CN102597242B - 新脂肪酸延伸组合物及其用途 - Google Patents
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Abstract
本发明提供编码新脂肪酸nECR的分离的核酸分子。本发明也提供含有nECR核酸分子的重组表达载体、已经引入了表达载体的宿主细胞,以及用于大规模产生长链多不饱和脂肪酸(LCPUFA),如ARA、ERA和DHA的方法。
Description
本发明主要涉及脂肪酸的重组产生领域。其提供编码新脂肪酸脱水酶/烯酰-CoA还原酶(nECR)家族成员的核酸分子。本发明也提供含有nECR核酸分子的重组表达载体、已经引入了表达载体的宿主细胞,以及用于大规模产生长链多不饱和脂肪酸(LCPUFA),如ARA、ERA和DHA的方法。
脂肪酸是具有长链碳氢化合物侧基的羧酸,其在许多生物学过程中发挥着重要的作用。在自然界中,很少发现脂肪酸是游离的,且相反,其以酯化形式存在,作为脂质的主要组分。因此,脂质/脂肪酸是能量的来源(例如,b-氧化)。此外,脂质/脂肪酸是细胞膜的组成部分,并因此对于处理生物学或生物化学信息是必不可少的。
可以将脂肪酸分为两组:单碳键形成的饱和脂肪酸和含有一个或多个顺式构型的碳双键的不饱和脂肪酸。不饱和脂肪酸通过末端去饱和酶产生,所述末端去饱和酶属于非血红素铁酶种类。这些酶的每一种都是电子传递系统的一部分,所述电子传递系统含有两种其他的蛋白质,命名为细胞色素b5和NADH-细胞色素b5还原酶。具体而言,此类酶例如,通过催化脂肪酸的氧依赖性脱氢作用,催化脂肪酸分子的碳原子之间双键的形成(Sperling等人,2003)。人类和其他哺乳动物具有有限的去饱和酶谱,所述去饱和酶对于在不饱和脂肪酸中形成特殊的双键是必需的,因此,对于合成必需脂肪酸,如长链多不饱和脂肪酸(LCPUFA)具有有限的能力。因而,人类不得不通过他们的饮食吸收一些脂肪酸。此类必需脂肪酸包括,例如亚油酸(C18:2),亚麻酸(C18:3)。相反,昆虫、微生物和植物能合成更多种类的不饱和脂肪酸及其衍生物。事实上,脂肪酸的生物合成是植物和微生物的主要活性。
长链多不饱和脂肪酸(LCPUFA)例如二十二碳六烯酸(DHA,22:6(4,7,10,13,16,19))在哺乳动物中是多种组织和器官(神经、视网膜、脑和免疫细胞)的细胞膜的必需组分。例如,在脑磷脂中,超过30%的脂肪酸是22:6(n-3)和20:4(n-6)(Crawford,M.A.,等人,(1997)Am.J.Clin.Nutr.66:1032S-1041S)。在视网膜中,DHA在视杆外段(rodoutersegment)(感光细胞的光敏感部分)中占总脂肪酸的60%以上(Giusto,N.M.,等人(2000)Prog.LipidRes.39:315-391)。临床研究显示,DHA对于婴儿脑的生长和发育,和对于成人中正常脑功能的维持是必需的(Martinetz,M.(1992)J.Pediatr.120:S129-S138)。DHA也对参与信号转导过程、视紫红质活化和视杆和视锥发育的光受体功能具有显著的影响(Giusto,N.M.,等人(2000)Prog.LipidRes.39:315-391)。此外,也发现DHA对疾病如高血压、关节炎、动脉硬化、抑郁、血栓形成和癌症具有一些积极的影响(Horrocks,L.A.和Yeo,Y.K.(1999)Pharmacol.Res.40:211-215)。因此,合适的饮食供应脂肪酸对于人类健康是重要的。由于婴儿、幼童和老人不能有效地合成此类脂肪酸,所以对于这些个体从饮食中足够地摄取这些脂肪酸是特别重要的(Spector,A.A.(1999)Lipids34:S1-S3)。
目前,DHA的主要来源是来自鱼和藻类的油。鱼油是这种脂肪酸的主要和常规来源,然而,其通常在销售时被氧化。另外,特别是由于鱼类种群缩减,鱼油的供应也变化不一。此外,由于提取的产量低和花费高,藻类来源的油比较昂贵。
EPA和ARA都是必需脂肪酸。它们形成了用于人类和动物的食物和饲料组分的独特种类。EPA属于在酰基链中具有5个双键的n-3系列。EPA见于海产品中,并在来自北大西洋的油质鱼中含量丰富。ARA属于具有4个双键的n-6系列。在ω-3位置中缺失的双键赋予ARA不同于EPA中所观察到的特性的特性。产生自AA的类二十烷酸具有强的炎症性和血小板聚集特性,然而那些衍生自EPA的类二十烷酸具有抗炎症和抗血小板聚集特性。ARA可以获自一些食物如肉、鱼和蛋,但浓度较低。
γ-亚麻酸(GLA)是在哺乳动物中发现的另一种必需脂肪酸。GLA是极长链n-6脂肪酸和多种活性分子的代谢中间物。在哺乳动物中,长链多不饱和脂肪酸的形成受Δ6去饱和作用的限速。许多生理和病理状况如衰老、应激、糖尿病、湿疹和一些传染病都显示出降低Δ6去饱和作用的步骤。此外,氧化和与某些疾病,如癌症或炎症相关的快速细胞分裂可以容易地分解代谢GLA。因此,饮食补充GLA可以降低这些疾病的风险。临床研究已经显示,饮食补充GLA可以有效地治疗一些病理疾病如特应性湿疹、经前期综合征、糖尿病、高胆固醇血症,以及炎症和心血管疾病。
尽管生物技术提供了用于产生特定脂肪酸的诱人途径,但当前技术还不能提供用于大规模生产不饱和脂肪酸的有效方法。因此,对于改进并有效的产生不饱和脂肪酸,例如DHA、EPA和ARA的方法,还存在需求。
因此,本发明涉及包含这样的核酸序列的多核苷酸,所述核酸序列选自:
a)具有SEQIDNO:1或3显示的核苷酸序列的核酸序列;
b)编码这样的多肽的核酸序列,所述多肽具有SEQIDNO:2或4显示的氨基酸序列;
c)与a)或b)的核酸序列具有至少50%同一性的核酸序列,其中所述核酸序列编码具有脱水酶/烯酰-CoA还原酶(nECR)活性的多肽;
d)编码具有nECR活性并与a)至c)任意之一的氨基酸序列具有至少50%同一性的氨基酸序列的多肽的核酸序列;和
e)能在严格条件下与a)至d)任意之一杂交的核酸序列,其中所述核酸序列编码具有nECR活性的多肽。
根据本发明,术语“多核苷酸”用于指包含这样的核酸序列的多核苷酸,所述核酸序列编码具有脱水酶/烯酰-CoA还原酶(nECR)活性的多肽。优选地,由本发明的多核苷酸编码的具有nECR活性的多肽,当在植物中表达时应当能增加例如,种子油或者整株植物或其部分中PUFA和特别是LCPUFA的量。当与产LCPUFA的转基因对照植物比较时,该增加优选地是统计学显著的,所述产LCPUFA的转基因对照植物表达极小集(minimalset)的对于LCPUFA合成所需的去饱和酶和延伸酶,但是并不表达本发明的多核苷酸。可以通过本领域熟知的统计学检验,包括例如Student′st-检验测定增加是否显著。更优选地,与所述对照比较,增加是含LCPUFA的甘油三酯的量的至少5%、至少10%、至少15%、至少20%或至少30%的增加。优选地,前面提及的LCPUFA是具有C-20、C-22或C24脂肪酸体的多不饱和脂肪酸,更优选是ARA、EPA或DHA。在所附实施例中描述了用于测量前述活性的合适测定法。
如本文中所用,术语“nECR活性”或“脱水酶/烯酰-CoA还原酶活性”指烯酰-CoA还原酶和脱水酶的组合活性,即该酶具有组合的活性,能从3-羟基-酰基-CoA中去除羟基,并还原所形成的双键(作为脂肪酸的延伸过程的一部分)。在四个步骤中催化脂肪酸延伸,由四种酶代表:KCS(酮基-酰基-CoA合酶)、KCR(酮基-酰基-CoA还原酶)、DH(脱水酶)、和ECR(烯酰-CoA还原酶)。在第一个步骤中,脂肪酸-CoA酯与丙二酰-CoA缩合产生酮-酰基-CoA中间物(其通过两个碳原子延伸)和CO2。然后KCR将中间物的酮基团还原成羟基基团。在接下来的步骤中,DH切割羟基基团(产生H2O),形成酰基-2-en-CoA酯(Δ-2-烯酰-CoA)。在最终步骤中,ECR还原位置2、3处的双键,形成延伸的酰基-CoA酯(Buchanan,Gruissem,Jones(2000)Biochemistry&Molecularbiologyofplants,AmericanSocietyofPlantPhysiologists)。在产生本发明的研究中,提供了天然存在的针对LCPUFA具有优良催化活性和特异性的DH和ECR的融合物。
更优选地,多核苷酸具有如在SEQIDNO:1或3中所示的核酸序列的多核苷酸,所述核酸序列编码具有如在SEQIDNO:2或4中所示的氨基酸序列或其变体,优选地显示出nECR活性的多肽。
根据本发明,如上所述的编码具有nECR活性的多肽的多核苷酸优选地获自假微型海链藻(Thalassiosirapseudonana)或三角褐指藻(Phaeodactylumtricornutum)。然而,可以从其他物种中鉴定直向同源物、旁系同源物或者其他同源物。优选地,它们获自植物如藻类,例如等鞭金藻属(Isochrysis)、Mantoniella、Ostreococcus或Crypthecodinium,藻类/硅藻类如褐指藻属(Phaeodactylum)、海链藻属(Thalassiosira)或破囊壶菌属(Thraustochytrium)、藓类植物如剑叶藓属(Physcomitrella)、角齿藓属(Ceratodon)或者高等植物如报春花科(Primulaceae)如石栗(Aleuritia)、Calendulastellata、Osteospermumspinescens或者Osteospermumhyoseroides,微生物如真菌,例如曲霉属(Aspergillus)、疫霉属(Phytophthora)、虫霉属(Entomophthora)、毛霉属(Mucor)或者被孢霉属(Mortierella),细菌如Shewanella,酵母或动物。优选的动物是线虫如新杆状线虫(Caenorhabditis),昆虫或者脊椎动物。在脊椎动物中,该核酸分子可以优选地来源于真骨类(Euteleostomi)、辐鳍鱼纲(Actinopterygii);新鳍类(Neopterygii);真骨鱼类(Teleostei);真真骨鱼(Euteleostei)、原棘鳍总目(Protacanthopterygii)、鲑目(Salmoniformes);鲑科(Salmonidae)或大麻哈鱼属(Oncorhynchus),更优选地来源鲑目,最优选地,鲑科,如鲑属(Salmo),例如来自下述的属和种:虹鳟(Oncorhynchusmykiss)、Truttatrutta或河鲑(Salmotruttafario)。此外,该核酸分子可以获自硅藻类如海链藻属(Thallasiosira)或褐指藻属。
因此,根据本发明所用的术语“多核苷酸”另外涵盖了上述特定多核苷酸的变体,其代表了本发明多核苷酸的直向同源物、旁系同源物或者其他同源物。此外,本发明多核苷酸的变体也包括人工改造的突变蛋白。所述突变蛋白包括,例如通过诱变技术产生的并且显示出改进的或者改变的底物特异性的酶、或者密码子优化的多核苷酸。多核苷酸变体优选地包含这样的核酸序列,所述核酸序列的特征在于,通过至少一个核苷酸取代、添加和/或缺失,其序列可以衍生自前述SEQIDNO:1或3的任意之一显示的特定核酸序列或者通过编码具有如在SEQIDNO:2或4的任意之一中所示的氨基酸序列的多肽的多核苷酸衍生,从而,变体核酸序列应当仍编码具有如上所指定的nECR活性的多肽。变体还包括这样的多核苷酸,其包含能与上述特定核酸序列杂交的核酸序列,优选在严格条件下能与上述特定核酸序列杂交的核酸序列。这些严格条件是技术人员已知的,并可见于CurrentProtocolsinMolecularBiology,JohnWiley&Sons,N.Y.(1989),6.3.1-6.3.6。严格杂交条件的优选实例是在约45℃的6x氯化钠/柠檬酸钠(=SSC)中杂交,然后在50-65℃下在0.2xSSC,0.1%SDS中进行一次或多次洗涤步骤。技术人员知道根据核酸类型和例如当存在有机溶剂时,在温度和缓冲液浓度方面,杂交条件中是不同的。在“标准杂交条件”下,在浓度为0.1至5×SSC(pH7.2)的水性缓冲溶液中,根据核酸类型,杂交温度在42℃和58℃之间。如果上述缓冲液中存在有机溶剂,例如50%甲酰胺,则标准条件下的杂交温度是大约42℃。对于DNA:DNA杂交体,杂交条件优选是0.1×SSC和20℃至45℃,优选在30℃至45℃之间。对于DNA:RNA杂交体,杂交条件优选是0.1×SSC和30℃至55℃,优选在45℃至55℃之间。上述杂交温度是针对例如长度约100bp(=碱基对),G+C含量为50%的核酸,在缺少甲酰胺的情况下确定的。技术人员在教科书帮助下知道如何确定需要的杂交条件,例如上述的,或者来自以下教科书:Sambrook等人,MolecularCloning,ColdSpringHarborLaboratory,1989;Hames和Higgins(编著)1985,“NucleicAcidsHybridization:APracticalApproach”,IRLPressatOxfordUniversityPress,Oxford;Brown(编著)1991,“EssentialMolecularBiology:APracticalApproach”,IRLPressatOxfordUniversityPress,Oxford。备选地,通过基于PCR的技术如基于混合的寡核苷酸引物的DNA扩增,即使用针对本发明多肽的保守结构域的简并引物,可以获得多核苷酸变体。本发明多肽的保守结构域可以通过序列比较本发明多核苷酸的核苷酸序列或者多肽的氨基酸序列来鉴定。在所附实施例中描述了适用作为PCR引物的寡核苷酸以及合适的PCR条件。对于模板,可以使用来自细菌、真菌、植物或动物的DNA或cDNA。此外,变体包括这样的多核苷酸,所述多核苷酸包含与SEQIDNO:1或3的任意之一中所示的核酸序列具有至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%同一性的核酸序列,优选地,其编码保留了如上所指定的nECR活性的多肽。此外,还包括这样的多核苷酸,所述多核苷酸包含编码具有与SEQIDNO:2或4的任意之一中所示的氨基酸序列具有至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%同一性的氨基酸序列的多肽的核酸序列,其中所述多肽,优选地其保留了如上所指定的nECR活性。优选地,在整个氨基酸或核酸序列区计算百分比同一性值。对于比较不同的序列,技术人员可以得到一系列基于多种算法的程序。在一个优选的实施方案中,使用已经整合到EMBOSS软件包中的needle程序(EMBOSS:TheEuropeanMolecularBiologyOpenSoftwareSuite,Rice,P.,Longden,I.,和Bleasby,A,TrendsinGenetics16(6),276-277,2000)中的Needleman和Wunsch算法(Needleman1970,J.Mol.Biol.48;443-453),使用用于远距离相关(distantlyrelated)蛋白质的BLOSUM45或PAM250评分矩阵或者用于密切相关蛋白质的BLOSUM62或PAM160评分矩阵,以及16、14、12、10、8、6或4的空位开放罚分和0.5、1、2、3、4、5或6的空位延伸罚分测定两个氨基酸序列之间的百分比同一性。可以在http://emboss.sourceforge.net发现EMBOSS包的本地安装以及与WEB-Services连接的指导。使用needle程序用于比对两个氨基酸序列的参数的优选非限制性实例是默认参数,包括EBLOSUM62评分矩阵、10的空位开放罚分和0.5的空位延伸罚分。又在另一优选的实施方案中,使用EMBOSS软件包中的needle程序(EMBOSS:TheEuropeanMolecularBiologyOpenSoftwareSuite,Rice,P.,Longden,I.,和Bleasby,A,TrendsinGenetics16(6),276-277,2000),使用EDNAFULL评分矩阵和16、14、12、10、8、6或4的空位开放罚分和0.5、1、2、3、4、5或6的空位延伸罚分测定两个核苷酸序列之间的百分比同一性。使用needle程序,同时用于比对两个序列的参数的优选的、非限制性实例是默认参数,包括EDNAFULL评分矩阵、10的空位开放罚分和0.5的空位延伸罚分。可以将本发明的核酸和蛋白质序列另外地用作为“查询序列”以对公共数据库进行检索,以例如鉴定其他家族成员或者相关序列。可以使用Altschul等人(Altschul1990,J.Mol.Biol.215:403-10)的BLAST系列程序(版本2.2)进行此类检索。使用本发明的nECR核酸序列作为查询序列,可以进行BLAST,使用默认参数的BLASTn、BLASTx或tBLASTx程序,以获得与本发明的nECR序列同源的核苷酸序列(BLASTn、tBLASTx)或氨基酸序列(BLASTx)。使用本发明的nECR蛋白质序列作为查询序列可以进行BLAST,使用默认参数的BLASTp或tBLASTn程序,以获得与本发明的nECR序列同源的氨基酸序列(BLASTp)或核酸序列(tBLASTn)。为了获得用于比较目的的空位比对,如在Altschul等人(Altschul1997,NucleicAcidsRes.25(17):3389-3402)中所述,可以应用使用默认参数的GappedBLAST。
表1:用于多种BLAST程序的查询和命中序列的序列类型的关系
输入查询序列 | 转换的查询 | 算法 | 转换的命中 | 实际数据库 |
DNA | BLASTn | DNA | ||
PRT | BLASTp | PRT | ||
DNA | PRT | BLASTx | PRT | |
PRT | tBLASTn | PRT | DNA | |
DNA | PRT | tBLASTx | PRT | DNA |
本发明的多核苷酸也涵盖包含任一上述核酸序列的片段的多核苷酸。该片段应当编码仍具有如上所指定的nECR活性的多肽。因此,该多肽可以包含赋予所述生物学活性的本发明多肽的结构域或者由上市结构域组成。本文中所指的片段,优选地包含上述核酸序列的任意之一的至少50、至少100、至少250或至少500个连续的核苷酸,或者编码包含上述氨基酸序列任意之一的至少20、至少30、至少50、至少80、至少100或至少150个连续氨基酸的氨基酸序列。
上述变体多核苷酸或片段,优选地编码了这样的多肽,所述多肽保留了显著程度的nECR活性,优选是至少10%、至少20%、至少30%、至少40%、至少50%、至少60%、至少70%、至少80%或至少90%的nECR活性,所述nECR活性由SEQIDNO:2或4的任意之一中所示的任一多肽显示。可以如在所附实施例中所述进行活性的测试。
本发明的多核苷酸基本上由上述核酸序列组成或者包含上述核酸序列。因此,它们也可以包含另外的核酸序列。优选地,除可读框外,本发明的多核苷酸可以另外包含在编码基因区的3’和5’末端的非翻译序列:编码区5’末端的上游序列的至少500,优选200,更优选100个核苷酸和编码基因区3’末端的下游序列的至少100,优选50,更优选20个核苷酸。此外,本发明的多核苷酸可以编码融合蛋白质,其中该融合蛋白质的一部分是由上述核苷酸序列编码的多肽。该融合蛋白还可以包含作为另一部分的脂肪酸或者PUFA生物合成通路中的其他酶,用于监测表达的多肽(例如,绿色、黄色、蓝色或红色荧光蛋白质、碱性磷酸酶和其他)或者所谓的“标签”,所述标签可以用作为可检测标记或者作为用于纯化目的的辅助措施。用于不同目的的标签是本领域熟知的,并包含FLAG标签、6-组氨酸标签、MYC标签和其它。
也可以将本发明的多核苷酸优选地作为分离的多核苷酸(即纯化或者至少从其天然背景例如其天然基因座中分离出来)或者以遗传修饰或者外源(即人工)操纵的形式提供。分离的多核苷酸可以例如包含少于约5kb、4kb、3kb、2kb、1kb、0.5kb或0.1kb的核苷酸序列,所述核苷酸序列天然地位于该核酸分子所来源的细胞的基因组DNA中该核酸分子的侧翼。该多核苷酸优选地以双链或单链分子形式提供。应当理解,本发明所提及的本发明的任一前述多核苷酸也指前述特定序列或其变体的互补链或反向互补链。该多核苷酸涵盖DNA,包括cDNA和基因组DNA或者RNA多核苷酸。
然而,本发明也涉及衍生自本发明的多核苷酸并能干扰本发明多核苷酸的转录或翻译的多核苷酸变体。此类变体多核苷酸包括反义核酸、核酶、siRNA分子、吗啉代核酸(磷酰二胺吗啉代寡核苷酸(phosphorodiamidatemorpholinooligos))、三股螺旋形式的寡核苷酸、抑制性寡核苷酸或者microRNA分子,由于存在互补的或基本上互补的序列,所述全部变体多核苷酸都可以特异识别本发明的多核苷酸。这些技术是本领域技术人员熟知的。基于本发明多核苷酸的结构,可以容易地设计前述种类的合适变体多核苷酸。
此外,也包含化学修饰的多核苷酸,包括天然存在的经修饰的多核苷酸如糖基化或者甲基化的多核苷酸或者人工修饰的多核苷酸如生物素化的多核苷酸。
在产生本发明的研究中,有利地,其中鉴定的多核苷酸编码来自假微型海链藻和三角褐指藻的脱水酶。具体而言,已经鉴定了假微型海链藻和三角褐指藻脱水酶/烯酰-CoA还原酶nECR[nECR(Tp)和nECR(Pt)]。每一这些nECR都能去除3-羟基-酰基-CoA的羟基并还原形成的双键(作为用于脂肪酸延伸过程的一部分)。例如,发现在不能延伸脂肪酸的酿酒酵母(Saccharomycescerevisae)突变体中表达nECR(Tp)和nECR(Pt)可以恢复延伸过程。本发明的多核苷酸特别适合于LCPUFA和特别是ARA、EPA和/或DHA的重组生产。
在本发明多核苷酸的优选实施方案中,所述多核苷酸另外包含与所述核酸序列有效连接的表达调控序列。
如本文中所用,术语“表达调控序列”指能支配,即起始和控制目的核酸序列(在当前情况下为上述核酸序列)的转录的核酸序列。该序列通常包含启动子或者启动子和增强子序列的组合或由其组成。多核苷酸的表达优选地包含核酸分子转录成可翻译的mRNA。其他调控元件包括转录以及翻译增强子。以下启动子和表达控制序列可以优选地用于根据本发明的表达载体。cos、tac、trp、tet、trp-tet、lpp、lac、lpp-lac、lacIq、T7、T5、T3、gal、trc、ara、SP6、λ-PR或λ-PL启动子优选地用于革兰氏阴性菌。对于革兰氏阳性菌,可以使用启动子amy和SPO2。对于酵母或真菌,优选地使用ADC1、AOX1r、GAL1、MFα、AC、P-60、CYC1、GAPDH、TEF、rp28、ADH。对于动物细胞或生物表达,优选地使用启动子CMV-、SV40-、RSV-启动子(劳氏肉瘤病毒)、CMV-增强子、SV40-增强子。对于植物,启动子优选CaMV/35S(Franck1980,Cell21:285-294)、PRP1(Ward1993,Plant.Mol.Biol.22)、SSU、OCS、lib4、usp、STLS1、B33、nos或者泛素或菜豆蛋白启动子。在本上下文中,优选的还有诱导型启动子,例如描述于下述现有技术中的启动子:EP0388186A1(即苯磺酰胺-诱导型启动子)、Gatz1992,PlantJ.2:397-404(即四环素-诱导型启动子)、EP0335528A1(即脱落酸-诱导型启动子)或WO93/21334(即乙醇-或环己烯醇-诱导型启动子)。其它合适的植物启动子是来自马铃薯的细胞质FBP酶启动子或ST-LSI启动子(Stockhaus1989,EMBOJ.8,2445)、来自大豆的磷酸核糖焦磷酸酰氨基转移酶启动子(Genbank登录号U87999)或在EP0249676A1中所描述的结节特异性启动子。特别优选的启动子是能在参与脂肪酸生物合成的组织中表达的启动子。根据实践,特别优选的是种子特异性启动子,例如USP启动子,以及其它启动子如LeB4、DC3、菜豆蛋白或油菜籽蛋白启动子。其它特别优选的启动子是种子特异性启动子,可用于单子叶或双子叶植物,并描述在US5,608,152(油菜的油菜籽蛋白启动子)、WO98/45461(拟南芥的油质蛋白启动子)、US5,504,200(菜豆(Phaseolusvulgaris)的菜豆蛋白启动子)、WO91/13980(芸苔的Bce4启动子)、Baeumlein等人,PlantJ.,2,2,1992:233-239(来自豆科植物的LeB4启动子)中,这些启动子适于双子叶植物。以下启动子适合于单子叶植物:大麦lpt-2或lpt-1启动子(WO95/15389和WO95/23230)、WO99/16890中所描述的大麦醇溶蛋白启动子和其它合适启动子。原则上,对于新方法,可以使用所有的天然启动子及其(例如上述的)调控序列。同时有利地,还可以单独或另外的使用合成启动子,特别是当它们介导种子特异性表达时,例如WO99/16890中所述。在特定的实施方案中,将种子特异性启动子应用于增强想要的PUFA或LCPUFA的产生。
如本文中所用,术语“有效连接”指表达调控序列和目的核酸是连接的,以使所述目的核酸的表达可以受所述表达调控序列支配,即表达调控序列应当与待表达的所述核酸序列功能性连接。因此,表达调控序列和待表达的核酸序列可以相互物理连接,例如,通过将表达调控序列插入到待表达核酸序列的5’末端。备选地,表达调控序列和待表达的核酸可以仅仅是物理邻近的,以使表达调控序列能支配至少一种目的核酸序列的表达。表达调控序列和待表达的核酸优选地被不多于500bp、300bp、100bp、80bp、60bp、40bp、20bp、10bp或5bp的(核苷酸)隔开。
在本发明多核苷酸另外的优选实施方案中,所述多核苷酸另外包含与核酸序列有效连接的终止子序列。
如本文中所用,术语“终止子”指能终止转录的核酸序列。这些序列可以引起转录机器从待转录的核酸序列上解离。优选地,终止子应当是在植物中和,特别地在植物种子中有活性的。合适的终止子在本领域是已知的,并优选地包括多腺苷酸化信号如SV40聚腺苷酸位点或tk聚腺苷酸位点或者在Loke等人(Loke2005,PlantPhysiol138,第1457-1468页)中指出的植物特异性信号之一,其位于待表达的核酸序列的下游。
本发明还涉及包含本发明的多核苷酸的载体。
术语“载体”优选地包括噬菌体、质粒、病毒载体以及人工染色体,例如细菌或酵母人工染色体。此外,该术语也涉及靶向构建体,其允许将靶向构建体随机或者定点地整合入基因组DNA中。此类靶向构建体优选地包含足够长度的DNA,以用于如下所详细描述的同源或异源重组。包含本发明多核苷酸的载体优选地另外包含用于在宿主中繁殖和/或选择的选择标记。可以通过本领域熟知的多种技术,将载体掺入到宿主细胞中。当导入到宿主细胞中时,载体可以定居在细胞质中或者可以整合到基因组中。在后者的情况下,应当理解,该载体可以另外包含允许同源重组或者异源插入的核酸序列。通过常规转化或转染技术,可以将载体引入到原核或真核细胞中。如本文上下文使用的,术语“转化”和“转染”、接合和转导,意在包含本领域已知的多种用于向宿主细胞导入外源核酸(如DNA)的方法,包括磷酸钙、氯化铷或氯化钙共沉淀、DEAE-葡聚糖-介导的转染、脂质体转染、天然感受态、基于碳的聚簇(carbon-basedcluster)、化学介导的转移、电穿孔或粒子轰击。转化或转染宿主细胞(包括植物细胞)的合适方法可见于Sambrook等人(MolecularCloning:ALaboratoryManual.,第2版,ColdSpringHarborLaboratory,ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY,1989)和其它的实验室教科书,例如MethodsinMolecularBiology,1995,第44卷,Agrobacteriumprotocols,Ed.:GartlandandDavey,HumanaPress,Totowa,NewJersey。备选地,可以通过热击或电穿孔技术引入质粒载体。如果载体是病毒,那么在应用于宿主细胞前,可以将其使用合适的包装细胞系进行体外包装。
优选地,可以将本文中提及的载体合适地作为克隆载体,即在微生物系统中是可复制的。此类载体确保在细菌和优选地,酵母或真菌中有效克隆且可能稳定转化植物。必须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。此类载体系统的特征通常是它们至少包含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)。优选的,这类载体系统还包括其它顺式调节区域,如启动子和终止子序列和/或选择标志物,通过所述选择标志物可以鉴别合适的转化宿主细胞或生物。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。这类双元载体包括来自pBIB-HYG、pPZP、pBecks、和pGreen系列的载体。根据本发明,Bin19、pBI101、pBinAR、pGPTV和pCAMBIA优选使用。双元载体和它们的用途的综述可在Hellens等,TrendsinPlantScience(2000)5,446-451中找到。此外,通过使用合适的克隆载体,可以将多核苷酸导入到宿主细胞或生物体内,例如植物或动物,并因而用于转化植物,例如那些适合该目的的载体公开和引用在:PlantMolecularBiologyandBiotechnology(CRCPress,BocaRaton,Florida),第6/7章,第71-119页(1993);F.F.White,VectorsforGeneTransferinHigherPlants;in:TransgenicPlants,第1卷,EngineeringandUtilization,编著:Kung和R.Wu,AcademicPress,1993,15-38;B.Jenes等人,TechniquesforGeneTransfer,in:TransgenicPlants,第1卷,EngineeringandUtilization,编著:Kung和R.Wu,AcademicPress(1993),128-143;Potrykus,Annu.Rev.PlantPhysiol.PlantMolec.Biol.42(1991),205-225。
更优选地,本发明载体是表达载体。在该表达载体(即包含本发明的多核苷酸载体)中允许在原核或真核细胞或其分离的部分中表达该多核苷酸,所述本发明的多核苷酸具有与表达调控序列有效连接的核酸序列(也称为“表达盒”)。合适的表达载体是本领域已知的,例如Okayama-BergcDNA表达载体pcDV1(Pharmacia)、pCDM8、pRc/CMV、pcDNA1、pcDNA3(Invitrogene)或pSPORT1(GIBCOBRL)。典型的融合表达载体的其他实例为pGEX(PharmaciaBiotechInc;Smith,1988,Gene67:31-40)、pMAL(NewEnglandBiolabs,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。合适的诱导型非融合大肠杆菌表达载体的例子尤其为pTrc(Amann1988,Gene69:301-315)和pET11d(Studier1990,MethodsinEnzymology185,60-89)。pTrc载体的靶基因表达是基于通过宿主RNA聚合酶的来自杂合trp-lac融合启动子的转录。来自pET11d载体的靶基因表达是基于经共表达的病毒RNA聚合酶(T7gn1)所介导的T7-gn10-lac融合启动子的转录。该病毒聚合酶由宿主菌株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lacUV5启动子转录控制的T7gn1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中的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。用于酿酒酵母中表达的载体的实例包括pYepSec1(Baldari1987,EmboJ.6:229-234)、pMFa(Kurjan1982,Cell30:933-943)、pJRY88(Schultz1987,Gene54:113-123)和pYES2(InvitrogenCorporation,SanDiego,CA)。适用于其它真菌(如丝状真菌)的载体和载体的构建方法,包括在vandenHondel,C.A.M.J.J.,&Punt,P.J.(1991)“Genetransfersystemsandvectordevelopmentforfilamentousfungi,in:AppliedMolecularGeneticsoffungi,J.F.Peberdy等人编著,第1-28页,CambridgeUniversityPress:Cambridge中,或者在MoreGeneManipulationsinFungi(J.W.Bennet&L.L.Lasure编著,第396-428页:AcademicPress:SanDiego)中详细描述的那些载体和载体的构建方法。其它合适的酵母载体是,例如pAG-1、YEp6、YEp13或pEMBLYe23。作为备选,本发明的多核苷酸还可以使用杆状病毒表达载体在昆虫细胞表达。可用于在培养昆虫细胞(例如Sf9细胞)中表达蛋白质的杆状病毒载体包括pAc系列(Smith1983,Mol.CellBiol.3:2156-2165)和pVL系列(Lucklow1989,Virology170:31-39)。
可以通过使用植物表达载体,可以在单细胞植物细胞(如藻类)(见Falciatore1999,MarineBiotechnology1(3):239-251及其中所引用的参考文献)和来自高等植物(例如种子植物(Spermatophyte),如作物)的植物细胞中表达本发明的多核苷酸。植物表达载体的实例包括在Becker1992,PlantMol.Biol.20:1195-1197;Bevan1984,Nucl.AcidsRes.12:8711-8721;VectorsforGeneTransferinHigherPlants;in:TransgenicPlants,第1卷,EngineeringandUtilization,编著:Kung和R.Wu,AcademicPress,1993,第15-38页中详述的那些植物表达载体。植物表达盒优选的包含能够在植物细胞中控制基因表达并有效连接的表达控制序列,使得每一种序列都可以实现其功能,例如多聚腺苷酸化信号的转录终止功能,所述多聚腺苷酸化信号是衍生自根癌农杆菌T-DNA,例如Ti质粒pTiACH5(Gielen1984,EMBOJ.3,835)的基因3的那些信号,所述基因3已知是章鱼氨酸合酶,或其功能性等同物,但是所有其它在植物中具有功能性活性的终止子序列也是合适的。由于植物基因表达通常不限于转录水平,植物表达盒优选地包含其它有效连接的序列,例如翻译增强子如超驱动序列,所述超驱动序列包括增加蛋白质/RNA比的烟草花叶病毒5’-非翻译前导序列(Gallie等人,1987,Nucl.AcidsResearch15:8693-8711)。如以上所述,植物基因表达必须与合适的启动子有效连接,所述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(Benfey1989,EMBOJ.8:2195-2202),例如源自植物病毒如35SCaMV(Franck1980,Cell21:285-294)、19SCaMV(参见US5352605和WO84/02913)的启动子,或植物启动子,如在US4,962,028中描述的小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序列是靶向序列,所述靶向序列是基因产物向其对应的细胞区室中导入所需的(参见综述Kermode1996,Crit.Rev.PlantSci.15,4:285-423和其中引用的参考文献),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。如上所述,还可以通过化学诱导型启动子促进植物基因表达(参见综述Gatz1997,Annu.Rev.PlantPhysiol.PlantMol.Biol.,48:89-108)。当希望基因表达以时间特异性方式发生时,化学诱导型启动子是特别适合的。此类启动子的实例是水杨酸诱导型启动子(WO95/19443)、四环素诱导型启动子(Gatz1992,PlantJ.2,397-404)和乙醇诱导型启动子。应答生物或非生物的胁迫条件的启动子也是合适的,例如病原诱导的PRP1基因启动子(Ward1993,Plant.Mol.Biol.22:361-366)、热诱导的番茄hsp80启动子(US5,187,267)、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(EP0375091A)。特别优选的是那些在发生脂肪酸、脂类和油生物合成的组织和器官中,在种子细胞中,如胚乳细胞和发育胚的细胞中引起基因表达的启动子。合适的启动子是油菜的油菜籽蛋白基因启动子(US5,608,152)、蚕豆(Viciafaba)的USP启动子(Baeumlein1991,MolGen.Genet.225(3):459-67)、拟南芥的油质蛋白启动子(WO98/45461)、菜豆的菜豆蛋白启动子(US5,504,200)、芸苔属的Bce4启动子(WO91/13980)或豆球蛋白B4启动子(LeB4;Baeumlein1992,PlantJournal,2(2):233-9),以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。值得注意的合适启动子是大麦的lpt2或lpt1基因启动子(WO95/15389和WO95/23230),或者在WO99/16890中描述的那些启动子(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、高粱的kasirin基因或黑麦的裸麦醇溶蛋白基因的启动子)。由于质体是合成脂类生物合成的前体和某些终产物的区室,因此,同样特别适合的启动子是引起质体特异性表达的启动子。合适的启动子,例如病毒RNA聚合酶启动子,描述在WO95/16783和WO97/06250中,以及来自拟南芥的clpP启动子,描述在WO99/46394中。
上述载体仅仅是对可能适合的载体的概略回顾。其它质粒是技术人员已知的,并描述在例如:CloningVectors(编著Pouwels,P.H.,等人,Elsevier,Amsterdam-NewYork-Oxford,1985,ISBN0444904018)。用于原核及真核细胞的其它合适表达系统,参见Sambrook,loccit的第16和17章。
由上可知,优选所述载体是表达载体。更优选地,本发明的所述多核苷酸是在本发明的载体中的种子特异性启动子的控制下的。本文中所指的优选的种子特异性启动子选自Conlinin1、Conlinin2、油菜籽蛋白、LuFad3、USP、LeB4、Arc、Fae、ACP、LuPXR和SBP。详细参见例如,US2003-0159174。
此外,本发明涉及包含本发明的多核苷酸或载体的宿主细胞。
优选地,所述宿主细胞是植物细胞和,更优选地,植物细胞获自油料作物(oilseedcrop)。更优选地,所述油料作物选自亚麻(亚麻属的种(Linumsp.))、油菜(芸苔属的种(Brassicasp.))、大豆(大豆属和黄豆属的种(Glycine和Sojasp.))、向日葵、(向日葵属的的种(Helianthussp.))、棉花(棉属的种(Gossypiumsp.))、玉米、橄榄(木犀榄属的种(Oleasp.))、红花(红蓝花属的种(Carthamussp.))、可可(可可树(Theobromacacao)、花生(落花生属的种(Arachissp.))、大麻、亚麻荠属(camelina)、两节荠属(crambe)、油棕榈、椰子、落花生、芝麻、蓖麻、lesquerella、乌桕、sheanuts、油桐果(tungnuts)、木棉花果实(kapokfruit)、罂粟籽、西蒙德木籽(jojobaseed)和紫苏属(perilla)。
还优选地,所述宿主细胞是微生物。更优选地,所述微生物是细菌、真菌或藻类。更优选地,其选自假丝酵母属(Candida)、隐球酵母属(Cryptococcus)、油脂酵母属(Lipomyces)、红冬孢酵母属(Rhodosporidium)、解脂耶氏酵母属(Yarrowia)和裂殖壶菌属(Schizochytrium)。
此外,根据本发明的宿主细胞也可以是动物细胞。优选地,所述动物宿主细胞是鱼的宿主细胞或者从其中获得的细胞系。更优选地,鱼宿主细胞来自鲱鱼、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。
通常,LCPUFA的产生,即长链不饱和脂肪酸生物合成途径的控制步骤由膜相关的脂肪酸延伸酶复合物催化。植物和大多数其他的真核生物具有特定的延伸酶系统,用于延长超过C18原子的脂肪酸。这些延伸酶反应物与脂肪酸合酶复合物(FAS)具有相同的几个重要特征。然而,延伸酶复合物与FAS复合物不同,因为该复合物定位在胞质中并且是膜结合的、ACP并不参与并且延伸酶3-酮-酰基-CoA合酶催化丙二酰-CoA与酰基引物的缩合。延伸酶复合物由4个具有不同催化功能的组分组成,酮-酰基CoA合酶(KCS,丙二酰-CoA与酰基-CoA的缩合反应,产生延长2C原子的酮-酰基-CoA脂肪酸)、酮-酰基-CoA还原酶(KCR,将3-酮基还原为3-羟基)、脱水酶(DH,脱水产生Δ-2-烯酰-酰基-CoA脂肪酸)和烯酰-CoA还原酶(ECR,还原位置2处的双键,从复合物中释放)。对于包括ARA、EPA和/或DHA的LCPUFA的产生,延伸反应是必需的。高等植物不具有用于产生LCPUFA(4个或更多的双键,20个或更多的C原子)所必需的酶。因此,需要对植物或植物细胞赋予催化活性。延伸过程中的一个关键步骤是脱水和还原反应。本发明的多核苷酸令人惊奇地通过一种酶催化脱水和还原活性。通过提供这种nECR,产生了增加水平的PUFA和LCPUFA。
然而,应当理解,取决于宿主细胞,可以例如通过重组技术另外地将酶活性赋予宿主细胞。因此,本发明优选地涉及如下宿主细胞,取决于选择的宿主细胞,除本发明的多核苷酸外,其还包含根据需要编码此类去饱和酶和/或延伸酶的多核苷酸。在宿主细胞中存在的优选的去饱和酶和/或延伸酶是选自以下的至少一种酶:Δ-4-去饱和酶、Δ-5-去饱和酶、Δ-5-延伸酶、Δ-6-去饱和酶、Δ12-去饱和酶、Δ15-去饱和酶、ω3-去饱和酶和Δ-6-延伸酶。特别优选的是来自卡氏棘阿米巴(Acanthamoebacastellanii)的双功能性d12d15-去饱和酶d12d15Des(Ac)(WO2007042510)、来自麦角菌(Clavicepspurpurea)的d12d15Des(Cp)(WO2008006202)和来自Lottiagigantea的d12d15Des(Lg)1(WO2009016202)、来自金盏菊的(Calendulaofficinalis)d12-去饱和酶d12Des(Co)(WO200185968)、来自双色蜡蘑(Laccariabicolor)的d12Des(Lb)(WO2009016202)、来自领鞭毛虫(Monosigabrevicollis)的d12Des(Mb)(WO2009016202)、来自禾生球腔菌(Mycosphaerellagraminicola)的d12Des(Mg)(WO2009016202)、来自Nectriahaematococca的d12Des(Nh)(WO2009016202)、来自Ostreococcuslucimarinus的d12Des(Ol)(WO2008040787)、来自布拉克须霉菌(Phycomycesblakesleeanus)的d12Des(Pb)(WO2009016202)、来自大豆疫霉菌(Phytophthorasojae)的d12Des(Ps)(WO2006100241)和来自假微型海链藻的d12Des(Tp)(WO2006069710)、来自Helobdellarobusta的d15-去饱和酶d15Des(Hr)(WO2009016202)、来自微鞘藻(Microcoleuschthonoplastes)的d15Des(Mc)(WO2009016202)、来自香蕉黑条叶斑病菌(Mycosphaerellafijiensis)的d15Des(Mf)(WO2009016202)、来自禾生球腔菌的d15Des(Mg)(WO2009016202)和来自Nectriahaematococca的d15Des(Nh)2(WO2009016202)、来自纤细裸藻(Euglenagracilis)的d4-去饱和酶d4Des(Eg)(WO2004090123)、来自破囊壶菌属(Thraustochytriumsp.)的d4Des(Tc)(WO2002026946)和来自假微型海链藻的d4Des(Tp)(WO2006069710)、来自Ostreococcuslucimarinus的d5-去饱和酶d5Des(Ol)2(WO2008040787)、来自球蒴藓(Physcomitrellapatens)的d5Des(Pp)(WO2004057001)、来自三角褐指藻的d5Des(Pt)(WO2002057465)、来自破囊壶菌属的d5Des(Tc)(WO2002026946)、来自假微型海链藻的d5Des(Tp)(WO2006069710)和来自角齿藓(Ceratodonpurpureus)的d6-去饱和酶d6Des(Cp)(WO2000075341)、来自Ostreococcuslucimarinus的d6Des(Ol)(WO2008040787)、来自Ostreococcustauri的d6Des(Ot)(WO2006069710)、来自粉报春(Primulafarinosa)的d6Des(Pf)(WO2003072784)、来自拉曼被孢霉菌(Pythiumirregulare)的d6Des(Pir)_BO(WO2002026946)、来自拉曼被孢霉的d6Des(Pir)(WO2002026946)、来自Primulaluteola的d6Des(Plu)(WO2003072784)、来自球蒴藓的d6Des(Pp)(WO200102591)、来自三角褐指藻的d6Des(Pt)(WO2002057465)、来自高穗花报春(Primulavialii)的d6Des(Pv)(WO2003072784和来自假微型海链藻的d6Des(Tp)(WO2006069710)、来自Acanthamoebacastellanii的d8-去饱和酶d8Des(Ac)(EP1790731)、来自纤细裸藻的d8Des(Eg)(WO200034439)和来自Perkinsusmarinus的d8Des(Pm)(WO2007093776)、来自马铃薯晚疫病菌(Phytophthorainfestans)的o3-去饱和酶o3Des(Pi)(WO2005083053)、来自拉曼被孢霉菌的o3Des(Pir)(WO2008022963)、来自拉曼被孢霉菌的o3Des(Pir)2(WO2008022963)和来自大豆疫霉菌的o3Des(Ps)(WO2006100241)、来自虹鳟(Oncorhynchusmykiss)的双功能d5d6-延伸酶d5d6Elo(Om)2(WO2005012316)、来自金黄色破囊壶菌(Thraustochytriumaureum)的d5d6Elo(Ta)(WO2005012316)和来自破囊壶菌属的d5d6Elo(Tc)(WO2005012316)、来自拟南芥(Arabidopsisthaliana)的d5-延伸酶d5Elo(At)(WO2005012316)、来自拟南芥的d5Elo(At)2(WO2005012316)、来自玻璃海鞘(Cionaintestinalis)的d5Elo(Ci)(WO2005012316)、来自Ostreococcuslucimarinus的d5Elo(Ol)(WO2008040787)、来自Ostreococcustauri的d5Elo(Ot)(WO2005012316)、来自假微型海链藻的d5Elo(Tp)(WO2005012316)和来自非洲爪蟾(Xenopuslaevis)的d5Elo(Xl)(WO2005012316)、来自Ostreococcuslucimarinus的d6-延伸酶d6Elo(Ol)(WO2008040787)、来自Ostreococcustauri的d6Elo(Ot)(WO2005012316)、来自马铃薯晚疫病菌的d6Elo(Pi)(WO2003064638)、来自畸雌腐霉菌(Pythiumirregulare)的d6Elo(Pir)(WO2009016208)、来自球蒴藓的d6Elo(Pp)(WO2001059128)、来自大豆疫霉菌的d6Elo(Ps)(WO2006100241)、来自大豆疫霉菌的d6Elo(Ps)2(WO2006100241)、来自大豆疫霉菌的d6Elo(Ps)3(WO2006100241)、来自三角褐指藻的d6Elo(Pt)(WO2005012316)、来自破囊壶菌属的d6Elo(Tc)(WO2005012316)、来自假微型海链藻的d6Elo(Tp)(WO2005012316)、来自绿光等鞭金藻(Isochrysisgalbana)的d9-延伸酶d9Elo(Ig)(WO2002077213)、来自Perkinsusmarinus的d9Elo(Pm)(WO2007093776)和来自米根霉(Rhizopusoryzae)的d9Elo(Ro)(WO2009016208)。特别地,如果涉及在高等植物中产生ARA,那么可以在宿主细胞中组合在下表3中列出的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-延伸酶、d5-去饱和酶、d12-去饱和酶和d6-延伸酶)或者具有基本上相同的活性的酶。如果涉及在高等植物中产生EPA,可以在宿主细胞中组合在下表4中列出的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-去饱和酶、d12-去饱和酶、d6-延伸酶、Ω3-去饱和酶和d15去饱和酶)或者具有基本上相同的活性的酶。如果涉及在高等植物中产生DHA,可以在宿主细胞中组合在下表5中列出的酶(即另外的d6-去饱和酶、d6-延伸酶、d5-去饱和酶、d12-去饱和酶、d6-延伸酶、Ω3-去饱和酶、d15去饱和酶、d5-延伸酶和d4-去饱和酶)或者具有基本上相同的活性的酶。
本发明还涉及细胞,优选如上指定的宿主细胞或者在本文中其他地方指定的非人类生物的细胞,所述细胞包含这样的多核苷酸,所述多核苷酸通过点突变、截断、倒位、缺失、添加、取代和同源重组获自本发明多核苷酸。如何对多核苷酸实施此类修饰是本领域技术人员熟知的并在本说明书中的其他地方进行了详细描述。
本发明还涉及用于产生由任一本发明多核苷酸编码的多肽的方法,包括
a)在允许所述多肽产生的条件下培养本发明的宿主细胞;和
b)从步骤a)的宿主细胞中获得多肽。
允许表达由宿主细胞包含的本发明多核苷酸的合适条件取决于宿主细胞以及用于支配所述多核苷酸的表达的表达调控序列。这些条件以及如何选择它们是本领域技术人员熟知的。表达的多肽可以例如通过所有常规纯化技术获得,所述常规纯化技术包括亲和层析、大小排阻层析、高压液相层析(HPLC)和包括抗体沉淀的沉淀技术。应当理解,尽管是优选的,但是该方法可能并不必定产生多肽的基本纯制备物。应当理解,取决于用于前述方法的宿主细胞,由此产生的多肽可以是翻译后修饰的或者另外加工的。
本发明包括由本发明的多核苷酸编码的或者由前述方法获得的多肽。
如本文中所用,术语“多肽”指基本纯的多肽或者包含其他蛋白质的多肽制品。另外,该术语还指融合蛋白和由以上提及的本发明多核苷酸至少部分编码的多肽片段。此外,其包括化学修饰的多肽。此类修饰可以是人工修饰或天然存在的修饰,例如磷酸化、糖基化、十四烷基化等(在Mann2003,Nat.Biotechnol.21,255-261,reviewwithfocusonplantsinHuber2004,Curr.Opin.PlantBiol.7,318-322中综述)。目前,已知300种以上的翻译后修饰(参见在http://www.abrf.org/index.cfm/dm.home中列出的fullABFRCDeltamass)。本发明的多肽应当显示出以上提及的nECR活性。
此外,本发明包括特异识别本发明多肽的抗体。
可以使用根据本发明的纯化多肽或其衍生的合适片段作为抗原,通过已知的方法制备抗本发明的多肽的抗体。可以通过本领域熟知的抗原性决定算法(antigenicitydeterminingalgorithm)鉴定适合作为抗原的片段。此类片段可以获自通过蛋白水解消化本发明的多肽或者可以是合成肽。优选地,本发明的抗体是单克隆抗体、多克隆抗体、单链抗体、嵌合抗体或者任一这些抗体的片段,例如Fab、Fv或scFv片段等。本发明还包含的抗体是双特异性抗体、合成抗体或者任一前述抗体的化学修饰衍生物。本发明的抗体应当是与本发明的多肽特异性结合的(即不与其它多肽或肽显著的交叉反应)。可以通过多种熟知的技术测试特异性结合。抗体或其片段可以通过使用例如在Harlow和Lane″Antibodies,ALaboratoryManual″,CSHPress,ColdSpringHarbor,1988中描述的方法获得。单克隆抗体可以通过最初在1975,Nature256,495,和Galfré1981,Meth.Enzymol.73,3中描述的技术制备,所述技术包括将小鼠骨髓瘤细胞与来源于免疫哺乳动物的脾细胞融合。可以将抗体例如,用于本发明多肽的免疫沉淀、免疫定位或纯化(例如,通过亲和层析),以及用于例如在重组生物中检测所述变体多肽的存在,和用于鉴定与本发明的蛋白质相互作用的蛋白质或化合物。
此外,本发明考虑了包含本发明的多核苷酸或载体的非人类转基因生物。
优选地,非人类转基因生物是植物、植物部分或植物种子。用于引入本发明的多核苷酸或载体的优选植物是所有能够合成脂肪酸的植物,例如所有的双子叶或单子叶植物、藻类或藓类。应当理解,也可以将来源于植物的宿主细胞用于产生根据本发明的植物。优选的植物选自以下植物科: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)、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)、绿枝藻纲(Prasinophyceae)或蔬菜植物或观赏植物例如万寿菊。提及的实例是选自以下的植物:Adelotheciaceae,例如剑叶藓属,如属和种球蒴藓(Physcomitrellapatens);漆树科如黄连木属(Pistacia)、芒果属(Mangifera)、腰果属(Anacardium)例如阿月混子(Pistaciavera[阿月混子实])、芒果(Mangiferindica[芒果])或腰果(Anacardiumoccidentale[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(Calendulaofficinalis[普通金盏花])、红花(Carthamustinctorius[红花])、矢车菊(Centaureacyanus[矢车菊])、菊苣(Cichoriumintybus[菊苣])、洋蓟(Cynarascolymus[朝鲜蓟])、向日葵(Helianthusannus[向日葵])、莴苣(Lactucasativa)、皱叶莴苣(Lactucacrispa)、芋(Lactucaesculenta)、毒莴苣的某些种(LactucascariolaL.ssp.Sativa)、LactucascariolaL.var.integrata、LactucascariolaL.var.integrifolia、莴苣romana亚种(Lactucasativasubsp.Romana)、Locustacommunis、莴苣缬草(Valerianalocusta[沙拉蔬菜])、香叶万寿菊(Tageteslucida)、万寿菊(Tageteserecta)或细叶万寿菊(Tagetestenuifolia)[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(Daucuscarota[胡萝卜])的属和种、桦木科如榛属(Corylus)例如欧洲榛(Corylusavellana)或榛子(Coryluscolurna[榛子])的属和种、紫草科如琉璃苣、例如,如下的属和种:Boragoofricinalis[琉璃苣]、十字花科如芸苔属(Brassica)、Melanosinapis、白芥属(Sinapis)、拟南芥属(Arabadopsis)例如欧洲油菜(Brassicanapus)、芜青属某些种(Brassicarapassp.[油菜])、野生欧白芥(Sinapisarvensis)、芥菜(Brassicajuncea)、Brassicajunceavar.juncea、皱叶芥菜(Brassicajunceavar.crispifolia)、大叶芥菜(Brassicajunceavar.foliosa)、黑芥(Brassicanigra)、Brassicasinapioides、芥菜(Melanosinapiscommunis[芥菜])、甘蓝(Brassicaoleracea[饲用甜菜])或拟南芥(Arabidopsisthaliana)的属和种、凤梨科如凤梨属(Anana)、Bromelia(菠萝)例如菠萝(Ananacomosus)、Ananasananas或Bromeliacomosa[菠萝]的属和种、番木瓜科如番木瓜属(Carica)例如番木瓜(Caricapapaya[番木瓜])的属和种、大麻科如大麻属(Cannabis)例如大麻(Cannabissativa[大麻])的属和种、旋花科如番薯属(Ipomea)、旋花属(Convolvulus)例如甘薯(Ipomoeabatatus)、提琴叶牵牛花(Ipomoeapandurata)、Convolvulusbatatas、Convolvulustiliaceus、Ipomoeafastigiata、Ipomoeatiliacea、三裂叶薯(Ipomoeatriloba)或Convolvuluspanduratus[甜薯、番薯]的属和种、藜科如甜菜属(Betavulgaris)例如甜菜(Betavulgaris)、Betavulgarisvar.altissima、甜菜普通变种(Betavulgarisvar.vulgaris)、Betamaritima、甜菜宿根变种Betavulgarisvar.perennis、Betavulgarisvar.Conditiva或Betavulgarisvar.esculenta[甜菜]的属和种、隐甲藻科如隐甲藻属例如寇氏隐甲藻(Cryptecodiniumcohnii)的属和种、葫芦科如南瓜属(Cucurbita)例如笋瓜(Cucurbitamaxima)、灰子南瓜(Cucurbitamixta)、西葫芦(Cucurbitapepo)或南瓜(Cucurbitamoschata[西葫芦/南瓜])的属和种,桥弯藻科(Cymbellaceae),如双眉藻属(Amphora),桥弯藻属(Cymbella),Okedenia,褐指藻属(Phaeodactylum),Reimeria,例如,属和种三角褐指藻(Phaeodactylumtricornutum),牛毛藓科(Ditrichaceae),如牛毛藓科(Ditrichaceae),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,例如属和种:Ceratodonantarcticus,Ceratodoncolumbiae,Ceratodonheterophyllus,Ceratodonpurpurascens,角齿藓台湾变种(Ceratodonpurpureus),Ceratodonpurpureusssp.convolutus,Ceratodonpurpureusssp.stenocarpus,角齿藓圆叶变种(Ceratodonpurpureusvar.Rotundifolius),Ceratodonratodon,疣蒴角齿藓(Ceratodonstenocarpus),Chrysoblastellachilensis,Ditrichumambiguum,Ditrichumbrevisetum,皱波牛毛藓(Ditrichumcrispatissimum),卷叶牛毛藓(Ditrichumdifficile),Ditrichumfalcifolium,扭叶牛毛藓(Ditrichumflexicaule),Ditrichumgiganteum,牛毛藓(Ditrichumheteromallum),Ditrichumlineare,Ditrichumlineare,Ditrichummontanum,Ditrichummontanum,黄牛毛藓(Ditrichumpallidum),Ditrichumpunctulatum,细叶牛毛藓(Ditrichumpusillum),细叶牛毛藓扭叶变种(Ditrichumpusillumvar.tortile),Ditrichumrhynchostegium,Ditrichumschimperi,Ditrichumtortile,对叶藓(Distichiumcapillaceum),小对叶藓(Distichiumhagenii),斜蒴对叶藓(Distichiuminclinatum),Distichiummacounii,Eccremidiumfloridanum,Eccremidiumwhiteleggei,Lophidionstrictus,尖叶丛毛藓(Pleuridiumacuminatum),Pleuridiumalternifolium,Pleuridiumholdridgei,Pleuridiummexicanum,Pleuridiumravenelii,丛毛藓(Pleuridiumsubulatum),石缝藓(Saelaniaglaucescens),Trichodonborealis,毛齿藓(Trichodoncylindricus)或Trichodoncylindricusvar.oblongus、胡颓子科如胡颓子属(Elaeagnus)例如油橄榄(Oleaeuropaea[橄榄])的属和种、杜鹃花科如山月桂属(Kalmia)例如阔叶山月桂(Kalmialatifolia)、狭叶山月桂(Kalmiaangustifolia)、小叶山月桂(Kalmiamicrophylla)、沼泽山月桂(Kalmiapolifolia)、西洋山月桂(Kalmiaoccidentalis)、Cistuschamaerhodendros或山月桂(Kalmialucida[山月桂])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(Manihotutilissima)、Janiphamanihot、Jatrophamanihot、Manihotaipil、甜木薯(Manihotdulcis)、Manihotmanihot、Manihotmelanobasis、木薯(Manihotesculenta[木薯])或蓖麻(Ricinuscommunis[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(Soja)例如豌豆(Pisumsativum)、饲料豌豆(Pisumarvense)、Pisumhumile[豌豆]、Albiziaberteriana、合欢(Albiziajulibrissin)、阔荚合欢(Albizialebbeck)、Acaciaberteriana、Acacialittoralis、Albiziaberteriana、Albizziaberteriana、Cathormionberteriana、Feuilleaberteriana、Ingafragrans、Pithecellobiumberterianum、Pithecellobiumfragrans、Pithecolobiumberterianum、Pseudalbizziaberteriana、Acaciajulibrissin、Acacianemu、Albizianemu、Feuilleeajulibrissin、Mimosajulibrissin、Mimosaspeciosa、Sericanrdajulibrissin、阔荚金合欢(Acacialebbeck)、Acaciamacrophylla、大叶合欢(Albizialebbeck)、Feuilleealebbeck、大叶含羞草(Mimosalebbeck)、Mimosaspeciosa[木棉树]、紫苜蓿(Medicagosativa)、野苜蓿(Medicagofalcata)、杂交苜蓿(Medicagovaria)[紫花苜蓿]、大豆(Glycinemax)、镰扁豆(Dolichossoja)、宽叶蔓豆(Glycinegracilis)、大豆(Glycinehispida)、大菜豆(Phaseolusmax)、Sojahispida或Sojamax[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),例如属和种Aphanorrhegmaserratum,Entosthodonattenuatus,Entosthodonbolanderi,Entosthodonbonplandii,Entosthodoncalifornicus,Entosthodondrummondii,Entosthodonjamesonii,Entosthodonleibergii,Entosthodonneoscoticus,Entosthodonrubrisetus,Entosthodonspathulifolius,Entosthodontucsoni,Funariaamericana,Funariabolanderi,齿叶葫芦藓(Funariacalcarea),Funariacalifornica,Funariacalvescens,Funariaconvoluta,Funariaflavicans,Funariagroutiana,葫芦藓(Funariahygrometrica),Funariahygrometricavar.arctica,葫芦藓暖地变种(Funariahygrometricavar.Calvescens),Funariahygrometricavar.convoluta,Funariahygrometricavar.muralis,Funariahygrometricavar.utahensis,小口葫芦藓(Funariamicrostoma),Funariamicrostomavar.obtusifolia,刺边葫芦藓(Funariamuhlenbergii),Funariaorcuttii,Funariaplano-convexa,Funariapolaris,Funariaravenelii,Funariarubriseta,Funariaserrata,Funariasonorae,Funariasublimbatus,Funariatucsoni,Physcomitrellacalifornica,球蒴藓(Physcomitrellapatens),Physcomitrellareaderi,Physcomitriumaustrale,Physcomitriumcalifornicum,Physcomitriumcollenchymatum,Physcomitriumcoloradense,Physcomitriumcupuliferum,Physcomitriumdrummondii,Physcomitriumeurystomum,Physcomitriumflexifolium,Physcomitriumhookeri,Physcomitriumhookerivar.serratum,Physcomitriumimmersum,Physcomitriumkellermanii,Physcomitriummegalocarpum,Physcomitriumpyriforme,Physcomitriumpyriformevar.serratum,Physcomitriumrufipes,Physcomitriumsandbergii,Physcomitriumsubsphaericum,Physcomitriumwashingtoniense,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(Pelargoniumgrossularioides)或椰子油(Oleumcocois[椰子])的属和种、禾本科如甘蔗属(Saccharum)例如甘蔗(Saccharumofficinarum)的属和种、胡桃科如胡桃属(Juglans)、Wallia例如胡桃(Juglansregia)、Juglansailanthifolia、山核桃(Juglanssieboldiana)、灰核桃(Juglanscinerea)、Walliacinerea、Juglansbixbyi、加州黑核桃(Juglanscalifornica)、印度黑核桃(Juglanshindsii)、Juglansintermedia、Juglansjamaicensis、大核桃(Juglansmajor)、Juglansmicrocarpa、黑核桃(Juglansnigra)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(Laurusnobilis[bay])、鳄梨(Perseaamericana)、鳄梨油(Perseagratissima)或Perseapersea[avocado]的属和种、豆科如落花生属(Arachis)例如落花生(Arachishypogaea[花生])的属和种、亚麻科如亚麻属、Adenolinum属例如亚麻(Linumusitatissimum)、Linumhumile、奥地利亚麻(Linumaustriacum)、Linumbienne、窄叶亚麻(Linumangustifolium)、泻亚麻(Linumcatharticum)、金黄亚麻(Linumflavum)、大花亚麻(Linumgrandiflorum)、Adenolinumgrandiflorum、刘易斯亚麻(Linumlewisii)、那旁亚麻(Linumnarbonense)、宿根亚麻(Linumperenne)、宿根亚麻刘易斯变种(Linumperennevar.lewisii)、Linumpratense或亚麻子(Linumtrigynum[亚麻子])的属和种、Lythrarieae如石榴属(Punica)例如石榴(Punicagranatum[pomegranate])的属和种、锦葵科(Malvaceae)如棉属(Gossypium)例如陆地棉(Gossypiumhirsutum)、树棉(Gossypiumarboreum)、海岛棉(Gossypiumbarbadense)、草棉(Gossypiumherbaceum)或瑟伯棉(Gossypiumthurberi[棉])的属和种、地钱科(Marchantiaceae)如地钱属(Marchantia)例如Marchantiaberteroana、Marchantiafoliacea、大孢地钱(Marchantiamacropora)的属和种、芭蕉科(Musaceae)如芭蕉属(Musa)例如香蕉(Musanana)、小果野蕉(Musaacuminata)、大蕉(Musaparadisiaca)、芭蕉属的某些种(Musaspp.[banana])的属和种、柳叶菜科(Onagraceae)如Camissonia属、月见草属(Oenothera)例如月见草(Oenotherabiennis)或夜来香(Camissoniabrevipes[月见草])的属和种、棕榈科(Palmae)如油棕属(Elaeis),例如油棕(Elaeisguineensis[油棕榈])的属和种、罂粟科(Papaveraceae)如罂粟属(Papaver),例如东方罂粟(Papaverorientale)、虞美人(Papaverrhoeas)、长荚罂粟(Papaverdubium[罂粟])的属和种、胡麻科(Pedaliaceae)如胡麻属(Sesamum)例如胡麻(Sesamumindicum[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(Piperaduncum)、Piperamalago、狭叶胡椒(Piperangustifolium)、大胡椒(Piperauritum)、萎叶(Piperbetel)、荜澄茄(Pipercubeba)、荜菝(Piperlongum)、胡椒(Pipernigrum)、假荜拔(Piperretrofractum)、Artantheadunca、Artantheelongata、长胡椒(Peperomiaelongata)、Piperelongatum、Steffensiaelongata[cayennepepper]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(Hordeumvulgare)、芒颖大麦草(Hordeumjubatum)、鼠大麦草(Hordeummurinum)、短芒大麦草(Hordeumsecalinum)、栽培二棱大麦(Hordeumdistichon)、Hordeumaegiceras、六列大麦(Hordeumhexastichon)、六棱大麦(Hordeumhexastichum)、不规则大麦(Hordeumirregulare)、大麦(Hordeumsativum)、短芒大麦草(Hordeumsecalinum)[大麦]、黑麦(Secalecereale[黑麦])、燕麦(Avenasativa)、野燕麦(Avenafatua)、地中海红燕麦(Avenabyzantina)、Avenafatuavar.sativa、杂种燕麦(Avenahybrida[燕麦])、二色高梁(Sorghumbicolor)、石茅高梁(Sorghumhalepense)、甜高梁(Sorghumsaccharatum)、高梁(Sorghumvulgare)、Andropogondrummondii、二色绒毛草(Holcusbicolor)、Holcussorghum、Sorghumaethiopicum、Sorghumarundinaceum、Sorghumcaffrorum、垂穗高梁草(Sorghumcernuum)、工艺高梁(Sorghumdochna)、Sorghumdrummondii、硬高粱草(Sorghumdurra)、Sorghumguineense、Sorghumlanceolatum、多脉高粱草(Sorghumnervosum)、甜高梁(Sorghumsaccharatum)、Sorghumsubglabrescens、垂叶高梁草(Sorghumverticilliflorum)、高梁(Sorghumvulgare)、石茅高梁(Holcushalepensis)、Sorghummiliaceum、黍粟(Panicummilitaceum[millet])、稻(Oryzasativa)、Oryzalatifolia[稻]、玉米(Zeamays[maize])、小麦(Triticumaestivum)、硬粒小麦(Triticumdurum)、圆柱小麦(Triticumturgidum)、Triticumhybernum、马卡小麦(Triticummacha)、普通小麦(Triticumsativum或Triticumvulgare)[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(Macadamiaintergrifolia[macadamia])的属和种、葱绿藻纲(Prasinophyceae),如肾藻属(Nephroselmis),Prasinococcus,Scherffelia,扁藻属(Tetraselmis),Mantoniella,Ostreococcus,例如属和种:绿肾藻(Nephroselmisolivacea),Prasinococcuscapsulatus,Scherffeliadubia,Tetraselmischui,扁藻(Tetraselmissuecica),Mantoniellasquamata,Ostreococcustauri,茜草科(Rubiaceae)如咖啡属(Coffea)例如咖啡属(Coffea)某些种、小果咖啡(Coffeaarabica)、中果咖啡(Coffeacanephora)或大果咖啡(Coffealiberica[coffee])、玄参科(Scrophulariaceae)如毛蕊花属(Verbascum)例如以下的属和种:毛瓣毛蕊花(Verbascumblattaria)、东方毛蕊花(Verbascumchaixii)、密叶毛蕊花(Verbascumdensiflorum)、Verbascumlagurus、长叶毛蕊花(Verbascumlongifolium)、Verbascumlychnitis、Verbascumnigrum、奥林匹克毛蕊花(Verbascumolympicum)、Verbascumphlomoides、紫毛蕊花(Verbascumphoenicum)、Verbascumpulverulentum或毛蕊花(Verbascumthapsus[mullein])、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(Capsicumannuum)、Capsicumannuumvar.glabriusculum、五色椒(Capsicumfrutescens[pepper])、红椒(Capsicumannuum[paprika])、烟草(Nicotianatabacum)、花烟草(Nicotianaalata)、长头烟草(Nicotianaattenuata)、光烟草(Nicotianaglauca)、Nicotianalangsdorffii、Nicotianaobtusifolia、Nicotianaquadrivalvis、Nicotianarepanda、黄花烟草(Nicotianarustica)、Nicotianasylvestris[烟草]、马铃薯(Solanumtuberosum[番茄])、茄(Solanummelongena[eggplant])、番茄(Lycopersiconesculentum)、Lycopersiconlycopersicum、梨形番茄(Lycopersiconpyriforme)、红茄(Solanumintegrifolium)或番茄(Solanumlycopersicum)[番茄]、梧桐科(Sterculiaceae)如可可树属(Theobroma)例如可可树(Theobromacacao[可可])的属和种或山茶科(Theaceae)如山茶属(Camellia)例如大叶茶(Camelliasinensis[茶])的属和种。根据本发明,特别优选的可用作为转基因植物的植物是包含大量的脂类化合物的油料作物,例如花生、油菜、卡诺拉油菜、向日葵、红花、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、扁桃、澳洲坚果、鳄梨、月桂(bay)、西葫芦/南瓜、亚麻子、大豆、阿月混子、琉璃苣、树(油棕榈树、椰子树或胡桃树)或作物例如玉米、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、野豌豆属的种、豌豆、紫花苜蓿或灌木(咖啡、可可、茶)、柳属的种和多年生牧草及饲料作物。根据本发明的优选的植物是油料作物植物如花生、油菜、卡诺拉油菜、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、大蓟或者红花、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。
优选的藓类是剑叶藓属或角齿藓属。优选的藻类是等鞭金藻属、Mantoniella、蚝球藻(Ostreococcus)或隐甲藻(Crypthecodinium),和藻类/硅藻类例如褐指藻属或破囊壶菌属(Thraustochytrium)。更优选地,所述藻类或藓类选自:希瓦菌(Shewanella)、剑叶藓属(Physcomitrella)、破囊壶菌、镰孢属(Fusarium)、疫霉属(Phytophthora)、角齿藓属(Ceratodon)、等鞭金藻属、Aleurita、Muscarioides、被孢霉属(Mortierella)、褐指藻属(Phaeodactylum)、Cryphthecodinium,尤其是来自以下的种和属:Thallasiosirapseudonona、纤细裸藻(Euglenagracilis)、球蒴藓(Physcomitrellapatens)、马铃薯晚疫病菌(Phytophtorainfestans)、Fusariumgraminaeum、Cryptocodiniumcohnii、角齿藓(Ceratodonpurpureus)、绿光等鞭金藻、Aleuritafarinosa、破囊壶菌属、Muscarioidesviallii、白被孢霉菌(Mortierellaalpina)、三角褐指藻或秀丽隐杆线虫(Caenorhabditiselegans)或者特别有利的马铃薯晚疫病菌。Thallasiosirapseudonona和Cryptocodiniumcohnii。
可以通过如在本说明书中其他地方的转化技术获得转基因植物。优选地,可以通过T-DNA介导的转化获得转基因植物。此类载体系统的特征通常是它们至少包含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)。合适的载体详细描述于本说明书中其他地方。
还包括包含本发明的载体或多核苷酸的转基因非人类动物。本发明涉及的优选的非人类转基因动物是鱼类,例如鲱鱼、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。
然而,应当理解,取决于上述非人类转基因生物,可以例如通过重组技术将另外的酶活性赋予所述生物。因此,本发明优选地涉及这样的上述非人类转基因生物,取决于选择的宿主细胞,除本发明的多核苷酸外,还根据需要包含编码此类去饱和酶和/或延伸酶的多核苷酸。在生物中存在的优选的去饱和酶和/或延伸酶是选自在本说明书其他地方(参见上面和表3、4和5)特定列出的去饱和酶和/或延伸酶的至少一种酶或者组合。
此外,本发明包括用于产生多不饱和脂肪酸的方法,包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培养本发明的宿主细胞;和
b)从所述宿主细胞中获得所述多不饱和脂肪酸。
如本文中所用,术语“多不饱和脂肪酸(PUFA)”指包含至少两个,优选地3个、4个、5个或6个双键的脂肪酸。此外,应当理解,此类脂肪酸在脂肪酸链中优选地包含18-24个碳原子。更优选地,该术语涉及在脂肪酸链中具有20-24个碳原子的长链PUFA(LCPUFA)。从本发明的意义上而言,优选的不饱和脂肪酸选自DGLA20:3(8,11,14)、ARA20:4(5,8,11,14)、iARA20:4(8,11,14,17)、EPA20:5(5,8,11,14,17)、DPA22:5(4,7,10,13,16)、DHA22:6(4,7,10,13,16,19)、20:4(8,11,14,17),更优选地,花生四烯酸(ARA)20:4(5,8,11,14)、二十碳五烯酸(EPA)20:5(5,8,11,14,17)、和二十二碳六烯酸(DHA)22:6(4,7,10,13,16,19)。因此,应当理解,最优选地,由本发明提供的方法涉及ARA、EPA或DHA的产生。此外,也包括在合成期间存在的LCPUFA的中间物。通过本发明多肽的nECR活性,此类中间物优选地形成自底物。优选地,底物包括LA18:2(9,12)、GLA18:3(6,9,12)、DGLA20:3(8,11,14)、ARA20:4(5,8,11,14)、二十碳二烯酸20:2(11,14)、二十碳四烯酸20:4(8,11,14,17)、二十碳五烯酸20:5(5,8,11,14,17)。
如本文中所用,术语“培养”指在允许细胞产生所述多不饱和脂肪酸,即上述PUFA和/或LCPUFA的培养条件下维持并生长宿主细胞。这意味着,在宿主细胞中表达本发明的多核苷酸,以存在nECR活性。以下详细描述了用于培养宿主细胞的合适培养条件。
如本文中所使用,术语“获得”包括提供细胞培养物(包括宿主细胞和培养基),以及提供其纯化的或部分纯化的制备物,所述制备物包含多不饱和脂肪酸,优选地以游离形式或者以-CoA结合形式,作为膜磷脂或者作为甘油三酯的ARA、EPA、DHA。更优选地,PUFA和LCPUFA作为甘油三酯而获得,例如以油的形式。在下文中其他地方可以找到更详细的纯化技术。
取决于宿主生物,可以以本领域技术人员熟知的方式生长或培养本发明方法中使用的宿主细胞。通常,宿主细胞在液体培养基中生长,所述培养基包含通常是糖形式的碳源、通常是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,温度在0℃和100℃之间,优选地在10℃和60℃之间,取决于生物的类型,在有氧或厌氧环境下。营养液的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提供。可以通过技术人员已知的方法,从上述的宿主细胞中分离所产生的PUFA或LCPUFA,所述方法例如通过提取、蒸馏、结晶、根据需要进行盐沉淀和/或层析。可能需要在纯化前破裂宿主细胞。为此,可事先破裂宿主细胞。使用的培养基必须符合所讨论的宿主细胞的要求。用于各种可用作本发明宿主细胞的微生物的培养基的描述可见于教材AmericanSocietyforBacteriology的“ManualofMethodsforGenaeralBacteriology”(WashingtonD.C.,USA,1981)。培养基也可以从多种商业供应商获得。所有的培养基成分均经加热或过滤灭菌法灭菌。所有的培养基成分可在培养开始时存在,或根据需要连续或分批式加入。如果已经将本发明的多核苷酸或载体引入到了另外包含可表达选择标记(例如抗生素抗性基因)的宿主细胞中,那么可能需要向培养物加入选择剂如抗生素,以维持引入的多核苷酸的稳定性。持续培养直至最大限度地形成目的产物。通常在10至160小时内实现所述目标。可以直接使用或者可以进一步加工该发酵培养基。根据需要,可以通过分离法例如离心、过滤、倾析或这些方法的组合,从发酵液中完全或部分地移出生物量,或者生物量完全滞留在所述培养液中。通过本发明方法获得的包含作为甘油三酯的期望的PUFA或LCPUFA的脂肪酸制备物,例如油,也适合作为用于化学合成另外的目的产物的原材料。例如,可以将它们相互组合或者单独地使用,用于制备药物或美容组合物、食品或者动物饲料。也可以通过上述方法产生包含期望的PUFA或LCPUFA的化学纯甘油三酯。为此,通过提取、蒸馏、结晶、层析或这些方法的组合进一步纯化脂肪酸制备物。为了从甘油三酯中释放脂肪酸部分,还需要进行水解。所述化学纯甘油三酯或游离脂肪酸特别适合于在食品工业中应用或者用于美容和药物组合物。
此外,本发明涉及用于产生多不饱和脂肪酸的方法,包括:
a)在允许在所述宿主细胞中产生多不饱和脂肪酸的条件下培养非人类转基因生物;和
b)从所述非人类转基因生物中获得所述多不饱和脂肪酸。
此外,由上可知,本发明还涉及用于产生油、脂质或脂肪酸组合物的方法,包括前述方法的任意之一的步骤和将PUFA或LCPUFA配制为油、脂质或脂肪酸组合物的步骤。优选地,可以将所述油、脂质或脂肪酸组合物用于饲料、食品、化妆品或药物。因此,对于各个涉及的产品,应当根据GMP标准实施PUFA或LCPUFA的配制。例如,可以通过油磨从植物种子中获得油。然而,对于生产安全原因,在适用的GMP标准下需要无菌。应当将类似的标准应用于待在美容或药物组合物中应用的脂质或脂肪酸组合物。前述生产包含了所有这些用于配制作为产品的油、脂质或脂肪酸组合物的措施。
本发明也涉及包含通过前述方法获得的多不饱和脂肪酸的油。
术语“油”指包含已酯化成甘油三酯的不饱和和/或饱和脂肪酸的脂肪酸混合物。优选地,本发明的油中的甘油三酯包含上述的PUFA或LCPUFA。酯化的PUFA和/或LCPUFA的量优选地为约30%,更优选的是50%的含量,甚至更优选的是60%、70%、80%或更高的含量。该油可以另外包含游离脂肪酸,优选上述PUFA和LCPUFA。对于分析,例如可以通过转酯作用将脂肪酸转化为甲酯后,通过GC分析确定脂肪酸的含量。油或脂肪中的多种脂肪酸含量可以变化,特别取决于来源。然而,关于PUFA和/或LCPUFA组合物和含量,该油应当具有非天然存在的组成。应当理解,只有通过应用上述的本发明方法才能获得在油的甘油三酯中PUFA和LCPUFA的此种独特的油组成和独特的酯化模式。此外,本发明的油还可以包含其他分子种类。具体而言,其可以包含较少的本发明多核苷酸或载体杂质。然而,只有通过高度敏感的技术如PCR,才能检测到此类杂质。
本申请中引用的所有参考文献的内容总体上并就其特定的上述公开内容而言通过引用作为参考。
附图
图1显示来自假微型海链藻的nECR的核苷酸(SEQIDNO:1)和氨基酸序列(SEQIDNO:2)。
图2显示来自三角褐指藻的nECR的核苷酸和氨基酸序列,如下:A)mRNA(SEQIDNO:3)的cDNA序列;B)翻译的氨基酸序列(SEQIDNO:4)。
图3显示来自假微型海链藻和三角褐指藻的nECR的氨基酸序列与来自酿酒酵母的脱水酶(YJL097W)和烯酰-CoA还原酶(YDL015C)的比对。
图4显示来自图3的比对的相似性表。通过使用来自VectorNTI软件包(Invitrogen)的Align程序的ClustalW比对完成同一性表。来自不同生物的两个nECR共有65%的同一性,而两种nECR都与来自酵母的已知脱水酶(YJL097W)或烯酰-CoA还原酶(YDL015C)具有低于30%的同一性。
图5显示通过酵母互补试验,nECR(Tp)和nECR(Pt)的功能性特征。互补试验用酵母Δydl015c中的nECR(Tp)和nECR(Pt)进行,所述酵母Δydl015c没有具备功能的烯酰-CoA还原酶。图注:YPD,完全培养基,SD-Ura,缺少尿嘧啶的培养基,SD-Leu,缺少亮氨酸的培养基,FOA,含亮氨酸和5-FOA的培养基;(1)tsc13ΔTrppTSC13URA:用含具备功能的YDL015C基因之载体pTSC13转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C;(2)phs1ΔKanpPHS1URA:用含具备功能的YJL097W基因之载体pPHS1转化的缺少脱水酶功能的酵母突变体Δyjl097w;(3)tsc13ΔTrppESC-nECR(Tp):用载体pESC-nECR(Tp)转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C;(4)sc13ΔTrppESC-nECR(Pt):用载体pESC-nECR(Pt)转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C。
图6显示具有nECR(Tp)的长链PUFA的增加产生。具有nECR(Tp)的长链PUFA的增加产生。在SD(-Ura-Leu)培养基中,用25μM亚麻酸(18:3Δ9,12,15)培养用pYES-d9Elo(Ig)(A)或YES-d9ELo(Ig)+pESC-nECR(Tp)(B)转化的酵母。温育48小时后,离心酵母细胞并将沉淀进行气相色谱分析。气相色谱显示不具有(A)和具有nECR(Tp)(B)的两种不同酵母菌株中不同的脂肪酸。如下进行转化率作图:(产物/底物-产物)×100。
图7显示假微型海链藻亚细胞部分的WesternBlot分析。M,蛋白质大小标记,1,总提取物,2,可溶性部分,3,膜部分。箭头指示两种型式的nECR(Tp)。
图8显示来自表达nECR(Tp)的酵母的亚细胞部分的WesternBlot分析。M,蛋白质大小标记,1,总提取物,2,可溶性部分,3,膜部分。箭头指示nECR(Tp)融合蛋白。
图9显示来自表达nECR(Tp)的酵母的亚细胞部分的WesternBlot分析。M,蛋白质大小标记,1,总提取物,2,可溶性部分,3,膜部分。箭头指示nECR(Tp)融合蛋白。
本发明通过以下实施例进一步说明,而不应将其解释为限制本发明。本申请中引用的所有参考文献、专利和公开的专利申请,以及附图都在此引入作为参考。
实施例
实施例1:生物和培养条件
为了产生单倍体,使用在Pan等人2004(MolecularCell16:487-496)中概述的方法。简言之,在DOB-尿嘧啶中过夜培养培养物,然后洗涤大约25OD600的每一培养物,并在新鲜培养基中重悬,并培养3小时。然后在孢子形成培养基(sporulationmedia)(1%乙酸钾、0.005%乙酸锌)中悬浮细胞,形成孢子5天并铺展在含200mg/LG418和60mg/L刀豆氨酸的单倍体选择魔法(magic)培养平板(2%半乳糖、氨基酸混合物-尿嘧啶-亮氨酸-组氨酸-精氨酸、0.17%没有氨基酸或硫酸铵的氮源(nitrogenbase)、0.1%谷氨酸钠)上。
实施例2:新的脱水酶/烯酰-CoA还原酶序列
筛查产PUFA藻类,以显示非PUFA产生者之间的差异。获得来自海链藻属(Thalassiosira)和褐指藻属(Phaeodactylum)的序列,其对于这些PUFA产生者是特异的(图1和2)。与来自酵母(YJL097W和YDL015C)的已知脱水酶和烯酰-CoA还原酶序列的比对显示出较低的同源性(图3和4)。因此,新产生的nECR代表了一种新的酶种类。通过互补试验(实施例3),这些序列显示出脱水酶和烯酰-CoA还原酶活性,并因此命名为nECR。
已鉴定的全长编码序列的列表显示于表1a和1b中。
表1a:全长编码序列的列表
SEQ ID NO: | 基因 | 生物 | bp长度 |
1 | nECR(Tp) | 假微型海链藻 | 2367 |
3 | nECR(Pt) | 三角褐指藻 | 2244 |
表1b:从表1中所述序列推断的氨基酸的列表
SEQ ID NO: | 基因 | 生物 | 氨基酸长度 |
2 | nECR(Tp) | 假微型海链藻 | 788 |
4 | nECR(Pt) | 三角褐指藻 | 747 |
根据厂商反应条件,将表1中所示的可读框克隆入来自Stratagene的pESC(Leu)载体中。将反应物转化到大肠杆菌DH5α中并分离质粒DNA。然后将质粒pESC-nECR(Tp)、pESC-nECR(Pt)用于酵母转化。由于突变酵母菌株Δydl015C(Δtsc13)和Δyjl097w(Δphs1)二者都是致死的,所以用有尿嘧啶-营养缺陷型标记URA互补这些突变体的质粒(pTSC13和pPHS1)转化菌株。通过使用5-FOA(5-fluorooroticacid;Sadowski等人Yeast.2008年8月;25(8):595-9),可以在酵母中除去含有URA标记的载体。
实施例3:酵母转化和生长条件
使用S.C.EasyCompTransformation试剂盒(Invitrogen,Carlsbad,California),用构建体pESC-nECR(Tp)、pESC-nECR(Pt)和pESC转化来自OpenBiosystems的酿酒酵母菌株YSC1021-674054,并在亮氨酸缺陷的培养基上选择。对于评估脱水酶/烯酰-CoA还原酶活性,进行互补研究。为此,使用来自OpenBiosystems的杂合魔法标记菌株YSC1021-674054。该菌株不显示出任一烯酰-CoA还原酶活性。由于烯酰-CoA还原酶活性递送延伸的脂肪酸,并且这些脂肪酸对于细胞生长和分裂是必需的,所以各个酵母菌株不能在不含所述延伸的脂肪酸的培养基上生长。
产生了以下的转化体:
1.tsc13ΔTrppTSC13URA:用含具备功能的YDL015C基因之载体pTSC13转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C。
2.phs1ΔKanpPHS1URA:用含具备功能的YJL097W基因之载体pPHS1转化的缺少脱水酶功能的酵母突变体Δyjl097w。
3.tsc13ΔTrppESC-nECR(Tp):用载体pESC-nECR(Tp)转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C。
4.sc13ΔTrppESC-nECR(Pt):用载体pESC-nECR(Pt)转化的缺少烯酰-CoA还原酶功能的酵母突变体Δydl015C。
转化后,在含所有氨基酸和核苷酸的完全培养基中培养酵母。然后,在不同的培养基上接种酵母,所述不同的培养基包括完全培养基(SD)、缺少尿嘧啶的完全培养基(SD-Ura)、缺少亮氨酸的完全培养基(SD-Leu)或者缺少亮氨酸并含有5-FOA的完全培养基(图5)。质粒1和2可以在SD和SD-Ura上生长,但由于它们不具有LEU标记,所以不能在SD-Leu上生长。质粒3和4可以在SD和SD-Leu上生长,但由于它们缺失URA标记,所以不能在SD-Ura上生长。在含FOA的平板上显示互补,所述平板去除了具有URA标记的质粒(1和2)。然而,即使在质粒1或2不存在的情况下,也存在具有质粒3和4的克隆的生长(图5,FOA)。
因此,序列nECR(Tp)和nECR(Pt)二者都能互补酵母中的致死无效突变3-羟酰基CoA脱水酶基因Δydl015C。
总之,通过缺陷型酵母突变体的互补,显示出序列nECR(Tp)和nECR(Pt)是具有生物学功能的并表现出烯酰-CoA还原酶活性。
实施例4:将nECR(Tp)与来自绿光等鞭金藻的d9-延伸酶组合表达
为了检查植物中nECR(Tp)在产生多不饱和脂肪酸中的用途,例如用于营养食品的用途,将该基因与PUFA延伸酶组分(自绿光等鞭金藻的d9-延伸酶(WO2002/077213))组合进行表达。该酶催化亚油酸或亚麻酸(18:2Δ9,12或18:3Δ9,12,15)的延伸。实验的目的是分析,如果添加nECR(Tp)是否会增加来自绿光等鞭金藻的d9-延伸酶的产生。为此,将用pESC-nECR(Tp)转化并培养在DOB(-亮氨酸)平板上的酵母细胞(来自Invitrogen的INVSC)如实施例1中所述用质粒pYES(Ura)-d9Elo(Ig)转化,并培养在DOB(-尿嘧啶、-亮氨酸)上。载体pYES(Ura)来源于Invitrogene并介导尿嘧啶的营养缺陷型。根据厂商条件,将如在WO2002/077213中所述的d9Elo(Ig)的可读框克隆入pYES2中。作为对照实验,将pYES-d9Elo(Ig)转化入仅含有pYES载体的对照酵母菌株中。
令人惊奇地,观察到对照(pESC-d9Elo(Ig))和含延伸复合物的双组份(pESC-d9Elo(Ig)+pYES-nECR(Tp))的酵母之间延伸产物20:3的量的差异。
图6中显示,nECR(Tp)基因的添加对长链PUFA的产率具有巨大的影响。与对照实验比较,产率增加了8倍。产率根据底物18:3(外源性添加到酵母培养基中)向延长的PUFA脂肪酸20:3的转变进行测量。
总之,nECR极大地改进了延长的脂肪酸,如对人类健康有益的长链PUFA的产生。
实施例5:酵母表达的nECR(Tp)和来自假微型海链藻的天然蛋白质的比较
根据厂商实践(Eurogentec,Belgium;肽抗体)产生针对nECR(Tp)的抗体。该抗体对于假微型海链藻中的nECR(Tp)和在酵母中异源表达的是高度特异的(图7和图8)。在使用抗体的情况下,可以观察到天然生物产生的和异源表达的之间的结构差异(图7和图8)。
使用标准步骤进行WesternBlot实验:
使用来自Biorad的预制胶,根据Laemmli(1970)进行SDS-PAGE。对于上样缓冲液,使用0.05MTris/HClpH6.8、0.1MDTT、2%(w/v)SDS、0.1%Bromphenolicblue和10%甘油。然后使用ProteanBA85硝酸纤维素膜(Schleicher&Schuell)在硝酸纤维素上印迹SDS-PAGE凝胶。使用含15mMNa2HPO4pH7.2,0.05%(w/v)SDS,20%(v/v)甲醇的缓冲液,在200mA、40V(ProteanII,Biorad)进行膜上的转移2小时。对于免疫学测试,将膜在PBS(0.14MNaCl,2.7mMKCl,10mMNa2HPO4,1,8mMKH2PO4pH7.4)、5%(w/v)奶粉中封闭1小时。将含针对nECR(Tp)的抗体的血清以1∶2000的浓度加入,并在4℃温育过夜。对于抗体的检测,将膜用PBS洗涤3次,并再次用PBS,5%(w/v)奶粉封闭30分钟。加入1个单位的第二抗体(Biorad抗兔辣根过氧化物酶(anti-rabbithorseradishperoxidase))并另外温育30分钟。用PBS洗涤3次后,将膜浸没在ECL溶液中并温育1分钟。然后,用手巾纸除去溶液并用Saran包裹膜。在BioIlluminator(LKB)中进行化学发光的检测。
对于分离自假微型海链藻的蛋白质,在F2培养基中在20℃温育500mL的培养物14天(使用如在Tonon等人(Tono2005,FEBSJ.272:3401-3412)中所述的用于假微型海链藻的生长条件和培养基)。通过离心(10分钟,5000×g)收集藻类,并将沉淀放入研钵中。使用pistil产生细粉末。将粉末用50mMTris/HClpH8.0、2mMEDTA悬浮,并经两层Miracloth(Merck)或任意其他过滤膜过滤。然后等分过滤产物,并用SDS-PAGE上样缓冲液(见上述)混合50uL的等份样品。
通过WesternBlot在假微型海链藻中检测的nECR(Tp)的分析(图7)显示,可以在细胞碎片、可溶性和膜级分中发现nECR(Tp)的蛋白质。令人惊奇地,在假微型海链藻中,主要形式是仅含烯酰-CoA还原酶活性(ECR)的切割形式。ECR结构域的证据推断自抗体在ECR结构域中的结合位点的位置。在可溶性级分中仅发现少量nECR(Tp)的较大融合蛋白质。在膜级分中没有发现nECR(Tp),甚至在更长的曝光时间后。
因此,可以断定,在假微型海链藻中存在两种形式的nECR(Tp),推断自cDNA(SEQIDNO1)的蛋白质和仅含ECR结构域的翻译后修饰的更短形式。只有ECR结构域是膜结合的,并因此正确地定位(ECR在延伸酶复合物中的功能在微粒体的膜上发生(Napier2007,AnnuRevPlantBiol58:295-319))。
随着在酵母中异源表达nECR(Tp),可以观察到不同的景象。如在实施例4中所述,将具有pESC-nECR(Tp)的酵母用于蛋白质提取。在28℃,在50ml培养物中培养酵母3天,并通过离心(10分钟,5000×g)收集沉淀。等分沉淀,用液氮冷冻,并加入与Qiagen/Tresch碾磨机系统兼容的钢珠。将沉淀在Tresch碾磨机中处理5分钟用于细胞破裂。通过100’000×g离心步骤30分钟,将总细胞提取物分离成可溶性和膜级分。沉淀构成了膜级分,上清液是可溶性级分。将所有3种级分进行如上所述的SDS-PAGE和Western分析,并进行分析(图8)。在酵母中,没有观察到nECR(Tp)的切割。相对于分子量,酵母表达的融合蛋白质大约为86kDa,其与假微型海链藻中的nECR(Tp)类似。不存在59kDaECR的切割形式。因此,可以断定,由于nECR(Tp)以其非切割形式发挥功能,互补了酵母KO突变体(实施例3),因此其代表了一种新的蛋白质类型。此外,详细分析了可溶性和微粒体级分,以检查是否存在任何nECR(Tp)的可溶性组分(图9)。再次将上清液和微粒体级分上样在凝胶上,并曝光更长的时间。在可溶性级分中没有发现nECR(Tp),这再次显示出与来自假微型海链藻的天然形式的结构性差异。
总之,发现了新的融合蛋白质,其令人惊奇地含有延伸复合物的两种酶活性(ECR和DH)。SEQIDNO.1产生了功能性融合蛋白质在异源系统中的表达,其与假微型海链藻中的天然蛋白质具有不同的结构特性。
实施例6:植物中nECR(Tp)和nECR(Pt)的表达
如在WO2003/093482、WO2005/083093或WO2007/093776中所述,将来自假微型海链藻和三角褐指藻的新nECR克隆到植物转化载体中。在表3、4和5中描述了基因的示例性合适组合。
表3:用于产生ARA的基因组合
表4:用于产生EPA的基因组合
表5:用于产生DHA的基因组合
如在Deblaere等人,1984,Nucl.Acids.Res.13,4777-4788中所述产生转基因油菜株系,并如在Qiu等人2001,J.Biol.Chem.276,31561-31566中所述分析转基因油菜植物的种子。
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Claims (8)
1.用于产生多不饱和脂肪酸的方法,包括:
a)在允许多不饱和脂肪酸在转基因植物细胞中产生的条件下培养所述转基因植物细胞,其中所述转基因植物细胞包含多核苷酸,所述多核苷酸包含选自以下的核酸序列:
i)由在SEQIDNO:1或3中所示的核苷酸序列组成的核酸序列;
ii)编码由在SEQIDNO:2或4中所示的氨基酸序列组成的多肽的核酸序列;和
b)从所述转基因植物细胞获得所述多不饱和脂肪酸。
2.用于产生多不饱和脂肪酸的方法,包括:
a)在允许多不饱和脂肪酸在转基因植物中产生的条件下培养所述转基因植物,其中所述转基因植物包含多核苷酸,所述多核苷酸包含选自以下的核酸序列:
i)由在SEQIDNO:1或3中所示的核苷酸序列组成的核酸序列;
ii)编码由SEQIDNO:2或4中所示的氨基酸序列组成的多肽的核酸序列;和
b)从所述转基因植物获得所述多不饱和脂肪酸。
3.权利要求1或2的方法,其中所述多核苷酸另外包含与所述核酸序列有效连接的表达调控序列。
4.权利要求1或2的方法,其中所述多核苷酸另外包含与所述核酸序列有效连接的终止子序列。
5.权利要求1或2的方法,其中所述的多核苷酸包含在载体中。
6.权利要求1或2的方法,其中所述多不饱和脂肪酸是花生四烯酸(ARA)、二十碳五烯酸(EPA)或二十二碳六烯酸(DHA)。
7.用于产生油、脂质或脂肪酸组合物的方法,包括权利要求1至6中任一项所述的方法的步骤和将所述多不饱和脂肪酸配制为油、脂质或脂肪酸组合物的另外步骤。
8.权利要求7的方法,其中将所述油、脂质或脂肪酸组合物用于饲料、食品、化妆品或药物。
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EP2176433B1 (de) | 2007-07-31 | 2011-10-12 | BASF Plant Science GmbH | Desaturasen und verfahren zur herstellung mehrfach ungesättigter fettsäuren in transgenen organismen |
WO2009016208A2 (de) | 2007-07-31 | 2009-02-05 | Basf Plant Science Gmbh | Elongasen und verfahren zur herstellung mehrfach ungesättigter fettsäuren in transgenen organismen |
WO2009133145A1 (en) * | 2008-04-30 | 2009-11-05 | Basf Plant Science Gmbh | Desaturase and method for the production of polyunsaturated fatty acids in transgenic organisms |
-
2010
- 2010-05-20 CN CN201080034907.XA patent/CN102597242B/zh not_active Expired - Fee Related
- 2010-05-20 CA CA2763107A patent/CA2763107A1/en not_active Abandoned
- 2010-05-20 JP JP2012514410A patent/JP2012529277A/ja active Pending
- 2010-05-20 BR BRPI1010614A patent/BRPI1010614A2/pt not_active Application Discontinuation
- 2010-05-20 DE DE112010002353T patent/DE112010002353T5/de not_active Ceased
- 2010-05-20 EP EP10723054.2A patent/EP2440662B1/en not_active Not-in-force
- 2010-05-20 AU AU2010257729A patent/AU2010257729B2/en not_active Ceased
- 2010-05-20 US US13/376,745 patent/US8993841B2/en not_active Expired - Fee Related
- 2010-05-20 WO PCT/EP2010/056936 patent/WO2010142522A2/en active Application Filing
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2011
- 2011-12-07 CL CL2011003104A patent/CL2011003104A1/es unknown
Also Published As
Publication number | Publication date |
---|---|
CL2011003104A1 (es) | 2012-06-01 |
CA2763107A1 (en) | 2010-12-16 |
DE112010002353T5 (de) | 2012-08-09 |
EP2440662B1 (en) | 2018-08-01 |
WO2010142522A3 (en) | 2011-04-14 |
US8993841B2 (en) | 2015-03-31 |
AU2010257729B2 (en) | 2016-03-24 |
EP2440662A2 (en) | 2012-04-18 |
CN102597242A (zh) | 2012-07-18 |
WO2010142522A2 (en) | 2010-12-16 |
BRPI1010614A2 (pt) | 2016-10-25 |
AU2010257729A1 (en) | 2012-01-12 |
US20120084889A1 (en) | 2012-04-05 |
JP2012529277A (ja) | 2012-11-22 |
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