基于遗传转化手段防治番茄黄化曲叶病毒的分析-analysis of controlling tomato yellow leaf curl virus based on genetic transformation.docxVIP

中 6 株进行 qRT-PCR 检测，5 株 T0 代转基因番茄苗 slIAA4 基因的表达水平比番茄9210 显著上调 10-50 倍。通过茎杆注射 TYLCV 侵染性克隆 2 周后，感病番茄 9210 和转基因番茄都有明显 TYLCV 发病症状，但 qRT-PCR 检测发现转基因番茄体内 TYLCV 病毒积累量是感病番茄的 6-8 倍。构建 TRV2-slIAA4 VIGS 载体转化农杆菌， 通过农杆菌侵润法注射抗病番茄子叶，20d 后发现番茄株型显著矮化且根系不发达， 后期生长畸形，qRT-PCR 检测发现侵润后的抗病番茄 slIAA4 基因表达量下降至对照 的 0.5-0.7 倍；进一步通过茎杆注射 TYLCV 侵染性克隆 2 周后，qRT-PCR 检测发现 slIAA4 基因沉默的抗病番茄病毒积累量是 slIAA4 基因未沉默的抗性番茄病毒积累量 的 20%-40%，病毒复制量减少。综合上述结果，slIAA4 基因过量表达导致番茄体内 TYLCV 含量增加，而 slIAA4 基因沉默则降低番茄体内 TYLCV 含量，表明 slIAA4 基 因是番茄黄化曲叶病毒的抗病相关基因。 关键词：番茄黄化曲叶病毒，烟粉虱，CYP6CM1，coe1，抗药性，slIAA4，抗病性AbstractTomato yellow leaf Curl diseases(TYLCD) cause serious damage to tomato production, which is caused by Tomato yellow leaf Curl Virus (TYLCV)----a geminivirus transmitted by Bemisia tabaci. At present, Control Bemisia tabaci and selecting breeds resistant to TYLCV are principal methods to control TYLCV. Therefore, understanding the mechanisms underlying insect-resistance and disease-resistance would be very important for improving disease resistance in tomatoes. We use the method of genetic transformation to study the possibility of control B.tabaci and TYLCV, integrating a RNAi cassette targetting Cytochrome P450 CYP6CM1 gene and carboxylesterase Coe1 gene from Bemisia tabaci into tobacco genome and transferring related TYLCV resistant gene slIAA4 into susceptible tomatoes.B.tabaci resistant to insecticides is closely related to stronger detoxification and metabolism of detoxifying enzymes such as Cytochrome P450 enzyme and carboxylesterase. A RNAi cassette targetting a P450 gene( CYP6CM1) and a Carboxylesterase gene(coe1) of Bemisia tobaci was integrated into tobacco genome through Agrobacterium transformation, and five independent T0 lines were confirmed by PCR.After 5 days of feeding Bemisia tabaci with tobaccos, the exprssion of CYP6CM1 and coe1 in Bemisia tabaci fed with transgenic tobacco (H1 and H2 which performed better kanamycin resistance) only accounted for 18%-24% and 17%-33% of that fed with non-transgenic tobac