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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。
The gene of 2a was used as the template of PStV-Hongan coat protein gene (CP), peanut dwarf virus light strain (PSV-Mi) and peanut cucumber mosaic virus (CMV-CA) , A 150 bp fragment of PStV-CP 5 ’end, PSV-Mi and CMV-CA2a 3’ end was obtained by PCR method. The fragment of 450 bp was obtained by PCR splicing with three fragment mixtures. The splicing fragment was recombined by Gateway system To the plant expression vector pK7GW1WG2 to obtain a plant expression vector pK450 containing an inverted repeat splicing fragment. The Agrobacterium tumefaciens strain GV3101 was transformed into Nicotiana benthamiana by freeze-thaw method. The transgenic plants were obtained by PCR assay. The T1 generation transgenic plants were inoculated with three kinds of viruses respectively, 66.7% of the plants were immunized against PStV, 9% of the plants showed resistance to CMV-CA, and all the plants were susceptible to PSV. Northern blot results of siRNA showed that all transgenic tobacco plants contained virus-specific siRNA, and the siRNA content was attenuated with time after inoculation.