反向重复RNA介导转基因烟草对花生条纹病毒抗性  

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

作　　者：晏立英[1,2] 许泽永[1] 陈坤荣[1] Rob Goldbach Marcel Prins 

机构地区：[1]中国农业科学院油料作物研究所,油料作物遗传改良农业部重点实验室,武汉430062 [2]Laboratory of Virology,Wageningen University,Netherlands

出　　处：《农业生物技术学报》2007年第4期702-707,共6页Journal of Agricultural Biotechnology

基　　金：中国农业科学院研究生院中荷博士联合培养项目;农业部油料作物遗传改良重点实验室开放课题(No.200404)资助

摘　　要：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 of the coat protein gene of Peanut stripe virus （PStV） was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens mediated transformation. The kanamycin resistant transgenic plants were tested for their resistance by inoculation with PStV at 5-6 leaf stage. By symptoms observation and ELISA detection, 87% transgenic plants were immune to PStV in To generation, 60%-100% T1 plants were immune to PStV of different resistant lines, and T2 plants were immune to PStV of most resistant line. Detection of small interfering RNA （siRNA） was carried out by Northern blot. Specific siRNA were detected in transgenic plants, but not in non-transgenic plants. Its amount was higher in resistant plants than that of susceptible plants in the leaves collected before inoculation, and the amount showed difference in different resistant plants. Further analysis showed that siRNA amount was presented equally in the leaves before inoculation, 15 and 50 days post inoculation of detected T2 resistant plants. In this experiment,double-stranded RNA （dsRNA） strategy was succeeded inducing high frequency oftransgenic resistant tobacco plants against PStV.

关 键 词：花生条纹病毒 DSRNA 反向重复序列 抗性 SIRNA 

分 类 号：S188[农业科学—农业基础科学]