霜霉菌侵染后葡萄叶片酵母双杂交cDNA文库构建

Construction of a yeast two-hybrid cDNA library from Vitis vinifera leaves infected by downy mildew

【目的】构建霜霉菌侵染后葡萄叶片的酵母双杂交cDNA文库,筛选致病因子在寄主葡萄体内的互作靶标,为葡萄霜霉病菌致病的分子机理研究提供材料基础。【方法】以一年生欧亚种葡萄西拉盆栽苗为试验材料,提取并等量混合霜霉菌侵染后0、12、18、24和36 h的葡萄叶片总RNA,利用基于Gateway技术的CloneMinerⅡcDNA文库构建试剂盒:首先将cDNA与pDONR222载体进行BP重组反应连接,连接产物转化大肠杆菌DH10B感受态细胞构建初级文库;然后初级文库质粒与pGADT7-DEST载体通过LR重组反应,重组反应产物转化大肠杆菌DH10B感受态细胞构建次级文库;再随机挑取次级文库转化Y187酵母菌株构建酵母双杂交cDNA文库;最后利用ImageJ软件和PCR反应分别统计库容量和鉴定重组率。【结果】构建的初级文库库容量为4.6×10^7 CFU,次级文库库容量为2.7×10^7 CFU,均达到构建cDNA文库的标准。酵母双杂交cDNA文库的插入片段主要集中在1000~2000 bp,且具有很好的多态性,文库质量较高。【结论】构建的霜霉菌诱导葡萄叶片酵母双杂交cDNA文库符合酵母双杂交筛选要求,可用于筛选霜霉菌致病效应蛋白在寄主体内的靶标及霜霉菌侵染寄主葡萄早期抑制寄主免疫过程中的致病机理研究。

【Objective】To construct yeast two-hybrid cDNA library of grape leaves after downy mildew infection,screen the target of pathogenic factors in host grapes and provide reference for molecular mechanism of pathogenic factors of Plasmopara viticola.【Method】Total RNA was extracted and mixed fromgrape leaves of Vitis vinifera Shiraz one-year potted seedlings at 0,12,18,24 and 36 h after downy mildew infection. The library was constructed using the CloneMiner Ⅱ cDNA library construction kit based on Gateway technology. The cDNA was ligated into the pDONR222 vector by BP recombination reaction,and the ligation product was transformed into Escherichia coli DH10B competent cell to construct a primary library. Then the primary library plasmid was introduced into the pGADT7-DEST vector by LR recombination reaction,recombinant reaction products transformed E. coli DH10 B competent cell to construct primary library,and secondary libraries were randomly selected to transform Y187 yeast strain to construct yeast two-hybrid c DNA library.The library capacity was counted by ImageJ software and the recombination rate was identified by PCR.【Result】The tests showed that the capacity of the primary library obtained by the experiment was 4.6×10~7 CFU,the capacity of secondary library was 2.7×10~7 CFU,reaching the standard to construct cDNA library. The inserts of the yeast twohybrid cDNA library were mainly concentrated at 1000-2000 bp and had good polymorphism. The library quality was high.【Conclusion】A high-quality yeast two-hybrid cDNA library from grape leaves infected with downy mildew is obtained in this experiment. It meets the requirements of yeast two-hybrid screening,is a target that can be used to screen downy mildew pathogenic effect proteins in the host,and study the pathogenic mechanism during the early inhibition on host immunity of downy mildew infected host grapes.