摘要
[Objective] This study was to construct RNAi expression vector targeting FaVRN1 gene. [Method] A 145 bp Arabidopsis actin intron was inserted into the expression vector to generate an intermediate vector pBI121-M-INT. And then two pairs of specific primers with enzyme restriction sites spanning a 351 bp cDNA conserved sequence fragment of FaVRN1 gene were designed for RT-PCR to construct RNAi expression cassette. The amplified fragment was inserted forwardly and reversely at two sides of the intron to construct an RNAi expression vector with hairpin structure. [Result] Double enzyme digestion（HindIII＋BamHI） showed the intron had been successfully into the vector pBI121. PCR amplification and double enzyme digestion indicated the success of forward and reverse ligation of target FaVRN1 fragment into the intermediate vector. [Conclusion] This study laid foundation for breeding novel flowering-inhibited tall fescue varieties.
[目的]用RNAi干扰技术沉默高羊茅FaVRN1。[方法]将拟南芥内肌动蛋白含子(145bp)插入到表达载体pBI121已构建能形成发夹的中间载体。设计带有限制性内切酶位点的特异性引物,扩增高羊茅春化基因351bp长的外显子靶标序列,限制性酶切后以正向和反向插入到RNA干扰中间载体内含子两端,构建带发夹结构的RNA干扰表达载体。[结果]双酶切结果表明内含子(145bp)已成功连入表达载体。PCR和酶切验证证实目的基因(351bp)已连入中间表达载体。[结论]该研究为培育RNAi转基因开花抑制型高羊茅新品种奠定基础。
基金
Research Fund from Guizhou Academy of Agricultural Sciences([2009]038)
Programs for Science and Technology Development in Guizhou Province([2009]3067)~~
