HbCoi1基因启动子酵母单杂pHIS载体的构建及互作蛋白筛选  

Construction of Yeast-one-hybrid Bait Vector and Screening of Interacted Proteins of HbCoi1

作　　者：洪灏[1] 刘小婷[1] 肖华[1] 黄惜[1] 袁红梅[1] 

机构地区：[1]海南大学农学院/热带生物资源教育部重点实验室/生物科学技术研究所,海南海口570228

出　　处：《热带生物学报》2015年第3期256-260,共5页Journal of Tropical Biology

基　　金：国家自然科学基金(31370608;31260170);海南省科技项目(ZDZX2013023)

摘　　要：茉莉酸是调节天然橡胶树产胶的主要激素,HbCoi1是茉莉酸信号的受体,研究HbCoi1基因的表达调控对了解橡胶树的产胶机理具有重要的意义。首先PCR扩增HbCoi1基因的启动子,然后将目标序列插入pHis2.1载体,构建酵母单杂诱饵载体。将该载体转入酵母Y187菌株感受态中,在缺失培养基上检测自转录激活,发现HbCoi1基因的启动子在3-AT浓度为10 mmol·L-1时自转录激活得到有效抑制。在此基础上,通过与橡胶cDNA文库进行酵母单杂筛选,得到了几个可能与Hbcoi1启动子发生互作的基因,其中包括DNA结合蛋白、核糖体蛋白和功能未知基因,旨在为深入研究Hbcoi1基因表达调控打下基础。Jasmonic acid（ JA） is one of the most important phytohormones for regulation of latex biosynthesis in Hevea brasiliensis. HbCoi1 is the receptor of JA signaling pathway. Analysis of the gene expression of HbCoi1 is of great significance to reveal molecular mechanism of latex biosynthesis in H. brasiliensis. In this context,the promoter of HbCoi1 was PCR amplified and inserted into pHis2. 1 vector to construct bait vector p HIS-HbCoi1 for yeast-one-hybrid. The vector was then transformed into Y187 yeast strain and cultured on the SD medium to test the auto-activity of the transcription. The growth of the strain containing bait vector was inhibited on the SD medium added with 10 mmol·L- 13-AT. Furthermore,p HIS-HbCoi1 vector and p GADT7-rec2 cDNA library of H.brasiliensis were co-transformed into Y187 strain,and several proteins interacted with HbCoi1 promoter were identified,including DNA binding protein,ribosomal proteins and uncharacterized proteins,which lays a foundation for further study of gene expression of Hb Coi1.

关 键 词：HbCoil pHIS载体 自转录激活 酵母单杂 

分 类 号：Q946.1[生物学—植物学]