非生物胁迫诱导的棉花酵母双杂交文库构建及GhJAZ1互作蛋白筛选  

Construction of Yeast Two-Hybrid Library of Cotton under Abiotic Stress and Screening of GhJAZ1 Interacting Proteins in Cotton

作　　者：蔡肖[1] 李兴河 王海涛 刘存敬[1] 唐丽媛[1] 张素君[1] 张建宏[1] CAI Xiao;LI Xinghe;WANG Haitao;LIU Cunjing;TANG Liyuan;ZHANG Sujun;ZHANG Jianhong(Institute of Cotton,Hebei Academy of Agriculture and Forestry Sciences/Key Laboratory of Cotton Biology and Genetic Breeding in Huanghuaihai Semiarid Area,Ministry of Agriculture and Rural Affairs/National Cotton Improvement Center Hebei Branch,Shijiazhuang,Hebei 050051)

机构地区：[1]河北省农林科学院棉花研究所/农业农村部黄淮海半干旱区生物学与遗传育种重点实验室/国家棉花改良中心河北分中心,河北石家庄050051

出　　处：《核农学报》2023年第11期2158-2165,共8页Journal of Nuclear Agricultural Sciences

基　　金：河北省自然科学基金项目(C2021301037);河北省农林科学院基本科研业务费项目(2021070202);河北省“三三三人才工程”资助项目(A202101057)。

摘　　要：非生物胁迫导致棉花生长发育受到抑制,严重影响棉花的产量和品质。为了深入探究棉花非生物逆境响应的分子作用机制并鉴定GhJAZ1的互作蛋白,本研究以陆地棉标准系TM-1为材料,采用Gateway重组技术构建了非生物胁迫诱导的陆地棉酵母双杂交cDNA文库,获得的初级文库总克隆数为1.2×10^(7)CFU,次级文库总克隆数为1.6×10^(7)CFU,重组阳性率100%,酵母文库滴度为5.0×10^(7)cells·mL^(-1),酵母克隆平均插入片段>1 000 bp,符合建库标准,可用于后续酵母双杂交筛选。以GhJAZ1为诱饵,构建pGBKT7-GhJAZ1诱饵蛋白表达载体,经毒性检测及自激活活性检测,明确了诱饵质粒在酵母系统中无毒性和自激活活性,可直接用于酵母文库筛选。利用酵母双杂交筛选构建的非生物胁迫诱导的酵母cDNA文库,得到72个初筛阳性克隆,经测序、序列比对和酵母回转验证,获得11个与GhJAZ1相互作用的候选蛋白。选取其中4个候选蛋白,利用酵母双杂交进一步确认了GhJAZ1与候选蛋白的相互作用关系。本研究结果为深入分析棉花非生物胁迫响应的调控网络奠定了良好的基础,为解析GhJAZ1低温应答分子机理提供了重要参考。Abiotic stress led to the inhibition of cotton growth and development,and significantly affected the yield and quality of cotton.To explore the molecular mechanism of cotton abiotic stress response and identify the interacting proteins of GhJAZ1,yeast two-hybrid cDNA library of upland cotton standard line TM-1 induced by abiotic stress was constructed by gateway recombination technology.The primary cDNA library and secondary cDNA library with 1.2×10^(7) CFU and 1.6×10^(7) CFU total clones respectively and 100%positive combination rate was constructed.The yeast library had a titer of 5.0×10^(7) cells/mL,and the average inserted fragment of yeast clone was more than 1000 bp.This constructed yeast library met the standard of library building and can be used for yeast two-hybrid screening.Taken GhJAZ1 as a bait,protein expression vector of pGBKT7-GhJAZ1 was constructed.The results of toxicity and self-activation activity assessments indicated the bait plasmid exhibited no toxicity and no self-activation ability in yeast system and thus could be used for yeast two-hybrid screening.A total of 72 positive clones were obtained by preliminary screening of the constructed abiotic stress induced yeast cDNA library using yeast two-hybrid technology.After sequencing,alignments and yeast verification,a total of 11 candidate proteins interacting with GhJAZ1 were obtained.The interacting relationship between four selected protein and GhJAZ1 were further confirmed by yeast two-hybrid assay.This study laid a good foundation for analysis of the regulatory network of cotton abiotic stress response,and provided an essential reference for understanding the molecular mechanism of GhJAZ1 in low-temperature response.

关 键 词：棉花 互作蛋白 酵母双杂交 非生物胁迫 GhJAZ1 

分 类 号：S562[农业科学—作物学]