反向重复RNA介导转基因烟草对花生条纹病毒抗性

构建携带花生条纹病毒（Peanut stripe virus, PStV）外壳蛋白基因（cp）反向重复序列植物表达载体pKcp，转化农杆菌菌株GV3101，叶盘法转化本生烟，卡那霉素筛选得到的转化植株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，T1代不同系植株的免疫比例为60~100%，T2代多数系植株免疫比例达到100%。利用PStVcp特异探针对转基因植株T1代进行特异siRNA的Northern blot检测，转基因植株都含有不同量的特异siRNA，而非转基因植株不含特异siRNA；接种前，免疫植株比感病植株体内siRNA含量高；不同转化系免疫植株在接种前、接种15天和50天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；不同转化系植株体内siRNA含量存在差异。本试验成功利用dsRNA获得对PStV具有较高比例抗性的转基因烟草植株。

Resistance to Peanut stripe virus Mediated by Inverted Repeat of the Coat Protein Gene in Transgenic Tobacco Plants

Inverted repeat plant expression vector pKcp was constructed with peanut stripe virus(PStV) coat protein gene, then transformed into Agrobacterium tumefaciens strain GV3101. Transgenic plants were obtained by transformation of Nicotiana benthamiana leaf discs by agrobacteirum and kanamycin screening. The transgenic plants were tested for its resistance by inoculation with PStV in 5~6 leaf stage. By symptom observation and ELISA detection, there was 87% transgenic plants immune to PStV in T0 , 60~100% immune to PStV in T1 different lines, most of T2 lines immune to PStV. siRNA was detected by Northern blot by PStV cp probe in T1 plants, different amount of siRNA was detected in transgenic plants, no siRNA was found in nontransgenic plants; siRNA amount was higher in resistant plants than susceptible plants in the leaves before inoculation; about same amount of siRNA of leaves were detected in different days post inoculation in the same resistant plant, different amount of siRNA was found in different resistant plants. In this experiment, dsRNA strategy were succeeded inducing high frequency of transgenic resistant tobacco plants to PStV.