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ClaⅠ和BamHⅠ位点,采用PCR技术扩增得到TMVCP基因,用ClaⅠ/BamHⅠ消化后重组到pBluescriptKS+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载体pKYLX7上获得双价CP基因表达载体pKDTC,并转入根癌农杆菌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Southern杂交,证实“丽春”的L-2、L-5、“京韩一号”的JH-6号为转TMVCP和CMVCP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。
The TMV CP gene was synthesized with Polymerase Chain Reaction （PCR） y using the cDNA of its genomic RNA as a template. A synthetic Cla Ⅰ site was included in T5 primer while a BamH Ⅰ site in T3 primer, the PCR product was cloned into the Cla Ⅰ/BamH 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 integrated into the transgenic plant lines L-2, L-5 of Lichun and JH-6 of Jinghan No 1. The transgenic plants grew normally and obviously resisted to the infection of corresponding viruses in greenhouse.
出处
《中国农学通报》
CSCD
2005年第8期28-32,共5页
Chinese Agricultural Science Bulletin
基金
汕头市科技攻关项目"番茄抗TMV和CMV的基因工程育种"(2002A108)
