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RNA干扰介导的基因沉默是植物抗病育种和大规模分析功能基因快速而有效的方法。构建烟草抗病毒病相关R基因的RNAi表达载体并导入烟草可获得抗病毒病烟草材料,并为大规模分析鉴定烟草功能基因提供新方法。本研究利用绒毛状烟草基因组测序获得的R基因序列设计引物,通过PCR技术扩增出带有特异性结合位点attB的R基因部分序列。采用GATEWAY方法将目的基因的干扰片段插入到表达载体pH 7 GWIWG2(I),成功构建了烟草抗病毒病相关R基因的RNAi表达载体。
RNA interference-mediated gene silencing is a fast and effective method for plant resistance breeding and large-scale analysis of functional genes. The construction of RNAi expression vector of tobacco R virus related R gene and introduction into tobacco can provide anti-viral tobacco material and provide a new method for large-scale analysis and identification of tobacco functional genes. In this study, primers were designed based on the R gene sequence obtained from the genome sequencing of villus tobacco, and partial sequence of R gene with specific binding site attB was amplified by PCR. The GATEWAY method was used to insert the interference fragment of the target gene into the expression vector pH 7 GWIWG2 (I), and the RNAi expression vector of R gene of tobacco antiviral disease was successfully constructed.