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将花生条纹病毒(Peanut stripe virus,PStV)外壳蛋白基因(cp)反向重复序列重组至植物表达载体pK7GW1WG2,通过根癌农杆菌(Agrobacterium tumefaciens)菌株GV3101导入本生烟(Nicotiana benthamiana)。卡那霉素抗性植株5～6叶期摩擦接种PStV,通过症状观察和ELISA检测,T0代转基因烟草植株87%表现免疫,不同抗性系T1代植株的免疫比例为60%～100%,多数抗性系T2代植株免疫比例达到100%。利用PStV cp基因特异探针对转基因植株T1代进行特异小干扰性RNA(siRNA)的Northern blot检测,转基因植株都检测出不同含量的特异siRNA,而非转基因植株未检测到特异siRNA;对病毒接种前的转基因植株进行特异siRNA检测,免疫植株比感病植株特异siRNA含量高;对免疫转基因植株进行不同时期的siRNA检测,植株在接种前、接种15和50d后的叶片中都检测到特异siRNA,同一植株不同时期siRNA含量相对稳定;各个转化系植株体内siRNA含量存在差异。实验成功地利用双链RNA(dsRNA)策略获得对PStV具有较高比例抗性的转基因烟草植株。
The inverted repeat sequence of the peanut stripe virus (PSV) coat protein gene (cp) was recombined into the plant expression vector pK7GW1WG2 and introduced into Nicotiana benthamiana via the Agrobacterium tumefaciens strain GV3101. The kanamycin-resistant plants were inoculated with PStV at the stage of 5 to 6 leaf stage, and immunized with 87% of T0 transgenic tobacco plants through symptom observation and ELISA. The immune ratios of the T1 plants of different resistant lines were 60% -100% Most resistant lines of T2 generation of plants reached 100% immune. Northern blot analysis of T1 generation of transgenic plants using PStV cp gene specific probe showed that different levels of specific siRNA were detected in transgenic plants but not in non-transgenic plants. Specific transgenic plants were detected by specific siRNA. The immune plants had higher specific siRNA than that of the susceptible plants. The siRNAs were detected in different stages of the transgenic plants. The specific siRNAs were detected in the leaves of the plants 15 and 50 days after inoculation, The same plant at different periods of siRNA content is relatively stable; each transformed plant in vivo siRNA content differences. Experiments successfully using double-stranded RNA (dsRNA) strategy to obtain PStV has a high proportion of transgenic tobacco plants.