番茄双抗烟草花叶病毒和黄瓜花叶病毒的基因工程研究.docx

Chinese Agricultural Science Bulletin V01．2 1 No．8 2005 August·28· http：／／znth．chinajournal．net．ca番茄双抗烟草花叶病毒和黄瓜花叶 病毒的基因工程研究廖俊杰-，李进进，，许继勇：(-广东轻工职业技术学院食品与生物工程系，广东广州510300；2汕头市中蔬花卉有限公司，广东汕头515041)摘要：在烟草花叶病毒(TMV)外壳蛋白(CP)基因的5’和3’端分别合成寡聚核苷酸T5和T3，并分别 引入Cla I和BamH I位点。采用PCR技术扩增得到TMV CP基因，用Cla I，BamH I消化后重组到 pBluescript KS+中．并将该基因与黄瓜花叶病毒(CMV)CP基因重组在同一个表达栽体pKYLX7上获 得双价CP基因表达栽体pKDTC，并转入根癌农杆茵LBA4404中，通过“叶盘法”转化番茄“丽春”、 “京韩一号”，获得了番茄再生植株。通过点杂交、PCR检测和Southem杂交，证实“丽春”的L-2、L一5、 “京韩一号”的JH一6号为转TMV CP和CMV CP基因的工程植株。工程植株在室温中表现正常，与对 照植株相比，转基因植株表现明显的抗TMV、CMV性能。 关键词：番茄；烟草花叶病毒：黄瓜花叶病毒；外壳蛋白基因；抗病毒基因工程Studies on Tmnsgenic Tomato Plants with Resistance to TobaccoMosaic Virus and Cucumber Mosaic VirusLiao Junjie 1，Li Jinjinl，Xu Jiyon92(1Department of Food and Bio-technology,G珊mgdD，lg Light Industry Technologic College，Guangzhou 510300；铆啪印z‘Zhon哥shu Flower Co．，Ltd，Shantou Guangdong 5 15041)Abstract：The TMV CP gene was synthesized with Polymerase Chain Reaction(PCR)Y using the cDNA of its genomic RNA as a template．A synthetic C／a I site Was included in T5 primer while a BamH I site in T3 primer．the PCR product was doned into the Cla I／B mH I site of pBluescript KS+．Then bivalent plant expression vector，pKDTC(TMV—CP+CMV-CP)，with double resistance to TMV and CMV，Was constructed，in which each CP gene Was controlled by a 35S promotor of CaMV．Transgenic tomato plants were regenerated via Agrobacterium transformation system，and analyzed by dot blot，PCR and Southern blot，these results showed that both genes were integrat ed into the transgenic plant lines L-2，L-5 of Lichun and JH一6 of JinghaIl No 1．The transgenic plants grew normally and obviously resisted to the infection of corresponding viruses in greenhouse．Key words：Tomato，Tobacco mosaic virus，Cucumber mosaic vires，Coat protein gene，Virus—resistantgenetic engineering番茄是主要的蔬菜作物，在生产中，烟草花叶病毒 难。植物抗病毒基因工程的发展，为培育抗性品种带 (TMV)、黄瓜花叶病毒(CMV)是危害番茄的主要病 来了希望。自1986年Powell等翻将TMV CP基因在 毒病害【l】，致使番茄严重减产，品质变劣。培育抗性品 烟草中表达，获得了抗TMV的烟草工程植株以来， 种是解决病毒危