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以感染百合无症病毒(LSV)的百合叶片总RNA为模板,利用反转录聚合酶链式反应(RT-PCR)扩增出了876bp的LSV CP基因,经Blast比对发现该片段与GenBank上发表的LSVCP基因序列高度同源,同源率98%,由该序列推导出的氨基酸序列相似性达到98%。应用Gateway技术将扩增的片段通过BP反应连接到入门载体pDONR201,并进行序列测定,再通过LR反应将目的片段插入到RNAi植物表达载体pH7GWIWG2(Ⅱ),成功构建了适合农杆菌介导的百合转化的RNAi载体。
The LSV CP gene of 876bp was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA of lily leaves infected by LSV as a template. The Blast comparison showed that the fragment was homologous to GenBank The published sequence of LSVCP gene was highly homologous with 98% homology. The amino acid sequence similarity deduced from this sequence was 98%. The amplified fragment was ligated into the entry vector pDONR201 by using the Gateway technique and sequenced. The target fragment was inserted into the RNAi plant expression vector pH7GWIWG2 (Ⅱ) by LR reaction, and the Agrobacterium tumefaciens-mediated lily Transformed RNAi vector.