香蕉条斑病毒ORF Ⅰ、ORF Ⅱ酵母双杂交系统诱饵质粒的构建及鉴定  

Construction and Test of the Bait Plasmid of ORF Ⅰ,ORF Ⅱ of Banana Streak Virus in the Yeast Two-hybrid System

作　　者：林湛松[1] 王小明[1] 龚殿[1] 刘志昕[2] 

机构地区：[1]海南大学环境与植物保护学院,海南儋州571737 [2]中国热带农业科学院热带生物技术研究所\农业部热带作物生物技术重点开放实验室,海南海口571101

出　　处：《热带农业科学》2011年第6期45-49,共5页Chinese Journal of Tropical Agriculture

基　　金：国家自然基金项目(No.31070131);中央级公益性科研院所基本科研业务费专项(No.ITBBZD0754)

摘　　要：以连接在pMD-18T载体上的香蕉条斑病毒(BSV)全基因组为模板,PCR扩增得到了带有Gateway R重组反应接头序列的香蕉条斑病毒ORFⅠ及ORFⅡ基因片段,经过BP、LR重组后,构建成带有目的片段的酵母双杂诱饵质粒pDEST32-O1及pDEST32-O2。将质粒转化E.coli DH5α感受态细胞,筛选序列和编码框均正确的重组质粒。将以上2种正确的重组质粒分别与用于构建诱饵质粒的pDEST22空质粒共转化酵母MaV203感受态细胞,利用营养缺陷培养基(SC-Leu-Trp)筛选转化子,并对转化子进行毒性鉴定、自激活鉴定以及3-AT抑制浓度鉴定。结果显示,2种转化子均生长正常,说明所构建的2个重组质粒表达蛋白对酵母无毒性作用,其在特异营养缺陷培养基(SC-Leu-Trp-Ura)中无法生长,不能产生自激活现象,3-AT抑制浓度分别为50、75 mmol/L。以上结果说明所构建的2种诱饵质粒均可用于后续酵母双杂工作,为BSV与宿主的蛋白互作奠定了基础。In the present study,the two target segments containing the Gateway recombinant sequences were firstly generated by two PCR amplifications,respectively,using the whole genome of BSV-Yunnan as the template.Then the two recombinant plasmids pDEST32-O1 and pDEST32-O2 were generated by two steps of Gateway recombination reactions,BP and LR respectively.The two plasmids were then transformed into the competent cells of E.coli DH5α,the plasmids with both accurate sequences and the reading-frames were selected by colony PCR amplification and sequencing.The two qualified plasmids were thereafter co-transformed with pDEST-22 empty plasmid respectively into the competent cells of MaV203 Yeast strains,and the tests of toxicity,self-activation and the inhibition concentration of 3-AT were performed by the utilization of selective plates.The results showed that there was no toxicity and self-activation among both of the recombinant plasmids,moreover,the inhibition concentration of 3-AT was 50 mmol/L and 75 mmol/L respectively for the two plasmids.These results suggested that both of the bait plasmids were qualified to be utilized in the subsequent yeast two-hybrid analysis and thus pave the way for the further investigation of interactions between proteins encoded by BSV and hosts.

关 键 词：香蕉条斑病毒 酵母双杂交 诱饵质粒 自激活鉴定 

分 类 号：Q503[生物学—生物化学] Q939.4