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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。
NS2 and NS3 are two post-transcriptional gene silencing suppressors encoded by rice stripe virus, and silencing suppressors are often associated with viral pathogenicity. In this study, partial cDNA fragments of NS2 and NS3 genes were recombined by overlapping PCR and ligated into the RNAi vector and recombined into plant expression vector PXQ by PstI. The calli were infected with Nipponbare seeds by Agrobacterium tumefaciens Thirty-one transgenic plants were obtained by neomycin (G418) resistance screening. The results of PCR and Southern blot analysis of T0 transgenic plants showed that the target gene has been successfully transferred into rice genome, and different transformed plants contain different copy number of the target gene. Disease resistance experiments showed that compared with wild type plants, T0 Generation of transgenic rice can delay the onset of RSV 10 ~ 20d, resulting in a 30% to 50% reduction in virus accumulation, and the decrease is related to the delay of onset.