玉米ZmMPK5酵母双杂交诱饵载体的构建及自激活作用检测  

Construction and self-activation detection of bait vector of ZmMPK5 in yeast two-hybrid system

作　　者：马芳芳[1,2] 王瑞云[1] 蒋明义[2] 

机构地区：[1]山西农业大学农学院,山西太谷030801 [2]南京农业大学生命科学学院,江苏南京210095

出　　处：《山西农业大学学报（自然科学版）》2016年第3期170-175,共6页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：国家自然科学基金(30970238);山西农业大学博士启动基金(2013YJ04);山西农业大学科技创新基金(2014022;2014YZ2-5)

摘　　要：玉米中ZmMPK5参与了ABA诱导的抗氧化防护从而增强了植物对干旱、盐渍以及氧化胁迫的忍受能力,然而截至目前对其潜在的分子机制却知之甚少。为构建玉米ZmMPK5酵母双杂交诱饵表达载体,并检测其在宿主酵母菌MaV203中的自激活活性和毒性,利用gateway技术,以实验室保存的重组质粒pMD19T-ZmMPK5为模板,扩增得到带有attB位点的ZmMPK5基因的ORF片段,通过BP重组反应和LR重组反应,最终将该片段构建到诱饵表达空载体pDEST32上,PCR及测序鉴定结果表明,成功构建了阅读框方向和序列均正确的重组诱饵载体pDEST32-ZmMPK5。用PEG/LiAc法将重组诱饵质粒pDEST32-ZmMPK5转化酵母菌株MaV203感受态细胞,通过缺陷型平板表型分析以及X-gal分析实验检测诱饵载体表达的蛋白对宿主细胞是否具有毒性和自激活作用。最终结果表明构建的诱饵载体对宿主酵母菌MaV203既无毒性也无自激活活性,可用于后续研究,为进一步利用酵母双杂交方法从玉米cDNA文库中筛选与ZmMPK5相互作用蛋白奠定了基础。In maize （Zea mays）, the mitogen-activated protein kinase ZmMPK5 has been shown to be involved in abscisie acid （ABA）-induced antioxidant defence, and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms were poorly understood. In order to construct the bait vector of ZmMPK5 in yeast two-hybrid system and detect its self-activation and toxicity in the host yeast strain MaV203, attB-flanked ORF fragment of ZmMPK5 was amplified using pMD19T-ZmMPK5 as DNA template. The fragment was then successfully fused into PDEST32 vector by BP and LR recombination reaction using gateway technology. The constructed bait plasmid of pDEST32-ZmMPK5 was transformed into MaV203 competent cells by PEG/LiAe method and the toxicity and self-activation of the bait vector was tested by the phenotype analysis and X-gal assay. Final results showed that the bait vector pDEST32-ZmMPK5 had no toxicity and self-activation to the host yeast strain MaV203, and could he used for further research of screening interacting protein of ZmMPK5 from maize cDNA library by yeast two-hybrid technology.

关 键 词：ZmMPK5 酵母双杂交 诱饵载体 MaV203 自激活 

分 类 号：S513[农业科学—作物学] Q78[生物学—分子生物学]