利用RNAi 介导的抗病性获得抗2 种花生病毒的转基因烟草

 分别以花生条纹病毒红安株系(PStV-Hongan)外壳蛋白基因(CP),花生矮化病毒轻型株系(PSV-Mi)和花生黄瓜花叶病毒(CMV-CA)复制酶基因2a为模板,通过PCR 方法分别得到PStV-CP 5′ 端,PSV-Mi 和CMV-CA 2a3′ 端150 bp 的片段,3 种片段混合物为模板经PCR 拼接得到450 bp 的片段,此拼接片段通过Gateway 系统重组至植物表达载体pK7GW1WG2,得到含反向重复拼接片段的植物表达载体pK450。冻融法导入根癌农杆菌(Agrobacterium tumefaciens)菌株GV3101 后,叶盘法转化本生烟(Nicotiana benthamiana),经PCR 检测,获得转基因植株。对T1 代转基因植株分别人工接种3 种病毒,66. 7% 的植株表现对PStV 免疫,9% 的植株表现对CMV-CA 的恢复抗性,全部植株对PSV 感病。siRNA 的Northern blot 结果表明,所有转基因烟草植株都含有病毒特异siRNA,siRNA 含量随接种后时间延长而衰减。

Transgenic tobacco plants resistant to two peanut viruses via RNA mediated virus resistance

Specific primers were designed according to the coat protein(CP) genes of Peanut stripe virus Hongan isolate(PStV-Hongan),2a gene of Peanut stunt virus-mild strin(PSV-Mi) and Cucumber mosaic virus peanut strain(CMV-CA),and cDNA fragments with length of 150 bp were obtained from the 5′ ends of CP gene of PStV-Hongan,3′ end of replicase 2a gene of PSV-Mi and CMV-CA.The three cDNA fragments were recombined into one chimeric cDNA by PCR amplification.The chimeric cDNA was then introduced into the plant expressing vector pK7GW1WG2 in a way of inverted repeats by Gateway recombination Kits.The identified plant-expression plasmid pK450 was introduced into Agrobacterium tumefaciens GV3101 by direct transferring,and the target cDNA with inverted repeats was introduced into Nicotiana benthamiana.Transgenic plants obtained by tissue culture and identified by PCR.The resistance assay indicated that about 66.7% of T1 transgenic plants were immune to PStV,9% of the transgenic plants were recovered after CMV inoculation and all the plants were susceptible to PSV.siRNA was found in all transgenic plants by Northern-blot test and its amount decreased with the time after inoculation.