CN1984994A - 重组蔗糖合酶的生产方法、及其在生产蔗糖测定试剂盒中的应用、生产腺苷二磷酸葡糖的方法和获得具有积累了高浓度的腺苷二磷酸葡萄糖和淀粉的叶片和储藏器官的转基因植物的方法 - Google Patents
重组蔗糖合酶的生产方法、及其在生产蔗糖测定试剂盒中的应用、生产腺苷二磷酸葡糖的方法和获得具有积累了高浓度的腺苷二磷酸葡萄糖和淀粉的叶片和储藏器官的转基因植物的方法 Download PDFInfo
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Abstract
本发明涉及一种用于大量可溶性重组的SS在其活性形式的有效生产的方法,其通过在一种大肠杆菌(Escherichia coli)菌株内表达编码SS的基因。所应用的表达载体使得这样生产的重组SS具有组氨酸尾,从而有助于其快速纯化。另外,本发明描述了SS基因的突变版本的序列,其编码适于产生ADPG的SS异构体。利用“野生型”和“突变型”版本的重组体SS,本发明还描述了一种有效用于生产ADPG和UDPG的方法。本发明进一步描述了将SS用于生产测定蔗糖的测试试剂盒的应用。最后,本发明描述了一种获得转基因植物的方法,所述转基因植物组成性地过量表达或在其储藏器官或叶片中过量表达SS基因,并且由于SS的高ADPG合成活性,该转基因植物具有高浓度的(在叶片和储藏组织中)蔗糖、ADPG、G6P和淀粉。
Description
发明所涉及的产业领域
本发明涉及利用适当的大肠杆菌(Escherichia coli)菌株最优化生产对可溶的、活性形式的重组蔗糖合酶(SS),SS用于制备测定蔗糖的试剂盒的应用,设计SS的最佳形式以便合成腺苷二磷酸葡萄糖(ADPG),和转基因植物的生产,所述叶片和贮藏组织积累高水平的ADPG和富含直链淀粉的淀粉,原因在于在过量表达SS的植物中胞质ADPG的过量生产。
现有技术
淀粉是植物中碳水化合物的主要贮存形式。它在诸如种子(小麦属、大麦属、玉蜀黍属、豌豆属等)和块茎(其中是马铃薯和薯蓣属)等器官中大量积累,而且是人类饮食的基本成分。并且,淀粉在纸张、化妆品、药品和食品工业中广泛应用,而且还用作制造生物可降解塑料和环境友好油漆的基本成分。由于它是由共价结合的葡萄糖分子组成,因此,在工业生产的多个领域中优先对这种多糖合成中所涉及的过程进行研究。
ADPG是植物中在异养器官(图1A)和叶片中(图2A)淀粉生物合成的通用前体,并且广泛地认为它的生产由ADPG焦磷酸化酶(AGPase)或ADPG合酶(EC 2.7.7.27)专一地控制(Okita,T.W.(1992)Is there analternative pathway for starch synthesis?Plant Physiol.100,560-56;Müller-Rber,B.,Sonnewald,U.Willmitzer,L.(1992)Inhibition of theADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storingtubers and influences tuber formation and expression of tuber storage proteingenes.EMBO J.11,1229-1238;Stark,D.M.,Timmerman,K.P.,Barry,G.F,Preiss,J.,Kishore,G.M.(1992)Regulation of the amount of starch in planttissues by ADPglucose pyrophosphorylase.Science 258,287-282;Neuhaus,E.H.,Husler,R.E.,Sonnewald,U.(2005)No time to shift the paradigm onthe metabolic pathway to transitory starch in leaves.Trends Plant Sci.atpress)。植物中所产生的淀粉的多种应用主要基于直链淀粉和支链淀粉的比例,它决定了淀粉颗粒的结构以及它在水性悬浮液中的黏性。所述直链淀粉和支链淀粉的比例,除了其它因素外,取决于植物细胞中ADPG的浓度(Clarke,B.R.,Denyer,K.,Jenner,C.F.,Smith,A.M.(1999)Therelationship between the rate of starch synthesis,the adenosine5’-diphosphoglucose concentration and the amylose content of starch indeveloping pea embryos.Planta 209,324-329)。
SS(EC 2.4.1.13,SS)(UDP-葡萄糖:D-果糖-2-葡萄糖苷转移酶)是一种催化从蔗糖和UDP到UDPG和果糖的生产的可逆酶。尽管,如图1A所示,传统上认为SS具有产生UDPG的作用,其代谢过程导致淀粉在诸如胚乳和块茎的异养组织中产生(Zrenner,R.,Salanoubat,M.,Willmitzer,L.,Sonnewald,U.(1995)Evidence for the crucial role of sucrose synthase forsink strength using transgenic potato plants.Plant J.7,97-107;Boroja-Fernández,E.,
F.J.,Saikusa,T.,Rodríguez-López,M.,Akazawa,T.,Pozueta-Romero,J.(2003)Sucrose synthase catalyzes the de novo productionof ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants.Plant Cell Physiol. 44,500-509;Pozueta-Romero,J.,
F.J.,Rodríguez-López,M.,Boroja-Fernández,E.,Akazawa,T.(August 2003)Newwaves in the starch field.Lett.Plant Cell Physiol.24-32),但是有参考文献报道该酶在体外应用其它的核苷酸二磷酸生产相应的糖核苷酸的潜能(Murata,T.,Sugiyama,T.,Minamikawa,T.,Akazawa,T.(1996)Enzymicmechanism of starch synthesis in ripening rice grains. Mechanism of thesucrose-starch conversion.Arch.Biochem.Biophys.113,34-44;Delmer,D.P.(1972)The purification and properties of sucrose synthase from etiolatedPhaseolus aureus seedlings.J.Biol.Chem.247,3822-3828)。尽管有可疑的生理相关性(Okita,T.W.(1992)Is there an alternative pathway for starchsynthesis?Plant Physiol.100,560-56;Müller-Rber,B.,Sonnewald,U.Willmitzer,L.(1992)Inhibition of the ADPglucose pyrophosphorylase intransgenic potatoes leads to sugar-storing tubers and influences tuberformation and expression of tuber storage protein genes. EMBO J. 11,1229-1238),已表明SS能够直接产生ADPG,其可以用于异养组织和光合组织(图1B和2B)中的淀粉生产(Pozueta-Romero,J.,Perata,P.,Akazawa,T.(1999)Sucrose-starch conversion in heterotrophic tissues of plants.Crit.Rev.Plant Sci.18,489-525;Baroja-Fernández,E.,
F.J.,Akazawa,T.,Pozueta-Romero,J.(2001)Reappraisal of the currently prevailing model ofstarch biosynthesis in photosynthetic tissues:a proposal involving thecytosolic production of ADPglucose by sucrose synthase and occurrence ofcyclic turnover of starch in the chloroplast.Plant Cell physiol.42,1311-1320;Baroja-Fern ández,E.,
F.J.,Saikusa,T.,Rodríguez-López,M.,Akazawa,T.,Pozueta-Romero,J.(2003)Sucrose synthase catalyzes the denovo production of ADPglucose linked to starch biosynthesis in heterotrophictissues of plants.Plant Cell Physiol.44,500-509;Baroja-Fernández,E.,F.J.,Zandueta-Criado,A.,Morán-Zorzano,M.T.,Viale,A.M.,Alonso-Casajús,N.,Pozueta-Romero,J.(2004)Most of ADPglucose linked tostarch biosynthesis occurs outside the chloroplast in source leaves.Proc.Natl.Acad.Sci.USA 101,13080-13085)。按照这一假说(完全和详尽地基于生物化学类型的证据),SS负责淀粉生物合成必需的重要的ADPG分子库的合成。然而,这一假说没有由基因工程或传统的作物改良技术在实验上得以证明,并且与表明AGPase是植物中ADPG的唯一来源的遗传和分子类型的无数的测试不相一致。(Okita,T.W.(1992)Is there an alternativepathway for starch synthesis?Plant Physiol.100,560-56;Müller-Rber,B.,Sonnewald,U.Willmitzer,L.(1992)Inhibition of the ADPglucosepyrophosphorylase in transgenic potatoes leads to sugar-storing tubers andinfluences tuber formation and expression of tuber storage protein genes.EMBO J.11,1229-1238;Neuhaus,E.H.,Husler,R.E.,Sonnewald,U.(2005)No time to shift the paradigm on the metabolic pathway to transitory starch inleaves.Trends Plant Sci.at press)
基于一种叫做葡萄糖-1-磷酸(G1P)的昂贵的物质的使用,诸如UDPG和ADPG的糖核苷酸在商业上分别由诸如UDPG焦磷酸化酶(UGPase)和AGPase的酶催化的焦磷酸化酶反应而生产。对实行糖核苷酸生产的备选方法是基于SS的使用,其发展很大程度上受到大肠杆菌(Escherichiacoli)对大量的真核蛋白表达和有效处理的局限性的妨碍。这种局限性激发一些研究者通过应用诸如酵母菌的真核类型的生物工厂(biologicalfactories)来生产重组的SS(Zervosen,A.,Rmer,U.,Elling,L.(1998)Application of recombinant sucrose synthase-large scale synthesis ofADP-glucose.J.Mol.Catalysis B:Enzymatic 5,25-28;Rmer,U.,Schrader,H.,Günther,N.,Nettelstroth,N.,Frommer,W.B.,Elling,L.(2004)Expression,purification and characterization of recombinant sucrose synthase I fromSolanum tuberosum L.for carbohydrate engineering.J.Biotechnology 107,135-149)。备选地,用于糖核苷酸生产的SS必须通过从植物提取液纯化蛋白的昂贵过程进行纯化(专利DE4221595(1993),纯化的蔗糖合酶用于核苷酸激活的糖或寡糖的生产)。所述从植物提取液中获得的SS具有如下缺点:它具有对UDP的偏好和对ADP的很低的亲和力(Pressey R(1969)Potato sucrose synthase:purification,properties,and changes in activityassociated with maturation.Plant Physiol.44,759-764;Nguyen-Quock,B.,Krivitzky,M.,Huber,S.C.,Lecharny,A.(1990)Sucrose synthase indeveloping maize leaves.Plant Physiol.94,516-523;Morell,M.,Copeland,L.(1985)Sucrose synthase of soybean nodules.Plant Physiol.78,149-154)。最近,已经完成了从大肠杆菌(E.coli)培养物中纯化重组SS(Nakai,T.,Tonouchi,N.,Tsuchida,T.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“Expression and characterization of sucrose synthase from mung bearseedling in Escherichia coli”Biosci.Biotech.Biochem.61,1500-1503;Nakai,T.,Konishi,T.,Zhang,Z-Q.,Chollet,R.,Tonouchi,N.,Tsuchida,T.,Yoshinaga,F.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“An increase inapparent affinity for sucrose of mung bean sucrose synthase is caused by invitro phosphorylation or directed mutagenesis of Serll”Plant Cell Physiol.39,1337-1341;Barratt,D.H.P.,Barber,L.,Kruger,N.J.,Smith,A.M.,Wang,T.L.,Martin,C.(2001)Multiple,distinct isoforms of sucrose synthase in pea.PlantPhysiol.127,655-664;Christopher,B.,William,B.,Robert,H.“Bacterialsucrose synthase compositions and methods of use”专利WO 9803637)。然而,在这种原核系统内生产SS与下述问题相关,如:(1)产生的SS的量很低(30毫克/克细菌,Nakai,T.Tonouchi,N.,Tsuchida,T.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“Expression and characterization of sucrose synthasefrom mung bean seedlings in Escherichia coli”Biosci.Biotech.Biochem.61,1500-1503;Li,C.R.,Zhang,X.B.,Hew,C.S.(2003)“Cloning,characterization and expression analysis of a sucrose synthase gene fromtropical epiphytic orehid Oncidium goldiana.Physiol.Plantarum 118,352-360),(2)获得的活性SS的量很低或等于零(0.05-1.5单位/mg,(Nakai,T.,Tonouchi,N.,Tsuchida T.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“Expression and characterization of sucrose synthase from mung beanseedlings in Escherichia coli”Biosci.Biotech.Biochem.61,1500-1503;Li,C.R.,Zhang,X.B.,Hew,C.S.(2003)“Cloning,characterization andexpression analysis of a sucrose synthase gene from tropcal epiphytic orchidOncidium goldiana.Physiol.Plantarum 118,352-360);5.6 U/mg(Rmer,U.,Schrader,H.,Günther,N.,Nettelstroth,N.,Frommer,W.B.,Elling,L.(2004)Expression,purification and characterization of recombinant sucrose synthaseI from Solanum tuberosum L.for carbohydrate engineering.J.Biotechnology107,135-149),(3)重组的SS必须通过诸如层析、电泳、等电聚焦等蛋白纯化的常规方法进行纯化,上述方法,结合起来,证明是昂贵的而且不能保证处于均一状态下的蛋白的纯化,和(4)由于细菌的正确折叠蛋白的机制失活,大部分的SS形成包含体或以无活性聚集体的形式聚集下来,(Miroux,B.,Walker,J.E.(1996)“Over-production of proteins in Escherichiacoli:mutant hosts that allow synthesis of some membrane proteins andglobular proteins at high levels”J.Mol.Biol.260,289-298)。
本发明描述一种系统的开发,该系统基于适宜的大肠杆菌(E.coli)的应用和合适的表达载体的应用,其中所述的表达载体允许大规模生产和对活性形式的重组SS的不同变种的快速而容易的纯化。一些所述的变种具有比从植物提取液中获得的那些蛋白更高的对ADP的亲和力,可以用于从诸如蔗糖、UDP和ADP的便宜物质生产UDPG和ADPG。
层析技术组成用于测定诸如植物提取液、血清、尿、果汁、酒、水果和食品的复合样品中蔗糖含量的有力工具(D’Aoust,M-A.,Yelle,S,Nguyen-Quock,B.(1999)Antisense inhibition of tomato fruit sucrosesynthase decreases fruit setting and the sucrose unloading capacity of youngfruit.Plant Cell 11,2407-2418;Tang,G-Q.,Sturm,A.(1999)Antisenserepression of sucrose synthase in carrot affects growth rather than sucrosepartitioning.Plant Mol.Biol.41,465-479;Frias,J.,Price,K.R.,Fenwich,G.R.,Hedley,C.L.,Sorensen,H.,Vidal-Valverde,C.(1996)J.Chromatogr.A 719,213-219)。此类技术要求高级专业技术人员并包括在设备上的大量投资。不幸地是,基于通过转化酶作用的蔗糖分子水解和后续的对葡萄糖和/或果糖分子的分光光度和荧光测定的备选方法(Sweetlove,L.J.,Burrell,M.M.,ap Rees,T.(1996)Starch metabolism in tubers of transgenic potatowith increased ADPglucose pyrophosphorylase.Biochem.J.320,493-498;Stitt,M.,Lilley,R.M.,Gerhardt,R.,Heldt,H.W.(1989)Metabolite levels inspecific cells and subcellular compartments of plant leaves.Methods Enzymol.174,518-552;Holmes,E.W.(1997)Coupled enzymatic assay for thedetermination of sucrose.Anal.Biochem.244,103-109;Methods of Analysis(1996)Code of Practice for Evaluation of Fruit and Vegetable Juices.Association of the Industry of Juices and Nectars from Fruits and Vegetablesofthe Eurcpean Economic Community)受到技术本质的局限,例如对应样品中存在的内源葡萄糖和/或果糖的测量的减少。样品中葡萄糖和/或果糖的丰度能够增加妨碍蔗糖的可靠而准确测定的本底噪声。在大多数情况下,在得出关于样品的真实蔗糖含量的可靠陈述前,有必要进行彻底的检验(Worrell,A.C.,Bruneau,J-M.,Summerfelt,K.,Boersig,M.,Voelker,T.A.(1991)Expression of a maize sucrose phosphate synthase in tomato alters leafcarbohydrate partitioning.Plant Cell 3,1121-1130)。基于转化酶的应用的测定蔗糖的试剂盒可以从诸如Sigma、Biopharm GmbH和Megazyrne的公司获得。备选地,已经开发了一种蔗糖测定的自动化方法,其基于对由于细菌源性的蔗糖磷酸化酶的作用而释放的葡萄糖-1-磷酸的测定(Vinet,B.,Panzini,B.,Boucher,M.,Massicotte,J.(1998)Automated enzymatic assayfor the determination of sucrose in serum and urine and its use as a marker ofgastric damage.Clin.Chem.44,2369-2371)。本发明描述开发一种简单的、可靠的和便宜的测定样品中蔗糖的备选方法,其基于SS和水解ADPG或UDPG的偶联酶的应用。
关于细胞内ADPG水平的控制因素的考虑主要围绕在合成酶AGPase的调节上(Preiss,(1988)Biosynthesis of starch and its regulation.TheBiochemistry of Plants.14卷,Academic Press,纽约,182-249页;Pozueta-Romero,J.,Perata,P.,Akazawa,T.(1999)Sucrose-starch conversionin heterotrophic tissues.Crit.Rev.Plant.Sci.18,489-525)。实际上,大部分关于ADPG生产和产生工业目的淀粉的植物的生产的专利和科学出版物围绕着AGPase的应用(Stark,D.M.,Timmerman,K.P.,Barry,G.F.,Preiss,J.,Kishore,G.M.(1992)Regulation of the amount of starch in plant tissues byADPglucose pyrophosphorylase.Science 258,287-282;Slattery,C.J.,Kavakli,H.,Okita,T.W.(2000)Engineering starch for increased quantity and quality.Trends Plant Sci.5,291-298)。然而,尽管它们还没有用遗传/分子类型的证据所证实,近来的生物化学类型的科学研究表明,如图1B和2B所示,SS可能参与淀粉生物合成必需的ADPG的直接合成中(Baroja-Fernández,F.,
F.J.,Saikusa,T.,Rodríguez-López,M.,Akazawa,T.,Pozueta-Romero,J.(2003)Sucrose synthase catalyzes the de novo productionof ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants.Plant Cell Physiol.44,500-509)。这一假说是非常有争议的,记住:(a)SS从未与叶片中淀粉的生产相关,(b)在质体膜上需要存在ADPG的易位体,该易位体将SS产生的ADPG胞质库与质体内部存在的淀粉合酶连系起来,和(c)包含SS作为ADPG生产来源是直接与许多生物化学/遗传/分子类型的测试相抵触的,所述测试看起来是表明AGPase是ADPG唯一的来源(Okita,T.W.(1992)Is there an alternative pathway for starch synthesis?Plant Physiol.100,560-56;Müllen-Rber,B.,Sonnewald,U.Willmitzer,L.(1992)Inhibition of the ADPglucose pyrophosphorylase in transgenicpotatoes leads to sugar-storing tubers and influences tuber formation andexpression of tuber storage protein genes.EMBO J.11,1229-1238;Stark,D.M.,Timmerman,K.P.,Barry,G.F.,Preiss,J.,Kishore,G.M.(1992)Regulation of the amount of starch in plant tissues by ADPglucosepyrophosphorylase.Science 258,287-282;Neuhaus,E.H.,Husler,R.E.,Sonnewald,U.(2005)No time to shift the paradigm on the metabolic pathwayto transitory starch in leaves.Trends Plant Sci.at press)。可能由于所有这些原因,直到现在从未设计过量表达SS用于生产高水平的淀粉的植物。然而,本发明第一次描述过量表达SS以增加它们的ADPG和淀粉的生产的转基因植物的生产。相反地,我们表明由于不存在AGPase而缺乏淀粉的植物具有正常的ADPG水平。所有这些都表明,如图1B和2B所示,SS参与淀粉生物合成所必需的ADPG的直接合成中,并且负责植物细胞中积聚的大部分ADPG的合成。
尽管基于图1A所展示的通路,按照该通路SS参与贮藏组织中的UDPG(但不是ADPG)合成中,许多工作描述了由于SS活性减少而引起淀粉含量减少的植物的生产(Chourey,P.S.,Nelson,O.E.(1976)Theenzymatic deficiency conditioned by the shrunken-1 mutations in maize.Bochem.Genet.14,1041-1055;Zrenner,R.,Salanoubat,M.,Willmitzer,L.,Sonnewald,U.(1995)Evidence for the crucial role of sucrose synthase forsink strength using transgenic potato plants.Plant J.7,97-107;Tang,G-Q.,Sturm,A.(1999)Antisense repression of sucrose synthase in carrot(Daucuscarota L.)affects growth rather than sucrose partitioning.Plant Mol.Biol.41,465-479)。就这种意义来说,按照图1B和2B所示的代谢方案,还没有SS的过量表达可能被用于生产由于ADPG水平增加而具有高淀粉含量的植物实验证据。然而,基于SS生产细胞壁多糖生物合成的前体分子(UDPG)的能力,描述由于SS的过量表达而具有高纤维含量的棉属植物或具有高纤维素含量的谷类植物的生产的工作已经公布并取得专利权(Timothy,H.J.,Xiamomu,N.,Kanwarpal,S.“Manipulation of sucrosesynthase genes to improve stalk and grain quality”专利WO 02067662;Robert,F.,Danny,L.,Yong-Ling,R.“Modification of sucrose synthase geneexpression in plant tissue and uses therefor”专利WO 0245485;Christopher,B.,William,B.,Robert,H.“Bacterial sucrose synthase compositions andmethods of use”专利WO 9803637)。
本发明首先涉及大量生产可溶的、易纯化的、具有高特异性活性的重组SS的方法的开发和最优化,其基于合适的大肠杆菌(E.coli)菌株的应用和使SS具有组氨酸尾从而可能获得SS的表达载体的应用。本发明进一步涉及制备测定蔗糖的试剂盒的步骤,其基于具有SS活性的与ADPG或UDPG代谢酶偶联的酶产物的应用。本发明还涉及起始于特意为所述目的而设计的SS变种的糖核苷酸如ADPG或UDPG的最优化生产。最后,详细给出随着SS的过量表达而具有高含量的蔗糖、ADPG和淀粉、和高直链淀粉/支链淀粉比例的转基因植物的设计。
发明详述
编码SS的cDNA的扩增
野生型蔗糖合酶SS4的核苷酸序列已知(Fu,H.,Park,W.D.(1995)Sink-and vascular-associated sucrose synthase functions are encoded bydifferent gene classes in potato.Plant Cell 7,1369-1385),对应基因的5’和3’端设计并制造2条特异的引物。应用上述引物,通过常规PCR技术从马铃薯叶片cDNA文库中扩增出一段2418碱基对的DNA片段,称为SSX。将所述PCR片段插入到pSK Bluescript质粒(Stratagene)中,制成pSS构建体(图3A),将其在宿主细菌XL1 Blue中扩增。
活性重组SS在大肠杆菌(E.coli)特殊菌株的生产
用限制性内切酶NcoI和NotI消化pSS。将释放出来的片段(其含有编码SS的cDNA,SSX)克隆到在多聚接头区域具有编码富含组氨酸序列的核苷酸序列的pET-28a(+)表达质粒(Novagen)的相同的限制酶切位点,其与重组蛋白融合。将由此得到的质粒(称为pET-SS,图3C)通过电穿孔插入到各种大肠杆菌(E.coli)的菌株中。将转化了pET-SS的大肠杆菌(E.coli)菌株BLR(DE3)(Novagen)于2003年10月29日保藏在位于Burjassot Campus,Burjassot 46100(巴伦西亚,西班牙)巴伦西亚大学的研究大厦的西班牙典型培养物保藏中心(Spanish Type Culture Collection),保藏号为CECT:5850。将所述细菌在LB培养基中在20℃孵化。SSX的过量表达通过在20℃生长的100ml细胞培养物中加入1mM异丙基-β-D-硫代半乳糖苷(IPTG)而实现。经过6小时的诱导培养后,将所述细菌收集下来并悬浮在4ml的结合缓冲液(Novagen,His-结合纯化试剂盒)中,然后超声处理并在40,000g离心20分钟。将上层清液流过Novagen His-结合纯化试剂盒的亲和柱,其中上层清液中含有在N-末端带有富含组氨酸残基的氨基酸序列的重组SS。遵循试剂盒的用法说明,用6ml推荐的洗脱缓冲液将SS洗脱下来,其中洗脱缓冲液中含有200mM咪唑而不是1M。洗脱后,将所述蛋白立即进行透析,以去除任何痕量的咪唑,其不可逆地使SS失活。
被最优化用于产生ADPG的SS异构体的生产
使用合适的引物,用pSS作模板,设计突变的变种SS5,制成构建体pSS5。这是使用QuikChange基因定点诱变试剂盒(Stratagene)进行的。将pSS5用NcoI和NotI消化。将释放出来的片段(其含有SS5)克隆到pET-28a(+)表达质粒的相同的限制酶切位点,制成pET-SS5,将其通过电穿孔插入到大肠杆菌(E.coli)BLR(DE3)菌株中。将转化了pSS5的大肠杆菌(E.coli)菌株XL1 Blue于2003年10月29日保藏在位于Burjassot Campus,Burjassot46100(巴伦西亚,西班牙)巴伦西亚大学的研究大厦的西班牙典型培养物保藏中心,保藏号为CECT:5849。
过量表达SS4的转基因植物的生产
在本发明中,SS以下述方式过量表达:(a)组成性地,(b)在叶片中特异表达和(c)在诸如块茎的贮藏器官中的特异表达。
对于组成性地过量表达SS的植物的生产,设计并制造由烟草花叶病毒的35S组成性启动子作用控制的构建体。在pSS中SSX的5’和3’区域连续插入35S启动子和NOS终止子,制成质粒p35S-SS-NOS,其限制性酶切图谱如图4B所示。
为了能够通过根癌农杆菌(Agrobacterium tumefaciens)将该构建体转移到植物的基因组,首先必须将它克隆到二元质粒(binary plasmid)中。为此,将p35S-SS-NOS连续用酶NotI、T4 DNA聚合酶和HindIII消化,并克隆到预先连续用酶EcoRI、T4 DNA聚合酶和HindIII消化的二元质粒pBIN20(图4A)中(Hennegan,K.P.,Danna,K.J.(1998)pBIN20:An improvedbinary vector for Agrobacterium-mediated transformation.Plant Mol.Biol.Rep.16,129-131)。由此获得的质粒称为pBIN35S-SS-NOS(图4C)。
为了特异地在光照的叶片中过量表达SS,用PCR扩增编码烟草RUBISCO(核酮糖-1,5-二磷酸羧化酶/加氧酶)小亚基的基因的启动子区域(称为RBCS)(Barnes,S.A.,Knight,J.S.,Gray,J.C.(1994)Alteration ofthe amount of the chloroplast phosphate translocator in transgenic tobaccoaffects the distribution of assimilate between starch and sugar.Plant Physiol.106,1123-1129)。将所述核苷酸序列(其赋予在光合活性细胞内的特异表达)插入到pGEMT-easy载体(Promega),制成pGEMT-RBCSprom(图5A)。将该构建体用HindIII和NcoI消化,并将释放出来的片段克隆到p35S-SS-NOS对应的限制酶切位点,制成pRBCS-SS-NOS(图5B)。将该构建体连续用HindIII、T4 DNA聚合酶和NotI消化。将释放出来的片段克隆到连续用HindIII、T4 DNA聚合酶和EcoRI消化的pBIN20中。由此得到的构建体称为pBINRBCS-SS-NOS(图5C)。
在大肠杆菌(E.coli)(XL1 Blue)中扩增后,将pBIN35S-SS-NOS和pBINRBCS-SS-NOS插入到根癌农杆菌(A.tumefaciens)C58:GV2260中(Debleare,R.,Rytebier,B.,de Greve,H.,Debroeck,F.,Schell,J.,vanMontagu,M.,Leemans,J.(1985)“Efficient octopine Ti plasmid-derivedvectors of Agrobacterium mediated gene transfer to plants”Nucl.Acids Res.13,4777-4788),其被用于通过常规技术转化诸如番茄(Lycopersiconsculentum)、烟草(Nicotiana tabacum)、马铃薯(Solanum tuberosum)和稻属的物种(Horsch,R.B.,Fry,J.E.,Hoffmann,N.L.,Eichholtz,D.,Rogers,S.G.,Fraley,R.T.(1985)“A simple and general method for transferring genesinto plants”Science 277,1229-1231;Pozueta-Romero,J.,Houlné,G.,Schantz,R.Chamarro,J.(2001)“Enhanced regeneration of tomato and pepper seedlingexplants for Agrobacterium-mediated transformation”Plant Cell Tiss.Org.Cult.67,173-180;Hiei,Y.,Ohta,S.,Komari,T.,Kumashiro.T.(1994)“Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacteriumand sequence analysis of the boundaries of the T-DNA.Plant J.6,271-282)。所述转化了pBIN35S-SS-NOS的根癌农杆菌C58:GV2260于2003年10月29日保藏在位于Burjassot Campus,Burjassot 46100(巴伦西亚,西班牙)巴伦西亚大学的研究大厦的西班牙典型培养物保藏中心,保藏号为CECT:5851。
测定蔗糖的测定试剂盒的制备
一种设计用于蔗糖测定的试剂盒,如下述方案I所示的包含在用于蔗糖的分光光度和荧光测定的试剂盒中的酶促反应,上述测定基于蔗糖到糖核苷酸的转化及随后糖核苷酸到葡萄糖-1-磷酸、葡萄糖-6-磷酸和NAD(P)H的转化。
所述试剂盒基于在核苷酸二磷酸(例如UDP或ADP)存在下SS对蔗糖分子的作用,释放出等摩尔量的果糖和相应的糖核苷酸。如果由所述反应产生的糖核苷酸是UDPG,该糖核苷酸受到诸如Nudix类型的UDPG焦磷酸化酶(EC 3.6.1.45)的UDPG水解酶的作用(Yagi,T.,Baroja-Fernández,E.,Yamamoto,R.,
F.J.,Akazawa,T.,Pozueta-Romero,J.(2003)Cloning,expression and characterization of a mammalian Nudix hydrolase-like enzymethat cleaves the pyrophosphate bond of UDP-glucose.Biochern.J.370,409-415)or UDPG hydrolase(Burns,D.M.,Beacham,I.R.(1986)Nucleotidesequence and transcriptional analysis of the E.coli ushA gene,encodingperiplasmic UDP-sugar hydrolase(5’-nucleotidase):regulation of the ushAgene,and the signal sequence of its encoded protein product.Nucl.Acids Res.14,4325-4342)。由上述水解酶作用释放出的G1P通过葡糖磷酸变位酶(PGM)作用而转化,产生葡萄糖-6-磷酸(G6P),其依次可以通过G6P脱氢酶(G6PDH)的作用而经受与NAD(P)+的偶联反应,产生在340nm处通过荧光测定法和分光光度法可以容易地测定的6-磷酸葡糖酸和NAD(P)H。依次地,释放出的NAD(P)H可以与FMN-氧化还原酶/荧光素酶作用偶联,产生可以由分光光度测定法定量的光。
备选地,如方案II所示,产生的UDPG可以与UDPG脱氢酶(EC 1.1.1.22)偶联,其在NAD的存在下给出等摩尔量的UDP-葡糖醛酸和NADH,所述UDP-葡糖醛酸和NADH在340nm下可以通过荧光测定法或分光光度法测定。依次地,释放出的NADH可以与FMN-氧化还原酶/荧光素酶作用偶联,产生可以由分光光度测定法定量的光。
产生可以由分光光度测定法定量的光。
方案II
如果SS催化反应的产物是ADPG,ADPG受到诸如细菌ADPG焦磷酸化酶(EC 3.6.1.21)的ADPG水解酶的作用(Moreno-Bruna,B.,Baroja-Fernández,E.,
F.J.,Bastarrica-Berasategui,A.,Zandueta--Criado,A.,Rodríguez-López,M.,Lasa,I.,Akazawa,T.,Pozueta-Romero,J.(2001)Adenosine diphosphate sugar pyrophosphatase prevents glycogenbiosynthesis in Escherichia coli.Proc.Natl.Acad.Sci.USA 98,8128-8132)。释放出的G1P通过葡糖磷酸变位酶作用而转化,产生葡萄糖-6-磷酸(G6P),其依次可以通过G6P脱氢酶作用而经受与NAD(P)+的偶联反应,产生在340nm处通过荧光测定法或分光光度法可以容易地测定的6-磷酸葡糖醛酸和NAD(P)H。
在任何情况中,与由SS调节的糖核苷酸的产生所偶联的酶促反应方案完美地适用于电流分析法检测。
实施本发明的实施例
下文描述实施例,其详细地显示将编码马铃薯SS的异构体的cDNA克隆到合适的表达载体中和对酶以其活性形式生产和积聚最优化的一种大肠杆菌(E.coli)菌株中的步骤。其它实施例描述重组SS用于制备测定植物样品、血清、尿、果汁、加糖果汁饮料、提神饮料等的蔗糖的测定试剂盒的应用。另一个实施例描述用于诸如UDPG和ADPG的糖核苷酸的大量生产的最优化的SS变种的应用。最后,另一个实施例描述由于在过量表达SS的植物中的高ADPG生产活性而具有高含量的蔗糖、ADPG和淀粉、和高直链淀粉/支链淀粉比例的植物的生产。
实施例1:在大肠杆菌(Escherichia coli)BLR(DE3)中表达带有组氨酸尾
的重组SS,其易于纯化并且具有高特异性活性
已知编码马铃薯SS异构体的SS4基因的核苷酸序列,可以设计2条特异性引物,其序列从5’-3’方向为SEQ ID NO:1和SEQ ID NO:2。应用上述引物,通过常规PCR方法从马铃薯块茎cDNA文库中扩增出一段称为SSX的DNA片段,将所述片段插入到pSK Bluescript质粒(Stratagene)中,将其在宿主细菌XL1 Blue中扩增。SSX的核苷酸序列为SEQ ID NO:3,其与SS4(GenBank入藏登记号U24087)有细微的不同。从序列SEQ ID NO:3推断的氨基酸序列与SS4细微不同,因此称为SSX。在pET-28a(+)质粒中表达SEQ ID NO:3后推断的氨基酸序列为SEQ ID NO:4,其包含一段融合到从SEQ ID NO:3推断的氨基酸序列氨末端的富含组氨酸的38个氨基酸的序列。
通过添加1mM IPTG来在转化了pET-SS的BL21(DE3)细菌中诱导生产SSX。在37℃继续培养6小时后,可以观察到转化了pET-SS的细菌积聚了一种聚集体形式的蛋白,其大小与SS相符。然而,所述细菌并不具有SS活性。这一在SS活性形式表达中的失败可以归因于大肠杆菌(E.coli)在对特定的大分子量的真核蛋白的正确折叠中存在的问题(Miroux,B.,Walker,J.E.(1996)“Over-production of proteins in Escherichia coli:mutanthosts that allow synthesis of some membrane proteins and globular proteins athigh levels”J.Mol.Biol.260,289-298)。为了克服这一问题的目的,研究了在其它细菌菌株中和在20℃温度下生产活性SS的能力。在所有的研究中,SSX的生产通过添加1mM IPTG而诱导。继续孵化6小时后,将所述细菌进行超声处理和离心。将由此获得的上层清液进行SS活性分析。在这些条件下,如图6所示,从生产可溶的、活性SS的观点来看,证明BLR(DE3)菌株是最有效的。所述转化了pET-SS的大肠杆菌(E.coli)菌株BLR(DE3)(Novagen)于2003年10月29日保藏在西班牙典型培养物保藏中心,保藏号为CECT:5850。与文献中描述的重组SS的非常低的生产力(30毫克每克细菌)(Nakai,T.,Tonouchi,N.,Tsuchida,T.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“Expression and characterization of sucrose synthase from mungbean seedlings in Escherichia coli”Biosci.Biotech.Biochem.61,1500-1503;Li,C.R.,Zhang,X.B.,Hew,C.S.(2003)“Cloning,characterization andexpression analysis of a sucrose synthase gene from tropical epiphytic orchidOncidium goldiana.Physiol.Plantarum 118,352-360)相比,在CECT:5850的全蛋白库中重组SSX的供量(contribution)大约为20%。将所述上层清液流经His-结合亲和柱(Novagen),具有组氨酸尾的重组蛋白特异性地保留在柱中。洗脱并将纯化的SS透析之后,将它与50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl/2mM UDP一起孵化。根据UDPG的生产测定的特异性活性是80单位/mg蛋白,远高于文献中描述的0.05-5单位/mg重组SS的活性(Nakai,T.,Tonouchi,N.,Tsuchida,T.,Mori,H.,Sakai,F.,Hayashi,T.(1997)“Expression andcharacterization of sucrose synthase from mung bean seedlings in Escherichiacoli”Biosci.Biotech.Biochem.61,1500-1503;Li,C.R.,Zhang,X.B.,Hew,C.S.(2003)“Cloning,characterization and expression analysis of a sucrosesynthase gene from tropical epiphytic orchid Oncidium goldiana.Physiol.Plantarum 118,352-360);Rmer,U.,Schrader,H.,Günther,N.,Nettelstroth,N.,Frommer,W.B.,Elling,L.(2004)Expression,purification andcharacterization of recombinant sucrose synthase I from Solanum tuberosum L.for carbohydrate engineering.J.Biotechnology 107,135-149),并且高于从植物提取液中纯化的SS对应的3单位/mg的活性(Pressey R(1969)Potato sucrose synthase:purification,properties,and changes in activityassociated with maturation.Plant Physiol.44,759-764.)。所述单位(unit)定义为每分钟催化产生1摩尔UDPG的酶的量。在500mM蔗糖存在下,对UDP的亲和力是Km(UDP)=0.25mM,而在1mM UDP存在下,对蔗糖的Km是30mM。在UDP存在下对蔗糖的亲和力显著地高于在酵母菌中获得的重组SS所表现出的亲和力(Km=95mM,Rmer,U.,Schrader,H.,Günther,N.,Nettelstroth,N.,Frommer,W.B.,Elling,L.(2004)Expression,purification and characterization of recombinant sucrose synthase I fromSolanum tuberosum L.for carbohydrate engineering.J.Biotechnology 107,135-149)。
实施例2:基于使用来自大肠杆菌(E.coli)的重组SS的UDPG和ADPG的
大规模生产
将100毫升含有1M蔗糖,50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl/100mM UDP和30单位的重组马铃薯SS的溶液在37℃孵化12小时之后,有效地和经济地生产出3克高纯度的UDPG,其中所述重组马铃薯SS通过在BLR(DE3)中表达pET-SS并随后进行纯化获得。将所述溶液在100℃加热90秒使反应终止,然后以10,000g转速离心10分钟。将上层清液应用到制备级HPLC色谱仪(WatersAssociates),并且按照文献(Rodríguez-López,M.,Baroja-Fernández,E.,Zandueta-Criado,A.,Pozueta-Romero,J.(2000)Adenosine diphosphateglucose pyrophosphatase:a plastidial phosphodiesterase that prevents starchbiosynthesis.Proc.Natl.Acad.Sci.美国97,8705-8710)所描述纯化UDPG。
ADPG的生产要求产生一种SS的突变形式,该突变形式对ADP的亲和力远高于所描述的从植物组织中提取的SS(Pnessey R(1969)Potatosucrose synthase:purification,properties,and changes in activity associatedwith maturaton.Plant Physiol.44,759-764;Nguyen-Quock,B.,Krivitzky,M.,Huber,S.C.,Lecharny,A.(1990)Sucrose synthase in developing maize leaves.Plant Physiol.94,516-523;Morell,M.,Copeland,L.(1985)Sucrose synthaseof soybean nodules.Plant Physiol.78,149-154)。
称为SS5的这一异构体通过使用QuikChange基因定点诱变试剂盒(Stratagene)对SSX进行点诱变和连续应用下述引物对而获得,其中所述引物对的序列为:[SEQ ID NO:5,SEQ ID NO:6],[SEQ ID NO:7,SEQ IDNO:8]和[SEQ ID NO:9,SEQ ID NO:10]。由此获得的被称为SS5的核苷酸序列为序列SEQ ID NO:11。SS5(Susy 5)氨基酸序列中相对于SS4-Susy4-(数据库中所示)的变化在表I中为阴影部分所示。将序列SEQ ID NO:11在pET-28a(+)质粒中表达后推断的氨基酸序列为SEQ ID NO:12,其包含一段融合到从SEQ ID NO:11推断的氨基酸序列氨末端的富含组氨酸的38个氨基酸的序列。
表I包含所述融合到SS5氨末端部分的富含组氨酸的38个氨基酸。
表I
表达pET-SS5之后获得的重组SS5在UDP和ADP存在下,分别具有80单位/mg蛋白和65单位/mg蛋白的Vmax。在500mM蔗糖存在下,对UDP和ADP的亲和力非常相似(Km=0.2mM,对ADP和UDP),而在饱和浓度的UDP和ADP存在下,对蔗糖的Km分别为30mM和100mM。这些动力学参数与那些对从马铃薯块茎和其它物种的其它组织提取的SS进行描述的参数有很大的不同,按照那些参数,所述酶的Vmax在UDP存在下比在ADP存在下高10倍(Pressey R(1969)Potato sucrose synthase:purification,properties,and changes in activity associated with maturation.Plant Physiol.44,759-764;Morell,M.,Copeland,L.(1985)Sucrose synthaseof soybean nodules.Plant Physiol.78,149-154;Nguyen-Quock,B.,Krivitzky,M.,Huber,S.C.,Lecharny,A.(1990)Sucrose synthase in developing maizeleaves.Plant Physiol.94,516-523)。转化了pSS5的大肠杆菌(E.coli)菌株XLl Blue保藏在西班牙典型培养物保藏中心,保藏号为CECT:5849。
将100毫升含有1M蔗糖,50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl/100mM ADP和30单位的重组马铃薯SS的溶液在37℃孵化12小时之后,有效地和经济地生产出3克高纯度的ADPG,其中所述重组马铃薯SS通过在BLR(DE3)中表达pET-SS5并随后在His-结合柱上进行纯化获得。将所述溶液在100℃加热90秒使反应终止,然后以10,000g转速离心10分钟。将上层清液应用到制各级HPLC色谱仪(Waters Associates),以纯化ADPG。
实施例3:测定蔗糖的酶促试剂盒的制备
为测定蔗糖,制备具有下述成分和最终量/浓度的下述反应混合物:
1.基于糖核苷酸水解酶的应用的试剂盒:
a.2单位的SS(重组或非重组)
b.2mM ADP或UDP(分别取决于是生产ADPG还是UDPG)
c.2单位ADPG焦磷酸化酶或2单位UDPG焦磷酸化酶(分别取决于它是否要包含在所述ADP或UDP反应混合物中)
d.2单位的PGM
e.2单位的G6PDH
f.0.5mM NAD(P)
g.反应缓冲液:50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl
h.预先过滤的测试样品
2.基于UDPG脱氢酶的应用的试剂盒:
a.2单位的SS(重组或非重组)
b.2mM UDP
c.2单位的UDPG脱氢酶
d.0.5mM NAD
e.反应缓冲液:50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl
f.预先过滤的测试样品
对测试样品中存在的蔗糖的量的测定是基于对按照方案I和方案II所示的偶联反应产生的NAD(P)H的荧光测定或分光光度测定(在340nm)。
为测定不同发育程度的大麦属种子的蔗糖含量(图7),反应发生在ELISA板的300微升的孔中,在37℃反应3分钟。测试样品的体积是20微升,由试剂a-g(试剂盒#1)和a-e(试剂盒#2)结合组成的混合物的体积是280微升。空白包含除SS外混合物的所有成分。用多扫描分光光度计进行测量。发现用类型“1”的试剂盒和类型“2”的试剂盒获得的数值与用导言中描述的层析技术测定的数值相当(Baroja-Fernández,E.,
Saikusa,T.,Rodríguez-López,M.,Akazawa,T.,Pozueta-Romero,J.(2003)Sucrose synthase catalyzes the de novo production of ADPglucose linked tostarch biosynthesis in heterotrophic tissues of plants.Plant Cell Physiol.44,500-509)。
实施例4:过量表达SS的转基因植物的生产
图8-10展示从组成性(35S-SS-NOS)和特异性(RBCS-SS-NOS)过量表达SS的马铃薯植物的叶片中获得的结果。
如图8所示,在任意所述植物的叶片中的SS活性比在野生型植物(WT)相同器官中的活性高2-10倍。所述叶片具有下述特征:
1.生产ADPG的SS活性(图8)与淀粉(图9)和ADPG(图10)的水平之间的明显的相关性。这一特征不仅在叶片中还在诸如块茎和种子的贮藏组织(参见下文)中观察到。
2.相对于野生型植物叶片的高淀粉含量(图9)。例如,在8小时光照/16小时黑暗的光周期和20℃生长的“野生型”马铃薯植物的叶片的淀粉含量是5微摩/克鲜重,而过量表达SS的转基因植物的叶片的淀粉含量是8微摩/克鲜重。当光周期变长时,将加强野生型植物和转基因植物之间的这种差别,以致过量表达SS的植物的叶片含有比野生型植物叶片多4倍的淀粉。
3.相对于非转化植物的相同组织或器官的高ADPG含量(图10)。在8小时光照/16小时黑暗的光周期和20℃生长的野生型马铃薯植物的叶片中平均含量是0.35纳摩/克鲜重,而过量表达SS的植物的叶片可以具有2.5纳摩/克鲜重的含量。
4.在光周期期间,ADPG和淀粉都表现出短暂的聚集(图11)。两种物质的聚集比例与SS活性保持正相关性,表明SS在ADPG生产和蔗糖代谢与淀粉代谢之间的联系中起着重要的作用,这与淀粉生物合成“传统”模型(图2A)所揭示的内容相反,并证实了图2B所示的“备选”模型假说,SS在ADPG产生中以及在蔗糖代谢和淀粉代谢的联系中起到重要的作用。
5.诸如葡萄糖和果糖的可溶性糖的正常水平。然而,在转基因叶片中葡萄糖-6-磷酸和蔗糖的水平高于在野生型马铃薯叶片中观察到的结果(表2)。
表2:在对照植物(WT)叶片中和35S-SuSy-NOS来源的叶片中,代谢物水平(用nmol/g鲜重表示)。用粗体字显示与在野生型(WT)中观察到的数值有显著区别的数值。
对照WT | 35S-SS-NOS | ||||||
6 | 5 | 12 | 3 | 4 | 7 | ||
葡萄糖 果糖 蔗糖 葡萄糖 -6-磷 酸 葡萄糖 -1-磷 酸 | 848±31996±431,012±27244±2822.7± 1.9 | 922±291,035±571,529±48309±1515.5± 2.1 | 860±301,094±171,402±68280±2510.3± 1.1 | 933±291,022±101,642±58271±279.9± 1.2 | 881±561067±581,307±35355±239.5± 1.5 | 895±321078±631,317±35298±1215.2± 1.9 | 871±60817±411,391±70325±9.811.4± 1.8 |
6.当与非转化的植物相比时,过量表达SS的植物外部形态学没有异常。
图12-14显示在组成性过量表达SS(35S-SS-NOS)的马铃薯块茎中获得的结果。所述结果与那些在特异性块茎启动子(patatina基因的启动子)控制下过量表达SS的块茎中观察到的结果基本上相同。
如图12所示,在任何一种所述植物的块茎中的SS活性高于在野生型植物的相同器官中的活性???倍。所述块茎具有下述特征:
1.生产ADPG的SS活性(图12)和淀粉(图13)与ADPG(图14)水平之间的明显的相关性。
2.相对于非转化植物块茎的高淀粉含量(图13)。例如,“野生型”植物的块茎中的淀粉含量是约300毫摩/克鲜重(相当于54mg淀粉/克鲜重),而在过量表达SS的块茎中,淀粉含量是450-600毫摩/克鲜重。
3.相对于野生型植物块茎的高ADPG含量(图14)。在野生型块茎中的平均含量是5纳摩/克鲜重,而过量表达SS的块茎具有7-9纳摩/克鲜重的含量。
在稻属种子、番茄属和烟草叶片、和番茄属果实中获得的结果,与图8-14显示的结果性质上相似。在所有情况下,存在淀粉含量的增加和直链淀粉/支链淀粉比例的增加。
按照目前关于淀粉的生物合成的观点(在图1A和2A中举例说明),随着SS的过量表达而具有高ADPG和淀粉含量的植物的生产是完全预料不到的,这或许可以解释为什么过量表达SS的植物设计早先没有被采用作为提高淀粉生产的策略。基于本工作所获得的结果表明SS,而不是AGPase,是植物中积聚的ADPG的基本来源。按照当前的模型,AGPase是ADPG的唯一来源。然而,令人吃惊地是,在AGPase-缺陷植物中的ADPG水平从未被研究过。为了探究本发明的重要性,我们第一次分析了在具有减少的AGPase活性的拟南芥属(Arabidopsis)和马铃薯植物中的ADPG和淀粉水平。如图15A所示,在AGPase-缺陷的TL25拟南芥(TL25Arabidopsis)植物中的淀粉水平(Lin,T.P.,Caspar,T.,Somerville,C.R.,Preiss,J.(1988)Isolation and characterization of a starchless mutant ofArabidopsis thaliana lacking ADPglucose pyrophosphorylase activity.PlantPhysiol.88,1131-1135)低于在野生型植物中观察到的结果。然而,ADPG水平是正常的(图15B)。但是,在AGP62和AGP85马铃薯植物中的淀粉水平(Miille-Rber,B.,Sonnewald,U.Willmitzer,L.(1992)Inhibition of theADPglucose pyrophosphorylase in transgenic potatoes leads to sugar-storingtubers and influences tuber formation and expression of tuber storage proteingenes.EMBO J.11,1229-1238)相对于在野生型植物叶片中观察到的结果是减少的(图16A)。然而,ADPG水平是完全正常的(图16B)。综合起来看,这些观察资料:(a)表明SS,而不是AGPase,是植物中ADPG的主要来源,和(b)在证明SS的过量表达引起植物具有高淀粉含量后,突出了本发明的重要性。
附图描述
图1:在异养器官中的淀粉生物合成机制。(A)“传统”机制,按照该机制SS参与UDPG的生产,在UDPG焦磷酸化酶(UGPase)、胞质葡糖磷酸变位酶(PGM)、质体葡糖磷酸变位酶、ADPG焦磷酸化酶(AGPase)和淀粉合酶的联合作用后,UDPG最终转化为淀粉。(B)“备选”机制,按照该机制SS参与胞质中的ADPG直接生产。然后ADPG通过易位体的作用被转运到造粉质体。一旦进入造粉质体,淀粉合酶利用ADPG生产淀粉。
图2:叶片中淀粉生物合成的机制。(A)“传统”机制,按照该机制,淀粉生物合成的全过程发生在叶绿体内。按照这一观点,淀粉新陈代谢和蔗糖是不相联系的。而且,SS不参与糖异生过程。(B)淀粉生物合成的“备选”机制,按照该机制,SS包含在胞质中的ADPG直接合成中。然后ADPG被转运到质体内部,在这里淀粉合酶利用它作为淀粉合成反应的底物。
图3:从pET-28a(+)和pSS构建pET-SS表达质粒的步骤。
图4:从pBIN20和p35S-SS-NOS构建pBIN35S-SS-NOS表达质粒的步骤。
图5:从pGEMT-RBCSprom、p35S-SS-NOS和pBIN20构建pRBCS-SS-NOS表达质粒的步骤。
图6:pET-SS在不同大肠杆菌菌株中的表达。(A)在转化了pET或pET-SS的细菌提取液中的SS活性(毫单位(mU)/毫克细菌蛋白)。反应发生在蔗糖降解和产生ADPG的方向。所述反应混合物包含50mMHEPES(pH7.0),1mM EDTA,20%聚乙二醇,1mM MgCl2,15mM KCl和2mM ADP。反应在37℃发生10分钟。(B)来自转化了pET和pET-SS的大肠杆菌(E.coli)不同菌株的蛋白提取液的SDS-PAGE。重组SSX的位置用星号表示。
图7:使用基于SS、ADPG(UDPG)焦磷酸化酶、PGM和G6PDH的偶联反应的试剂盒对大麦属胚乳不同发育阶段的蔗糖的测定。所述结果与下述实验平行获得的结果一致:(a)通过使用基于SS和ADPG(UDPG)脱氢酶偶联反应的试剂盒,和(b)通过使用在与Carbo-Pac PA1柱相连的DX-500 Dionex系统中带有电泳检测的高效液相色谱(HPLC)。
横坐标:开花后的天数
纵坐标:蔗糖含量(μmol/gFW)
图8:在野生型(WT)马铃薯植物和在将构建体35S-SS-NOS(通过根癌农杆菌CECT:5851菌株的作用)或RBCS-SS-NOS整合入其基因组后过量表达SSX的马铃薯植物的叶片中的SS活性。活性用毫单位(mU)每克鲜重表示。所述单位定义为每分钟产生1微摩尔ADPG所需的SS的量。
图9:在野生型(WT)马铃薯植物和在将构建体35S-SS-NOS(通过根癌农杆菌CECT:5851菌株的作用)或RBCS-SS-NOS整合入其基因组后过量表达SSX的马铃薯植物的叶片中的淀粉含量。
图10:在野生型(WT)马铃薯和在将构建体35S-SS-NOS(通过根癌农杆菌CECT:5851菌株的作用)或RBCS-SS-NOS整合入其基因组后过量表达SSX的马铃薯植物的叶片中的ADPG含量。
图11:(A)淀粉和(B)ADPG在8小时光照和16小时黑暗的光周期期间在野生型植物(●)、35S-SS-NOS(■)和RBCS-SS-NOS(▲)的叶片中的短暂积聚。
图12:在野生型马铃薯植物(WT)、再生对照(RG)和在构建体35S-SS-NOS整合到其基因组(通过根癌农杆菌(Agrobacterium tumefaciens)CECT:5851菌株的作用)后过量表达SSX的马铃薯植物的块茎(线条4,5,6和12)中的SS活性(参考于鲜重,FW)。活性用毫单位(mU)每克鲜重表示。所述单位定义为每分钟产生1微摩尔ADPG所需的SS的量。
图13:在野生型马铃薯植物(WT)、再生对照(RG)和在构建体35S-SS-NOS整合到其基因组(通过根癌农杆菌(Agrobacterium tumefaciens)CECT:5851菌株的作用)后过量表达SSX的马铃薯植物的块茎(线条4,5,6和12)中的淀粉含量(参考于鲜重,FW)。
图14:在野生型马铃薯植物(WT)和在构建体35S-SS-NOS整合到其基因组(通过根癌农杆菌(Agrobacterium tumefaciens)CECT:5851菌株的作用)后过量表达SSX的马铃薯植物的块茎中的ADPG含量(参考于鲜重,FW)。
图15:在AGPase-缺陷的拟南芥(Arabidopsis thaliana)TL25的叶片中(A)淀粉和(B)ADPG含量。
图16:在AGPase-缺陷的马铃薯AGP62和AGP85的叶片中(A)淀粉和(B)ADPG含量。
序列表
<110>纳瓦拉公立大学
<120>重组蔗糖合酶的生产方法、及其在生产蔗糖测定试剂盒中的应用、生产腺苷二磷酸葡糖的方法和获得具有积累了高浓度的腺苷二磷酸葡萄糖和淀粉的叶片和储藏器官的转基因植物的方法
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tct cgc ttt gaa gtg tgg cca tac atg gag aca ttc att gag 1176
gat gtt gca aaa gaa att tct gca gaa ctg cag gcc aag cca 1218
gat ttg ata att gga aac tac agt gag ggc aat ctt gct gct 1260
tct ttg cta gct cac aag tta ggc gta act cag tgc acc att 1302
gcc cac gcg ttg gag aaa acg aag tat cct gat tcc gac att 1344
tac tgg aaa aag ttt gat gaa aaa tac cat ttc tcg tcc cag 1386
ttt acc gct gat ctc att gca atg aat cac act gat ttc atc 1428
atc acc agc acc ttc cag gag ata gca gga agc aag gac act 1470
gtg gga caa tat gag agc cat atg gca ttc aca atg cct gga 1512
ttg tac aga gtt gtt cat ggc att aat gtg ttc gac ccc aaa 1554
ttc aac att gtc tca cct gga gct gat att aac ctc tacttc 1596
tcg tac tcc gaa acg gaa aag aga ctt aca gca tct cac cct 1638
gaa att gat gag ctg ctg tat agt gac gtt gag aat gac gaa 1680
cat ctg tgt gtg ctc aag gat agg act aaa cca att tta ttc 1722
aca atg gca agg ttg gat cgt gtg aag aat tta act gga ctt 1764
gtt gag tgg tac gcc aag aat cca cga cta agg gga ttg gtt 1806
aac ctg gtt gta gtt ggc gga gat cga agg aag gaa tcc aaa 1848
gat ttg gaa gag cag gca gag atg aag aag atg tat gag cta 1890
ata gag act cat aat ttg aat ggc caa ttc aga tgg att tct 1932
tcc cag atg aac cga gtg agg aat ggt gag ctc tac cga tac 1974
att gct gac act aag gga gct ttc gtt cag cct gca ttc tac 2016
gag gct ttt ggt ctg act gtt gtc gaa gca atg act tgt ggt 2058
ttg cct aca ttt gca act aat cac ggt ggt cca gct gag atc 2100
atc gtt cat gga aag tcc ggc ttc cac att gat cca tat cac 2142
ggt gag caa gct gct gat ctg cta gct gat ttc ttt gag aaa 2184
tgc aag aga gag cct tca cat tgg gaa acc att tcg acg gat 2226
ggc ctg aag cgc atc caa gag aag tac act tgg caa atc tac 2268
tcc gaa agg cta ttg aca ctg gct gct gtt tat ggg ttc tgg 2310
aaa cat gtt tct aag ctt gat cgt cta gaa atc cgt cgc tat 2352
ctt gaa atg ttt tat gct ctc aag tac cgt aag atg gct gaa 2394
gct gtt cca ttg gct gct gag tga atg aag 2418
<210>12
<211>841
<212>蛋白
<213>马铃薯
<223>与富含组氨酸氨基酸序列融合的SS5
<400>
Met gly ser ser his his his his his his ser ser gly leu val pro arg gly ser his
5 10 15 20
met ala ser met thr gly gly gln gln met gly arg gly ser glu phe met ala glu arg
25 30 35 40
val leu thr arg val his ser leu arg glu arg val asp ala thr leu ala ala his arg
45 50 55 60
asn glu ile leu leu phe leu ser arg ile glu ser his gly lys gly ile leu lys pro
65 70 75 80
his glu leu leu ala glu phe asp ala ile arg gln asp asp lys asn lys leu asn glu
85 90 95 100
his ala phe glu glu pro leu lys ser thr gln glu ala ile val leu pro pro trp val
105 110 115 120
ala leu ala ile arg leu arg pro gly val trp glu tyr ile arg val asn val asn ala
125 130 135 140
leu val val glu glu leu ser val pro glu tyr leu gln phe lys glu glu leu val asp
145 150 155 160
gly ala ser asn gly ash phe val leu glu leu asp phe glu pro phe thr ala ser phe
165 170 175 180
pro lys pro thr leu thr lys ser ila gly asn gly val glu phe leu ash arg his leu
185 190 195 200
ser ala lys met phe his asp lys glu ser met thr pro leu leu glu phe leu arg ala
205 210 215 220
his his tyr lys gly lys thr met met leu asn asp arg ile gln asn ser asn thr leu
225 230 235 240
gln asn val leu arg lys ala glu glu tyr leu ile met leu ser pro asp thr pro tyr
245 250 255 260
phe glu phe glu his lys phe gln glu ile gly leu glu lys gly trp gly asp thr ala
265 270 275 280
glu arg val leu glu met val cys met leu leu asp leu leu glu ala pro asp ser cys
285 290 295 300
thr leu glu lys phe leu gly arg ile pro met val phe asn val val lie leu ser pro
305 310 315 320
hls gly tyr phe ala gln glu asn val leu gly tyr pro asp thr gly gly gln val val
325 330 335 340
tyr ile leu asp gln val pro ala leu glu arg glu met leu lys arg ile lys glu gln
345 350 355 360
gly leu asp ile ile pro arg ile leu ile val thr arg leu leu pro asp ala val gly
365 370 375 380
thr thr cys gly gln arg ile glu lys val tyr gly ala glu his ser his ile leu arg
385 390 395 400
val pro phe arg thr glu lys gly ile val arg lys trp ile ser arg phe glu val trp
405 410 415 420
pro tyr met glu thr phe ile glu asp val ala lys glu ile ser ala glu leu gln ala
425 430 435 440
lys pro asp leu ile ile gly asn tyr ser glu gly asn leu ala ala ser leu leu ala
445 450 455 460
his lys leu gly val thr gln cys thr ile aia his ala leu glu lys thr lys tyr pro
465 470 475 480
asp ser asp ile tyr trp lys lys phe asp glu lys tyr his phe ser ser gln phe thr
485 490 495 500
ala asp leu ile ala met asn his thr asp phe ile ile thr ser thr phe gln glu ile
505 510 515 520
ala gly ser lys asp thr val gly gln tyr glu ser his met ala phe thr met pro gly
525 530 535 540
leu tyr arg val val his gly ile asn val phe asp pro lys phe asn ile val ser pro
545 550 555 560
gly ala asp ile asn leu tyr phe ser tyr ser glu thr glu lys arg leu thr ala ser
565 570 575 580
his pro glu ile asp glu leu leu tyr ser asp val glu asn asp glu his leu cys val
585 590 595 600
leu lys asp arg thr lys pro ile leu phe thr met ala arg leu asp arg val lys asn
605 610 615 620
leu thr gly leu val glu trp tyr ala lys asn pro arg leu arg gly leu val asn leu
625 630 635 640
val val val gly gly asp arg arg lys glu ser lys asp leu glu glu gln ala glu met
645 650 655 660
lys lys met tyr glu leu ile glu thr his ash leu asn gly gln phe arg trp ile ser
665 670 675 680
ser gln met asn arg val arg asn gly glu leu tyr arg tyr ile ala asp thr lys gly
685 690 695 700
ala phe val gln pro ala phe tyr glu ala phe gly leu thr val val glu ala met thr
705 710 715 720
cys gly leu pro thr phe ala thr asn his gly gly pro ala glu ile ile val his gly
725 730 735 740
lys ser gly phe his ile asp pro tyr his gly glu gln ala ala asp leu leu ala asp
745 750 755 760
phe phe glu lys cys lys arg glu pro ser his trp glu thr ile ser thr asp gly leu
765 770 775 780
lys arg ile gln glu lys tyr thr trp gln ile tyr ser glu arg leu leu thr leu ala
785 790 795 800
ala val tyr gly phe trp lys his val ser lys leu asp arg leu glu ile arg arg tyr
805 810 815 820
leu glu met phe tyr ala leu lys tyr arg lys met ala glu ala val pro leu ala ala
825 830 835 840
glu
841
Claims (43)
1.-一种以其可溶、活性形式有效生产高水平的重组蔗糖合酶(SS)酶的方法,其特征在于其包括:
a)从编码马铃薯SS异构体SS4的基因的核苷酸序列获得2个由SEQID NO:1和SEQ ID NO:2序列代表的引物,基于马铃薯叶片cDNA文库,使用该引物通过PCR扩增由SEQ ID NO:3序列代表的2418碱基对的cDNA片段,
b)将所述cDNA片段插入到第一载体,
c)将所述第一载体插入到第一宿主微生物,所述载体在其中被扩增,
d)用至少2个限制性内切酶消化扩增的构建体,
e)在上述消化后,获得包含编码SSX的cDNA的DNA片段,其推断的氨基酸序列由SEQ ID NO:4代表,
f)在与其相同的限制酶切位点处,将所述片段克隆到包含编码富含组氨酸序列的核苷酸序列的载体中,将所述富含组氨酸的尾融合到SS,得到第二表达载体,
g)将第二载体插入到第二宿主微生物中,所述载体在其中被表达,
h)在适宜的培养条件下孵化所述转化的微生物,以合成可溶、活性形式的SSX,
i)分离并纯化活性形式的SSX。
2.-前述权利要求中要求的生产重组SS的方法,其特征在于步骤b)所用的第一表达载体是质粒pSK,当插入SEQ ID NO:3序列时,得到图3A的构建体pSS。
3.-前述权利要求中要求的生产重组SS的方法,其特征在于步骤c)所用的扩增pSK质粒的第一宿主微生物是大肠杆菌(E.coli)细菌XL1 Blue。
4.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于步骤d)中所用的限制性内切酶是NcoI和NotI。
5.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于,步骤f)中,步骤d)后释放的DNA片段克隆的限制酶切位点与图3B所示的质粒pET-28a(+)相同,得到图3C所示的pET-SS质粒。
6.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于步骤g)中所用的第二宿主微生物是大肠杆菌的BLR(DE3)菌株。
7.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于步骤g)中转化的菌株是CECT5850,其在步骤h)中在适宜的培养条件下孵化,以合成可溶的、活性形式的SSX。
8.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于合成SSX的适宜的培养条件包括将所述细菌培养物置于20℃的温度。
9.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于SSX的纯化优选地通过用于富含组氨酸残基的氨基酸序列的亲和层析和使用含有优选浓度为200mM的咪唑的洗脱缓冲液实现。
10.-前述权利要求任一项中要求的生产重组SS的方法,其特征在于,为保持纯化的SSX酶处于其活性形式,将所述从亲和层析洗脱下来的纯化酶立即进行透析,以去除任何痕量的咪唑。
11.-一种生产重组马铃薯SS5异构体的方法,其特征在于其包括:
a)应用构建体pSS作为模板,和,通过连续地应用序列为[SEQ IDNO:5,SEQ ID NO:6]、[SEQ ID NO:7,SEQ ID NO:8]和[SEQ ID NO:9,SEQ ID NO:10]的引物对后进行定向诱变,获得由序列SEQ ID NO:11代表的2418碱基对的DNA片段,
b)将所述DNA片段插入到第一载体,
c)将所述第一载体插入到第一宿主微生物,所述载体在其中被扩增,
d)用至少2个限制性内切酶消化扩增的构建体,
e)在上述消化后,获得编码SS5的DNA片段,其氨基酸序列由SEQID NO:12代表,
f)在与其相同的限制酶切位点处,将所述片段克隆到包含编码富含组氨酸序列的核苷酸序列的载体中,将所述富含组氨酸的尾融合到SS5,得到第二表达载体,
g)将所述第二载体插入到第二宿主微生物中,所述载体在其中被表达,
h)在适宜的培养条件孵化所述转化的微生物,以合成可溶、活性形式的SS5,
i)分离并纯化活性形式的SS5。
12.-权利要求11中要求的生产重组SS5的方法,其特征在于步骤b)得到构建体pSS5。
13.-权利要求11或12任一项中要求的生产重组SS5的方法,其特征在于步骤c)中所用的用于扩增pSS5的第一宿主微生物是大肠杆菌(E.coli)细菌XL1 Blue。
14.-权利要求11至13任一项中要求的生产重组SS5的方法,其特征在于步骤d)中所用的限制性内切酶是NcoI和NotI。
15.-权利要求11至14任一项中要求的生产重组SS5的方法,其特征在于,步骤f)中,步骤d)后释放的DNA片段克隆的限制酶切位点与质粒pET-28a(+)相同,得到pET-SS5质粒。
16.-权利要求11至15任一项中要求的生产重组SS5的方法,其特征在于步骤g)中所用的第二宿主微生物是大肠杆菌BLR(DE3)菌株。
17.-权利要求11至16任一项中要求的生产重组SS5的方法,其特征在于步骤g)中转化的菌株是CECT5849,其在步骤h)中在适宜的培养条件下孵化,以合成可溶的、活性形式的SS5。
18.-权利要求11至17任一项中要求的生产重组SS5的方法,其特征在于合成SS5的适宜的培养条件包括将所述细菌培养物置于20℃的温度。
19.-权利要求11至18任一项中要求的生产重组SS5的方法,其特征在于SS5的纯化优选地通过亲和层析和使用含有优选浓度为200mM的咪唑的洗脱缓冲液实现。
20.-权利要求11至19任一项中要求的生产重组SS5的方法,其特征在于,为保持纯化的SS5酶处于活性形式,将所述从亲和层析洗脱下来的纯化酶立即进行透析,以去除任何痕量的咪唑。
21.-按照权利要求1至10的方法的要求可获得的一种可溶的、活性的重组SSX酶产品,其特征在于其具有由SEQ ID NO:4序列代表的推断序列,并显示出蔗糖合酶(SS)活性。
22.-权利要求21中要求的可溶的、活性的重组SSX酶产品,其特征在于,在蔗糖和UDP存在下,它具有80U/mg蛋白的特异活性,和Km(UDP)=0.25mM和Km(蔗糖)=30mM的动力学参数。
23.-权利要求21和22的可溶的、活性的重组酶产品的SS5异构体,其可按照权利要求11至20的方法获得,其特征在于其具有SEQ ID NO:12序列所示的推断的氨基酸序列,并显示出蔗糖合酶(SS)活性。
24.-权利要求23中要求的重组SS5异构体,其特征在于,在UDP和ADP存在下,它分别具有80U/mg蛋白和60U/mg蛋白的特异活性,和关于UDP和ADP的Km(UDP)=Km(ADP)=0.3mM的动力学参数。
25.-权利要求21和22的酶产品在生产UDPG中的应用,其特征在于通过在适宜的条件下孵化UDP和SSX,接着分离和纯化产生的UDPG。
26.-权利要求25中要求的应用,其特征在于其包括:
a)将100ml下述溶液在37℃孵化12h:
蔗糖 1M
HEPES,pH7.0 50mM
EDTA 1mM
聚乙二醇 20%
MgCl2 1mM
KCl 15mM
UDP 100mM
SSX 30U
b)通过加热终止反应,优选地在100℃加热90s,
c)10000g离心10min,
d)将上述上层清液通过HPLC进行色谱分析,通过常规方法洗脱和纯化UDPG。
27.-权利要求23和24的酶产品在生产ADPG中的应用,其特征在于通过在适宜的条件下孵化ADP和SS5,接着分离和纯化产生的ADPG。
28.-权利要求27中要求的应用,其特征在于其包括:
e)将100ml下述溶液在37℃孵化12h:
蔗糖 1M
HEPES,pH7.0 50mM
EDTA 1mM
聚乙二醇 20%
MgCl2 1mM
KCl 15mM
ADP 100mM
f)通过加热终止反应,优选地在100℃加热90s,
g)10000g离心10min,
h)将上述上层清液通过HPLC进行色谱分析,通过常规方法洗脱和纯化ADPG。
29.-具有蔗糖合酶(SS)活性的酶产品在制造用于蔗糖的分光光度/荧光/电流测定的测定试剂盒中的应用。
30.-权利要求29中要求的应用,其特征在于其包含下述孵化培养基:
a.2单位的SS
b.2 mM ADP
c.2单位的植物、动物或微生物源性的ADPG焦磷酸化酶
d.2单位的PGM
e.2单位的G6PDH
f.0.5mM的NAD(P)
g.100ml的反应缓冲液:50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl
h.先前过滤的测试样品。
31.-权利要求29中要求的应用,其特征在于其包含下述孵化培养基:
a.2单位的SS
b.2mM UDP
c.2单位的植物、动物或微生物源性的UDPG焦磷酸化酶
d.2单位的PGM
e.2单位的G6PDH
f.0.5mM的NAD(P)
g.100ml的反应缓冲液:50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl
h.先前过滤的测试样品。
32.-权利要求29中要求的应用,其特征在于其包含下述孵化培养基:
a.2单位的SS
b.2mM UDP
c.2单位的UDPG脱氢酶
d.0.5mM的NAD
e.100ml的反应缓冲液:50mM HEPES,pH7.0/1mM EDTA/20%聚乙二醇/1mM MgCl2/15mM KCl
f.先前过滤的测试样品。
33.-权利要求29至32任一项中要求的应用,其特征在于存在于测定试剂盒中的SS不加区别地是SS4、SS5或SSX或其结合。
34.-编码SS的DNA在生产表达SS的转基因植物中的应用,其特征在于将包含并表达所述DNA的基因构建体插入到合适的载体,并将所述基因构建体转入植物的基因组。
35.-权利要求34中要求的应用,其特征在于所用的cDNA编码SSX。
36.-权利要求35中要求的应用,其特征在于其包含下述步骤:
a)在pSS质粒中分别在5’和3’区域连续插入启动子35S和终止子NOS,插入编码SS的SSX基因或任意其它版本,以产生质粒p35S-SS-NOS,其限制性酶切图谱如图4B所示,
b)将p35S-SS-NOS用酶NotI、T4 DNA聚合酶和HindIII连续消化,
c)将所产生的片段克隆到预先用EcoRI、T4 DNA聚合酶和HindIII连续消化的二元质粒pBIN20中,获得图4C所示的质粒pBIN35S-SS-NOS,
d)在大肠杆菌(E.coli)(XL1 Blue)中扩增pBIN35S-SS-NOS,
e)将前述步骤扩增的基因构建体插入到根癌农杆菌C58:GV2260中,获得转化的菌株CECT 5851
f)用转化的菌株CECT 5851转染植物。
37.-权利要求35中要求的应用,其特征在与它包含下述步骤:
a)在pGEMT质粒中连续插入编码RUBISCO小亚基的基因的启动子,以产生质粒pGEMT-RBCSprom,其限制性酶切图谱如图5A所示,
b)将pGEMT-RBCS用酶HindIII和NcoI消化,以将释放出的片段插入到p35S-SS-NOS对应的限制酶切位点,得到pRBCS-SS-NOS,其限制性酶切图谱如图5B所示,
c)将pRBCS-SS-NOS用酶HindIII、T4 DNA聚合酶和NotI连续消化,并将释放出的片段克隆到用HindIII、T4 DNA聚合酶和EcoRI连续消化的pBIN20中,得到pBINRBCS-SS-NOS,其限制性酶切图谱如图5C所示,
d)在大肠杆菌(E.coli)(XL1 Blue)中扩增pBINRBCS-SS-NOS,
g)将前述步骤扩增的基因构建体插入到根癌农杆菌C58:GV2260中,并用转化的菌株转染植物。
38.-通过权利要求33至37的应用的方法可获得的转基因植物,其特征在于过量表达SS酶活性。
39.-权利要求37中要求的转基因植物,其特征在于所述过量表达认为SS酶活性水平比处于非转基因的野生型植物的相同组织中的SS酶活性水平高约2-10倍。
40.-权利要求38或39中要求的转基因植物,其特征在于其们优选地选自烟草、马铃薯、番茄或稻属植物。
41.-权利要求38至40之任一项中要求的转基因植物,其特征在于,另外,它们具有更高的蔗糖、G6P、ADPG和淀粉含量,高于在同样条件下生长的对应的野生型植物的相同组织或器官中观察到的结果。
42.-权利要求40中要求的转基因植物,其叶片具有的蔗糖、G6P、ADPG和淀粉含量、以及直链淀粉/支链淀粉比例,高于在对应的野生型植物的叶片中观察到的结果。
43.-权利要求40中要求的转基因植物,其根、块茎和/或种子具有的蔗糖、G6P、ADPG和淀粉含量、以及直链淀粉/支链淀粉比例,高于在对应的野生型植物的相同组织或器官中观察到的结果。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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ES200400257A ES2245867B1 (es) | 2004-02-05 | 2004-02-05 | Procedimiento produccion sacarosa sintasa recombinante a partir de escherichia coli y uso en fabricacion de kits de determinacion de sacarosa, produccion de azucares-nucleotidos y obtencion de plantas transgenicas con alto contenido de almidon y alto balance amilosa/amilopectina. |
ESP200400257 | 2004-02-05 | ||
PCT/ES2005/070010 WO2005075649A1 (es) | 2004-02-05 | 2005-01-27 | Procedimiento para la producción de sacarosa sintasa recombinante su uso en la fabricación de kits de determinación de sacarosa producción de adpglucosa y obtención de plantas transgénicas cuyas hojas y órganos de reserva acumulen alto contenido en adpglucosa y almidón |
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CN1984994A true CN1984994A (zh) | 2007-06-20 |
CN1984994B CN1984994B (zh) | 2013-11-20 |
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CN200580004166XA Expired - Fee Related CN1984994B (zh) | 2004-02-05 | 2005-01-27 | 重组蔗糖合酶的生产方法、及其在生产蔗糖测定试剂盒中的应用、生产腺苷二磷酸葡糖的方法和获得具有积累了高浓度的腺苷二磷酸葡萄糖和淀粉的叶片和储藏器官的转基因植物的方法 |
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US (3) | USRE46642E1 (zh) |
EP (1) | EP1721980B1 (zh) |
JP (1) | JP4738351B2 (zh) |
KR (1) | KR101244638B1 (zh) |
CN (1) | CN1984994B (zh) |
AT (1) | ATE502107T1 (zh) |
AU (1) | AU2005210055B2 (zh) |
BR (1) | BRPI0507477B1 (zh) |
CA (2) | CA2554780C (zh) |
DE (1) | DE602005026917D1 (zh) |
ES (3) | ES2245867B1 (zh) |
IL (1) | IL176847A (zh) |
MX (1) | MXPA06008811A (zh) |
PL (1) | PL1721980T3 (zh) |
RU (1) | RU2369637C2 (zh) |
WO (1) | WO2005075649A1 (zh) |
ZA (1) | ZA200606235B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106834343A (zh) * | 2017-02-21 | 2017-06-13 | 中国农业大学 | 蔗糖合成酶在调控植物果实发育中的应用 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2354895B1 (es) * | 2008-09-12 | 2012-01-23 | Iden Carbohydrate Biotechnology, S.L | Procedimiento para la producción de plantas transgénicas que presentan alto contenido y rendimiento en almidón y alto balance amilosa/amilopectina. |
ES2339094B1 (es) * | 2008-11-13 | 2011-03-18 | Iden Carbohydrate Biotechnology, S.L. | Procedimiento para la produccion de plantas transgenicas que presentan alto contenido en compuestos antioxidantes, alta capacidad antioxidante y resistencia al empardecimiento. |
KR101985668B1 (ko) * | 2017-10-30 | 2019-06-04 | 명지대학교산학협력단 | 벼 유래 OsSUS4 유전자를 이용한 도열병 저항성이 조절된 형질전환 식물체의 제조방법 및 그에 따른 식물체 |
CN111172237A (zh) * | 2019-10-24 | 2020-05-19 | 新疆农业科学院农产品贮藏加工研究所 | 一种测定植物中蔗糖合成酶和蔗糖磷酸合成酶含量的方法 |
DE102020111560A1 (de) | 2020-04-28 | 2021-10-28 | Rheinisch-Westfälische Technische Hochschule (Rwth) Aachen | Enzymkaskaden auf Basis von Saccharose Synthase und Pyrophosphorylase zur Umsetzung von ADP zu ATP |
CN111793641B (zh) * | 2020-07-20 | 2022-07-19 | 中国农业科学院郑州果树研究所 | 甜樱桃PavSS或PavSPS基因在调控果实着色或果实成熟软化中的用途 |
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CA1340522C (en) * | 1987-03-10 | 1999-05-04 | Heinz Dobeli | Fusion proteins containing neighbouring histidines for improved purification |
WO1994028146A2 (en) * | 1993-05-24 | 1994-12-08 | Hoechst Schering Agrevo Gmbh | Dna sequences and plasmids for the preparation of sugar beet with changed sucrose concentration |
US6682918B1 (en) * | 1996-07-19 | 2004-01-27 | Arch Development Corporation | Bacterial sucrose synthase compositions and methods of use |
DE19736343B4 (de) * | 1997-08-21 | 2006-02-09 | Forschungszentrum Jülich GmbH | Verfahren zur Erhöhung der Genexpression von Saccharose Synthase |
US7598361B2 (en) * | 1997-11-24 | 2009-10-06 | Monsanto Technology Llc | Nucleic acid molecules and other molecules associated with the sucrose pathway |
JP2001335600A (ja) * | 2000-05-25 | 2001-12-04 | Funakoshi Co Ltd | タンパク質の精製方法 |
JP2002065265A (ja) * | 2000-08-25 | 2002-03-05 | Sumitomo Electric Ind Ltd | 複合タグ |
BR0115782A (pt) * | 2000-12-08 | 2004-01-20 | Commonwealh Scient And Ind Res | Modificação de expressão de gene de sacarose sintase em tecido de planta e usos |
US7091398B2 (en) * | 2001-02-22 | 2006-08-15 | Pioneer Hi-Bred International, Inc. | Isolated sucrose sythase polynucleotides and uses thereof |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106834343A (zh) * | 2017-02-21 | 2017-06-13 | 中国农业大学 | 蔗糖合成酶在调控植物果实发育中的应用 |
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