CN1053469C - 利用△6－去饱和酶生产γ亚麻酸 - Google Patents
利用△6－去饱和酶生产γ亚麻酸 Download PDFInfo
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- CN1053469C CN1053469C CN92113085A CN92113085A CN1053469C CN 1053469 C CN1053469 C CN 1053469C CN 92113085 A CN92113085 A CN 92113085A CN 92113085 A CN92113085 A CN 92113085A CN 1053469 C CN1053469 C CN 1053469C
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- desaturase
- nucleic acid
- seq
- isolated nucleic
- acid
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Abstract
亚油酸在Δ6-去饱和酶作用下转变成γ-亚麻酸。本发明涉及一种分离的含有Δ6-去饱和酶基因的核酸。更具体地说，分离的核酸含有Δ6-去饱和酶基因的启动子、编码区和终止区。本发明提供包括与异源调节顺序功能性结合的Δ6-去饱和酶编码区的重组体构建体。本发明的核酸和重组体构建体对在转基因生物体中GLA的生产有用。
Description
亚油酸(18∶2)(LA)经Δ6-去饱和酶的作用转变成γ亚麻酸(18∶3)(GLA)。当Δ6-去饱和酶或者是编码这种酶的核酸被转入到产LA的细胞中后即可产生GLA。本发明提供了含有Δ6-去饱和酶基因的核酸。更具体地说，本发明的核酸包括Δ6-去饱和酶基因的启动子、编码区和终止区。本发明进一步还涉及含有与异源调节顺序进行功能性结合的Δ6-去饱和酶编码区的重组构建体。本发明的核酸和重组体构建体可用在转基因生物体中生产GLA。
不饱和脂肪酸，如亚油酸(C18Δ9，12)和α-亚麻酸(C18Δ9，12，15)是每日食物摄入的必需成分，它不能被脊椎动物自身合成，因为脊椎动物细胞只能在脂肪酸的Δ9位置引入双键，但不能在Δ9双键和脂肪酸链的甲基末端之间引入另外双键。亚油酸和α-亚麻酸是其它产物的前体，所以它们是必需脂肪酸，并且通常是从植物中获取。亚油酸可以由哺乳动物转变为γ-亚麻酸(GLA，C18Δ6，9，12)，再转变为花生四烯酸(20∶4)。花生四烯酸是一种相当重要的脂肪酸，因为它是大多数前列腺素必不可少的前体。
通过转变为GLA和花生四烯酸，亚油酸食品中满足了食物中的GLA和花生四烯酸的需要。然而，已经证明了饱和脂肪酸的消耗和疾病的发生如高胆固醇血症、粥样硬化以及与冠脉疾病易感性相关的化学物质紊乱之间的关系。而消耗不饱和脂肪与血胆固醇的降低有关，患粥样硬化的危险性就减小。GLA饮食的治疗性优点来自于GLA可作为花生四烯酸的前体，继而有助于前列腺素的合成。因此，更多的不饱和的GLA而不是亚油酸将益于健康，但是，在任何商业性生产的谷类植物中实际上都不存在GLA。
亚油酸在Δ6-去饱和酶的作用下转变为GLA。Δ6-去饱和酶具有约359个氨基酸，有一个膜结合区和一个脂肪酸去饱和作用的活化位点。当Δ6-去饱和酶转入一个本身只产生亚油酸而不产生GLA的细胞中时，该细胞就可以产生GLA。本发明通过提供Δ6-去饱和酶的基因编码，产生含有功能性Δ6-去饱和酶并产生GLA的转基因生物体。除了产生大量的GLA外，本发明还提供了新的GLA饮食来源。
本发明涉及一种被分离的Δ6-去饱和酶基因，具体地说，这种被分离的基因含有Δ6-去饱和酶启动子、编码区和终止密码区。
本发明还涉及含有Δ6-去饱和酶启动子、编码区、终止密码区的表达载体。
本发明还涉及包括Δ6-去饱和酶编码区与外源调节区即非源于Δ6-去饱和酶基因的单元进行功能性结合的表达载体。
本发明还提供含有本发明载体的细胞和生物体以及这类生物体的子代。
本发明还提供了被分离的细菌性Δ6-去饱和酶，进一步还涉及被分离的编码细菌性Δ6-去饱和酶的核酸。
本发明提供了产生增高γ-亚麻酸(GLA)含量的植物的方法，通过用本发明的分离的核酸转化植物细胞，以及用上述高GLA含量的植物细胞再生植物。
本发明提供了一种产生耐寒植物的方法。
图1描绘了集胞藻属菌(synechocystis)Δ6-去饱和酶(图1A)和Δ12-去饱和酶(图1B)的推测氨基酸顺序的水疗法图。用实线表示推测的膜距区(membrane spanning regions)。用19个氨基酸残基的窗格大小计算疏水指数(kyte，et al，1982，J.Molec.Biol.157)。
图2表示野生型鱼腥藻属细菌(图2A)和转基因鱼腥藻属细菌(图2B)的气液色谱图。
图3是带有重叠区和亚克隆的粘性质粒cSy75、cSy13和cSy7的简图。cSy75、cSy75-3.5和cSy7的亚克隆的起始点用带斜条的框线表示。还未被活化的限制性位点写在括号内。
图4表示野生型(图A)和转移基因型烟草(图B)的气液色谱图。
本发明提供了一种分离的编码Δ6-去饱和酶的核酸，为了鉴定编码Δ6-去饱和酶的核酸，从能产生GLA的生物体中分离DNA。例如，所说的生物体可以是动物细胞、某些真菌(如被孢霉属)，某些细菌(例如集胞藻属)或某些植物(琉璃苣、月见草属、茶藨子属植物)。基因DNA的分离可以用本领域普通技术人员已知的各种方法进行，例如用Sambrook等人举证的方法(Molecular Cloning：A Laboratory Manual，Cold Spring Harbor，NY.1989)。用物理方法或酶消化作用将分离的DNA裂解，然后再通过参考文献中的(例如Sambrook et al，1989)各种已知方法把裂解片段克隆到合适的载体中，如噬菌体或粘性质粒载体。本文特别设计了含有本发明DNA的表达载体。通过获得的功能性分析结果鉴定编码Δ6-去饱和酶的DNA。含有裂解的DNA片段的载体通过感染、转结合和转染等方式转移到产亚油酸但不产GLA的宿主生物体中。本文所说的“转化”通常指的是将外源DNA掺入到宿主细胞中，将重组DNA导入宿主生物体中的方法是本领域普通技术人员已知的。例如可以参考的文献有Sambrook et al.，1989。这些有机体的GLA的生产(如功能的获得)诸如气液色谱图或其它普通技术人员所熟悉的方法鉴定。
经诱导产生GLA的生物体，亦即通过载体的导入而获得上述功能的生物体被证明为表达了编码Δ6-去饱和酶的DNA，再从生物体中回收所说的DNA。回收的DNA可再次裂解，用表达载体进行克隆，并用上述过程进行功能性评估，以便更为精确地弄清楚编码Δ6-去饱和酶的DNA。
本发明的一个例子是从蓝藻细菌集胞藻属菌(PasteurCulture Collection，(PCC)6803，American Type Culture Collection，(ATCC)27184)中分离随机的DNA，克隆到粘性质粒载体中，并通过转结合作用导入到GLA缺陷的蓝藻细菌鱼腥藻属菌菌株PCC7120，ATCC 27893中。用气相色谱监测从鱼腥藻属菌的亚油酸生成GLA，并分离相应的DNA片段。
用本领域普通技术人员已知的方法，例如Sam brook等人(1989)描述的方法对分离的DNA排序。
根据本发明，已经分离了含有Δ6-去饱和酶基因的DNA。更具体是说，一段含有Δ6-去饱和酶基因的3.588kb的DNA已从蓝藻细菌集胞藻属菌中分离出来.测定3.588kb DNA的核苷酸顺序并示于SEQ ID NO：1中。表明有效编码区的开放读码存在于核苷酸317-1507和核苷酸2002-3081。为了明确负责编码Δ6-去饱和酶的核苷酸，把提供Δ6-去饱和酶活性的3.588kb片段裂解为二个亚片段，其中的每一个亚片段都仅含有一个开放读码。片段ORF1含有核苷酸1-1704，片段ORF2含有核苷酸1705-3588。每一个片段都以向前和逆向两种方向亚克隆到结合的表达载体中(AM542，Wolk et al，1984 Proc.Natl.Acad.Sci.USA 81，1561)，该载体含有一种蓝藻细菌羧化酶启动子。所得到的构建体[即ORF1(F)，ORF1(R)，ORF2(F)和ORF2(R)]通过标准方法(例如参见Wolk，et al，1984，Proc.Natl.Acad.Sci.USA 81，1561)结合到野生型鱼腥藻属菌PCC7120中。在选择性培养基(含有30μg/ml新霉素的标准无机培养基BG11N，参见Rippka et al，1979，J.Gen Microbiol.111，1)生长二周后，从死亡的非结合性细胞的棕色背景中被结合的鱼腥藻属细胞为NeoR绿色菌落。筛选出绿色菌落，并在选择性液体培养基(含有15μg/ml新霉素的BG11N)中生长。用标准方法(例如Dahmer et al，1989，Journal of AmericanOil Chemical Society 66，543)从所得的含有向前和逆向定位的ORF1和ORF2构建体的转结合体中提取脂类，作为对比，同样也从鱼腥藻属菌和集胞藻属菌的野生型培养物中提取脂类。通过气液色谱(GLC)，例如用装有氢火焰电离检测器和毛细管柱的Tracor-560气液色谱分析脂肪酸甲基酯。GLC分析结果示于表1中。
表1：野生型和转基因蓝藻细菌中C18脂肪酸
| 来源 | 18∶0 18∶1 18∶2 γ18∶3 α18∶3 18∶4 |
| 鱼腥藻属菌(野生型)鱼腥藻属菌+ORF1(F)鱼腥藻属菌+ORF1(R)鱼腥藻属菌+ORF2(F)鱼腥藻属菌+ORF2(R)集胞藻属菌(野生型) | + + + - + -+ + + - + -+ + + - + -+ + + + + ++ + + - + -+ + + + - - |
如GLC分析所确定的，当含有以向前方向定位的ORF2的构建体通过转结合，GLA缺陷的鱼腥藻属菌便获得了生产GLA的功能。带有以相对于羧化酶启动子而言相反的方向定位的ORF2或以正或反两个方向定位的ORF1的构建体的转结合体未显示出GLA的生成。这一分析结果表明，位于1884bp片段中的单个开放读码编码Δ6-去饱和酶。1884bp片段示于SEQ.ID NO：3中。这也分别由示于图1(A)和(B)中的Δ6-去饱和酶和Δ12-去饱和酶[Wada et al，1990，Nature，347]之间水疗法图完全相似性所证实。
通过上述功能性分析，或通过用分离编码鱼腥藻属菌Δ6-去饱和酶的核酸作为杂交探针的核酸杂交技术从其它的产GLA生物体鉴定分离的编码Δ6-去饱和酶的核酸。本发明设想的基因组克隆和cDNA克隆的方法是技术人员已知的。杂交探针可以含有公开于SEQ ID NO：1中的完整DNA序列或者是限制性片段或是其它的DNA片段，包括寡核苷酸探针，用交叉杂交来克隆同源基因的方法是本领域普通技术人员已知的，例如可见于Sambrook(1989)和Bcltz et al，1983，Methods inEnzymology 100，266。
通过导入编码Δ6-去饱和酶DNA而获得生产GLA功能的转基因生物体也获得了产十八碳四烯酸(octadecatetraenoicacid)(18：4Δ6，9，12，15的功能。十八碳四烯酸(Octadecate-traenoic acid)正常存在于鱼油和紫草科的一些植物种中(Craig etal，1964，J.Amer.Oil Chem.Soc.41，209-211；Gross et al；1976，Can.J.Plant Sci.56，659-664)。在本发明的转基因生物体中，十八碳四烯酸是在Δ6-去饱和酶的作用下α-亚麻酸进一步去饱和或是在Δ15-去饱和酶作用下GLA进一步去饱和而得到的。
由ORF2编码的359个氨基酸，即编码Δ6-去饱和酶的开放读码用SEQ.ID NO：2表示，本发明进一步设想了编码SEQID NO：2的氨基酸的其它核苷酸顺序。鉴定例如由遗传密码简并性而产生的这类顺序是普通专业技术人员已知的，而且，通过上述得到的功能性分析，本领域的普通技术人员能确定含有编码Δ6-去饱和酶的ORF2的1884bp片段的更次一级片段。
本发明设计了任何Δ6-去饱和酶多肽片段以及保留有将LA转变成GLA活性的核酸。
在本发明的另外一方面，含有1884bp片段的载体或含有Δ6-去饱和酶基因启动子、编码顺序和终止区的更小的片段被转移到一种生物体中，例如蓝藻细菌中，在所说的生物体中，Δ6-去饱和酶启动子和终止区是能发挥功能的。因此，本发明提供了生产重组的Δ6-去饱和酶的生物体。另外一方面，本发明提供了分离的Δ6-去饱和酶，它能通过标准的蛋白质纯化技术(例如可参见Ausubel et al，1987，Current Protocols in MolecularBiology，Green Publishing Associates，New York)从重组生物体中纯化出来。
本发明还提供了含有编码Δ6-去饱和酶的DNA的载体。在各种生物体中构建适合的载体指导表达Δ6-去饱和酶编码顺序是本领域普通技术人员显而易见的。特别优选的是可复制性表达载体，本文所述的可复制性表达载体指的是经过处理的用于调控目的基因即Δ6-去饱和酶基因表达的DNA或RNA的分子。优选的载体是质粒、噬菌体、粘性质粒或病毒。诸如wolk et al.(1984)Proc.Natl.Acad.Sci.USA.1561-1565和Bustos et al(1991)J.Bacteriol.174，7525-7533所述的往复性载体证实与本发明相符。一些文献(Sambrook et al，(1989)；Goeddel，ed.(1990)Methods in Enzymology 185 Academic Press；Perbal 1988，A Practical Guide to Molecular Cloning，John Wiley andSons，Inc.)对能够插入和表达编码所说Δ6-去饱和酶核酸的载体进行了详细论述。这类载体还含有能有效表达编码Δ6-去饱和酶核酸的核酸顺序。能够有效进行基因产物表达的顺序单元包括启动子、增强子成分；上游活化顺序、转录终止信号和多腺苷酸化位点，基本的和组织特异性的启动子本发明都考虑到了。为了转化植物细胞，花椰菜花叶病毒(CaMV)35s启动子和在植物种子突变过程中被调整过的启动子都特别值得感兴趣。所有这类启动子和转录调节成分的单个体或结合体用于本发明的可复制性表达载体，并为本领域普通技术人员所了解。例如Restrepo等人(Plant Cell，2，987，1990)描述过CaMV35S启动子。还设计了经遗传工程处理的和遗传突变的调节顺序。
普通的专业技术人员能够决定适于在特定宿主细胞中进行表达的载体和调节成分。例如，含有来自编码鱼腥藻属菌的羧化酶基因启动子的载体可连接到Δ6-去饱和酶编码区，并进一步可连接到来自集胞藻属菌的终止信号，这些载体在蓝藻细菌中适合Δ6-去饱和酶的表达。本文中的“可连接”(Operablylinked)指的是启动子和终止子顺序有效地发挥调节转录的作用。另一个例子是适于在转基因植物中表达Δ6-去饱和酶的载体能含有一个衍生于半日花素。napin或glycin种子特异性的启动子顺序，该启动子可连接到Δ6-去饱和酶编码区，并进一步可行性连接到种子终止信号或nopaline合成酶终止信号。
本发明特别考虑了将半日花素(helianthinin)调节成分作为指导本发明Δ6-去饱和酶表达的启动子成分，调节成分在申请号为628、354申请日为1991，4，8的美国末决申请中公开，该申请并入本文作为参考。
对本文公开的核苷酸顺序或具有所设计功能的调节成分的修饰也包括在本发明范围内。这类修饰作用包括插入、替代和缺失以及反映遗传密码简并性的特异性替代。
构建这种杂交载体的标准技术是本领域普通技术人员已知的，并可参见文献，如Sambrook et al，1989或大量可随处可得的关于重组DNA技术的实验室手册。多种设计可用于DNA片段的连接，所做的选择取决于DNA片段的末端性质，根据本发明，杂交载体包括便于克隆、表达或加工的其它核苷酸顺序成分，例如编码信号肽的顺序，编码使蛋白质在内质网中滞留所要求的KDEL的顺序，或是编码引导Δ6-去饱和酶进入叶绿体的转运肽的顺序。这些顺序是本领域普通专业人员已知的。例如，Van den Broeck等人(Nature 313，358，1985)已描述过比较理想的转运肽。又如，Michaelis等人(Ann.Rev.Microbiol，36，425，1982)公开了原核的和真核的信号顺序。
本发明的另外一个方面是提供了含有编码本发明的Δ6-去饱和酶DNA的生物体而不是蓝藻细菌。根据本发明的设计，转基因生物体包括细菌、蓝藻细菌、真菌、植物和动物。本发明分离的DNA可以通过现有技术已知的方法，如感染、转染、转化或转结合而导入宿主细胞中。将本发明的DNA转移到上述生物体中的技术是普遍知晓的，可见于参考文献中，如Sambrook etal，1989。
已知多种植物转化方法。根据Horsch等人(Science 227，1229，1985)描述的叶花盘转化一再生方法可将Δ6-去饱和酶基因导入到植物中。其它的转化方法，如原生质体培养(Horschet al，1984，Science 223，496；De Block et al(1984)EMBO J.2，2143，Barton et al，(1983)Cell 32，(1033)也可以应用，且包括于本发明的范围内.在一个优选的实施方案中，用土壤杆菌衍生的载体转化植物。而且，其它的方法可用来将本发明的Δ6-去饱和酶基因插入到植物细胞中。这类可选择的方法包括biolistic方法(Klein et al，1987，Nature 327，70)、电穿孔、化学诱导DNA摄入以及用病毒或花粉作为载体的应用。
当转化的方法需要时，本发明的Δ6-去饱和酶基因可以插入到植物转化载体中，例如Bevan(1984，Nucleic Acids Res.12，8111)描述过的二元载体。植物转化载体可以通过修饰根癌土壤杆菌的天然基因转移系统进行衍生，天然系统包括含有称作T-DNA的大片段的大Ti(肿瘤诱生性)质粒，它可被转移到转化的植物中。Ti质粒的另外一个片段Vir区是负责T-DNA转移的。T-DNA区是由末端重复区接界的。在经修饰的二元载体中，缺失了肿瘤诱导基因，并利用Vir区的功能转移由T-DNA边界顺序接界的外源DNA。T区还含有一个用于抗生素抗性的可选择性标记和一个作为转移的插入顺序的多克隆位点。这种工程菌被称为“去武装”根癌土壤杆菌菌株，并且允许由T-区接界的顺序的有效转化到植物的核基因组中。表面无菌的叶花盘与去武装的含外源DNA的根癌土壤杆菌一起孵育，培养2天，然后转移到含抗生素的培养基中。当培养物在含有合适的抗生素的培养基中生根之后，筛选被转化的嫩芽，再转移到泥土中进行再生。
本发明的另一方面是提供了含有本发明分离的DNA的转基因植物或这些植物的子代，单子叶和双子叶植物都考虑到了。借助上述任何一种植物转化方法分离的编码Δ6-去饱和酶的DNA转化植物细胞。被转化的植物细胞通常是在胼胝体培养物或叶花盘中，通过本领域技术人员已知的方法(例如，Horschet al，1985，Science，227，1129)再生成一个完全的转基因植物在一个优选的实施方案中，转基因植物是向日葵、油籽油菜、玉米、烟草、花生或大豆。因为转基因植物的子代遗传编码Δ6-去饱和酶DNA，得自被转化植物的种子或插技常用于保持转基因的植物系。
本发明进一步提供了生产GLA含量提高了的转基因植物的方法。该方法包括将编码Δ6-去饱和酶的DNA导入缺乏GLA或GLA水平较低但含有LA的植物细胞中，和从转基因细胞再生提高了GLA含量的植物。特别考虑了商业性生产的农作物植物作为转基因生物体，它包括但不限于向日葵、大豆、油籽油菜、玉米、花生和烟草。
本发明进一步提供了生产含有GLA转基因生物体的方法。该方法包括将编码Δ6-去饱和酶DNA导入缺乏GLA或具有低水平GLA但含有LA的生物体中，在另一个实施方案中，该方法包括将含有编码Δ12-去饱和酶和Δ6-去饱和酶DNA的一种或多种表达载体导入缺乏GIA和LA的生物体中。因此，通过表达Δ12-去饱和酶，在缺乏LA和GLA的生物体中诱导LA的产生，然后因Δ6-去饱和酶的表达而产生GLA。通过本领域技术人员已知的重组技术方法(Sambrook，et al，1989)和已公开的Δ12-去饱和酶顺序(Wada et al，1990，Nature 347，200-203)可以构建含有编码Δ12-去饱和酶或是含有编码Δ12-去饱和酶和Δ6-去饱和酶的DNA的表达载体。
此外，根据本发明，已发现SEQ.ID NO：1的核苷酸2002-3081编码蓝藻细菌的Δ12-去饱和酶，据此。该顺序能用于构建有关本题的表达载体。特别考虑于商业性生产的农作物植物的作为转基因生物体，它包括但不限于向日葵、大豆、油籽菜油、玉米、花生和烟草。
本发明进一步涉及一种在植物中诱导抗寒力的方法。寒冷敏感性可能产生于细胞膜中脂质的相变。膜脂中相变温度取决于脂肪酸的不饱和度。因此，通过导入Δ6-去饱和酶使LA转变成GLA以增加不饱和度即可诱生或改善抗寒能力。鉴此，本发明的方法包括将编码Δ6-去饱和酶的DNA导入植物细胞，并从所说的被转化植物细胞再生改善了抗寒力的植物，在一个优选的实施方案中，所说的植物是向日葵、大豆、油籽油菜、玉米、花生或烟草。
下面的实施例将进一步说明本发明。
实施例1
菌株和培养条件
集胞藻属菌、鱼腥藻属菌(PCC7120，ATCC 27893)和聚胞藻属菌(Synechococcus)(PCC 7942，ATCC 33912)置BG11N+培养基(Rippka et al，1979，J.Gen.Microbiol.111，1-61)中在白炽灯照明条件下(60μE·m-2·s-1)于30℃行光能自养性生长。选择粘性质粒和质粒并使之在Maniatis et al，(1982)[Molecular Cloning：A Laboratory Manual，Cold spring Harbor Laboratory，Cold Spring，New York]等人所描述的在标准浓度的含抗生素的LB介质的大肠杆菌菌株DH5α中生长。
实施例2
集胞藻属菌粘性质粒基因组文库的构建
用Sau 3A部分消化得自集胞藻属菌(PCC 6803)的总的基因组DNA，并在蔗糖梯度上进行分离(Ausubel et al，1987，CurrentProtocols in Molecular Biology，Greene Publishing Associates andWiley Inter scinec，New York)。筛选含有30-40kbDNA片段的馏份，并连接到粘性质粒载体的脱磷酸BamHI位点PDUCA7(Buikema et al(1991)J.Bacteriol.173，1879-1885)。如Ausubel等人所述(1987)在体外包装连接的DNA，并包装的噬菌体在含有AVal和E-co4711甲基酶辅助质粒pRL528(Buikema et al.1991)的E.Col-iDH5α中繁殖。随机分离共1152克隆，并单个维持于12个96孔微量滴定板中。
实施例3
鱼腥藻属菌获得表达GLA的功能
鱼腥藻属菌(PCC 7120)是一种花丝状蓝藻细菌，缺乏GLA但含有足够量的生成GLA的前体亚油酸(图2；表2)。实施例2中所述的集胞藻属菌粘性质粒文库结合到鱼腥藻属菌(PCC7120)中，鉴定生产GLA的转结合体。在BG11N+液态介质中，鱼腥藻属菌细胞生长至对数中期，之后在相同介质中悬浮至终浓度约2×108细胞/ml.生长于含氨苄青霉素的LB中的E.Coli Rp4(Burkardt et al，1979，J Gen，Microbiol.114.341-348)对数中期培养物经洗涤后重悬于新鲜的LB培养基中。然后将鱼腥藻属菌与RP4混合，并均匀地分散于含有5％LB的BG11N+平板上。粘性质粒基因组文库经平板印影培养到含50μg/ml卡那霉素和17.5μg/ml氯霉素的LB平板上，继尔将上述培养物切成小片置于含有鱼腥藻属菌和RP4和BG11N+平板上，于30℃孵育24小时后，30μg/ml的新霉素加至平板底层于30℃继续孵育直至出现转结合体。
结合作用完成后分离单个的转结合体，并置于含15μg/ml新霉素的2ml BG11N+液体培养基中生长。如下所述，从野生型培养物和含有10个转结合体库的培养物中制备脂肪酸甲基酯。通过离心收集野生型和转基因型蓝藻细菌培养物。用蒸馏水洗涤二次。按Dahmer等人所述方法(1989，J.Amer.Oil Chem.Soc.66，543-548)从这些培养物中提取脂肪酸甲基酯，并通过气液色谱(GLC)用装有氢火焰电离检测仪和毛细管柱(30m×0.25mm，结合了FSOT Superox II，Alltech Associatelnc.，IL)的Tra-cor-560进行分析。以滞留时间和标样(得自Sigma化学公司)的共同色谱分析来鉴定脂肪酸。平均脂肪酸组成通过各个C18脂肪酸峰值区与内标区的比值归一化确定。
有代表性的GLC图谱示于图2中，还显示了C18脂肪酸甲基酯。通过与已知的脂肪酸甲基酯标准物的洗脱时间进行比较鉴别出各个峰，并用气相色谱质谱光谱测定法进行证实。图2(A)描述了野生型鱼腥藻属菌脂肪酸的GLC分析。箭头表示GLA的迁移时间。图2(B)是带有pAM542+1。8F的鱼腥藻属菌转结合体脂肪酸的GLC图谱。证明了2个产GLA库(从代表250个转结合体的25个库中)能产生GLA。对每个GLA阳性库的单个转结合体进行产GLA的分析，两个来自不同库的独立的转结合体AS13和AS75被鉴定为能表达很高水平的GLA并分别含有粘性质粒cSy13和cSy75(图3)。粘性质粒在约7.5kb长度的区域重叠。cSy75的一个3.5kb NheI片段再克隆入载体pDUCA7中，并转移到鱼腥藻属菌中从而获得GLA表达功能(图2)。
cSy75-3.5的3.5kb NheI片段的两个NheI/Hind III亚片段(1.8和1.7kb)被亚克隆到“pBLUESCRIPT”中(Stratagene)(图3)用于测序。按照Maniatis等人(1982)和Ausubel等人(1987)所述的方法进行标准的分子生物学技术操作。通过用Advaneed DNA Technologies Laboratory(BiologyDepartment，Texas A & M University)合成特殊的低聚核苷酸引物用“SEQUENASE”(United States Biochemical)进行pBS1.8的双链双脱氧测序(Sanger et al.1977，Proc.Natl.Acad.Sci.USA，74，5463-5467)。用Devereux等人(1984，Nucleic Acids Res.12.387-395)描述的GCG(Madison WI)软件进行DNA序列分析。
两个Nhe I/Hind III片段都以相对于蓝藻细菌羧化酶启动子的向前和逆向两个方向转移到结合性表达载体AM542中，再经结合作用导入鱼腥藻属菌。含有向前方向定位的1.8kb片段(AM542-1.8F)的转结合体产生大量的GLA和十八碳四烯酸，(图2，表2)。含有其它构建体(无论是逆向定位的1.8kb片段还是向前和逆向定位的1.7kb片段)的转结合体不能产生可检测水平的GLA(表2)。
图2将从野生型鱼腥藻属菌中提取的C18脂肪酸图谱(图2A)与含有向前方向定位的cSy75-3.5的1.8kb片段的转基因鱼腥藻属菌提取物中的C18脂肪酸图谱(图2B)进行了比较。得自AM542-1.8F的脂肪酸甲基酯的GLC分析显示了一个与可靠的GLA标准物滞留时间一致的峰。经气相色谱质谱光谱测定法进行的有关该峰的分析证实，它的质谱裂解方式与GLA参照样品的相同。改变了多不饱和脂肪酸含量的转基因鱼腥藻属菌在生长速度和形态学方面与野生型鱼腥藻属菌相似。
表2：野生型和转基因蓝藻细菌中
C18脂肪酸的比较
18∶0，硬脂酸；18∶1，油酸；18∶2，亚油酸；18∶3(α)，α-亚麻酸，18∶3(γ)，γ-亚麻酸；18∶4，十八碳四烯酸。
| 菌株 | 脂肪酸 | |||||
| 18∶0 | 18∶1 | 18∶2 | 18∶3(α) | 18∶3(γ) | 18∶4 | |
| 野生型集胞藻属菌(PCC 6803)鱼腥藻属菌(PCC 7120)聚胞藻属(蓝藻)(PCC 7942)鱼腥藻属菌转结合体cSy75cSy75-3.5pAM542-1.8FpAM542-1.8RpAM542-1.7FpAM542-1.7R聚胞藻属菌转结合体pAM854pAM854-Δ12pAM854-Δ6pAM854-Δ6和Δ12 | 13.62.920.63.84.34.27.72.82.827.84.018.242.7 | 4.524.879.424.427.613.923.127.825.472.243.281.825.3 | 54.537.1-22.318.112.138.436.142.3-46.0-19.5 | -35.2-9.13.219.130.833.329.6---- | 27.3--27.940.425.4------16.5 | ---12.56.425.4------- |
实施例5
Δ6-去饱和酶的核苷酸顺序
测定了包括功能性Δ6-去饱和酶基因的cSy75-3.5的1.8kb片段的核苷酸顺序。一个编码359个氨基酸多肽的开放读码被鉴定(图4)。由Kyte-Doolittle(Kyte et al.[1982]J.Mol.Biol.157.105-132水疗分析鉴定疏水氨基酸的两个区可代表转换膜区(transmembrane domains)(图1A)，并且Δ6-去饱和酶基因与Δ12-去饱和酶基因(图1B，Wada et al.)和Δ9去饱和酶基因(Thiede et al.)[1986]J.Biol Chem.261，13230-13235)的水疗图相似。
但是，集胞藻属菌的Δ6-去饱和酶和Δ12-去饱和酶之间的顺序相似性在核苷酸水平小于40％，在氨基酸水平约小于18％。
实施例6
蓝藻细菌的Δ6-去饱和酶转移到烟草中
将蓝藻细菌的Δ6-去饱和酶基因转移到植物表达载体中，再通过土壤杆菌介导的基因转移技术转移到烟草中。为了保证被转移的去饱和酶在叶及发育的种子中合适地表达及内质网和叶绿素中去饱和基因产物的确定。安装带有集胞藻属菌去饱和酶开放读码(ORF)的各种表达盒。这些盒的成分包括：(i)35S启动子或衍生于向日葵半日花素基因的种子特异性启动子，它分别启动Δ6-去饱和酶基因在所有植物组织中表达或仅在发育的种子中表达；(ii)一种推测的信号肽，它来自胡萝卜延伸蛋白基因或向日葵半日花素基因，引导新合成的Δ6-去饱和酶进入ER；(iii)位于Δ6-去饱和酶ORF羧基末端的ER腔滞留信号顺序(KDEL)；(iv)引导Δ6-去饱和酶进入叶绿体的理想转运肽。35S启动子是Restrepo等人(1990)所述质粒pRTL 2衍生物。Vande Broeck等人(1985)描述了理想的转移肽顺序。Chen等人(1985，EMBO J.9，2145)描述了胡萝卜延伸蛋白信号肽。
生产含有嵌合蓝藻细菌性去饱和酶基因的转基因烟草植物，所说的嵌合体由融合到内质网滞留顺序(KDEL)中的集胞藻属菌Δ6-去饱和酶基因和由CaMv 35S启动子启动的延伸蛋白信号肽组成。转基因烟草基因组DNA的PCR扩增反应表明Δ6去饱和酶基因已被掺入到烟草基因组中，从这些转基因烟草植物叶中提取脂肪酸甲基酯，并用气液色谱(GLC)进行分析。这些转基因烟草集聚了大量的GLA(图4)。图4表示通过GLC确定的脂肪酸甲基酯。与已知的脂肪酸甲基酯标准品的洗脱时间进行比较鉴定出各个峰。因此，涉及脂肪酸代谢的蓝藻细菌的基因可用于生产改变了脂肪酸组成的转基因植物。
序列表(1)概况(i)申请人：Thomas，Terry L.
Reddy，Avutu S.
Nuccie，Michael
Freyssinet，Georges L.(ii)发明名称：利用Δ6-去饱和酶生产γ亚麻酸(iii)顺序数目：3(iv)通信地址：
(A)地址：Scully，Scott，Murphy & Presser
(B)街道：400 Garden City Plaza
(C)城市：Garden市
(D)州：纽约
(E)国家：美国
(F)邮政编码：1(V)计算机可读形式：
(A)介质类型：Floppy盘
(B)计算机：IBM PC可容性
(C)操作系统：PC-DOS/MS-DOS
(D)软件：Patent In Release #1.0 Version#1.25(Vi)目前的申请资料：
(A)申请号：待定
(B)申请日：1992.1.8
(C)分类(Viii)代理人情况
(A)姓名：McNulty，Willian E.
(B)登记号：22，606
(C)参考/标记号：8383Z(ix)电信信息：
(A)电话：(516)742-4343
(B)传真：(516)742-4366
(C)电传：230 901 SANS UR(2)SEQ. ID NO：1(i)顺序特征：
(A)长度：3588bp
(B)类型：核酸
(C)链股数：双
(D)拓扑学：线性(ii)分子类型：DNA(基因组的)(ix)性质：
(A)名称/关键：CDS
(B)定位：2002..3081(xi)顺序描述：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 Ala 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(i)顺序特征：
(A)长度：359个氨基酸
(B)类型：氨基酸
(D)拓扑学：线性(ii)分子类型：蛋白质(xi)顺序描述：SEQ ID NO：2Met 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(i)顺序特征：
(A)长度：1884bp
(B)类型：核酸
(C)链股数：双
(D)拓扑学：线性(ii)分子类型：DNA(基因组的)(xi)顺序描述：SEQ ID NO：3AGCTTCACTT 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
Claims (13)
1.一种在γ-亚麻酸缺陷或缺乏的细菌或植物中诱导γ-亚麻酸产生的方法，它包括用一种SEQ ID No：1或SEQ ID No：3的分离的核酸，或者用一种具有一个或多个插入、缺失或取代的碱基、同时保持所述分离核酸的亚麻酸产生功能的SEQ ID No：1或SEQ ID No：3的核酸转化所述细菌或植物。
2.一种在γ-亚麻酸缺陷或缺乏的细菌或植物中诱导γ-亚麻酸产生的方法，它包括用一种表达载体转化所述细菌或植物，所述表达载体包含一种SEQ ID No：1或SEQ ID No：3的分离的核酸，或者一种具有一个或多个插入、缺失或取代的碱基、同时保持所述分离核酸的亚麻酸产生功能的SEQ ID No：1或SEQ ID No：3的核酸。
3.一种产生高γ-亚麻酸含量的植物的方法，它包括：
(a)用一种表达载体转化植物细胞，所述表达载体含有一种SEQID No：1或SEQ ID No：3的分离的核酸或者一种具有一个或多个插入、缺失或取代的碱基、同时保持所述分离核酸的亚麻酸产生功能的SEQ ID No：1或SEQ ID No：3的核酸，其中所述核酸可操作地与能够影响所述分离核酸的基因产物表达的启动子和终止信号相连接；和
(b)由所述植物细胞再生具有高亚麻酸含量的植物。
4.权利要求3的方法，其中所述植物是烟草。
5.权利要求1的方法，其中所述分离的核酸包含在一种穿梭载体中，该载体具有整合外DNA到聚胞藻属(蓝藻属)(synechocystis)的基因组所必需的顺序。
6.权利要求5的方法，其中所述穿梭载体是pAM854Δ6。
7.权利要求5的方法，其中所述穿梭载体是pAM854Δ6&Δ12。
8.权利要求2的方法，其中所述表达载体是AM542-1.8F。
9.权利要求2的方法，其中所述表达载体是pAM854-6。
10.权利要求1或2的方法，其中所述细菌是鱼腥藻属菌(Anabaena)。
11.一种在γ-亚麻酸和亚油酸缺陷或缺乏的细菌中诱导γ-亚麻酸产生的方法，它包括用一种编码蓝藻细菌的Δ6-去饱和酶的分离的核酸和一种编码Δ12-去饱和酶的分离的核酸转化所说的细菌。
12.一种在γ-亚麻酸和亚油酸缺陷或缺乏的细菌中诱导γ-亚麻酸产生的方法，它包括用至少一种表达载体转化所说的细菌，所述表达载体含有编码蓝藻细菌的Δ6-去饱和酶的分离的核酸和编码Δ12-去饱和酶的分离的核酸。
13.权利要求11或12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：1的2002-3081核苷酸。
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| US77447591A | 1991-10-10 | 1991-10-10 | |
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| US81791992A | 1992-01-08 | 1992-01-08 | |
| US817,919 | 1992-01-08 |
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| CN97104566A Division CN1121499C (zh) | 1991-10-10 | 1997-03-21 | 诱导产生十八碳四烯酸的方法 |
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| CN92113085A Expired - Lifetime CN1053469C (zh) | 1991-10-10 | 1992-10-10 | 利用△6－去饱和酶生产γ亚麻酸 |
| CN97104566A Expired - Lifetime CN1121499C (zh) | 1991-10-10 | 1997-03-21 | 诱导产生十八碳四烯酸的方法 |
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| US5968809A (en) * | 1997-04-11 | 1999-10-19 | Abbot Laboratories | Methods and compositions for synthesis of long chain poly-unsaturated fatty acids |
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