水稻条纹病毒NS2和NS3基因共干扰转基因水稻的培育及抗病性分析

Construction of Rice Stripe Virus NS2 and NS3 Co-RNAi Transgenic Rice and Disease-resistance Analysis

NS2和NS3是水稻条纹病毒编码的两个转录后基因沉默抑制子,沉默抑制子往往与病毒致病性相关。本研究利用重叠PCR方法将NS2和NS3基因的部分cDNA片段重组后连接到RNAi载体,并借助PstⅠ将其重组到植物表达载体PXQ上;通过农杆菌介导法侵染水稻日本晴种子的愈伤组织,经新霉素（G418）抗性筛选获得31株转基因植株。对T0代转基因植株进行PCR及Southern blot分析结果显示,目的基因已成功转入水稻基因组中,并且不同转化植株含有目的基因的拷贝数不同;抗病性实验表明,与野生型植株相比,T0代转基因水稻能推迟RSV发病时间10~20d,导致病毒积累量下降30%~50%,且其下降量与发病时间延迟有相关性。

NS2and NS3 are two post-transcriptional gene silencing suppressors that are encoded by Rice stripe virus.Gene silencing suppressors are always related to the pathogenicity of viruses.In this study,the cDNA of NS2 and NS3were recombined by overlapping PCR assays,ligated to the RNAi vector,and inserted into the PXQ expression vector using PstⅠ;the expressed vector was transferred into calluses induced from seeds of the japonica rice cultivar,‘Nipponbare’,using an Agrobacterium-mediated method.Thirty-one T0 transgenic plants were selected by G418 screening.PCR and southern blot analyses confirmed that the target gene was transformed into transgenic rice successfully,and different transgenic plants contained various copies of the gene.The disease resistance assay revealed that T0 transgenic rice had a delayed onset of RSV for approximately 10-20 d,and the accumulation of virus in the transgenic plants was reduced by 30%-50%.This was related to the delayed onset of disease.