烟草花叶病毒siRNA设计及其植物表达载体构建(英文)

Design of siRNAs and Construction of Plant Expression Vector for Interfering with Tobacco Mosaic Virus

RNA干扰技术已广泛应用于沉默基因表达的研究.本文分析烟草花叶病毒(TMV)基因序列,选择设计编码小分子发卡结构RNA(hpRNA)的cDNA;并根据农杆菌双元载体质粒p2355多克隆位点区限制性酶切位点,两端分别加入XbaI和BamHI酶切位点;分别合成单链DNA复性后插入到p2355上,经PCR、测序验证,表明已成功构建了具有潜在表达烟草花叶病毒siRNA的植物表达载体.

RNA interference （RNAi） is now established as a powerful tool to silence gene expression in a variety of organisms. Depending on the gene sequence of tobacco mosaic virus （TMV） , 3 cDNA sequences that could generate short hairpin RNA were chosen and designed. Corresponding to the multiple cloning sites of binary plasmid p2355, Xbal and BamHI were added at the end of each strand, respectively. After being synthesized and annealed, the fragments were inserted into p2355 driven by CaMV35S promotor. Identified by PCR and sequencing, the results showed that 3 vectors with double -stranded RNA-expressing construct were constructed and had potency to produce RNAi against the virus in tobacco. This could be an antiviral strategy in plant. Fig 3, Tab 1, Ref 26.