CN1177379A - 通过△6-去饱和酶生产γ亚麻酸 - Google Patents
通过△6-去饱和酶生产γ亚麻酸 Download PDFInfo
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- CN1177379A CN1177379A CN95197728A CN95197728A CN1177379A CN 1177379 A CN1177379 A CN 1177379A CN 95197728 A CN95197728 A CN 95197728A CN 95197728 A CN95197728 A CN 95197728A CN 1177379 A CN1177379 A CN 1177379A
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Abstract
亚油酸被△6去饱和酶转化为γ－亚麻酸。本发明涉及分离的包括△6去饱和酶基因的核酸。更具体地,分离的核酸包括△6去饱和酶基因的启动子,编码区和终止区。本发明提供了包含与异源性调节序列功能性组合的△6去饱和酶编码区的重组构建体。本发明的核酸和重组构建体在转基因生物体GLA的产生中有用。
Description
通过Δ6-去饱和酶作用，亚油酸(18∶2)(LA)转化为γ亚麻酸(18∶3)(GLA)。当此酶或者编码此酶的核苷酸转移进生产LA的细胞时，生产出GLA。本发明提供含有Δ6-去饱和酶基因的核苷酸。尤其是此核苷酸包含Δ6-去饱和酶基因的启动子、编码区域和终止区域。本发明进一步目标是建立重组结构，它包含Δ6-去饱和酶编码区域并与异源调节序列功能上相组合。本发明的核苷酸和重组结构在转基因组织中生产GLA是有用的。
不饱和脂肪酸比如亚油酸(C18Δ9,12)和α-亚麻酸(C18Δ9,12,15)是必需的饮食组成成份，它们不能被脊椎动物合成，因为脊椎动物细胞能在饱和酸Δ9位置引入双键，但是不能在Δ9双键和脂肪酸链甲基末端之间引入其它双键。因为它们是其它产物的前体，亚油酸和α-亚麻酸是必需的脂肪酸，经常从植物中获取。亚油酸被哺乳动物转变成γ-亚麻酸(GLA，C18Δ6,9,12)，γ-亚麻酸接着被转变成花生四烯酸(20∶4)，一个非常关键的脂肪酸，因为它是大多数前列腺素的必需前体。
饮食提供的亚油酸，因为可以转变为GLA和花生四烯酸，满足了饮食上对GLA和花生四烯酸的需要。然而已表明在饱和脂肪的消耗和健康危险方面存在一个关系，这些健康危险比如血胆固醇过多，动脉粥样硬化和对冠状疾病敏感有关的其它临床疾病。而消耗未饱和脂肪则与降低的血胆固醇浓度以及降低动脉动脉粥样硬化的危险有联系。食用GLA治疗的好处也许在于GLA是花生四烯酸的前体，因此有助于前列腺素的合成。相应地，消耗更多的未饱和GLA而不是亚油酸，有潜在的健康益处。然而，GLA并不存在于任何商业性生长的作物中。
亚油酸通过Δ6-去饱和酶转变为GLA。Δ6-去饱和酶，超过350个氨基酸，有一个膜固定区域和一个脂肪酸去饱和的活性位点。如果细胞内源性生产亚油酸而不是GLA，当Δ6-去饱和酶转化进细胞时，则会产生GLA。通过提供编码Δ6-去饱和酶的基因，本发明允许转基因组织的生成，它含有功能性Δ6-去饱和酶，而且能生产GLA。除了允许生产大量GLA外，本发明提供GLA的新的饮食来源。
本发明的目的在于分离Δ6-去饱和酶基因。尤其是分离的基因含有Δ6-去饱和酶启动子，编码区域和终止区域。
本发明进一步目的在于组建含有Δ6-去饱和酶启动子，编码区域和终止区域的表达载体。
本发明另一方面是组建含有Δ6-去饱和酶编码区域并与异源调节区域功能上相联合的表达载体，异源调节区域即不起源于Δ6-去饱和酶基因的组份。
含有本发明载体的细胞和组织以及这些组织的后代，也可以由本发明提供。
本发明更进一步在于提供分离的细菌的Δ6-去饱和酶。也可以提供分离的植物的Δ6-去饱和酶。
本发明的另一方面是提供一种方法，用于生产具有增高γ亚麻酸成份的植物。
本发明也提供一种方法用于生产耐寒性植物。
图1描述了集胞藻属Δ6-去饱和酶(A组)和Δ12-去饱和酶(B组)推断的氨基酸序列的水疗(hydropathy)外形图。推断的跨膜区用实体棒表示，疏水指数经过计算是19个氨基酸残基的窗口大小[Kyte，et al.(1982)《分子生物学杂志》157]。
图2提供了野生型(A组)和转基因鱼腥藻属(B组)的气液相色谱分布图。
图3是带有重叠区域和亚克隆的粘粒Csy75，Csy13和Csy7的图的图解。Csy75，Csy75-3.5和Csy7的亚克隆区域用添加的斜线表示出来。失活的限制性酶切位点放于括号内。
图4提供了野生型(A组)和转基因烟草(B组)的气液相色谱分布图。
图5A描述了从琉璃苣中分离的Δ6-去饱和酶的DNA序列。
图5B描述了分离出的琉璃苣的Δ6-去饱和酶cDNA中开读框架的蛋白序列。去饱和酶三个特有的氨基酸功能域显示出来，依照顺序分别是脂类框，金属框1和金属框2
图6是一个树状图，显示了琉璃苣的Δ6-去饱和酶与其它膜固定的去饱和酶的类似性。通过Gene Works(IntelliGenetics)比较琉璃苣Δ6-去饱和酶与其它已知去饱和酶的氨基酸序列。各亚属之间与相对系统发育距离有关的数值得到比较。
图7是221.Δ6.NOS和121.Δ6.NOS的限制性酶切图谱。在221.Δ6.NOS中，质粒余下的部分pBI221，在121.Δ6.NOS，质粒余下的部分是pBI121。
图8提供了模拟转化(A组)和221.Δ6.NOS转化(B组)的胡萝卜细胞的气液相色谱图。18∶2，18∶3α和18∶3γ(GLA)的位置得到显示。
图9提供了未转化的烟草叶子(A组)和用121.Δ6.NOS转化的烟草叶子(B组)的气液相色谱图。18∶2，18∶3α，18∶3γ(GLA)和18∶4的位置得到显示。
图10提供了未转化的烟草种子(A组)和用121.Δ6.NOS转化的烟草种子(B组)的气液相色谱图。18∶2，18∶3α，18∶3γ(GLA)的位置得到显示。
本发明提供分离的编码Δ6-去饱和酶的核酸。为了确认编码Δ6-去饱和酶的核酸，DNA从生产GLA的组织中分离出来。所说的组织可以是，例如动物细胞，某些真菌(如被孢霉属)，某些细菌(如集胞藻属)或某些植物(琉璃苣、月见草属、茶藨子类)。基因组DNA的分离可以通过许多方法来完成，这些方法是本领域中熟知的常规技术之一，举例说明见Sambrook et al.(1989)《分子克隆：实验室手册》，冷泉港，纽约。分离的DNA通过物理方法或酶切方法片段化，克隆进合适的载体，比如噬菌体或粘粒载体，这些都可以通过一系列熟知方法中的任一种来进行，这些方法在文献中可以找到，比如Sambrook et al(1989)。含有本发明DNA的表达载体在此得到重点考虑。编码Δ6-去饱和酶的DNA通过功能获得性分析得到证实。含有片段DNA的载体通过传染，转结合(transconjugation)，转染等方法转移进产亚油酸而不产GLA的宿主组织。用在这里，“转化”总的来说是指吸收外源DNA进入宿主细胞。诱导重组DNA进入宿主组织的方法对本领域中普通技术人员是熟知的常规技术之一，能在Sambrook et al(1989)中找到。这些组织生产的GLA(即功能的获取)可以通过比如气相色谱法或本领域普通技术人员所熟知的其它方法来测定。被诱导产生GLA，即通过导入载体而获得功能的组织被确认为表达DNA编码的Δ6-去饱和酶，所说的DNA从此组织中获取。获取的DNA再一次片段化，克隆进表达载体，通过上述程序进行功能性测试以更精确确认编码Δ6-去饱和酶的DNA。
做为本发明的一个例子，随机DNA从蓝细菌集胞藻属(PasteurCulture Collection(PCC)6803，美国典型培养物保藏中心(ATCC)27184)中分离出来，克隆进粘粒载体，通过转结合诱导进入GLA缺陷的蓝细菌鱼腥藻属菌株PCC 7120，ATCC 27893。从鱼腥藻属的亚油酸中生产的GLA通过气相色谱法来监测，对应的DNA片段被分离出来。
分离的DNA进行测序，此方法是本领域技术人员中熟知的常规技术之一，见Sambrook et al(1989)。
依据本发明，含有Δ6-去饱和酶基因的DNA分子已被分离。更确切地说，一个3.588kb含有Δ6-去饱和酶基因的DNA已从蓝细菌集胞藻属中分离出来。3.588kb DNA的核苷酸序列已确定，显示于SEQ ID NO：1。定义潜在编码区域的开读框架位于核苷酸317至1507和2002至3081。为了确定编码Δ6-去饱和酶的核苷酸，赋于Δ6-去饱和酶活性的3.588kb的片段被切成两个亚片段，每一个只含有一个开读框架。片段ORF1含有核苷酸1至1704，片段ORF2含有核苷酸1705至3588。每一片段都以正反两个方向亚克隆进一个共同的表达载体(AM 542，Wolk et al.[1984]《美国国家科学院院刊》81，1561)此载体含有一个蓝细菌1羧化酶启动子。产生的重组(即ORF1(F)，ORF1(R)，ORF2(F)和ORF2(R))通过标准方法(见Wolketal.(1984)《美国国家科学院院刊》81，1561)结合进野生型鱼腥藻属PCC 7120；在选择培养基(依据Rippka et al.，(1979)普通微生物学杂志.111.1，标准矿物培养基BG11N+30ug/ml新霉素)上生长两周以后，在将要死亡的非结合细胞的棕色背景上，鱼腥藻属的结合细胞被确认为NeoR绿色克隆。挑出绿色克隆，培养于选择性液体培养基(BG11N+15ug/ml新霉素)内。通过标准方法(如Dahmer et al.(1989)美国石油化学联合物杂志66，543)脂类从含有正反向ORF1和ORF2重组的转结合物中抽提出来。做为对照，脂类也从野生型的鱼腥藻属和集胞藻属培养物中抽提出来。用气液相色谱(GLC)分析脂肪酸甲酯，例如用Tracor-560气液相色谱仪并配备一个氢火焰离子检测器和一个毛细管柱。GLC分析结果显示于表1表1：在野生型和转基因的蓝细菌中C18脂肪酸的出现
| 来源 | 18∶0 | 18∶1 | 18∶2 | γ18∶3 | α18∶3 | 18∶4 |
| 鱼腥藻属(野生型) | + | + | + | - | + | - |
| 鱼腥藻属+ORF1(F) | + | + | + | - | + | - |
| 鱼腥藻属+ORF1(R) | + | + | + | - | + | - |
| 鱼腥藻属+ORF2(F) | + | + | + | + | + | + |
| 鱼腥藻属+ORF2(R) | + | + | + | - | + | - |
| 集胞藻属(野生型) | + | + | + | + | - | - |
通过GLC分析，当含有ORF2正方向的重组物通过转结合被诱导进细胞时，GLA缺陷的鱼腥藻属获得产GLA的功能。转结合物中含有对于羟化酶启动子呈反方向的ORF2或ORF1正反方向重组物，未有GLA产生。此分析表明在1884bp片段的单个开读框架(ORF2)中编码Δ6-去饱和酶。此1884bp片段显示于SEQ ID NO：3。通过Δ6-去饱和酶和Δ12-去饱和酶(Wada et al.(1990)《自然》347)水疗图全面相似性。这些得到具体证明，此图分别显示于图1(A)和(B)。
依据本发明，包含Δ6-去饱和酶基因的cDNA从琉璃苣中分离。此1.685kb cDNA的核苷酸序列得到确认并显示于图5A(SEQ ID NO：4)ATG起始密码子和终止密码子下面划线。对应于琉璃苣Δ6-去饱和酶中开读框架的氨基酸序列显示于图5B(SEQ ID NO：5)。
分离的编码Δ6-去饱和酶的核苷酸也可以通过上面描述的功能获得分析法从其它生产GLA的组织中得到确认，或者用分离出的编码集胞藻属或琉璃苣Δ6-去饱和酶的核苷酸作杂交探针，通过核酸杂交技术来确认。基因组或cDNA克隆的方法对于熟练的技术人员是已知的，并在本发明中得到研究。杂交探针可以含有显示于SEQ ID NO：1或SEQID NO：4全长的DNA序列，或一个限制性片段或其它片段，并且包括寡核苷酸探针。通过交叉杂交克隆同源基因的方法对于普通的技术人员是已知的，可以在Sambrook(1989)和Beltz et al(1983)《酶学方法》100,266中找到。
在另一个从生产GLA组织中确定Δ6-去饱和酶的方法中，从Poly-A+RNA制备cDNA文库，而Poly-A+RNA是从多核糖体RNA中分离。为了从cDNA群中去除超丰富表达的基因，cDNA或其相对于超丰富cDNA基因的片段被用作对cDNA文库杂交的探针。未杂交的噬菌斑切除下来，产生的细菌克隆孵育在液体培养基中并测序。例如，为了把从琉璃苣多核糖体RNA中制备的cDNA文库中去除其它种子贮存蛋白cDNAs，对应于丰富表达的种子贮存蛋白cDNA首先与cDNA文库杂交。“差减”DNA文库接着用于产生表达的序列标签(ETSs)，这些标签用于扫描数据库，比如GenBank来确认潜在的去饱和酶。
通过导入编码Δ6-去饱和酶DNA，转基因组织获得生产GLA功能的同时，也获得了生产十八碳四烯酸(18∶4Δ6,9,12,15)的功能。十八碳四烯酸目前通常存在于鱼油中和紫草科家族的某些植物种属中(Craig et al[1964]，美国石油化学联合会杂志，41,209-211；Gross etal.[1976]Can.J.Plant.Sci 56,659-664)。在本发明的转基因组织中，十八碳四烯酸产生于Δ6-去饱和酶对α亚麻酸进一步去饱和作用，或者通过Δ15-去饱和酶对GLA去饱和作用。
被ORF2即编码集胞藻属Δ6-去饱和酶的开读框架所编码的359个氨基酸，显示于SEQ ID NO：2。编码琉璃苣Δ6-去饱和酶的开读框架显示于SEQ ID NO：5。本发明进一步重点考虑编码SEQ ID NO：2和SEQ ID NO：5中氨基酸的别的核苷酸序列。确认这些序列是在普通的熟练的技术人员的知识范围之内，例如这些序列产生于遗传密码的破译。此外，本领域常规技术之一是通过下面描述的功能获得的分析，来决定含有编码Δ6-去饱和酶开读框架的片段中更少的亚片段。
本发明密切注视Δ6-去饱和酶的任何多肽片段和仍保持将LA转变为GLA活性的核苷酸。
本发明的另一方面，一个含有本发明核苷酸的载体，或者一个含有Δ6-去饱和酶基因启动子，编码序列和终止区域的小片段被转化进一个组织，比如蓝细菌，其中Δ6-去饱和酶启动子和终止区域都是功能性的，因此，本发明提供了产生重组Δ6-去饱和酶的组织。本发明另一方面是提供分离Δ6-去饱和酶，通过标准的蛋白纯化方法，(比如见Ausubel et al(1987)《当气分子生物学方法》Green publishingAssociates，New York)可以从重组组织中纯化出来。
包含编码Δ6-去饱和酶DNA的载体也可以由本发明指供。构建合适的载体在各种组织中表达Δ6-去饱和酶的编码序列明显是本领域常规技术之一。复制表达载体尤其得到优选。在此描述的复制表达载体是工程化为了控制目的基因即Δ6-去饱和酶表达的DNA或RNA分子。优选的载体是质粒，噬菌体，粘粒或病毒。穿梭载体如描述于Wolk et al.(1984)《美国国家科学院院刊》1561-1565和Bustos et al.(1991)J.Bacterol.174,7525-7533。依据本发明也可以考虑。Sambrook et al.(1989)，Goeddel，ed.(1990)《酶学方法》185，Academic.Press，和Perbal(1988)分子克隆操作指南，John wiley和Sons，Inc.提供了载体的详细论著，编码Δ6-去饱和酶的核苷酸可以插入并表达。这些载体还包含能够影响编码Δ6-去饱和酶的核苷酸表达的核苷酸序列。能够影响一个基因产物表达的序列元件包括启动子、增强子、上游激活序列、转录终止信号和多腺苷酸位点。组成性的和组织特异的启动子都可以考虑。为了转化植物细胞，花椰菜花斑病毒(CaMV)355启动子和在植物种子成熟过程中被调节的启动子尤其令人感兴趣。所有这些启动子或转录调节元件，单个或组合形式，都可以在本复制表达载体中被考虑得到使用，并且是本领域技术人员已知的。CaMV 35S启动子被Restrepo et al(1990)所描述，《植物细胞》2，987，遗传工程和突变的调节序列也可以考虑。
普通的技术人员能够决定适合于在特定宿主细胞中表达的载体和调节元件。例如，一个载体含有来源于编码鱼腥藻属羧化酶基因的启动子可操作性连接到Δ6-去饱和酶区域，进一步可操作性连接到来源于集胞藻属的终止信号上，此载体适合在蓝细菌中表达去饱和酶。“可操作性连接”在此文中指启动子和终止子序列能够有效对转录调控行使功能，再举一个例子，一个适合于在转基因植物中表达Δ6-去饱和酶的载体可以含有来源于半日花素、napin、大豆球蛋白的种子特异性序列，可操作性连接到Δ6-去饱和酶的编码区域，进一步可操作性连接到一个种子终止信号或胭脂碱合成酶的终止信号上。再举一例，一个在植物中表达Δ6-去饱和酶的载体，可以含有一个组成性的启动子或一个组织特异性启动子可操作性连接到Δ6-去饱和酶的编码区域。并进一步可操作性连接到一个基本的或组织特异的终止子或胭脂碱合成酶的终止信号上。
尤其是半日花素调节元件显示于申请人共同未决的美国专利申请，申请流水号682,354，1991年4月8号提交，并引入本文作为参考，可以做为在本发明中指导Δ6-去饱和酶表达的启动子元件来考虑。
修改显示于此的核苷酸序列或调控元件，并同时维持在此所考虑的功能，是在本发明范围之内的。这些修饰包括插入，替换或删除。特别是删除，它反映了遗传密码的简并性。
组建这些杂种载体的标准技术是本领域中普通技术人员是众所周知的，可以在许多文献中找到，此如Sambrook et al(1989)，或者是广泛所找到的关于重组DNA技术的无数实验室手册的任一种，有许多关于连接DNA片段的策略，选择其中的策略依靠于DNA片段的性质。依据本发明，进一步考虑的是在杂种载体中包括一些有助于克隆，表达或者加工的其它核苷酸序列元件，例如编码信号肽的序列，编码KDEL的序列，此序列对于固定蛋白于内质网上是必需的，或者编码转运多肽的序列，它能够定向Δ6-去饱和酶至叶绿体中。这些序列是本领域中的技术人员已知的最优。Van den Broeck et al(1985)《自然》313,358描述了最优的转还多肽。Michaelis et al(1982)《Ann.Rev.Microbiol》36,425显示了原核和真核的信号肽。
本发明另一方面是提供除了蓝细菌或植物之外的其它含有编码本发明的Δ6-去饱和酶的组织。依据本发明考虑的转基因组织包括细菌、蓝细菌、真菌、植物和动物。本发明所分离的DNA可以通过本领域已知的方法导入宿主，比如传染、感染、转化或转结合。把本发明的DNA转移进入这些组织的技术是广泛熟知的，在文献中可以找到，比如Sambrooket al.(1989)。
一系列植物转化方法也已熟知。Δ6-去饱和酶基因可以通过Horshet al.(1985)《科学》227,1229所描述的叶盘转化-再生程序导入植物。其它的转化方法，比如原生质体培养(Horsch et al.(1984)《科学》223,496；DeBlock et al.(1984)EMBO J.2,2143，Barton et al.(1983)《细胞》32,1033)也可以使用，属于本发明的范围。一个优选的具体化的植物可以用土壤杆菌属起源的载体来转化，然而把本发明的Δ6-去饱和酶插入植物细胞也可以使用其它方法。这些替代方法包括biolistic方法(Klein et al.(1987)《自然》327,70)，电穿孔，化学诱导DNA的吸收，使用病毒或花粉做为载体。
对于转化方法所必要的是，本发明的Δ6-去饱和酶基因可以插入植物转化载体，如Bevan(1984)《核酸研究》12,8111，上所描述的二元载体。植物转化载体来源于修改根癌生杆菌的自然基因转移系统。自然系统包含一个大Ti(肿瘤诱导)质粒，此质粒中含有一个大片段，已知为T-DNA，它可以转移到转化的植物中。Ti质粒的另一个片段，Vir区域，负责T-DNA的转移。T-DNA区域与未端重复序列接界。在修饰的二元载体中，删除了肿瘤诱导基因，Vir区域的功能用于转移被T-DNA边界序列所接界的外源DNA。T区域还含有一个抗生素抗性的选择性标记，和一个用于插入要转移序列的多克隆位点。这些工程化的菌种已知为“去除武装”的(“disarmed”)的根癌生杆菌菌种，允许有效地转化T区域接界的序列进入植物的核基因组。
表面无菌叶盘与“去除武装”(“disarmed”)含有外源DNA的根癌生杆菌孵育，培养两天，然后转移到含有抗生素的培养基中。等到在含有抗生素的培养基中长出根后，选择长出的转化的嫩芽，转移到土壤中再生。
本发明的另一方面是提供含有本发明分离DNA的转基因植物或这些植物的后代。单子叶和双子叶植物都可以考虑。通过上面描述的任一种植物转化方法，用分离的编码Δ6-去饱和酶DNA转化植物细胞。转化的植物细胞，经常在愈伤组织培养物或叶盘中，通过本领域熟知的常规技术之一(如Horsh et al.(1985)《科学》227,1129)再生成一个完整的转基因植物。优选的转基因植物是向日葵、油籽油菜、玉米、烟草、花生或大豆。因为转化植物的后代遗传了编码Δ6-去饱和酶的DNA，来源于转化植物的种子或剪枝可以用做保持转基因的植物系。
本发明提供了一种方法用于提供具有增高的GLA含量的转基因植物。这些方法包括诱导编码Δ6-去饱和酶的DNA进入植物细胞，此细胞缺少或只含有低含量的GLA，但含有LA，再生的植物从转基因细胞中具有增开的GLA含量。尤其是，商业中生长的粮食植物做为转基因组织可以考虑的有，向日葵、大豆、油籽油菜、玉米、花生和烟草，但并不限于此。
本发明进一步提供了方法用于提供含有GLA的转基因组织。此方法包括诱导编码Δ6-去饱和酶的DNA进入组织，此组织缺少或含有低水平的GLA，但含有LA。在另一实施方案中，方法包括诱导一个或多个含有编码Δ12-去饱和酶和Δ6-去饱和酶的表达载体进入缺乏GLA和LA的组织。相应的，通过Δ12-去饱和酶的表达，缺乏LA和GLA的组织被诱导产生LA，由于Δ6-去饱和酶的表达，接着产生GLA。含有编码Δ12-去饱和酶或Δ12-去饱和酶和Δ6-去饱和酶的表达载体，可以通过本领域普通技术人员已知重组技术的方法来构建(Sambrook et al.(1989)发表的Δ12-去饱和酶序列见(Sambrooket al.(1990)《自然》(伦敦)347,200-2030。此外，已发现对应于本发明，SEQ ID NO：1的核苷酸2002-3081编码蓝细菌Δ12-去饱和酶。因此，此序列用于构建目标作物表达载体。尤其是，商业生长的作物可以考虑做为转基因的组织包括，但不限于此的有向日葵、大豆、油籽油菜、玉米、花生和烟草。
本发明进一步目标是提供在植物中诱导抗冻的方法。冷的敏感性也许是由于细胞膜中脂类的相的转换。相的转换温度依赖于细胞膜脂类中脂肪酸的不饱和程度。这样，例如通过导入Δ6-去饱和酶把LA转变GLA，提高了不饱和的程度，就能诱导或增进抗冻性。因此，本发明包括诱导编码Δ6-去饱和酶的DNA进八植物细胞，从所说植物细胞中再生的植物具有增强的抗冻能力。在优选的实施方案中，植物可以是向日葵、大豆、油籽油菜、玉米、花生，或烟草植物。
下面的实施例进一步说明本发明实施例1菌株和培养条件
集胞藻属(PCC 6803，ATCC 27184)，鱼腥藻属(PCC 7120，ATCC 27893)和聚球藻属(PCC 7942，ATCC 33912)光自养生长于白炽灯照射下(60uE.m-2.S-1)，30℃ BG11N+培养基中(Rippka et al(1979)J.Gen.Microbiol 111.1-61) 选择粘粒和质粒，在大肠杆菌DH5α中扩增，DH5α生长于加入标准抗生素浓度的LB培养基中，描述见Maniatis et al(1982)《分子克隆：实验手册)》，冷泉港实验室，冷泉，纽约。实施例2集胞藻属粘粒基因组文库的建立集胞藻属(PCC 6803)总的基因组DNA用Sau 3A部分酶切，在蔗糖梯度中分级分离(Ausubel et al(1987)《当气分子生物学方法》Greenepublishing Associate and Wiley interscience，纽约)。含有30至40Kb DNA片段的部分选择出来，连接进粘粒载体pDUCA7(Buikem et al.(1991)细菌学杂志173,1879-1885)的去磷酸化的BamHI位点。连接的DNA体外进行包装，见Ausubel et al.(1987)。包装的噬菌体在含有AvaI和Eco 4711甲基化酶辅助质粒，pRL528(见Buikema et al(1991)的大肠杆菌DH5α中扩增。随机分离1152个克隆，分别培养于12个96孔微滴度板中。
例3
GLA在鱼腥藻属中功能获得性表达
鱼腥藻属(PCC 7120)，一种丝状蓝细菌，缺乏GLA，但含有显著量的亚油酸，即GLA的前体(图2；表2)。实施例2中描述的集胞藻属粘粒文库结合进鱼腥藻属(PCC 7120)以确认能够生产GLA的转结合物。鱼腥藻属细胞在BG11N液体培养基中生长至对数中期，重新悬浮于相同的培养基中至终浓度约为每毫升2×109个细胞。生长于含有氨苄青霉素LB中的大肠杆菌RP4(Burkavdt et al(1979)普通微生物学杂志114,341-348)的对数中期培养物被洗涤，重新悬浮于新鲜的LB培养基中。鱼腥藻属和RP4混合，均匀涂布于含有5％LB的BG11N平皿中。粘粒基因组文库影印铺板于含有50ug/ml卡那霉素和17.5ug/ml的氯霉素的LB板上，然后挑斑放于含有鱼腥藻属和RP4的BG11N平皿上。30℃培养24小时后，加入30ug/ml新霉素，30℃下培养直至转结合物出现。
结合后单个转结合物分离出来，生长在加有15ug/ml新霉素的2mlBG11N液体培养基中。脂肪酸甲酯从野生型培养物以及10个转结合物的细胞群培养物中参照如下制备。野生型和转基因的蓝细菌培养物通过离心收获，用蒸馏水洗涤两次。脂肪酸甲酯从这些培养物中抽提出来，见Dahmer et al(1989)美国石油化学联合会杂志，66,543-548，通过气液相色谱法分析(GLC)。GLC使用配备有氢火焰离子探测器和毛细柱(30m×0.25mm bonded FSOT SuperoxII，Alltech Associates Inc.IL)的Tracor-560。保留时间和标准的共色谱(从Sigma化学公司获得)用于确认脂肪酸。平均脂肪酸成份由每个C18脂肪酸的峰区对内在标准进行标准化的比率决定。
典型GLC图形显示于图2。显示C18脂肪酸甲酯。通过与已知标准的脂肪酸甲酯比较洗脱时间确定峰值，并通过气相色谱-质谱加以确定。A组描述了野生型鱼腥藻属的脂肪酸GLC分析，箭头表示GLA迁移时间。B组是用pAM542+1.8F转化的鱼腥藻属转结合物中脂肪酸的GLC图。两个生产GLA的混合细胞群(代表250个转结合物的25个混合细胞群之中的)被确认可以产生GLA。每个GLA阳性混合细胞群中的单个转结合物用于分析GLA的产生；两个独立的转结合物AS13和AS75，分别来自此两个混合细胞群被确认可以表达显著水平的GLA，并分别含有粘粒cSy13和cSy75(图3)。粘粒重叠于一个大约7.5kb长的区域内，cSy75的一个3.5kb的Nhe I片段被重新克隆进载体PDUCA7，并转化进鱼腥藻属产生一个GLA功能获得性表达(表2)。
cSy75-3.535kb Nhe I片段的两个Nhe I/Hind III亚片段(1.8和1.7kb)重新亚克隆进入pBLUESCR1PT(Stratagene)(图3)进行测序。执行标准的分子生物学技术，见Maniatis et al(1982)和Ausubel etal(1987)。通过由先进DNA技术实验室(生物系，Texas ACM大学)合成的特定寡核苷酸引物，在两条链上用“测序酶”(“SEQUENASE”)(美国生化)对pBS1.8进行双脱氧测序(Sanger et al.(1977)《美国国家科学院院刊》74,5463-5467)。DNA序列分析采用GCG(Madison，WI)软件，见Devereux et al(1984)《核酸研究》12,387-395。
两个Nhe I/Hind III亚片段相对于蓝细菌羧化酶启动子分别以正反两个方向连接进一个共同的表达载体AM542，并通过结合诱导进入鱼腥藻属。以正向进入含1.8kb片段《AM542-1.8F)的转结合物产生显著量的GLA和十八碳四烯酸(图2，表2)。含有其它重组的转结物，如1.8kb片段的反向1.7kb的正反向，并不产生可检测水平的GLA(表2)。
图2比较从野生型鱼腥藻属(图2A)和从含有cSy75-3.5中1.8kb片段正向重组的转基因鱼腥藻属(图2B)中抽提的C18脂肪酸图。从AM542-1.8F的脂肪酸甲酯GLC分析中揭示，一个具有保留时间的峰与真正的GLA标准的峰是相同的。通过气相色谱-质谱[GC-MS]分析此峰证实它有GLA参考样品同样的物质片段形式。具有改变的多不饱和脂肪酸水平的转基因鱼腥藻属在生长速率和形态上与野生型相似。表2在野生型和转基因蓝细菌中C18脂肪酸的组成
脂肪酸(％)菌株
18∶0 18∶1 18∶2 18.3(α) 18.3(γ) 18.4野生型集胞藻属 13.6 4.5 54.5 - 27.3 -(种.PCC6803)鱼腥藻属 2.9 24.8 37.1 35.2 - -(种.PCC7120)聚球藻属 20.6 79.4 - - - -(种PCC7942)鱼腥藻属的转结合物cSy75 3.8 24.4 22.3 9.1 27.9 12.5cSy75-3.5 4.3 27.6 18.1 3.2 40.4 6.4pAM542-1.8R 4.2 13.9 12.1 19.1 25.4 25.4pAM542-1.8R 7.7 23.1 38.4 30.8 - -pAM542-1.7R 2.8 27.8 36.1 33.3 - -pAM542-1.7R 2.8 25.4 42.3 29.6 - -聚球藻属转化子pAM854 27.8 72.2 - - - -pAM854-Δ12 4.0 43.2 46.0 - - -pAM854-Δ6 18.2 81.8 - - - -pAM854-Δ12&Δ12 42.7 25.3 19.5 - 16.5 -18∶0，硬脂酸；18∶1油酸；18∶2亚油酸；18∶3(α)亚麻酸；18∶3(γ)，γ亚麻酸；18∶4，十八碳四烯酸
实施例4
用Δ6和Δ12去饱和酶基因转化聚球藻属
用已发表的集胞藻属Δ12去饱和酶基因序列(Wada et al.[1990]《自然》(伦敦)347,200-203)合成寡核苷酸，以此筛选集胞藻属基因组文库，分离到含有Δ12去饱和酶基因的第三个粘粒cSy70。以含有Δ12去饱和酶的此粘粒上确认一个1.7kb Ava I片段，用做探针表明cSy13不仅含有Δ6-去饱和酶基因，而且还有Δ12-去饱和酶基因(图3)。基因组Southern点印迹分析进一步表明Δ6和Δ12-去饱和酶基因在集胞藻属基因组中是单一的，两个与C18脂肪酸去饱和相关的功能性基因在集胞藻属基因组中是紧密相连在一起的。
单细胞的蓝细菌聚球藻属(PCC 7942)在亚油酸和GLA上的缺乏的，Δ12和Δ6-去饱和酶基因分别或一起克隆进pAM854(Bustos etal.[1991]细菌学杂志，174,7525-7533)，pAM854是一个穿梭载体，含有把外源DNA整合进入聚球藻属基因组所需的序列(Golden et al.[1987]《酶学方法》153,215-231)用这些基因重组物转化聚球藻属，挑选克隆，从转基因的聚球藻属中抽提脂肪酸甲酯，并且GLC分析。
表2显示野生型聚球藻属中主要的脂肪酸是硬脂酸(18∶0)和油酸(18∶1)。用pAM854Δ12转化的聚球藻属除了主要的脂肪酸外，还表达亚油酸(18∶2)。用pAM854Δ6和Δ12转化子产生亚油酸和GLA(表1)。这些结果表明，含有Δ12和Δ6去饱和酶基因的聚球藻属已获得了在C18脂肪酸Δ12位置上引入第二个双键和在Δ6的位置上引入第三个双键的能力。然而在含有pAM854-Δ6转化子中没有观察到脂肪酸组份的变化，表明在缺少由Δ12去饱和酶合成底物的情况下，Δ6去饱和酶是无活性的。这个实验进一步证实1.8kb Nhe I/Hind III片段(图3)含有集胞藻属Δ6-去饱和酶基因的编码区域和启动子区域。具有改变的多不饱和脂肪酸水平的转基因聚球藻属在生长速率和形态上与野生型类似。
实施例5Δ6-去饱和酶的核苷酸序列
鉴定包含功能性Δ6-去饱和酶基因的cSy75-3.5中1.8kb片段的核苷酸序列。确定了一个编码359个氨基酸多肽的开读框架(图4)。Kyte-Doolittle水疗图分析(Kyte et al.(1982)J.Mol.Biol.157,105-132)证实两个疏水氨基酸区域代表了跨膜区(图1A)；此外，Δ6-去饱和酶的水疗图与Δ12-去饱和酶(图1B；Wada et al)和Δ9-去饱和酶(Thiede et al.[1986]J.Biol.Chem 261,13230-13235)的类似。然而，集胞藻属Δ6和Δ12-去饱和酶序列相似性在核苷酸水平少于40％，在氨基酸水平大约有18％
例6
转移蓝细菌1Δ6-去饱和酶进入烟草
蓝细菌Δ6-去饱和酶基因移动至植物表达载体，用土壤杆菌属介导的基因转移技术转移至烟草。为了确保转移的去饱和酶在叶中和正发育的种子中表达，以及去饱和酶基因产生定向于内质网或叶绿体中，构建各种带有集胞藻属Δ6-去饱和酶开读框架(ORF)的表达盒。这些盒的成份包括：(i)35S启动子或来源于向日葵半日花素基因的种子特异性启动子去驱动Δ6-去饱和酶基因在所有的植物组织或只在发育的种子中表达(ii)一个推断的信号肽，或者来源于胡萝卜伸展蛋白基因或者是向日葵半日花素基因，以定向新合成的Δ6-去饱和酶进入内质网(iii)一个位于Δ6-去饱和酶开读框架羧基端的内质网腔保留信号序列(KDEL)，(iv)一个最优的转移多肽以定向Δ6-去饱和酶进入叶绿体。35S启动子是PRTL2的衍生物，见Restrepo et al(1990)。优化的转移多肽序列见Van de Broeck et al(1985)。胡萝卜伸展蛋白信号肽见Chen et al(1985)EMBO J.9,2145
转基因烟草植物产生，含有嵌合的蓝细菌去饱和酶基因，组成有集胞藻属Δ6-去饱和酶基因与内质网保留序列(KDEL)融合，以及由CaMV 35S启动子启动的伸展蛋白信号肽。PCR扩增转基因烟草基因组DNA显示Δ6-去饱和酶基因已整合进烟草基因组。抽提转基因烟草叶中脂肪酸甲酯，并用气液色谱法(GLC)分析。这些转基因烟草中聚集了显著数量的GLA(图4)。图4显示了由GLC决定的脂肪酸甲酯。通过与脂肪酸甲酯比较已知标准确定峰值。相应的，参与脂肪酸代谢的蓝细菌1基因用于产生具有改变的脂肪酸组份的转基因植物。
例7构建琉璃苣的cDNA文库
用Larkins和Davies(1975植物生理学，55：749-756)为豌豆所建立的方法，从受粉后12天(12DPP)的琉璃苣种子中分离出膜结合的多核糖体。RNA被从多核糖体中抽提出来，见Mechler，(1987《酶学方法》152：241-248，Academic press)所述。
用oligotex-dT小珠(Qiagen)将poly-A RNA从膜结合的核糖体RNA中分离出来。用Stratagene′s ZAP cDNA合成试剂盒制备相应的cDNA。用λZAPII载体试剂盒在λZAPII载体中构建cDNA文库。原始文库在Gigapack II金包装提取物(Stratagene)中包装，文库用于产生表达序列标签(ESTs)，对应于标准的序列用于扫描Gen Bank数据库。
例8杂交方法
用于筛选琉璃苣cDNA文库的杂交探针通过随机引物DNA合成法产生，见Ausubel et al(1994《分子生物学现行方法》Wiley intezscience，N.Y)。此探针并与先前确认的丰富表达的种子贮存蛋白cDNA相对应。未掺入的核苷酸通过G-50旋转柱(Boehringer Manheim)除去。为了杂交，探针在沸水中煮5分钟变性，然后迅速放入冰中冷却。杂交膜于60℃在预杂交液[6XSSC(maniatis et al 1984，《分子克隆实验室手册》冷泉港实验室)。1XDenharts溶液，0.05％焦磷酸钠，100ug/ml变性鲑鱼精子DNA]杂交2-4小时，变性探针加入杂交液中(6xSSC，1xDenharts溶液，0.05％焦磷酸钠，100ug/ml变性鲑鱼精子DNA)60℃搅拌孵育过夜。膜在4x，2x和1xSET中60℃下分别洗涤15分钟。20xSET储液是3M Wacl，0.4M Tris碱，20mM Na2EDTA-2H2O。4xSET洗涤是4xSET，12.5mM PO4，pH6.8和0.2％SDS，2×SET洗涤是2xSET，12.5mM PO4，pH6.8和0.2％SDS，1xSET洗涤是1xSET，12.5mM PO4，pH6.8和0.2％ SDS。膜可以在空气中干燥，然后用增感屏于-80℃下曝光X-光胶片24小时。
实施例9
从琉璃苣种子(12DPP)膜结合的核糖体
文库而来的cDNA的随机测序
琉璃苣cDNA文库以低密度铺板(5000pfu于150mm petri细菌培养皿中)。用以前确认的相应的cDNA，通过筛选，差减去广泛存在的种子贮存蛋白cDNA。未杂交的噬菌斑用Stratagene′s切除方法和试剂切除。产生的细菌克隆孵育于液基培养基中，手工测序或者用ABI自动测序仪测序。每个cDNA测序一次，从200至300个碱基对产生一个序列标签。所有测序通过循环测序(Epicentre)来执行。产生超过300个ESTs。每个序列标签用BLASTX计算机程序与GenBank数据库相比较，许多脂类代谢的基因，包括Δ6去饱酶得以确认。
采用BLASTX在GenBank数据库中搜索命名为mbp-65的cDNA克隆，发现与集胞藻属的Δ6-去饱和酶有显著的匹配。然而发现它并不是全长cDNA。用mbp-65筛选琉璃苣膜结合的多核糖体文库，分离出全长cDNA。确定分离出的cDNA序列(图5A，SEQ ID NO：4)，开读框架的蛋白质序列(图5B，SEQ ID NO：5)用Gene works(Intellig Genetics)蛋白排列程序(图2)与已知去饱和酶相比较。排列表明此cDNA是琉璃苣Δ6-去饱和酶基因。
尽管与其它已知植物去饱和酶类似，琉璃苣Δ6-去饱和酶是独特的，即在图6树状图中所显示的。此外，进一步比较去饱和酶特征性的氨基酸序列，特别是那些参与金属结合的氨基酸序列(金属框1和金属框2)，表明了琉璃苣Δ6-去饱和酶和别的植物去饱和酶的区别(表3)。
琉璃苣Δ6-去饱和酶与蓝细菌1的不仅所有序列不同(图6)，而且在于脂类框，金属框1，金属框2这些氨基酸框(表3)。依表3所显示，所有3个基序在序列上是新的。只有琉璃苣Δ6-去饱和酶的金属框2显示出与集胞藻属Δ6-去饱和酶的金属框2有一些关系。
此外，琉璃苣Δ6-去饱和酶与另一个琉璃苣去饱和酶基因，Δ12-去饱和酶也是有区别的。P1-81是通过EST分析确认的全长cDNA，表明与拟南芥属Δ12-去饱和酶(Fad2)有高度类似。比较琉璃苣Δ6和Δ12-去饱和酶中脂类框，金属框1和2这些氨基酸基序(表3)，显示在这些区域有极少的同源性。把此这个序列放入图6的树状图中表明这两个基因关系有多远。表3.比较膜结合的去饱和酶中普通的氨基酸基序
氨基酸基序
去饱和酶 脂类框 金属框1 金属框2破璃苣Δ6 WIGHDAGH(SEQ.ID.NO：6) HNAHH(SEQ.ID.NO：12) FQIEHH(SEQ.ID.NO：20)集胞藻属Δ6 NVGHDANH(SEQ.ID.NO：7) HNYLHH(SEQ.ID.NO：13) HQVTHH(SEQ.ID.NO：21)拟南芥属叶绿体Δ15 VLGHDCGH(SEQ.ID.NO：8) HRTHH(SEQ.ID.NO：14) HVIHH(SEQ.ID.NO：22)稻Δ15 VLGHDCGH(SEQ.ID.NO：8) HRTHH(SEQ.ID.NO：14) HVIHH(SEQ.ID.NO：22)甘氨酸叶绿体Δ15 VLGHDCGH(SEQ.ID.NO：8) HRTHH(SEQ.ID.NO：14) HVIHH(SEQ.ID.NO：22)拟南芥.fad3(Δ15) VLGHDCGH(SEQ.ID.NO：8) HRTHH(SEQ.ID.NO：14) HVIHH(SEQ.ID.NO：22)芸苔属fad3(Δ15) VLGHDCGH(SEQ.ID.NO：8) HRTHH(SEQ.ID.NO：14) HVIHH(SEQ.ID.NO：22)玻璃苣Δ12(P1-81)* VIAHECGH(SEQ.ID.NO：9) HRRHH(SEQ.ID.NO：15) HVAHH(SEQ.ID.NO：23)拟南芥fad2(Δ12) VIAHECGH(SEQ.ID.NO：9) HRRHH(SEQ.ID.NO：15) HVAHH(SEQ.ID.NO：23)拟南芥属叶绿体Δ12 VIGHDCAH(SEQ.ID.NO：10) HDRHH(SEQ.ID.NO：16) HIPHH(SEQ.ID.NO：24)甘氨酸质体Δ12 VIGHDCAH(SEQ.ID.NO：10) HDRHH(SEQ.ID.NO：16) HIPHH(SEQ.ID.NO：24)菠菜质体n-6 VIGHDCAH(SEQ.ID.NO：10) HDQHH(SEQ.ID.NO：17) HIPHH(SEQ.ID.NO：24)集胞藻属Δ12 VVGHDCGH(SEQ.ID.NO：11) HDHHH(SEQ.ID.NO：18) HIPHH(SEQ.ID.NO：24)鱼腥藻属Δ12 VLGHDCGH(SEQ.1D.NO：8) HNHHH(SEQ.ID.NO：19) HVPHH(SEQ ID.NO：25)*P1-81是通过EST分析确认的全长cDNA，显示出与拟南芥属Δ12去饱和酶(fad2)有高度类似性
实施例10
构建221.Δ6.NOS的瞬时表达
载体pBI221(Jefferson et al 1987 EMBO J.6：3901-3907)用BamHI和EcoICRI(promega)酶切去除GUS编码区域剩于完整的35S启动子和NOS完整的终止子，以制备连接片段。琉璃苣Δ6-去饱和酶cDNA用BamHI和xhoI酶切从Bluescript质粒(Stratagene)上切下。XhoI末端用Klenow大片段补齐，此片段克隆进pBI221的BamHI/EcoICRI位点，产生221Δ6.NOS(图7)。在221.Δ6NOS中，图7的限制性图谱余下的部分骨架是pBI221。
实施例11建立121Δ6.NOS的稳定转化
载体pBI121(Jefferson et al.1981 EMBO J.6：3901-3907)通过Bam HI和EcoI CRI(Promega)酶切除去GUS编码区域，剩下完整的35S启动子和NOS终止子，以制备连接片段。琉璃苣的Δ6-去饱酶cDNA用Bam HI和XhoI酶切从Bluesoript质粒(Stratagene)上切下。xhoI未端用Klenow大片段补平，此片段克隆进pBI121的Bam HI/EcoICRI位点，产生121.Δ6NOS(图7)。在121.Δ6NOS中，图7的限制性图谱余下的部分(骨架)是pBI121。
实施例12瞬时表达
所有涉及原生质体的工作全在无菌通风橱中进行。1ml浓集的胡萝卜悬浮细胞在30ml质壁分离液中过夜消化(25g/l kol，3.5g/lCacl2·H2O，10mM MES，pH5.6和0.2M甘露醇)，此质壁分离液中还含有1％纤维素酶，0.1％果胶酶和0.1％ dreisalase，此消化于室温下反应，并避光。释放出来的原生质体通过150uM网过滤，然后离心沉淀(100xg，5分)。用质壁分离液洗涤两次，原生质体用双室血细胞计数器计数。把106原生质体和50-70ug质粒DNA(221.Δ6NOS)用PEG处理，DNA转化进原生质体，方法见Nunberg和Thomos(1993《植物分子生物学和生物技术的方法》B.R.Glick and J.E.Thompson，eds，pp241-248)原生质体在加有0.2M甘露醇和3uM2，4-D的5ml MS培养基中振荡培养48小时，并避光。
实施例13烟草的稳定转化
除了开始的转化子在100ug/ml卡那霉素中选择，依据标准程序(Horsh et al.，1985《科学》227：1229-1231；Bogue et al.，1990 Mol.Gen.Genet.221；49-57)，121.Δ6 NOS质粒通过土壤杆菌属转化烟草(Nicotiana tabacum cv.xanthi)。
实施例14制备与分析脂肪酸甲酯(FAMEs)
从转化原生质体和转化烟草植物中来源的组织冻于液氮中，过夜冷冻干燥。制备FAMEs，方法见Dahmer et al(1989 J.Amer.Oil Chem.Soc.66：543-548)。在一些情况下，溶剂再一次蒸发，FAMEs悬浮于乙酸乙酯中，并且无离子水抽提一次去除任何水溶性污染物。FAMEs通过气相色谱法(GC)在一个J&W Scientific DB-wax柱(长30m，内径0.25mm，0.25um膜)上分析。
瞬时测定的实例显示于图8，它代表混合在一起的三个独立的转化子。琉璃苣Δ6-去饱和酶的增加对应于γ-亚麻酸(GLA)的出现，而GLA是Δ6-去饱和酶可能的产物之一。
图9和10描述了来源于对照和转化烟草植物叶子和种子组织中FAMEs的GC图。图9A提供了野生型烟草叶组织的图(Xanthi)，图9B提供了在35S CaMV启动子(pBI 121.Δ6NOS)转录调控下琉璃苣Δ6-去饱和酶转化的烟草叶组织图。峰值对应于18∶2，18∶3γ(GLA)，18∶3α和18∶4(十八碳酸)。图10A表明了野生型烟草种子的GC图；图10B表明用pBI 121.Δ6NOS转化的烟草种子组织的图。峰值对应于18∶2，18∶3γ(GLA)和18∶3α。
C18脂肪酸在对照和转基因烟草种子中的相对分布显示于表4中。
表4
| 脂肪酸 | Xanthi | pBI121Δ6NOS |
| 18∶0 | 4.0％ | 2.5％ |
| 18∶1 | 13％ | 13％ |
| 18∶2 | 82％ | 82％ |
| 18∶3γ(GLA) | - | 2.7％ |
| 18∶3α | 0.82％ | 1.4％ |
上述结果显示，GLA已掺入含有琉璃苣Δ6-去饱和酶的转基因烟草的叶与种子的三酰基甘油酯中。
序列表(1)总的信息：(i)申请人：Rhone-Poulenc Agrochimie(ii)发明题目：通过Δ6-去饱和酶生产γ-亚麻酸(iii)序列数目：25(iv)通信地址：
(A)收信人： Scully，Scott，Murphy & Presser
(B)街道：400 Garden City Plaza
(C)城市：Garden City
(D)州：New York
(E)国家：美国
(F)邮区：11530(v)机读形式：
(A)媒质形式： Floppy disk
(B)计算机：兼容IBM PC机
(C)操作系统：PC-DOS/MS-DOS
(D)软件：Patentln Release#1.0，Version#1.25(vi)目前申请资料：
(A)申请号：
(B)申请日期：1994.12.30.
(C)分类：(Viii)委托代理人信息：
(A)名字：Presser，Leopold
(B)登记号：19,827
(C)文献/档案号码：8383ZYXW(ix)通信信息：
(A)电话：(516)742-4343
(B)传真：(516)742-4366
(C)电传：230 901 SANS UR(2)SEQ ID NO：1的信息：(i)序列特征：
(A)长度：3588个碱基对 (B)类型：核酸(C)链型：22(D)拓扑结构：线性(ii)分子类型：DNA(基因组)(ix)特征：(A)名字/键：CDS(B)位置：2002..3081(xi)序列说明；SEQ ID NO：1：GCTAGCCACC AGTGACGATG CCTTGAATTT GGCCATTCTG ACCCAGGCCC GTATTCTGAA 60TCCCCGCATT CGCATTGTTA ATCGTTTGTT CAACCATGCC CTGGGTAAAC GTTTAGACAC 120CACCTTGCCA GACCACGTTA GTTTGAGTGT TTCCGCCCTG GCGGCCCCGA TTTTTTCCTT 180TGCGGCTTTG GGCAATCAGG CGATCGGGCA ATTGCGTTTG TTTGACCAGA CTTGGCCCAT 240TCAGGAAATT GTCATTCACC AAGACCATCC CTGGCTCAAT TTACCCCTGG CGGATTTATG 300GGATGATCCG AGCCGAATGT TGATCTATTA CCTACCGGCC CACAGTGAAA CGGATTTAGT 360AGGCGCAGTG GTGAATAATT TAACGTTGCA ATCTGGGGAC CATTTAATAG TGGGACAAAA 420ACCCCAACCC AAGACCAAAC GGCGATCGCC TTGGCGCAAA TTTTCCAAAC TGATTACCAA 480CCTGCGGGAG TATCAGCGGT ATGTCCAACA GGTGATATGG GTGGTGTTGT TTTTATTGTT 540GATGATTTTT CTGGCCACCT TCATCTACGT TTCCATTGAT CAACATATTG CCCCAGTGGA 600CGCGTTGTAT TTTTCCGTGG GCATGATTAC CGGGGCCGGT GGCAAGGAAG AGGTGGCCGA 660AAAGTCCCCC GATATCATCA AAGTATTCAC AGTGGTGATG ATGATCGCCG GGGCGGGGGT 720GATTGGTATT TGTTATGCCC TACTGAATGA TTTCATCCTT GGCAGTCGCT TTAGTCAGTT 780TTTGGATGCG GCCAAGTTAC CCGATCGCCA TCACATCATC ATTTGTGGGC TGGGGGGAGT 840GAGCATGGCC ATTATTGAAG AGTTAATTCA CCAGGGCCAT GAAATTGTGG TAATCGAAAA 900GGATACAGAT AATCGTTTCT TGCATACGGC CCGCTCCCTG GGGGTGCCCG TAATTGTGGA 960GGATGCCCGC CTAGAAAGAA CGTTGGCCTG CGCCAATATC AACCGAGCCG AAGCCATTGT 1020GGTGGCCACC AGCGACGACA CCGTTAACTT GGAAATTGGC CTAACTGCCA AGGCGATCGC 1080CCCTAGCCTG CCAGTGGTGT TGCGTTGCCA GGATGCCCAG TTTAGCCTGT CCCTGCAGGA 1140AGTATTTGAA TTTGAAACGG TGCTTTGTCC GGCGGAATTG GCCACCTATT CCTTTGCGGC 1200GGCGGCCCTG GGGGGCAAAA TTTTGGGCAA CGGCATGACC GATGATTTGC TGTGGGTAGC 1260CCTAGCCACC TTAATCACTC CTAACCATCC CTTTGCCGAC CAATTGGTTA AAATTGCAGC 1320CCAAAAGTCT GATTTCGTTC CCCTCTATCT AGAACGGGGT GGCAAAACCA TCCATAGCTG 1380GGAATTATTG GGTACCCATC TCGACTCTGG AGACGTGTTG TATTTAACCA TGCCCGCCAC 1440TGCCCTAGAG CAACTTTGGC GATCGCCCCG TGCCACTGCT GATCCTCTGG ACTCTTTTTT 1500GGTTTAGCAT GGGGGGATGG AACTCTTGAC TCGGCCCAAT GGTGATCAAG AAAGAACGCT 1560TTGTCTATGT TTAGTATTTT TAAGTTAACC AACAGCAGAG GATAACTTCC AAAAGAAATT 1620AAGCTCAAAA AGTAGCAAAA TAAGTTTAAT TCATAACTGA GTTTTACTGC TAAACAGCGG 1680TGCAAAAAAG TCAGATAAAA TAAAAGCTTC ACTTCGGTTT TATATTGTGA CCATGGTTCC 1740CAGGCATCTG CTCTAGGGAG TTTTTCCGCT GCCTTTAGAG AGTATTTTCT CCAAGTCGGC 1800TAACTCCCCC ATTTTTAGGC AAAATCATAT ACAGACTATC CCAATATTGC CAGAGCTTTG 1860ATGACTCACT GTAGAAGGCA GACTAAAATT CTAGCAATGG ACTCCCAGTT GGAATAAATT 1920TTTAGTCTCC CCCGGCGCTG GAGTTTTTTT GTAGTTAATG GCGGTATAAT GTGAAAGTTT 1980TTTATCTATT TAAATTTATA A ATG CTA ACA GCG GAA AGA ATT AAA TTT ACC 2031
Met Leu Thr Ala Glu Arg Ile Lys Phe Thr
1 5 10CAG AAA CGG GGG TTT CGT CGG GTA CTA AAC CAA CGG GTG GAT GCC TAC 2079Gln Lys Arg Gly Phe Arg Arg Val Leu Asn Gln Arg Val Asp Ala Tyr
15 20 25TTT GCC GAG CAT GGC CTG ACC CAA AGG GAT AAT CCC TCC ATG TAT CTG 2127Phe Ala Glu His Gly Leu Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu
30 35 40AAA ACC CTG ATT ATT GTG CTC TGG TTG TTT TCC GCT TGG GCC TTT GTG 2175Lys Thr Leu Ile Ile Val Leu Trp Leu Phe Ser Aia Trp Ala Phe Val
45 50 55CTT TTT GCT CCA GTT ATT TTT CCG GTG CGC CTA CTG GGT TGT ATG GTT 2223Leu Phe Ala Pro Val Ile Phe Pro Val Arg Leu Leu Gly Cys Met Val
60 65 70TTG GCG ATC GCC TTG GCG GCC TTT TCC TTC AAT GTC GGC CAC GAT GCC 2271Leu Ala Ile Ala Leu Ala Ala Phe Ser Phe Asn Val Gly His Asp Ala75 80 85 90AAC CAC AAT GCC TAT TCC TCC AAT CCC CAC ATC AAC CGG GTT CTG GGC 2319Asn His Asn Ala Tyr Ser Ser Asn Pro His Ile Asn Arg Val Leu Gly
95 100 105ATG ACC TAC GAT TTT GTC GGG TTA TCT AGT TTT CTT TGG CGC TAT CGC 2367Met Thr Tyr Asp Phe Val Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg
110 115 120CAC AAC TAT TTG CAC CAC ACC TAC ACC AAT ATT CTT GGC CAT GAC GTG 2415His Asn Tyr Leu His His Thr Tyr Thr Asn Ile Leu Gly His Asp Val
125 130 135GAA ATC CAT GGA GAT GGC GCA GTA CGT ATG AGT CCT GAA CAA GAA CAT 2463Glu Ile His Gly Asp Gly Ala Val Arg Met Ser Pro Glu Gln Glu His
140 145 150GTT GGT ATT TAT CGT TTC CAG CAA TTT TAT ATT TGG GGT TTA TAT CTT 2511Val Gly Ile Tyr Arg Phe Gln Gln Phe Tyr Ile Trp Gly Leu Tyr Leu155 160 165 170TTC ATT CCC TTT TAT TGG TTT CTC TAC GAT GTC TAC CTA GTG CTT AAT 2559Phe Ile Pro Phe Tyr Trp Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn
175 180 185AAA GGC AAA TAT CAC GAC CAT AAA ATT CCT CCT TTC CAG CCC CTA GAA 2607Lys Gly Lys Tyr His Asp His Lys Ile Pro Pro Phe Gln Pro Leu Glu
190 195 200TTA GCT AGT TTG CTA GGG ATT AAG CTA TTA TGG CTC GGC TAC GTT TTC 2655Leu Ala Ser Leu Leu Gly Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe
205 210 215GGC TTA CCT CTG GCT CTG GGC TTT TCC ATT CCT GAA GTA TTA ATT GGT 2703Gly Leu Pro Leu Ala Leu Gly Phe Ser Ile Pro Glu Val Leu Ile Gly
220 225 230GCT TCG GTA ACC TAT ATG ACC TAT GGC ATC GTG GTT TGC ACC ATC TTT 2751Ala Ser Val Thr Tyr Met Thr Tyr Gly Ile Val Val Cys Thr Ile Phe235 240 245 250ATG CTG GCC CAT GTG TTG GAA TCA ACT GAA TTT CTC ACC CCC GAT GGT 2799Met Leu Ala His Val Leu Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly
255 260 265GAA TCC GGT GCC ATT GAT GAC GAG TGG GCT ATT TGC CAA ATT CGT ACC 2847Glu Ser Gly Ala Ile Asp Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr
270 275 280ACG GCC AAT TTT GCC ACC AAT AAT CCC TTT TGG AAC TGG TTT TGT GGC 2895Thr Ala Asn Phe Ala Thr Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly
285 290 295GGT TTA AAT CAC CAA GTT ACC CAC CAT CTT TTC CCC AAT ATT TGT CAT 2943Gly Leu Asn His Gln Val Thr His His Leu Phe Pro Asn Ile Cys His
300 305 310ATT CAC TAT CCC CAA TTG GAA AAT ATT ATT AAG GAT GTT TGC CAA GAG 2991Ile His Tyr Pro Gln Leu Glu Asn Ile Ile Lys Asp Val Cys Gln Glu315 320 325 330TTT GGT GTG GAA TAT AAA GTT TAT CCC ACC TTC AAA GCG GCG ATC GCC 3039Phe Gly Val Glu Tyr Lys Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala
335 340 345TCT AAC TAT CGC TGG CTA GAG GCC ATG GGC AAA GCA TCG TGACATTGCC 3088Ser Asn Tyr Arg Trp Leu Glu Ala Met Gly Lys Ala Ser
350 355 360TTGGGATTGA AGCAAAATGG CAAAATCCCT CGTAAATCTA TGATCGAAGC CTTTCTGTTG 3148CCCGCCGACC AAATCCCCGA TGCTGACCAA AGGTTGATGT TGGCATTGCT CCAAACCCAC 3208TTTGAGGGGG TTCATTGGCC GCAGTTTCAA GCTGACCTAG GAGGCAAAGA TTGGGTGATT 3268TTGCTCAAAT CCGCTGGGAT ATTGAAAGGC TTCACCACCT TTGGTTTCTA CCCTGCTCAA 3328TGGGAAGGAC AAACCGTCAG AATTGTTTAT TCTGGTGACA CCATCACCGA CCCATCCATG 3388TGGTCTAACC CAGCCCTGGC CAAGGCTTGG ACCAAGGCCA TGCAAATTCT CCACGAGGCT 3448AGGCCAGAAA AATTATATTG GCTCCTGATT TCTTCCGGCT ATCGCACCTA CCGATTTTTG 3508AGCATTTTTG CCAAGGAATT CTATCCCCAC TATCTCCATC CCACTCCCCC GCCTGTACAA 3568AATTTTATCC ATCAGCTAGC 3588(2)SEQ ID NO：2的信息：(I)序列特征：
(A)长度：359个氨基酸
(B)类型：氨基酸
(C)拓扑构型：线性(ii)分子类型：蛋白质(xi)序列说明：SEQ ID NO：2：Met Leu Thr Ala Glu Arg Ile Lys Phe Thr Gln Lys Arg Gly Phe Arg1 5 10 15Arg Val Leu Asn Gln Arg Val Asp Ala Tyr Phe Ala Glu His Gly Leu
20 25 30Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu Lys Thr Leu Ile Ile Val
35 40 45Leu Trp Leu Phe Ser Ala Trp Ala Phe Val Leu Phe Ala Pro Val Ile
50 55 60Phe Pro Val Arg Leu Leu Gly Cys Met Val Leu Ala Ile Ala Leu Ala65 70 75 80Ala Phe Ser Phe Asn Val Gly His Asp Ala Asn His Asn Ala Tyr Ser
85 90 95Ser Asn Pro His Ile Asn Arg Val Leu Gly Met Thr Tyr Asp Phe Val
100 105 110Gly Leu Ser Ser Phe Leu Trp Arg Tyr Arg His Asn Tyr Leu His His
115 120 125Thr Tyr Thr Asn Ile Leu Gly His Asp Val Glu Ile His Gly Asp Gly
130 135 140Ala Val Arg Met Ser Pro Glu Gln Glu His Val Gly Ile Tyr Arg Phe145 150 155 160Gln Gln Phe Tyr Ile Trp Gly Leu Tyr Leu Phe Ile Pro Phe Tyr Trp
165 170 175Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn Lys Gly Lys Tyr His Asp
180 185 190His Lys Ile Pro Pro Phe Gln Pro Leu Glu Leu Ala Ser Leu Leu Gly
195 200 205Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe Gly Leu Pro Leu Ala Leu
210 215 220Gly Phe Ser Ile Pro Glu Val Leu Ile Gly Ala Ser Val Thr Tyr Met225 230 235 240Thr Tyr Gly Ile Val Val Cys Thr Ile Phe Met Leu Ala His Val Leu
245 250 255Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu Ser Gly Ala Ile Asp
260 265 270Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr Thr Ala Asn Phe Ala Thr
275 280 285Asn Asn Pro Phe Trp Asn Trp Phe Cys Gly Gly Leu Asn His Gln Val
290 295 300Thr His His Leu Phe Pro Asn Ile Cys His Ile His Tyr Pro Gln Leu305 310 315 320Glu Asn Ile Ile Lys Asp Val Cys Gln Glu Phe Gly Val Glu Tyr Lys
325 330 335Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala Ser Asn Tyr Arg Trp Leu
340 345 350Glu Ala Met Gly Lys Ala Ser
355(2)SEQ ID NO：3的信息：(i)序列特征：
(A)长度：1884个碱基对
(B)类型：核酸
(C)链型：22
(D)拓扑构型：线性(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：3：AGCTTCACTT CGGTTTTATA TTGTGACCAT GGTTCCCAGG CATCTGCTCT AGGGAGTTTT 60TCCGCTGCCT TTAGAGAGTA TTTTCTCCAA GTCGGCTAAC TCCCCCATTT TTAGGCAAAA 120TCATATACAG ACTATCCCAA TATTGCCAGA GCTTTGATGA CTCACTGTAG AAGGCAGACT 180AAAATTCTAG CAATGGACTC CCAGTTGGAA TAAATTTTTA GTCTCCCCCG GCGCTGGAGT 240TTTTTTGTAG TTAATGGCGG TATAATGTGA AAGTTTTTTA TCTATTTAAA TTTATAAATG 300CTAACAGCGG AAAGAATTAA ATTTACCCAG AAACGGGGGT TTCGTCGGGT ACTAAACCAA 360CGGGTGGATG CCTACTTTGC CGAGCATGGC CTGACCCAAA GGGATAATCC CTCCATGTAT 420CTGAAAACCC TGATTATTGT GCTCTGGTTG TTTTCCGCTT GGGCCTTTGT GCTTTTTGCT 480CCAGTTATTT TTCCGGTGCG CCTACTGGGT TGTATGGTTT TGGCGATCGC CTTGGCGGCC 540TTTTCCTTCA ATGTCGGCCA CGATGCCAAC CACAATGCCT ATTCCTCCAA TCCCCACATC 600AACCGGGTTC TGGGCATGAC CTACGATTTT GTCGGGTTAT CTAGTTTTCT TTGGCGCTAT 660CGCCACAACT ATTTGCACCA CACCTACACC AATATTCTTG GCCATGACGT GGAAATCCAT 720GGAGATGGCG CAGTACGTAT GAGTCCTGAA CAAGAACATG TTGGTATTTA TCGTTTCCAG 780CAATTTTATA TTTGGGGTTT ATATCTTTTC ATTCCCTTTT ATTGGTTTCT CTACGATGTC 840TACCTAGTGC TTAATAAAGG CAAATATCAC GACCATAAAA TTCCTCCTTT CCAGCCCCTA 900GAATTAGCTA GTTTGCTAGG GATTAAGCTA TTATGGCTCG GCTACGTTTT CGGCTTACCT 960CTGGCTCTGG GCTTTTCCAT TCCTGAAGTA TTAATTGGTG CTTCGGTAAC CTATATGACC 1020TATGGCATCG TGGTTTGCAC CATCTTTATG CTGGCCCATG TGTTGGAATC AACTGAATTT 1080CTCACCCCCG ATGGTGAATC CGGTGCCATT GATGACGAGT GGGCTATTTG CCAAATTCGT 1140ACCACGGCCA ATTTTGCCAC CAATAATCCC TTTTGGAACT GGTTTTGTGG CGGTTTAAAT 1200CACCAAGTTA CCCACCATCT TTTCCCCAAT ATTTGTCATA TTCACTATCC CCAATTGGAA 1260AATATTATTA AGGATGTTTG CCAAGAGTTT GGTGTGGAAT ATAAAGTTTA TCCCACCTTC 1320AAAGCGGCGA TCGCCTCTAA CTATCGCTGG CTAGAGGCCA TGGGCAAAGC ATCGTGACAT 1380TGCCTTGGGA TTGAAGCAAA ATGGCAAAAT CCCTCGTAAA TCTATGATCG AAGCCTTTCT 1440GTTGCCCGCC GACCAAATCC CCGATGCTGA CCAAAGGTTG ATGTTGGCAT TGCTCCAAAC 1500CCACTTTGAG GGGGTTCATT GGCCGCAGTT TCAAGCTGAC CTAGGAGGCA AAGATTGGGT 1560GATTTTGCTC AAATCCGCTG GGATATTGAA AGGCTTCACC ACCTTTGGTT TCTACCCTGC 1620TCAATGGGAA GGACAAACCG TCAGAATTGT TTATTCTGGT GACACCATCA CCGACCCATC 1680CATGTGGTCT AACCCAGCCC TGGCCAAGGC TTGGACCAAG GCCATGCAAA TTCTCCACGA 1740GGCTAGGCCA GAAAAATTAT ATTGGCTCCT GATTTCTTCC GGCTATCGCA CCTACCGATT 1800TTTGAGCATT TTTGCCAAGG AATTCTATCC CCACTATCTC CATCCCACTC CCCCGCCTGT 1860ACAAAATTTT ATCCATCAGC TAGC 1884(2)SEQ ID NO：4的信息：
(i)序列特征：
(A)长度1685个碱基对
(B)类型：核酸
(C)链型：22
(D)拓扑构型：线性(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：4：AATATCTGCC TACCCTCCCA AAGAGAGTAG TCATTTTTCA TCAATGGCTG CTCAAATCAA 60GAAATACATT ACCTCAGATG AACTCAAGAA CCACGATAAA CCCGGAGATC TATGGATCTC 120GATTCAAGGG AAAGCCTATG ATGTTTCGGA TTGGGTGAAA GACCATCCAG GTGGCAGCTT 180TCCCTTGAAG AGTCTTGCTG GTCAAGAGGT AACTGATGCA TTTGTTGCAT TCCATCCTGC 240CTCTACATGG AAGAATCTTG ATAAGTTTTT CACTGGGTAT TATCTTAAAG ATTACTCTGT 300TTCTGAGGTT TCTAAAGATT ATAGGAAGCT TGTGTTTGAG TTTTCTAAAA TGGGTTTGTA 360TGACAAAAAA GGTCATATTA TGTTTGCAAC TTTGTGCTTT ATAGCAATGC TGTTTGCTAT 420GAGTGTTTAT GGGGTTTTGT TTTGTGAGGG TGTTTTGGTA CATTTGTTTT CTGGGTGTTT 480GATGGGGTTT CTTTGGATTC AGAGTGGTTG GATTGGACAT GATGCTGGGC ATTATATGGT 540AGTGTCTGAT TCAAGGCTTA ATAAGTTTAT GGGTATTTTT GCTGCAAATT GTCTTTCAGG 600AATAAGTATT GGTTGGTGGA AATGGAACCA TAATGCACAT CACATTGCCT GTAATAGCCT 660TGAATATGAC CCTGATTTAC AATATATACC ATTCCTTGTT GTGTCTTCCA AGTTTTTTGG 720TTCACTCACC TCTCATTTCT ATGAGAAAAG GTTGACTTTT GACTCTTTAT CAAGATTCTT 780TGTAAGTTAT CAACATTGGA CATTTTACCC TATTATGTGT GCTGCTAGGC TCAATATGTA 840TGTACAATCT CTCATAATGT TGTTGACCAA GAGAAATGTG TCCTATCGAG CTCAGGAACT 900CTTGGGATGC CTAGTGTTCT CGATTTGGTA CCCGTTGCTT GTTTCTTGTT TGCCTAATTG 960GGGTGAAAGA ATTATGTTTG TTATTGCAAG TTTATCAGTG ACTGGAATGC AACAAGTTCA 1020GTTCTCCTTG AACCACTTCT CTTCAAGTGT TTATGTTGGA AAGCCTAAAG GGAATAATTG 1080GTTTGAGAAA CAAACGGATG GGACACTTGA CATTTCTTGT CCTCCTTGGA TGGATTGGTT 1140TCATGGTGGA TTGCAATTCC AAATTGAGCA TCATTTGTTT CCCAAGATGC CTAGATGCAA 1200CCTTAGGAAA ATCTCGCCCT ACGTGATCGA GTTATGCAAG AAACATAATT TGCCTTACAA 1260TTATGCATCT TTCTCCAAGG CCAATGAAAT GACACTCAGA ACATTGAGGA ACACAGCATT 1320GCAGGCTAGG GATATAACCA AGCCGCTCCC GAAGAATTTG GTATGGGAAG CTCTTCACAC 1380TCATGGTTAA AATTACCCTT AGTTCATGTA ATAATTTGAG ATTATGTATC TCCTATGTTT 1440GTGTCTTGTC TTGGTTCTAC TTGTTGGAGT CATTGCAACT TGTCTTTTAT GGTTTATTAG 1500ATGTTTTTTA ATATATTTTA GAGGTTTTGC TTTCATCTCC ATTATTGATG AATAAGGAGT 1560TGCATATTGT CAATTGTTGT GCTCAATATC TGATATTTTG GAATGTACTT TGTACCACTG 1620TGTTTTCAGT TGAAGCTCAT GTGTACTTCT ATAGACTTTG TTTAAATGGT TATGTCATGT 1680TATTT 1685(2)SEQ ID NO：5的信息：
(i)序列特征：
(A)长度：448个氨基酸
(B)类型：氨基酸
(C)拓扑构型：线性
(ii)分子类型：DNA(基因组)
(xi)序列说明：SEQ ID NO：5：Met Ala Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn1 5 10 15His Asp Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr
20 25 30Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu
35 40 45Lys Ser Leu Ala G1y Gln Glu Val Thr Asp Ala Phe Val Ala Phe His
50 55 60Pro Ala Ser Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr65 70 75 80Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu
85 90 95Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile
100 105 110Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met Ser Val
115 120 125Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu Phe Ser Gly
130 135 140Cys Leu Met Gly Phe Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp145 150 155 160Ala Gly His Tyr Met Val Val Ser Asp Ser Arg Leu Asn Lys Phe Met
165 170 175Gly Ile Phe Ala Ala Asn Cys Leu Ser Gly Ile Ser Ile Gly Trp Trp
180 185 190Lys Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr
195 200 205Asp Pro Asp Leu Gln Tyr Ile Pro Phe Leu Val Val Ser Ser Lys Phe
210 215 220Phe Gly Ser Leu Thr Ser His Phe Tyr Glu Lys Arg Leu Thr Phe Asp225 230 235 240Ser Leu Ser Arg Phe Phe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro
245 250 255Ile Met Cys Ala Ala Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met
260 265 270Leu Leu Thr Lys Arg Asn Val Ser Tyr Arg Ala Gln Glu Leu Leu Gly
275 280 285Cys Leu Val Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro
290 295 300Asn Trp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr305 310 315 320Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val
325 330 335Tyr Val Gly Lys Pro Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp
340 345 350Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe His Gly
355 360 365Gly Ser Gln Phe Gln Ile Glu His His Leu Phe Pro Lys Met Pro Arg
370 375 380Cys Asn Leu Arg Lys Ile Ser Pro Tyr Val Ile Glu Leu Cys Lys Lys385 390 395 400His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe Ser Lys Ala Asn Glu Met
405 410 415Thr Leu Arg Thr Leu Arg Asn Thr Ala Leu Gln Ala Arg Asp Ile Thr
420 425 430Lys Pro Leu Pro Lys Asn Leu Val Trp Glu Ala Leu His Thr His Gly
435 440 445(2)SEQ ID NO：6的信息：
(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：线性
(ii)分子类型：DNA(基因组)
(xi)序列说明：SEQ ID NO：6：Trp Ile Gly His Asp Ala Gly His1 5(2)SEQ ID NO：7的信息：
(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线
(ii)分子类型：DNA(基因组) (xi)序列说明：SEQ ID NO：7：Asn Val Gly His Asp Ala Asn His1 5(2)SEQ ID NO：8的信息：(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：8：Val Leu Gly His Asp Cys Gly His1 5(2)SEQ ID NO：9的信息：(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：9：Val Ile Ala His Glu Cys Gly His1 5(2)SEQ ID NO：10的信息：(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：10：Val Ile Gly His Asp Cys Ala His1 5(2)SEQ ID NO：11的信息：(i)序列特征：
(A)长度：8个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：11：Val Val Gly His Asp Cys Gly His1 5(2)SEQ ID NO：12的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：12：His Asn Ala His His1 5(2)SEQ ID NO：13的信息：(i)序列特征：
(A)长度：6个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：13：His Asn Tyr Leu His His1 5(2)SEQ ID NO：14的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：14：His Arg Thr His His1 5(2)SEQ ID NO：15的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：15：His Arg Arg His His1 5(2)SEQ ID NO：16的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：16：His Asp Arg His His1 5(2)SEQ ID NO：17的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：17：His Asp Gln His His1 5(2)SEQ ID NO：18的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：18：His Asp His His His1 5(2)SEQ ID NO：19的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：19：His Asn His His His1 5(2)SEQ ID NO：20的信息：(i)序列特征：
(A)长度：6个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：20：Phe Gln Ile Glu His His1 5(2)SEQ ID NO：21的信息：(i)序列特征：
(A)长度：6个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：21：His Gln Val Thr His His1 5(2)SEQ ID NO：22的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：22：His Val Ile His His1 5(2)SEQ ID NO：23的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组) (xi)序列说明：SEQ ID NO：23：His Val Ala His His1 5(2)SEQ ID NO：24的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：24：His Ile Pro His His1 5(2)SEQ ID NO：25的信息：(i)序列特征：
(A)长度：5个氨基酸
(B)类型：氨基酸
(D)拓扑构型：直线(ii)分子类型：DNA(基因组)(xi)序列说明：SEQ ID NO：25：His Val Pro His His1 5
Claims (28)
1.编码琉璃苣Δ6-去饱和酶的分离的核酸。
2.权利要求1的分离出的核酸，包括SEQ ID NO：4的核苷酸序列。
3.编码SEQ ID NO：5的氨基酸序列的分离出的核苷酸。
4.包含权利要求1-3中任何一个核酸的载体。
5.一种表达载体，包含权利要求1-3任一项的分离的核酸，其中该核酸可操作性地与启动子和任选的能够影响所述分离核酸基因产物表达的终止序列连接。
6.权利要求5的表达载体，其中所说的启动子是Δ6去饱和酶启动子，鱼腥藻属羧化酶启动子，半日花素启动子，大豆球蛋白启动子，napin启动子，来源于CaMV的35S启动子或半日花素组织特异性启动子。
7.权利要求5的表达载体，在此所说的启动子是组成性的或组织特异性的。
8.权利要求5的表达载体，在此所说的终止信号是一个集胞藻属终止信号，胭脂碱合成酶终止信号或种子终止信号。
9.包含权利要求4-8中任一项的载体的细胞。
1O.权利要求9的细胞，其中所说细胞是动物细胞，细菌细胞，植物细胞，或真菌细胞。
11.包含权利要求1-3中任一项的分离的核酸的转基因生物体。
12.包含权利要求4-8中任一项载体的转基因生物体。
13.权利要求11或12的转基因生物体，其中所说的生物体是细菌，真菌，植物或动物。
14.从权利要求10的植物细胞再生而来的植物或所说植物的后代。
15.权利要求14的植物，其中所说的植物是向日葵，大豆，玉米，烟草，花生，胡萝卜或油籽油菜。
16.生产具有增高的γ亚麻酸(GLA)含量的植物的方法，包括：
(a).用权利要求1-3任一项中分离的核酸转化植物细胞；和
(b).从所说植物细胞再生具有增加的GLA含量的植物。
17.生产具有增高的γ亚麻酸(GLA)含量的植物的方法，包括：
(a).用权利要求4-8中任一项的载体转化植物细胞；和
(b).从所说的植物细胞中再生具有增高的GLA含量的植物。
18.权利要求16或17的方法，其中所说的植物是向日葵，大豆，玉米，烟草，花生，胡萝卜和油籽油菜。
19.在GLA缺陷或缺乏的生物体中诱导生产γ亚麻酸(GLA)的方法，包括用权利要求1-3任一项中分离核酸转化所说生物体。
20.在GLA缺陷或缺乏的生物体中诱导生产γ亚麻酸(GLA)的方法，包括用权利要求4-8任一项中的载体转化所说的生物体。
21.在GLA和亚油酸缺陷或缺乏的生物体中诱导生产γ亚麻酸(GLA)的方法，包括用编码琉璃苣Δ6-去饱和酶和编码Δ12-去饱和酶的分离的核酸转化所说的生物体。
22.权利要求21的方法，其中所说的编码Δ6-去饱和酶的分离的核酸包括SEQ ID NO：4的44至1390核苷酸。
23.在γ-亚麻酸缺陷或缺乏的生物体中诱导生产十八碳四烯酸的方法，包括用权利要求1-3中任一项的分离的核酸转化所说的生物体。
24在γ-亚麻酸缺陷或缺乏的生物体中诱导生产十八碳四烯酸的方法，包括用权利要求4-8中任一项的载体转化所说的生物体。
25.权利要求23或24的方法，其中所说的生物体是细菌，真菌，植物或动物。
26.生产具有增强抗冻性植物的方法，包括：
(a).用权利要求1-3中任一项的分离的核酸转化植物细胞；和
(b).从所说的转化的植物细胞中再生具有增强抗冻性的所说植物。
27.生产具有增强抗冻性植物的方法，包括：
(a).用权利要求4-8中任一项的载体转化植物细胞；
(b).从所说转化的植物细胞中再生具有增强抗冻性的所说的植物。
28.权利要求26或27的方法，其中所说植物是向日葵，大豆，玉米，烟草，花生，胡萝卜或者油籽油菜。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/366,779 US5614393A (en) | 1991-10-10 | 1994-12-30 | Production of γ-linolenic acid by a Δ6-desaturase |
| US08/366,779 | 1994-12-30 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1177379A true CN1177379A (zh) | 1998-03-25 |
| CN1117864C CN1117864C (zh) | 2003-08-13 |
Family
ID=23444458
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95197728A Expired - Lifetime CN1117864C (zh) | 1994-12-30 | 1995-12-28 | 通过△6－去饱和酶生产γ亚麻酸 |
Country Status (14)
| Country | Link |
|---|---|
| US (2) | US5614393A (zh) |
| EP (1) | EP0801680B1 (zh) |
| JP (2) | JP4422211B2 (zh) |
| CN (1) | CN1117864C (zh) |
| AR (1) | AR000582A1 (zh) |
| AU (1) | AU707061B2 (zh) |
| BR (1) | BR9510411A (zh) |
| CA (1) | CA2207906C (zh) |
| DE (1) | DE69535064T2 (zh) |
| ES (1) | ES2262146T3 (zh) |
| RO (1) | RO121387B1 (zh) |
| RU (1) | RU2181772C2 (zh) |
| UA (1) | UA61880C2 (zh) |
| WO (1) | WO1996021022A2 (zh) |
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| CN102250928A (zh) * | 2011-04-07 | 2011-11-23 | 中国农业科学院北京畜牧兽医研究所 | Δ-6脂肪酸脱氢酶突变基因及其表达载体和应用 |
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| CN112048460B (zh) * | 2020-08-07 | 2023-06-16 | 中国科学院水生生物研究所 | 工程化细鞘丝藻及其生产gla的应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| DE69535064T2 (de) | 2006-12-07 |
| CN1117864C (zh) | 2003-08-13 |
| EP0801680B1 (en) | 2006-06-14 |
| RU2181772C2 (ru) | 2002-04-27 |
| CA2207906C (en) | 2009-12-01 |
| BR9510411A (pt) | 1998-05-19 |
| AU707061B2 (en) | 1999-07-01 |
| EP0801680A2 (en) | 1997-10-22 |
| US5789220A (en) | 1998-08-04 |
| WO1996021022A3 (en) | 1996-09-12 |
| US5614393A (en) | 1997-03-25 |
| JP4422211B2 (ja) | 2010-02-24 |
| JPH10511848A (ja) | 1998-11-17 |
| WO1996021022A2 (en) | 1996-07-11 |
| UA61880C2 (en) | 2003-12-15 |
| DE69535064D1 (de) | 2006-07-27 |
| JP2007330267A (ja) | 2007-12-27 |
| AU4673596A (en) | 1996-07-24 |
| ES2262146T3 (es) | 2006-11-16 |
| AR000582A1 (es) | 1997-07-10 |
| CA2207906A1 (en) | 1996-07-11 |
| RO121387B1 (ro) | 2007-04-30 |
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