海藻糖-6-磷酸合酶基因转化小麦的研究
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摘要
本文以TPS1基因的CDNA,质粒pWM101为研究对象，运用基因克隆技术，成功构建了植物表达载体pWM102。将质粒PWM102转化农杆菌菌株EHA105，经kana霉素，Rif霉素筛选，PCR鉴定，获得含有重组质粒PWM102的农杆菌。通过对诱导培养基，分化培养基中2，4-D浓度调整，得到了一组对成熟胚诱导与分化较好的培养基，并确定以小麦温6的成熟胚与幼胚作为基因转化的外植体，利用农杆菌介导的方法，将海藻糖-6-磷酸合成酶基因（TPS1）导入小麦温6，幼胚与成熟胚产生的愈伤组织中，在含有20-60mg/l潮霉素的继代培养基上，分别获得了一批抗性愈伤组织，经过组织分化，获得了一批再生苗。并且在其中四株再生苗中检测到TPS1基因，结果表明四株扩增出TPS1基因片段的植株为转基因植株。
The fragment of tpsl was digested by two restriction enzyme Kpnl/Xbal ,and then was successfully joined with Pwml01 plasmid which was digested by Kpnl/Xbal too.The outcome was expression vector Pwml02,which wastransformed into Agrobacterium tumefaciens EHA105.By changing the concentration of the 2,4-D,we get a good inducing and differentiation medias, and deciding using the mature and immature embryos of Wen 6 as explants.Agrobacteriurn mediates transforming the TPS1 gene into the callus forming by mature and immature embryos of wen 6.Then we get some resistencing callus which could growing on medias containing 20-60mg/1 hygromycin.We get some regernerating wheats and four of them are positive by PCR analysis.These four plants were indentified to be
the transgenic wheats.
The fragment of tpsl was digested by two restriction enzyme Kpnl/Xbal ,and then was successfully joined with Pwml01 plasmid which was digested by Kpnl/Xbal too.The outcome was expression vector Pwml02,which wastransformed into Agrobacterium tumefaciens EHA105.By changing the concentration of the 2,4-D,we get a good inducing and differentiation medias, and deciding using the mature and immature embryos of Wen 6 as explants.Agrobacteriurn mediates transforming the TPS1 gene into the callus forming by mature and immature embryos of wen 6.Then we get some resistencing callus which could growing on medias containing 20-60mg/1 hygromycin.We get some regernerating wheats and four of them are positive by PCR analysis.These four plants were indentified to be
the transgenic wheats.
引文
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[18] Christeopher,j.Lamb,et.al(1992):Bio/Technology 10，1436-1444
[19]黄大年，朱冰，杨炜. 抗菌肽B基因导入水稻及转基因植株的鉴定，中国科学C辑，1997，27（1）：55-62
[20] 石强，刘飞鹏，抗菌肽克隆基因的表达和转基因研究现状，生物工程进展，2000，20,
37-40
[21] 原亚萍，母秋华，付荣昭等，用基因枪法将防御素基因转入玉米并再生植株初报，吉林农业大学学报 1997,19(4):113～115
[22] 钱前，抗白叶枯病基因Xa-21成功转入我国杂交水稻 ，中国稻米，2000（1），5
[23] 舒群芳，赵路，李文彬等，植物基因克隆的策略和方法，植物学通报，1999,16(1):80～85
[24] 王小军，刘玉乐，HuangYong等，可育的抗除草剂溴苯腈转基因小麦，植物学报 1996,38(12):942～948
[25] 李德森.，渠荣达.，基因枪法获得可育抗除草剂转基因小麦,南开大学学报（自然科学版），1997，30（3）：69-75
[26] 梁雪莲，孙毅，郭平毅，农杆菌介导转化小麦幼胚获得抗除草剂再生植株，植物生理与分子生物学报，2003,29(6):501-506
[27] Potrykus I.Bio/Technology,1990,6:535 ~542
[28] Potrykus I. Nature,1992，355(2):568~569
[29]Delbreil B, Guerche P,Jullien M. Plant Cell Rep,1993, 12:129~ 132
[30]Chan M,Chang H, Tong W, et al.Plant Mol Biol,1993, 22:491~506
[31] Hiei Y,Ohta S,Komari T.Plant J,1994，6:271 ~ 282
[32]Ishida Y, Saito H, Ohta S, Hiei Y, Komari T and Kumashiro T.Na t-Biotechnol,1996，14:745 ~750
[33] Hess D, Dressler K, Nimmrichter R.Plant Sci Lim,1990, 72(20):2 33~244
[34] Pukhal'skii VA,Smirnov SP, korostyleva TV, Bilinskaya EN, Elis eeva AA.Genetika,1996，32(110):1596
[35] Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan AR, Conn er TW, Wan Y. Plant Physiol，1997, 115(3): 971~981
[36]周文麟，倪建福.作物学报,1992,18（6）:418 ~ 423
[37] Xia GM,Li Zy,He CX,Chen HM,Brettell R.植物生理学报，1999，25( 1): 22~28
[38] Trick HN, Finer JJ. Transgenic Res,1997，6(5):329~336
[39] Singh N,Chawla HS.Current Science 1999, 76(11):1483~1486
[40] Serik O,Ainur I.Plant Cell Rep,1996，16:133 ~ 136
[41]ChibbarRN,KarthaKK,LeungN,etal.Transientexpressionofmarkergenesinimmaturezygoticem-brosofspringwheat(Triticumaestivum)throughmicroprojectilebombardment.Genome,1991,34:453～460
[42]IglesiasVA,GiselA,PotrykusI,etal.Invitrogerminationofwheatproembryostofertileplants.PlantCellReports,1994,13:377～280
[43]VasilV,CastilloAM,FrommME,etal.Herbicideresistantfertiletransgenicwheatplantsobtainedbymicroprojectilebombardmentofregenerableembryogeniccallus.Bio/Techno
logy,1992,10:667～674
[44]VasilV,SrivastavaV,CastilloAM,etal.Rapidproductionoftransgenicwheatplantsbydirectbombard-mentofcullturedimmatureembryos.Bio/Technology,1993,11:1553～1558
[45]WeeksJT,AndersonOD,BlechlAE.Rapidproductionofmultipleindependentlinesoffertiletransgenicwheat(Triticumaestivum).PlantPhysiol,1993,102:1077～1084
[46]VasilV,BrownSM,ReD,etal.Stabletransformedcalluslinesfrommicroprojectilebombardmentofcellsuspensionculturesofwheat.Bio/Technology,1991,9:743～747
[47]WangYC,KleinTM,FrommM,etal.Transientexpressionofforeigngenesinrice,wheatandsoybeancellsfollowingparticlebombardment.PlantMolBiol,1988,11(4):433～439
[48] 梁辉， 唐顺学， 张弛， 赵铁汉， 李良材， 欧阳俊闻， 田文忠， 王道文， 贾旭， 提高小麦基因枪法转化频率的研究，遗传学报，1999，26（6）：643-648
[49] 梁辉， 郑近， 段霞瑜， 盛宝钦，贾双娥，李义文，唐顺学，用基因枪法获得抗白粉病转芪合酶基因小麦，科学通报，1999，44（24）：2644-2648
[50]LonsdaleD,OndeS,CumingA.TransientexpressionofexogenousDNAinintact,viablewheatembryosfollowingparticlebombardment.JExpBot,1990,41:1161～1165
[51]OardJH,PaigeDF,SimmondsJA,etal.Transientgeneexpressioninmaize,andwheatcellsusinganair-gunapparatus.PlantPhysiol,1990,92:334～339
[52] 贾士荣.植物遗传转化的研究进展.见:魏建昆主编.农业高新技术论.北京:科学技术文献出版社,1992,121～128
[53] 成卓敏，何小源,等.自然科学进展－国家重点实验室通讯，1993，3（ 6）：560 ~ 564
[54] 曾君祉，王东江.中国科学（B辑），1993,23（3）：256 ~ 261
[55] 刘根齐，张孔恬. 遗传学报，1994，21（6）：463 ~ 467
[56] Zeng JZ, Wu YQ.Chinese Sci Bull, 1998，43(10):798
[57] Vasil V,Castillo AM,Fromm ME,Vasil IK.Bio/Technology, 1992,10:66 7~674
[58]??Langridge P,Brettschneider R,Lazzeri P,Loez H.The Plant Journal,1 992，2(4):631~638
[59] Vasil IK.Plant Mol Biol,1994,25:925 ~ 937
[60]??Maheshwari N,Rajyalakshmi K, Baweja K, Dhir SK, Maheshari SC.Crit ical reviews in plant sciences,1995，14 (2):149
[61]??Nehra NS,Chibber RN，Kutty KK.Plant Breeding Abstracts, 1995，65( 6):803~808
[62]??Vasil IK,Vasil V.Plant Biobechnology for Sustainable Development of Agriculture.1996,pp1~4
[63]??李艳红, 张爱民, 肖兴国,中国青年农业科学学术年报,1997，B：551~ 556
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