利用RNAi介导的抗病性获得抗2种花生病毒的转基因烟草

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

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-CA2a 3'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。

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 replicase2a 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 pK7GWIWG2 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 inoculatior 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.