Zheng Lu-ping, Lin Chen, Xie Li-yan, Wu Zu-jian, Xie Lian-hui
Bing Du Xue Bao. 2014 Nov;30(6):661-7.
NS2 and 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 NS3 were recombined by overlapping PCR assays, ligated to the RNAi vector, and inserted into the PXQ expression vector using Pst I; 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.
NS2和NS3是由水稻条纹病毒编码的两种转录后基因沉默抑制因子。基因沉默抑制因子总是与病毒的致病性相关。在本研究中,通过重叠PCR法将NS2和NS3的cDNA进行重组,连接到RNAi载体上,并使用Pst I酶切插入到PXQ表达载体中;采用农杆菌介导的方法将表达载体导入粳稻品种‘日本晴’种子诱导产生的愈伤组织中。通过G418筛选获得了31株T0代转基因植株。PCR和Southern杂交分析证实目标基因已成功转化到转基因水稻中,且不同转基因植株含有该基因的不同拷贝数。抗病性检测表明,T0代转基因水稻对水稻条纹病毒病的发病时间延迟了约10 - 20天,转基因植株中病毒的积累量减少了30% - 50%。这与发病时间的延迟有关。