【作者】 包常华；

【导师】 哈斯阿古拉；

【作者基本信息】 内蒙古大学 ， 微生物学， 2006， 硕士

【摘要】 本实验将苜蓿花叶病毒（Alfalfa mosaic virus，AlMV）复制酶P₂亚基基因全长cDNA序列及3′端非编码区序列构建到植物双元表达载体pROKⅡ中，分别得到重组表达载体pROKAMVF、pROKAMV3N。在实验室前期工作中已将AlMV复制酶基因的5′端1／2cDNA序列、3′端1／2 cDNA序列分别构建到植物双元表达载体pROKⅡ中，得到重组植物表达载体pROKAMV5、pROKAMV3。 将上述四种重组表达载体经农杆菌LBA4404介导转化烟草，获得相应的转化植株，进行PCR检测，结果证明目的基因已整合到烟草基因组中。pROKAMV5转化烟草获得24个转基因株系共90株植株；pROKAMV3转化烟草获得18个转基因株系共69株植株；pROKAMVF转化烟草获得4株转基因植株；pROKAMV3N转化烟草获3株转基因植株。 AlMV接种后检测抗病性结果为：pROKAMV5转基因植株中10个株系对AlMV接种表现完全抗性，pROKAMV3转化植株中12个株系对AlMV接种表现完全抗性。研究结果表明转AlMV复制酶基因不同片断的转基因植株对AlMV的侵染具有抗性，但3′端1／2 cDNA片断介导的抗病性优于5′端1／2cDNA片断。更多还原

【Abstract】 The full length of AlMV replicase gene cDNA sequence, as well as 168bp cDNA sequence at the 3’-end were respectively fused with the plant binary expression vector pROKII, correspondingly, the recombinated vectors pROKAMVF pR0KAMV3N were obtained, The half part of cDNA at 5’-end & 3’-end have been recombinated into binary vector pROK II during the former work of our lab, and recombinated vectors pR0KAMV5 pR0KAMV3 were obtained.According to this experiment, the four recombined expression vectors were mated into Agrobacterium tumefacient LBA4404 and then transformed into tobacco. PCR analysis was employed to detect the four ontogenious sequence of the gained transformants. The result show that 90 pROKAMV5 transgenic plants, 69 pROKAMV3 transgenic plants, 4 pROKAMVF transgenic plants 3 pROKAMV5 transgenic plants were obtained.Detected the transgenic plants by ELISA after inoculate AlMV 60 days, 10 thereinto 24 transgenic plants chains transformed with pROKAMV5 confer AlMV virus resistance, 12 thereinto 18 transgenic plants chains transformed with pROKAMV3 confer AlMV virus resistance.The research indicated that different sequence of AlMV replicase gene transformants perform resistance to virus infection in different level.更多还原