小麦Ta-SKP2A克隆及其酵母双杂交诱饵载体的构建  

Cloning and Construction of Yeast Two-hybrid Bait Vector of Wheat Ta-SKP2A Gene

作　　者：许媛[1,2] 李铃仙 魏春茹[1,2] 魏新燕[3] 于秀梅[1,4,2] 刘大群 

机构地区：[1]河北农业大学生命科学学院,河北保定071001 [2]河北省植物生理与分子病理学重点实验室,河北保定071001 [3]河北农业大学国家北方山区农业工程技术研究中心,河北保定071001 [4]河北省农作物病虫害生物防治工程技术研究中心,河北保定071001

出　　处：《华北农学报》2016年第2期17-22,共6页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31301649);高等学校博士学科点专项科研基金项目(20121302120010)

摘　　要：SKP2A是调控细胞周期转录因子降解的F-box蛋白。为研究该蛋白在小麦-叶锈菌互作过程的作用,首先克隆获得中国春小麦Ta-SKP2A全长序列,然后将其与pGBKT7载体连接,转入酵母Y187感受态细胞中,构建酵母诱饵表达载体,并检测其表达产物对酵母细胞有无毒性和自激活效应。结果表明,Ta-SKP2A编码区序列长度为1 146 bp,其ORF区编码一条由382个氨基酸组成的多肽。在ORF区两侧连接酶切位点(EcoRⅠ和Bam HⅠ),并将其与载体pGBKT7连接,成功构建了包含目的基因的诱饵载体pGBKT7-Ta-SKP2A,且含重组诱饵质粒的酵母菌株在SD/-Trp营养缺陷平板上生长良好,其过夜培养物OD600>0.8,表明重组诱饵质粒表达产物对酵母细胞无毒性。含重组诱饵质粒的酵母菌株在二缺、三缺及四缺营养缺陷平板上不能生长,表明诱饵质粒的表达产物不能自激活报告基因。成功构建了一个包含Ta-SKP2A的重组诱饵质粒,为深入筛选与其互作的靶蛋白,研究其在小麦-叶锈菌互作体系中的作用机制奠定了基础。SKP2A is an F-box protein that regulates the proteolysis of cell cycle transcription factors. In order to understand the role of SKP2 A in the interaction between wheat and leaf rust pathogen,a full-length sequence of TaSKP2 A was firstly obtained from wheat cv. China Spring,and ligated with pGBKT7 vector. Recombinant vector was transformed into yeast Y187 competent cell,further detected its self-activated activity and toxicity effect for yeast cells. Results showed that Ta-SKP2 A was 1 146 bp and encoded a polypeptide of 382 amino acids. The ORF with restriction enzyme sites of Bam H I and EcoR I was inserted into expression vector pGBKT7. The results showed that the bait vector pGBKT7-Ta-SKP2 A was successfully constructed by sequencing. The recombinant bait plasmid grew well on SD /-Trp plate,which showed that the bait plasmid was not toxic to yeast cell. The yeast strains containing bait plasmid couldn＇t grow on SD /-Leu /-Trp,SD /-Leu /-Trp /-His and SD /-Trp /-Leu /-His /-Ade plate,so the bait plasmid didn＇ t have transcriptional activation. A recombinant bait plasmid with Ta-SKP2 A was successfully constructed,which would lay foundation for screening for targeted protein interacted with Ta-SKP2 A,and would help to understand its function in the interaction of wheat and leaf rust pathogen.

关 键 词：小麦 F-box基因SKP2A 诱饵载体构建 毒性检测 自激活效应 

分 类 号：Q784[生物学—分子生物学]