Tyr Pro Leu Ala Asn Phe Ser Ser Val Tyr Ala
35 40 45
Ile Cys Val Gly Tyr Leu Leu Phe Val Ile Phe Gly Thr Ala Leu Met
50 55 60
Lys Met Gly Val Pro Ala Ile Lys Thr Ser Pro Leu Gln Phe Val Tyr
65 70 75 80
Asn Pro Ile Gln Val Ile Ala Cys Ser Tyr Met Cys Val Glu Ala Ala
85 90 95
Ile Gln Ala Tyr Arg Asn Gly Tyr Thr Ala Ala Pro Cys Asn Ala Phe
100 105 110
Lys Ser Asp Asp Pro Val Met Gly Asn Val Leu Tyr Leu Phe Tyr Leu
115 120 125
Ser Lys Met Leu Asp Leu Cys Asp Thr Val Phe Ile Ile Leu Gly Lys
130 135 140
Lys Trp Lys Gln Leu Ser Ile Leu His Val Tyr His His Leu Thr Val
145 150 155 160
Leu Phe Val Tyr Tyr Val Thr Phe Arg Ala Ala Gln Asp Gly Asp Ser
165 170 175
Tyr Ala Thr Ile Val Leu Asn Gly Phe Val His Thr Ile Met Tyr Thr
180 185 190
Tyr Tyr Phe Val Ser Ala His Thr Arg Asn Ile Trp Trp Lys Lys Tyr
195 200 205
Leu Thr Arg Ile Gln Leu Ile Gln Phe Val Thr Met Asn Val Gln Gly
210 215 220
Tyr Leu Thr Tyr Ser Arg Gln Cys Pro Gly Met Pro Pro Lys Val Pro
225 230 235 240
Leu Met Tyr Leu Val Tyr Val Gln Ser Leu Phe Trp Leu Phe Met Asn
245 250 255
Phe Tyr Ile Arg Ala Tyr Val Phe Gly Pro Lys Lys Pro Ala Val Glu
260 265 270
Glu Ser Lys Lys Lys Leu
275
<210>33
<211>954
<212>DNA
<213>高山被孢霉
<220>
<221>CDS
<222>(1)..(954)
<223>Δ6-延伸酶
<400>33
atg gcc gcc gca atc ttg gac aag gtc aac ttc ggc att gat cag ccc 48
Met Ala Ala Ala Ile Leu Asp Lys Val Asn Phe Gly Ile Asp Gln Pro
1 5 10 15
ttc gga atc aag ctc gac acc tac ttt gct cag gcc tat gaa ctc gtc 96
Phe Gly Ile Lys Leu Asp Thr Tyr Phe Ala Gln Ala Tyr Glu Leu Val
20 25 30
acc gga aag tcc atc gac tcc ttc gtc ttc cag gag ggc gtc acg cct 144
Thr Gly Lys Ser Ile Asp Ser Phe Val Phe Gln Glu Gly Val Thr Pro
35 40 45
ctc tcg acc cag aga gag gtc gcc atg tgg act atc act tac ttc gtc 192
Leu Ser Thr Gln Arg Glu Val Ala Met Trp Thr Ile Thr Tyr Phe Val
50 55 60
gtc atc ttt ggt ggt cgc cag atc atg aag agc cag gac gcc ttc aag 240
Val Ile Phe Gly Gly Arg Gln Ile Met Lys Ser Gln Asp Ala Phe Lys
65 70 75 80
ctc aag ccc ctc ttc atc ctc cac aac ttc ctc ctg acg atc gcg tcc 288
Leu Lys Pro Leu Phe Ile Leu His Asn Phe Leu Leu Thr Ile Ala Ser
85 90 95
gga tcg ctg ttg ctc ctg ttc atc gag aac ctg gtc ccc atc ctc gcc 336
Gly Ser Leu Leu Leu Leu Phe Ile Glu Asn Leu Val Pro Ile Leu Ala
100 105 110
aga aac gga ctt ttc tac gcc atc tgc gac gac ggt gcc tgg acc cag 384
Arg Asn Gly Leu Phe Tyr Ala Ile Cys Asp Asp Gly Ala Trp Thr Gln
115 120 125
cgc ctc gag ctc ctc tac tac ctc aac tac ctg gtc aag tac tgg gag 432
Arg Leu Glu Leu Leu Tyr Tyr Leu Asn Tyr Leu Val Lys Tyr Trp Glu
130 135 140
ttg gcc gac acc gtc ttt ttg gtc ctc aag aag aag cct ctt gag ttc 480
Leu Ala Asp Thr Val Phe Leu Val Leu Lys Lys Lys Pro Leu Glu Phe
145 150 155 160
ctg cac tac ttc cac cac tcg atg acc atg gtt ctc tgc ttt gtc cag 528
Leu His Tyr Phe His His Ser Met Thr Met Val Leu Cys Phe Val Gln
165 170 175
ctt gga gga tac act tca gtg tcc tgg gtc cct att acc ctc aac ttg 576
Leu Gly Gly Tyr Thr Ser Val Ser Trp Val Pro Ile Thr Leu Asn Leu
180 185 190
act gtc cac gtc ttc atg tac tac tac tac atg cgc tcc gct gcc ggt 624
Thr Val His Val Phe Met Tyr Tyr Tyr Tyr Met Arg Ser Ala Ala Gly
195 200 205
gtt cgc atc tgg tgg aag cag tac ttg acc act ctc cag atc gtc cag 672
Val Arg Ile Trp Trp Lys Gln Tyr Leu Thr Thr Leu Gln Ile Val Gln
210 215 220
ttc gtt ctt gac ctc gga ttc atc tac ttc tgc gcc tac acc tac ttc 720
Phe Val Leu Asp Leu Gly Phe Ile Tyr Phe Cys Ala Tyr Thr Tyr Phe
225 230 235 240
gcc ttc acc tac ttc ccc tgg gct ccc aac gtc ggc aag tgc gcc ggt 768
Ala Phe Thr Tyr Phe Pro Trp Ala Pro Asn Val Gly Lys Cys Ala Gly
245 250 255
acc gag ggt gct gct ctc ttt ggc tgc gga ctc ctc tcc agc tat ctc 816
Thr Glu Gly Ala Ala Leu Phe Gly Cys Gly Leu Leu Ser Ser Tyr Lau
260 265 270
ttg ctc ttt atc aac ttc tac cgc att acc tac aat gcc aag gcc aag 864
Leu Leu Phe Ile Asn Phe Tyr Arg Ile Thr Tyr Asn Ala Lys Ala Lys
275 280 285
gca gcc aag gag cgt gga agc aac ttt acc ccc aag act gtc aag tcc 912
Ala Ala Lys Glu Arg Gly Ser Asn Phe Thr Pro Lys Thr Val Lys Ser
290 295 300
ggc gga tcg ccc aag aag ccc tcc aag agc aag cac atc taa 954
Gly Gly Ser Pro Lys Lys Pro Ser Lys Ser Lys His Ile
305 310 315
<210>34
<211>317
<212>PRT
<213>高山被孢霉
<400>34
Met Ala Ala Ala Ile Leu Asp Lys Val Asn Phe Gly Ile Asp Gln Pro
1 5 10 15
Phe Gly Ile Lys Leu Asp Thr Tyr Phe Ala Gln Ala Tyr Glu Leu Val
20 25 30
Thr Gly Lys Ser Ile Asp Ser Phe Val Phe Gln Glu Gly Val Thr Pro
35 40 45
Leu Ser Thr Gln Arg Glu Val Ala Met Trp Thr Ile Thr Tyr Phe Val
50 55 60
Val Ile Phe Gly Gly Arg Gln Ile Met Lys Ser Gln Asp Ala Phe Lys
65 70 75 80
Leu Lys Pro Leu Phe Ile Leu His Asg Phe Leu Leu Thr Ile Ala Ser
85 90 95
Gly Ser Leu Leu Leu Leu Phe Ile Glu Asn Leu Val Pro Ile Leu Ala
100 105 110
Arg Asn Gly Leu Phe Tyr Ala Ile Cys Asp Asp Gly Ala Trp Thr Gln
115 120 125
Arg Leu Glu Leu Leu Tyr Tyr Leu Asn Tyr Leu Val Lys Tyr Trp Glu
130 135 140
Leu Ala Asp Thr Val Phe Leu Val Leu Lys Lys Lys Pro Leu Glu Phe
145 150 155 160
Leu His Tyr Phe His His Ser Met Thr Met Val Leu Cys Phe Val Gln
165 170 175
Leu Gly Gly Tyr Thr Ser Val Ser Trp Val Pro Ile Thr Leu Asn Leu
180 185 190
Thr Val His Val Phe Met Tyr Tyr Tyr Tyr Met Arg Ser Ala Ala Gly
195 200 205
Val Arg Ile Trp Trp Lys Gln Tyr Leu Thr Thr Leu Gln Ile Val Gln
210 215 220
Phe Val Leu Asp Leu Gly Phe Ile Tyr Phe Cys Ala Tyr Thr Tyr Phe
225 230 235 240
Ala Phe Thr Tyr Phe Pro Trp Ala Pro Asn Val Gly Lys Cys Ala Gly
245 250 255
Thr Glu Gly Ala Ala Leu Phe Gly Cys Gly Leu Leu Ser Ser Tyr Leu
260 265 270
Leu Leu Phe Ile Asn Phe Tyr Arg Ile Thr Tyr Asn Ala Lys Ala Lys
275 280 285
Ala Ala Lys Glu Arg Gly Ser Asn Phe Thr Pro Lys Thr Val Lys Ser
290 295 300
Gly Gly Ser Pro Lys Lys Pro Ser Lys Ser Lys His Ile
305 310 315
<210>35
<211>957
<212>DNA
<213>高山被孢霉
<220>
<221>CDS
<222>(1)..(957)
<113>Δ6-延伸酶
<400>35
atg gag tcg att gcg cca ttc ctc cca tca aag atg ccg caa gat ctg 48
Met Glu Ser Ile Ala Pro Phe Leu Pro Ser Lys Met Pro Gln Asp Leu
1 5 10 15
ttt atg gac ctt gcc acc gct atc ggt gtc cgg gcc gcg ccc tat gtc 96
Phe Met Asp Leu Ala Thr Ala Ile Gly Val Arg Ala Ala Pro Tyr Val
20 25 30
gat cct ctc gag gcc gcg ctg gtg gcc cag gcc gag aag tac atc ccc 144
Asp Pro Leu Glu Ala Ala Leu Val Ala Gln Ala Glu Lys Tyr Ile Pro
35 40 45
acg att gtc cat cac acg cgt ggg ttc ctg gtc gcg gtg gag tcg cct 192
Thr Ile Val His His Thr Arg Gly Phe Leu Val Ala Val Glu Ser Pro
50 55 60
ttg gcc cgt gag ctg ccg ttg atg aac ccg ttc cac gtg ctg ttg atc 240
Leu Ala Arg Glu Leu Pro Leu Met Asn Pro Phe His Val Leu Leu Ile
65 70 75 80
gtg ctc gct tat ttg gtc acg gtc ttt gtg ggc atg cag atc atg aag 288
Val Leu Ala Tyr Leu Val Thr Val Phe Val Gly Met Gln Ile Met Lys
85 90 95
aac ttt gag cgg ttc gag gtc aag acg ttt tcg ctc ctg cac aac ttt 336
Asn Phe Glu Arg Phe Glu Val Lys Thr Phe Ser Leu Leu His Asn Phe
100 105 110
tgt ctg gtc tcg atc agc gcc tac atg tgc ggt ggg atc ctg tac gag 384
Cys Leu Val Ser Ile Ser Ala Tyr Met Cys Gly Gly Ile Leu Tyr Glu
115 120 125
gct tat cag gcc aac tat gga ctg ttt gag aac gct gct gat cat acc 432
Ala Tyr Gln Ala Asn Tyr Gly Leu Phe Glu Asn Ala Ala Asp His Thr
130 135 140
ttc aag ggt ctt cct atg gcc aag atg atc tgg ctc ttc tac ttc tcc 480
Phe Lys Gly Leu Pro Met Ala Lys Met Ile Trp Leu Phe Tyr Phe Ser
145 150 155 160
aag atc atg gag ttt gtc gac acc atg atc atg gtc ctc aag aag aac 528
Lys Ile Met Glu Phe Val Asp Thr Met Ile Met Val Leu Lys Lys Asn
165 170 175
aac cgc cag atc tcc ttc ttg cac gtt tac cac cac agc tcc atc ttc 576
Asn Arg Gln Ile Ser Phe Leu His Val Tyr His His Ser Ser Ile Phe
180 185 190
acc atc tgg tgg ttg gtc acc ttt gtt gca ccc aac ggt gaa gcc tac 624
Thr Ile Trp Trp Leu Val Thr Phe Val Ala Pro Asn Gly Glu Ala Tyr
195 200 205
ttc tct gct gcg ttg aac tcg ttc atc cat gtg atc atg tac ggc tac 672
Phe Ser Ala Ala Leu Asn Ser Phe Ile His Val Ile Met Tyr Gly Tyr
210 215 220
tac ttc ttg tcg gcc ttg ggc ttc aag cag gtg tcg ttc atc aag ttc 720
Tyr Phe Leu Ser Ala Leu Gly Phe Lys Gln Val Ser Phe Ile Lys Phe
225 230 235 240
tac atc acg cgc tcg cag atg aca cag ttc tgc atg atg tcg gtc cag 768
Tyr Ile Thr Arg Ser Gln Met Thr Gln Phe Cys Met Met Ser Val Gln
245 250 255
tct tcc tgg gac atg tac gcc atg aag gtc ctt ggc cgc ccc gga tac 816
Ser Ser Trp Asp Met Tyr Ala Met Lys Val Leu Gly Arg Pro Gly Tyr
260 265 270
ccc ttc ttc atc acg gct ctg ctt tgg ttc tac atg tgg acc atg ctc 864
Pro Phe Phe Ile Thr Ala Leu Leu Trp Phe Tyr Met Trp Thr Met Leu
275 280 285
ggt ctc ttc tac aac ttt tac aga aag aac gcc aag ttg gcc aag cag 912
Gly Leu Phe Tyr Asn Phe Tyr Arg Lys Asn Ala Lys Leu Ala Lys Gln
290 295 300
gcc aag gcc gac gct gcc aag gag aag gca agg aag ttg cag taa 957
Ala Lys Ala Asp Ala Ala Lys Glu Lys Ala Arg Lys Leu Gln
305 310 315
<210>36
<211>318
<212>PRT
<213>高山被孢霉
<400>36
Met Glu Ser Ile Ala Pro Phe Leu Pro Ser Lys Met Pro Gln Asp Leu
1 5 10 15
Phe Met Asp Leu Ala Thr Ala Ile Gly Val Arg Ala Ala Pro Tyr Val
20 25 30
Asp Pro Leu Glu Ala Ala Leu Val Ala Gln Ala Glu Lys Tyr Ile Pro
35 40 45
Thr Ile Val His His Thr Arg Gly Phe Leu Val Ala Val Glu Ser Pro
50 55 60
Leu Ala Arg Glu Leu Pro Leu Met Asn Pro Phe His Val Leu Leu Ile
65 70 75 80
Val Leu Ala Tyr Leu Val Thr Val Phe Val Gly Met Gln Ile Met Lys
85 90 95
Asn Phe Glu Arg Phe Glu Val Lys Thr Phe Ser Leu Leu His Asn Phe
100 105 110
Cys Leu Val Ser Ile Ser Ala Tyr Met Cys Gly Gly Ile Leu Tyr Glu
115 120 125
Ala Tyr Gln Ala Asn Tyr Gly Leu Phe Glu Asn Ala Ala Asp His Thr
130 135 140
Phe Lys Gly Leu Pro Met Ala Lys Met Ile Trp Leu Phe Tyr Phe Ser
145 150 155 160
Lys Ile Met Glu Phe Val Asp Thr Met Ile Met Val Leu Lys Lys Asn
165 170 175
Asn Arg Gln Ile Ser Phe Leu His Val Tyr His His Ser Ser Ile Phe
180 185 190
Thr Ile Trp Trp Leu Val Thr Phe Val Ala Pro Asn Gly Glu Ala Tyr
195 200 205
Phe Ser Ala Ala Leu Asn Ser Phe Ile His Val Ile Met Tyr Gly Tyr
210 215 220
Tyr Phe Leu Ser Ala Leu Gly Phe Lys Gln Val Ser Phe Ile Lys Phe
225 230 235 240
Tyr Ile Thr Arg Ser Gln Met Thr Gln Phe Cys Met Met Ser Val Gln
245 250 255
Ser Ser Trp Asp Met Tyr Ala Met Lys Val Leu Gly Arg Pro Gly Tyr
260 265 270
Pro Phe Phe Ile Thr Ala Leu Leu Trp Phe Tyr Met Trp Thr Met Leu
275 280 285
Gly Leu Phe Tyr Asn Phe Tyr Arg Lys Asn Ala Lys Leu Ala Lys Gln
290 295 300
Ala Lys Ala Asp Ala Ala Lys Glu Lys Ala Arg Lys Leu Gln
305 310 315
<210>37
<211>867
<212>DNA
<213>秀丽隐杆线虫
<220>
<221>CDS
<222>(1)..(867)
<223>Δ6-延伸酶
<400>37
atg gct cag cat ccg ctc gtt caa cgg ctt ctc gat gtc aaa ttc gac 48
Met Ala Gln His Pro Leu Val Gln Arg Leu Leu Asp Val Lys Phe Asp
1 5 10 15
acg aaa cga ttt gtg gct att gct act cat ggg cca aag aat ttc cct 96
Thr Lys Arg Phe Val Ala Ile Ala Thr His Gly Pro Lys Asn Phe Pro
20 25 30
gac gca gaa ggt cgc aag ttc ttt gct gat cac ttt gat gtt act att 144
Asp Ala Glu Gly Arg Lys Phe Phe Ala Asp His Phe Asp Val Thr Ile
35 40 45
cag gct tca atc ctg tac atg gtc gtt gtg ttc gga aca aaa tgg ttc 192
Gln Ala Ser Ile Leu Tyr Met Val Val Val Phe Gly Thr Lys Trp Phe
50 55 60
atg cgt aat cgt caa cca ttc caa ttg act att cca ctc aac atc tgg 240
Met Arg Asn Arg Gln Pro Phe Gln Leu Thr Ile Pro Leu Asn Ile Trp
65 70 75 80
aat ttc atc ctc gcc gca ttt tcc atc gca gga gct gtc aaa atg acc 288
Asn Phe Ile Leu Ala Ala Phe Ser Ile Ala Gly Ala Val Lys Met Thr
85 90 95
cca gag ttc ttt gga acc att gcc aac aaa gga att gtc gca tcc tac 336
Pro Glu Phe Phe Gly Thr Ile Ala Asn Lys Gly Ile Val Ala Ser Tyr
100 105 110
tgc aaa gtg ttt gat ttc acg aaa gga gag aat gga tac tgg gtg tgg 384
Cys Lys Val Phe Asp Phe Thr Lys Gly Glu Asn Gly Tyr Trp Val Trp
115 120 125
ctc ttc atg gct tcc aaa ctt ttc gaa ctt gtt gac acc atc ttc ttg 432
Leu Phe Met Ala Ser Lys Leu Phe Glu Leu Val Asp Thr Ile Phe Leu
130 135 140
gtt ctc cgt aaa cgt cca ctc atg ttc ctt cac tgg tat cac cat att 480
Val Leu Arg Lys Arg Pro Leu Met Phe Leu His Trp Tyr His His Ile
145 150 155 160
ctc acc atg atc tac gcc tgg tac tct cat cca ttg acc cca gga ttc 528
Leu Thr Met Ile Tyr Ala Trp Tyr Ser His Pro Leu Thr Pro Gly Phe
165 170 175
aac aga tac gga att tat ctt aac ttt gtc gtc cac gcc ttc atg tac 576
Asn Arg Tyr Gly Ile Tyr Leu Asn Phe Val Val His Ala Phe Met Tyr
180 185 190
tct tac tac ttc ctt cgc tcg atg aag att cgc gtg cca gga ttc atc 624
Ser Tyr Tyr Phe Leu Arg Ser Met Lys Ile Arg Val Pro Gly Phe Ile
195 200 205
gcc caa gct atc aca tct ctt caa atc gtt caa ttc atc atc tct tgc 672
Ala Gln Ala Ile Thr Ser Leu Gln Ile Val Gln Phe Ile Ile Ser Cys
210 215 220
gcc gtt ctt gct cat ctt ggt tat ctc atg cac ttc acc aat gcc aac 720
Ala Val Leu Ala His Leu Gly Tyr Leu Met His Phe Thr Asn Ala Asn
225 230 235 240
tgt gat ttc gag cca tca gta ttc aag ctc gca gtt ttc atg gac aca 768
Cys Asp Phe Glu Pro Ser Val Phe Lys Leu Ala Val Phe Met Asp Thr
245 250 255
aca tac ttg gct ctt ttc gtc aac ttc ttc ctc caa tca tat gtt ctc 816
Thr Tyr Leu Ala Leu Phe Val Asn Phe Phe Leu Gln Ser Tyr Val Leu
260 265 270
cgc gga gga aaa gac aag tac aag gca gtg cca aag aag aag aac aac 864
Arg Gly Gly Lys Asp Lys Tyr Lys Ala Val Pro Lys Lys Lys Asn Asn
275 280 285
taa 867
<210>38
<211>288
<212>PRT
<213>秀丽隐杆线虫
<400>38
Met Ala Gln His Pro Leu Val Gln Arg Leu Leu Asp Val Lys Phe Asp
1 5 10 15
Thr Lys Arg Phe Val Ala Ile Ala Thr His Gly Pro Lys Asn Phe Pro
20 25 30
Asp Ala Glu Gly Arg Lys Phe Phe Ala Asp His Phe Asp Val Thr Ile
35 40 45
Gln Ala Ser Ile Leu Tyr Met Val Val Val Phe Gly Thr Lys Trp Phe
50 55 60
Met Arg Asn Arg Gln Pro Phe Gln Leu Thr Ile Pro Leu Asn Ile Trp
65 70 75 80
Asn Phe Ile Leu Ala Ala Phe Ser Ile Ala Gly Ala Val Lys Met Thr
85 90 95
Pro Glu Phe Phe Gly Thr Ile Ala Asn Lys Gly Ile Val Ala Ser Tyr
100 105 110
Cys Lys Val Phe Asp Phe Thr Lys Gly Glu Asn Gly Tyr Trp Val Trp
115 120 125
Leu Phe Met Ala Ser Lys Leu Phe Glu Leu Val Asp Thr Ile Phe Leu
130 135 140
Val Leu Arg Lys Arg Pro Leu Met Phe Leu His Trp Tyr His His Ile
145 150 155 160
Leu Thr Met Ile Tyr Ala Trp Tyr Ser His Pro Leu Thr Pro Gly Phe
165 170 175
Asn Arg Tyr Gly Ile Tyr Leu Asn Phe Val Val His Ala Phe Met Tyr
180 185 190
Ser Tyr Tyr Phe Leu Arg Ser Met Lys Ile Arg Val Pro Gly Phe Ile
195 200 205
Ala Gln Ala Ile Thr Ser Leu Gln Ile Val Gln Phe Ile Ile Ser Cys
210 215 220
Ala Val Leu Ala His Leu Gly Tyr Leu Met His Phe Thr Asn Ala Asn
225 230 235 240
Cys Asp Phe Glu Pro Ser Val Phe Lys Leu Ala Val Phe Met Asp Thr
245 250 255
Thr Tyr Leu Ala Leu Phe Val Asn Phe Phe Leu Gln Ser Tyr Val Leu
260 265 270
Arg Gly Gly Lys Asp Lys Tyr Lys Ala Val Pro Lys Lys Lys Asn Asn
275 280 285
<210>39
<211>1626
<212>DNA
<213>细小裸藻
<220>
<221>CDS
<222>(1)..(1626)
<223>Δ4-去饱和酶
<400>39
atg ttg gtg ctg ttt ggc aat ttc tat gtc aag caa tac tcc caa aag 48
Met Leu Val Leu Phe Gly Asn Phe Tyr Val Lys Gln Tyr Ser Gln Lys
1 5 10 15
aac ggc aag ccg gag aac gga gcc acc cct gag aac gga gcg aag ccg 96
Asn Gly Lys Pro Glu Asn Gly Ala Thr Pro Glu Asn Gly Ala Lys Pro
20 25 30
caa cct tgc gag aac ggc acg gtg gaa aag cga gag aat gac acc gcc 144
Gln Pro Cys Glu Asn Gly Thr Val Glu Lys Arg Glu Asn Asp Thr Ala
35 40 45
aac gtt cgg ccc acc cgt cca gct gga ccc ccg ccg gcc acg tac tac 192
Asn Val Arg Pro Thr Arg Pro Ala Gly Pro Pro Pro Ala Thr Tyr Tyr
50 55 60
gac tcc ctg gca gtg tcg ggg cag ggc aag gag cgg ctg ttc acc acc 240
Asp Ser Leu Ala Val Ser Gly Gln Gly Lys Glu Arg Leu Phe Thr Thr
65 70 75 80
gat gag gtg agg cgg cac atc ctc ccc acc gat ggc tgg ctg acg tgc 288
Asp Glu Val Arg Arg His Ile Leu Pro Thr Asp Gly Trp Leu Thr Cys
85 90 95
cac gaa gga gtc tac gat gtc act gat ttc ctt gcc aag cac cct ggt 336
His Glu Gly Val Tyr Asp Val Thr Asp Phe Leu Ala Lys His Pro Gly
100 105 110
ggc ggt gtc atc acg ctg ggc ctt gga agg gac tgc aca atc ctc atc 384
Gly Gly Val Ile Thr Leu Gly Leu Gly Arg Asp Cys Thr Ile Leu Ile
115 120 125
gag tca tac cac cct gct ggg cgc ccg gac aag gtg atg gag aag tac 432
Glu Ser Tyr His Pro Ala Gly Arg Pro Asp Lys Val Met Glu Lys Tyr
130 135 140
cgc att ggt acg ctg cag gac ccc aag acg ttc tat gct tgg gga gag 480
Arg Ile Gly Thr Leu Gln Asp Pro Lys Thr Phe Tyr Ala Trp Gly Glu
145 150 155 160
tcc gat ttc tac cct gag ttg aag cgc cgg gcc ctt gca agg ctg aag 528
Ser Asp Phe Tyr Pro Glu Leu Lys Arg Arg Ala Leu Ala Arg Leu Lys
165 170 175
gag gct ggt cag gcg cgg cgc ggc ggc ctt ggg gtg aag gcc ctc ctg 576
Glu Ala Gly Gln Ala Arg Arg Gly Gly Leu Gly Val Lys Ala Leu Leu
180 185 190
gtg ctc acc ctc ttc ttc gtg tcg tgg tac atg tgg gtg gcc cac aag 624
Val Leu Thr Leu Phe Phe Val Ser Trp Tyr Met Trp Val Ala His Lys
195 200 205
tcc ttc ctc tgg gcc gcc gtc tgg ggc ttc gcc ggc tcc cac gtc ggg 672
Ser Phe Leu Trp Ala Ala Val Trp Gly Phe Ala Gly Ser His Val Gly
210 215 220
ctg agc atc cag cac gat ggc aac cac ggc gcg ttc agc cgc aac aca 720
Leu Ser Ile Gln His Asp Gly Asn His Gly Ala Phe Ser Arg Asn Thr
225 230 235 240
ctg gtg aac cgc ctg gcg ggg tgg ggc atg gac ttg atc ggc gcg tcg 768
Leu Val Asn Arg Leu Ala Gly Trp Gly Met Asp Leu Ile Gly Ala Ser
245 250 255
tcc acg gtg tgg gag tac cag cac gtc atc ggc cac cac cag tac acc 816
Ser Thr Val Trp Glu Tyr Gln His Val Ile Gly His His Gln Tyr Thr
260 265 270
aac ctc gtg tcg gac acg cta ttc agt ctg cct gag aac gat ccg gac 864
Asn Leu Val Ser Asp Thr Leu Phe Ser Leu Pro Glu Asn Asp Pro Asp
275 280 285
gtc ttc tcc agc tac ccg ctg atg cgc atg cac ccg gat acg gcg tgg 912
Val Phe Ser Ser Tyr Pro Leu Met Arg Met His Pro Asp Thr Ala Trp
290 295 300
cag ccg cac cac cgc ttc cag cac ctg ttc gcg ttc cca ctg ttc gcc 960
Gln Pro His His Arg Phe Gln His Leu Phe Ala Phe Pro Leu Phe Ala
305 310 315 320
ctg atg aca atc agc aag gtg ctg acc agc gat ttc gct gtc tgc ctc 1008
Leu Met Thr Ile Ser Lys Val Leu Thr Ser Asp Phe Ala Val Cys Leu
325 330 335
agc atg aag aag ggg tcc atc gac tgc tcc tcc agg ctc gtc cca ctg 1056
Ser Met Lys Lys Gly Ser Ile Asp Cys Ser Ser Arg Leu Val Pro Leu
340 345 350
gag ggg cag ctg ctg ttc tgg ggg gcc aag ctg gcg aac ttc ctg ttg 1104
Glu Gly Gln Leu Leu Phe Trp Gly Ala Lys Leu Ala Asn Phe Leu Leu
355 360 365
cag att gtg ttg cca tgc tac ctc cac ggg aca gct atg ggc ctg gcc 1152
Gln Ile Val Leu Pro Cys Tyr Leu His Gly Thr Ala Met Gly Leu Ala
370 375 380
ctc ttc tct gtt gct cac ctt gtg tcg ggg gag tac ctc gcg atc tgc 1200
Leu Phe Ser Val Ala His Leu Val Ser Gly Glu Tyr Leu Ala Ile Cys
385 390 395 400
ttc atc atc aac cac atc agc gag tct tgt gag ttt atg aat aca agc 1248
Phe Ile Ile Asn His Ile Ser Glu Ser Cys Glu Phe Met Asn Thr Ser
405 410 415
ttt caa acc gcc gcc cgg agg aca gag atg ctt cag gca gca cat cag 1296
Phe Gln Thr Ala Ala Arg Arg Thr Glu Met Leu Gln Ala Ala His Gln
420 425 430
gca gcg gag gcc aag aag gtg aag ccc acc cct cca ccg aac gat tgg 1344
Ala Ala Glu Ala Lys Lys Val Lys Pro Thr Pro Pro Pro Asn Asp Trp
435 440 445
gct gtg aca cag gtc caa tgc tgc gtg aat tgg aga tca ggt ggc gtg 1392
Ala Val Thr Gln Val Gln Cys Cys Val Asn Trp Arg Ser Gly Gly Val
450 455 460
ttg gcc aat cac ctc tct gga ggc ttg aac cac cag atc gag cat cat 1440
Leu Ala Asn His Leu Ser Gly Gly Leu Asn His Gln Ile Glu His His
465 470 475 480
ctg ttc ccc agc atc tcg cat gcc aac tac ccc acc atc gcc cct gtt 1488
Leu Phe Pro Ser Ile Ser His Ala Asn Tyr Pro Thr Ile Ala Pro Val
485 490 495
gtg aag gag gtg tgc gag gag tac ggg ttg ccg tac aag aat tac gtc 1536
Val Lys Glu Val Cys Glu Glu Tyr Gly Leu Pro Tyr Lys Asn Tyr Val
500 505 510
acg ttc tgg gat gca gtc tgt ggc atg gtt cag cac ctc cgg ttg atg 1584
Thr Phe Trp Asp Ala Val Cys Gly Met Val Gln His Leu Arg Leu Met
515 520 525
ggt gct cca ccg gtg cca acg aac ggg gac aaa aag tca taa 1626
Gly Ala Pro Pro Val Pro Thr Asn Gly Asp Lys Lys Ser
530 535 540
<210>40
<211>541
<212>PRT
<213>细小裸藻
<400>40
Met Leu Val Leu Phe Gly Asn Phe Tyr Val Lys Gln Tyr Ser Gln Lys
1 5 10 15
Asn Gly Lys Pro Glu Asn Gly Ala Thr Pro Glu Asn Gly Ala Lys Pro
20 25 30
Gln Pro Cys Glu Asn Gly Thr Val Glu Lys Arg Glu Asn Asp Thr Ala
35 40 45
Asn Val Arg Pro Thr Arg Pro Ala Gly Pro Pro Pro Ala Thr Tyr Tyr
50 55 60
Asp Ser Leu Ala Val Ser Gly Gln Gly Lys Glu Arg Leu Phe Thr Thr
65 70 75 80
Asp Glu Val Arg Arg His Ile Leu Pro Thr Asp Gly Trp Leu Thr Cys
85 90 95
His Glu Gly Val Tyr Asp Val Thr Asp Phe Leu Ala Lys His Pro Gly
100 105 110
Gly Gly Val Ile Thr Leu Gly Leu Gly Arg Asp Cys Thr Ile Leu Ile
115 120 125
Glu Ser Tyr His Pro Ala Gly Arg Pro Asp Lys Val Met Glu Lys Tyr
130 135 140
Arg Ile Gly Thr Leu Gln Asp Pro Lys Thr Phe Tyr Ala Trp Gly Glu
145 150 155 160
Ser Asp Phe Tyr Pro Glu Leu Lys Arg Arg Ala Leu Ala Arg Leu Lys
165 170 175
Glu Ala Gly Gln Ala Arg Arg Gly Gly Leu Gly Val Lys Ala Leu Leu
180 185 190
Val Leu Thr Leu Phe Phe Val Ser Trp Tyr Met Trp Val Ala His Lys
195 200 205
Ser Phe Leu Trp Ala Ala Val Trp Gly Phe Ala Gly Ser His Val Gly
210 215 220
Leu Ser Ile Gln His Asp Gly Asn His Gly Ala Phe Ser Arg Asn Thr
225 230 235 240
Leu Val Asn Arg Leu Ala Gly Trp Gly Met Asp Leu Ile Gly Ala Ser
245 250 255
Ser Thr Val Trp Glu Tyr Gln His Val Ile Gly His His Gln Tyr Thr
260 265 270
Asn Leu Val Ser Asp Thr Leu Phe Ser Leu Pro Glu Asn Asp Pro Asp
275 280 285
Val Phe Ser Ser Tyr Pro Leu Met Arg Met His Pro Asp Thr Ala Trp
290 295 300
Gln Pro His His Arg Phe Gln His Leu Phe Ala Phe Pro Leu Phe Ala
305 310 315 320
Leu Met Thr Ile Ser Lys Val Leu Thr Ser Asp Phe Ala Val Cys Leu
325 330 335
Ser Met Lys Lys Gly Ser Ile Asp Cys Ser Ser Arg Leu Val Pro Leu
340 345 350
Glu Gly Gln Leu Leu Phe Trp Gly Ala Lys Leu Ala Asn Phe Leu Leu
355 360 365
Gln Ile Val Leu Pro Cys Tyr Leu His Gly Thr Ala Met Gly Leu Ala
370 375 380
Leu Phe Ser Val Ala His Leu Val Ser Gly Glu Tyr Leu Ala Ile Cys
385 390 395 400
Phe Ile Ile Asn His Ile Ser Glu Ser Cys Glu Phe Met Asn Thr Ser
405 410 415
Phe Gln Thr Ala Ala Arg Arg Thr Glu Met Leu Gln Ala Ala His Gln
420 425 430
Ala Ala Glu Ala Lys Lys Val Lys Pro Thr Pro Pro Pro Asn Asp Trp
435 440 445
Ala Val Thr Gln Val Gln Cys Cys Val Asn Trp Arg Ser Gly Gly Val
450 455 460
Leu Ala Asn His Leu Ser Gly Gly Leu Asn His Gln Ile Glu His His
465 470 475 480
Leu Phe Pro Ser Ile Ser His Ala Asn Tyr Pro Thr Ile Ala Pro Val
485 490 495
Val Lys Glu Val Cys Glu Glu Tyr Gly Leu Pro Tyr Lys Asn Tyr Val
500 505 510
Thr Phe Trp Asp Ala Val Cys Gly Met Val Gln His Leu Arg Leu Met
515 520 525
Gly Ala Pro Pro Val Pro Thr Asn Gly Asp Lys Lys Ser
530 535 540
<210>41
<211>1548
<212>DNA
<213>破囊壶菌
<220>
<221>CDS
<222>(1)..(1548)
<223>Δ4-去饱和酶
<400>41
atg acg gtc ggg ttt gac gaa acg gtg act atg gac acg gtc cgc aac 48
Met Thr Val Gly Phe Asp Glu Thr Val Thr Met Asp Thr Val Arg Asn
1 5 10 15
cac aac atg ccg gac gac gcc tgg tgc gcg atc cac ggc acc gtg tac 96
His Asn Met Pro Asp Asp Ala Trp Cys Ala Ile His Gly Thr Val Tyr
20 25 30
gac atc acc aag ttc agc aag gtg cac ccc ggc ggg gac atc atc atg 144
Asp Ile Thr Lys Phe Ser Lys Val His Pro Gly Gly Asp Ile Ile Met
35 40 45
ctg gcc gct ggc aag gag gcc acc atc ctg ttc gag acc tac cac atc 192
Leu Ala Ala Gly Lys Glu Ala Thr Ile Leu Phe Glu Thr Tyr His Ile
50 55 60
aag ggc gtc ccg gac gcg gtg ctg cgc aag tac aag gtc ggc aag ctc 240
Lys Gly Val Pro Asp Ala Val Leu Arg Lys Tyr Lys Val Gly Lys Leu
65 70 75 80
ccc cag ggc aag aag ggc gaa acg agc cac atg ccc acc ggg ctc gac 288
Pro Gln Gly Lys Lys Gly Glu Thr Ser His Met Pro Thr Gly Leu Asp
85 90 95
tcg gcc tcc tac tac tcg tgg gac agc gag ttt tac agg gtg ctc cgc 336
Ser Ala Ser Tyr Tyr Ser Trp Asp Ser Glu Phe Tyr Arg Val Leu Arg
100 105 110
gag cgc gtc gcc aag aag ctg gcc gag ccc ggc ctc atg cag cgc gcg 384
Glu Arg Val Ala Lys Lys Leu Ala Glu Pro Gly Leu Met Gln Arg Ala
115 120 125
cgc atg gag ctc tgg gcc aag gcg atc ttc ctc ctg gca ggt ttc tgg 432
Arg Met Glu Leu Trp Ala Lys Ala Ile Phe Leu Leu Ala GlV Phe Trp
130 135 140
ggc tcc ctt tac gcc atg tgc gtg cta gac ccg cac ggc ggt gcc atg 480
Gly Ser Leu Tyr Ala Met Cys Val Leu Asp Pro His Gly Gly Ala Met
145 150 155 160
gta gcc gcc gtt acg ctc ggc gtg ttc gct gcc ttt gtc gga act tgc 528
Val Ala Ala Val Thr Leu Gly Val Phe Ala Ala Phe Val Gly Thr Cys
165 170 175
atc cag cac gac ggc agc cac ggc gcc ttc tcc aag tcg cga ttc atg 576
Ile Gln His Asp Gly Ser His Gly Ala Phe Ser Lys Ser Arg Phe Met
180 185 190
aac aag gcg gcg ggc tgg acc ctc gac atg atc ggc gcg agt gcg atg 624
Asn Lys Ala Ala Gly Trp Thr Leu Asp Met Ile Gly Ala Ser Ala Met
195 200 205
acc tgg gag atg cag cac gtt ctt ggc cac cac ccg tac acc aac ctc 672
Thr Trp Glu Met Gln His Val Leu Gly His His Pro Tyr Thr Asn Leu
210 215 220
atc gag atg gag aac ggt ttg gcc aag gtc aag ggc gcc gac gtc gac 720
Ile Glu Met Glu Asn Gly Leu Ala Lys Val Lys Gly Ala Asp Val Asp
225 230 235 240
ccg aag aag gtc gac cag gag agc gac ccg gac gtc ttc agt acg tac 768
Pro Lys Lys Val Asp Gln Glu Ser Asp Pro Asp Val Phe Ser Thr Tyr
245 250 255
ccg atg ctt cgc ctg cac ccg tgg cac cgc cag cgg ttt tac cac aag 816
Pro Met Leu Arg Leu His Pro Trp His Arg Gln Arg Phe Tyr His Lys
260 265 270
ttc cag cac ctg tac gcc ccg ttt atc ttt ggg tct atg acg att aac 864
Phe Gln His Leu Tyr Ala Pro Phe Ile Phe Gly Ser Met Thr Ile Asn
275 280 285
aag gtg att tcc cag gat gtc ggg gtt gtg ctg cgc aag cgc ctg ttc 912
Lys Val Ile Ser Gln Asp Val Gly Val Val Leu Arg Lys Arg Leu Phe
290 295 300
cag atc gac gcc aac tgc cgg tat ggc agc ccc tgg tac gtg gcc cgc 960
Gln Ile Asp Ala Asn Cys Arg Tyr Gly Ser Pro Trp Tyr Val Ala Arg
305 310 315 320
ttc tgg atc atg aag ctc ctc acc acg ctc tac atg gtg gcg ctt ccc 1008
Phe Trp Ile Met Lys Leu Leu Thr Thr Leu Tyr Met Val Ala Leu Pro
325 330 335
atg tac atg cag ggg cct gct cag ggc ttg aag ctt ttc ttc atg gcc 1056
Met Tyr Met Gln Gly Pro Ala Gln Gly Leu Lys Leu Phe Phe Met Ala
340 345 350
cac ttc acc tgc gga gag gtc ctc gcc acc atg ttt att gtc aac cac 1104
His Phe Thr Cys Gly Glu Val Leu Ala Thr Met Phe Ile Val Asn His
355 360 365
atc atc gag ggc gtc agc tac gct tcc aag gac gcg gtc aag ggc gtc 1152
Ile Ile Glu Gly Val Ser Tyr Ala Ser Lys Asp Ala Val Lys Gly Val
370 375 380
atg gct ccg ccg cgc act gtg cac ggt gtc acc ccg atg cag gtg acg 1200
Met Ala Pro Pro Arg Thr Val His Gly Val Thr Pro Met Gln Val Thr
385 390 395 400
caa aag gcg ctc agt gcg gcc gag tcg gcc aag tcg gac gcc gac aag 1248
Gln Lys Ala Leu Ser Ala Ala Glu Ser Ala Lys Ser Asp Ala Asp Lys
405 410 415
acg acc atg atc ccc ctc aac gac tgg gcc gct gtg cag tgc cag acc 1296
Thr Thr Met Ile Pro Leu Asn Asp Trp Ala Ala Val Gln Cys Gln Thr
420 425 430
tct gtg aac tgg gct gtc ggg tcg tgg ttt tgg aac cac ttt tcg ggc 1344
Ser Val Asn Trp Ala Val Gly Ser Trp Phe Trp Asn His Phe Ser Gly
435 440 445
ggc ctc aac cac cag att gag cac cac tgc ttc ccc caa aac ccc cac 1392
Gly Leu Asn His Gln Ile Glu His His Cys Phe Pro Gln Asn Pro His
450 455 460
acg gtc aac gtc tac atc tcg ggc atc gtc aag gag acc tgc gaa gaa 1440
Thr Val Asn Val Tyr Ile Ser Gly Ile Val Lys Glu Thr Cys Glu Glu
465 470 475 480
tac ggc gtg ccg tac cag gct gag atc agc ctc ttc tct gcc tat ttc 1488
Tyr Gly Val Pro Tyr Gln Ala Glu Ile Ser Leu Phe Ser Ala Tyr Phe
485 490 495
aag atg ctg tcg cac ctc cgc acg ctc ggc aac gag gac ctc acg gcc 1536
Lys Met Leu Ser His Leu Arg Thr Leu Gly Asn Glu Asp Leu Thr Ala
500 505 510
tgg tcc acg tga 1548
Trp Ser Thr
515
<210>42
<211>515
<212>PRT
<213>破囊壶菌
<400>42
Met Thr Val Gly Phe Asp Glu Thr Val Thr Met Asp Thr Val Arg Asn
1 5 10 15
His Asn Met Pro Asp Asp Ala Trp Cys Ala Ile His Gly Thr Val Tyr
20 25 30
Asp Ile Thr Lys Phe Ser Lys Val His Pro Gly Gly Asp Ile Ile Met
35 40 45
Leu Ala Ala Gly Lys Glu Ala Thr Ile Leu Phe Glu Thr Tyr His Ile
50 55 60
Lys Gly Val Pro Asp Ala Val Leu Arg Lys Tyr Lys Val Gly Lys Leu
65 70 75 80
Pro Gln Gly Lys Lys Gly Glu Thr Ser His Met Pro Thr Gly Leu Asp
85 90 95
Ser Ala Ser Tyr Tyr Ser Trp Asp Ser Glu Phe Tyr Arg Val Leu Arg
100 105 110
Glu Arg Val Ala Lys Lys Leu Ala Glu Pro Gly Leu Met Gln Arg Ala
115 120 125
Arg Met Glu Leu Trp Ala Lys Ala Ile Phe Leu Leu Ala Gly Phe Trp
130 135 140
Gly Ser Leu Tyr Ala Met Cys Val Leu Asp Pro His Gly Gly Ala Met
145 150 155 160
Val Ala Ala Val Thr Leu Gly Val Phe Ala Ala Phe Val Gly Thr Cys
165 170 175
Ile Gln His Asp Gly Ser His Gly Ala Phe Ser Lys Ser Arg Phe Met
180 185 190
Asn Lys Ala Ala Gly Trp Thr Leu Asp Met Ile Gly Ala Ser Ala Met
195 200 205
Thr Trp Glu Met Gln His Val Leu Gly His His Pro Tyr Thr Asn Leu
210 215 220
Ile Glu Met Glu Asn Gly Leu Ala Lys Val Lys Gly Ala Asp Val Asp
225 230 235 240
Pro Lys Lys Val Asp Gln Glu Ser Asp Pro Asp Val Phe Ser Thr Tyr
245 250 255
Pro Met Leu Arg Leu His Pro Trp His Arg Gln Arg Phe Tyr His Lys
260 265 270
Phe Gln His Leu Tyr Ala Pro Phe Ile Phe Gly Ser Met Thr Ile Asn
275 280 285
Lys Val Ile Ser Gln Asp Val Gly Val Val Leu Arg Lys Arg Leu Phe
290 295 300
Gln Ile Asp Ala Asn Cys Arg Tyr Gly Ser Pro Trp Tyr Val Ala Arg
305 310 315 320
Phe Trp Ile Met Lys Leu Leu Thr Thr Leu Tyr Met Val Ala Leu Pro
325 330 335
Met Tyr Met Gln Gly Pro Ala Gln Gly Leu Lys Leu Phe Phe Met Ala
340 345 350
His Phe Thr Cys Gly Glu Val Leu Ala Thr Met Phe Ile Val Asn His
355 360 365
Ile Ile Glu Gly Val Ser Tyr Ala Ser Lys Asp Ala Val Lys Gly Val
370 375 380
Met Ala Pro Pro Arg Thr Val His Gly Val Thr Pro Met Gln Val Thr
385 390 395 400
Gln Lys Ala Leu Ser Ala Ala Glu Ser Ala Lys Ser Asp Ala Asp Lys
405 410 415
Thr Thr Met Ile Pro Leu Asn Asp Trp Ala Ala Val Gln Cys Gln Thr
420 425 430
Ser Val Asn Trp Ala Val Gly Ser Trp Phe Trp Asn His Phe Ser Gly
435 440 445
Gly Leu Asn His Gln Ile Glu His His Cys Phe Pro Gln Asn Pro His
450 455 460
Thr Val Asn Val Tyr Ile Ser Gly Ile Val Lys Glu Thr Cys Glu Glu
465 470 475 480
Tyr Gly Val Pro Tyr Gln Ala Glu Ile Ser Leu Phe Ser Ala Tyr Phe
485 490 495
Lys Met Leu Ser His Leu Arg Thr Leu Gly Asn Glu Asp Leu Thr Ala
500 505 510
Trp Ser Thr
515
<210>43
<211>960
<212>DNA
<213>假矮海链藻(Thalassiosira pseudonana)
<220>
<221>CDS
<222>(1)..(960)
<223>Δ5-延伸酶
<400>43
atg gtg ttg tac aat gtg gcg caa gtg ctg ctc aat ggg tgg acg gtg 48
Met Val Leu Tyr Asn Val Ala Gln Val Leu Leu Asn Gly Trp Thr Val
1 5 10 15
tat gcg att gtg gat gcg gtg atg aat aga gac cat ccg ttt att gga 96
Tyr Ala Ile Val Asp Ala Val Met Asn Arg Asp His Pro Phe Ile Gly
20 25 30
agt aga agt ttg gtt ggg gcg gcg ttg cat agt ggg agc tcg tat gcg 144
Ser Arg Ser Leu Val Gly Ala Ala Leu His Ser Gly Ser Ser Tyr Ala
35 40 45
gtg tgg gtt cat tat tgt gat aag tat ttg gag ttc ttt gat acg tat 192
Val Trp Val His Tyr Cys Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr
50 55 60
ttt atg gtg ttg agg ggg aaa atg gac cag atg gta ctt ggt gaa gtt 240
Phe Met Val Leu Arg Gly Lys Met Asp Gin Met Val Leu Gly Glu Val
65 70 75 80
ggt ggc agt gtg tgg tgt ggc gtt gga tat atg gat atg gag aag atg 288
Gly Gly Ser Val Trp Cys Gly Val Gly Tyr Met Asp Met Glu Lys Met
85 90 95
ata cta ctc agc ttt gga gtg cat cgg tct gct cag gga acg ggg aag 336
Ile Leu Leu Ser Phe Gly Val His Arg Ser Ala Gln Gly Thr Gly Lys
100 105 110
gct ttc acc aac aac gtt acc aat cca cat ctc acg ctt cca cct cat 384
Ala Phe Thr Asn Asn Val Thr Asn Pro His Leu Thr Leu Pro Pro His
115 120 125
tct aca aaa aca aaa aaa cag gtc tcc ttc ctc cac atc tac cac cac 432
Ser Thr Lys Thr Lys Lys Gln Val Ser Phe Leu His Ile Tyr His His
130 135 140
acg acc ata gcg tgg gca tgg tgg atc gcc ctc cgc ttc tcc ccc ggt 480
Thr Thr Ile Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly
145 150 155 160
gga gac att tac ttc ggg gca ctc ctc aac tcc atc atc cac gtc ctc 528
Gly Asp Ile Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu
165 170 175
atg tat tcc tac tac gcc ctt gcc cta ctc aag gtc agt tgt cca tgg 576
Met Tyr Ser Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp
180 185 190
aaa cga tac ctg act caa gct caa tta ttg caa ttc aca agt gtg gtg 624
Lys Arg Tyr Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val
195 200 205
gtt tat acg ggg tgt acg ggt tat act cat tac tat cat acg aag cat 672
Val Tyr Thr Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His
210 215 220
gga gcg gat gag aca cag cct agt tta gga acg tat tat ttc tgt tgt 720
Gly Ala Asp Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys
225 230 235 240
gga gtg cag gtg ttt gag atg gtt agt ttg ttt gta ctc ttt tcc atc 768
Gly Val Gln Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile
245 250 255
ttt tat aaa cga tcc tat tcg aag aag aac aag tca gga gga aag gat 816
Phe Tyr Lys Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp
260 265 270
agc aag aag aat gat gat ggg aat aat gag gat caa tgt cac aag gct 864
Ser Lys Lys Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala
275 280 285
atg aag gat ata tcg gag ggt gcg aag gag gtt gtg ggg cat gca gcg 912
Met Lys Asp Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala
290 295 300
aag gat gct gga aag ttg gtg gct acg aga gta agg tgt aag gtg taa 960
Lys Asp Ala Gly Lys Leu Val Ala Thr Arg Val Arg Cys Lys Val
305 310 315
<210>44
<211>319
<212>PRT
<213>假矮海链藻
<400>44
Met Val Leu Tyr Asn Val Ala Gln Val Leu Leu Asn Gly Trp Thr Val
l 5 10 15
Tyr Ala Ile Val Asp Ala Val Met Asn Arg Asp His Pro Phe Ile Gly
20 25 30
Ser Arg Ser Leu Val Gly Ala Ala Leu His Ser Gly Ser Ser Tyr Ala
35 40 45
Val Trp Val His Tyr Cys Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr
50 55 60
Phe Met Val Leu Arg Gly Lys Met Asp Gln Met Val Leu Gly Glu Val
65 70 75 80
Gly Gly Ser Val Trp Cys Gly Val Gly Tyr Met Asp Met Glu Lys Met
85 90 95
Ile Leu Leu Ser Phe Gly Val His Arg Ser Ala Gln Gly Thr Gly Lys
100 105 110
Ala Phe Thr Asn Asn Val Thr Asn Pro His Leu Thr Leu Pro Pro His
115 120 125
Ser Thr Lys Thr Lys Lys Gln Val Ser Phe Leu His Ile Tyr His His
130 135 140
Thr Thr Ile Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly
145 150 155 160
Gly Asp Ile Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu
165 170 175
Met Tyr Ser Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp
180 185 190
Lys Arg Tyr Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val
195 200 205
Val Tyr Thr Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His
210 215 220
Gly Ala Asp Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys
225 230 235 240
Gly Val Gln Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile
245 250 255
Phe Tyr Lys Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp
260 265 270
Ser Lys Lys Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala
275 280 285
Met Lys Asp Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala
290 295 300
Lys Asp Ala Gly Lys Leu Val Ala Thr Arg Val Arg Cys Lys Val
305 310 315
<210>45
<211>819
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(819)
<223>Δ5-延伸酶
<400>45
atg gac gcc tac aac gct gca atg gat aag atc ggt gcc gcc atc atc 48
Met Asp Ala Tyr Asn Ala Ala Met Asp Lys Ile Gly Ala Ala Ile Ile
1 5 10 15
gat tgg tct gat ccc gat gga aag ttc cgt gcc gat aga gag gac tgg 96
Asp Trp Ser Asp Pro Asp Gly Lys Phe Arg Ala Asp Arg Glu Asp Trp
20 25 30
tgg ctc tgc gsc ttc cgt agc gcc atc acc atc gcc ctc atc tac atc 144
Trp Leu Cys Asp Phe Arg Ser Ala Ile Thr Ile Ala Leu Ile Tyr Ile
35 40 45
gcc ttc gtc atc ctc ggt tcc gcc gtc atg caa tcc ctc ccc gca atg 192
Ala Phe Val Ile Leu Gly Ser Ala Val Met Gln Ser Leu Pro Ala Met
50 55 60
gat ccc tac ccc atc aaa ttc ctc tac aac gtc tcc caa atc ttc ctt 240
Asp Pro Tyr Pro Ile Lys Phe Leu Tyr Asn Val Ser Gln Ile Phe Leu
65 70 75 80
tgt gcc tac atg act gtc gag gcg gga ttt ttg gcc tac cgc aat gga 288
Cys Ala Tyr Met Thr Val Glu Ala Gly Phe Leu Ala Tyr Alg Asn Gly
85 90 95
tat acc gtc atg cct tgc aat cat ttc aat gtg aat gat cct ccc gtg 336
Tyr Thr Val Met Pro Cys Asn His Phe Asn Val Asn Asp Pro Pro Val
100 105 110
gcg aat ctt ctt tgg ttg ttt tat att tcc aag gtg tgg gac ttt tgg 384
Ala Asn Leu Leu Trp Leu Phe Tyr Ile Ser Lys Val Trp Asp Phe Trp
115 120 125
gat acc att ttc att gtg ttg ggg aag aag tgg cgt caa tta tct ttc 432
Asp Thr Ile Phe Ile Val Leu Gly Lys Lys Trp Arg Gln Leu Ser Phe
130 135 140
ttg cat gta tac cat cac acc acc atc ttt cta ttc tat tgg ctg aat 480
Leu His Val Tyr His His Thr Thr Ile Phe Leu Phe Tyr Trp Leu Asn
145 150 155 160
gcc aat gtc ttg tac gat ggt gac atc ttc ctt acc atc ttg ctc aat 528
Ala Asn Val Leu Tyr Asp Gly Asp Ile Phe Leu Thr Ile Leu Leu Asn
165 170 175
gga ttc atc cac acg gtg atg tac acg tat tac ttc atc tgt atg cat 576
Gly Phe Ile His Thr Val Met Tyr Thr Tyr Tyr Phe Ile Cys Met His
180 185 190
acc aaa gat tcc aag acg ggc aag agt ctt cct ata tgg tgg aag tcg 624
Thr Lys Asp Ser Lys Thr Gly Lys Ser Leu Pro Ile Trp Trp Lys Ser
195 200 205
agt ttg acg gcg ttt cag ttg ttg caa ttc act atc atg atg agt cag 672
Ser Leu Thr Ala Phe Gln Leu Leu Gln Phe Thr Ile Met Met Ser Gln
210 215 220
gct acc tac ctt gtc ttc cae ggg tgt gat aag gtg tcg ctt cgt atc 720
Ala Thr Tyr Leu Val Phe His Gly Cys Asp Lys Val Ser Leu Arg Ile
225 230 235 240
acg att gtg tac ttt gtg tcc ctt ttg agt ttg ttc ttc ctt ttt gct 768
Thr Ile Val Tyr Phe Val Ser Lau Leu Ser Leu Phe Phe Leu Phe Ala
245 250 255
cag ttc ttt gtg caa tca tac atg gca ccc aaa aag aag aag agt gct 816
Gln Phe Phe Val Gln Ser Tyr Met Ala Pro Lys Lys Lys Lys Ser Ala
260 265 270
tag 819
<210>46
<211>272
<212>PRT
<213>假矮海链藻
<400>46
Met Asp Ala Tyr Asn Ala Ala Met Asp Lys Ile Gly Ala Ala Ile Ile
1 5 10 15
Asp Trp Ser Asp Pro Asp Gly Lys Phe Arg Ala Asp Arg Glu Asp Trp
20 25 30
Trp Leu Cys Asp Phe Arg Ser Ala Ile Thr Ile Ala Leu Ile Tyr Ile
35 40 45
Ala Phe Val Ile Leu Gly Ser Ala Val Met Gln Ser Leu Pro Ala Met
50 55 60
Asp Pro Tyr Pro Ile Lys Phe Leu Tyr Asn Val Ser Gln Ile Phe Leu
65 70 75 80
Cys Ala Tyr Met Thr Val Glu Ala Gly Phe Leu Ala Tyr Arg Asn Gly
85 90 95
Tyr Thr Val Met Pro Cys Asn His Phe Asn Val Asn Asp Pro Pro Val
100 105 110
Ala Asn Leu Leu Trp Leu Phe Tyr Ile Ser Lys Val Trp Asp Phe Trp
115 120 125
Asp Thr Ile Phe Ile Val Leu Gly Lys Lys Trp Arg Gln Leu Ser Phe
130 135 140
Leu His Val Tyr His His Thr Thr Ile Phe Leu Phe Tyr Trp Leu Asn
145 150 155 160
Ala Asn Val Leu Tyr Asp Gly Asp Ile Phe Leu Thr Ile Leu Leu Asn
165 170 175
Gly Phe Ile His Thr Val Met Tyr Thr Tyr Tyr Phe Ile Cys Met His
180 185 190
Thr Lys Asp Ser Lys Thr Gly Lys Ser Leu Pro Ile Trp Trp Lys Ser
195 200 205
Ser Leu Thr Ala Phe Gln Leu Leu Gln Phe Thr Ile Met Met Ser Gln
210 215 220
Ala Thr Tyr Leu Val Phe His Gly Cys Asp Lys Val Ser Leu Arg Ile
225 230 235 240
Thr Ile Val Tyr Phe Val Ser Leu Leu Ser Leu Phe Phe Leu Phe Ala
245 250 255
Gln Phe Phe Val Gln Ser Tyr Met Ala Pro Lys Lys Lys Lys Ser Ala
260 265 270
<210>47
<211>936
<212>DNA
<213>寇氏隐甲藻(Crypthecodinium cohnii)
<220>
<221>CDS
<222>(1)..(936)
<223>Δ5-延伸酶
<400>47
atg tct gcc ttc atg act ctc cca cag gct ctc tcc gat gtg acc tcg 48
Met Ser Ala Phe Met Thr Leu Pro Gln Ala Leu Ser Asp Val Thr Ser
1 5 10 15
gcc ttg gtc acg ctg gga aag gat gtc tcc agc cct tca gct ttt caa 96
Ala Leu Val Thr Leu Gly Lys Asp Val Ser Ser Pro Ser Ala Phe Gln
20 25 30
gct gtc act ggc ttc tgc agg gag cag tgg ggg att ccg aca gta ttc 144
Ala Val Thr Gly Phe Cys Arg Glu Gln Trp Gly Ile Pro Thr Val Phe
35 40 45
tgc ctg ggc tac ttg gcc atg gtc tac gcg gcc aga aga ccc ctc ccg 192
Cys Leu Gly Tyr Leu Ala Met Val Tyr Ala Ala Arg Arg Pro Leu Pro
50 55 60
cag cac ggc tac atg gtt gcg gtg gac cgt tgc ttc gct gct tgg aac 240
Gln His Gly Tyr Met Val Ala Val Asp Arg Cys Phe Ala Ala Trp Asn
65 70 75 80
ttg gct ctc tct gtc ttc agc act tgg ggc ttc tac cac atg gct gtc 288
Leu Ala Leu Ser Val Phe Ser Thr Trp Gly Phe Tyr His Met Ala Val
85 90 95
ggg ctc tac aac atg aca gag acg agg ggc ttg caa ttc acc atc tgc 336
Gly Leu Tyr Asn Met Thr Glu Thr Arg Gly Leu Gln Phe Thr Ile Cys
100 105 110
ggt tcg act ggg gag ctc gtg cag aac ctt cag act ggc cca acc gct 384
Gly Ser Thr Gly Glu Leu Val Gln Asn Leu Gln Thr Gly Pro Thr Ala
115 120 125
ctg gcg ctc tgc ctc ttc tgc ttc agc aag atc ccc gag ttg atg gac 432
Leu Ala Leu Cys Leu Phe Cys Phe Ser Lys Ile Pro Glu Leu Met Asp
130 135 140
acg gtg ttt ctc atc ctg aag gcc aag aag gtc cgc ttc ttg cag tgg 480
Thr Val Phe Leu Ile Leu Lys Ala Lys Lys Val Arg Phe Leu Gln Trp
145 150 155 160
tac cac cat gcc aca gtc atg ctc ttc tgt tgg ctc gcc ctc gcg acg 528
Tyr His His Ala Thr Val Met Leu Phe Cys Trp Leu Ala Leu Ala Thr
165 170 175
gag tac act cct ggc ttg tgg ttt gcg gcg acg aac tac ttc gtg cac 576
Glu Tyr Thr Pro Gly Leu Trp Phe Ala Ala Thr Asn Tyr Phe Val His
180 185 190
tcc atc atg tac atg tac ttc ttc ctc atg acc ttc aag tcg gcc gcg 624
Ser Ile Met Tyr Met Tyr Phe Phe Leu Met Thr Phe Lys Ser Ala Ala
195 200 205
aag gtg gtg aag ccc atc gcc cct ctc atc aca gtt atc cag att gct 672
Lys Val Val Lys Pro Ile Ala Pro Leu Ile Thr Val Ile Gln Ile Ala
210 215 220
cag atg gtc tgg ggc ctc atc gtc aac ggc atc gcc atc acc acc ttc 720
Gln Met Val Trp Gly Leu Ile Val Asn Gly Ile Ala Ile Thr Thr Phe
225 230 235 240
ttc acg act ggt gcc tgc cag atc cag tct gtg act gtg tat tcg gcc 768
Phe Thr Thr Gly Ala Cys Gln Ile Gln Ser Val Thr Val Tyr Ser Ala
245 250 255
atc atc atg tac gct tcg tac ttc tac ctg ttc tcc cag ctc ttc ttc 816
Ile Ile Met Tyr Ala Ser Tyr Phe Tyr Leu Phe Ser Gln Leu Phe Phe
260 265 270
gag gcc cat ggt gcc gct ggc aag aac aag aag aag ttg acc cgc gag 864
Glu Ala His Gly Ala Ala Gly Lys Asn Lys Lys Lys Leu Thr Arg Glu
275 280 285
ctc tct cga aaa atc tcg gag gct ctc ctg aac acc ggt gac gag gtt 912
Leu Ser Arg Lys Ile Ser Glu Ala Leu Leu Asn Thr Gly Asp Glu Val
290 295 300
tcc aag cac ctg aag gtg aat tga 936
Ser Lys His Leu Lys Val Asn
305 310
<210>48
<211>311
<212>PRT
<213>寇氏隐甲藻
<400>48
Met Ser Ala Phe Met Thr Leu Pro Gln Ala Leu Ser Asp Val Thr Ser
1 5 10 15
Ala Leu Val Thr Leu Gly Lys Asp Val Ser Ser Pro Ser Ala Phe Gln
20 25 30
Ala Val Thr Gly Phe Cys Arg Glu Gln Trp Gly Ile Pro Thr Val Phe
35 40 45
Cys Leu Gly Tyr Lau Ala Met Val Tyr Ala Ala Arg Arg Pro Leu Pro
50 55 60
Gln His Gly Tyr Met Val Ala Val Asp Arg Cys Phe Ala Ala Trp Asn
65 70 75 80
Leu Ala Leu Ser Val Phe Ser Thr Trp Gly Phe Tyr His Met Ala Val
85 90 95
Gly Leu Tyr Asn Met Thr Glu Thr Arg Gly Leu Gln Phe Thr Ile Cys
100 105 110
Gly Ser Thr Gly Glu Leu Val Gln Asn Leu Gln Thr Gly Pro Thr Ala
115 120 125
Leu Ala Leu Cys Leu Phe Cys Phe Ser Lys Ile Pro Glu Leu Met Asp
130 135 140
Thr Val Phe Leu Ile Leu Lys Ala Lys Lys Val Arg Phe Leu Gln Trp
145 150 155 160
Tyr His His Ala Thr Val Met Leu Phe Cys Trp Leu Ala Leu Ala Thr
165 170 175
Glu Tyr Thr Pro Gly Leu Trp Phe Ala Ala Thr Asn Tyr Phe Val His
180 185 190
Ser Ile Met Tyr Met Tyr Phe Phe Leu Met Thr Phe Lys Ser Ala Ala
195 200 205
Lys Val Val Lys Pro Ile Ala Pro Leu Ile Thr Val Ile Gln Ile Ala
210 215 220
Gln Met Val Trp Gly Leu Ile Val Asn Gly Ile Ala Ile Thr Thr Phe
225 230 235 240
Phe Thr Thr Gly Ala Cys Gln Ile Gln Ser Val Thr Val Tyr Ser Ala
245 250 255
Ile Ile Met Tyr Ala Ser Tyr Phe Tyr Leu Phe Ser Gln Leu Phe Phe
260 265 270
Glu Ala His Gly Ala Ala Gly Lys Asn Lys Lys Lys Leu Thr Arg Glu
275 280 285
Leu Ser Arg Lys Ile Ser Glu Ala Leu Leu Asn Thr Gly Asp Glu Val
290 295 300
Ser Lys His Leu Lys Val Asn
305 310
<210>49
<211>927
<212>DNA
<213>寇氏隐甲藻
<220>
<221>CDS
<222>(1)..(927)
<223>Δ5-延伸酶
<400>49
atg gct tcc tac caa caa gca ttc tcc gaa ttg gct aga gct ttg tcc 48
Met Ala Ser Tyr Gln Gln Ala Phe Ser Glu Leu Ala Arg Ala Leu Ser
1 5 10 15
act ttg aac cac gac ttc tcc agc gtc gag cca ttc aaa gtc gtg acg 96
Thr Leu Asn His Asp Phe Ser Ser Val Glu Pro Phe Lys Val Val Thr
20 25 30
cag ttc tgc agg gac cag tgg gcg atc ccg aca gtc ttt tgc atc ggt 144
Gln Phe Cys Arg Asp Gln Trp Ala Ile Pro Thr Val Phe Cys Ile Gly
35 40 45
tac ttg gca atg gtc tac gcc acg cga aga cct atc gcg aag cac ccc 192
Tyr Leu Ala Met Val Tyr Ala Thr Arg Arg Pro Ile Ala Lys His Pro
50 55 60
tac atg tct ctc gtg gat cgc tgc ttt gcg gcc tgg aac ttg ggc ctc 240
Tyr Met Ser Leu Val Asp Arg Cys Phe Ala Ala Trp Asn Leu Gly Leu
65 70 75 80
tcg ctc ttc agt tgc tgg ggc ttc tac cac atg gca gtg gga ctc tcc 288
Ser Leu Phe Ser Cys Trp Gly Phe Tyr His Met Ala Val Gly Leu Ser
85 90 95
cac acc act tgg aat ttc ggg ctc cag ttc acc atc tgc ggc agc acc 336
His Thr Thr Trp Asn Phe Gly Leu Gln Phe Thr Ile Cys Gly Ser Thr
100 105 110
acg gag ctt gtg aat ggc ttc cag aag ggc ccg gcg gcc ctc gcc ctc 384
Thr Glu Leu Val Asn Gly Phe Gln Lys Gly Pro Ala Ala Leu Ala Leu
115 120 125
atc ctg ttc tgc ttc tcc aag atc ccg gag ttg ggc gac acc gtc ttc 432
Ile Leu Phe Cys Phe Ser Lys Ile Pro Glu Leu Gly Asp Thr Val Phe
130 135 140
ttg atc ttg aag gga aag aag gtc cgc ttc ttg cag tgg tac cac cac 480
Leu Ile Leu Lys Gly Lys Lys Val Arg Phe Leu Gln Trp Tyr His His
145 150 155 160
acg acc gtg atg ctc ttc tgt tgg atg gcc ttg gcg act gag tac act 528
Thr Thr Val Met Leu Phe Cys Trp Met Ala Leu Ala Thr Glu Tyr Thr
165 170 175
cct gga ttg tgg ttc gcg gcc acg aac tac ttc gtg cac tcc atc atg 576
Pro Gly Leu Trp Phe Ala Ala Thr Asn Tyr Phe Val His Ser Ile Met
180 185 190
tac atg tac ttc ttc ctc atg acc ttc aag acg gcc gcc ggc atc atc 624
Tyr Met Tyr Phe Phe Leu Met Thr Phe Lys Thr Ala Ala Gly Ile Ile
195 200 205
aag ccc atc gcg cct ctc atc acc atc atc cag atc tcc cag atg gtc 672
Lys Pro Ile Ala Pro Leu Ile Thr Ile Ile Gln Ile Ser Gln Met Val
210 215 220
tgg ggc ttg gtc gtg aac gcc atc gcc gtc ggc acc ttc ttc acc aca 720
Trp Gly Leu Val Val Asn Ala Ile Ala Val Gly Thr Phe Phe Thr Thr
225 230 235 240
ggc aac tgc cag atc cag gca gtg aca gtc tac tcc gcc atc gtg atg 768
Gly Asn Cys Gln Ile Gln Ala Val Thr Val Tyr Ser Ala Ile Val Met
245 250 255
tac gcc tcc tac ttc tac ctc ttc ggc cag ctc ttc ttc gag gcc cag 816
Tyr Ala Ser Tyr Phe Tyr Leu Phe Gly Gln Leu Phe Phe Glu Ala Gln
260 265 270
ggt tcg gct gga aag gac aag aag aag ttg gcc cga gag ctg agc cga 864
Gly Ser Ala Gly Lys Asp Lys Lys Lys Leu Ala Arg Glu Leu Ser Arg
275 280 285
aag gtc tcg cgg gct ctc aca gca acg ggc gaa gag gtg tcg aag cac 912
Lys Val Ser Arg Ala Leu Thr Ala Thr Gly Glu Glu Val Ser Lys His
290 295 300
atg aag gtg aat tga 927
Met Lys Val Asn
305
<210>50
<211>308
<212>PRT
<213>寇氏隐甲藻
<400>50
Met Ala Ser Tyr Gln Gln Ala Phe Ser Glu Leu Ala Arg Ala Leu Ser
1 5 10 15
Thr Leu Asn His Asp Phe Ser Ser Val Glu Pro Phe Lys Val Val Thr
20 25 30
Gln Phe Cys Arg Asp Gln Trp Ala Ile Pro Thr Val Phe Cys Ile Gly
35 40 45
Tyr Leu Ala Met Val Tyr Ala Thr Arg Arg Pro Ile Ala Lys His Pro
50 55 60
Tyr Met Ser Leu Val Asp Arg Cys Phe Ala Ala Trp Asn Leu Gly Leu
65 70 75 80
Ser Leu Phe Ser Cys Trp Gly Phe Tyr His Met Ala Val Gly Leu Ser
85 90 95
His Thr Thr Trp Asn Phe Gly Leu Gln Phe Thr Ile Cys Gly Ser Thr
100 105 110
Thr Glu Leu Val Asn Gly Phe Gln Lys Gly Pro Ala Ala Leu Ala Leu
115 120 125
Ile Leu Phe Cys Phe Ser Lys Ile Pro Glu Leu Gly Asp Thr Val Phe
130 135 140
Leu Ile Leu Lys Gly Lys Lys Val Arg Phe Leu Gln Trp Tyr His His
145 150 155 160
Thr Thr Val Met Leu Phe Cys Trp Met Ala Leu Ala Thr Glu Tyr Thr
165 170 175
Pro Gly Leu Trp Phe Ala Ala Thr Asn Tyr Phe Val His Ser Ile Met
180 185 190
Tyr Met Tyr Phe Phe Leu Met Thr Phe Lys Thr Ala Ala Gly Ile Ile
195 200 205
Lys Pro Ile Ala Pro Leu Ile Thr Ile Ile Gln Ile Ser Gln Met Val
210 215 220
Trp Gly Leu Val Val Asn Ala Ile Ala Val Gly Thr Phe Phe Thr Thr
225 230 235 240
Gly Asn Cys Gln Ile Gln Ala Val Thr Val Tyr Ser Ala Ile Val Met
245 250 255
Tyr Ala Ser Tyr Phe Tyr Leu Phe Gly Gln Leu Phe Phe Glu Ala Gln
260 265 270
Gly Ser Ala Gly Lys Asp Lys Lys Lys Leu Ala Arg Glu Leu Ser Arg
275 280 285
Lys Val Ser Arg Ala Leu Thr Ala Thr Gly Glu Glu Val Ser Lys His
290 295 300
Met Lys Val Asn
305
<210>51
<211>795
<212>DNA
<213>虹鳟(Oncorhynchus mykiss)
<220>
<221>CDS
<222>(1)..(795)
<223>Δ5-延伸酶
<400>51
atg gct tca aca tgg caa agc gtt cag tcc atg cgc cag tgg att tta 48
Met Ala Ser Thr Trp Gln Ser Val Gln Ser Met Arg Gln Trp Ile Leu
1 5 10 15
gag aat gga gat aaa agg aca gac cca tgg cta ctg gtc tac tcc cct 96
Glu Asn Gly Asp Lys Arg Thr Asp Pro Trp Leu Leu Val Tyr Ser Pro
20 25 30
atg cca gtg gcc att ata ttc ctc ctc tat ctt ggt gtg gtc tgg gct 144
Met Pro Val Ala Ile Ile Phe Leu Leu Tyr Leu Gly Val Val Trp Ala
35 40 45
ggg ccc aag ctg atg aaa cgc agg gaa cca gtt gat ctc aag gct gta 192
Gly Pro Lys Leu Met Lys Arg Arg Glu Pro Val Asp Leu Lys Ala Val
50 55 60
ctc att gtc tac aac ttc gcc atg gtc tgc ctg tct gtc tac atg ttc 240
Leu Ile Val Tyr Asn Phe Ala Met Val Cys Leu Ser Val Tyr Met Phe
65 70 75 80
cat gag ttc ttg gtc acg tcc ttg ctg tct aac tac agt tac ctg tgt 288
His Glu Phe Leu Val Thr Ser Leu Leu Ser Asn Tyr Ser Tyr Leu Cys
85 90 95
caa cct gtg gat tac agc act agt cca ctg gcg atg agg atg gcc aaa 336
Gln Pro Val Asp Tyr Ser Thr Ser Pro Leu Ala Met Arg Met Ala Lys
100 105 110
gta tgc tgg tgg ttt ttc ttc tcc aag gtc ata gaa ttg gct gac acg 384
Val Cys Trp Trp Phe Phe Phe Ser Lys Val Ile Glu Leu Ala Asp Thr
115 120 125
gtg ttc ttc atc ctg agg aag aag aac agt cag ctg act ttc ctg cat 432
Val Phe Phe Ile Leu Arg Lys Lys Asn Ser Gln Leu Thr Phe Leu His
130 135 140
gtc tat cac cat ggc acc atg atc ttc aac tgg tgg gca ggg gtc aag 480
Val Tyr His His Gly Thr Met Ile Phe Asn Trp Trp Ala Gly Val Lys
145 150 155 160
tat ctg gct gga ggc caa tcg ttc ttc atc ggc ctg ctc aat acc ttt 528
Tyr Leu Ala Gly Gly Gln Ser Phe Phe Ile Gly Leu Leu Asn Thr Phe
165 170 175
gtg cac atc gtg atg tac tct tac tac gga ctg gct gcc ctg ggg cct 576
Val His Ile Val Met Tyr Ser Tyr Tyr Gly Leu Ala Ala Leu Gly Pro
180 185 190
cac acg cag aag tac tta tgg tgg aag cgc tat ctg acc tca ctg cag 624
His Thr Gln Lys Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Ser Leu Gln
195 200 205
ctg ctc cag ttt gtc ctg ttg acc act cac act ggc tac aac ctc ttc 672
Leu Leu Gln Phe Val Leu Leu Thr Thr His Thr Gly Tyr Asn Leu Phe
210 215 220
act gag tgt gac ttc ccg gac tcc atg aac gct gtg gtg ttt gcc tac 720
Thr Glu Cys Asp Phe Pro Asp Ser Met Asn Ala Val Val Phe Ala Tyr
225 230 235 240
tgt gtc agt ctc att gct ctc ttc agc aac ttc tac tat cag agc tac 768
Cys Val Ser Leu Ile Ala Leu Phe Ser Asn Phe Tyr Tyr Gln Ser Tyr
245 250 255
ctc aac agg aag agc aag aag aca taa 795
Leu Asn Arg Lys Ser Lys Lys Thr
260
<210>52
<211>264
<212>PRT
<213>虹鳟
<400>52
Met Ala Ser Thr Trp Gln Ser Val Gln Ser Met Arg Gln Trp Ile Leu
1 5 10 15
Glu Asn Gly Asp Lys Arg Thr Asp Pro Trp Leu Leu Val Tyr Ser Pro
20 25 30
Met Pro Val Ala Ile Ile Phe Leu Leu Tyr Leu Gly Val Val Trp Ala
35 40 45
Gly Pro Lys Leu Met Lys Arg Arg Glu Pro Val Asp Leu Lys Ala Val
50 55 60
Leu Ile Val Tyr Asn Phe Ala Met Val Cys Leu Ser Val Tyr Met Phe
65 70 75 80
His Glu Phe Leu Val Thr Ser Leu Leu Ser Asn Tyr Ser Tyr Leu Cys
85 90 95
Gln Pro Val Asp Tyr Ser Thr Ser Pro Leu Ala Met Arg Met Ala Lys
100 105 110
Val Cys Trp Trp Phe Phe Phe Ser Lys Val Ile Glu Leu Ala Asp Thr
115 120 125
Val Phe Phe Ile Leu Arg Lys Lys Asn Ser Gln Leu Thr Phe Leu His
130 135 140
Val Tyr His His Gly Thr Met Ile Phe Asn Trp Trp Ala Gly Val Lys
145 150 155 160
Tyr Leu Ala Gly Gly Gln Ser Phe Phe Ile Gly Leu Leu Asn Thr Phe
165 170 175
Val His Ile Val Met Tyr Ser Tyr Tyr Gly Leu Ala Ala Leu Gly Pro
180 185 190
His Thr Gln Lys Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Ser Leu Gln
195 200 205
Leu Leu Gln Phe Val Leu Leu Thr Thr His Thr Gly Tyr Asn Leu Phe
210 215 220
Thr Glu Cys Asp Phe Pro Asp Ser Met Asn Ala Val Val Phe Ala Tyr
225 230 235 240
Cys Val Ser Leu Ile Ala Leu Phe Ser Asn Phe Tyr Tyr Gln Ser Tyr
245 250 255
Leu Asn Arg Lys Ser Lys Lys Thr
260
<210>53
<211>885
<212>DNA
<213>虹鳟
<220>
<221>CDS
<222>(1)..(885)
<223>Δ5-延伸酶
<400>53
atg gag act ttt aat tat aaa cta aac atg tac ata gac tca tgg atg 48
Met Glu Thr Phe Asn Tyr Lys Leu Asn Met Tyr Ile Asp Ser Trp Met
1 5 10 15
ggt ccc aga gat gag cgg gta cag gga tgg ctg ctt ctg gac aac tac 96
Gly Pro Arg Asp Glu Arg Val Gln Gly Trp Leu Leu Leu Asp Asn Tyr
20 25 30
cct cca acc ttt gca cta aca gtc atg tac ctg ctg atc gta tgg atg 144
Pro Pro Thr Phe Ala Leu Thr Val Met Tyr Leu Leu Ile Val Trp Met
35 40 45
ggg ccc aag tac atg aga cac aga cag ccg gtg tct tgc cgg ggt ctc 192
Gly Pro Lys Tyr Met Arg His Arg Gln Pro Val Ser Cys Arg Gly Leu
50 55 60
ctc ttg gtc tac aat ctg ggc ctc acg atc ttg tcc ttc tat atg ttc 240
Leu Leu Val Tyr Asn Leu Gly Leu Thr Ile Leu Ser Phe Tyr Met Phe
65 70 75 80
tat gag atg gtg tct gct gtg tgg cac ggg gat tat aac ttc ttt tgc 288
Tyr Glu Met Val Ser Ala Val Trp His Gly Asp Tyr Asn Phe Phe Cys
85 90 95
caa gac aca cac agt gca gga gaa acc gat acc aag atc ata aat gtg 336
Gln Asp Thr His Ser Ala Gly Glu Thr Asp Thr Lys Ile Ile Asn Val
100 105 110
ctg tgg tgg tac tac ttc tcc aag ctc ata gag ttt atg gat acc ttc 384
Leu Trp Trp Tyr Tyr Phe Ser Lys Leu Ile Glu Phe Met Asp Thr Phe
115 120 125
ttc ttc atc ctg cgg aag aac aac cat caa atc acg ttt ctg cac atc 432
Phe Phe Ile Leu Arg Lys Asn Asn His Gln Ile Thr Phe Leu His Ile
130 135 140
tac cac cat gct agc atg ctc aac atc tgg tgg ttc gtc atg aac tgg 480
Tyr His His Ala Ser Met Leu Asn Ile Trp Trp Phe Val Met Asn Trp
145 150 155 160
gtg ccc tgt ggt cac tcc tac ttt ggt gcc tcc ctg aac agc ttc atc 528
Val Pro Cys Gly His Ser Tyr Phe Gly Ala Ser Leu Asn Ser Phe Ile
165 170 175
cat gtc ctg atg tac tct tac tat ggg ctc tct gct gtc ccg gcc ttg 576
His Val Leu Met Tyr Ser Tyr Tyr Gly Leu Ser Ala Val Pro Ala Leu
180 185 190
cgg ccc tat cta tgg tgg aag aaa tac atc aca caa gta cag ctg att 624
Arg Pro Tyr Leu Trp Trp Lys Lys Tyr Ile Thr Gln Val Gln Leu Ile
195 200 205
cag ttc ttt ttg acc atg tcc cag acg ata tgt gca gtc att tgg cca 672
Gln Phe Phe Leu Thr Met Ser Gln Thr Ile Cys Ala Val Ile Trp Pro
210 215 220
tgt gat ttc ccc aga ggg tgg ctg tat ttc cag ata ttc tat gtc atc 720
Cys Asp Phe Pro Arg Gly Trp Leu Tyr Phe Gln Ile Phe Tyr Val Ile
225 230 235 240
aca ctt att gcc ctt ttc tca aac ttc tac att cag act tac aag aaa 768
Thr Leu Ile Ala Leu Phe Ser Asn Phe Tyr Ile Gln Thr Tyr Lys Lys
245 250 255
cac ctt gtt tca caa aag aag gag tat cat cag aat ggc tct gtt gct 816
His Leu Val Ser Gln Lys Lys Glu Tyr His Gln Asn Gly Ser Val Ala
260 265 270
tca ttg aat ggc cat gtg aat ggg gtg aca ccc acg gaa acc att aca 864
Ser Leu Asn Gly His Val Asn Gly Val Thr Pro Thr Glu Thr Ile Thr
275 280 285
cac agg aaa gtg agg ggg gac 885
His Arg Lys Val Arg Gly Asp
290 295
<210>54
<211>295
<212>PRT
<213>虹鳟
<400>54
Met Glu Thr Phe Asn Tyr Lys Leu Asn Met Tyr Ile Asp Ser Trp Met
1 5 10 15
Gly Pro Arg Asp Glu Arg Val Gln Gly Trp Leu Leu Leu Asp Asn Tyr
20 25 30
Pro Pro Thr Phe Ala Leu Thr Val Met Tyr Leu Leu Ile Val Trp Met
35 40 45
Gly Pro Lys Tyr Met Arg His Arg Gln Pro Val Ser Cys Arg Gly Leu
50 55 60
Leu Leu Val Tyr Asn Leu Gly Leu Thr Ile Leu Ser Phe Tyr Met Phe
65 70 75 80
Tyr Glu Met Val Ser Ala Val Trp His Gly Asp Tyr Asn Phe Phe Cys
85 90 95
Gln Asp Thr His Ser Ala Gly Glu Thr Asp Thr Lys Ile Ile Asn Val
100 105 110
Leu Trp Trp Tyr Tyr Phe Ser Lys Leu Ile Glu Phe Met Asp Thr Phe
115 120 125
Phe Phe Ile Leu Arg Lys Asn Asn His Gln Ile Thr Phe Leu His Ile
130 135 140
Tyr His His Ala Ser Met Leu Asn Ile Trp Trp Phe Val Met Asn Trp
145 150 155 160
Val Pro Cys Gly His Ser Tyr Phe Gly Ala Ser Leu Asn Ser Phe Ile
165 170 175
His Val Leu Met Tyr Ser Tyr Tyr Gly Leu Ser Ala Val Pro Ala Leu
180 185 190
Arg Pro Tyr Leu Trp Trp Lys Lys Tyr Ile Thr Gln Val Gln Leu Ile
195 200 205
Gln Phe Phe Leu Thr Met Ser Gln Thr Ile Cys Ala Val Ile Trp Pro
210 215 220
Cys Asp Phe Pro Arg Gly Trp Leu Tyr Phe Gln Ile Phe Tyr Val Ile
225 230 235 240
Thr Leu Ile Ala Leu Phe Ser Asn Phe Tyr Ile Gln Thr Tyr Lys Lys
245 250 255
His Leu Val Ser Gln Lys Lys Glu Tyr His Gln Asn Gly Ser Val Ala
260 265 270
Ser Leu Asn Gly His Val Asn Gly Val Thr Pro Thr Glu Thr Ile Thr
275 280 285
His Arg Lys Val Arg Gly Asp
290 295
<210>55
<211>6753
<212>DNA
<213>虹鳟
<220>
<221>CDS
<222>(513)..(1397)
<223>Δ5-延伸酶
<400>55
acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60
cctcgtcctc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120
acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180
ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240
ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300
taacagatat ataaatgcaa aaactgcatt aaccacttta actaatactt tcaacatttt 360
cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata 420
cctctatact ttaacgtcaa ggagaaaaaa ccccggatcg gactactagc agctgtaata 480
cgactcacta tagggaatat taagcttaca ta atg gag act ttt aat tat aaa 533
Met Glu Thr Phe Asn Tyr Lys
1 5
cta aac atg tac ata gac tca tgg atg ggt ccc aga gat gag cgg gta 581
Leu Asn Met Tyr Ile Asp Set Trp Met Gly Pro Arg Asp Glu Arg Val
10 15 20
cag gga tgg ctg ctt ctg gac aac tac cct cca acc ttt gca cta aca 629
Gln Gly Trp Leu Leu Leu Asp Asn Tyr Pro Pro Thr Phe Ala Leu Thr
25 30 35
gtc atg tac ctg ctg atc gta tgg atg ggg ccc aag tac atg aga cac 677
Val Met Tyr Leu Leu Ile Val Trp Met Gly Pro Lys Tyr Met Arg His
40 45 50 55
aga cag ccg gtg tct tgc cgg ggt ctc ctc ttg gtc tac aat ctg ggc 725
Arg Gln Pro Val Ser Cys Arg Gly Leu Leu Leu Val Tyr Asn Leu Gly
60 65 70
ctc acg atc ttg tcc ttc tat atg ttc tat gag atg gtg tct gct gtg 773
Leu Thr Ile Leu Ser Phe Tyr Met Phe Tyr Glu Met Val Ser Ala Val
75 80 85
tgg cac ggg gat tat aac ttc ttt tgc caa gac aca cac agt gca gga 821
Trp His Gly Asp Tyr Asn Phe Phe Cys Gln Asp Thr His Ser Ala Gly
90 95 100
gaa acc gat acc aag atc ata aat gtg ctg tgg tgg tac tac ttc tcc 869
Glu Thr Asp Thr Lys Ile Ile Asn Val Leu Trp Trp Tyr Tyr Phe Ser
105 110 115
aag ctc ata gag ttt atg gat acc ttc ttc ttc atc ctg cgg aag aac 917
Lys Leu Ile Glu Phe Met Asp Thr Phe Phe Phe Ile Leu Arg Lys Asn
120 125 130 135
aac cat caa atc acg ttt ctg cac atc tac cac cat gct agc atg ctc 965
Asn His Gln Ile Thr Phe Leu His Ile Tyr His His Ala Ser Met Leu
140 145 150
aac atc tgg tgg ttc gtc atg aac tgg gtg ccc tgt ggt cac tcc tac 1013
Asn Ile Trp Trp Phe Val Met Asn Trp Val Pro Cys Gly His Ser Tyr
155 160 165
ttt ggt gcc tcc ctg aac agc ttc atc cat gtc ctg atg tac tct tac 1061
Phe Gly Ala Ser Leu Asn Ser Phe Ile His Val Leu Met Tyr Ser Tyr
170 175 180
tat ggg ctc tct gct gtc ccg gcc ttg cgg ccc tat cta tgg tgg aag 1109
Tyr Gly Leu Ser Ala Val Pro Ala Leu Arg Pro Tyr Leu Trp Trp Lys
185 190 195
aaa tac arc aca caa gta cag ctg att cag ttc ttt ttg acc atg tcc 1157
Lys Tyr Ile Thr Gln Val Gln Leu Ile Gln Phe Phe Leu Thr Met Ser
200 205 210 215
cag acg ata tgt gca gtc att tgg cca tgt gat ttc ccc aga ggg tgg 1205
Gln Thr Ile Cys Ala Val Ile Trp Pro Cys Asp Phe Pro Arg Gly Trp
220 225 230
ctg tat ttc cag ata ttc tat gtc atc aca ctt att gcc ctt ttc tca 1253
Leu Tyr Phe Gln Ile Phe Tyr Val Ile Thr Leu Ile Ala Leu Phe Ser
235 240 245
aac ttc tac att cag act tac aag aaa cac ctt gtt tca caa aag aag 1301
Asn Phe Tyr Ile Gln Thr Tyr Lys Lys His Leu Val Ser Gln Lys Lys
250 255 260
gag tat cat cag aat ggc tct gtt gct tca ttg aat ggc cat gtg aat 1349
Glu Tyr His Gln Asn Gly Set Val Ala Ser Leu Asn Gly His Val Asn
265 270 275
ggg gtg aca ccc acg gaa acc att aca cac agg aaa gtg agg ggg gac 1397
Gly Val Thr Pro Thr Glu Thr Ile Thr His Arg Lys Val Arg Gly Asp
280 285 290 295
tgaaggatcc actagtaacg gccgccagtg tgctggaatt ctgcagatat ccagcacagt 1457
ggcggccgct cgagtctaga gggcccttcg aaggtaagcc tatccctaac cctctcctcg 1517
gtctcgattc tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat 1577
cctagagggc cgcatcatgt aattagttat gtcacgctta cattcacgcc ctccccccac 1637
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 1697
ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 1757
gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 1817
acgctcgaag gctttaattt gcaagctgcg gccctgcatt aatgaatcgg ccaacgcgcg 1877
gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 1937
tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 1997
acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaagcccagg 2057
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 2117
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 2177
gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2237
tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 2297
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2357
cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2417
gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2477
ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 2537
ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 2597
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 2657
agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 2717
aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 2777
atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 2837
tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 2897
tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gcgcttacca 2957
tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 3017
gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 3077
tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 3137
ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 3197
gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 3257
aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 3317
ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 3377
tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 3437
ccgagttgct cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta 3497
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 3557
ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 3617
ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 3677
agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 3737
tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 3797
ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 3857
atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaaatt 3917
cggtcgaaaa aagaaaagga gagggccaag agggagggca ttggtgacta ttgagcacgt 3977
gagtatacgt gattaagcac acaaaggcag cttggagtat gtctgttatt aatttcacag 4037
gtagttctgg tccattggtg aaagtttgcg gcttgcagag cacagaggcc gcagaatgtg 4097
ctctagattc cgatgctgac ttgctgggta ttatatgtgt gcccaataga aagagaacaa 4157
ttgacccggt tattgcaagg aaaatttcaa gtcttgtaaa agcatataaa aatagttcag 4217
gcactccgaa atacttggtt ggcgtgtttc gtaatcaacc taaggaggat gttttggctc 4277
tggtcaatga ttacggcatt gatatcgtcc aactgcacgg agatgagtcg tggcaagaat 4337
accaagagtt cctcggtttg ccagttatta aaagactcgt atttccaaaa gactgcaaca 4397
tactactcag tgcagcttca cagaaacctc attcgtttat tcccttgttt gattcagaag 4457
caggtgggac aggtgaactt ttggattgga actcgatttc tgactgggtt ggaaggcaag 4517
agagccccga gagcttacat tttatgttag ctggtggact gacgccagaa aatgttggtg 4577
atgcgcttag attaaatggc gttattggtg ttgatgtaag cggaggtgtg gagacaaatg 4637
gtgtaaaaga ctctaacaaa atagcaaatt tcgtcaaaaa tgctaagaaa taggttatta 4697
ctgagtagta tttatttaag tattgtttgt gcacttgccc tagcttatcg atgataagct 4757
gtcaaagatg agaattaatt ccacggacta tagactatac tagatactcc gtctactgta 4817
cgatacactt ccgctcaggt ccttgtcctt taacgaggcc ttaccactct tttgttactc 4877
tattgatcca gctcagcaaa ggcagtgtga tctaagattc tatcttcgcg atgtagtaaa 4937
actagctaga ccgagaaaga gactagaaat gcaaaaggca cttctacaat ggctgccatc 4997
attattatcc gatgtgacgc tgcagcttct caatgatatt cgaatacgct ttgaggagat 5057
acagcctaat atccgacaaa ctgttttaca gatttacgat cgtacttgtt acccatcatt 5117
gaattttgaa catccgaacc tgggagtttt ccctgaaaca gatagtatat ttgaacctgt 5177
ataataatat atagtctagc gctttacgga agacaatgta tgtatttcgg ttcctggaga 5237
aactattgca tctattgcat aggtaatctt gcacgtcgca tccccggttc attttctgcg 5297
tttccatctt gcacttcaat agcatatctt tgttaacgaa gcatctgtgc ttcattttgt 5357
agaacaaaaa tgcaacgcga gagcgctaat ttttcaaaca aagaatctga gctgcatttt 5417
tacagaacag aaatgcaacg cgaaagcgct attttaccaa cgaagaatct gtgcttcatt 5477
tttgtaaaac aaaaatgcaa cgcgacgaga gcgctaattt ttcaaacaaa gaatctgagc 5537
tgcattttta cagaacagaa atgcaacgcg agagcgctat tttaccaaca aagaatctat 5597
acttcttttt tgttctacaa aaatgcatcc cgagagcgct atttttctaa caaagcatct 5657
tagattactt tttttctcct ttgtgcgctc tataatgcag tctcttgata actttttgca 5717
ctgtaggtcc gttaaggtta gaagaaggct actttggtgt ctattttctc ttccataaaa 5777
aaagcctgac tccacttccc gcgtttactg attactagcg aagctgcggg tgcatttttt 5837
caagataaag gcatccccga ttatattcta taccgatgtg gattgcgcat actttgtgaa 5897
cagaaagtga tagcgttgat gattcttcat tggtcagaaa attatgaacg gtttcttcta 5957
ttttgtctct atatactacg tataggaaat gtttacattt tcgtattgtt ttcgattcac 6017
tctatgaata gttcttacta caattttttt gtctaaagag taatactaga gataaacata 6077
aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc gaaaggtgga tgggtaggtt 6137
atatagggat atagcacaga gatatatagc aaagagatac ttttgagcaa tgtttgtgga 6197
agcggtattc gcaatgggaa gctccacccc ggttgataat cagaaaagcc ccaaaaacag 6257
gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa aattcgcgtt 6317
aaatttttgt taaatcagct cattttttaa cgaatagccc gaaatcggca aaatccctta 6377
taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttcca acaagagtcc 6437
actattaaag aacgtggact ccaacgtcaa agggcgaaaa agggtctatc agggcgatgg 6497
cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcagt 6557
aaatcggaag ggtaaacgga tgcccccatt tagagcttga cggggaaagc cggcgaacgt 6617
ggcgagaaag gaagggaaga aagcgaaagg agcgggggct agggcggtgg gaagtgtagg 6677
ggtcacgctg ggcgtaacca ccacacccgc cgcgcttaat ggggcgctac agggcgcgtg 6737
gggatgatcc actagt 6753
<210>56
<2l1>295
<212>PRT
<213>虹鳟
<400>56
Met Glu Thr Phe Asn Tyr Lys Leu Asn Met Tyr Ile Asp Ser Trp Met
1 5 10 15
Gly Pro Arg Asp Glu Arg Val Gln Gly Trp Leu Leu Leu Asp Asn Tyr
20 25 30
Pro Pro Thr Phe Ala Leu Thr Val Met Tyr Leu Leu Ile Val Trp Met
35 40 45
Gly Pro Lys Tyr Met Arg His Arg Gln Pro Val Ser Cys Arg Gly Leu
50 55 60
Leu Leu Val Tyr Asn Leu Gly Leu Thr Ile Leu Ser Phe Tyr Met Phe
65 70 75 80
Tyr Glu Met Val Ser Ala Val Trp His Gly Asp Tyr Asn Phe Phe Cys
85 90 95
Gln Asp Thr His Ser Ala Gly Glu Thr Asp Thr Lys Ile Ile Asn Val
100 105 110
Leu Trp Trp Tyr Tyr Phe Ser Lys Leu Ile Glu Phe Met Asp Thr Phe
115 120 125
Phe Phe Ile Leu Arg Lys Asn Asn His Gln Ile Thr Phe Leu His Ile
130 135 140
Tyr His His Ala Ser Met Leu Asn Ile Trp Trp Phe Val Met Asn Trp
145 150 155 160
Val Pro Cys Gly His Ser Tyr Phe Gly Ala Ser Leu Asn Ser Phe Ile
165 170 175
His Val Leu Met Tyr Ser Tyr Tyr Gly Leu Ser Ala Val Pro Ala Leu
180 185 190
Arg Pro Tyr Leu Trp Trp Lys Lys Tyr Ile Thr Gln Val Gln Leu Ile
195 200 205
Gln Phe Phe Leu Thr Met Ser Gln Thr Ile Cys Ala Val Ile Trp Pro
210 215 220
Cys Asp Phe Pro Arg Gly Trp Leu Tyr Phe Gln Ile Phe Tyr Val Ile
225 230 235 240
Thr Leu Ile Ala Leu Phe Ser Asn Phe Tyr Ile Gln Thr Tyr Lys Lys
245 250 255
His Leu Val Ser Gln Lys Lys Glu Tyr His Gln Asn Gly Ser Val Ala
260 265 270
Ser Leu Asn Gly His Val Asn Gly Val Thr Pro Thr Glu Thr Ile Thr
275 280 285
His Arg Lys Val Arg Gly Asp
290 295
<210>57
<211>6645
<212>DNA
<213>虹鳟
<220>
<221>CDS
<222>(513)..(1304)
<223>Δ5-延伸酶
<400>57
acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60
cctcgtcctc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120
acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180
ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240
ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300
taacagatat ataaatgcaa aaactgcatt aaccacttta actaatactt tcaacatttt 360
cggtttgtat tacttcttat tcaaatgtaa taaaagtatc aacaaaaaat tgttaatata 420
cctctatact ttaacgtcaa ggagaaaaaa ccccggatcg gactactagc agctgtaata 480
cgactcacta tagggaatat taagcttaca ta atg gct tca aca tgg caa agc 533
Met Ala Ser Thr Trp Gln Ser
1 5
gtt cag tcc atg cgc cag tgg att tta gag aat gga gat aaa agg aca 581
Val Gln Ser Met Arg Gln Trp Ile Leu Glu Asn Gly Asp Lys Arg Thr
10 15 20
gac cca tgg cta ctg gtc tac tcc cct atg cca gtg gcc att ata ttc 629
Asp Pro Trp Leu Leu Val Tyr Ser Pro Met Pro Val Ala Ile Ile Phe
25 30 35
ctc ctc tat ctt ggt gtg gtc tgg gct ggg ccc aag ctg atg aaa cgc 677
Leu Leu Tyr Leu Gly Val Val Trp Ala Gly Pro Lys Leu Met Lys Arg
40 45 50 55
agg gaa cca gtt gat ctc aag gct gta ctc att gtc tac aac ttc gcc 725
Arg Glu Pro Val Asp Leu Lys Ala Val Leu Ile Val Tyr Asn Phe Ala
60 65 70
atg gtc tgc ctg tct gtc tac atg ttc cat gag ttc ttg gtc acg tcc 773
Met Val Cys Leu Ser Val Tyr Met Phe His Glu Phe Leu Val Thr Ser
75 80 85
ttg ctg tct aac tac agt tac ctg tgt caa cct gtg gat tac agc act 821
Leu Leu Ser Asn Tyr Ser Tyr Leu Cys Glu Pro Val Asp Tyr Ser Thr
90 95 100
agt cca ctg gcg atg agg atg gcc aaa gta tgc tgg tgg ttt ttc ttc 869
Ser Pro Leu Ala Met Arg Met Ala Lys Val Cys Trp Trp Phe Phe Phe
105 110 115
tcc aag gtc ata gaa ttg gct gac acg gtg ttc ttc atc ctg agg aag 917
Ser Lys Val Ile Glu Leu Ala Asp Thr Val Phe Phe Ile Leu Arg Lys
120 125 130 135
aag aac agt cag ctg act ttc ctg cat gtc tat cac cat ggc acc atg 965
Lys Asn Ser Gln Leu Thr Phe Leu His Val Tyr His His Gly Thr Met
140 145 150
atc ttc aac tgg tgg gca ggg gtc aag tat ctg gct gga ggc caa tcg 1013
Ile Phe Asn Trp Trp Ala Gly Val Lys Tyr Leu Ala Gly Gly Gln Ser
155 160 165
ttc ttc atc ggc ctg ctc aat acc ttt gtg cac atc gtg atg tac tct 1061
Phe Phe Ile Gly Leu Leu Asn Thr Phe Val His Ile Val Met Tyr Ser
170 175 180
tac tac gga ctg gct gcc ctg ggg cct cac acg cag aag tac tta tgg 1109
Tyr Tyr Gly Leu Ala Ala Leu Gly Pro His Thr Gln Lys Tyr Leu Trp
185 190 195
tgg aag cgc tat ctg acc tca ctg cag ctg ctc cag ttt gtc ctg ttg 1157
Trp Lys Arg Tyr Leu Thr Ser Leu Gln Leu Leu Gln Phe Val Leu Leu
200 205 210 215
acc act cac act ggc tac aac ctc ttc act gag tgt gac ttc ccg gac 1205
Thr Thr His Thr Gly Tyr Asn Leu Phe Thr Glu Cys Asp Phe Pro Asp
220 225 230
tcc atg aac gct gtg gtg ttt gcc tac tgt gtc agt ctc att gct ctc 1253
Ser Met Asn Ala Val Val Phe Ala Tyr Cys Val Ser Leu Ile Ala Leu
235 240 245
ttc agc aac ttc tac tat cag agc tac ctc aac agg aag agc aag aag 1301
Phe Ser Asn Phe Tyr Tyr Gln Ser Tyr Leu Asn Arg Lys Ser Lys Lys
250 255 260
aca taaggatcca ctagtaacgg ccgccagtgt gctggaattc tgcagatatc 1354
Thr
catcacactg gcggccgctc gagcatgcat ctagagggcc gcatcatgta attagttatg 1414
tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga 1474
caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 1534
tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 1594
ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cggccctgca 1654
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 1714
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 1774
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 1834
aaaaggccag caaaagccca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 1894
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 1954
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2014
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2074
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2134
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatc9tct 2194
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2254
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2314
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2374
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2434
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2494
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 2554
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 2614
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 2674
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 2734
tacgatacgg gagcgcttac catctggccc cagtgctgca atgataccgc gagacccacg 2794
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 2854
tggtcctgca actttatccg cctccattca gtctattaat tgttgccggg aagctagagt 2914
aagtagttcg ccagttaata gtttgcgcaa cgttgttggc attgctacag gcatcgtggt 2974
gtcactctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3034
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3094
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3154
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3214
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatag 3274
tgtatcacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3334
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3394
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 3454
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 3514
ttttcaatgg gtaataactg atataattaa attgaagctc taatttgtga gtttagtata 3574
catgcattta cttataatac agttttttag ttttgctggc cgcatcttct caaatatgct 3634
tcccagcctg cttttctgta acgttcaccc tctaccttag catcccttcc ctttgcaaat 3694
agtcctcttc caacaataat aatgtcagat cctgtagaga ccacatcatc cacggttcta 3754
tactgttgac ccaatgcgtc tcccttgtca tctaaaccca caccgggtgt cataatcaac 3814
caatcgtaac cttcatctct tccacccatg tctctttgag caataaagcc gataacaaaa 3874
tctttgtcgc tcttcgcaat gtcaacagta cccttagtat attctccagt agatagggag 3934
cccttgcatg acaattctgc taacatcaaa aggcctctag gttcctttgt tacttcttct 3994
gccgcctgct tcaaaccgct aacaatacct gggcccacca caccgtgtgc attcgtaatg 4054
tctgcccatt ctgctattct gtatacaccc gcagagtact gcaatttgac tgtattacca 4114
atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt acttggcgga taatgccttt 4174
agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga tatccacatg tgtttttagt 4234
aaacaaattt tgggacctaa tgcttcaact aactccagta attccttggt ggtacgaaca 4294
tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga tattaaatag cttggcagca 4354
acaggactag gatgagtagc agcacgttcc ttatatgtag ctttcgacat gatttatctt 4414
cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga atactgggca atttcatgtt 4474
tcttcaacac tacatatgcg tatatatacc aatctaagtc tgtgctcctt ccttcgttct 4534
tccttctgtt cggagattac cgaatcaaaa aaatttcaaa gaaaccgaaa tcaaaaaaaa 4594
gaataaaaaa aaaatgatga attgaattga aaagctagct tatcgatgat aagctgtcaa 4654
agatgagaat taattccacg gactatagac tatactagat actccgtcta ctgtacgata 4714
cacttccgct caggtccttg tcctttaacg aggccttacc actcttttgt tactctattg 4774
atccagctca gcaaaggcag tgtgatctaa gattctatct tcgcgatgta gtaaaactag 4834
ctagaccgag aaagagacta gaaatgcaaa aggcacttct acaatggctg ccatcattat 4894
tatccgatgt gacgctgcag cttctcaatg atattcgaat acgctttgag gagatacagc 4954
ctaatatccg acaaactgtt ttacagattt acgatcgtac ttgttaccca tcattgaatt 5014
ttgaacatcc gaacctggga gttttccctg aaacagatag tatatttgaa cctgtataat 5074
aatatatagt ctagcgcttt acggaagaca atgtatgtat ttcggttcct ggagaaacta 5134
ttgcatctat tgcataggta atcttgcacg tcgcatcccc ggttcatttt ctgcgtttcc 5194
atcttgcact tcaatagcat atctttgtta acgaagcatc tgtgcttcat tttgtagaac 5254
aaaaatgcaa cgcgagagcg ctaatttttc aaacaaagaa tctgagctgc atttttacag 5314
aacagaaatg caacgcgaaa gcgctatttt accaacgaag aatctgtgct tcatttttgt 5374
aaaacaaaaa tgcaacgcga cgagagcgct aatttttcaa acaaagaatc tgagctgcat 5434
ttttacagaa cagaaatgca acgcgagagc gctattttac caacaaagaa tctatacttc 5494
ttttttgttc tacaaaaatg catcccgaga gcgctatttt tctaacaaag catcttagat 5554
tacttttttt ctcctttgtg cgctctataa tgcagtctct tgataacttt ttgcactgta 5614
ggtccgttaa ggttagaaga aggctacttt ggtgtctatt ttctcttcca taaaaaaagc 5674
ctgactccac ttcccgcgtt tactgattac tagcgaagct gcgggtgcat tttttcaaga 5734
taaaggcatc cccgattata ttctataccg atgtggattg cgcatacttt gtgaacagaa 5794
agtgatagcg ttgatgattc ttcattggtc agaaaattat gaacggtttc ttctattttg 5854
tctctatata ctacgtatag gaaatgttta cattttcgta ttgttttcga ttcactctat 5914
gaatagttct tactacaatt tttttgtcta aagagtaata ctagagataa acataaaaaa 5974
tgtagaggtc gagtttagat gcaagttcaa ggagcgaaag gtggatgggt aggttatata 6034
gggatatagc acagagatat atagcaaaga gatacttttg agcaatgttt gtggaagcgg 6094
tattcgcaat gggaagctcc accccggttg ataatcagaa aagccccaaa aacaggaaga 6154
ttgtataagc aaatatttaa attgtaaacg ttaatatttt gttaaaattc gcgttaaatt 6214
tttgttaaat cagctcattt tttaacgaat agcccgaaat cggcaaaatc ccttataaat 6274
caaaagaata gaccgagata gggttgagtg ttgttccagt ttccaacaag agtccactat 6334
taaagaacgt ggactccaac gtcaaagggc gaaaaagggt ctatcagggc gatggcccac 6394
tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcagtaaatc 6454
ggaagggtaa acggatgccc ccatttagag cttgacgggg aaagccggcg aacgtggcga 6514
gaaaggaagg gaagaaagcg aaaggagcgg gggctagggc ggtgggaagt gtaggggtca 6574
cgctgggcgt aaccaccaca cccgccgcgc ttaatggggc gctacagggc gcgtggggat 6634
gatccactag t 6645
<210>58
<211>264
<212>PRT
<213>虹鳟
<400>58
Met Ala Ser Thr Trp Gln Ser Val Gln Ser Met Arg Gln Trp Ile Leu
1 5 10 15
Glu Asn Gly Asp Lys Arg Thr Asp Pro Trp Leu Leu Val Tyr Ser Pro
20 25 30
Met Pro Val Ala Ile Ile Phe Leu Leu Tyr Leu Gly Val Val Trp Ala
35 40 45
Gly Pro Lys Leu Met Lys Arg Arg Glu Pro Val Asp Leu Lys Ala Val
50 55 60
Leu Ile Val Tyr Asn Phe Ala Met Val Cys Leu Ser Val Tyr Met Phe
65 70 75 80
His Glu Phe Leu Val Thr Ser Leu Leu Ser Asn Tyr Ser Tyr Leu Cys
85 90 95
Gln Pro Val Asp Tyr Ser Thr Ser Pro Leu Ala Met Arg Met Ala Lys
100 105 110
Val Cys Trp Trp Phe Phe Phe Ser Lys Val Ile Glu Leu Ala Asp Thr
115 120 125
Val Phe Phe Ile Leu Arg Lys Lys Asn Ser Gln Leu Thr Phe Leu His
130 135 140
Val Tyr His His Gly Thr Met Ile Phe Asn Trp Trp Ala Gly Val Lys
145 150 155 160
Tyr Leu Ala Gly Gly Gln Ser Phe Phe Ile Gly Leu Leu Asn Thr Phe
165 170 175
Val His Ile Val Met Tyr Ser Tyr Tyr Gly Leu Ala Ala Leu Gly Pro
180 185 190
His Thr Gln Lys Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Ser Leu Gln
195 200 205
Leu Leu Gln Phe Val Leu Leu Thr Thr His Thr Gly Tyr Asn Leu Phe
210 215 220
Thr Glu Cys Asp Phe Pro Asp Ser Met Asn Ala Val Val Phe Ala Tyr
225 230 235 240
Cys Val Ser Leu Ile Ala Leu Phe Ser Asn Phe Tyr Tyr Gln Ser Tyr
245 250 255
Leu Asn Arg Lys Ser Lys Lys Thr
260
<210>59
<211>1077
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1077)
<223>Δ5-延伸酶
<400>59
atg tgc tca tca ccg ccg tca caa tcc aaa aca aca tcc ctc cta gca 48
Met Cys Ser Ser Pro Pro Ser Gln Ser Lys Thr Thr Ser Leu Leu Ala
1 5 10 15
cgg tac acc acc gcc gcc ctc ctc ctc ctc acc ctc aca aca tgg tgc 96
Arg Tyr Thr Thr Ala Ala Leu Leu Leu Leu Thr Leu Thr Thr Trp Cys
20 25 30
cac ttc gcc ttc cca gcc gcc acc gcc aca ccc ggc ctc acc gcc gaa 144
His Phe Ala Phe Pro Ala Ala Thr Ala Thr Pro Gly Leu Thr Ala Glu
35 40 45
atg cac tcc tac aaa gtc cca ctc ggt ctc acc gta ttc tac ctg ctg 192
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
50 55 60
agt cta ccg tca cta aag tac gtt acg gac aac tac ctt gcc aaa aag 240
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
65 70 75 80
tat gat atg aag tca ctc cta acg gaa tca atg gtg ttg tac aat gtg 288
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
85 90 95
gcg caa gtg ctg ctc aat ggg tgg acg gtg tat gcg att gtg gat gcg 336
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
100 105 110
gtg atg aat aga gac cat ccg ttt att gga agt aga agt ttg gtt ggg 384
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
115 120 125
gcg gcg ttg cat agt ggg agc tcg tat gcg gtg tgg gtt cat tat tgt 432
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
130 135 140
gat aag tat ttg gag ttc ttt gat acg tat ttt atg gtg ttg agg ggg 480
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
145 150 155 160
aaa atg gac cag gtc tcc ttc ctc cac atc tac cac cac acg acc ata 528
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
165 170 175
gcg tgg gca tgg tgg atc gcc ctc cgc ttc tcc ccc ggt gga gac att 576
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
180 185 190
tac ttc ggg gca ctc ctc aac tcc atc atc cac gtc ctc atg tat tcc 624
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
195 200 205
tac tac gcc ctt gcc cta ctc aag gtc agt tgt cca tgg aaa cga tac 672
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
210 215 220
ctg act caa gct caa tta ttg caa ttc aca agt gtg gtg gtt tat acg 720
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
225 230 235 240
ggg tgt acg ggt tat act cat tac tat cat acg aag cat gga gcg gat 768
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
245 250 255
gag aca cag cct agt tta gga acg tat tat ttc tgt tgt gga gtg cag 816
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
260 265 270
gtg ttt gag atg gtt agt ttg ttt gta ctc ttt tcc atc ttt tat aaa 864
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
275 280 285
cga tcc tat tcg aag aag aac aag tca gga gga aag gat agc aag aag 912
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
290 295 300
aat gat gat ggg aat aat gag gat caa tgt cac aag gct atg aag gat 960
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
305 310 315 320
ata tcg gag ggt gcg aag gag gtt gtg ggg cat gca gcg aag gat gct 1008
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
325 330 335
gga aag ttg gtg gct acg gcg agt aag gct gta aag agg aag gga act 1056
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
340 345 350
cgt gtt act ggt gcc atg tag 1077
Arg Val Thr Gly Ala Met
355
<210>60
<211>358
<212>PRT
<213>假矮海链藻
<400>60
Met Cys Ser Ser Pro Pro Ser Gln Ser Lys Thr Thr Ser Leu Leu Ala
1 5 10 15
Arg Tyr Thr Thr Ala Ala Leu Leu Leu Leu Thr Leu Thr Thr Trp Cys
20 25 30
His Phe Ala Phe Pro Ala Ala Thr Ala Thr Pro Gly Leu Thr Ala Glu
35 40 45
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
50 55 60
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
65 70 75 80
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
85 90 95
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
100 105 110
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
115 120 125
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
130 135 140
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
145 150 155 160
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
165 170 175
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
180 185 190
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
195 200 205
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
210 215 220
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
225 230 235 240
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
245 250 255
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
260 265 270
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
275 280 285
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
290 295 300
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
305 310 315 320
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
325 330 335
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
340 345 350
Arg Val Thr Gly Ala Met
355
<210>61
<211>933
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(933)
<223>Δ5-延伸酶
<400>61
atg cac tcc tac aaa gtc cca ctc ggt ctc acc gta ttc tac ctg ctg 48
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
1 5 10 15
agt cta ccg tca cta aag tac gtt acg gac aac tac ctt gcc aaa aag 96
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
20 25 30
tat gat atg aag tca ctc cta acg gaa tca atg gtg ttg tac aat gtg 144
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
35 40 45
gcg caa gtg ctg ctc aat ggg tgg acg gtg tat gcg att gtg gat gcg 192
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
50 55 60
gtg atg aat aga gac cat ccg ttt att gga agt aga agt ttg gtt ggg 240
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
65 70 75 80
gcg gcg ttg cat agt ggg agc tcg tat gcg gtg tgg gtt cat tat tgt 288
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
85 90 95
gat aag tat ttg gag ttc ttt gat acg tat ttt atg gtg ttg agg ggg 336
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
100 105 110
aaa atg gac cag gtc tcc ttc ctc cac atc tac cac cac acg acc ata 384
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
115 120 125
gcg tgg gca tgg tgg atc gcc ctc cgc ttc tcc ccc ggt gga gac att 432
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
130 135 140
tac ttc ggg gca ctc ctc aac tcc atc atc cac gtc ctc atg tat tcc 480
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
145 150 155 160
tac tac gcc ctt gcc cta ctc aag gtc agt tgt cca tgg aaa cga tac 528
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
165 170 175
ctg act caa gct caa tta ttg caa ttc aca agt gtg gtg gtt tat acg 576
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
180 185 190
ggg tgt acg ggt tat act cat tac tat cat acg aag cat gga gcg gat 624
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
195 200 205
gag aca cag cct agt tta gga acg tat tat ttc tgt tgt gga gtg cag 672
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
210 215 220
gtg ttt gag atg gtt agt ttg ttt gta ctc ttt tcc atc ttt tat aaa 720
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
225 230 235 240
cga tcc tat tcg aag aag aac aag tca gga gga aag gat agc aag aag 768
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
245 250 255
aat gat gat ggg aat aat gag gat caa tgt cac aag gct atg aag gat 816
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
260 265 270
ata tcg gag ggt gcg aag gag gtt gtg ggg cat gca gcg aag gat gct 864
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
275 280 285
gga aag ttg gtg gct acg gcg agt aag gct gta aag agg aag gga act 912
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
290 295 300
cgt gtt act ggt gcc atg tag 933
Arg Val Thr Gly Ala Met
305 310
<210>62
<211>310
<212>PRT
<213>假矮海链藻
<400>62
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
1 5 10 15
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
20 25 30
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
35 40 45
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
50 55 60
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
65 70 75 80
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
85 90 95
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
100 105 110
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
115 120 125
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
130 135 140
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
145 150 155 160
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
165 170 175
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
180 185 190
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
195 200 205
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
210 215 220
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
225 230 235 240
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
245 250 255
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
260 265 270
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
275 280 285
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
290 295 300
Arg Val Thr Gly Ala Met
305 310
<210>63
<211>933
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(933)
<223>Δ5-延伸酶
<400>63
atg cac tcc tac aaa gtc cca ctc ggt ctc acc gta ttc tac ctg ctg 48
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
1 5 10 15
agt cta ccg tca cta aag tac gtt acg gac aac tac ctt gcc aaa aag 96
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
20 25 30
tat gat atg aag tca ctc cta acg gaa tca atg gtg ttg tac aat gtg 144
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
35 40 45
gcg caa gtg ctg ctc aat ggg tgg acg gtg tat gcg att gtg gat gcg 192
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
50 55 60
gtg atg aat aga gac cat ccg ttt att gga agt aga agt ttg gtt ggg 240
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
65 70 75 80
gcg gcg ttg cat agt ggg agc tcg tat gcg gtg tgg gtt cat tat tgt 288
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
85 90 95
gat aag tat ttg gag ttc ttt gat acg tat ttt atg gtg ttg agg ggg 336
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
100 105 110
aaa atg gac cag gtc tcc ttc ctc cac atc tac cac cac acg acc ata 384
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
115 120 125
gcg tgg gca tgg tgg atc gcc ctc cgc ttc tcc ccc ggt gga gac att 432
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
130 135 140
tac ttc ggg gca ctc ctc aac tcc atc atc cac gtc ctc atg tat tcc 480
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
145 150 155 160
tac tac gcc ctt gcc cta ctc aag gtc agt tgt cca tgg aaa cga tac 528
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
165 170 175
ctg act caa gct caa tta ttg caa ttc aca agt gtg gtg gtt tat acg 576
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
180 185 190
ggg tgt acg ggt tat act cat tac tat cat acg aag cat gga gcg gat 624
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
195 200 205
gag aca cag cct agt tta gga acg tat tat ttc tgt tgt gga gtg cag 672
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
210 215 220
gtg ttt gag atg gtt agt ttg ttt gta ctc ttt tcc atc ttt tat aaa 720
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
225 230 235 240
cga tcc tat tcg aag aag aac aag tca gga gga aag gat agc aag aag 768
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
245 250 255
aat gat gat ggg aat aat gag gat caa tgt cac aag gct atg aag gat 816
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
260 265 270
ata tcg gag ggt gcg aag gag gtt gtg ggg cat gca gcg aag gat gct 864
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
275 280 285
gga aag ttg gtg gct acg gcg agt aag gct gta aag agg aag gga act 912
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
290 295 300
cgt gtt act ggt gcc atg tag 933
Arg Val Thr Gly Ala Met
305 310
<210>64
<211>310
<212>PRT
<213>假矮海链藻
<400>64
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
1 5 10 15
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
20 25 30
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
35 40 45
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
50 55 60
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
65 70 75 80
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
85 90 95
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
100 105 110
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
115 120 125
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
130 135 140
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
145 150 155 160
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
165 170 175
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
180 185 190
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
195 200 205
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
210 215 220
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
225 230 235 240
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
245 250 255
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
260 265 270
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
275 280 285
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
290 295 300
Arg Val Thr Gly Ala Met
305 310
<210>65
<2ll>825
<212>DNA
<213>鲜黄破囊壶菌(Thraustochytrium aureum)
<220>
<221>CDS
<222>(1)..(825)
<223>Δ5-延伸酶
<400>65
atg acg agc aac atg agc gcg tgg ggc gtc gcc gtc gac cag acg cag 48
Met Thr Ser Asn Met Ser Ala Trp Gly Val Ala Val Asp Gln Thr Gln
1 5 10 15
cag gtc gtc gac cag atc atg ggc ggc gcc gag ccg tac aag ctg aca 96
Gln Val Val Asp Gln Ile Met Gly Gly Ala Glu Pro Tyr Lys Leu Thr
20 25 30
gaa ggg cgc atg acg aac gtc gag acg atg atg gcg atc gag tgc ggc 144
Glu Gly Arg Met Thr Asn Val Glu Thr Met Leu Ala Ile Glu Cys Gly
35 40 45
tac gcc gcc atg ctg ctg ttc ctg acc ccg atc atg aag cag gcc gag 192
Tyr Ala Ala Met Leu Leu Phe Leu Thr Pro Ile Met Lys Gln Ala Glu
50 55 60
aag ccc ttc gag ctc aag tcc ttc aag ctc gcc cac aac ctg ttc ctg 240
Lys Pro Phe Glu Leu Lys Ser Phe Lys Leu Ala His Asn Leu Phe Leu
65 70 75 80
ttc gtc ctg tcc gcc tac atg tgc ctc gag acc gtc cgc cag gcc tac 288
Phe Val Leu Ser Ala Tyr Met Cys Leu Glu Thr Val Arg Gln Ala Tyr
85 90 95
ctt gcg ggc tac tcg gtg ttc ggc aac gac atg gag aag ggc agc gag 336
Leu Ala Gly Tyr Ser Val Phe Gly Asn Asp Met Glu Lys Gly Ser Glu
100 105 110
ccg cac gcg cac ggc atg gcc caa atc gtg tgg atc ttt tac gtg tcc 384
Pro His Ala His Gly Met Ala Gln Ile Val Trp Ile Phe Tyr Val Ser
115 120 125
aag gcg tac gag ttc gtg gac acg ctg atc atg atc ctg tgc aaa aag 432
Lys Ala Tyr Glu Phe Val Asp Thr Leu Ile Met Ile Leu Cys Lys Lys
130 135 140
ttc aac cag gtc tcc gtc ctg cac gtg tac cac cac gcc acc atc ttt 480
Phe Asn Gln Val Ser Val Leu His Val Tyr His His Ala Thr Ile Phe
145 150 155 160
gct atc tgg ttt atg atc gcc aag tac gcc ccg ggc ggc gac gca tac 528
Ala Ile Trp Phe Met Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr
165 170 175
ttt agc gtc atc ctg aac tcg ttc gtg cac acc gtc atg tac gcg tac 576
Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr
180 185 190
tac ttc ttc tcg tcg cag ggc ttc ggg ttc gtc aag ccg atc aag ccg 624
Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro
195 200 205
tac atc acc tcg ctg cag atg acg cag ttc atg gcg atg ctc gtg cag 672
Tyr Ile Thr Ser Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln
210 215 220
tcg ctg tac gac tac ctt tac ccg tgc gac tac ccg cag ggg ctc gtc 720
Ser Leu Tyr Asp Tyr Leu Tyr Pro Cys Asp Tyr Pro Gln Gly Leu Val
225 230 235 240
aag ctc ctc ggc gtg tac atg ctc acc ctg ctt gcg ctc ttc ggc aac 768
Lys Leu Leu Gly Val Tyr Met Leu Thr Leu Leu Ala Leu Phe Gly Asn
245 250 255
ttt ttc gtg cag agc tac ctc aag aag tcg aac aag ccc aag gcc aag 816
Phe Phe Val Gln Ser Tyr Leu Lys Lys Ser Asn Lys Pro Lys Ala Lys
260 265 270
tcg gcc taa 825
Ser Ala
<210>66
<211>274
<212>PRT
<213>鲜黄破囊壶菌
<400>66
Met Thr Ser Asn Met Ser Ala Trp Gly Val Ala Val Asp Gln Thr Gln
1 5 10 15
Gln Val Val Asp Gln Ile Met Gly Gly Ala Glu Pro Tyr Lys Leu Thr
20 25 30
Glu Gly Arg Met Thr Asn Val Glu Thr Met Leu Ala Ile Glu Cys Gly
35 40 45
Tyr Ala Ala Met Leu Leu Phe Leu Thr Pro Ile Met Lys Gln Ala Glu
50 55 60
Lys Pro Phe Glu Leu Lys Ser Phe Lys Leu Ala His Asn Leu Phe Leu
65 70 75 80
Phe Val Leu Ser Ala Tyr Met Cys Leu Glu Thr Val Arg Gln Ala Tyr
85 90 95
Leu Ala Gly Tyr Ser Val Phe Gly Asn Asp Met Glu Lys Gly Ser Glu
100 105 110
Pro His Ala His Gly Met Ala Gln Ile Val Trp Ile Phe Tyr Val Ser
115 120 125
Lys Ala Tyr Glu Phe Val Asp Thr Leu Ile Met Ile Leu Cys Lys Lys
130 135 140
Phe Asn Gln Val Ser Val Leu His Val Tyr His His Ala Thr Ile Phe
145 150 155 160
Ala Ile Trp Phe Met Ile Ala Lys Tyr Ala Pro Gly Gly Asp Ala Tyr
165 170 175
Phe Ser Val Ile Leu Asn Ser Phe Val His Thr Val Met Tyr Ala Tyr
180 185 190
Tyr Phe Phe Ser Ser Gln Gly Phe Gly Phe Val Lys Pro Ile Lys Pro
195 200 205
Tyr Ile Thr Ser Leu Gln Met Thr Gln Phe Met Ala Met Leu Val Gln
210 215 220
Ser Leu Tyr Asp Tyr Leu Tyr Pro Cys Asp Tyr Pro Gln Gly Leu Val
225 230 235 240
Lys Leu Leu Gly Val Tyr Met Leu Thr Leu Leu Ala Leu Phe Gly Asn
245 250 255
Phe Phe Val Gln Ser Tyr Leu Lys Lys Ser Asn Lys Pro Lys Ala Lys
260 265 270
Ser Ala
<210>67
<211>903
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(903)
<223>Δ5-延伸酶
<400>67
atg agc gcc tcc ggt gcg ctg ctg ccc gcg atc gcg ttc gcc gcg tac 48
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
gcg tac gcg acg tac gcc tac gcc ttt gag tgg tcg cac gcg aat ggc 96
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
atc gac aac gtc gac gcg cgc gag tgg atc ggt gcg ctg tcg ttg agg 144
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
ctc ccg gcg atc gcg acg acg atg tac ctg ttg ttc tgc ctg gtc gga 192
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
ccg agg ttg atg gcg aag cgc gag gcg ttc gac ccg aag ggg ttc atg 240
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
ctg gcg tac aat gcg tat cag acg gcg ttc aac gtc gtc gtg ctc ggg 288
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
atg ttc gcg cga gag atc tcg ggg ctg ggg cag ccc gtg tgg ggg tca 336
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
acc atg ccg tgg agc gat aga aaa tcg ttt aag atc ctc ctc ggg gtg 384
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
tgg ttg cac tac aac aac caa tat ttg gag cta ttg gac act gtg ttc 432
Trp Leu His Tyr Asn Asn Gln Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
atg gtt gcg cgc aag aag acg aag cag ttg agc ttc ttg cac gtt tat 480
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
cat cac gcc ctg ttg atc tgg gcg tgg tgg ttg gtg tgt cac ttg atg 528
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
gcc acg aac gat tgt atc gat gcc tac ttc ggc gcg gcg tgc aac tcg 576
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
ttc att cac atc gtg atg tac tcg tat tat ctc atg tcg gcg ctc ggc 624
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
att cga tgc ccg tgg aag cga tac atc acc cag gct caa atg ctc caa 672
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
ttc gtc att gtc ttc gcg cac gcc gtg ttc gtg ctg cgt cag aag cac 720
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
tgc ccg gtc acc ctt cct tgg gcg caa atg ttc gtc atg acg aac atg 768
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
ctc gtg ctc ttc ggg aac ttc tac ctc aag gcg tac tcg aac aag tcg 816
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
cgc ggc gac ggc gcg agt tcc gtg aaa cca gcc gag acc acg cgc gcg 864
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
ccc agc gtg cga cgc acg cga tct cga aaa att gac taa 903
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>68
<211>300
<212>PRT
<213>Ostreococcus tauri
<400>68
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
Trp Leu His Tyr Asn Asn Gln Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>69
<211>879
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(879)
<223>Δ6-延伸酶
<400>69
atg agt ggc tta cgt gca ccc aac ttt tta cac aga ttc tgg aca aag 48
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
tgg gac tac gcg att tcc aaa gtc gtc ttc acg tgt gcc gac agt ttt 96
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
cag tgg gac atc ggg cca gtg agt tcg agt acg gcg cat tta ccc gcc 144
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
att gaa tcc cct acc cca ctg gtg act agc ctc ttg ttc tac tta gtc 192
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
aca gtt ttc ttg tgg tat ggt cgt tta acc agg agt tca gac aag aaa 240
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
att aga gag cct acg tgg tta aga aga ttc ata ata tgt cat aat gcg 288
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
ttc ttg ata gtc ctc agt ctt tac atg tgc ctt ggt tgt gtg gcc caa 336
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
gcg tat cag aat gga tat act tta tgg ggt aat gaa ttc aag gcc acg 384
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
gaa act cag ctt gct ctc tac att tac att ttt tac gta agt aaa ata 432
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
tac gag ttt gta gat act tac att atg ctt ctc aag aat aac ttg cgg 480
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Asn Leu Arg
145 150 155 160
caa gta agt ttc cta cac att tat cac cac agc acg att tcc ttt att 528
Gln Val Ser Phe Leu His Ile Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
tgg tgg atc att gct cgg agg gct ccg ggt ggt gat gct tac ttc agc 576
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
gcg gcc ttg aac tca tgg gta cac gtg tgc atg tac acc tat tat cta 624
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
tta tca acc ctt att gga aaa gaa gat cct aag cgt tcc aac tac ctt 672
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
tgg tgg ggt cgc cac cta acg caa atg cag atg ctt cag ttt ttc ttc 720
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
aac gta ctt caa gcg ttg tac tgc gct tcg ttc tct acg tat ccc aag 768
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
ttt ttg tcc aaa att ctg ctc gtc tat atg atg agc ctt ctc ggc ttg 816
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
ttt ggg cat ttc tac tat tcc aag cac ata gca gca gct aag ctc cag 864
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
aaa aaa cag cag tga 879
Lys Lys Gln Gln
290
<210>70
<211>292
<212>PRT
<213>Ostreococcus tauri
<400>70
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Asn Leu Arg
145 150 155 160
Gln Val Ser Phe Leu His Ile Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
Lys Lys Gln Gln
290
<210>7l
<211>1362
<212>DNA
<213>有粉报春(Primula farinosa)
<220>
<221>CDS
<222>(1)..(1362)
<223>Δ6-去饱和酶
<400>71
atg gct aac aaa tct cca cca aac ccc aaa aca ggt tac ata acc agc 48
Met Ala Asn Lys Ser Pro Pro Asn Pro Lys Thr Gly Tyr Ile Thr Ser
1 5 10 15
tca gac ctg aaa tcc cac aac aag gca ggt gac cta tgg ata tca atc 96
Ser Asp Leu Lys Ser His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile
20 25 30
cac ggc caa gtc tac gac gtg tcc tct tgg gcc gcc ctt cat ccg ggg 144
His Gly Gln Val Tyr Asp Val Ser Ser Trp Ala Ala Leu His Pro Gly
35 40 45
ggc act gcc cct ctc atg gcc ctt gca gga cac gac gtg acc gat gct 192
Gly Thr Ala Pro Leu Met Ala Leu Ala Gly His Asp Val Thr Asp Ala
50 55 60
ttc ctc gcg tac cat ccc cct tcc act gcc cgt ctc ctc cct cct ctc 240
Phe Leu Ala Tyr His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu
65 70 75 80
tct acc aac ctc ctt ctt caa aac cac tcc gtc tcc ccc acc tcc tca 288
Ser Thr Asn Leu Leu Leu Gln Asn His Ser Val Ser Pro Thr Ser Ser
85 90 95
gac tac cgc aaa ctc ctc gac aac ttc cat aaa cat ggc ctt ttc cgc 336
Asp Tyr Arg Lys Leu Leu Asp Asn Phe His Lys His Gly Leu Phe Arg
100 105 110
gcc agg ggc cac act gct tac gcc acc ttc gtc ttc atg ata gcg atg 384
Ala Arg Gly His Thr Ala Tyr Ala Thr Phe Val Phe Met Ile Ala Met
115 120 125
ttt cta atg agc gtg act gga gtc ctt tgc agc gac agt gcg tgg gtc 432
Phe Leu Met Ser Val Thr Gly Val Leu Cys Ser Asp Ser Ala Trp Val
130 135 140
cat ttg gct agc ggc gga gca atg ggg ttc gcc tgg atc caa tgc gga 480
His Leu Ala Ser Gly Gly Ala Met Gly Phe Ala Trp Ile Gln Cys Gly
145 150 155 160
tgg ata ggt cac gac tct ggg cat tac cgg att atg tct gac agg aaa 528
Trp Ile Gly His Asp Ser Gly His Tyr Arg Ile Met Ser Asp Arg Lys
165 170 175
tgg aac tgg ttc gcg caa atc cta agc aca aac tgc ctc cag ggg att 576
Trp Asn Trp Phe Ala Gln Ile Leu Ser Thr Asn Cys Leu Gln Gly Ile
180 185 190
agt atc ggg tgg tgg aag tgg aac cat aat gcg cac cac atc gct tgc 624
Ser Ile Gly Trp Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys
195 200 205
aat agc ctg gat tac gac ccc gac ctc cag tat atc cct ttg ctc gtc 672
Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val
210 215 220
gtc tcc ccc aag ttc ttc aac tcc ctt act tct cgt ttc tac gac aag 720
Val Ser Pro Lys Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asp Lys
225 230 235 240
aag ctg aac ttc gac ggc gtg tcg agg ttt ctg gtt tgc tac cag cac 768
Lys Leu Asn Phe Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His
245 250 255
tgg acg ttt tat ccg gtc atg tgt gtc gct agg ctg aac atg ctc gcg 816
Trp Thr Phe Tyr Pro Val Met Cys Val Ala Arg Leu Asn Met Leu Ala
260 265 270
cag tca ttt ata acg ctt ttc tcg agt agg gag gtg tgc cat agg gcg 864
Gln Ser Phe Ile Thr Leu Phe Ser Ser Arg Glu Val Cys His Arg Ala
275 280 285
caa gag gtt ttc gga ctt gcc gtg ttt tgg gtt tgg ttt ccg ctt tta 912
Gln Glu Val Phe Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu
290 295 300
ctt tct tgt tta cct aat tgg ggc gag agg att atg ttt ttg ctt gcg 960
Leu Ser Cys Leu Pro Asn Trp Gly Glu Arg Ile Met Phe Leu Leu Ala
305 310 315 320
agc tat tcc gtt acg ggg ata caa cac gtg cag ttc agc ttg aac cat 1008
Ser Tyr Ser Val Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His
325 330 335
ttt tct tcg gac gtc tat gtg ggc ccg cca gta ggt aat gac tgg ttc 1056
Phe Ser Ser Asp Val Tyr Val Gly Pro Pro Val Gly Asn Asp Trp Phe
340 345 350
aag aaa cag act gcc ggg aca ctt aac ata tcg tgc ccg gcg tgg atg 1104
Lys Lys Gln Thr Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met
355 360 365
gat tgg ttc cat ggc ggg tta cag ttt cag gtc gag cac cac ttg ttt 1152
Asp Trp Phe His Gly Gly Leu Gln Phe Gln Val Glu His His Lau Phe
370 375 380
ccg cgg atg cct agg ggt cag ttt agg aag att tct cct ttt gtg agg 1200
Pro Arg Met Pro Arg Gly Gln Phe Arg Lys Ile Ser Pro Phe Val Arg
385 390 395 400
gat ttg tgt aag aaa cac aac ttg cct tac aat atc gcg tct ttt act 1248
Asp Leu Cys Lys Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr
405 410 415
aaa gcg aat gtg ttt acg ctt aag acg ctg aga aat acg gcc att gag 1296
Lys Ala Asn Val Phe Thr Leu Lys Thr Leu Arg Asn Thr Ala Ile Glu
420 425 430
gct cgg gac ctc tct aat ccg ctc cca aag aat atg gtg tgg gaa gct 1344
Ala Arg Asp Leu Ser Asn Pro Leu Pro Lys Asn Met Val Trp Glu Ala
435 440 445
ctt aaa act ctc ggg tga 1362
Leu Lys Thr Leu Gly
450
<210>72
<211>453
<212>PRT
<213>有粉报春
<400>72
Met Ala Asn Lys Ser Pro Pro Asn Pro Lys Thr Gly Tyr Ile Thr Ser
1 5 10 15
Ser Asp Leu Lys Ser His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile
20 25 30
His Gly Gln Val Tyr Asp Val Ser Ser Trp Ala Ala Leu His Pro Gly
35 40 45
Gly Thr Ala Pro Leu Met Ala Leu Ala Gly His Asp Val Thr Asp Ala
50 55 60
Phe Leu Ala Tyr His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu
65 70 75 80
Ser Thr Asn Leu Leu Leu Gln Asn His Ser Val Ser Pro Thr Ser Ser
85 90 95
Asp Tyr Arg Lys Leu Leu Asp Asn Phe His Lys His Gly Leu Phe Arg
100 105 110
Ala Arg Gly His Thr Ala Tyr Ala Thr Phe Val Phe Met Ile Ala Met
115 120 125
Phe Leu Met Ser Val Thr Gly Val Leu Cys Ser Asp Ser Ala Trp Val
130 135 140
His Leu Ala Ser Gly Gly Ala Met Gly Phe Ala Trp Ile Gln Cys Gly
145 150 155 160
Trp Ile Gly His Asp Ser Gly His Tyr Arg Ile Met Ser Asp Arg Lys
165 170 175
Trp Asn Trp Phe Ala Gln Ile Leu Ser Thr Asn Cys Leu Gln Gly Ile
180 185 190
Ser Ile Gly Trp Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys
195 200 205
Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val
210 215 220
Val Ser Pro Lys Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asp Lys
225 230 235 240
Lys Leu Asn Phe Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His
245 250 255
Trp Thr Phe Tyr Pro Val Met Cys Val Ala Arg Leu Asn Met Leu Ala
260 265 270
Gln Ser Phe Ile Thr Leu Phe Ser Ser Arg Glu Val Cys His Arg Ala
275 280 285
Gln Glu Val Phe Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu
290 295 300
Leu Ser Cys Leu Pro Asn Trp Gly Glu Arg Ile Met Phe Leu Leu Ala
305 310 315 320
Ser Tyr Ser Val Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His
325 330 335
Phe Ser Ser Asp Val Tyr Val Gly Pro Pro Val Gly Asn Asp Trp Phe
340 345 350
Lys Lys Gln Thr Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met
355 360 365
Asp Trp Phe His Gly Gly Leu Gln Phe Gln Val Glu His His Leu Phe
370 375 380
Pro Arg Met Pro Arg Gly Gln Phe Arg Lys Ile Ser Pro Phe Val Arg
385 390 395 400
Asp Leu Cys Lys Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr
405 410 415
Lys Ala Asn Val Phe Thr Leu Lys Thr Leu Arg Asn Thr Ala Ile Glu
420 425 430
Ala Arg Asp Leu Ser Asn Pro Leu Pro Lys Asn Met Val Trp Glu Ala
435 440 445
Leu Lys Thr Leu Gly
450
<210>73
<211>1362
<212>DNA
<213>高穗花报春(Primula vialii)
<220>
<221>CDS
<222>(1)..(1362)
<223>Δ6-去饱和酶
<400>73
atg gct aac aaa tct cca cca aac ccc aaa aca ggt tac att acc agc 48
Met Ala Asn Lys Ser Pro Pro Asn Pro Lys Thr Gly Tyr Ile Thr Ser
1 5 10 15
tca gac ctg aaa ggg cac aac aaa gca gga gac cta tgg ata tca atc 96
Ser Asp Leu Lys Gly His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile
20 25 30
cac ggg gag gta tac gac gtg tcc tcg tgg gcc ggc ctt cac ccg ggg 144
His Gly Glu Val Tyr Asp Val Ser Ser Trp Ala Gly Leu His Pro Gly
35 40 45
ggc agt gcc ccc ctc atg gcc ctc gca gga cac gac gta acc gac gct 192
Gly Ser Ala Pro Leu Met Ala Leu Ala Gly His Asp Val Thr Asp Ala
50 55 60
ttt cta gcg tat cat cct cct tct acc gcc cgc ctc ctc cct ccc ctc 240
Phe Leu Ala Tyr His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu
65 70 75 80
tcc acc aac ctc ctc ctt caa aac cac tcc gtc tcc ccc acc tcc tct 288
Ser Thr Asn Leu Leu Leu Gln Asn His Ser Val Ser Pro Thr Ser Ser
85 90 95
gac tac cgc aaa ctc ctc cac aac ttc cat aaa att ggt atg ttc cgc 336
Asp Tyr Arg Lys Leu Leu His Asn Phe His Lys Ile Gly Met Phe Arg
100 105 110
gcc agg ggc cac act gct tac gcc acc ttc gtc atc atg ata gtg atg 384
Ala Arg Gly His Thr Ala Tyr Ala Thr Phe Val Ile Met Ile Val Met
115 120 125
ttt cta acg agc gtg acc gga gtc ctt tgc agc gac agt gcg tgg gtc 432
Phe Leu Thr Ser Val Thr Gly Val Leu Cys Ser Asp Ser Ala Trp Val
130 135 140
cat ctg gct agc ggc gca gca atg ggg ttc gcc tgg atc cag tgc gga 480
His Leu Ala Ser Gly Ala Ala Met Gly Phe Ala Trp Ile Gln Cys Gly
145 150 155 160
tgg ata ggt cac gac tct ggg cat tac cgg att atg tct gac agg aaa 528
Trp Ile Gly His Asp Ser Gly His Tyr Arg Ile Met Ser Asp Arg Lys
165 170 175
tgg aac tgg ttc gcg cag gtc ctg agc aca aac tgc ctc cag ggg atc 576
Trp Asn Trp Phe Ala Gln Val Leu Ser Thr Asn Cys Leu Gln Gly Ile
180 185 190
agt atc ggg tgg tgg aag tgg aac cat aac gcc cac cac att gct tgc 624
Ser Ile Gly Trp Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys
195 200 205
aat agc ctg gac tac gac ccc gac ctc cag tat atc cct ttg ctc gtg 672
Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val
210 215 220
gtc tcc ccc aag ttc ttc aac tcc ctt act tct cgt ttc tac gac aag 720
Val Ser Pro Lys Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asp Lys
225 230 235 240
aag ctg aat ttc gac ggc gtg tca agg ttt ctg gtt tgc tac cag cac 768
Lys Leu Asn Phe Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His
245 250 255
tgg acg ttt tat cca gtc atg tgt gtc gct agg cta aac atg atc gca 816
Trp Thr Phe Tyr Pro Val Met Cys Val Ala Arg Leu Asn Met Ile Ala
260 265 270
cag tcg ttt ata acg ctt ttc tcg agc agg gag gtg ggt cat agg gcg 864
Gln Ser Phe Ile Thr Leu Phe Ser Ser Arg Glu Val Gly His Arg Ala
275 280 285
caa gag att ttc gga ctt gct gtg ttt tgg gtt tgg ttt ccg ctc ctg 912
Gln Glu Ile Phe Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu
290 295 300
ctc tct tgc tta cct aat tgg agc gag agg att atg ttt ctg cta gcg 960
Leu Ser Cys Leu Pro Asn Trp Ser Glu Arg Ile Met Phe Leu Leu Ala
305 310 315 320
agc tat tcc gtt acg ggg ata cag cac gtg cag ttc agc ttg aac cat 1008
Ser Tyr Ser Val Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His
325 330 335
ttt tct tcg gac gtc tac gtg ggc ccg cca gta gct aac gac tgg ttc 1056
Phe Ser Ser Asp Val Tyr Val Gly Pro Pro Val Ala Asn Asp Trp Phe
340 345 350
aag aaa cag act gct ggg aca ctt aac ata tcg tgc ccg gcg tgg atg 1104
Lys Lys Gln Thr Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met
355 360 365
gac tgg ttc cat ggc ggg ttg cag ttt cag gtc gag cac cac ttg ttt 1152
Asp Trp Phe His Gly Gly Leu Gln Phe Gln Val Glu His His Leu Phe
370 375 380
ccg cgg atg cct agg ggt cag ttt agg aag att tct cct ttt gtg agg 1200
Pro Arg Met Pro Arg Gly Gln Phe Arg Lys Ile Ser Pro Phe Val Arg
385 390 395 400
gat ttg tgt aag aaa cac aac ttg cct tac aat atc gcg tct ttt act 1248
Asp Leu Cys Lys Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr
405 410 415
aaa gca aac gtg ttg acg ctt aag acg ctg aga aat acg gcc att gag 1296
Lye Ala Asn Val Leu Thr Leu Lys Thr Leu Arg Asn Thr Ala Ile Glu
420 425 430
gct cgg gac ctc tct aat ccg acc cca aag aat atg gtg tgg gaa gcc 1344
Ala Arg Asp Leu Ser Asn Pro Thr Pro Lys Asn Met Val Trp Glu Ala
435 440 445
gtc cac aca cac ggc tag 1362
Val His Thr His Gly
450
<210>74
<211>453
<212>PRT
<213>高穗花报春
<400>74
Met Ala Asn Lys Ser Pro Pro Asn Pro Lys Thr Gly Tyr Ile Thr Ser
1 5 10 15
Ser Asp Leu Lys Gly His Asn Lys Ala Gly Asp Leu Trp Ile Ser Ile
20 25 30
His Gly Glu Val Tyr Asp Val Ser Ser Trp Ala Gly Leu His Pro Gly
35 40 45
Gly Ser Ala Pro Leu Met Ala Leu Ala Gly His Asp Val Thr Asp Ala
50 55 60
Phe Leu Ala Tyr His Pro Pro Ser Thr Ala Arg Leu Leu Pro Pro Leu
65 70 75 80
Ser Thr Asn Leu Leu Leu Gln Asn His Ser Val Ser Pro Thr Ser Ser
85 90 95
Asp Tyr Arg Lys Leu Leu His Asn Phe His Lys Ile Gly Met Phe Arg
100 105 110
Ala Arg Gly His Thr Ala Tyr Ala Thr Phe Val Ile Met Ile Val Met
115 120 125
Phe Leu Thr Ser Val Thr Gly Val Leu Cys Ser Asp Ser Ala Trp Val
130 135 140
His Leu Ala Ser Gly Ala Ala Met Gly Phe Ala Trp Ile Gln Cys Gly
145 150 155 160
Trp Ile Gly His Asp Ser Gly His Tyr Arg Ile Met Ser Asp Arg Lys
165 170 175
Trp Asn Trp Phe Ala Gln Val Leu Ser Thr Asn Cys Leu Gln Gly Ile
180 185 190
Ser Ile Gly Trp Trp Lys Trp Asn His Asn Ala His His Ile Ala Cys
195 200 205
Asn Ser Leu Asp Tyr Asp Pro Asp Leu Gln Tyr Ile Pro Leu Leu Val
210 215 220
Val Ser Pro Lys Phe Phe Asn Ser Leu Thr Ser Arg Phe Tyr Asp Lys
225 230 235 240
Lys Leu Asn Phe Asp Gly Val Ser Arg Phe Leu Val Cys Tyr Gln His
245 250 255
Trp Thr Phe Tyr Pro Val Met Cys Val Ala Arg Leu Asn Met Ile Ala
260 265 270
Gln Ser Phe Ile Thr Leu Phe Ser Ser Arg Glu Val Gly His Arg Ala
275 280 285
Gln Glu Ile Phe Gly Leu Ala Val Phe Trp Val Trp Phe Pro Leu Leu
290 295 300
Leu Ser Cys Leu Pro Asn Trp Ser Glu Arg Ile Met Phe Leu Leu Ala
305 310 315 320
Ser Tyr Ser Val Thr Gly Ile Gln His Val Gln Phe Ser Leu Asn His
325 330 335
Phe Ser Ser Asp Val Tyr Val Gly Pro Pro Val Ala Asn Asp Trp Phe
340 345 350
Lys Lys Gln Thr Ala Gly Thr Leu Asn Ile Ser Cys Pro Ala Trp Met
355 360 365
Asp Trp Phe His Gly Gly Leu Gln Phe Gln Val Glu His His Leu Phe
370 375 380
Pro Arg Met Pro Arg Gly Gln Phe Arg Lys Ile Ser Pro Phe Val Arg
385 390 395 400
Asp Leu Cys Lys Lys His Asn Leu Pro Tyr Asn Ile Ala Ser Phe Thr
405 410 415
Lys Ala Asn Val Leu Thr Leu Lys Thr Leu Arg Asn Thr Ala Ile Glu
420 425 430
Ala Arg Asp Leu Ser Asn Pro Thr Pro Lys Asn Met Val Trp Glu Ala
435 440 445
Val His Thr His Gly
450
<210>75
<211>903
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(903)
<223>Δ5-延伸酶
<400>75
atg agc gcc tcc ggt gcg ctg ctg ccc gcg atc gcg tcc gcc gcg tac 48
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Ser Ala Ala Tyr
1 5 10 15
gcg tac gcg acg tac gcc tac gcc ttt gag tgg tcg cac gcg aat ggc 96
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
atc gac aac gtc gac gcg cgc gag tgg atc ggt gcg ctg tcg ttg agg 144
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
ctc ccg gcg atc gcg acg acg atg tac ctg ttg ttc tgc ctg gtc gga 192
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
ccg agg ttg atg gcg aag cgc gag gcg ttc gac ccg aag ggg ttc atg 240
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
ctg gcg tac aat gcg tat cag acg gcg ttc aac gtc gtc gtg ctc ggg 288
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
atg ttc gcg cga gag atc tcg ggg ctg ggg cag ccc gtg tgg ggg tca 336
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
acc atg ccg tgg agc gat aga aaa tcg ttt aag atc ctc ctc ggg gtg 384
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
tgg ttg cac tac aac aac aaa tat ttg gag cta ttg gac act gtg ttc 432
Trp Leu His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
atg gtt gcg cgc aag aag acg aag cag ttg agc ttc ttg cac gtt tat 480
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
cat cac gcc ctg ttg atc tgg gcg tgg tgg ttg gtg tgt cac ttg atg 528
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
gcc acg aac gat tgt atc gat gcc tac ttc ggc gcg gcg tgc aac tcg 576
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
ttc att cac atc gtg atg tac tcg tat tat ctc atg tcg gcg ctc ggc 624
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
att cga tgc ccg tgg aag cga tac atc acc cag gct caa atg ctc caa 672
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
ttc gtc att gtc ttc gcg cac gcc gtg ttc gtg ctg cgt cag aag cac 720
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
tgc ccg gtc acc ctt cct tgg gcg caa atg ttc gtc atg acg aac atg 768
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
ctc gtg ctc ttc ggg aac ttc tac ctc aag gcg tac tcg aac aag tcg 816
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
cgc ggc gac ggc gcg agt tcc gtg aaa cca gcc gag acc acg cgc gcg 864
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
ccc agc gtg cga cgc acg cga tct cga aaa att gac taa 903
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>76
<211>300
<212>PRT
<213>Ostreococcus tauri
<400>76
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Ser Ala Ala Tyr
1 5 10 15
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
Trp Leu His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>77
<211>903
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(903)
<223>Δ5-延伸酶
<400>77
atg agc gcc tcc ggt gcg ctg ctg ccc gcg atc gcg ttc gcc gcg tac 48
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
gcg tac gcg acg tac gcc tac gcc ttt gag tgg tcg cac gcg aat ggc 96
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
atc gac aac gtc gac gcg cgc gag tgg atc ggt gcg ctg tcg ttg agg 144
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
ctc ccg gcg atc gcg acg acg atg tac ctg ttg ttc tgc ctg gtc gga 192
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
ccg agg ttg atg gcg aag cgc gag gcg ttc gac ccg aag ggg ttc atg 240
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
ctg gcg tac aat gcg tat cag acg gcg ttc aac gtc gtc gtg ctc ggg 288
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
atg ttc gcg cga gag atc tcg ggg ctg ggg cag ccc gtg tgg ggg tca 336
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
acc atg ccg tgg agc gat aga aaa tcg ttt aag atc ctc ctc ggg gtg 384
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
tgg ttg cac tac aac aac aaa tat ttg gag cta ttg gac act gtg ttc 432
Trp Ley His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
atg gtt gcg cgc aag aag acg aag cag ttg agc ttc ttg cac gtt tat 480
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
cat cac gcc ctg ttg atc tgg gcg tgg tgg ttg gtg tgt cac ttg atg 528
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
gcc acg aac gat tgt atc gat gcc tac ttc ggc gcg gcg tgc aac tcg 576
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
ttc att cac atc gtg atg tac tcg tat tat ctc atg tcg gcg ctc ggc 624
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
att cga tgc ccg tgg aag cga tac atc acc cag gct caa atg ctc caa 672
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
ttc gtc att gtc ttc gcg cac gcc gtg ttc gtg ctg cgt cag aag cac 720
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
tgc ccg gtc acc ctt cct tgg gcg caa atg ttc gtc atg acg aac atg 768
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
ctc gtg ctc ttc ggg aac ttc tac ctc aag gcg tac tcg aac aag tcg 816
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
cgc ggc gac ggc gcg agt tcc gtg aaa cca gcc gag acc acg cgc gcg 864
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
ccc agc gtg cga cgc acg cga tct cga aaa att gac taa 903
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>78
<211>300
<212>PRT
<213>Ostreococcus tauri
<400>78
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
Trp Leu His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>79
<211>903
<212>DNA
<213>Ostreococcus tauri
<220>
<22l>CDS
<222>(1)..(903)
<223>Δ5-延伸酶
<400>79
atg agc gcc tcc ggt gcg ctg ctg ccc gcg atc gcg tcc gcc gcg tac 48
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Ser Ala Ala Tyr
1 5 10 15
gcg tac gcg acg tac gcc tac gcc ttt gag tgg tcg cac gcg aat ggc 96
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
atc gac aac gtc gac gcg cgc gag tgg atc ggt gcg ctg tcg ttg agg 144
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
ctc ccg gcg atc gcg acg acg atg tac ctg ttg ttc tgc ctg gtc gga 192
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
ccg agg ttg atg gcg aag cgc gag gcg ttc gac ccg aag ggg ttc atg 240
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
ctg gcg tac aat gcg tat cag acg gcg ttc aac gtc gtc gtg ctc ggg 288
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
atg ttc gcg cga gag atc tcg ggg ctg ggg cag ccc gtg tgg ggg tca 336
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
acc atg ccg tgg agc gat aga aaa tcg ttt aag atc ctc ctc ggg gtg 384
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
tgg ttg cac tac aac aac caa tat ttg gag cta ttg gac act gtg ttc 432
Trp Leu His Tyr Asn Asn Gln Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
atg gtt gcg cgc aag aag acg aag cag ttg agc ttc ttg cac gtt tat 480
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
cat cac gcc ctg ttg atc tgg gcg tgg tgg ttg gtg tgt cac ttg atg 528
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
gcc acg aac gat tgt atc gat gcc tac ttc ggc gcg gcg tgc aac tcg 576
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
ttc att cac atc gtg atg tac tcg tat tat ctc atg tcg gcg ctc ggc 624
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
att cga tgc ccg tgg aag cga tac atc acc cag gct caa atg ctc caa 672
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
ttc gtc att gtc ttc gcg cac gcc gtg ttc gtg ctg cgt cag aag cac 720
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
tgc ccg gtc acc ctt cct tgg gcg caa atg ttc gtc atg acg aac atg 768
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
ctc gtg ctc ttc ggg aac ttc tac ctc aag gcg tac tcg aac aag tcg 816
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
cgc ggc gac ggc gcg agt tcc gtg aaa cca gcc gag acc acg cgc gcg 864
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
ccc agc gtg cga cgc acg cga tct cga aaa att gac taa 903
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>80
<211>300
<212>PRT
<213>Ostreococcus tauri
<400>80
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Ser Ala Ala Tyr
1 5 10 15
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
Trp Leu His Tyr Asn Asn Gln Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>81
<211>879
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(879)
<223>Δ6-延伸酶
<400>81
atg agt ggc tta cgt gca ccc aac ttt tta cac aga ttc tgg aca aag 48
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
tgg gac tac gcg att tcc aaa gtc gtc ttc acg tgt gcc gac agt ttt 96
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
cag tgg gac atc ggg cca gtg agt tcg agt acg gcg cat tta ccc gcc 144
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
att gaa tcc cct acc cca ctg gtg act agc ctc ttg ttc tac tta gtc 192
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
aca gtt ttc ttg tgg tat ggt cgt tta acc agg agt tca gac aag aaa 240
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
att aga gag cct acg tgg tta aga aga ttc ata ata tgt cat aat gcg 288
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
ttc ttg ata gtc ctc agt ctt tac atg tgc ctt ggt tgt gtg gcc caa 336
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
gcg tat cag aat gga tat act tta tgg ggt aat gaa ttc aag gcc acg 384
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
gaa act cag ctt gct ctc tac att tac att ttt tac gta agt aaa ata 432
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
tac gag ttt gta gat act tac att atg ctt ctc aag aat aac ttg cgg 480
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Ann Leu Arg
145 150 155 160
caa gta aga ttc cta cac act tat cac cac agc acg att tcc ttt att 528
Gln Val Arg Phe Leu His Thr Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
tgg tgg atc att gct cgg agg gct ccg ggt ggt gat gct tac ttc agc 576
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
gcg gcc ttg aac tca tgg gta cac gtg tgc atg tac acc tat tat cta 624
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
tta tca acc ctt att gga aaa gaa gat cct aag cgt tcc aac tac ctt 672
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
tgg tgg ggt cgc cac cta acg caa atg cag atg ctt cag ttt ttc ttc 720
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
aac gta ctt caa gcg ttg tac tgc gct tcg ttc tct acg tat ccc aag 768
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
ttt ttg tcc aaa att ctg ctc gtc tat atg atg agc ctt ctc ggc ttg 816
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
ttt ggg cat ttc tac tat tcc aag cac ata gca gca gct aag ctc cag 864
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
aaa aaa cag cag tga 879
Lys Lys Gln Gln
290
<210>82
<211>292
<212>PRT
<213>Ostreococcus tauri
<400>82
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Asn Leu Arg
145 150 155 160
Gln Val Arg Phe Leu His Thr Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
Lys Lys Gln Gln
290
<210>83
<211>831
<212>DNA
<213>破囊壶菌属中的种(Thraustochytrium sp.)
<220>
<221>CDS
<222>(1)..(831)
<223>Δ5-延伸酶
<400>83
atg gac gtc gtc gag cag caa tgg cgc cgc ttc gtg gac gcc gtg gac 48
Met Asp Val Val Glu Gln Gln Trp Arg Arg Phe Val Asp Ala Val Asp
1 5 10 15
aac gga atc gtg gag ttc atg gag cat gag aag ccc aac aag ctg aac 96
Asn Gly Ile Val Glu Phe Met Glu His Glu Lys Pro Asn Lys Leu Asn
20 25 30
gag ggc aag ctc ttc acc tcg acc gag gag atg atg gcg ctt atc gtc 144
Glu Gly Lys Leu Phe Thr Ser Thr Glu Glu Met Met Ala Leu Ile Val
35 40 45
ggc tac ctg gcg ttc gtg gtc ctc ggg tcc gcc ttc atg aag gcc ttt 192
Gly Tyr Leu Ala Phe Val Val Leu Gly Ser Ala Phe Met Lys Ala Phe
50 55 60
gtc gat aag cct ttc gag ctc aag ttc ctc aag ctc gtg cac aac atc 240
Val Asp Lys Pro Phe Glu Leu Lys Phe Leu Lys Leu Val His Asn Ile
65 70 75 80
ttc ctc acc ggt ctg tcc atg tac atg gcc acc gag tgc gcg cgc cag 288
Phe Leu Thr Gly Leu Ser Met Tyr Met Ala Thr Glu Cys Ala Arg Gln
85 90 95
gca tac ctc ggc ggc tac aag ctc ttt ggc aac ccg atg gag aag ggc 336
Ala Tyr Leu Gly Gly Tyr Lys Leu Phe Gly Asn Pro Met Glu Lys Gly
100 105 110
acc gag tcg cac gcc ccg ggc atg gcc aac atc atc tac atc ttc tac 384
Thr Glu Ser His Ala Pro Gly Met Ala Asn Ile Ile Tyr Ile Phe Tyr
115 120 125
gtg agc aag ttc ctc gaa ttc ctc gac acc gtc ttc atg atc ctc ggc 432
Val Ser Lys Phe Leu Glu Phe Leu Asp Thr Val Phe Met Ile Leu Gly
130 135 140
aag aag tgg aag cag ctc agc ttt ctc cac gtc tac cac cac gcg agc 480
Lys Lys Trp Lys Gln Leu Ser Phe Leu His Val Tyr His His Ala Ser
145 150 155 160
atc agc ttc atc tgg ggc atc atc gcc cgc ttc gcg ccc ggt ggc gac 528
Ile Ser Phe Ile Trp Gly Ile Ile Ala Arg Phe Ala Pro Gly Gly Asp
165 170 175
gcc tac ttc tct acc atc ctc aac agc agc gtg cat gtc gtg ctc tac 576
Ala Tyr Phe Ser Thr Ile Leu Asn Ser Ser Val His Val Val Leu Tyr
180 185 190
ggc tac tac gcc tcg acc acc ctc ggc tac acc ttc atg cgc ccg ctg 624
Gly Tyr Tyr Ala Ser Thr Thr Leu Gly Tyr Thr Phe Met Arg Pro Leu
195 200 205
cgc ccg tac att acc acc att cag ctc acg cag ttc atg gcc atg gtc 672
Arg Pro Tyr Ile Thr Thr Ile Gln Leu Thr Gln Phe Met Ala Met Val
210 215 220
gtc cag tcc gtc tat gac tac tac aac ccc tgc gac tac ccg cag ccc 720
Val Gln Ser Val Tyr Asp Tyr Tyr Asn Pro Cys Asp Tyr Pro Gln Pro
225 230 235 240
ctc gtc aag ctg ctc ttc tgg tac atg ctc acc atg ctc ggc ctc ttc 768
Leu Val Lys Leu Leu Phe Trp Tyr Met Leu Thr Met Leu Gly Leu Phe
245 250 255
ggc aac ttc ttc gtg cag cag tac ctc aag ccc aag gcg ccc aag aag 816
Gly Asn Phe Phe Val Gln Gln Tyr Leu Lys Pro Lys Ala Pro Lys Lys
260 265 270
cag aag acc atc taa 831
Gln Lys Thr Ile
275
<210>84
<211>276
<212>PRT
<213>破囊壶菌属中的种
<400>84
Met Asp Val Val Glu Gln Gln Trp Arg Arg Phe Val Asp Ala Val Asp
1 5 10 15
Asn Gly Ile Val Glu Phe Met Glu His Glu Lys Pro Asn Lys Leu Asn
20 25 30
Glu Gly Lys Leu Phe Thr Ser Thr Glu Glu Met Met Ala Leu Ile Val
35 40 45
Gly Tyr Leu Ala Phe Val Val Leu Gly Ser Ala Phe Met Lys Ala Phe
50 55 60
Val Asp Lys Pro Phe Glu Leu Lys Phe Leu Lys Leu Val His Asn Ile
65 70 75 80
Phe Leu Thr Gly Leu Ser Met Tyr Met Ala Thr Glu Cys Ala Arg Gln
85 90 95
Ala Tyr Leu Gly Gly Tyr Lys Leu Phe Gly Asn Pro Met Glu Lys Gly
100 105 110
Thr Glu Ser His Ala Pro Gly Met Ala Asn Ile Ile Tyr Ile Phe Tyr
115 120 125
Val Ser Lys Phe Leu Glu Phe Leu Asp Thr Val Phe Met Ile Leu Gly
130 135 140
Lys Lys Trp Lys Gln Leu Ser Phe Leu His Val Tyr His His Ala Ser
145 150 155 160
Ile Ser Phe Ile Trp Gly Ile Ile Ala Arg Phe Ala Pro Gly Gly Asp
165 170 175
Ala Tyr Phe Ser Thr Ile Leu Asn Ser Ser Val His Val Val Leu Tyr
180 185 190
Gly Tyr Tyr Ala Ser Thr Thr Leu Gly Tyr Thr Phe Met Arg Pro Leu
195 200 205
Arg Pro Tyr Ile Thr Thr Ile Gln Leu Thr Gln Phe Met Ala Met Val
210 215 220
Val Gln Ser Val Tyr Asp Tyr Tyr Asn Pro Cys Asp Tyr Pro Gln Pro
225 230 235 240
Leu Val Lys Leu Leu Phe Trp Tyr Met Leu Thr Met Leu Gly Leu Phe
245 250 255
Gly Asn Phe Phe Val Gln Gln Tyr Leu Lys Pro Lys Ala Pro Lys Lys
260 265 270
Gln Lys Thr Ile
275
<210>85
<211>1077
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1077)
<223>Δ5-延伸酶
<400>85
atg tgc tca cca ccg ccg tca caa tcc aaa aca aca tcc ctc cta gca 48
Met Cys Ser Pro Pro Pro Ser Gln Ser Lys Thr Thr Ser Leu Leu Ala
1 5 10 15
cgg tac acc acc gcc gcc ctc ctc ctc ctc acc ctc aca acg tgg tgc 96
Arg Tyr Thr Thr Ala Ala Leu Leu Leu Leu Thr Leu Thr Thr Trp Cys
20 25 30
cac ttc gcc ttc cca gcc gcc acc gcc aca ccc ggc ctc acc gcc gaa 144
His Phe Ala Phe Pro Ala Ala Thr Ala Thr Pro Gly Leu Thr Ala Glu
35 40 45
atg cac tcc tac aaa gtc cca ctc ggt ctc acc gta ttc tac ctg ctg 192
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
50 55 60
agt cta ccg tca cta aag tac gtt acg gac aac tac ctt gcc aaa aag 240
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
65 70 75 80
tat gat atg aag tca ctc ctg acg gaa tca atg gtg ttg tac aat gtg 288
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
85 90 95
gcg caa gtg ctg ctc aat ggg tgg acg gtg tat gcg att gtg gat gcg 336
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
100 105 110
gtg atg aat aga gac cat cct ttt att gga agt aga agt ttg gtt ggg 384
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
115 120 125
gcg gcg ttg cat agt ggg agc tcg tat gcg gtg tgg gtt cat tat tgt 432
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
130 135 140
gat aag tat ttg gag ttc ttt gat acg tat ttt atg gtg ttg agg ggg 480
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
145 150 155 160
aaa atg gac cag gtc tcc ttc ctc cac atc tac cac cac acg acc ata 528
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
165 170 175
gcg tgg gca tgg tgg atc gcc ctc cgc ttc tcc ccc ggc gga gac att 576
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
180 185 190
tac ttc ggg gca ctc ctc aac tcc atc atc cac gtc ctc atg tat tcc 624
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
195 200 205
tac tac gcc ctt gcc cta ctc aag gtc agt tgt cca tgg aaa cga tac 672
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
210 215 220
ttg act caa gct caa tta ttg caa ttc aca agt gtg gtg gtt tat acg 720
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
225 230 235 240
ggg tgt acg ggt tat act cat tac tat cat acg aag cat gga gcg gat 768
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
245 250 255
gag aca cag cct agt tta gga acg tat tat ttc tgt tgt gga gtg cag 816
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
260 265 270
gtg ttt gag atg gtt agt ttg ttt gta ctc ttt tcc atc ttt tat aaa 864
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
275 280 285
cga tcc tat tcg aag aag aac aag tca gga gga aag gat agc aag aag 912
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly GLy Lys Asp Ser Lys Lys
290 295 300
aat gat gat ggg aat aat gag gat caa tgt cac aag gct atg aag gat 960
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
305 310 315 320
ata tcg gag ggt gcg aag gag gtt gtg ggg cat gca gcg aag gat gct 1008
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
325 330 335
gga aag ttg gtg gct acg gcg agt aag gct gta aag agg aag gga act 1056
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
340 345 350
cgt gtt act ggt gcc atg tag 1077
Arg Val Thr Gly Ala Met
355
<210>86
<211>358
<212>PRT
<213>假矮海链藻
<400>86
Met Cys Ser Pro Pro Pro Ser Gln Ser Lys Thr Thr Ser Leu Leu Ala
1 5 10 15
Arg Tyr Thr Thr Ala Ala Leu Leu Leu Leu Thr Leu Thr Thr Trp Cys
20 25 30
His Phe Ala Phe Pro Ala Ala Thr Ala Thr Pro Gly Leu Thr Ala Glu
35 40 45
Met His Ser Tyr Lys Val Pro Leu Gly Leu Thr Val Phe Tyr Leu Leu
50 55 60
Ser Leu Pro Ser Leu Lys Tyr Val Thr Asp Asn Tyr Leu Ala Lys Lys
65 70 75 80
Tyr Asp Met Lys Ser Leu Leu Thr Glu Ser Met Val Leu Tyr Asn Val
85 90 95
Ala Gln Val Leu Leu Asn Gly Trp Thr Val Tyr Ala Ile Val Asp Ala
100 105 110
Val Met Asn Arg Asp His Pro Phe Ile Gly Ser Arg Ser Leu Val Gly
115 120 125
Ala Ala Leu His Ser Gly Ser Ser Tyr Ala Val Trp Val His Tyr Cys
130 135 140
Asp Lys Tyr Leu Glu Phe Phe Asp Thr Tyr Phe Met Val Leu Arg Gly
145 150 155 160
Lys Met Asp Gln Val Ser Phe Leu His Ile Tyr His His Thr Thr Ile
165 170 175
Ala Trp Ala Trp Trp Ile Ala Leu Arg Phe Ser Pro Gly Gly Asp Ile
180 185 190
Tyr Phe Gly Ala Leu Leu Asn Ser Ile Ile His Val Leu Met Tyr Ser
195 200 205
Tyr Tyr Ala Leu Ala Leu Leu Lys Val Ser Cys Pro Trp Lys Arg Tyr
210 215 220
Leu Thr Gln Ala Gln Leu Leu Gln Phe Thr Ser Val Val Val Tyr Thr
225 230 235 240
Gly Cys Thr Gly Tyr Thr His Tyr Tyr His Thr Lys His Gly Ala Asp
245 250 255
Glu Thr Gln Pro Ser Leu Gly Thr Tyr Tyr Phe Cys Cys Gly Val Gln
260 265 270
Val Phe Glu Met Val Ser Leu Phe Val Leu Phe Ser Ile Phe Tyr Lys
275 280 285
Arg Ser Tyr Ser Lys Lys Asn Lys Ser Gly Gly Lys Asp Ser Lys Lys
290 295 300
Asn Asp Asp Gly Asn Asn Glu Asp Gln Cys His Lys Ala Met Lys Asp
305 310 315 320
Ile Ser Glu Gly Ala Lys Glu Val Val Gly His Ala Ala Lys Asp Ala
325 330 335
Gly Lys Leu Val Ala Thr Ala Ser Lys Ala Val Lys Arg Lys Gly Thr
340 345 350
Arg Val Thr Gly Ala Met
355
<210>87
<211>1086
<212>DNA
<213>致病疫霉
<220>
<221>CDS
<222>(1)..(1086)
<223>ω3-去饱和酶
<400>87
atg gcg acg aag gag gcg tat gtg ttc ccc act ctg acg gag atc aag 48
Met Ala Thr Lys Glu Ala Tyr Val Phe Pro Thr Leu Thr Glu Ile Lys
1 5 10 15
cgg tcg cta cct aaa gac tgt ttc gag gct tcg gtg cct ctg tcg ctc 96
Arg Ser Leu Pro Lys Asp Cys Phe Glu Ala Ser Val Pro Leu Ser Leu
20 25 30
tac tac acc gtg cgt tgt ctg gtg atc gcg gtg gct cta acc ttc ggt 144
Tyr Tyr Thr Val Arg Cys Leu Val Ile Ala Val Ala Leu Thr Phe Gly
35 40 45
ctc aac tac gct cgc gct ctg ccc gag gtc gag agc ttc tgg gct ctg 192
Leu Asn Tyr Ala Arg Ala Leu Pro Glu Val Glu Ser Phe Trp Ala Leu
50 55 60
gac gcc gca ctc tgc acg ggc tac atc ttg ctg cag ggc atc gtg ttc 240
Asp Ala Ala Leu Cys Thr Gly Tyr Ile Leu Leu Gln Gly Ile Val Phe
65 70 75 80
tgg ggc ttc ttc acg gtg ggc cac gat gcc ggc cac ggc gcc ttc tcg 288
Trp Gly Phe Phe Thr Val Gly His Asp Ala Gly His Gly Ala Phe Ser
85 90 95
cgc tac cac ctg ctt aac ttc gtg gtg ggc act ttc atg cac tcg ctc 336
Arg Tyr His Leu Leu Asn Phe Val Val Gly Thr Phe Met His Ser Leu
100 105 110
atc ctc acg ccc ttc gag tcg tgg aag ctc acg cac cgt cac cac cac 384
Ile Leu Thr Pro Phe Glu Ser Trp Lys Leu Thr His Arg His His His
115 120 125
aag aac acg ggc aac att gac cgt gac gag gtc ttc tac ccg caa cgc 432
Lys Asn Thr Gly Asn Ile Asp Arg Asp Glu Val Phe Tyr Pro Gln Arg
130 135 140
aag gcc gac gac cac ccg ctg tct cgc aac ctg att ctg gcg ctc ggg 480
Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu Ile Leu Ala Leu Gly
145 150 155 160
gca gcg tgg ctc gcc tat ttg gtc gag ggc ttc cct cct cgt aag gtc 528
Ala Ala Trp Leu Ala Tyr Leu Val Glu Gly Phe Pro Pro Arg Lys Val
165 170 175
aac cac ttc aac ccg ttc gag cct ctg ttc gtg cgt cag gtg tca gct 576
Asn His Phe Asn Pro Phe Glu Pro Leu Phe Val Arg Gln Val Ser Ala
180 185 190
gtg gta atc tct ctt ctc gcc cac ttc ttc gtg gcc gga ctc tcc atc 624
Val Val Ile Ser Leu Leu Ala His Phe Phe Val Ala Gly Leu Ser Ile
195 200 205
tat ctg agc ctc cag ctg ggc ctt aag acg atg gca atc tac tac tat 672
Tyr Leu Ser Leu Gln Leu Gly Leu Lys Thr Met Ala Ile Tyr Tyr Tyr
210 215 220
gga cct gtt ttt gtg ttc ggc agc atg ctg gtc att acc acc ttc cta 720
Gly Pro Val Phe Val Phe Gly Ser Met Leu Val Ile Thr Thr Phe Leu
225 230 235 240
cac cac aat gat gag gag acc cca tgg tac gcc gac tcg gag tgg acg 768
His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp Ser Glu Trp Thr
245 250 255
tac gtc aag ggc aac ctc tcg tcc gtg gac cga tcg tac ggc gcg ctc 816
Tyr Val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr Gly Ala Leu
260 265 270
att gac aac ctg agc cac aac atc ggc acg cac cag atc cac cac ctt 864
Ile Asp Asn Leu Ser His Asn Ile Gly Thr His Gln Ile His His Leu
275 280 285
ttc cct atc att ccg cac tac aaa ctc aag aaa gcc act gcg gcc ttc 912
Phe Pro Ile Ile Pro His Tyr Lys Leu Lys Lys Ala Thr Ala Ala Phe
290 295 300
cac cag gct ttc cct gag ctc gtg cgc aag agc gac gag cca att atc 960
His Gln Ala Phe Pro Glu Leu Val Arg Lys Ser Asp Glu Pro Ile Ile
305 310 315 320
aag gct ttc ttc cgg gtt gga cgt ctc tac gca aac tac ggc gtt gtg 1008
Lys Ala Phe Phe Arg Val Gly Arg Leu Tyr Ala Asn Tyr Gly Val Val
325 330 335
gac cag gag gcg aag ctc ttc acg cta aag gaa gcc aag gcg gcg acc 1056
Asp Gln Glu Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala Ala Thr
340 345 350
gag gcg gcg gcc aag acc aag tcc acg taa 1086
Glu Ala Ala Ala Lys Thr Lys Ser Thr
355 360
<210>88
<211>361
<212>PRT
<213>致病疫霉
<400>88
Met Ala Thr Lys Glu Ala Tyr Val Phe Pro Thr Leu Thr Glu Ile Lys
1 5 10 15
Arg Ser Leu Pro Lys Asp Cys Phe Glu Ala Ser Val Pro Leu Ser Leu
20 25 30
Tyr Tyr Thr Val Arg Cys Leu Val Ile Ala Val Ala Leu Thr Phe Gly
35 40 45
Leu Asn Tyr Ala Arg Ala Leu Pro Glu Val Glu Ser Phe Trp Ala Leu
50 55 60
Asp Ala Ala Leu Cys Thr Gly Tyr Ile Leu Leu Gln Gly Ile Val Phe
65 70 75 80
Trp Gly Phe Phe Thr Val Gly His Asp Ala Gly His Gly Ala Phe Ser
85 90 95
Arg Tyr His Leu Leu Asn Phe Val Val Gly Thr Phe Met His Ser Leu
100 105 110
Ile Leu Thr Pro Phe Glu Ser Trp Lys Leu Thr His Arg His His His
115 120 125
Lys Asn Thr Gly Asn Ile Asp Arg Asp Glu Val Phe Tyr Pro Gln Arg
130 135 140
Lys Ala Asp Asp His Pro Leu Ser Arg Asn Leu Ile Leu Ala Leu Gly
145 150 155 160
Ala Ala Trp Leu Ala Tyr Leu Val Glu Gly Phe Pro Pro Arg Lys Val
165 170 175
Asn His Phe Asn Pro Phe Glu Pro Leu Phe Val Arg Gln Val Ser Ala
180 185 190
Val Val Ile Ser Leu Leu Ala His Phe Phe Val Ala Gly Leu Ser Ile
195 200 205
Tyr Leu Ser Leu Gln Leu Gly Leu Lys Thr Met Ala Ile Tyr Tyr Tyr
210 215 220
Gly Pro Val Phe Val Phe Gly Ser Met Leu Val Ile Thr Thr Phe Leu
225 230 235 240
His His Asn Asp Glu Glu Thr Pro Trp Tyr Ala Asp Ser Glu Trp Thr
245 250 255
Tyr Val Lys Gly Asn Leu Ser Ser Val Asp Arg Ser Tyr Gly Ala Leu
260 265 270
Ile Asp Asn Leu Ser His Asn Ile Gly Thr His Gln Ile His His Leu
275 280 285
Phe Pro Ile Ile Pro His Tyr Lys Leu Lys Lys Ala Thr Ala Ala Phe
290 295 300
His Gln Ala Phe Pro Glu Leu Val Arg Lys Ser Asp Glu Pro Ile Ile
305 310 315 320
Lys Ala Phe Phe Arg Val Gly Arg Leu Tyr Ala Asn Tyr Gly Val Val
325 330 335
Asp Gln Glu Ala Lys Leu Phe Thr Leu Lys Glu Ala Lys Ala Ala Thr
340 345 350
Glu Ala Ala Ala Lys Thr Lys Ser Thr
355 360
<210>89
<211>1371
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(1371)
<223>Δ6-去饱和酶
<400>89
atg tgc gtg gag acg gaa aat aac gat ggg atc ccc acg gtg gag atc 48
Met Cys Val Glu Thr Glu Asn Asn Asp Gly Ile Pro Thr Val Glu Ile
1 5 10 15
gcg ttc gac ggt gag cgc gag cgg gcg gag gca aac gtg aag ctg tcc 96
Ala Phe Asp Gly Glu Arg Glu Arg Ala Glu Ala Asn Val Lys Leu Ser
20 25 30
gcg gag aag atg gag ccg gcg gcg ctg gcg aag acg ttc gcg agg cgg 144
Ala Glu Lys Met Glu Pro Ala Ala Leu Ala Lys Thr Phe Ala Arg Arg
35 40 45
tac gtc gtg atc gag ggg gtg gag tac gat gtg acg gat ttt aag cac 192
Tyr Val Val Ile Glu Gly Val Glu Tyr Asp Val Thr Asp Phe Lys His
50 55 60
ccg gga gga acg gtt att ttc tat gcg ttg tca aac acc ggg gcg gac 240
Pro Gly Gly Thr Val Ile Phe Tyr Ala Leu Ser Asn Thr Gly Ala Asp
65 70 75 80
gcg acg gaa gcg ttc aag gag ttt cat cat cgg tcg aga aag gcg agg 288
Ala Thr Glu Ala Phe Lys Glu Phe His His Arg Ser Arg Lys Ala Arg
85 90 95
aaa gcc ttg gcg gcg ctc ccg tct cga ccg gcc aag acg gcc aag gtg 336
Lys Ala Leu Ala Ala Leu Pro Ser Arg Pro Ala Lys Thr Ala Lys Val
100 105 110
gac gac gcg gag atg ctc caa gat ttc gcc aag tgg cgg aaa gaa ttg 384
Asp Asp Ala Glu Met Leu Gln Asp Phe Ala Lys Trp Arg Lys Glu Leu
115 120 125
gag aga gat gga ttc ttc aag ccc tct ccg gcg cac gtg gcg tat cgc 432
Glu Arg Asp Gly Phe Phe Lys Pro Ser Pro Ala His Val Ala Tyr Arg
130 135 140
ttc gcc gag ctc gcg gcg atg tac gct ctc ggg acg tac ctg atg tac 480
Phe Ala Glu Leu Ala Ala Met Tyr Ala Leu Gly Thr Tyr Leu Met Tyr
145 150 155 160
gct cga tac gtc gtc tcc tcg gtg ctc gtg tac gct tgc ttt ttc ggc 528
Ala Arg Tyr Val Val Ser Ser Val Leu Val Tyr Ala Cys Phe Phe Gly
165 170 175
gcc cga tgc ggt tgg gtg cag cac gag ggc gga cac agc tcg ctg acg 576
Ala Arg Cys Gly Trp Val Gln His Glu Gly Gly His Ser Ser Leu Thr
180 185 190
ggc aac att tgg tgg gac aag cgc atc cag gcc ttc aca gcc ggg ttc 624
Gly Asn Ile Trp Trp Asp Lys Arg Ile Gln Ala Phe Thr Ala Gly Phe
195 200 205
ggt ctc gcc ggt agc ggc gac atg tgg aac tcg atg cac aac aag cat 672
Gly Leu Ala Gly Ser Gly Asp Met Trp Asn Ser Met His Asn Lys His
210 215 220
cac gcg acg cct caa aag gtt cgt cac gac atg gat ctg gac acc acc 720
His Ala Thr Pro Gln Lys Val Arg His Asp Met Asp Leu Asp Thr Thr
225 230 235 240
ccc gcg gtg gcg ttc ttc aac acc gcg gtg gaa gac aat cgt ccc cgt 768
Pro Ala Val Ala Phe Phe Asn Thr Ala Val Glu Asp Asn Arg Pro Arg
245 250 255
ggc ttt agc aag tac tgg ttg cgc ctt cag gcg tgg acc ttc atc ccc 816
Gly Phe Ser Lys Tyr Trp Leu Arg Leu Gln Ala Trp Thr Phe Ile Pro
260 265 270
gtg acg tcc ggc ttg gtg ctc ctt ttc tgg atg ttt ttc ctc cac ccc 864
Val Thr Ser Gly Leu Val Leu Leu Phe Trp Met Phe Phe Leu His Pro
275 280 285
tcc aag gct ttg aag ggt ggc aag tac gaa gag ttg gtg tgg atg ctc 912
Ser Lys Ala Leu Lys Gly Gly Lys Tyr Glu Glu Leu Val Trp Met Leu
290 295 300
gcc gcg cac gtc atc cgc acg tgg acg atc aag gcg gtg acc gga ttc 960
Ala Ala His Val Ile Arg Thr Trp Thr Ile Lys Ala Val Thr Gly Phe
305 310 315 320
acc gcg atg cag tcc tac ggc tta ttt ttg gcg acg agc tgg gtg agc 1008
Thr Ala Met Gln Ser Tyr Gly Leu Phe Leu Ala Thr Ser Trp Val Ser
325 330 335
ggc tgc tat ctg ttt gca cac ttc tcc acg tcg cac acg cac ctg gat 1056
Gly Cys Tyr Leu Phe Ala His Phe Ser Thr Ser His Thr His Leu Asp
340 345 350
gtg gtg ccc gcg gac gag cat ctc tcc tgg gtt cga tac gcc gtc gat 1104
Val Val Pro Ala Asp Glu His Leu Ser Trp Val Arg Tyr Ala Val Asp
355 360 365
cac acg atc gac atc gat ccg agt caa ggt tgg gtg aac tgg ttg atg 1152
His Thr Ile Asp Ile Asp Pro Ser Gln Gly Trp Val Asn Trp Leu Met
370 375 380
ggc tac ctc aac tgc caa gtc atc cac cac ctc ttt ccg agc atg ccg 1200
Gly Tyr Leu Asn Cys Gln Val Ile His His Leu Phe Pro Ser Met Pro
385 390 395 400
cag ttc cgc cag ccc gag gta tct cgc cgc ttc gtc gcc ttt gcg aaa 1248
Gln Phe Arg Gln Pro Glu Val Ser Arg Arg Phe Val Ala Phe Ala Lys
405 410 415
aag tgg aac ctc aac tac aag gtc atg acc tac gcc ggt gcg tgg aag 1296
Lys Trp Asn Leu Asn Tyr Lys Val Met Thr Tyr Ala Gly Ala Trp Lys
420 425 430
gca acg ctc gga aac ctc gac aac gtg ggt aag cac tac tac gtg cac 1344
Ala Thr Leu Gly Asn Leu Asp Asn Val Gly Lys His Tyr Tyr Val His
435 440 445
ggc caa cac tcc gga aag acg gcg taa 1371
Gly Gln His Ser Gly Lys Thr Ala
450 455
<210>90
<211>456
<212>PRT
<213>Ostreococcus tauri
<400>90
Met Cys Val Glu Thr Glu Asn Asn Asp Gly Ile Pro Thr Val Glu Ile
1 5 10 15
Ala Phe Asp Gly Glu Arg Glu Arg Ala Glu Ala Asn Val Lys Leu Ser
20 25 30
Ala Glu Lys Met Glu Pro Ala Ala Leu Ala Lys Thr Phe Ala Arg Arg
35 40 45
Tyr Val Val Ile Glu Gly Val Glu Tyr Asp Val Thr Asp Phe Lys His
50 55 60
Pro Gly Gly Thr Val Ile Phe Tyr Ala Leu Ser Asn Thr Gly Ala Asp
65 70 75 80
Ala Thr Glu Ala Phe Lys Glu Phe His His Arg Ser Arg Lys Ala Arg
85 90 95
Lys Ala Leu Ala Ala Leu Pro Ser Arg Pro Ala Lys Thr Ala Lys Val
100 105 110
Asp Asp Ala Glu Met Leu Gln Asp Phe Ala Lys Trp Arg Lys Glu Leu
115 120 125
Glu Arg Asp Gly Phe Phe Lys Pro Ser Pro Ala His Val Ala Tyr Arg
130 135 140
Phe Ala Glu Leu Ala Ala Met Tyr Ala Leu Gly Thr Tyr Leu Met Tyr
145 150 155 160
Ala Arg Tyr Val Val Ser Ser Val Leu Val Tyr Ala Cys Phe Phe Gly
165 170 175
Ala Arg Cys Gly Trp Val Gln His Glu Gly Gly His Ser Ser Leu Thr
180 185 190
Gly Asn Ile Trp Trp Asp Lys Arg Ile Gln Ala Phe Thr Ala Gly Phe
195 200 205
Gly Leu Ala Gly Ser Gly Asp Met Trp Asn Ser Met His Asn Lys His
210 215 220
His Ala Thr Pro Gln Lys Val Arg His Asp Met Asp Leu Asp Thr Thr
225 230 235 240
Pro Ala Val Ala Phe Phe Asn Thr Ala Val Glu Asp Asn Arg Pro Arg
245 250 255
Gly Phe Ser Lys Tyr Trp Leu Arg Leu Gln Ala Trp Thr Phe Ile Pro
260 265 270
Val Thr Ser Gly Leu Val Leu Leu Phe Trp Met Phe Phe Leu His Pro
275 280 285
Ser Lys Ala Leu Lys Gly Gly Lys Tyr Glu Glu Leu Val Trp Met Leu
290 295 300
Ala Ala His Val Ile Arg Thr Trp Thr Ile Lys Ala Val Thr Gly Phe
305 310 315 320
Thr Ala Met Gln Ser Tyr Gly Leu Phe Leu Ala Thr Ser Trp Val Ser
325 330 335
Gly Cys Tyr Leu Phe Ala His Phe Ser Thr Ser His Thr His Leu Asp
340 345 350
Val Val Pro Ala Asp Glu His Leu Ser Trp Val Arg Tyr Ala Val Asp
355 360 365
His Thr Ile Asp Ile Asp Pro Ser Gln Gly Trp Val Asn Trp Leu Met
370 375 380
Gly Tyr Leu Asn Cys Gln Val Ile His His Leu Phe Pro Ser Met Pro
385 390 395 400
Gln Phe Arg Gln Pro Clu Val Ser Arg Arg Phe Val Ala Phe Ala Lys
405 410 415
Lys Trp Asn Leu Asn Tyr Lys Val Met Thr Tyr Ala Gly Ala Trp Lys
420 425 430
Ala Thr Leu Gly Asn Leu Asp Asn Val Gly Lys His Tyr Tyr Val His
435 440 445
Gly Gln His Ser Gly Lys Thr Ala
450 455
<210>91
<211>606
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(606)
<223>Δ5-去饱和酶
<400>91
atg tac ggt ttg cta tcg ctc aag tcg tgc ttc gtc gac gat ttc aac 48
Met Tyr Gly Leu Leu Ser Leu Lys Ser Cys Phe Val Asp Asp Phe Asn
1 5 10 15
gcc tac ttc tcc gga cgc atc ggc tgg gtc aag gtg atg aag ttc acc 96
Ala Tyr Phe Ser Gly Arg Ile Gly Trp Val Lys Val Met Lys Phe Thr
20 25 30
cgc ggc gag gcg atc gca ttt tgg ggc acc aag ctc ttg tgg gcc gcg 144
Arg Gly Glu Ala Ile Ala Phe Trp Gly Thr Lys Leu Leu Trp Ala Ala
35 40 45
tat tac ctc gcg ttg ccg cta aag atg tcg cat cgg ccg ctc gga gaa 192
Tyr Tyr Leu Ala Leu Pro Leu Lys Met Ser His Arg Pro Leu Gly Glu
50 55 60
ctc ctc gca ctc tgg gcc gtc acc gag ttc gtc acc gga tgg ctg ttg 240
Leu Leu Ala Leu Trp Ala Val Thr Glu Phe Val Thr Gly Trp Leu Leu
65 70 75 80
gcg ttc atg ttc caa gtc gcc cac gtc gtc ggc gag gtt cac ttc ttc 288
Ala Phe Met Phe Gln Val Ala His Val Val Gly Glu Val His Phe Phe
85 90 95
acc ctc gac gcg aag aac cgc gtg aac ttg gga tgg gga gag gca cag 336
Thr Leu Asp Ala Lys Asn Arg Val Asn Leu Gly Trp Gly Glu Ala Gln
100 105 110
ctc atg tcg agc gcg gat ttc gcc cac gga tcc aag ttt tgg acg cac 384
Leu Met Ser Ser Ala Asp Phe Ala His Gly Ser Lys Phe Trp Thr His
115 120 125
ttc tcc gga ggc tta aac tac caa gtc gtc cac cat ctc ttc ccg ggc 432
Phe Ser Gly Gly Leu Asn Tyr Gln Val Val His His Leu Phe Pro Gly
130 135 140
gtc tgc cac gtg cac tat ccc gcg ctc gcg cca att att aag gcg gca 480
Val Cys His Val His Tyr Pro Ala Leu Ala Pro Ile Ile Lys Ala Ala
145 150 155 160
gct gag aag cac ggc ctc cac tac cag att tac ccc acg ttt tgg tcc 528
Ala Glu Lys His Gly Leu His Tyr Gln Ile Tyr Pro Thr Phe Trp Ser
165 170 175
gcc ctg cgc gcg cac ttc cgg cac ctc gcc aac gtc ggc cgc gcc gcg 576
Ala Leu Arg Ala His Phe Arg His Leu Ala Asn Val Gly Arg Ala Ala
180 185 190
tac gta ccg tcc ctc caa acc gtc gga tga 606
Tyr Val Pro Ser Leu Gln Thr Val Gly
195 200
<210>92
<211>201
<212>PRT
<213>Ostreococcus tauri
<400>92
Met Tyr Gly Leu Leu Ser Leu Lys Ser Cys Phe Val Asp Asp Phe Asn
1 5 10 15
Ala Tyr Phe Ser Gly Arg Ile Gly Trp Val Lys Val Met Lys Phe Thr
20 25 30
Arg Gly Glu Ala Ile Ala Phe Trp Gly Thr Lys Leu Leu Trp Ala Ala
35 40 45
Tyr Tyr Leu Ala Leu Pro Leu Lys Met Ser His Arg Pro Leu Gly Glu
50 55 60
Leu Leu Ala Leu Trp Ala Val Thr Glu Phe Val Thr Gly Trp Leu Leu
65 70 75 80
Ala Phe Met Phe Gln Val Ala His Val Val Gly Glu Val His Phe Phe
85 90 95
Thr Leu Asp Ala Lys Asn Arg Val Asn Leu Gly Trp Gly Glu Ala Gln
100 105 110
Leu Met Ser Ser Ala Asp Phe Ala His Gly Ser Lys Phe Trp Thr His
115 120 125
Phe Ser Gly Gly Leu Asn Tyr Gln Val Val His His Leu Phe Pro Gly
130 135 140
Val Cys His Val His Tyr Pro Ala Leu Ala Pro Ile Ile Lys Ala Ala
145 150 155 160
Ala Glu Lys His Gly Leu His Tyr Gln Ile Tyr Pro Thr Phe Trp Ser
165 170 175
Ala Leu Arg Ala His Phe Arg His Leu Ala Asn Val Gly Arg Ala Ala
180 185 190
Tyr Val Pro Ser Leu Gln Thr Val Gly
195 200
<210>93
<211>714
<212>DNA
<213>Ostreococcus tauri
<220>
<22l>CDS
<222>(1)..(714)
<223>Δ5-去饱和酶
<400>93
atg gtg agc cat cac tcg tac tgt aac gac gcg gat ttg gat cag gat 48
Met Val Ser His His Ser Tyr Cys Asn Asp Ala Asp Leu Asp Gln Asp
1 5 10 15
gtg tac acc gca ctg ccg ctc ctg cgc ctg gac ccg tct cag gag ttg 96
Val Tyr Thr Ala Leu Pro Leu Leu Arg Leu Asp Pro Ser Gln Glu Leu
20 25 30
aag tgg ttt cat cga tac cag gcg ttt tac gcc ccg ctc atg tgg ccg 144
Lys Trp Phe His Arg Tyr Gln Ala Phe Tyr Ala Pro Leu Met Trp Pro
35 40 45
ttt ttg tgg ctc gcg gcg cag ttt ggc gac gcg cag aac atc ctg atc 192
Phe Leu Trp Leu Ala Ala Gln Phe Gly Asp Ala Gln Asn Ile Leu Ile
50 55 60
gac cga gcg tcg ccg ggc gtc gcg tac aag gga ttg atg gcg aac gag 240
Asp Arg Ala Ser Pro Gly Val Ala Tyr Lys Gly Leu Met Ala Asn Glu
65 70 75 80
gtc gcg ctg tac gtt ctc ggt aag gtt tta cac ttt ggt ctt ctc ctc 288
Val Ala Leu Tyr Val Leu Gly Lys Val Leu His Phe Gly Leu Leu Leu
85 90 95
ggc gtt cct gcg tac ttg cac gga ttg tcc aac gcg atc gtt cca ttc 336
Gly Val Pro Ala Tyr Leu His Gly Leu Ser Asn Ala Ile Val Pro Phe
100 105 110
ttg gcg tac ggc gca ttc ggc tcc ttc gtc ctg tgc tgg ttc ttc atc 384
Leu Ala Tyr Gly Ala Phe Gly Ser Phc Val Leu Cys Trp Phe Phe Ile
115 120 125
gtc agc cat aac ctc gaa gcg ctg aca ccc gtt aac ctt aac aag tcc 432
Val Ser His Asn Leu Glu Ala Leu Thr Pro Val Asn Leu Asn Lys Ser
130 135 140
acg aag aac gac tgg ggg gcg tgg cag atc gag aca tcg gcg tct tgg 480
Thr Lys Asn Asp Trp Gly Ala Trp Gln Ile Glu Thr Ser Ala Ser Trp
145 150 155 160
ggc aac gcg ttc tgg agc ttc ttc tct gga ggt ctg aac ctg caa atc 528
Gly Asn Ala Phe Trp Ser Phe Phe Ser Gly Gly Leu Asn Leu Gln Ile
165 170 175
gag cac cac ctc ttc ccg ggc atg gcg cac aac ctg tac ccg aag atg 576
Glu His His Leu Phe Pro Gly Met Ala His Asn Leu Tyr Pro Lys Met
180 185 190
gtg ccg atc atc aag gac gag tgt gcg aaa gcg ggc gtt cgc tac acc 624
Val Pro Ile Ile Lys Asp Glu Cys Ala Lys Ala Gly Val Arg Tyr Thr
195 200 205
ggt tac ggt ggc tac acc ggc ctg ctc ccg atc acc cgc gac atg ttc 672
Gly Tyr Gly Gly Tyr Thr Gly Leu Leu Pro Ile Thr Arg Asp Met Phe
210 215 220
tcc tac ctc cat aag tgt ggc cga acg gcg aaa cta gcc taa 714
Ser Tyr Leu His Lys Cys Gly Arg Thr Ala Lys Leu Ala
225 230 235
<210>94
<211>237
<212>PRT
<213>Ostreococcus tauri
<400>94
Met Val Ser His His Ser Tyr Cys Asn Asp Ala Asp Leu Asp Gln Asp
1 5 10 15
Val Tyr Thr Ala Leu Pro Leu Leu Arg Leu Asp Pro Ser Gln Glu Leu
20 25 30
Lys Trp Phe His Arg Tyr Gln Ala Phe Tyr Ala Pro Leu Met Trp Pro
35 40 45
Phe Leu Trp Leu Ala Ala Gln Phe Gly Asp Ala Gln Asn Ile Leu Ile
50 55 60
Asp Arg Ala Ser Pro Gly Val Ala Tyr Lys Gly Leu Met Ala Asn Glu
65 70 75 80
Val Ala Leu Tyr Val Leu Gly Lys Val Leu His Phe Gly Leu Leu Leu
85 90 95
Gly Val Pro Ala Tyr Leu His Gly Leu Ser Asn Ala Ile Val Pro Phe
100 105 110
Leu Ala Tyr Gly Ala Phe Gly Ser Phe Val Leu Cys Trp Phe Phe Ile
115 120 125
Val Ser His Asn Leu Glu Ala Leu Thr Pro Val Asn Leu Asn Lys Ser
130 135 140
Thr Lys Asn Asp Trp Gly Ala Trp Gln Ile Glu Thr Ser Ala Ser Trp
145 150 155 160
Gly Asn Ala Phe Trp Ser Phe Phe Ser Gly Gly Leu Asn Leu Gln Ile
165 170 175
Glu His His Leu Phe Pro Gly Met Ala His Asn Leu Tyr Pro Lys Met
180 185 190
Val Pro Ile Ile Lys Asp Glu Cys Ala Lys Ala Gly Val Arg Tyr Thr
195 200 205
Gly Tyr Gly Gly Tyr Thr Gly Leu Leu Pro Ile Thr Arg Asp Met Phe
210 215 220
Ser Tyr Leu His Lys Cys Gly Arg Thr Ala Lys Leu Ala
225 230 235
<210>95
<211>1611
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(1611)
<223>Δ4-去饱和酶
<400>95
atg tac ctc gga cgc ggc cgt ctc gag agc ggg acg acg cga ggg atg 48
Met Tyr Leu GIy Arg Gly Arg Leu Glu Ser Gly Thr Thr Arg Gly Met
1 5 10 15
atg cgg acg cac gcg cgg cga ccg tcg acg acg tcg aat ccg tgc gcg 96
Met Arg Thr His Ala Arg Arg Pro Ser Thr Thr Ser Asn Pro Cys Ala
20 25 30
cgg tca cgc gtg cgt aag acg acg gag cga tcg ctc gcg cga gtg cga 144
Arg Ser Arg Val Arg Lys Thr Thr Glu Arg Ser Leu Ala Arg Val Arg
35 40 45
cga tcg acg agt gag aag gga agc gcg ctc gtg ctc gag cga gag agc 192
Arg Ser Thr Ser Glu Lys Gly Ser Ala Leu Val Leu Glu Arg Glu Ser
50 55 60
gaa cgg gag aag gag gag gga ggg aaa gcg cga gcg gag gga ttg cga 240
Glu Arg Glu Lys Glu Glu Gly Gly Lys Ala Arg Ala Glu Gly Leu Arg
65 70 75 80
ttc caa cgc ccg gac gtc gcc gcg ccg ggg gga gcg gat cct tgg aac 288
Phe Gln Arg Pro Asp Val Ala Ala Pro Gly Gly Ala Asp Pro Trp Asn
85 90 95
gac gag aag tgg aca aag acc aag tgg acg gta ttc aga gac gtc gcg 336
Asp Glu Lys Trp Thr Lys Thr Lys Trp Thr Val Phe Arg Asp Val Ala
100 105 110
tac gat ctc gat cct ttc ttc gct cga cac ccc gga gga gac tgg ctc 384
Tyr Asp Leu Asp Pro Phe Phe Ala Arg His Pro Gly Gly Asp Trp Leu
115 120 125
ctg aac ttg gcc gtg gga cga gac tgc acc gcg ctc atc gaa tcc tat 432
Leu Asn Leu Ala Val Gly Arg Asp Cys Thr Ala Leu Ile Glu Ser Tyr
130 135 140
cac ttg cga cca gag gtg gcg acg gct cgt ttc aga atg ctg ccc aaa 480
His Leu Arg Pro Glu Val Ala Thr Ala Arg Phe Arg Met Leu Pro Lys
145 150 155 160
ctc gag gat ttt ccc gtc gag gcc gtg ccc aag tcc ccg aga ccg aac 528
Leu Glu Asp Phe Pro Val Glu Ala Val Pro Lys Ser Pro Arg Pro Asn
165 170 175
gat tcg ccg tta tac aac aac att cgc aac cga gtc cgc gaa gag ctc 576
Asp Ser Pro Leu Tyr Asn Asn Ile Arg Asn Arg Val Arg Glu Glu Leu
180 185 190
ttc cca gag gag gga aag aat atg cac aga cag ggc ggc gac cac ggc 624
Phe Pro Glu Glu Gly Lys Asn Met His Arg Gln Gly Gly Asp His Gly
195 200 205
gac ggt gac gat tct ggg ttt cgc cgc ctt ttg ctt atg ccg tgt acc 672
Asp Gly Asp Asp Ser Gly Phe Arg Arg Leu Leu Leu Met Pro Cys Thr
210 215 220
tat tcc ctt ccg ggg gtt cct ttc cgg ctg cct cct cgg gtc tcg cgg 720
Tyr Ser Leu Pro Gly Val Pro Phe Arg Leu Pro Pro Arg Val Ser Arg
225 230 235 240
ggg cgt gga ttg gtc tca cga ttc agg cac tgc gcc aac cac ggc gcg 768
Gly Arg Gly Leu Val Ser Arg Phe Arg His Cys Ala Asn His Gly Ala
245 250 255
atg tct cct tcg ccg gcc gtt aac ggc gtc ctc ggt ttg acg aac gat 816
Met Ser Pro Ser Pro Ala Val Asn Gly Val Leu Gly Leu Thr Asn Asp
260 265 270
ctc atc ggc ggc tcg tcc ttg atg tgg aga tat cac cac caa gtc agc 864
Leu Ile Gly Gly Ser Ser Leu Met Trp Arg Tyr His His Gln Val Ser
275 280 285
cac cac att cat tgc aac gac aac gcc atg gat caa gac gtg tac acg 912
His His Ile His Cys Asn Asp Asn Ala Met Asp Gln Asp Val Tyr Thr
290 295 300
gcg atg cca tta ttg cgt ttc gac gct cgc cgg ccc aag tcc tgg tac 960
Ala Met Pro Leu Leu Arg Phe Asp Ala Arg Arg Pro Lys Ser Trp Tyr
305 310 315 320
cat cgc ttc cag cag tgg tac atg ttt tta gcg ttc ccg ttg ttg cag 1008
His Arg Phe Gln Gln Trp Tyr Met Phe Leu Ala Phe Pro Leu Leu Gln
325 330 335
gtt gcc ttc caa gtc gga gac att gcc gca ctg ttc acg cgt gat acc 1056
Val Ala Phe Gln Val Gly Asp Ile Ala Ala Leu Phe Thr Arg Asp Thr
340 345 350
gaa ggc gct aag ctt cac ggg gcg acg acg tgg gag ctt acc acg gtt 1104
Glu Gly Ala Lys Leu His Gly Ala Thr Thr Trp Glu Leu Thr Thr Val
355 360 365
gtc ctc ggt aag att gtg cac ttc ggt ctt ttg ttg ggg ccg ttg atg 1152
Val Leu Gly Lys Ile Val His Phe Gly Leu Leu Leu Gly Pro Leu Met
370 375 380
aac cac gcg gtg agt tct gtt ttg ctg ggg atc gtc ggt ttc atg gcg 1200
Asn His Ala Val Ser Ser Val Leu Leu Gly Ile Val Gly Phe Met Ala
385 390 395 400
tgc caa ggt ata gtt ctg gcg tgc acg ttt gct gtg agt cac aat gtc 1248
Cys Gln Gly Ile Val Leu Ala Cys Thr Phe Ala Val Ser His Asn Val
405 410 415
gcg gag gcg aag ata cct gag gac acc gga gga gaa gcc tgg gag aga 1296
Ala Glu Ala Lys Ile Pro Glu Asp Thr Gly Gly Glu Ala Trp Glu Arg
420 425 430
gat tgg ggt gtc cag cag ttg gtg act agc gcc gac tgg ggt gga aag 1344
Asp Trp Gly Val Gln Gln Leu Val Thr Ser Ala Asp Trp Gly Gly Lys
435 440 445
ata ggt aac ttc ttc acg ggt ggc ctc aac ttg caa gtt gag cac cac 1392
Ile Gly Asn Phe Phe Thr Gly Gly Leu Asn Leu Gln Val Glu His His
450 455 460
ttg ttt ccg gcg att tgc ttc gtc cac tac ccg gac atc gcg aag atc 1440
Leu Phe Pro Ala Ile Cys Phe Val His Tyr Pro Asp Ile Ala Lys Ile
465 470 475 480
gtg aag gaa gaa gcg gcc aag ctc aac atc cct tac gcg tct tac agg 1488
Val Lys Glu Glu Ala Ala Lys Leu Asn Ile Pro Tyr Ala Ser Tyr Arg
485 490 495
act ctt cct ggt att ttc gtc caa ttc tgg aga ttt atg aag gac atg 1536
Thr Leu Pro Gly Ile Phe Val Gln Phe Trp Arg Phe Met Lys Asp Met
500 505 510
ggc acg gct gag caa att ggt gaa gtt cca ttg ccg aag att ccc aac 1584
Gly Thr Ala Glu Gln Ile Gly Glu Val Pro Leu Pro Lys Ile Pro Asn
515 520 525
ccg cag ctc gcg ccg aag ctc gct tag 1611
Pro Gln Leu Ala Pro Lys Leu Ala
530 535
<210>96
<211>536
<212>PRT
<213>Ostreococcus tauri
<400>96
Met Tyr Leu Gly Arg Gly Arg Leu Glu Ser Gly Thr Thr Arg Gly Met
1 5 10 15
Met Arg Thr His Ala Arg Arg Pro Ser Thr Thr Ser Asn Pro Cys Ala
20 25 30
Arg Ser Arg Val Arg Lys Thr Thr Glu Arg Ser Leu Ala Arg Val Arg
35 40 45
Arg Ser Thr Ser Glu Lys Gly Ser Ala Leu Val Leu Glu Arg Glu Ser
50 55 60
Glu Arg Glu Lys Glu Glu Gly Gly Lys Ala Arg Ala Glu Gly Leu Arg
65 70 75 80
Phe Gln Arg Pro Asp Val Ala Ala Pro Gly Gly Ala Asp Pro Trp Asn
85 90 95
Asp Glu Lys Trp Thr Lys Thr Lys Trp Thr Val Phe Arg Asp Val Ala
100 105 110
Tyr Asp Leu Asp Pro Phe Phe Ala Arg His Pro Gly Gly Asp Trp Leu
115 120 125
Leu Asn Leu Ala Val Gly Arg Asp Cys Thr Ala Leu Ile Glu Ser Tyr
130 135 140
His Leu Arg Pro Glu Val Ala Thr Ala Arg Phe Arg Met Leu Pro Lys
145 150 155 160
Leu Glu Asp Phe Pro Val Glu Ala Val Pro Lys Ser Pro Arg Pro Asn
165 170 175
Asp Ser Pro Leu Tyr Asn Asn Ile Arg Asr Arg Val Arg Glu Glu Leu
180 185 190
Phe Pro Glu Glu Gly Lys Asn Met His Arg Gln Gly Gly Asp His Gly
195 200 205
Asp Gly Asp Asp Ser Gly Phe Arg Arg Leu Leu Leu Met Pro Cys Thr
210 215 220
Tyr Ser Leu Pro Gly Val Pro Phe Arg Leu Pro Pro Arg Val Ser Arg
225 230 235 240
Gly Arg Gly Leu Val Ser Arg Phe Arg His Cys Ala Asn His Gly Ala
245 250 255
Met Ser Pro Ser Pro Ala Val Asn Gly Val Leu Gly Leu Thr Asn Asp
260 265 270
Leu Ile Gly Gly Ser Ser Leu Met Trp Arg Tyr His His Gln Val Ser
275 280 285
His His Ile His Cys Asn Asp Asn Ala Met Asp Gln Asp Val Tyr Thr
290 295 300
Ala Met Pro Leu Leu Arg Phe Asp Ala Arg Arg Pro Lys Ser Trp Tyr
305 310 315 320
His Arg Phe Gln Gln Trp Tyr Met Phe Leu Ala Phe Pro Leu Leu Gln
325 330 335
Val Ala Phe Gln Val Gly Asp Ile Ala Ala Leu Phe Thr Arg Asp Thr
340 345 350
Glu Gly Ala Lys Leu His Gly Ala Thr Thr Trp Glu Leu Thr Thr Val
355 360 365
Val Leu Gly Lys Ile Val His Phe Gly Leu Leu Leu Gly Pro Leu Met
370 375 380
Asn His Ala Val Ser Ser Val Leu Leu Gly Ile Val Gly Phe Met Ala
385 390 395 400
Cys Gln Gly Ile Val Leu Ala Cys Thr Phe Ala Val Ser His Asn Val
405 410 415
Ala Glu Ala Lys Ile Pro Glu Asp Thr Gly Gly Glu Ala Trp Glu Arg
420 425 430
Asp Trp Gly Val Gln Gln Leu Val Thr Ser Ala Asp Trp Gly Gly Lys
435 440 445
Ile Gly Asn Phe Phe Thr Gly Gly Leu Asn Leu Gln Val Glu His His
450 455 460
Leu Phe Pro Ala Ile Cys Phe Val His Tyr Pro Asp Ile Ala Lys Ile
465 470 475 480
Val Lys Glu Glu Ala Ala Lys Leu Asn Ile Pro Tyr Ala Ser Tyr Arg
485 490 495
Thr Leu Pro Gly Ile Phe Val Gln Phe Trp Arg Phe Met Lys Asp Met
500 505 510
Gly Thr Ala Glu Gln Ile Gly Glu Val Pro Leu Pro Lys Ile Pro Asn
515 520 525
Pro Gln Leu Ala Pro Lys Leu Ala
530 535
<210>97
<211>1455
<212>DNA
<213>假矮海链藻
<220>
<22l>CDS
<222>(1)..(1455)
<223>Δ6-去饱和酶
<400>97
atg gga aaa gga gga gac gca gcc gca gct acc aag cgt agt gga gca 48
Met Gly Lys Gly Gly Asp Ala Ala Ala Ala Thr Lys Arg Ser Gly Ala
1 5 10 15
ttg aaa ttg gcg gag aag ccg cag aag tac act tgg cag gag gtg aag 96
Leu Lys Leu Ala Glu Lys Pro Gln Lys Tyr Thr Trp Gln Glu Val Lys
20 25 30
aag cac atc acc ccc gac gat gcc tgg gta gtc cac caa aac aaa gtc 144
Lys His Ile Thr Pro Asp Asp Ala Trp Val Val His Gln Asn Lys Val
35 40 45
tac gac gtc tcc aac tgg tac gac cac ccc ggt gga gcc gtg gtg ttc 192
Tyr Asp Val Ser Asn Trp Tyr Asp His Pro Gly Gly Ala Val Val Phe
50 55 60
acc cac gcc gga gac gac atg acg gac atc ttc gcc gcc ttc cac gcc 240
Thr His Ala Gly Asp Asp Met Thr Asp Ile Phe Ala Ala Phe His Ala
65 70 75 80
caa ggc tct cag gcc atg atg aag aag ttt tac att gga gat ttg att 288
Gln Gly Ser Gln Ala Met Met Lys Lys Phe Tyr Ile Gly Asp Leu Ile
85 90 95
Ccg gag agt gtg gag cat aag gat caa aga cag ttg gat ttc gag aag 336
Pro Glu Ser Val Glu His Lys Asp Gln Arg Gln Leu Asp Phe Glu Lys
100 105 110
gga tat cgt gat tta cgg gcc aag ctt gtc atg atg ggg atg ttc aag 384
Gly Tyr Arg Asp Leu Arg Ala Lys Leu Val Met Met Gly Met Phe Lys
115 120 125
tcg agt aag atg tat tat gca tac aag tgc tcg ttc aat atg tgc atg 432
Ser Ser Lys Met Tyr Tyr Ala Tyr Lys Cys Ser Phe Asn Met Cys Met
130 135 140
tgg ttg gtg gcg gtg gcc atg gtg tac tac tcg gac agt ttg gca atg 480
Trp Leu Val Ala Val Ala Met Val Tyr Tyr Ser Asp Ser Leu Ala Met
145 150 155 160
cac att gga tcg gct ctc ttg ttg gga ttg ttc tgg cag cag tgt gga 528
His Ile Gly Ser Ala Leu Leu Leu Gly Leu Phe Trp Gln Gln Cys Gly
165 170 175
tgg ctt gcg cac gac ttt ctt cac cac caa gtc ttt aag caa cga aag 576
Trp Leu Ala His Asp Phe Leu His His Gln Val Phe Lys Gln Arg Lys
180 185 190
tac gga gat ctc gtt ggc atc ttt tgg gga gat ctc atg cag ggg ttc 624
Tyr Gly Asp Leu Val Gly Ile Phe Trp Gly Asp Leu Met Gln Gly Phe
195 200 205
tcg atg cag tgg tgg aag aac aag cac aat ggc cac cat gct gtt ccc 672
Ser Met Gln Trp Trp Lys Asn Lys His Asn Gly His His Ala Val Pro
210 215 220
aac ttg cac aac tct tcc ttg gac agt cag gat ggt gat ccc gat att 720
Asn Leu His Asn Ser Ser Leu Asp Ser Gln Asp Gly Asp Pro Asp Ile
225 230 235 240
gat acc atg cca ctc ctt gct tgg agt ctc aag cag gct cag agt ttc 768
Asp Thr Met Pro Leu Leu Ala Trp Ser Leu Lys Gln Ala Gln Ser Phe
245 250 255
aga gag atc aat aag gga aag gac agt acc ttc gtc aag tac gct atc 816
Arg Glu Ile Asn Lys Gly Lys Asp Ser Thr Phe Val Lys Tyr Ala Ile
260 265 270
aaa ttc cag gca ttc aca tac ttc ccc atc ctc ctc ttg gct cgc atc 864
Lys Phe Gln Ala Phe Thr Tyr Phe Pro Ile Leu Leu Leu Ala Arg Ile
275 280 285
tct tgg ttg aat gaa tcc ttc aaa act gca ttc gga ctc gga gct gcc 912
Ser Trp Leu Asn Glu Ser Phe Lys Thr Ala Phe Gly Leu Gly Ala Ala
290 295 300
tcg gag aat gcc aag ttg gag ttg gag aag cgt gga ctt cag tac cca 960
Ser Glu Asn Ala Lys Leu Glu Leu Glu Lys Arg Gly Leu Gln Tyr Pro
305 310 315 320
ctt ttg gag aag ctt gga atc acc ctt cat tac act tgg atg ttc gtc 1008
Leu Leu Glu Lys Leu Gly Ile Thr Leu His Tyr Thr Trp Met Phe Val
325 330 335
ctc tct tcc gga ttt gga agg tgg tct ctt cca tat tcc atc atg tat 1056
Leu Ser Ser Gly Phe Gly Arg Trp Ser Leu Pro Tyr Ser Ile Met Tyr
340 345 350
ttc ttc act gcc aca tgc tcc tcg gga ctt ttc ctc gca ttg gtc ttt 1104
Phe Phe Thr Ala Thr Cys Ser Ser Gly Leu Phe Leu Ala Leu Val Phe
355 360 365
gga ttg gga cac aac ggt atg tca gtg tac gat gcc acc acc cga cct 1152
Gly Leu Gly His Asn Gly Met Ser Val Tyr Asp Ala Thr Thr Arg Pro
370 375 380
gac ttc tgg caa ctc caa gtc acc act aca cgt aac atc att ggt gga 1200
Asp Phe Trp Gln Leu Gln Val Thr Thr Thr Arg Asn Ile Ile Gly Gly
385 390 395 400
cac ggc att ccc caa ttc ttt gtg gat tgg ttc tgc ggt gga ttg caa 1248
His Gly Ile Pro Gln Phe Phe Val Asp Trp Phe Cys Gly Gly Leu Gln
405 410 415
tac caa gtg gat cac cac ctc ttc ccc atg atg cct aga aac aat atc 1296
Tyr Gln Val Asp His His Leu Phe Pro Met Met Pro Arg Asn Asn Ile
420 425 430
gcg aaa tgc cac aag ctt gtg gag tca ttc tgt aag gag tgg ggt gtg 1344
Ala Lys Cys His Lys Leu Val Glu Ser Phe Cys Lys Glu Trp Gly Val
435 440 445
aag tac cat gag gcc gat atg tgg gat ggt acc gtg gaa gtg ttg caa 1392
Lys Tyr His Glu Ala Asp Met Trp Asp Gly Thr Val Glu Val Leu Gln
450 455 460
cat ctc tcc aag gtg tcg gat gat ttc ctt gtg gag atg gtg aag gat 1440
His Leu Ser Lys Val Ser Asp Asp Phe Leu Val Glu Met Val Lys Asp
465 470 475 480
ttc cct gcc atg taa 1455
Phe Pro Ala Met
<210>98
<211>484
<212>PRT
<213>假矮海链藻
<400>98
Met Gly Lys Gly Gly Asp Ala Ala Ala Ala Thr Lys Arg Ser Gly Ala
1 5 10 15
Leu Lys Leu Ala Glu Lys Pro Gln Lys Tyr Thr Trp Gln Glu Val Lys
20 25 30
Lys His Ile Thr Pro Asp Asp Ala Trp Val Val His Gln Asn Lys Val
35 40 45
Tyr Asp Val Ser Asn Trp Tyr Asp His Pro Gly Gly Ala Val Val Phe
50 55 60
Thr His Ala Gly Asp Asp Met Thr Asp Ile Phe Ala Ala Phe His Ala
65 70 75 80
Gln Gly Ser Gln Ala Met Met Lys Lys Phe Tyr Ile Gly Asp Leu Ile
85 90 95
Pro Glu Ser Val Glu His Lys Asp Gln Arg Gln Leu Asp Phe Glu Lys
100 105 110
Gly Tyr Arg Asp Leu Arg Ala Lys Leu Val Met Met Gly Met Phe Lys
115 120 125
Ser Ser Lys Met Tyr Tyr Ala Tyr Lys Cys Ser Phe Asn Met Cys Met
130 135 140
Trp Leu Val Ala Val Ala Met Val Tyr Tyr Ser Asp Ser Leu Ala Met
145 150 155 160
His Ile Gly Ser Ala Leu Leu Leu Gly Leu Phe Trp Gln Gln Cys Gly
165 170 175
Trp Leu Ala His Asp Phe Leu His His Gln Val Phe Lys Gln Arg Lys
180 185 190
Tyr Gly Asp Leu Val Gly Ile Phe Trp Gly Asp Leu Met Gln Gly Phe
195 200 205
Ser Met Gln Trp Trp Lys Asn Lys His Asn Gly His His Ala Val Pro
210 215 220
Asn Leu His Asn Ser Ser Leu Asp Ser Gln Asp Gly Asp Pro Asp Ile
225 230 235 240
Asp Thr Met Pro Leu Leu Ala Trp Ser Leu Lys Gln Ala Gln Ser Phe
245 250 255
Arg Glu Ile Asn Lys Gly Lys Asp Ser Thr Phe Val Lys Tyr Ala Ile
260 265 270
Lys Phe Gln Ala Phe Thr Tyr Phe Pro Ile Leu Leu Leu Ala Arg Ile
275 280 285
Ser Trp Leu Asn Glu Ser Phe Lys Thr Ala Phe Gly Leu Gly Ala Ala
290 295 300
Ser Glu Asn Ala Lys Leu Glu Leu Glu Lys Arg Gly Leu Gln Tyr Pro
305 310 315 320
Leu Leu Glu Lys Leu Gly Ile Thr Leu His Tyr Thr Trp Met Phe Val
325 330 335
Leu Ser Ser Gly Phe Gly Arg Trp Ser Leu Pro Tyr Ser Ile Met Tyr
340 345 350
Phe Phe Thr Ala Thr Cys Ser Ser Gly Leu Phe Leu Ala Leu Val Phe
355 360 365
Gly Leu Gly His Asn Gly Met Ser Val Tyr Asp Ala Thr Thr Arg Pro
370 375 380
Asp Phe Trp Gln Leu Gln Val Thr Thr Thr Arg Asn Ile Ile Gly Gly
385 390 395 400
His Gly Ile Pro Gln Phe Phe Val Asp Trp Phe Cys Gly Gly Leu Gln
405 410 415
Tyr Gln Val Asp His His Leu Phe Pro Met Met Pro Arg Asn Asn Ile
420 425 430
Ala Lys Cys His Lys Leu Val Glu Ser Phe Cys Lys Glu Trp Gly Val
435 440 445
Lys Tyr His Glu Ala Asp Met Trp Asp Gly Thr Val Glu Val Leu Gln
450 455 460
His Leu Ser Lys Val Ser Asp Asp Phe Leu Val Glu Met Val Lys Asp
465 470 475 480
Phe Pro Ala Met
<210>99
<211>1431
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1431)
<223>Δ5-去饱和酶
<400>99
atg ccc ccc aac gcc gat atc tcc cgc atc cgc aac cgc atc ccc acc 48
Met Pro Pro Asn Ala Asp Ile Ser Arg Ile Arg Asn Arg Ile Pro Thr
1 5 10 15
aaa aca ggt acc gtt gcc tct gcc gac aac aac gac ccc gcc acc caa 96
Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala Thr Gln
20 25 30
tcc gtc cga acc ctc aaa tct ctc aag ggc aac gag gtc gtc atc aac 144
Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn
35 40 45
ggc aca att tat gac att gct gac ttt gtc cat cct gga gga gag gtt 192
Gly Thr Ile Tyr Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val
50 55 60
gtc aag ttc ttt ggt ggg aat gat gtt act att cag tat aat atg att 240
Val Lys Phe Phe Gly Gly Asn Asp Val Thr Ile Gln Tyr Asn Met Ile
65 70 75 80
cat ccg tat cat acg ggg aaa cat ctg gag aag atg aag gct gtt gga 288
His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met Lys Ala Val Gly
85 90 95
aag gtt gta gat tgg cag tcg gac tac aag ttc gac acc ccc ttt gaa 336
Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu
100 105 110
cga gag atc aaa tca gaa gtg ttc aag atc gta cgt cgc ggg cgt gag 384
Arg Glu Ile Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu
115 120 125
ttc ggc aca aca ggc tac ttc ctc cgt gcc ttt ttc tac atc gct ctc 432
Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala Phe Phe Tyr Ile Ala Leu
130 135 140
ttc ttc acc atg caa tac act ttc gcc aca tgc acc acc ttc acc acc 480
Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr Phe Thr Thr
145 150 155 160
tac gat cac tgg tat cag agt ggt gta ttc atc gca att gtg ttt ggt 528
Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly
165 170 175
att tca cag gca ttc att ggg ttg aat gtc cag cac gat gcc aat cac 576
Ile Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His
180 185 190
gga gct gcc agt aag cgt ccc tgg gtg aat gac ttg ttg gga ttt gga 624
Gly Ala Ala Ser Lys Arg Pro Trp Val Asn Asp Leu Leu Gly Phe Gly
195 200 205
acg gat ttg att gga tct aac aaa tgg aat tgg atg gca cag cat tgg 672
Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln His Trp
210 215 220
act cat cac gct tac act aac cat agt gag aag gat ccc gat agc ttc 720
Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe
225 230 235 240
agc tcg gaa cct atg ttt gca ttc aat gac tat ccc att gga cac ccg 768
Ser Ser Glu Pro Met Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro
245 250 255
aag aga aag tgg tgg cat agg ttc cag gga ggg tac ttc ctc ttc atg 816
Lys Arg Lys Trp Trp His Arg Phe Gln Gly Gly Tyr Phe Leu Phe Met
260 265 270
ctt gga ctt tac tgg ctc tcg act gta ttc aat ccg caa ttc att gat 864
Leu Gly Leu Tyr Trp Leu Ser Thr Val Phe Asn Pro Gln Phe Ile Asp
275 280 285
ctt cgt caa cgt ggg gct cag tac gtc gga att caa atg gag aat gat 912
Leu Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp
290 295 300
ttc att gtc aag agg agg aag tac gcc gtt gca ttg agg atg atg tac 960
Phe Ile Val Lys Arg Arg Lys Tyr Ala Val Ala Leu Arg Met Met Tyr
305 310 315 320
att tac ttg aac att gtc agc ccc ttc atg aac aat ggt ttg agc tgg 1008
Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn Asn Gly Leu Ser Trp
325 330 335
tct acc ttt gga atc atc atg ttg atg gga atc agc gag agt ctc act 1056
Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr
340 345 350
ctc agt gtg ctc ttc tcg ttg tct cac aac ttc atc aat tcg gat cgt 1104
Leu Ser Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg
355 360 365
gat cct acg gct gac ttc aaa aag acc gga gaa caa gtg tgc tgg ttc 1152
Asp Pro Thr Ala Asp Phe Lys Lys Thr Gly Glu Gln Val Cys Trp Phe
370 375 380
aag tcg cag gtg gag act tcg tct acc tat ggg ggt ttt att tcc gga 1200
Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly
385 390 395 400
tgt ctt acg gga gga ctc aac ttt cag gtg gaa cat cat ctc ttt ccc 1248
Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro
405 410 415
cgt atg agc agt gct tgg tat cct tac att gca cct acg gtt cgt gag 1296
Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu
420 425 430
gtt tgc aag aag cac ggg gtg aac tac gct tat tat cct tgg att ggg 1344
Val Cys Lys Lys His Gly Val Asn Tyr Ala Tyr Tyr Pro Trp Ile Gly
435 440 445
cag aat ttg gta tca aca ttc aaa tac atg cat cgc gct ggt agt gga 1392
Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala Gly Ser Gly
450 455 460
gcc aac tgg gag ctc aag ccg ttg tct gga agt gcc taa 1431
Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala
465 470 475
<210>100
<211>476
<212>PRT
<213>假矮海链藻
<400>100
Met Pro Pro Asn Ala Asp Ile Ser Arg Ile Arg Asn Arg Ile Pro Thr
1 5 10 15
Lys Thr Gly Thr Val Ala Ser Ala Asp Asn Asn Asp Pro Ala Thr Gln
20 25 30
Ser Val Arg Thr Leu Lys Ser Leu Lys Gly Asn Glu Val Val Ile Asn
35 40 45
Gly Thr Ile Tyr Asp Ile Ala Asp Phe Val His Pro Gly Gly Glu Val
50 55 60
Val Lys Phe Phe Gly Gly Asn Asp Val Thr Ile Gln Tyr Asn Met Ile
65 70 75 80
His Pro Tyr His Thr Gly Lys His Leu Glu Lys Met Lys Ala Val Gly
85 90 95
Lys Val Val Asp Trp Gln Ser Asp Tyr Lys Phe Asp Thr Pro Phe Glu
100 105 110
Arg Glu Ile Lys Ser Glu Val Phe Lys Ile Val Arg Arg Gly Arg Glu
115 120 125
Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala Phe Phe Tyr Ile Ala Leu
130 135 140
Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr Cys Thr Thr Phe Thr Thr
145 150 155 160
Tyr Asp His Trp Tyr Gln Ser Gly Val Phe Ile Ala Ile Val Phe Gly
165 170 175
Ile Ser Gln Ala Phe Ile Gly Leu Asn Val Gln His Asp Ala Asn His
180 185 190
Gly Ala Ala Ser Lys Arg Pro Trp Val Asn Asp Leu Leu Gly Phe Gly
195 200 205
Thr Asp Leu Ile Gly Ser Asn Lys Trp Asn Trp Met Ala Gln His Trp
210 215 220
Thr His His Ala Tyr Thr Asn His Ser Glu Lys Asp Pro Asp Ser Phe
225 230 235 240
Ser Ser Glu Pro Met Phe Ala Phe Asn Asp Tyr Pro Ile Gly His Pro
245 250 255
Lys Arg Lys Trp Trp His Arg Phe Gln Gly Gly Tyr Phe Leu Phe Met
260 265 270
Leu Gly Leu Tyr Trp Leu Ser Thr Val Phe Asn Pro Gln Phe Ile Asp
275 280 285
Leu Arg Gln Arg Gly Ala Gln Tyr Val Gly Ile Gln Met Glu Asn Asp
290 295 300
Phe Ile Val Lys Arg Arg Lys Tyr Ala Val Ala Leu Arg Met Met Tyr
305 310 315 320
Ile Tyr Leu Asn Ile Val Ser Pro Phe Met Asn Asn Gly Leu Ser Trp
325 330 335
Ser Thr Phe Gly Ile Ile Met Leu Met Gly Ile Ser Glu Ser Leu Thr
340 345 350
Leu Ser Val Leu Phe Ser Leu Ser His Asn Phe Ile Asn Ser Asp Arg
355 360 365
Asp Pro Thr Ala Asp Phe Lys Lys Thr Gly Glu Gln Val Cys Trp Phe
370 375 380
Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr Gly Gly Phe Ile Ser Gly
385 390 395 400
Cys Leu Thr Gly Gly Leu Asn Phe Gln Val Glu His His Leu Phe Pro
405 410 415
Arg Met Ser Ser Ala Trp Tyr Pro Tyr Ile Ala Pro Thr Val Arg Glu
420 425 430
Val Cys Lys Lys His Gly Val Asn Tyr Ala Tyr Tyr Pro Trp Ile Gly
435 440 445
Gln Asn Leu Val Ser Thr Phe Lys Tyr Met His Arg Ala Gly Ser Gly
450 455 460
Ala Asn Trp Glu Leu Lys Pro Leu Ser Gly Ser Ala
465 470 475
<210>101
<211>1449
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1449)
<223>Δ5-去饱和酶
<400>101
atg cca ccc aac gcc gag gtc aaa aac ctc cgt tca cgt tcc atc cca 48
Met Pro Pro Asn Ala Glu Val Lys Asn Leu Arg Ser Arg Ser Ile Pro
1 5 10 15
acg aag aag tcc agt tca tcg tca tcc acc gcg aac gac gat ccg gct 96
Thr Lys Lys Ser Ser Ser Ser Ser Ser Thr Ala Asn Asp Asp Pro Ala
20 25 30
acc caa tcc acc tca cct gtg aac cga acc ctc aag tct ttg aat gga 144
Thr Gln Ser Thr Ser Pro Val Asn Arg Thr Leu Lys Ser Leu Asn Gly
35 40 45
aac gaa ata gct att gac ggt gtc atc tat gat att gat ggc ttt gtc 192
Asn Glu Ile Ala Ile Asp Gly Val Ile Tyr Asp Ile Asp Gly Phe Val
50 55 60
cat cct gga gga gag gtt att agc ttc ttt gga ggc aac gat gtg act 240
His Pro Gly Gly Glu Val Ile Ser Phe Phe Gly Gly Asn Asp Val Thr
65 70 75 80
gta cag tac aaa atg att cat ccg tat cat aat agt aag cat ctc gag 288
Val Gln Tyr Lys Met Ile His Pro Tyr His Asn Ser Lys His Leu Glu
85 90 95
aag atg aga gcc gtt gga aag att gca gac tac tcc aca gag tac aag 336
Lys Met Arg Ala Val Gly Lys Ile Ala Asp Tyr Ser Thr Glu Tyr Lys
100 105 110
ttc gac aca ccc ttt gaa cga gag atc aaa tcc gaa gtg ttc aaa atc 384
Phe Asp Thr Pro Phe Glu Arg Glu Ile Lys Ser Glu Val Phe Lys Ile
115 120 125
gtc cgt cga gga cgt gaa ttc ggt aca aca gga tat ttc ctc cgt gcc 432
Val Arg Arg Gly Arg Glu Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala
130 135 140
ttc ttc tac att gct ctc ttc ttc acc atg caa tac acc ttc gcc aca 480
Phe Phe Tyr Ile Ala Leu Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr
145 150 155 160
tgc act acc ttc acc acc tac gat cat tgg tat caa agt ggt gta ttc 528
Cys Thr Thr Phe Thr Thr Tyr Asp His Trp Tyr Gln Ser Gly Val Phe
165 170 175
atc gcc att gtg ttt ggt atc tca caa gct ttc att ggg ttg aat gta 576
Ile Ala Ile Val Phe Gly Ile Ser Gln Ala Phe Ile Gly Leu Asn Val
180 185 190
caa cat gat gcc aat cac gga gct gct agc aaa cga cct tgg gtg aat 624
Gln His Asp Ala Asn His Gly Ala Ala Ser Lys Arg Pro Trp Val Asn
195 200 205
gat ctc ctt gga tct gga gct gat ctc atc ggt gga tgc aaa tgg aac 672
Asp Leu Leu Gly Ser Gly Ala Asp Leu Ile Gly Gly Cys Lys Trp Asn
210 215 220
tgg ttg gct cag cat tgg act cat cat gcg tat acc aat cac gct gat 720
Trp Leu Ala Gln His Trp Thr His His Ala Tyr Thr Asn His Ala Asp
225 230 235 240
aaa gat cct gat agc ttt agt tcc gag ccg gtc ttc aac ttt aac gat 768
Lys Asp Pro Asp Ser Phe Ser Ser Glu Pro Val Phe Asn Phe Asn Asp
245 250 255
tat ccc att ggt cac ccc aaa aga aag tgg tgg cat agg ttc caa ggg 816
Tyr Pro Ile Gly His Pro Lys Arg Lys Trp Trp His Arg Phe Gln Gly
260 265 270
ctc tac ttc cta atc atg ctg agt ttc tat tgg gta tcg atg gta ttc 864
Leu Tyr Phe Leu Ile Met Leu Ser Phe Tyr Trp Val Ser Met Val Phe
275 280 285
aac cca caa gtt atc gac ctc cgt cat gct gga gct gcc tac gtt gga 912
Asn Pro Gln Val Ile Asp Leu Arg His Ala Gly Ala Ala Tyr Val Gly
290 295 300
ttt cag atg gag aac gac ttt atc gtc aaa cgg aga aag tat gca atg 960
Phe Gln Met Glu Asn Asp Phe Ile Val Lys Arg Arg Lys Tyr Ala Met
305 310 315 320
gca ctt cgt gca atg tac ttc tat ttc aac atc tat tgt ccg att gtc 1008
Ala Leu Arg Ala Met Tyr Phe Tyr Phe Asn Ile Tyr Cys Pro Ile Val
325 330 335
aac aat gga ttg act tgg tcg aca gtt gga atc atc ctc tta atg gga 1056
Asn Asn Gly Leu Thr Trp Ser Thr Val Gly Ile Ile Leu Leu Met Gly
340 345 350
gtt agc gaa agc ttc atg ctc tcc ggt cta ttc gta ctc tca cac aac 1104
Val Ser Glu Ser Phe Met Leu Ser Gly Leu Phe Val Leu Ser His Asn
355 360 365
ttt gaa aat tcc gaa cgt gat cct acc tct gag tat cgc aag act ggt 1152
Phe Glu Asn Ser Glu Arg Asp Pro Thr Ser Glu Tyr Arg Lys Thr Gly
370 375 380
gag caa gta tgt tgg ttc aag tct caa gtg gag act tct tct acc tac 1200
Glu Gln Val Cys Trp Phe Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr
385 390 395 400
gga ggt atc gtt gct ggg tgt ctc act ggt gga ctc aac ttt caa gtg 1248
Gly Gly Ile Val Ala Gly Cys Leu Thr Gly Gly Leu Asn Phe Gln Val
405 410 415
gag cat cat ttg ttc ccg agg atg agc agt gct tgg tat cct ttc atc 1296
Glu His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Phe Ile
420 425 430
gcg ccg aag gtt aga gag att tgt aag aag cat gga gtt aga tac gct 1344
Ala Pro Lys Val Arg Glu Ile Cys Lys Lys His Gly Val Arg Tyr Ala
435 440 445
tac tat ccg tac atc tgg cag aac ttg cat tct acc gtg agt tac atg 1392
Tyr Tyr Pro Tyr Ile Trp Gln Asn Leu His Ser Thr Val Ser Tyr Met
450 455 460
cat ggg acg gga acg gga gct aga tgg gag ctt cag ccg ttg tct gga 1440
His Gly Thr Gly Thr Gly Ala Arg Trp Glu Leu Gln Pro Leu Ser Gly
465 470 475 480
agg gcg tag 1449
Arg Ala
<210>102
<211>482
<212>PRT
<213>假矮海链藻
<400>102
Met Pro Pro Asn Ala Glu Val Lys Asn Leu Arg Ser Arg Ser Ile Pro
1 5 10 15
Thr Lys Lys Ser Ser Ser Ser Ser Ser Thr Ala Asn Asp Asp Pro Ala
20 25 30
Thr Gln Ser Thr Ser Pro Val Asn Arg Thr Leu Lys Ser Leu Asn Gly
35 40 45
Asn Glu Ile Ala Ile Asp Gly Val Ile Tyr Asp Ile Asp Gly Phe Val
50 55 60
His Pro Gly Gly Glu Val Ile Ser Phe Phe Gly Gly Asn Asp Val Thr
65 70 75 80
Val Gln Tyr Lys Met Ile His Pro Tyr His Asn Ser Lys His Leu Glu
85 90 95
Lys Met Arg Ala Val Gly Lys Ile Ala Asp Tyr Ser Thr Glu Tyr Lys
100 105 110
Phe Asp Thr Pro Phe Glu Arg Glu Ile Lys Ser Glu Val Phe Lys Ile
115 120 125
Val Arg Arg Gly Arg Glu Phe Gly Thr Thr Gly Tyr Phe Leu Arg Ala
130 135 140
Phe Phe Tyr Ile Ala Leu Phe Phe Thr Met Gln Tyr Thr Phe Ala Thr
145 150 155 160
Cys Thr Thr Phe Thr Thr Tyr Asp His Trp Tyr Gln Ser Gly Val Phe
165 170 175
Ile Ala Ile Val Phe Gly Ile Ser Gln Ala Phe Ile Gly Leu Asn Val
180 185 190
Gln His Asp Ala Asn His Gly Ala Ala Ser Lys Arg Pro Trp Val Asn
195 200 205
Asp Leu Leu Gly Ser Gly Ala Asp Leu Ile Gly Gly Cys Lys Trp Asn
210 215 220
Trp Leu Ala Gln His Trp Thr His His Ala Tyr Thr Asn His Ala Asp
225 230 235 240
Lys Asp Pro Asp Ser Phe Ser Ser Glu Pro Val Phe Asn Phe Asn Asp
245 250 255
Tyr Pro Ile Gly His Pro Lys Arg Lys Trp Trp His Arg Phe Gln Gly
260 265 270
Leu Tyr Phe Leu Ile Met Leu Ser Phe Tyr Trp Val Ser Met Val Phe
275 280 285
Asn Pro Gln Val Ile Asp Leu Arg His Ala Gly Ala Ala Tyr Val Gly
290 295 300
Phe Gln Met Glu Asn Asp Phe Ile Val Lys Arg Arg Lys Tyr Ala Met
305 310 315 320
Ala Leu Arg Ala Met Tyr Phe Tyr Phe Asn Ile Tyr Cys Pro Ile Val
325 330 335
Asn Asn Gly Leu Thr Trp Ser Thr Val Gly Ile Ile Leu Leu Met Gly
340 345 350
Val Ser Glu Ser Phe Met Leu Ser Gly Leu Phe Val Leu Ser His Asn
355 360 365
Phe Glu Asn Ser Glu Arg Asp Pro Thr Ser Glu Tyr Arg Lys Thr Gly
370 375 380
Glu Gln Val Cys Trp Phe Lys Ser Gln Val Glu Thr Ser Ser Thr Tyr
385 390 395 400
Gly Gly Ile Val Ala Gly Cys Leu Thr Gly Gly Leu Asn Phe Gln Val
405 410 415
Glu His His Leu Phe Pro Arg Met Ser Ser Ala Trp Tyr Pro Phe Ile
420 425 430
Ala Pro Lys Val Arg Glu Ile Cys Lys Lys His Gly Val Arg Tyr Ala
435 440 445
Tyr Tyr Pro Tyr Ile Trp Gln Asn Leu His Ser Thr Val Ser Tyr Met
450 455 460
His Gly Thr Gly Thr Gly Ala Arg Trp Glu Leu Gln Pro Leu Ser Gly
465 470 475 480
Arg Ala
<210>103
<211>1512
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1512)
<223>Δ4-去饱和酶
<400>103
atg tgc aac ggc aac ctc cca gca tcc acc gca cag ctc aag tcc acc 48
Met Cys Asn Gly Asn Leu Pro Ala Ser Thr Ala Gln Leu Lys Ser Thr
1 5 10 15
tcg aag ccc cag cag caa cat gag cat cgc acc atc tcc aag tcc gag 96
Ser Lys Pro Gln Gln Gln His Glu His Arg Thr Ile Ser Lys Ser Glu
20 25 30
ctc gcc caa cac aac acg ccc aaa tca gca tgg tgt gcc gtc cac tcc 144
Leu Ala Gln His Asn Thr Pro Lys Ser Ala Trp Cys Ala Val His Ser
35 40 45
act ccc gcc acc gac cca tcc cac tcc aac aac aaa caa cac gca cac 192
Thr Pro Ala Thr Asp Pro Ser His Ser Asn Asn Lys Gln His Ala His
50 55 60
cta gtc ctc gac att acc gac ttt gcg tcc cgc cat cca ggg gga gac 240
Leu Val Leu Asp Ile Thr Asp Phe Ala Ser Arg His Pro Gly Gly Asp
65 70 75 80
ctc atc ctc ctc gct tcc ggc aaa gac gcc tcg gtg ctg ttt gaa aca 288
Leu Ile Leu Leu Ala Ser Gly Lys Asp Ala Ser Val Leu Phe Glu Thr
85 90 95
tac cat cca cgt gga gtt ccg acg tct ctc att caa aag ctg cag att 336
Tyr His Pro Arg Gly Val Pro Thr Ser Leu Ile Gln Lys Leu Gln Ile
100 105 110
gga gtg atg gag gag gag gcg ttt cgg gat tcg ttt tac agt tgg act 384
Gly Val Met Glu Glu Glu Ala Phe Arg Asp Ser Phe Tyr Ser Trp Thr
115 120 125
gat tct gac ttt tat act gtg ttg aag agg agg gtt gtg gag cgg ttg 432
Asp Ser Asp Phe Tyr Thr Val Leu Lys Arg Arg Val Val Glu Arg Leu
130 135 140
gag gag agg ggg ttg gac agg agg gga tcg aaa gag att tgg atc aag 480
Glu Glu Arg Gly Leu Asp Arg Arg Gly Ser Lys Glu Ile Trp Ile Lys
145 150 155 160
gct ttg ttc ttg ttg gtt gga ttt tgg tac tgt ttg tac aag atg tat 528
Ala Leu Phe Leu Leu Val Gly Phe Trp Tyr Cys Leu Tyr Lys Met Tyr
165 170 175
act acg tcg gat atc gat cag tac ggt att gcc att gcc tat tct att 576
Thr Thr Ser Asp Ile Asp Gln Tyr Gly Ile Ala Ile Ala Tyr Ser Ile
180 185 190
gga atg gga acc ttt gcg gca ttc atc ggc acg tgt att caa cac gat 624
Gly Met Gly Thr Phe Ala Ala Phe Ile Gly Thr Cys Ile Gln His Asp
195 200 205
gga aat cac ggt gca ttc gct cag aac aag tta ctc aac aag ttg gct 672
Gly Asn His Gly Ala Phe Ala Gln Asn Lys Leu Leu Asn Lys Leu Ala
210 215 220
ggg tgg acg ttg gat atg att ggt gcg agt gcg ttt acg tgg gag ctt 720
Gly Trp Thr Leu Asp Met Ile Gly Ala Ser Ala Phe Thr Trp Glu Leu
225 230 235 240
cag cac atg ctg ggg cst cat cca tat acg aat gtg ttg gat ggg gtg 768
Gln His Met Leu Gly His His Pro Tyr Thr Asn Val Leu Asp Gly Val
245 250 255
gag gag gag agg aag gag agg ggg gag gat gtt gct ttg gaa gaa aag 816
Glu Glu Glu Arg Lys Glu Arg Gly Glu Asp Val Ala Leu Glu Glu Lys
260 265 270
gat cag gat ttt gaa gtt gcc aca tcc gga cga tta tat cat att gat 864
Asp Gln Asp Phe Glu Val Ala Thr Ser Gly Arg Leu Tyr His Ile Asp
275 280 285
gcc aat gta cgt tat ggt tcg gta tgg aat gtc atg agg ttt tgg gct 912
Ala Asn Val Arg Tyr Gly Ser Val Trp Asn Val Met Arg Phe Trp Ala
290 295 300
atg aag gtc att acg atg gga tat atg atg gga tta cca atc tac ttt 960
Met Lys Val Ile Thr Met Gly Tyr Met Met Gly Leu Pro Ile Tyr Phe
305 310 315 320
cat gga gta ctg agg gga gtt gga ttg ttt gtt att ggg cat ttg gcg 1008
His Gly Val Leu Arg Gly Val Gly Leu Phe Val Ile Gly His Leu Ala
325 330 335
tgt gga gag ttg ttg gcg acg atg ttt att gtg aat cac gtc att gag 1056
Cys Gly Glu Leu Leu Ala Thr Met Phe Ile Val Asn His Val Ile Glu
340 345 350
ggt gtg agt tat gga acg aag gat ttg gtt ggt ggt gcg agt cat gta 1104
Gly Val Ser Tyr Gly Thr Lys Asp Leu Val Gly Gly Ala Ser His Val
355 360 365
gat gag aag aag att gtc aag cca acg act gta ttg gga gat aca cca 1152
Asp Glu Lys Lys Ile Val Lys Pro Thr Thr Val Leu Gly Asp Thr Pro
370 375 380
atg gta aag act cgc gag gag gca ttg aaa agc aac agc aat aac aac 1200
Met Val Lys Thr Arg Glu Glu Ala Leu Lys Ser Asn Ser Asn Asn Asn
385 390 395 400
aag aag aag gga gag aag aac tcg gta cca tcc gtt cca ttc aac gac 1248
Lys Lys Lys Gly Glu Lys Asn Ser Val Pro Ser Val Pro Phe Asn Asp
405 410 415
tgg gca gca gtc caa tgc cag acc tcc gtg aat tgg tct cca ggc tca 1296
Trp Ala Ala Val Gln Cys Gln Thr Ser Val Asn Trp Ser Pro Gly Ser
420 425 430
tgg ttc tgg aat cac ttt tct ggg gga ctc tct cat cag att gag cat 1344
Trp Phe Trp Asn His Phe Ser Gly Gly Leu Ser His Gln Ile Glu His
435 440 445
cac ttg ttc ccc agc att tgt cat aca aac tac tgt cat atc cag gat 1392
His Leu Phe Pro Ser Ile Cys His Thr Asn Tyr Cys His Ile Gln Asp
450 455 460
gtt gtg gag agt acg tgt gct gag tac gga gtt ccg tat cag agt gag 1440
Val Val Glu Ser Thr Cys Ala Glu Tyr Gly Val Pro Tyr Gln Ser Glu
465 470 475 480
agt aat ttg ttt gtt gct tat gga aag atg att agt cat ttg aag ttt 1488
Ser Asn Leu Phe Val Ala Tyr Gly Lys Met Ile Ser His Leu Lys Phe
485 490 495
ttg ggt aaa gcc aag tgt gag tag 1512
Leu Gly Lys Ala Lys Cys Glu
500
<210>104
<211>503
<212>PRT
<213>假矮海链藻
<400>104
Met Cys Asn Gly Asn Leu Pro Ala Ser Thr Ala Gln Leu Lys Ser Thr
1 5 10 15
Ser Lys Pro Gln Gln Gln His Glu His Arg Thr Ile Ser Lys Ser Glu
20 25 30
Leu Ala Gln His Asn Thr Pro Lys Ser Ala Trp Cys Ala Val His Ser
35 40 45
Thr Pro Ala Thr Asp Pro Ser His Ser Asn Asn Lys Gln His Ala His
50 55 60
Leu Val Leu Asp Ile Thr Asp Phe Ala Ser Arg His Pro Gly Gly Asp
65 70 75 80
Leu Ile Leu Leu Ala Ser Gly Lys Asp Ala Ser Val Leu Phe Glu Thr
85 90 95
Tyr His Pro Arg Gly Val Pro Thr Ser Leu Ile Gln Lys Leu Gln Ile
100 105 110
Gly Val Met Glu Glu Glu Ala Phe Arg Asp Ser Phe Tyr Ser Trp Thr
115 120 125
Asp Ser Asp Phe Tyr Thr Val Leu Lys Arg Arg Val Val Glu Arg Leu
130 135 140
Glu Glu Arg Gly Leu Asp Arg Arg Gly Ser Lys Glu Ile Trp Ile Lys
145 150 155 160
Ala Leu Phe Leu Leu Val Gly Phe Trp Tyr Cys Leu Tyr Lys Met Tyr
165 170 175
Thr Thr Ser Asp Ile Asp Gln Tyr Gly Ile Ala Ile Ala Tyr Ser Ile
180 185 190
Gly Met Gly Thr Phe Ala Ala Phe Ile Gly Thr Cys Ile Gln His Asp
195 200 205
Gly Asn His Gly Ala Phe Ala Gln Asn Lys Leu Leu Asn Lys Leu Ala
210 215 220
Gly Trp Thr Leu Asp Met Ile Gly Ala Ser Ala Phe Thr Trp Glu Leu
225 230 235 240
Gln His Met Leu Gly His His Pro Tyr Thr Asn Val Leu Asp Gly Val
245 250 255
Glu Glu Glu Arg Lys Glu Arg Gly Glu Asp Val Ala Leu Glu Glu Lys
260 265 270
Asp Gln Asp Phe Glu Val Ala Thr Ser Gly Arg Leu Tyr His Ile Asp
275 280 285
Ala Asn Val Arg Tyr Gly Ser Val Trp Asn Val Met Arg phe Trp Ala
290 295 300
Met Lys Val Ile Thr Met Gly Tyr Met Met Gly Leu Pro Ile Tyr Phe
305 310 315 320
His Gly Val Leu Arg Gly Val Gly Leu Phe Val Ile Gly His Leu Ala
325 330 335
Cys Gly Glu Leu Leu Ala Thr Met Phe Ile Val Asn His Val Ile Glu
340 345 350
Gly Val Ser Tyr Gly Thr Lys Asp Leu Val Gly Gly Ala Ser His Val
355 360 365
Asp Glu Lys Lys Ile Val Lys Pro Thr Thr Val Leu Gly Asp Thr Pro
370 375 380
Met Val Lys Thr Arg Glu Glu Ala Leu Lys Ser Asn Ser Asn Asn Asn
385 390 395 400
Lys Lys Lys Gly Glu Lys Asn Ser Val Pro Ser Val Pro Phe Asn Asp
405 410 415
Trp Ala Ala Val Gln Cys Gln Thr Ser Val Asn Trp Ser Pro Gly Ser
420 425 430
Trp Phe Trp Asn His Phe Ser Gly Gly Leu Ser His Gln Ile Glu His
435 440 445
His Leu Phe Pro Ser Ile Cys His Thr Asn Tyr Cys His Ile Gln Asp
450 455 460
Val Val Glu Ser Thr Cys Ala Glu Tyr Gly Val Pro Tyr Gln Ser Glu
465 470 475 480
Ser Asn Leu Phe Val Ala Tyr Gly Lys Met Ile Ser His Leu Lys Phe
485 490 495
Leu Gly Lys Ala Lys Cys Glu
500
<210>105
<2ll>1257
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1257)
<223>ω3-去饱和酶
<400>105
atg tac aga tta aca tcc acc ttc ctc atc gca ttg gca ttc tcc tcc 48
Met Tyr Arg Leu Thr Ser Thr Phe Leu Ile Ala Leu Ala Phe Ser Ser
1 5 10 15
tcc atc aat gcc ttc tct cca caa cgg cca cca cgt act atc acc aaa 96
Ser Ile Asn Ala Phe Ser Pro Gln Arg Pro Pro Arg Thr Ile Thr Lys
20 25 30
agt aaa gtc caa agc acc gtg cta ccc ata ccg acc aag gat gat ctg 144
Ser Lys Val Gln Ser Thr Val Leu Pro Ile Pro Thr Lys Asp Asp Leu
35 40 45
aac ttt ctc caa cca caa ctc gat gag aat gat ctc tac ctc gac gat 192
Asn Phe Leu Gln Pro Gln Leu Asp Glu Asn Asp Leu Tyr Leu Asp Asp
50 55 60
gtc aac act cca cca aga gca ggt acc atc atg aag atg ttg ccg aag 240
Val Asn Thr Pro Pro Arg Ala Gly Thr Ile Met Lys Met Leu Pro Lys
65 70 75 80
gaa acg ttc aac att gat aca gca act tca ttg ggt tac ttt ggt atg 288
Glu Thr Phe Asn Ile Asp Thr Ala Thr Ser Leu Gly Tyr Phe Gly Met
85 90 95
gat atg gca gcg gtt gta tcg tcc atg acg ttg cta aat gct att gta 336
Asp Met Ala Ala Val Val Ser Ser Met Thr Leu Leu Asn Ala Ile Val
100 105 110
act tcg gat cag tac cat gct ctt cca ctt cct ctc caa gca gca aca 384
Thr Ser Asp Gln Tyr His Ala Leu Pro Leu Pro Leu Gln Ala Ala Thr
115 120 125
gtg att ccc ttt cag cta ttg gct ggg ttc gcc atg tgg tgt atg tgg 432
Val Ile Pro Phe Gln Leu Leu Ala Gly Phe Ala Met Trp Cys Met Trp
130 135 140
tgc att gga cac gat gct gga cat tct act gtt tcg aag aca aag tgg 480
Cys Ile Gly His Asp Ala Gly His Ser Thr Val Ser Lys Thr Lys Trp
145 150 155 160
atc aac cga gtc gtt ggt gaa gtg gct cat tct gtt gtt tgt ctc acg 528
Ile Asn Arg Val Val Gly Glu Val Ala His Ser Val Val Cys Leu Thr
165 170 175
ccg ttc gtg cct tgg cag atg tcg cat agg aaa cac cat ttg aat cac 576
Pro Phe Val Pro Trp Gln Met Ser His Arg Lys His His Leu Asn His
180 185 190
aat cat att gaa aag gac tac tct cat aag tgg tac agt cgc gac gag 624
Asn His Ile Glu Lys Asp Tyr Ser His Lys Trp Tyr Ser Arg Asp Glu
195 200 205
ttt gat gat atc cca caa ctc tat aag aca ttt ggc tac aac cca aga 672
Phe Asp Asp Ile Pro Gln Leu Tyr Lys Thr Phe Gly Tyr Asn Pro Arg
210 215 220
atg atg caa ctt cca ttc ctc tac ttc atg tat ctt gca ttg gga att 720
Met Met Gln Leu Pro Phe Leu Tyr Phe Met Tyr Leu Ala Leu Gly Ile
225 230 235 240
cca gat ggt ggg cat gtt gtg ttc tac gga aga atg tgg gaa gga gtg 768
Pro Asp Gly Gly His Val Val Phe Tyr Gly Arg Met Trp Glu Gly Val
245 250 255
tca ttg cag aag aag ttt gat gct gct att tct gtg gcc gta tca tgt 816
Ser Leu Gln Lys Lys Phe Asp Ala Ala Ile Ser Val Ala Val Ser Cys
260 265 270
gca act gct gga tcg ctt tgg atg aat atg ggt aca gca gac ttc acg 864
Ala Thr Ala Gly Ser Leu Trp Met Asn Met Gly Thr Ala Asp Phe Thr
275 280 285
gtg gta tgc atg gtt cct tgg cta gtt cta tcg tgg tgg ctc ttc atg 912
Val Val Cys Met Val Pro Trp Leu Val Leu Ser Trp Trp Leu Phe Met
290 295 300
gta aca tac ctt cag cat cat tca gaa gac gga aag cta tac act gat 960
Val Thr Tyr Leu Gln His His Ser Glu Asp Gly Lys Leu Tyr Thr Asp
305 310 315 320
gaa acg ttt aca ttt gaa aag gga gcc ttc gag acc gtg gat cgt tcg 1008
Glu Thr Phe Thr Phe Glu Lys Gly Ala Phe Glu Thr Val Asp Arg Ser
325 330 335
tac ggc aag ttg atc aac cga atg tcg cat cac atg atg gac ggt cac 1056
Tyr Gly Lys Leu Ile Asn Arg Met Ser His His Met Met Asp Gly His
340 345 350
gtg gtg cac cac ttg ttc ttt gaa cgt gta cct cac tac aga tta gag 1104
Val Val His His Leu Phe Phe Glu Arg Val Pro His Tyr Arg Leu Glu
355 360 365
gca gct acc gaa gct ctt gtg aaa gga atg gat gaa acg gga cag aaa 1152
Ala Ala Thr Glu Ala Leu Val Lys Gly Met Asp Glu Thr Gly Gln Lys
370 375 380
cat ttg tac aaa tac att gat act cct gat ttc aat gcc gag att gtc 1200
His Leu Tyr Lys Tyr Ile Asp Thr Pro Asp Phe Asn Ala Glu Ile Val
385 390 395 400
aac gga ttt cgc gac aat tgg ttc ctt gtt gaa gag gag aac atc aaa 1248
Asn Gly Phe Arg Asp Asn Trp Phe Leu Val Glu Glu Glu Asn Ile Lys
405 410 415
agg gag tag 1257
Arg Glu
<210>106
<211>418
<212>PRT
<213>假矮海链藻
<400>106
Met Tyr Arg Leu Thr Ser Thr Phe Leu Ile Ala Leu Ala Phe Set Ser
1 5 10 15
Ser Ile Asn Ala Phe Ser Pro Gln Arg Pro Pro Arg Thr Ile Thr Lys
20 25 30
Ser Lys Val Gln Ser Thr Val Leu Pro Ile Pro Thr Lys Asp Asp Leu
35 40 45
Asn Phe Leu Gln Pro Gln Leu Asp Glu Asn Asp Leu Tyr Leu Asp Asp
50 55 60
Val Asn Thr Pro Pro Arg Ala Gly Thr Ile Met Lys Met Leu Pro Lys
65 70 75 80
Glu Thr Phe Asn Ile Asp Thr Ala Thr Ser Leu Gly Tyr Phe Gly Met
85 90 95
Asp Met Ala Ala Val Val Ser Ser Met Thr Leu Leu Asn Ala Ile Val
100 105 110
Thr Ser Asp Gln Tyr His Ala Leu Pro Leu Pro Leu Gln Ala Ala Thr
115 120 125
Val Ile Pro Phe Gln Leu Leu Ala Gly Phe Ala Met Trp Cys Met Trp
130 135 140
Cys Ile Gly His Asp Ala Gly His Ser Thr Val Ser Lys Thr Lys Trp
145 150 155 160
Ile Asn Arg Val Val Gly Glu Val Ala His Ser Val Val Cys Leu Thr
165 170 175
Pro Phe Val Pro Trp Gln Met Ser His Arg Lys His His Leu Asn His
180 185 190
Asn His Ile Glu Lys Asp Tyr Ser His Lys Trp Tyr Ser Arg Asp Glu
195 200 205
Phe Asp Asp Ile Pro Gln Leu Tyr Lys Thr Phe Gly Tyr Asn Pro Arg
210 215 220
Met Met Gln Leu Pro Phe Leu Tyr Phe Met Tyr Leu Ala Leu Gly Ile
225 230 235 240
Pro Asp Gly Gly His Val Val Phe Tyr Gly Arg Met Trp Glu Gly Val
245 250 255
Ser Leu Gln Lys Lys Phe Asp Ala Ala Ile Ser Val Ala Val Ser Cys
260 265 270
Ala Thr Ala Gly Ser Leu Trp Met Asn Met Gly Thr Ala Asp Phe Thr
275 280 285
Val Val Cys Met Val Pro Trp Leu Val Leu Ser Trp Trp Leu Phe Met
290 295 300
Val Thr Tyr Leu Gln His His Ser Glu Asp Gly Lys Leu Tyr Thr Asp
305 310 315 320
Glu Thr Phe Thr Phe Glu Lys Gly Ala Phe Glu Thr Val Asp Arg Ser
325 330 335
Tyr Gly Lys Leu Ile Asn Arg Met Ser His His Met Met Asp Gly His
340 345 350
Val Val His His Leu Phe Phe Glu Arg Val Pro His Tyr Arg Leu Glu
355 360 365
Ala Ala Thr Glu Ala Leu Val Lys Gly Met Asp Glu Thr Gly Gln Lys
370 375 380
His Leu Tyr Lys Tyr Ile Asp Thr Pro Asp Phe Asn Ala Glu Ile Val
385 390 395 400
Asn Gly Phe Arg Asp Asn Trp Phe Leu Val Glu Glu Glu Asn Ile Lys
405 410 415
Arg Glu
<210>107
<211>1086
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(1086)
<223>Δ12-去饱和酶
<400>107
atg cag gag ggg gtg cga aac att ccg aac gag tgc ttt gag acg gga 48
Met Gln Glu Gly Val Arg Asn Ile Pro Asn Glu Cys Phe Glu Thr Gly
1 5 10 15
cat ctt gaa aga ccc tgg cgt tcc ggc cgg tgt ggg cgc gat ccc ggt 96
His Leu Glu Arg Pro Trp Arg Ser Gly Arg Cys Gly Arg Asp Pro Gly
20 25 30
tcg aat tgg ggc gct ggc ttc cgc ttt ttt tcg ctc aag ggg ttt tgg 144
Ser Asn Trp Gly Ala Gly Phe Arg Phe Phe Ser Leu Lys Gly Phe Trp
35 40 45
tgg ccg gcg tgg tgg gcg tac gcg ttc gtg acg ggg acg gcg gcc act 192
Trp Pro Ala Trp Trp Ala Tyr Ala Phe Val Thr Gly Thr Ala Ala Thr
50 55 60
ggg tgt tgg gtc gcc gcg cac gag tgc ggg cac ggc gcg ttc agc gat 240
Gly Cys Trp Val Ala Ala His Glu Cys Gly His Gly Ala Phe Ser Asp
65 70 75 80
aac aag acg ttg caa gat gcg gtt gga tac gtg ttg cac tcg ttg ctc 288
Asn Lys Thr Leu Gln Asp Ala Val Gly Tyr Val Leu His Ser Leu Leu
85 90 95
ttg gtg ccg tac ttt tct tgg cag cga tca cac gcg gtg cat cac tcg 336
Leu Val Pro Tyr Phe Ser Trp Gln Arg Ser His Ala Val His His Ser
100 105 110
agg acg aat cac gtt ctt gag ggc gag acg cac gtg ccg gcg cgc ttg 384
Arg Thr Asn His Val Leu Glu Gly Glu Thr His Val Pro Ala Arg Leu
115 120 125
ggg acg gaa gac gcc aac gtc gtg ttc aag ctt cgc gaa ttg atc ggt 432
Gly Thr Glu Asp Ala Asn Val Val Phe Lys Leu Arg Glu Leu Ile Gly
130 135 140
gaa ggg ccg ttc acg ttt ttc aac ctc gtc ggc gtc ttc gcg ctc gga 480
Glu Gly Pro Phe Thr Phe Phe Asn Leu Val Gly Val Phe Ala Leu Gly
145 150 155 160
tgg ccg att tac ttg ctc acc ggc gcg agc ggc gga ccg gtg cgc ggt 528
Trp Pro Ile Tyr Leu Leu Thr Gly Ala Ser Gly Gly Pro Val Arg Gly
165 170 175
aac acg aac cac ttc tta ccc ttc atg ggc gag aaa ggt aag cac gcg 576
Asn Thr Asn His Phe Leu Pro Phe Met Gly Glu Lys Gly Lys His Ala
180 185 190
ctg ttc ccg ggt aag tgg gcg aag aag gtg tgg cag tct gac atc ggc 624
Leu Phe Pro Gly Lys Trp Ala Lys Lys Val Trp Gln Ser Asp Ile Gly
195 200 205
gtt gtt gcc gtc ctg ggc gcg ctc gcg gct tgg gcg gcg cac agc ggg 672
Val Val Ala Val Leu Gly Ala Leu Ala Ala Trp Ala Ala His Ser Gly
210 215 220
att gcc aca gtg atg gca ctc tac gtc ggc ccg tac atg gtg acc aac 720
Ile Ala Thr Val Met Ala Leu Tyr Val Gly Pro Tyr Met Val Thr Asn
225 230 235 240
ttt tgg ctc gtc ttg tac acg tgg tta cag cac acc gac gtt gac gtg 768
Phe Trp Leu Val Leu Tyr Thr Trp Leu Gln His Thr Asp Val Asp Val
245 250 255
ccg cac ttc gag ggc gac gat tgg aac ttg gtc aag ggg gca ttc atg 816
Pro His Phe Glu Gly Asp Asp Trp Asn Leu Val Lys Gly Ala Phe Met
260 265 270
acg atc gat cgc ccg tac ggc cca gtt ttt gat ttc ttg cac cac cgc 864
Thr Ile Asp Arg Pro Tyr Gly Pro Val Phe Asp Phe Leu His His Arg
275 280 285
atc ggc agc acg cac gtc gcg cac cac atc aac aca cca ttc ccg cat 912
Ile Gly Ser Thr His Val Ala His His Ile Asn Thr Pro Phe Pro His
290 295 300
tac aag gct caa atg gcg acg gat gcg cta aag gag gcg tat ccc gac 960
Tyr Lys Ala Gln Met Ala Thr Asp Ala Leu Lys Glu Ala Tyr Pro Asp
305 310 315 320
ctc tac ctt tac gat cca act ccg atc gcg acc gct acg tgg cgc gtg 1008
Leu Tyr Leu Tyr Asp Pro Thr Pro Ile Ala Thr Ala Thr Trp Arg Val
325 330 335
ggg agc aag tgc atc gcc gtc gtg aag aag gga gac gaa tgg gtg ttc 1056
Gly Ser Lys Cys Ile Ala Val Val Lys Lys Gly Asp Glu Trp Val Phe
340 345 350
acg gat aag caa ctc ccg gtc gcg gcg tga 1086
Thr Asp Lys Gln Leu Pro Val Ala Ala
355 360
<210>108
<211>361
<212>PRT
<213>Ostreococcus tauri
<400>108
Met Gln Glu Gly Val Arg Asn Ile Pro Asn Glu Cys Phe Glu Thr Gly
1 5 10 15
His Leu Glu Arg Pro Trp Arg Ser Gly Arg Cys Gly Arg Asp Pro Gly
20 25 30
Ser Asn Trp Gly Ala Gly Phe Arg Phe Phe Ser Leu Lys Gly Phe Trp
35 40 45
Trp Pro Ala Trp Trp Ala Tyr Ala Phe Val Thr Gly Thr Ala Ala Thr
50 55 60
Gly Cys Trp Val Ala Ala His Glu Cys Gly His Gly Ala Phe Ser Asp
65 70 75 80
Asn Lys Thr Leu Gln Asp Ala Val Gly Tyr Val Leu His Ser Leu Leu
85 90 95
Leu Val Pro Tyr Phe Ser Trp Gln Arg Ser His Ala Val His His Ser
100 105 110
Arg Thr Asn His Val Leu Glu Gly Glu Thr His Val Pro Ala Arg Leu
115 120 125
Gly Thr Glu Asp Ala Asn Val Val Phe Lys Leu Arg Glu LeuIle Gly
130 135 140
Glu Gly Pro Phe Thr Phe Phe Asn Leu Val Gly Val Phe Ala Leu Gly
145 150 155 160
Trp Pro Ile Tyr Leu Leu Thr Gly Ala Ser Gly Gly Pro Val Arg Gly
165 170 175
Asn Thr Asn His Phe Leu Pro Phe Met Gly Glu Lys Gly Lys His Ala
180 185 190
Leu Phe Pro Gly Lys Trp Ala Lys Lys Val Trp Gln Ser Asp Ile Gly
195 200 205
Val Val Ala Val Leu Gly Ala Leu Ala Ala Trp Ala Ala His Ser Gly
210 215 220
Ile Ala Thr Val Met Ala Leu Tyr Val Gly Pro Tyr Met Val Thr Asn
225 230 235 240
Phe Trp Leu Val Leu Tyr Thr Trp Leu Gln His Thr Asp Val Asp Val
245 250 255
Pro His Phe Glu Gly Asp Asp Trp Asn Leu Val Lys Gly Ala Phe Met
260 265 270
Thr Ile Asp Arg Pro Tyr Gly Pro Val Phe Asp Phe Leu His His Arg
275 280 285
Ile Gly Ser Thr His Val Ala His His Ile Asn Thr Pro Phe Pro His
290 295 300
Tyr Lys Ala Gln Met Ala Thr Asp Ala Leu Lys Glu Ala Tyr Pro Asp
305 310 315 320
Leu Tyr Leu Tyr Asp Pro Thr Pro Ile Ala Thr Ala Thr Trp Arg Val
325 330 335
Gly Ser Lys Cys Ile Ala Val Val Lys Lys Gly Asp Glu Trp Val Phe
340 345 350
Thr Asp Lys Gln Leu Pro Val Ala Ala
355 360
<210>109
<211>1305
<212>DNA
<213>假矮海链藻
<220>
<221>CDS
<222>(1)..(1305)
<223>Δ12-去饱和酶
<400>109
atg gga aag gga gga aga tca gta acc cgc gct caa aca gca gaa aag 48
Met Gly Lys Gly Gly Arg Ser Val Thr Arg Ala Gln Thr Ala Glu Lys
1 5 10 15
tca gca cac acc atc caa acc ttc acc gac ggc cga tgg gtc tcc ccc 96
Ser Ala His Thr Ile Gln Thr Phe Thr Asp Gly Arg Trp Val Ser Pro
20 25 30
tac aac ccc ctc gca aaa gat gca cct gaa ctc ccc tcc aag ggt gaa 144
Tyr Asn Pro Leu Ala Lys Asp Ala Pro Glu Leu Pro Ser Lys Gly Glu
35 40 45
atc aag gcg gtc atc ccc aaa gag tgc ttc gaa cga agc tac ctc cac 192
Ile Lys Ala Val Ile Pro Lys Glu Cys Phe Glu Arg Ser Tyr Leu His
50 55 60
tcc atg tac ttc gtc ctc cgt gac acc gtc atg gcc gtg gcc tgc gcc 240
Ser Met Tyr Phe Val Leu Arg Asp Thr Val Met Ala Val Ala Cys Ala
65 70 75 80
tac atc gcc cac tca acg ctc tcc acc gat att ccc tcc gag tta ctg 288
Tyr Ile Ala His Ser Thr Leu Ser Thr Asp Ile Pro Ser Glu Leu Leu
85 90 95
agc gtg gac gca ctc aaa tgg ttc ctc gga tgg aac acc tac gcc ttt 336
Ser Val Asp Ala Leu Lys Trp Phe Leu Gly Trp Asn Thr Tyr Ala Phe
100 105 110
tgg atg ggg tgc att ctc acc gga cac tgg gtc cta gcc cat gaa tgt 384
Trp Met Gly Cys Ile Leu Thr Gly His Trp Val Leu Ala His Glu Cys
115 120 125
gga cat ggt gca ttc tct ccc tct cag acg ttt aat gac ttt tgg ggg 432
Gly His Gly Ala Phe Ser Pro Ser Gln Thr Phe Asn Asp Phe Trp Gly
130 135 140
ttc att atg cat cag gcg gtg ttg gtt ccg tat ttc gcc tgg cag tsc 480
Phe Ile Met His Gln Ala Val Leu Val Pro Tyr Phe Ala Trp Gln Tyr
145 150 155 160
tct cat gcg aag cat cat cga cgt acc aac aac att atg gat ggg gag 528
Ser His Ala Lys His His Arg Arg Thr Asn Asn Ile Met Asp Gly Glu
165 170 175
agc cat gtg ccc aat atc gcc aag gaa atg gga ttg aac gag aag aat 576
Ser His Val Pro Asn Ile Ala Lys Glu Met Gly Leu Asn Glu Lys Asn
180 185 190
gag cgc agt gga gga tat gcc gcc att cat gag gct att gga gat gga 624
Glu Arg Ser Gly Gly Tyr Ala Ala Ile His Glu Ala Ile Gly Asp Gly
195 200 205
ccc ttt gcg atg ttt caa atc ttt gct cac ttg gtg atc ggg tgg cct 672
Pro Phe Ala Met Phe Gln Ile Phe Ala His Leu Val Ile Gly Trp Pro
210 215 220
att tac ttg atg gga ttt gct tcc act gga cgt ctc ggt cag gat ggg 720
11e Tyr Leu Met Gly Phe Ala Ser Thr Gly Arg Leu Gly Gln Asp Gly
225 230 235 240
aag gaa ctt cag gct gga gag atc atc gac cat tac cgt cct tgg agt 768
Lys Glu Leu Gln Ala Gly Glu Ile Ile Asp His Tyr Arg Pro Trp Ser
245 250 255
aag atg ttc ccc acc aag ttg cga ttc aaa att gct ctt tcg aca ctt 816
Lys Met Phe Pro Thr Lys Leu Arg Phe Lys Ile Ala Leu Ser Thr Leu
260 265 270
gga gtg att gcc gcc tgg gtt ggg ttg tac ttt gct gca caa gag tat 864
Gly Val Ile Ala Ala Trp Val Gly Leu Tyr Phe Ala Ala Gln Glu Tyr
275 280 285
gga gtc ttg ccc gtg gtt ctt tgg tac att ggc cca ctc atg tgg aat 912
Gly Val Leu Pro Val Val Leu Trp Tyr Ile Gly Pro Leu Met Trp Asn
290 295 300
cag gcg tgg ctt gtg ctc tac act tgg ctt cag cac aat gat ccc tcc 960
Gln Ala Trp Leu Val Leu Tyr Thr Trp Leu Gln His Asn Asp Pro Ser
305 310 315 320
gtg cct caa tat gga agt gac gaa tgg aca tgg gtc aag gga gct ttg 1008
Val Pro Gln Tyr Gly Ser Asp Glu Trp Thr Trp Val Lys Gly Ala Leu
325 330 335
tcg acg att gat cgc ccg tat ggt atc ttt gac ttc ttc cat cac aag 1056
Ser Thr Ile Asp Arg Pro Tyr Gly Ile Phe Asp Phe Phe His His Lys
340 345 350
att gga agc act cac gta gct cat cat ttg ttc cac gag atg cca ttt 1104
Ile Gly Ser Thr His Val Ala His His Leu Phe His Glu Met Pro Phe
355 360 365
tac aag gcg gat gtg gct act gcg tcg atc aag ggt ttc ttg gag ccg 1152
Tyr Lys Ala Asp Val Ala Thr Ala Ser Ile Lys Gly Phe Leu Glu Pro
370 375 380
aag gga ctt tac aac tat gat cca acg cct tgg tat gtg gcc atg tgg 1200
Lys Gly Leu Tyr Asn Tyr Asp Pro Thr Pro Trp Tyr Val Ala Met Trp
385 390 395 400
agg gtg gcc aag act tgt cat tat att gag gat gtg gat gga gtt cag 1248
Arg Val Ala Lys Thr Cys His Tyr Ile Glu Asp Val Asp Gly Val Gln
405 410 415
tat tat aag agt ttg gag gat gtg cct ttg aag aag gat gcc aag aag 1296
Tyr Tyr Lys Ser Leu Glu Asp Val Pro Leu Lys Lys Asp Ala Lys Lys
420 425 430
tct gat tag 1305
Ser Asp
<210>110
<211>434
<212>PRT
<213>假矮海链藻
<400>110
Met Gly Lys Gly Gly Arg Ser Val Thr Arg Ala Gln Thr Ala Glu Lys
1 5 10 15
Ser Ala His Thr Ile Gln Thr Phe Thr Asp Gly Arg Trp Val Ser Pro
20 25 30
Tyr Asn Pro Leu Ala Lys Asp Ala Pro Glu Leu Pro Ser Lys Gly Glu
35 40 45
Ile Lys Ala Val Ile Pro Lys Glu Cys Phe Glu Arg Ser Tyr Leu His
50 55 60
Ser Met Tyr Phe Val Leu Arg Asp Thr Val Met Ala Val Ala Cys Ala
65 70 75 80
Tyr Ile Ala His Ser Thr Leu Ser Thr Asp Ile Pro Ser Glu Leu Leu
85 90 95
Ser Val Asp Ala Leu Lys Trp Phe Leu Gly Trp Asn Thr Tyr Ala Phe
100 105 110
Trp Met Gly Cys Ile Leu Thr Gly His Trp Val Leu Ala His Glu Cys
115 120 125
Gly His Gly Ala Phe Ser Pro Ser Gln Thr Phe Asn Asp Phe Trp Gly
130 135 140
Phe Ile Met His Gln Ala Val Leu Val Pro Tyr Phe Ala Trp Gln Tyr
145 150 155 160
Ser His Ala Lys His His Arg Arg Thr Asn Asn Ile Met Asp Gly Glu
165 170 175
Ser His Val Pro Asn Ile Ala Lys Glu Met Gly Leu Asn Glu Lys Asn
180 185 190
Glu Arg Ser Gly Gly Tyr Ala Ala Ile His Glu Ala Ile Gly Asp Gly
195 200 205
Pro Phe Ala Met Phe Gln Ile Phe Ala His Leu Val Ile Gly Trp Pro
210 215 220
Ile Tyr Leu Met Gly Phe Ala Ser Thr Gly Arg Leu Gly Gln Asp Gly
225 230 235 240
Lys Glu Leu Gln Ala Gly Glu Ile Ile Asp His Tyr Arg Pro Trp Ser
245 250 255
Lys Met Phe Pro Thr Lys Leu Arg Phe Lys Ile Ala Leu Ser Thr Leu
260 265 270
Gly Val Ile Ala Ala Trp Val Gly Leu Tyr Phe Ala Ala Gln Glu Tyr
275 280 285
Gly Val Leu Pro Val Val Leu Trp Tyr Ile Gly Pro Leu Met Trp Asn
290 295 300
Gln Ala Trp Leu Val Leu Tyr Thr Trp Leu Gln His Asn Asp Pro Ser
305 310 315 320
Val Pro Gln Tyr Gly Ser Asp Glu Trp Thr Trp Val Lys Gly Ala Leu
325 330 335
Ser Thr Ile Asp Arg Pro Tyr Gly Ile Phe Asp Phe Phe His His Lys
340 345 350
Ile Gly Ser Thr His Val Ala His His Leu Phe His Glu Met Pro Phe
355 360 365
Tyr Lys Ala Asp Val Ala Thr Ala Ser Ile Lys Gly Phe Leu Glu Pro
370 375 380
Lys Gly Leu Tyr Asn Tyr Asp Pro Thr Pro Trp Tyr Val Ala Met Trp
385 390 395 400
Arg Val Ala Lys Thr Cys His Tyr Ile Glu Asp Val Asp Gly Val Gln
405 410 415
Tyr Tyr Lys Ser Leu Glu Asp Val Pro Leu Lys Lys Asp Ala Lys Lys
420 425 430
Ser Asp
<210>111
<211>879
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(879)
<223>Δ6-延伸酶
<400>111
atg agt ggc tta cgt gca ccc aac ttt tta cac aga ttc tgg aca aag 48
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
tgg gac tac gcg att tcc aaa gtc gtc ttc acg tgt gcc gac agt ttt 96
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
cag tgg gac atc ggg cca gtg agt tcg agt acg gcg cat tta ccc gcc 144
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
att gaa tcc cct acc cca ctg gtg act agc ctc ttg ttc tac tta gtc 192
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
aca gtt ttc ttg tgg tat ggt cgt tta acc agg agt tca gac aag aaa 240
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
att aga gag cct acg tgg tta aga aga ttc ata ata tgt cat aat gcg 288
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
ttc ttg ata gtc ctc agt ctt tac atg tgc ctt ggt tgt gtg gcc caa 336
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
gcg tat cag aat gga tat act tta tgg ggt aat gaa ttc aag gcc acg 384
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
gaa act cag ctt gct ctc tac att tac att ttt tac gta agt aaa ata 432
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
tac gag ttt gta gat act tac att atg ctt ctc aag aat aac ttg cgg 480
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Asn Leu Arg
145 150 155 160
caa gta agt ttc cta cac att tat cac cac agc acg att tcc ttt att 528
Gln Val Ser Phe Leu His Ile Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
tgg tgg atc att gct cgg agg gct ccg ggt ggt gat gct tac ttc agc 576
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
gcg gcc ttg aac tca tgg gta cac gtg tgc atg tac acc tat tat cta 624
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
tta tca acc ctt att gga aaa gaa gat cct aag cgt tcc aac tac ctt 672
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
tgg tgg ggt cgc cac cta acg caa atg cag atg ctt cag ttt ttc ttc 720
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
aac gta ctt caa gcg ttg tac tgc gct tcg ttc tct acg tat ccc aag 768
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
ttt ttg tcc aaa att ctg ctc gtc tat atg atg agc ctt ctc ggc ttg 816
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
ttt ggg cat ttc tac tat tcc aag cac ata gca gca gct aag ctc cag 864
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
aaa aaa cag cag tga 879
Lys Lys Gln Gln
290
<210>112
<211>292
<212>PRT
<213>Ostreococcus tauri
<400>112
Met Ser Gly Leu Arg Ala Pro Asn Phe Leu His Arg Phe Trp Thr Lys
1 5 10 15
Trp Asp Tyr Ala Ile Ser Lys Val Val Phe Thr Cys Ala Asp Ser Phe
20 25 30
Gln Trp Asp Ile Gly Pro Val Ser Ser Ser Thr Ala His Leu Pro Ala
35 40 45
Ile Glu Ser Pro Thr Pro Leu Val Thr Ser Leu Leu Phe Tyr Leu Val
50 55 60
Thr Val Phe Leu Trp Tyr Gly Arg Leu Thr Arg Ser Ser Asp Lys Lys
65 70 75 80
Ile Arg Glu Pro Thr Trp Leu Arg Arg Phe Ile Ile Cys His Asn Ala
85 90 95
Phe Leu Ile Val Leu Ser Leu Tyr Met Cys Leu Gly Cys Val Ala Gln
100 105 110
Ala Tyr Gln Asn Gly Tyr Thr Leu Trp Gly Asn Glu Phe Lys Ala Thr
115 120 125
Glu Thr Gln Leu Ala Leu Tyr Ile Tyr Ile Phe Tyr Val Ser Lys Ile
130 135 140
Tyr Glu Phe Val Asp Thr Tyr Ile Met Leu Leu Lys Asn Asn Leu Arg
145 150 155 160
Gln Val Ser Phe Leu His Ile Tyr His His Ser Thr Ile Ser Phe Ile
165 170 175
Trp Trp Ile Ile Ala Arg Arg Ala Pro Gly Gly Asp Ala Tyr Phe Ser
180 185 190
Ala Ala Leu Asn Ser Trp Val His Val Cys Met Tyr Thr Tyr Tyr Leu
195 200 205
Leu Ser Thr Leu Ile Gly Lys Glu Asp Pro Lys Arg Ser Asn Tyr Leu
210 215 220
Trp Trp Gly Arg His Leu Thr Gln Met Gln Met Leu Gln Phe Phe Phe
225 230 235 240
Asn Val Leu Gln Ala Leu Tyr Cys Ala Ser Phe Ser Thr Tyr Pro Lys
245 250 255
Phe Leu Ser Lys Ile Leu Leu Val Tyr Met Met Ser Leu Leu Gly Leu
260 265 270
Phe Gly His Phe Tyr Tyr Ser Lys His Ile Ala Ala Ala Lys Leu Gln
275 280 285
Lys Lys Gln Gln
290
<210>113
<211>903
<212>DNA
<213>Ostreococcus tauri
<220>
<221>CDS
<222>(1)..(903)
<223>Δ5-延伸酶
<400>113
atg agc gcc tcc ggt gcg ctg ctg ccc gcg atc gcg ttc gcc gcg tac 48
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
gcg tac gcg acg tac gcc tac gcc ttt gag tgg tcg cac gcg aat ggc 96
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
atc gac aac gtc gac gcg cgc gag tgg atc ggt gcg ctg tcg ttg agg 144
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
ctc ccg gcg atc gcg acg acg atg tac ctg ttg ttc tgc ctg gtc gga 192
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
ccg agg ttg atg gcg aag cgc gag gcg ttc gac ccg aag ggg ttc atg 240
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
ctg gcg tac aat gcg tat cag acg gcg ttc aac gtc gtc gtg ctc ggg 288
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
atg ttc gcg cga gag atc tcg ggg ctg ggg cag ccc gtg tgg ggg tca 336
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
acc atg ccg tgg agc gat aga aaa tcg ttt aag atc ctc ctc ggg gtg 384
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
tgg ttg cac tac aac aac aaa tat ttg gag cta ttg gac act gtg ttc 432
Trp Leu His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
atg gtt gcg cgc aag aag acg aag cag ttg agc ttc ttg cac gtt tat 480
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
cat cac gcc ctg ttg atc tgg gcg tgg tgg ttg gtg tgt cac ttg atg 528
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
gcc acg aac gat tgt atc gat gcc tac ttc ggc gcg gcg tgc aac tcg 576
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
ttc att cac atc gtg atg tac tcg tat tat ctc atg tcg gcg ctc ggc 624
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
att cga tgc ccg tgg aag cga tac atc acc cag gct caa atg ctc caa 672
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
ttc gtc att gtc ttc gcg cac gcc gtg ttc gtg ctg cgt cag aag cac 720
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
tgc ccg gtc acc ctt cct tgg gcg caa atg ttc gtc atg acg aac atg 768
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
ctc gtg ctc ttc ggg aac ttc tac ctc aag gcg tac tcg aac aag tcg 816
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
cgc ggc gac ggc gcg agt tcc gtg aaa cca gcc gag acc acg cgc gcg 864
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
ccc agc gtg cga cgc acg cga tct cga aaa att gac taa 903
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>114
<211>300
<212>PRT
<213>Ostreococcus tauri
<400>114
Met Ser Ala Ser Gly Ala Leu Leu Pro Ala Ile Ala Phe Ala Ala Tyr
1 5 10 15
Ala Tyr Ala Thr Tyr Ala Tyr Ala Phe Glu Trp Ser His Ala Asn Gly
20 25 30
Ile Asp Asn Val Asp Ala Arg Glu Trp Ile Gly Ala Leu Ser Leu Arg
35 40 45
Leu Pro Ala Ile Ala Thr Thr Met Tyr Leu Leu Phe Cys Leu Val Gly
50 55 60
Pro Arg Leu Met Ala Lys Arg Glu Ala Phe Asp Pro Lys Gly Phe Met
65 70 75 80
Leu Ala Tyr Asn Ala Tyr Gln Thr Ala Phe Asn Val Val Val Leu Gly
85 90 95
Met Phe Ala Arg Glu Ile Ser Gly Leu Gly Gln Pro Val Trp Gly Ser
100 105 110
Thr Met Pro Trp Ser Asp Arg Lys Ser Phe Lys Ile Leu Leu Gly Val
115 120 125
Trp Leu His Tyr Asn Asn Lys Tyr Leu Glu Leu Leu Asp Thr Val Phe
130 135 140
Met Val Ala Arg Lys Lys Thr Lys Gln Leu Ser Phe Leu His Val Tyr
145 150 155 160
His His Ala Leu Leu Ile Trp Ala Trp Trp Leu Val Cys His Leu Met
165 170 175
Ala Thr Asn Asp Cys Ile Asp Ala Tyr Phe Gly Ala Ala Cys Asn Ser
180 185 190
Phe Ile His Ile Val Met Tyr Ser Tyr Tyr Leu Met Ser Ala Leu Gly
195 200 205
Ile Arg Cys Pro Trp Lys Arg Tyr Ile Thr Gln Ala Gln Met Leu Gln
210 215 220
Phe Val Ile Val Phe Ala His Ala Val Phe Val Leu Arg Gln Lys His
225 230 235 240
Cys Pro Val Thr Leu Pro Trp Ala Gln Met Phe Val Met Thr Asn Met
245 250 255
Leu Val Leu Phe Gly Asn Phe Tyr Leu Lys Ala Tyr Ser Asn Lys Ser
260 265 270
Arg Gly Asp Gly Ala Ser Ser Val Lys Pro Ala Glu Thr Thr Arg Ala
275 280 285
Pro Ser Val Arg Arg Thr Arg Ser Arg Lys Ile Asp
290 295 300
<210>115
<211>13
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(13)
<223>序列中第2,3,4,6,7,8和9位的Xaa具有表A中给出的含义
<400>115
Asn Xaa Xaa Xaa His Xaa Xaa Met Tyr Xaa Tyr Tyr Xaa
1 5 10
<210>116
<211>10
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(10)
<223>序列中第3,4,5和6位的Xaa具有表A中给出的含义
<400>116
His His Xaa Xaa Xaa Xaa Trp Ala Trp Trp
1 5 10
<210>117
<211>909
<212>DNA
<213>非洲爪蟾(Xenopus laevis)
<220>
<221>CDS
<222>(1)..(909)
<223>Δ5-延伸酶
<400>117
atg gcc ttc aag gag ctc aca tca agg gca gtg ctc ctg tat gat gaa 48
Met Ala Phe Lys Glu Leu Thr Ser Arg Ala Val Leu Leu Tyr Asp Glu
1 5 10 15
tgg att aaa gat gct gat cct agg gtt gaa gac tgg cca ctc atg tcc 96
Trp Ile Lys Asp Ala Asp Pro Arg Val Glu Asp Trp Pro Leu Met Ser
20 25 30
tct cct atc cta caa acc atc atc atc ggc gct tac atc tac ttt gtc 144
Ser Pro Ile Leu Gln Thr Ile Ile Ile Gly Ala Tyr Ile Tyr Phe Val
35 40 45
aca tca ttg ggc cca agg atc atg gag aac agg aag ccg ttt gct ctg 192
Thr Ser Leu Gly Pro Arg Ile Met Glu Asn Arg Lys Pro Phe Ala Leu
50 55 60
aag gag atc atg gca tgt tac aac tta ttc atg gtt ctg ttt tct gtg 240
Lys Glu Ile Met Ala Cys Tyr Asn Leu Phe Met Val Leu Phe Ser Val
65 70 75 80
tac atg tgc tat gag ttt ctc atg tcg ggc tgg gct act gga tat tcc 288
Tyr Met Cys Tyr Glu Phe Leu Met Ser Gly Trp Ala Thr Gly Tyr Ser
85 90 95
ttt aga tgt gac att gtt gac tac tct cag tca cct cag gcg tta cgg 336
Phe Arg Cys Asp Ile Val Asp Tyr Ser Gln Ser Pro Gln Ala Leu Arg
100 105 110
atg gcc tgg acc tgc tgg ctc ttc tat ttt tca aag ttc att gaa tta 384
Met Ala Trp Thr Cys Trp Leu Phe Tyr Phe Ser Lys Phe Ile Glu Leu
115 120 125
tta gac act gtt ttc ttt gtg ctg cgt aag aag aac agc cag att aca 432
Leu Asp Thr Val Phe Phe Val Leu Arg Lys Lys Asn Ser Gln Ile Thr
130 135 140
ttc ctg cac gtc tat cac cac tcc att atg cct tgg acg tgg tgg ttt 480
Phe Leu His Val Tyr His His Ser Ile Met Pro Trp Thr Trp Trp Phe
145 150 155 160
gga gtc aaa ttt gct cca ggt ggt ttg ggc aca ttc cat gca ctg gtg 528
Gly Val Lys Phe Ala Pro Gly Gly Leu Gly Thr Phe His Ala Leu Val
165 170 175
aac tgt gtg gtc cat gtt atc atg tac agc tac tac ggc ctg tca gcc 576
Asn Cys Val Val His Val Ile Met Tyr Ser Tyr Tyr Gly Leu Ser Ala
180 185 190
ttg ggg cct gcc tac cag aag tac ctg tgg tgg aaa aag tac atg acg 624
Leu Gly Pro Ala Tyr Gln Lys Tyr Leu Trp Trp Lys Lys Tyr Met Thr
195 200 205
tct atc caa ctg acc cag ttc ttg atg gtt act ttt cac atc ggc cag 672
Ser Ile Gln Leu Thr Gln Phe Leu Met Val Thr Phe His Ile Gly Gln
210 215 220
ttc ttc ttc atg gag aat tgc ccg tac cag tat ccc gtc ttc ttg tat 720
Phe Phe Phe Met Glu Asn Cys Pro Tyr Gln Tyr Pro Val Phe Leu Tyr
225 230 235 240
gtc att tgg ctg tac ggg ttc gtt ttc tta atc ttg ttc ctc aac ttc 768
Val Ile Trp Leu Tyr Gly Phe Val Phe Leu Ile Leu Phe Leu Asn Phe
245 250 255
tgg ttc cac gct tac atc aaa gga cag agg ctg ccg aaa gcc gtc caa 816
Trp Phe His Ala Tyr Ile Lys Gly Gln Arg Leu Pro Lys Ala Val Gln
260 265 270
aat ggc cac tgc aag aac aac aac aac caa gaa aac act tgg tgc aag 864
Asn Gly His Cys Lys Asn Asn Asn Asn Gln Glu Asn Thr Trp Cys Lys
275 280 285
aac aaa aac cag aaa aac ggt gca ttg aaa agc aaa aac cat tga 909
Asn Lys Asn Gln Lys Asn Gly Ala Leu Lys Ser Lys Asn His
290 295 300
<210>118
<211>302
<212>PRT
<213>非洲爪蟾
<400>118
Met Ala Phe Lys Glu Leu Thr Ser Arg Ala Val Leu Leu Tyr Asp Glu
1 5 10 15
Trp Ile Lys Asp Ala Asp Pro Arg Val Glu Asp Trp Pro Leu Met Ser
20 25 30
Ser Pro Ile Leu Gln Thr Ile Ile Ile Gly Ala Tyr Ile Tyr Phe Val
35 40 45
Thr Ser Leu Gly Pro Arg Ile Met Glu Asn Arg Lys Pro Phe Ala Leu
50 55 60
Lys Glu Ile Met Ala Cys Tyr Asn Leu Phe Met Val Leu Phe Ser Val
65 70 75 80
Tyr Met Cys Tyr Glu Phe Leu Met Ser Gly Trp Ala Thr Gly Tyr Ser
85 90 95
Phe Arg Cys Asp Ile Val Asp Tyr Ser Gln Ser Pro Gln Ala Leu Arg
100 105 110
Met Ala Trp Thr Cys Trp Leu Phe Tyr Phe Ser Lys Phe Ile Glu Leu
115 120 125
Leu Asp Thr Val Phe Phe Val Leu Arg Lys Lys Asn Ser Gln Ile Thr
130 135 140
Phe Leu His Val Tyr His His Ser Ile Met Pro Trp Thr Trp Trp Phe
145 150 155 160
Gly Val Lys Phe Ala Pro Gly Gly Leu Gly Thr Phe His Ala Leu Val
165 170 175
Asn Cys Val Val His Val Ile Met Tyr Ser Tyr Tyr Gly Leu Ser Ala
180 185 190
Leu Gly Pro Ala Tyr Gln Lys Tyr Leu Trp Trp Lys Lys Tyr Met Thr
195 200 205
Ser Ile Gln Leu Thr Gln Phe Leu Met Val Thr Phe His Ile Gly Gln
210 215 220
Phe Phe Phe Met Glu Asn Cys Pro Tyr Gln Tyr Pro Val Phe Leu Tyr
225 230 235 240
Val Ile Trp Leu Tyr Gly Phe Val Phe Leu Ile Leu Phe Leu Asn Phe
245 250 255
Trp Phe His Ala Tyr Ile Lys Gly Gln Arg Leu Pro Lys Ala Val Gln
260 265 270
Asn Gly His Cys Lys Asn Asn Asn Asn Gln Glu Asn Thr Trp Cys Lys
275 280 285
Asn Lys Asn Gln Lys Asn Gly Ala Leu Lys Ser Lys Asn His
290 295 300
<210>119
<211>870
<212>DNA
<213>玻璃海鞘(Ciona intestinalis)
<220>
<221>CDS
<222>(1)..(870)
<223>Δ5-延伸酶
<400>119
atg gac gta ctt cat cgt ttc tta gga ttc tac gaa tgg acg ctg act 48
Met Asp Val Leu His Arg Phe Leu Gly Phe Tyr Glu Trp Thr Leu Thr
1 5 10 15
ttc gcg gac ccc cga gtg gca aaa tgg cct tta ata gaa aac ccc ctt 96
Phe Ala Asp Pro Arg Val Ala Lys Trp Pro Leu Ile Glu Asn Pro Leu
20 25 30
cct aca att gct att gtg ttg ctg tac ctg gcg ttt gtt ctg tat att 144
Pro Thr Ile Ala Ile Val Leu Leu Tyr Leu Ala Phe Val Leu Tyr Ile
35 40 45
ggg ccg cgt ttt atg cga aaa aga gca cca gtt gac ttt ggt tta ttc 192
Gly Pro Arg Phe Met Arg Lys Arg Ala Pro Val Asp Phe Gly Leu Phe
50 55 60
ctc cct gga tat aac ttt gct ttg gtt gca tta aat tat tat atc ctg 240
Leu Pro Gly Tyr Asn Phe Ala Leu Val Ala Leu Asn Tyr Tyr Ile Leu
65 70 75 80
caa gaa gtg gtc act ggg agt tat ggg gct ggg tat gat ttg gtt tgc 288
Gln Glu Val Val Thr Gly Ser Tyr Gly Ala Gly Tyr Asp Leu Val Cys
85 90 95
aca cca ctt cga agt gat tcc tac gat ccc aat gaa atg aag gtt gca 336
Thr Pro Leu Arg Ser Asp Ser Tyr Asp Pro Asn Glu Met Lys Val Ala
100 105 110
aac gct gta tgg tgg tat tat gta tcc aag ata ata gag ttg ttt gat 384
Asn Ala Val Trp Trp Tyr Tyr Val Ser Lys Ile Ile Glu Leu Phe Asp
115 120 125
act gtg ttg ttc act cta cgc aaa cga gac cga caa gta act ttc ctt 432
Thr Val Leu Phe Thr Leu Arg Lys Arg Asp Arg Gln Val Thr Phe Leu
130 135 140
cat gtt tat cac cat tct acc atg ccc ctg ttg tgg tgg att ggg gca 480
His Val Tyr His His Ser Thr Met Pro Leu Leu Trp Trp Ile Gly Ala
145 150 155 160
aag tgg gtg cct ggt ggg caa tca ttt gtt ggc atc ata ctg aac tcc 528
Lys Trp Val Pro Gly Gly Gln Ser Phe Val Gly Ile Ile Leu Asn Ser
165 170 175
agt gtt cat gtt atc atg tat acg tac tat gga ttg tca gcc ttg ggg 576
Ser Val His Val Ile Met Tyr Thr Tyr Tyr Gly Leu Ser Ala Leu Gly
180 185 190
cct cac atg cag aag ttt cta tgg tgg aag aaa tat atc aca atg ttg 624
Pro His Met Gln Lys Phe Leu Trp Trp Lys Lys Tyr Ile Thr Met Leu
195 200 205
caa ctg gtt caa ttt gtt ctt gcc atc tac cat act gct cga tca ttg 672
Gln Leu Val Gln Phe Val Leu Ala Ile Tyr His Thr Ala Arg Ser Leu
210 215 220
tac gtt aaa tgt ccc tcg cct gtt tgg atg cac tgg gca ctt atc ttg 720
Tyr Val Lys Cys Pro Ser Pro Val Trp Met His Trp Ala Leu Ile Leu
225 230 235 240
tac gct ttc tca ttc att ttg ctt ttc tca aac ttc tac atg cat gcc 768
Tyr Ala Phe Ser Phe Ile Leu Leu Phe Ser Asn Phe Tyr Met His Ala
245 250 255
tat atc aag aaa tca aga aaa ggg aaa gag aat ggc agt cga gga aaa 816
Tyr Ile Lys Lys Ser Arg Lys Gly Lys Glu Asn Gly Ser Arg Gly Lys
260 265 270
ggt ggt gta agt aat gga aag gaa aag ctg cac gct aat ggt aaa acc 864
Gly Gly Val Ser Asn Gly Lys Glu Lys Leu His Ala Asn Gly Lys Thr
275 280 285
gat taa 870
Asp
<210>120
<211>289
<212>PRT
<213>玻璃海鞘
<400>120
Met Asp Val Leu His Arg Phe Leu Gly Phe Tyr Glu Trp Thr Leu Thr
1 5 10 15
Phe Ala Asp Pro Arg Val Ala Lys Trp Pro Leu Ile Glu Asn Pro Leu
20 25 30
Pro Thr Ile Ala Ile Val Leu Leu Tyr Leu Ala Phe Val Leu Tyr Ile
35 40 45
Gly Pro Arg Phe Met Arg Lys Arg Ala Pro Val Asp Phe Gly Leu Phe
50 55 60
Leu Pro Gly Tyr Asn Phe Ala Leu Val Ala Leu Asn Tyr Tyr Ile Leu
65 70 75 80
Gln Glu Val Val Thr Gly Ser Tyr Gly Ala Gly Tyr Asp Leu Val Cys
85 90 95
Thr Pro Leu Arg Ser Asp Ser Tyr Asp Pro Asn Glu Met Lys Val Ala
100 105 110
Asn Ala Val Trp Trp Tyr Tyr Val Ser Lys Ile Ile Glu Leu Phe Asp
115 120 125
Thr Val Leu Phe Thr Leu Arg Lys Arg Asp Arg Gln Val Thr Phe Leu
130 135 140
His Val Tyr His His Ser Thr Met Pro Leu Leu Trp Trp Ile Gly Ala
145 150 155 160
Lys Trp Val Pro Gly Gly Gln Ser Phe Val Gly Ile Ile Leu Asn Ser
165 170 175
Ser Val His Val Ile Met Tyr Thr Tyr Tyr Gly Leu Ser Ala Leu Gly
180 185 190
Pro His Met Gln Lys Phe Leu Trp Trp Lys Lys Tyr Ile Thr Met Leu
195 200 205
Gln Leu Val Gln Phe Val Leu Ala Ile Tyr His Thr Ala Arg Ser Leu
210 215 220
Tyr Val Lys Cys Pro Ser Pro Val Trp Met His Trp Ala Leu Ile Leu
225 230 235 240
Tyr Ala Phe Ser Phe Ile Leu Leu Phe Ser Asn Phe Tyr Met His Ala
245 250 255
Tyr Ile Lys Lys Ser Arg Lys Gly Lys Glu Asn Gly Ser Arg Gly Lys
260 265 270
Gly Gly Val Ser Asn Gly Lys Glu Lys Leu His Ala Asn Gly Lys Thr
275 280 285
Asp
<210>121
<211>30
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(30)
<223>
<400>121
aggatccatg gccttcaagg agctcacatc 30
<210>122
<211>35
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(35)
<223>
<400>122
cctcgagtca atggtttttg cttttcaatg caccg 35
<210>123
<211>25
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>
<400>123
taagcttatg gacgtacttc atcgt 25
<210>124
<211>26
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>
<400>124
tcagatcttt aatcggtttt accatt 26
<210>125
<211>34
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(34)
<223>
<400>125
gcggccgcac catggccttc aaggagctca catc 34
<210>126
<211>38
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(38)
<223>
<400>126
gcggccgcct tcaatggttt ttgcttttca atgcaccg 38
<210>127
<211>29
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(29)
<223>
<400>127
gcggccgcac catggacgta cttcatcgt 29
<210>128
<211>27
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(27)
<223>
<400>128
gcggccgctt taatcggttt taccatt 27
<210>129
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>129
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>130
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>130
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>131
<211>789
<212>DNA
<213>细小裸藻
<220>
<221>CDS
<222>(1)..(789)
<223>Δ5-延伸酶
<400>131
atg ctg ggg gcc atc gcg gac gtc gtg ctc cgg ggg ccc gcc gca ttc 48
Met Leu Gly Ala Ile Ala Asp Val Val Leu Arg Gly Pro Ala Ala Phe
1 5 10 15
cac tgg gac cct gcc acc acc ccg ctc gca tcg atc gtc agc ccc tgt 96
His Trp Asp Pro Ala Thr Thr Pro Leu Ala Ser Ile Val Ser Pro Cys
20 25 30
gtg gcc tcc gtg gcg tac ctg ggg gcc atc ggg ctg ctg aag cgc cgc 144
Val Ala Ser Val Ala Tyr Leu Gly Ala Ile Gly Leu Leu Lys Arg Arg
35 40 45
act gga ccg gag gtc cgc tcc aag ccc ttc gag ctg cta cac aac ggg 192
Thr Gly Pro Glu Val Arg Ser Lys Pro Phe Glu Leu Leu His Asn Gly
50 55 60
ctg ctg gtg ggc tgg tcc ctc gtg gtg ctg ctc ggg acg ctg tac ggc 240
Leu Leu Val Gly Trp Ser Leu Val Val Leu Leu Gly Thr Leu Tyr Gly
65 70 75 80
gcg ttc cag cgc gtg cag gag gac ggc cgg ggg gtg cag gcc ctc ctg 288
Ala Phe Gln Arg Val Gln Glu Asp Gly Arg Gly Val Gln Ala Leu Leu
85 90 95
tgc acc cag cgg cca cca tct cag atc tgg gac ggc ccg gtg ggg tac 336
Cys Thr Gln Arg Pro Pro Ser Gln Ile Trp Asp Gly Pro Val Gly Tyr
100 105 110
ttc acg tac ctc ttc tac ctc gcg aag tac tgg gag ctg gcg gac act 384
Phe Thr Tyr Leu Phe Tyr Leu Ala Lys Tyr Trp Glu Leu Ala Asp Thr
115 120 125
gtc atc ctc gcc ctc cgc cag aag ccc acc atc ccc ctc cac gtc tac 432
Val Ile Leu Ala Leu Arg Gln Lys Pro Thr Ile Pro Leu His Val Tyr
130 135 140
cat cac gcc gtc atg ctg ttc atc gtg tgg tcg tgg ttc gcg cac ccc 480
His His Ala Val Met Leu Phe Ile Val Trp Ser Trp Phe Ala His Pro
145 150 155 160
tgg ctc gag ggg agc tgg tgg tgc tcc ctg gtc aac tct ttc atc cac 528
Trp Leu Glu Gly Ser Trp Trp Cys Ser Leu Val Asn Ser Phe Ile His
165 170 175
acg gtg atg tac tcg tac tac acc ctg acg gtg gtt ggc atc aac cct 576
Thr Val Met Tyr Ser Tyr Tyr Thr Leu Thr Val Val Gly Ile Asn Pro
180 185 190
tgg tgg aag aag tgg atg acc acc atg cag atc atc cag ttc atc acg 624
Trp Trp Lys Lys Trp Met Thr Thr Met Gln Ile Ile Gln Phe Ile Thr
195 200 205
ggc tgc gtg tac gtc atg gcg ttc ttc ggc cta tat tat gcc ggg gcg 672
Gly Cys Val Tyr Val Met Ala Phe Phe Gly Leu Tyr Tyr Ala Gly Ala
210 215 220
ggc tgc acc tcc aac gtg tac act gcc tgg ttc tcg atg ggg gtc aac 720
Gly Cys Thr Ser Asn Val Tyr Thr Ala Trp Phe Ser Met Gly Val Asn
225 230 235 240
ctc agc ttt ctg tgg ctc ttc gct ctt ttc ttc cgc cgg tca tac agc 768
Leu Ser Phe Leu Trp Leu Phe Ala Leu Phe Phe Arg Arg Ser Tyr Ser
245 250 255
aaa cct agc cgg aag gag tag 789
Lys Pro Ser Arg Lys Glu
260
<210>132
<211>262
<212>PRT
<213>细小裸藻
<400>132
Met Leu Gly Ala Ile Ala Asp Val Val Leu Arg Gly Pro Ala Ala Phe
1 5 10 15
His Trp Asp Pro Ala Thr Thr Pro Leu Ala Ser Ile Val Ser Pro Cys
20 25 30
Val Ala Ser Val Ala Tyr Leu Gly Ala Ile Gly Leu Leu Lys Arg Arg
35 40 45
Thr Gly Pro Glu Val Arg Ser Lys Pro Phe Glu Leu Leu His Asn Gly
50 55 60
Leu Leu Val Gly Trp Ser Leu Val Val Leu Leu Gly Thr Leu Tyr Gly
65 70 75 80
Ala Phe Gln Arg Val Gln Glu Asp Gly Arg Gly Val Gln Ala Leu Leu
85 90 95
Cys Thr Gln Arg Pro Pro Ser Gln Ile Trp Asp Gly Pro Val Gly Tyr
100 105 110
Phe Thr Tyr Leu Phe Tyr Leu Ala Lys Tyr Trp Glu Leu Ala Asp Thr
115 120 125
Val Ile Leu Ala Leu Arg Gln Lys Pro Thr Ile Pro Leu His Val Tyr
130 135 140
His His Ala Val Met Leu Phe Ile Val Trp Ser Trp Phe Ala His Pro
145 150 155 160
Trp Leu Glu Gly Ser Trp Trp Cys Ser Leu Val Asn Ser Phe Ile His
165 170 175
Thr Val Met Tyr Ser Tyr Tyr Thr Leu Thr Val Val Gly Ile Asn Pro
180 185 190
Trp Trp Lys Lys Trp Met Thr Thr Met Gln Ile Ile Gln Phe Ile Thr
195 200 205
Gly Cys Val Tyr Val Met Ala Phe Phe Gly Leu Tyr Tyr Ala Gly Ala
210 215 220
Gly Cys Thr Ser Asn Val Tyr Thr Ala Trp Phe Ser Met Gly Val Asn
225 230 235 240
Leu Ser Phe Leu Trp Leu Phe Ala Leu Phe Phe Arg Arg Ser Tyr Ser
245 250 255
Lys Pro Ser Arg Lys Glu
260
<210>133
<211>789
<212>DNA
<213>细小裸藻
<220>
<221>CDS
<222>(1)..(789)
<223>Δ5-延伸酶
<400>133
atg ctg ggg gcc atc gcg gac gtc gtg ctc cgg ggg ccc gcc gca ttc 48
Met Leu Gly Ala Ile Ala Asp Val Val Leu Arg Gly Pro Ala Ala Phe
1 5 10 15
cac tgg gac cct gcc acc acc ccg ctc gca tcg atc gtc agc ccc tgt 96
His Trp Asp Pro Ala Thr Thr Pro Leu Ala Ser Ile Val Ser Pro Cys
20 25 30
gtg gcc tcc gtg gcg tac ctg ggg gcc atc ggg ctg ctg aag cgc cgc 144
Val Ala Ser Val Ala Tyr Leu Gly Ala Ile Gly Leu Leu Lys Arg Arg
35 40 45
act gga ccg gag gtc cgc tcc aag ccc ttc gag ctg cta cac aac ggg 192
Thr Gly Pro Glu Val Arg Ser Lys Pro Phe Glu Leu Leu His Asn Glv
50 55 60
ctg ctg gtg ggc tgg tcc ctc gtg gtg ctg ctc ggg acg ctg tac ggc 240
Leu Leu Val Gly Trp Ser Leu Val Val Leu Leu Gly Thr Leu Tyr Gly
65 70 75 80
gcg tac cag cgc gtg cag gag gac ggc cgg ggg gtg cag gcc ctg ctg 288
Ala Tyr Gln Arg Val Gln Glu Asp Gly Arg Gly Val Gln Ala Leu Leu
85 90 95
tgc acc cag cgg cca cca tct cag atc tgg gac ggc ccg gtg ggg tac 336
Cys Thr Gln Arg Pro Pro Ser Gln Ile Trp Asp Gly Pro Val Gly Tyr
100 105 110
ttc acg tac ctt ttc tac ctc gcg aag tac tgg gag ctg gtg gac act 384
Phe Thr Tyr Leu Phe Tyr Leu Ala Lys Tyr Trp Glu Leu Val Asp Thr
115 120 125
gtc atc ctc gcc ctc cgc cag aag ccc acc atc ccc ctc cac gtc tac 432
Val Ile Leu Ala Leu Arg Gln Lys Pro Thr Ile Pro Leu His Val Tyr
130 135 140
cat cac gcc gtc atg ctg ttc att gtg tgg tcg tgg ttc gcg cac ccc 480
His His Ala Val Met Leu Phe Ile Val Trp Ser Trp Phe Ala His Pro
145 150 155 160
tgg ctc gag ggg agc tgg tgg tgc tcc ctg gtc aac tct ttc atc cac 528
Trp Leu Glu Gly Ser Trp Trp Cys Ser Leu Val Asn Ser Phe Ile His
165 170 175
acg gtg atg tac tcg tat tac acc ctg acg gtg gtt ggc atc aac cct 576
Thr Val Met Tyr Ser Tyr Tyr Thr Leu Thr Val Val Gly Ile Asn Pro
180 185 190
tgg tgg aag aag tgg atg acc acc atg cag atc atc cag ttc atc acg 624
Trp Trp Lys Lys Trp Met Thr Thr Met Gln Ile Ile Gln Phe Ile Thr
195 200 205
ggc tgc gtg tac gtc acg gcg ttc ttc ggc cta tac tat gcc ggg gcg 672
Gly Cys Val Tyr Val Thr Ala Phe Phe Gly Leu Tyr Tyr Ala Gly Ala
210 215 220
ggc tgc acc tcc aac gtg tac act gcc tgg ttc tcg atg ggg gtc aac 720
Gly Cys Thr Ser Asn Val Tyr Thr Ala Trp Phe Ser Met Gly Val Asn
225 230 235 240
ctc agc ttt ctg tgg ctc ttc gct ctt ttc ttc cgc cgg tcg tac agc 768
Leu Ser Phe Leu Trp Leu Phe Ala Leu Phe Phe Arg Arg Ser Tyr Ser
245 250 255
aaa cct agc cgg aag gag tag 789
Lys Pro Ser Arg Lys Glu
260
<210>134
<211>262
<212>PRT
<213>细小裸藻
<400>134
Met Leu Gly Ala Ile Ala Asp Val Val Leu Arg Gly Pro Ala Ala Phe
1 5 10 15
His Trp Asp Pro Ala Thr Thr Pro Leu Ala Ser Ile Val Ser Pro Cys
20 25 30
Val Ala Ser Val Ala Tyr Leu Gly Ala Ile Gly Leu Leu Lys Arg Arg
35 40 45
Thr Gly Pro Glu Val Arg Ser Lys Pro Phe Glu Leu Leu His Asn Gly
50 55 60
Leu Leu Val Gly Trp Ser Leu Val Val Leu Leu Gly Thr Leu Tyr Gly
65 70 75 80
Ala Tyr Gln Arg Val Gln Glu Asp Gly Arg Gly Val Gln Ala Leu Leu
85 90 95
Cys Thr Gln Arg Pro Pro Ser Gln Ile Trp Asp Gly Pro Val Gly Tyr
100 105 110
Phe Thr Tyr Leu Phe Tyr Leu Ala Lys Tyr Trp Glu Leu Val Asp Thr
115 120 125
Val Ile Leu Ala Leu Arg Gln Lys Pro Thr Ile Pro Leu His Val Tyr
130 135 140
His His Ala Val Met Leu Phe Ile Val Trp Ser Trp Phe Ala His Pro
145 150 155 160
Trp Leu Glu Gly Ser Trp Trp Cys Ser Leu Val Asn Ser Phe Ile His
165 170 175
Thr Val Met Tyr Ser Tyr Tyr Thr Leu Thr Val Val Gly Ile Asn Pro
180 185 190
Trp Trp Lys Lys Trp Met Thr Thr Met Gln Ile Ile Gln Phe Ile Thr
195 200 205
Gly Cys Val Tyr Val Thr Ala Phe Phe Gly Leu Tyr Tyr Ala Gly Ala
210 215 220
Gly Cys Thr Ser Asn Val Tyr Thr Ala Trp Phe Ser Met Gly Val Asn
225 230 235 240
Leu Ser Phe Leu Trp Leu Phe Ala Leu Phe Phe Arg Arg Ser Tyr Ser
245 250 255
Lys Pro Ser Arg Lys Glu
260
<210>135
<211>897
<212>DNA
<213>拟南芥菜(Arabidopsis thaliana)
<220>
<221>CDS
<222>(1)..(897)
<223>Δ5-延伸酶
<400>135
atg gca tct gtt tac tcc acc cta acc tac tgg ctc gtc cac cac ccc 48
Met Ala Ser Val Tyr Ser Thr Leu Thr Tyr Trp Leu Val His His Pro
1 5 10 15
tac att gcc aac ttc acg tgg acc gaa ggt gaa aca cta ggc tcc acc 96
Tyr Ile Ala Asn Phe Thr Trp Thr Glu Gly Glu Thr Leu Gly Ser Thr
20 25 30
gtt ttc ttt gtc ttt gtc gtc gtc tcc ctt tac ctc tcc gcc aca ttc 144
Val Phe Phe Val Phe Val Val Val Ser Leu Tyr Leu Ser Ala Thr Phe
35 40 45
ctc ctc cga tac acc gtc gat tca ctc ccc aca ctc ggt ccc cgc att 192
Leu Leu Arg Tyr Thr Val Asp Ser Leu Pro Thr Leu Gly Pro Arg Ile
50 55 60
ctc aaa cca atc aca gcc gtt cac agc ctc att ctc ttc ctc ctc tcc 240
Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Phe Leu Leu Ser
65 70 75 80
tta acc atg gcc gtt ggt tgc act ctc tcc cta atc tct tcc tcg gac 288
Leu Thr Met Ala Val Gly Cys Thr Leu Ser Leu Ile Ser Ser Ser Asp
85 90 95
ccg aag gcg cgt ctc ttc gac gcc gtt tgt ttc ccc ctc gac gtg aaa 336
Pro Lys Ala Arg Leu Phe Asp Ala Val Cys Phe Pro Leu Asp Val Lys
100 105 110
cct aag gga ccg ctt ttc ttt tgg gct caa gtc ttt tac ctc tcg aag 384
Pro Lys Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr Leu Ser Lys
115 120 125
atc ctt gag ttc gta gac aca ctt ctc atc ata ctc aac aaa tca atc 432
Ile Leu Glu Phe Val Asp Thr Leu Leu Ile Ile Leu Asn Lvs Ser Ile
130 135 140
caa cgg ctc tcg ttc ctc cac gtc tac cac cac gca acg gtt gtg att 480
Gln Arg Leu Ser Phe Leu His Val Tyr His His Ala Thr Val Val Ile
145 150 155 160
ttg tgc tac ctc tgg tta cga aca cgt caa tcg atg ttt cct gtt ggg 528
Leu Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe Pro Val Gly
165 170 175
ctc gtg ttg aac tcg acg gtc cat gtg att atg tac ggg tac tat ttc 576
Leu Val Leu Asn Ser Thr Val His Val Ile Met Tyr Gly Tyr Tyr Phe
180 185 190
ctc tgc gct atc gga tcg agg ccc aag tgg aag aag ttg gtg acg aat 624
Leu Cys Ala Ile Gly Ser Arg Pro Lys Trp Lys Lys Leu Val Thr Asn
195 200 205
ttt caa atg gtt cag ttt gct ttc ggc atg ggg tta gga gcc gct tgg 672
Phe Gln Met Val Gln Phe Ala Phe Gly Met Gly Leu Gly Ala Ala Trp
210 215 220
atg ctc cca gag cat tat ttc ggg tcg ggt tgc gcc ggg att tgg aca 720
Met Leu Pro Glu His Tyr Phe Gly Ser Gly Cys Ala Gly Ile Trp Thr
225 230 235 240
gtt tat ttc aat ggt gtg ttt act gct tct cta ttg gct ctc ttc tac 768
Val Tyr Phe Asn Gly Val Phe Thr Ala Ser Leu Leu Ala Leu Phe Tyr
245 250 255
aac ttc cac tcc aag aac tat gag aag act aca acg tcg cct ttg tat 816
Asn Phe His Ser Lys Asn Tyr Glu Lys Thr Thr Thr Ser Pro Leu Tyr
260 265 270
aag atc gaa tcc ttt ata ttt att cac gga gag agg tgg gca aat aaa 864
Lys Ile Glu Ser Phe Ile Phe Ile His Gly Glu Arg Trp Ala Asn Lys
275 280 285
gcg att aca tta ttt tcc aag aaa aac gat taa 897
Ala Ile Thr Leu Phe Ser Lys Lys Asn Asp
290 295
<210>136
<211>298
<212>PRT
<213>拟南芥菜
<400>136
Met Ala Ser Val Tyr Ser Thr Leu Thr Tyr Trp Leu Val His His Pro
1 5 10 15
Tyr Ile Ala Asn Phe Thr Trp Thr Glu Gly Glu Thr Leu Gly Ser Thr
20 25 30
Val Phe Phe Val Phe Val Val Val Ser Leu Tyr Leu Ser Ala Thr Phe
35 40 45
Leu Leu Arg Tyr Thr Val Asp Ser Leu Pro Thr Leu Gly Pro Arg Ile
50 55 60
Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Phe Leu Leu Ser
65 70 75 80
Leu Thr Met Ala Val Gly Cys Thr Leu Ser Leu Ile Ser Ser Ser Asp
85 90 95
Pro Lys Ala Arg Leu Phe Asp Ala Val Cys Phe Pro Leu Asp Val Lys
100 105 110
Pro Lys Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr Leu Ser Lys
115 120 125
Ile Leu Glu Phe Val Asp Thr Leu Leu Ile Ile Leu Asn Lys Ser Ile
130 135 140
Gln Arg Leu Ser Phe Leu His Val Tyr His His Ala Thr Val Val Ile
145 150 155 160
Leu Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe Pro Val Gly
165 170 175
Leu Val Leu Asn Ser Thr Val His Val Ile Met Tyr Gly Tyr Tyr Phe
180 185 190
Leu Cys Ala Ile Gly Ser Arg Pro Lys Trp Lys Lys Leu Val Thr Asn
195 200 205
Phe Gln Met Val Gln Phe Ala Phe Gly Met Gly Leu Gly Ala Ala Trp
210 215 220
Met Leu Pro Glu His Tyr Phe Gly Ser Gly Cys Ala Gly Ile Trp Thr
225 230 235 240
Val Tyr Phe Asn Gly Val Phe Thr Ala Ser Leu Leu Ala Leu Phe Tyr
245 250 255
Asn Phe His Ser Lys Asn Tyr Glu Lys Thr Thr Thr Ser Pro Leu Tyr
260 265 270
Lys Ile Glu Ser Phe Ile Phe Ile His Gly Glu Arg Trp Ala Asn Lys
275 280 285
Ala Ile Thr Leu Phe Ser Lys Lys Asn Asp
290 295
<210>137
<211>837
<212>DNA
<213>拟南芥菜
<220>
<221>CDS
<222>(1)..(837)
<223>Δ5-延伸酶
<400>137
atg gca tca att tac tcc tct tta acc tac tgg ctc gtt aac cac ccc 48
Met Ala Ser Ile Tyr Ser Ser Leu Thr Tyr Trp Leu Val Asn His Pro
1 5 10 15
tac atc tcc aat ttt act tgg atc gaa ggt gaa acc cta ggc tcc acc 96
Tyr Ile Ser Asn Phe Thr Trp Ile Glu Gly Glu Thr Leu Gly Ser Thr
20 25 30
gtc ttt ttc gta tcc gtc gta gtc tcc gtt tac ctc tcc gcc acg ttc 144
Val Phe Phe Val Ser Val Val Val Ser Val Tyr Leu Ser Ala Thr Phe
35 40 45
ctc ctc cga tcc gcc atc gat tca ctc cca tca ctc agt cca cgt atc 192
Leu Leu Arg Ser Ala Ile Asp Ser Leu Pro Ser Leu Ser Pro Arg Ile
50 55 60
ctc aaa ccg atc aca gcc gtc cac agc cta atc ctc tgt ctc ctc tcc 240
Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Cys Leu Leu Ser
65 70 75 80
tta gtc atg gcc gtc ggt tgc act ctc tca ata acc tca tct cac gcg 288
Leu Val Met Ala Val Gly Cys Thr Leu Ser Ile Thr Ser Ser His Ala
85 90 95
tct tca gat ccg atg gcg cgt ttc ctt cac gcg att tgc ttt ccc gtc 336
Ser Ser Asp pro Met Ala Arg Phe Leu His Ala Ile Cys Phe Pro Val
100 105 110
gac gtt aaa cct aac gga ccg ctt ttc ttc tgg gct caa gtc ttc tac 384
Asp Val Lys Pro Asn Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr
115 120 125
ctc tcg aag atc ctc gag ttc gga gac acg atc ctc atc ata ctc ggc 432
Leu Ser Lys Ile Leu Glu Phe Gly Asp Thr Ile Leu Ile Ile Leu Gly
130 135 140
aaa tca atc caa cgg cta tcc ttc ctc cac gtg tac cac cac gcg acg 480
Lys Ser Ile Gln Arg Leu Ser Phe Leu His Val Tyr His His Ala Thr
145 150 155 160
gtt gtg gtc atg tgt tat ctc tgg ctc cga act cgc caa tcg atg ttt 528
Val Val Val Met Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe
165 170 175
ccg att gcg ctc gtg acg aat tcg acg gta cac gtc atc atg tac ggt 576
Pro Ile Ala Leu Val Thr Asn Ser Thr Val His Val Ile Met Tyr Gly
180 185 190
tac tac ttc ctc tgc gcc gtt gga tcg agg ccc aag tgg aag aga ttg 624
Tyr Tyr Phe Leu Cys Ala Val Gly Ser Arg Pro Lys Trp Lys Arg Leu
195 200 205
gtg acg gat tgt cag att gtt cag ttt gtt ttc agt ttc ggg tta tcc 672
Val Thr Asp Cys Gln Ile Val Gln Phe Val Phe Ser Phe Gly Leu Ser
210 215 220
ggt tgg atg ctc cga gag cac tta ttc ggg tcg ggt tgc acc ggg att 720
Gly Trp Met Leu Arg Glu His Leu Phe Gly Ser Gly Cys Thr Gly Ile
225 230 235 240
tgg gga tgg tgt ttc aac gct gca ttt aat gct tct ctt ttg gct ctc 768
Trp Gly Trp Cys Phe Asn Ala Ala Phe Asn Ala Ser Leu Leu Ala Leu
245 250 255
ttt tcc aac ttc cat tca aag aat tat gtc aag aag cca acg aga gag 816
Phe Ser Asn Phe His Ser Lys Asn Tyr Val Lys Lys Pro Thr Arg Glu
260 265 270
gat ggc aaa aaa agc gat tag 837
Asp Gly Lys Lys Ser Asp
275
<210>138
<211>278
<212>PRT
<213>拟南芥菜
<400>138
Met Ala Ser Ile Tyr Ser Ser Leu Thr Tyr Trp Leu Val Asn His Pro
1 5 10 15
Tyr Ile Ser Asn Phe Thr Trp Ile Glu Gly Glu Thr Leu Gly Ser Thr
20 25 30
Val Phe Phe Val ser Val Val Val Ser Val Tyr Leu Ser Ala Thr Phe
35 40 45
Leu Leu Arg Ser Ala Ile Asp Ser Leu Pro Ser Leu Ser Pro Arg Ile
50 55 60
Leu Lys Pro Ile Thr Ala Val His Ser Leu Ile Leu Cys Leu Leu Ser
65 70 75 80
Leu Val Met Ala Val Gly Cys Thr Leu Ser Ile Thr Ser Ser His Ala
85 90 95
Ser Ser Asp Pro Met Ala Arg Phe Leu His Ala Ile Cys Phe Pro Val
100 105 110
Asp Val Lys Pro Asn Gly Pro Leu Phe Phe Trp Ala Gln Val Phe Tyr
115 120 125
Leu Ser Lys Ile Leu Glu Phe Gly Asp Thr Ile Leu Ile Ile Leu Gly
130 135 140
Lys Ser Ile Gln Arg Leu Ser Phe Leu His Val Tyr His H1s Ala Thr
145 150 155 160
Val Val Val Met Cys Tyr Leu Trp Leu Arg Thr Arg Gln Ser Met Phe
165 170 175
Pro Ile Ala Leu Val Thr Asn Ser Thr Val His Val Ile Met Tyr Gly
180 185 190
Tyr Tyr Phe Leu Cys Ala Val Gly Ser Arg Pro Lys Trp Lys Arg Leu
195 200 205
Val Thr Asp Cys Gln Ile Val Gln Phe Val Phe Ser Phe Gly Leu Ser
210 215 220
Gly Trp Met Leu Arg Glu His Leu Phe Gly Ser Gly Cys Thr Gly Ile
225 230 235 240
Trp Gly Trp Cys Phe Asn Ala Ala Phe Asn Ala Ser Leu Leu Ala Leu
245 250 255
Phe Ser Asn Phe His Ser Lys Asn Tyr Val Lys Lys Pro Thr Arg Glu
260 265 270
Asp Gly Lys Lys Ser Asp
275
<210>139
<211>6
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(6)
<223>序列中在第3和4位的Xaa具有表A中给出的含义
<400>139
Leu His Xaa Xaa His His
1 5
<210>140
<211>8
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(8)
<223>序列中在第2,3,5和6位的Xaa具有表A中给出的含义
<400>140
Thr Xaa Xaa Gln Xaa Xaa Gln Phe
1 5
<210>141
<211>6
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(6)
<223>序列中在第3位的Xaa具有表A中给出的含义
<400>141
Asp Thr Xaa Phe Met Val
1 5
<210>142
<211>8
<212>PRT
<213>共有序列
<220>
<221>MISC_FEATURE
<222>(1)..(8)
<223>序列中在第5和6位的Xaa具有表A中给出的含义
<400>142
Thr Gln Ala Gln Xaa Xaa Gln Phe
1 5
<210>143
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>143
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>144
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>144
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>145
<211>36
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(36)
<223>
<400>145
ggtaccacat aatgtgcgtg gagacggaaa ataacg 36
<210>146
<211>33
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(33)
<223>
<400>146
ctcgagttac gccgtctttc cggagtgttg gcc 33
<210>147
<211>24
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>
<400>147
gcggccgctt acgtggactt ggtc 24
<210>148
<211>24
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>
<400>148
gcggccgcat ggcgacgaag gagg 24
<210>149
<211>25
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>
<400>149
taagcttaca tggcgacgaa ggagg 25
<210>150
<211>24
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>
<400>150
tggatccact tacgtggact tggt 24
<210>151
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>151
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>152
<211>31
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(31)
<223>
<400>152
gcggccgcac catgtgctca ccaccgccgt c 31
<210>153
<211>26
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>
<400>153
gcggccgcct acatggcacc agtaac 26
<210>154
<211>31
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(31)
<223>
<400>154
gcggccgcac catgtgctca tcaccgccgt c 31
<210>155
<211>26
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>
<400>155
gcggccgcct acatggcacc agtaac 26
<210>156
<211>31
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(31)
<223>
<400>156
gcggccgcac catggacgcc tacaacgctg c 31
<210>157
<211>27
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(27)
<223>
<400>157
gcggccgcct aagcactctt cttcttt 27
<210>158
<211>23
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(23)
<223>
<400>158
accatgtgct caccaccgcc gtc 23
<210>159
<211>18
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(18)
<223>
<400>159
ctacatggca_ccagtaac 18
<210>160
<211>23
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(23)
<223>
<400>160
accatgtgct catcaccgcc gtc 23
<210>161
<211>18
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(18)
<223>
<400>161
ctacatggca ccagtaac 18
<210>162
<211>23
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(23)
<223>
<400>162
accatggacg cctacaacgc tgc 23
<210>163
<211>19
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(19)
<223>
<400>163
ctaagcactc ttcttcttt 19
<210>164
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>164
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>165
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>165
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>166
<211>29
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(29)
<223>
<400>166
gcggccgcat aatgacgagc aacatgagc 29
<210>167
<211>29
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(29)
<223>
<400>167
gcggccgctt aggccgactt ggccttggg 29
<210>168
<211>34
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(34)
<223>
<400>168
gcggccgcac catggacgtc gtcgagcagc aatg 34
<210>169
<211>36
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(36)
<223>
<400>169
gcggccgctt agatggtctt ctgcttcttg ggcgcc 36
<210>170
<211>23
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(23)
<223>
<400>170
gacataatga cgagcaacat gag 23
<210>171
<211>25
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(25)
<223>
<400>171
cggcttaggc cgacttggcc ttggg 25
<210>172
<211>30
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(30)
<223>
<400>172
agacataatg gacgtcgtcg agcagcaatg 30
<210>173
<211>28
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(28)
<223>
<400>173
ttagatggtc ttctgcttct tgggcgcc 28
<210>174
<211>60
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(60)
<223>
<400>174
gtcgacccgc ggactagtgg gccctctaga cccgggggat ccggatctgc tggctatgaa 60
<210>175
<211>29
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(29)
<223>
<400>175
gcggccgcat aatggcttca acatggcaa 29
<210>176
<211>32
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(32)
<223>
<400>176
gcggccgctt atgtcttctt gctcttcctg tt 32
<210>177
<211>26
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>
<400>177
gcggccgcat aatggagact tttaat 26
<210>178
<211>28
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(28)
<223>
<400>178
gcggccgctc agtcccccct cactttcc 28
<210>179
<211>29
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(29)
<223>
<400>179
aagcttacat aatggcttca acatggcaa 29
<210>180
<211>30
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(30)
<223>
<400>180
ggatccttat gtcttcttgc tcttcctgtt 30
<210>181
<211>26
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(26)
<223>
<400>181
aagcttacat aatggagact tttaat 26
<210>182
<211>27
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(27)
<223>
<400>182
ggatccttca gtcccccctc actttcc 27
<210>183
<211>993
<212>DNA
<213>三角褐指藻
<220>
<221>CDS
<222>(103)..(939)
<223>Δ6-延伸酶
<400>183
ggtcttttgt ggtagctatc gtcatcacac gcaggtcgtt gctcactatc gtgatccgta 60
tattgaccgt gcacttgtgt aaaacagaga tatttcaaga gt atg atg gta cct 114
Met Met Val Pro
1
tca agt tat gac gag tat atc gtc atg gtc aac gac ctt ggc gac tct 162
Ser Ser Tyr Asp Glu Tyr Ile Val Met Val Asn Asp Leu Gly Asp Ser
5 10 15 20
att ctg agc tgg gcc gac cct gat cac tat cgt gga cat acc gag gga 210
Ile Leu Ser Trp Ala Asp Pro Asp His Tyr Arg Gly His Thr Glu Gly
25 30 35
tgg gag ttc act gac ttt tct gct gct ttt agc att gcc gtc gcg tac 258
Trp Glu Phe Thr Asp Phe Ser Ala Ala Phe Ser Ile Ala Val Ala Tyr
40 45 50
ctc ctg ttt gtc ttt gtt gga tct ctc att atg agt atg gga gtc ccc 306
Leu Leu Phe Val Phe Val Gly Ser Leu Ile Met Ser Met Gly Val Pro
55 60 65
gca att gac cct tat ccg ctc aag ttt gtc tac aat gtt tca cag att 354
Ala Ile Asp Pro Tyr Pro Leu Lys Phe Val Tyr Asn Val Ser Gln Ile
70 75 80
atg ctt tgt gct tac atg acc att gaa gcc agt ctt cta gct tat cgt 402
Met Leu Cys Ala Tyr Met Thr Ile Glu Ala Ser Leu Leu Ala Tyr Arg
85 90 95 100
aac ggc tac aca ttc tgg cct tgc aac gat tgg gac ttt gaa aag ccg 450
Asn Gly Tyr Thr Phe Trp Pro Cys Asn Asp Trp Asp Phe Glu Lys Pro
105 110 115
cct atc gct aag ctc ctc tgg ctc ttt tac gtt tcc aaa att tgg gat 498
Pro Ile Ala Lys Leu Leu Trp Leu Phe Tyr Val Ser Lys Ile Trp Asp
120 125 130
ttt tgg gac acc atc ttt att gtt ctc ggg aag aag tgg cgt caa ctt 546
Phe Trp Asp Thr Ile Phe Ile Val Leu Gly Lys Lys Trp Arg Gln Leu
135 140 145
tcc ttc ctg cac gtc tac cat cac acc acc atc ttt ctc ttc tac tgg 594
Ser Phe Leu His Val Tyr His His Thr Thr Ile Phe Leu Phe Tyr Trp
150 155 160
ttg aat gca cat gta aac ttt gat ggt gat att ttc ctc acc atc gtc 642
Leu Asn Ala His Val Asn Phe Asp Gly Asp Ile Phe Leu Thr Ile Val
165 170 175 180
ttg aac ggt ttc atc cac acc gtc atg tac acg tac tac ttc att tgc 690
Leu Asn Gly Phe Ile His Thr Val Met Tyr Thr Tyr Tyr Phe Ile Cys
185 190 195
atg cac acc aag gtc cca gag acc ggc aaa tcc ttg ccc att tgg tgg 738
Met His Thr Lys Val Pro Glu Thr Gly Lys Ser Leu Pro Ile Trp Trp
200 205 210
aaa tct agt ttg aca agc atg cag ctg gtg cag ttc atc acg atg atg 786
Lys Ser Ser Leu Thr Ser Met Gln Leu Val Gln Phe Ile Thr Met Met
215 220 225
acg cag gct atc atg atc ttg tac aag ggc tgt gct gct ccc cat agc 834
Thr Gln Ala Ile Met Ile Leu Tyr Lys Gly Cys Ala Ala Pro His Ser
230 235 240
cgg gtg gtg aca tcg tac ttg gtt tac att ttg tcg ctc ttt att ttg 882
Arg Val Val Thr Ser Tyr Leu Val Tyr Ile Leu Ser Leu Phe Ile Leu
245 250 255 260
ttc gcc cag ttc ttt gtc agc tca tac ctc aag ccg aag aag aag aag 930
Phe Ala Gln Phe Phe Val Ser Ser Tyr Leu Lys Pro Lys Lys Lys Lys
265 270 275
aca gct taa gcgaaatttg ggtctacgtt aaaacaatta cgttacaaaa 979
Thr Ala
aaaaaaaaaa aaaa 993
<210>184
<211>278
<212>PRT
<213>三角褐指藻
<400>184
Met Met Val Pro Ser Ser Tyr Asp Glu Tyr Ile Val Met Val Asn Asp
1 5 10 15
Leu Gly Asp Ser Ile Leu Ser Trp Ala Asp Pro Asp His Tyr Arg Gly
20 25 30
His Thr Glu Gly Trp Glu Phe Thr Asp Phe Ser Ala Ala Phe Ser Ile
35 40 45
Ala Val Ala Tyr Leu Leu Phe Val Phe Val Gly Ser Leu Ile Met Ser
50 55 60
Met Gly Val Pro Ala Ile Asp Pro Tyr Pro Leu Lys Phe Val Tyr Asn
65 70 75 80
Val Ser Gln Ile Met Leu Cys Ala Tyr Met Thr Ile Glu Ala Ser Leu
85 90 95
Leu Ala Tyr Arg Asn Gly Tyr Thr Phe Trp Pro Cys Asn Asp Trp Asp
100 105 110
Phe Glu Lys Pro Pro Ile Ala Lys Leu Leu Trp Leu Phe Tyr Val Ser
115 120 125
Lys Ile Trp Asp Phe Trp Asp Thr Ile Phe Ile Val Leu Gly Lys Lys
130 135 140
Trp Arg Gln Leu Ser Phe Leu His Val Tyr His His Thr Thr Ile Phe
145 150 155 160
Leu Phe Tyr Trp Leu Asn Ala His Val Asn Phe Asp Gly Asp Ile Phe
165 170 175
Leu Thr Ile Val Leu Asn Gly Phe Ile His Thr Val Met Tyr Thr Tyr
180 185 190
Tyr Phe Ile Cys Met His Thr Lys Val Pro Glu Thr Gly Lys Ser Leu
195 200 205
Pro Ile Trp Trp Lys Ser Ser Leu Thr Ser Met Gln Leu Val Gln Phe
210 215 220
Ile Thr Met Met Thr Gln Ala Ile Met Ile Leu Tyr Lys Gly Cys Ala
225 230 235 240
Ala Pro His Ser Arg Val Val Thr Ser Tyr Leu Val Tyr Ile Leu Ser
245 250 255
Leu Phe Ile Leu Phe Ala Gln Phe Phe Val Ser Ser Tyr Leu Lys Pro
260 265 270
Lys Lys Lys Lys Thr Ala
275
<210>185
<211>20
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(20)
<223>在第3和18位的N为C或T.
<400>185
aanctuctut ggctuttnta 20
<210>186
<211>23
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(23)
<223>在第3和15位的N为C或T;在第9,12和21位的N为A或G.
<400>186
gantguacna anaantgugc naa 23
<210>187
<211>446
<212>DNA
<213>PCR-片段
<220>
<221>misc_feature
<222>(1)..(446)
<223>PCR-片段
<400>187
aagctcctct ggctctttta cgtttccaaa atttgggatt tttgggacac catctttatt 60
gttctcggga agaagtggcg tcaactttcc ttcctgcacg tctaccatca caccaccatc 120
tttctcttct actggttgaa tgcacatgta aactttgatg gtgatatttt cctcaccatc 180
gtcttgaacg gtttcatcca caccgtcatg tacacgtact acttcatttg catgcacacc 240
aaggtcccag agaccggcaa atccttgccc atttggtgga aatctagttt gacaagcatg 300
cagctggtgc agttcatcac gatgatgacg caggctatca tgatcttgta caagggctgt 360
gctgctcccc atagccgggt ggtgacatcg tacttggttt acattttgtc gctctttatt 420
ttgttcgccc agttctttgt cagctc 446
<210>188
<211>30
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(30)
<223>
<400>188
gcggccgcac ataatgatgg taccttcaag 30
<210>189
<211>22
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(22)
<223>
<400>189
gaagacagct taatagacta gt 22
<210>190
<211>31
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(31)
<223>
<400>190
gcggccgcac catgatggta ccttcaagtt a 31
<210>191
<211>24
<212>DNA
<213>引物
<220>
<221>misc_feature
<222>(1)..(24)
<223>
<400>191
gaagacagct taataggcgg ccgc 24
<210>192
<211>859
<212>DNA
<213>PcR产物
<400>192
gcggccgcac ataatgatgg taccttcaag ttatgacgag tatatcgtca tggtcaacga 60
ccttggcgac tctattctga gctgggccga ccctgatcac tatcgtggac ataccgaggg 120
atgggagttc actgactttt ctgctgcttt tagcattgcc gtcgcgtacc tcctgtttgt 180
ctttgttgga tctctcatta tgagtatggg agtccccgca attgaccctt atccgctcaa 240
gtttgtctac aatgtttcac agattatgct ttgtgcttac atgaccattg aagccagtct 300
tctagcttat cgtaacggct acacattctg gccttgcaac gattgggact ttgaaaagcc 360
gcctatcgct aagctcctct ggctctttta cgtttccaaa atttgggatt tttgggacac 420
catctttatt gttctcggga agaagtggcg tcaactttcc ttcctgcacg tctaccatca 480
caccaccatc tttctcttct actggttgaa tgcacatgta aactttgatg gtgatatttt 540
cctcaccatc gtcttgaacg gtttcatcca caccgtcatg tacacgtact acttcatttg 600
catgcacacc aaggtcccag agaccggcaa atccttgccc atttggtgga aatctagttt 660
gacaagcatg cagctggtgc agttcatcac gatgatgacg caggctatca tgatcttgta 720
caagggctgt gctgctcccc atagccgggt ggtgacatcg tacttggttt acattttgtc 780
gctctttatt ttgttcgccc agttctttgt cagctcatac ctcaagccga agaagaagaa 840
gacagcttaa tagactagt 859
Claims (34)
1.用于在转基因生物中产生式I化合物的方法,
其中基于所述转基因生物的总脂类含量,这种转基因生物具有至少1%重量的式I化合物含量,所述方法包括如下步骤:
a)将至少一个编码Δ9延伸酶或Δ6去饱和酶活性的核酸序列导入生物体,以及
b)将至少一个编码Δ8去饱和酶或Δ6延伸酶活性的核酸序列导入生物体,以及
c)将至少一个编码Δ5去饱和酶活性的核酸序列导入生物体,以及
d)将至少一个编码Δ5延伸酶活性的核酸序列导入生物体,以及
e)将至少一个编码Δ4去饱和酶活性的核酸序列导入生物体,
并且其中式I的变量和取代基的含义如下:
R1=羟基、辅酶A(硫酯)、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇、鞘碱或式II的基团,
其中
R2=氢、溶血磷脂酰胆碱、溶血磷脂酰乙醇胺、溶血磷脂酰甘油、溶血二磷脂酰甘油、溶血磷脂酰丝氨酸、溶血磷脂酰肌醇或者饱和或不饱和的C2-C24烷基羰基,
R3=氢、饱和或不饱和的C2-C24烷基羰基,或R2和R3彼此独立地是式Ia的基团:
其中n=2、3、4、5、6、7或9,m=2、3、4、5或6并且p=0或3。
2.根据权利要求1所述的方法,其中编码具有Δ9-延伸酶、Δ6-去饱和酶、Δ8-去饱和酶、Δ6-延伸酶、Δ5-去饱和酶、Δ5-延伸酶或Δ4-去饱和酶活性的多肽的核酸序列选自:
a)具有SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可由SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132或SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:11、SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25、SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41、SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ ID NO:49、SEQ ID NO:51、SEQ ID NO:53、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:99、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:117、SEQ ID NO:119、SEQ ID NO:131、SEQ ID NO:133、SEQ ID NO:135、SEQ ID NO:137或SEQ ID NO:183中所示核酸序列的衍生物,其编码与SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:12、SEQ ID NO:14、、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40、SEQ ID NO:42、SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:54、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQ ID NO:66、SEQ ID NO:68、SEQ ID NO:70、SEQ ID NO:72、SEQ ID NO:74、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:88、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:98、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:112、SEQ ID NO:114、SEQ ID NO:118、SEQ ID NO:120、SEQ ID NO:132、SEQ ID NO:134、SEQ ID NO:136、SEQ ID NO:138或SEQ ID NO:184在氨基酸水平具有至少40%同一性并且具有Δ9延伸酶、Δ6去饱和酶、Δ8去饱和酶、Δ6延伸酶、Δ5去饱和酶、Δ5延伸酶或Δ4去饱和酶活性的多肽。
3.根据权利要求1或2所述的方法,其中向生物中额外地导入编码具有ω3去饱和酶活性的多肽的核酸序列,其选自:
a)具有SEQ ID NO:87或SEQ ID NO:105中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可由SEQ ID NO:88或SEQ ID NO:106中所示的氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:87或SEQ ID NO:105中所示核酸序列的衍生物,其编码与SEQ ID NO:88或SEQ ID NO:106在氨基酸水平具有至少60%同一性并且具有ω3去饱和酶活性的多肽。
4.根据权利要求1至3中任意一项所述的方法,其中向生物中额外地导入编码具有Δ12去饱和酶活性的多肽的核酸序列,其选自:
a)具有SEQ ID NO:107或SEQ ID NO:109中所示序列的核酸序列,或
b)作为遗传密码简并性的结果,可由SEQ ID NO:108或SEQ IDNO:110中所示的氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:107或SEQ ID NO:110中所示核酸序列的衍生物,其编码与SEQ ID NO:108或SEQ ID NO:110在氨基酸水平具有至少60%同一性并且具有Δ12去饱和酶活性的多肽。
5.根据权利要求1至4中任意一项所述的方法,其中取代基R2或R3彼此独立地是饱和或不饱和的C18-C22烷基羰基。
6.根据权利要求1至5中任意一项所述的方法,其中取代基R2或R3彼此独立地是具有至少两个双键的不饱和C18、C20或C22烷基羰基。
7.根据权利要求1至6中任意一项所述的方法,其中转基因生物是转基因微生物或转基因植物。
8.根据权利要求1至7中任意一项所述的方法,其中转基因生物是产油植物、蔬菜植物或装饰植物。
9.根据权利要求1至8中任意一项所述的方法,其中转基因生物是选自Adelotheciaceae、漆树科(Anacardiaceae)、菊科(Asteraceae)、伞形科(Apiaceae)、桦木科(Betulaceae)、紫草科(Boraginaceae)、十字花科(Brassicaceae)、凤梨科(Bromeliaceae)、番木瓜科(Caricaceae)、大麻科(Cannabaceae)、旋花科(Convolvulaceae)、藜科(Chenopodiaceae)、隐甲藻科(Crypthecodiniaceae)、葫芦科(Cucurbitaceae)、牛毛藓科(Ditrichaceae)、胡颓子科(Elaeagnaceae)、杜鹃花科(Ericaceae)、大戟科(Euphorbiaceae)、豆科(Fabaceae)、牻牛儿苗科(Geraniaceae)、禾本科(Gramineae)、核桃科(Juglandaceae)、樟科(Lauraceae)、豆科(Leguminosae)、亚麻科(Linaceae)或草绿藻科(Prasinophyceae)的转基因植物。
10.根据权利要求1至9中任意一项所述的方法,其中式I化合物是从生物中以其油类、脂类或游离脂肪酸的形式分离的。
11.根据权利要求1至10中任意一项所述的方法,其中式I化合物基于转基因生物的总脂类含量,以至少5%重量的浓度分离。
12.油、脂类或脂肪酸或者它们的级分,其为通过权利要求1至11中任意一项所述的方法产生的。
13.油、脂类或脂肪酸的组合物,其包含通过权利要求1至11中任意一项所述的方法产生的PUFA并源自转基因植物。
14.用于产生油类、脂类或脂肪酸的组合物的方法,其通过将根据权利要求12所述的油类、脂类或脂肪酸或者根据权利要求13所述的油、脂类或脂肪酸的组合物与动物的油类、脂类或脂肪酸混合。
15.根据权利要求12所述的油类、脂类或脂肪酸类或者根据权利要求13所述的油类、脂类或脂肪酸的组合物或者根据权利要求14所产生的油类、脂类或脂肪酸的组合物的用途,用在饲料、食品、化妆品或药品中。
16.经分离的核酸序列,其编码具有Δ5-延伸酶活性的多肽并且所述多肽包含选自具有SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:139、SEQ ID NO:140、SEQ ID NO:141或SEQ ID NO:142中所示序列的氨基酸序列。
17.根据权利要求16所述的经分离的核酸序列,其中所述核酸序列编码这样的具有Δ5-延伸酶活性的多肽,所述多肽包含选自如下的氨基酸序列的组合:
a)SEQ ID NO:115和SEQ ID NO:139、SEQ ID NO:115和SEQID NO:140、或SEQ ID NO:139和SEQ ID NO:140;或
b)SEQ ID NO:116和SEQ ID NO:141、SEQ ID NO:116和SEQID NO:142、或SEQ ID NO:141和SEQ ID NO:142;或
c)SEQ ID NO:115、SEQ ID NO:139和SEQ ID NO:140;或SEQID NO:116、SEQ ID NO:141和SEQ ID NO:142。
18.根据权利要求16或17所述的编码具有Δ5-延伸酶活性的多肽的经分离核酸序列,其中所述核酸序列选自:
a)具有SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQID NO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQ ID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ IDNO:113、SEQ ID NO:131或SEQ ID NO:133中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:44,SEQ ID NO:46,SEQ ID NO:48,SEQ ID NO:50,SEQ ID NO:60,SEQ IDNO:62,SEQ ID NO:64,SEQ ID NO:66,SEQ ID NO:68,SEQID NO:76,SEQ ID NO:78,SEQ ID NO:80,SEQ ID NO:84,SEQ ID NO:86,SEQ ID NO:114,SEQ ID NO:132或SEQ ID NO:134中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:43、SEQ ID NO:45、SEQ ID NO:47、SEQ IDNO:49、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63;SEQID NO:65、SEQ ID NO:67、SEQ ID NO:75、SEQ ID NO:77、SEQ ID NO:79、SEQ ID NO:83、SEQ ID NO:85、SEQ ID NO:113、SEQ ID NO:131或SEQ ID NO:133中所示核酸序列的衍生物,其编码与SEQ ID NO:44、SEQ ID NO:46、SEQ ID NO:48、SEQ IDNO:50、SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:64、SEQID NO:66、SEQ ID NO:68、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:86、SEQ ID NO:114、SEQ ID NO:132或SEQ ID NO:134在氨基酸水平具有至少40%同源性并且具有Δ5延伸酶活性的多肽。
19.编码具有Δ6延伸酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ ID NO:184中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:69、SEQ ID NO:81、SEQ ID NO:111或SEQ ID NO:183中所示核酸序列的衍生物,其编码与SEQ ID NO:70、SEQ ID NO:82、SEQ ID NO:112或SEQ ID NO:184在氨基酸水平具有至少40%同源性并且具有Δ6延伸酶活性的多肽。
20.编码具有ω3去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:87或SEQ ID NO:105中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:88或SEQ ID NO:106中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:87或SEQ ID NO:105中所示核酸序列的衍生物,其编码与SEQ ID NO:88或SEQ ID NO:106在氨基酸水平具有至少60%同一性并且具有ω3去饱和酶活性的多肽。
21.编码具有Δ6去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:89或SEQ ID NO:97中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:90或SEQ ID NO:98中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:89或SEQ ID NO:97中所示核酸序列的衍生物,其编码与SEQ ID NO:90或SEQ ID NO:98在氨基酸水平具有至少40%同源性并且具有Δ6去饱和酶活性的多肽。
22.编码具有Δ5去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQ ID NO:101中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:100或SEQ ID NO:102中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:99或SEQ ID NO:101中所示核酸序列的衍生物,其编码与SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:100或SEQ ID NO:102在氨基酸水平具有至少40%同源性并且具有Δ5去饱和酶活性的多肽。
23.编码具有Δ4去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:95或SEQ ID NO:103中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:96或SEQ ID NO:104中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:95或SEQ ID NO:103中所示核酸序列的衍生物,其编码与SEQ ID NO:96或SEQ ID NO:104在氨基酸水平具有至少40%同源性并且具有Δ4去饱和酶活性的多肽。
24.编码具有Δ12去饱和酶活性的多肽的经分离核酸序列,其选自:
a)具有SEQ ID NO:107或SEQ ID NO:109中所示序列的核酸序列,
b)作为遗传密码简并性的结果,可从SEQ ID NO:108或SEQ ID NO:110中所示氨基酸序列衍生的核酸序列,或
c)SEQ ID NO:107或SEQ ID NO:109中所示核酸序列的衍生物,其编码与SEQ ID NO:108或SEQ ID NO:110在氨基酸水平具有至少50%同一性并且具有Δ12去饱和酶活性的多肽。
25.根据权利要求16至24中任意一项所述的经分离的核酸序列,其中所述核酸序列源自藻类、真菌、微生物、植物或非人类动物。
26.根据权利要求16至25中任意一项所述的经分离的核酸序列,其中所述核酸序列源自鲑形目、硅藻的海链藻属或隐甲藻属或者源自草绿藻科、裸藻科或腐霉科。
27、氨基酸序列,其为由权利要求16至26中任意一项所述的经分离核酸序列编码的氨基酸序列。
28.基因构建体,其包含根据权利要求16至26中任意一项所述的经分离核酸序列,其中所述核酸序列与一个或多个调节信号有效地连接。
29.根据权利要求28所述的基因构建体,其中所述核酸构建体包含脂肪酸或脂类代谢中的选自酰基辅酶A脱氢酶、酰基ACP[酰基载体蛋白]去饱和酶、酰基ACP硫酯酶、脂肪酸酰基转移酶、酰基辅酶A:溶血磷脂酰基转移酶、脂肪酸合成酶、脂肪酸羟化酶、乙酰基辅酶A羧化酶、酰基辅酶A氧化酶、脂肪酸去饱和酶、脂肪酸炔属酶、脂加氧酶、三酰甘油酯脂肪酶、环丙烯合酶、氢过氧化物裂合酶或脂肪酸延伸酶的额外生物合成基因。
30.根据权利要求28或29所述的基因构建体,其中所述核酸构建体包含脂肪酸或脂类代谢中的选自Δ4去饱和酶、Δ5去饱和酶、Δ6去饱和酶、Δ8去饱和酶、Δ9去饱和酶、Δ12去饱和酶、Δ6延伸酶或Δ9延伸酶的额外生物合成基因。
31.载体,其包含根据权利要求16至26中任意一项所述的核酸或者根据权利要求28至30中任意一项所述的基因构建体。
32.转基因非人类生物,其包含根据权利要求16至26中任意一项所述的至少一种核酸、根据权利要求28至30中任意一项所述的基因构建体或者根据权利要求31中所述的载体。
33.根据权利要求32所述的转基因非人类生物,其中所述生物是微生物、非人类动物或植物。
34.根据权利要求32或33所述的转基因非人类生物,其中所述生物是植物。
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