拟南芥MAPKKK16酵母双杂交诱饵载体的构建及毒性和自激活效应检测  

Construction of Bait Vector Containing MAPKKK16 Gene from Arabidopsis thaliana in Yeast Two-hybrid System and Identification of Its Toxicity and Autonomous Activation Effects

作　　者：雷柯煜 邓治[2] 吴文冠 程汉[2] LEI Keyu;DENG Zhi;WU Wenguan;CHENG Han(College of Horticulture,Hainan University,Haikou,Hainan 570228,China;Rubber Research Institute,CATAS,Haikou,Hainan 571101,China)

机构地区：[1]海南大学园艺学院,海南海口570228 [2]中国热带农业科学院橡胶研究所,海南海口571101

出　　处：《热带农业科学》2021年第4期43-48,共6页Chinese Journal of Tropical Agriculture

基　　金：国家自然科学基金项目(No.31870646);中国热带农业科学院创新团队项目(No.16300220017004)。

摘　　要：前期研究发现,橡胶树MAPKKK基因与抗寒相关。通过同源比对发现,拟南芥MAPKKK16与橡胶树抗寒基因MAPKKK高度同源,但拟南芥MAPKKK16的功能及其调控机制仍不清楚。为了筛选拟南芥MAPKKK16基因的互作蛋白,利用PCR技术从拟南芥基因组DNA中扩增出拟南芥MAPKKK16基因的目的片段,随后亚克隆到pGBKT7表达载体,通过菌液PCR和测序鉴定序列正确后,将重组质粒pGBKT7-MAPKKK16质粒转化至酵母Y2HGold感受态细胞中,检测到诱饵蛋白无毒性且无自主激活报告基因的功能。本研究为后续筛选MAPKKK16互作蛋白,以及为进一步研究该激酶在植物抗寒机制中的作用打下基础。Previous studies have identified a rubber tree MAPKKK gene whose expression is correlated with cold resistance.Homology search results showed that Hevea MAPKKK16 is highly homologous to Arabidopsis MAPKKK16,whose functions are yet to be unravelled.To reveal its roles in regulating plant cold resistance,Arabidopsis MAPKKK16 interacting proteins were screened.First,Arabidopsis MAPKKK16 gene was amplified and sub-cloned into pGBKT7 expression vector.After verifying the correct gene sequence by sequencing,the recombinant plasmid pGBKT7-MAPKKK16 was transformed into yeast Y2HGold competent cells,in which the bait protein was proved to be non-toxic,and non-self-activated.This study provides some fundamental reference for screening of the interaction proteins of MAPKKK16,and for further unraveling its role in regulating cold resistance in plants.

关 键 词：拟南芥 MAPKKK16 酵母双杂交系 毒性检测 自激活检测 

分 类 号：S794.4[农业科学—林木遗传育种]