利用酵母单杂交筛选拟南芥AtSTK基因表达调控蛋白的研究  

Study on the Expression Regulatory Proteins of Arabidopsis Gene AtSTK by Yeast One Hybrid System

作　　者：杨歌[1] 惠颖 赵翠[1] 田梦涵 任媛媛[1] 黄占景[1] 葛荣朝[1] 

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024 [2]石药集团中奇制药技术有限公司,河北石家庄050035

出　　处：《华北农学报》2017年第4期73-77,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(30900104);河北省自然科学基金项目(C2016205158)

摘　　要：拟南芥丝氨酸/苏氨酸蛋白激酶基因AtSTK的过量表达可以明显提高拟南芥的耐盐性。为进一步研究AtSTK基因表达的调控机制,以拟南芥基因组DNA为模板设计引物,扩增获得AtSTK基因的启动子序列,并构建到报告载体pAbAi,将重组报告载体质粒pAbAi-HYT利用BstbⅠ进行单酶切线性化,转化酵母菌株Y1H Gold,线性化的pAbAi-HYT整合到基因组中。然后,将纯化的拟南芥双链cDNA、pGADT7-Rec载体共转化含有报告载体pAbAi-HYT的酵母菌,将菌液涂布到含有100 ng/m L Ab A的SD/-Leu培养基上进行阳性克隆的酵母单杂交筛选。通过回复鉴定、序列测定,最终筛选获得了可能参与AtSTK基因表达调控的4个拟南芥基因。测序结果表明,酵母单杂交筛选获得的拟南芥基因AT3G32090含有WRKY转录因子保守结构域,为WRKY类转录因子的一员。因此,AtSTK基因的表达很可能接受MAPK信号传导途径中AT3G32090基因编码的WRKY类转录因子的调控,从而影响拟南芥植株的耐盐性。The overexpression of Serine/Threonine protein kinase gene AtSTK can significantly enhance the salt tolerance of Arabidopsis thaliana. In order to study the regulation mechanism of AtSTK gene expression,the promoter sequences of AtSTK were amplified from genomic DNA and constructed into the reporter vector pAbAi. The recombinant plasmid pAbAi-HYT was linearized by Bstb Ⅰ and transformed into the yeast strain Y1 H Gold,the linear pAbAi-HYT integrated into the genomic DNA. Then,the purified double strand c DNA of Arabidopsis and the linearized library vector p GADT7-Rec were co-transformed into the yeast cell which containing reporter vector pAbAiHYT. The positive clones were screened on the SD/-Leu medium containing 100 ng/m L Ab A by the yeast one hybrid method. Through the identification,it was found that four genes might be involved in the regulation of AtSTK gene expression. The sequencing results indicated that the Arabidopsis thaliana gene AT3G32090,which contained a conserved domain of WRKY transcription factor,was a member of the WRKY transcription factor. Therefore,the expression level of AtSTK might affect the salt tolerance of Arabidopsis plants through the MAPK signaling pathway transcription factor which coded by the gene AT3G32090.

关 键 词：AtSTK 启动子 表达调控蛋白 酵母单杂交 

分 类 号：Q756[生物学—分子生物学]