马铃薯酵母双杂交cDNA文库构建及StIPT互作蛋白筛选鉴定  

Construction of yeast two-hybrid cDNA library and interacting protein screening and identification for StIPT in potato

作　　者：石源睿 吴美玲 单虔文 贾小云[1] 冯雪 段永红 白小东[2,5] 唐锐敏 SHI Yuanrui;WU Meiling;SHAN Qianwen;JIA Xiaoyun;FENG Xue;DUAN Yonghong;BAI Xiaodong;TANG Ruimin(College of Life Sciences,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;Key Laboratory of Potato Genetic Improvement and Germplasm Innovation in Shanxi Province,Datong,Shanxi 037008,China;College of Plant Protection,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;College of Agriculture,Shanxi Agricultural University,Jinzhong,Shanxi 030801,China;High Latitude Crops Institute to Shanxi Academy,Shanxi Agricultural University,Datong,Shanxi 037008,China)

机构地区：[1]山西农业大学生命科学学院,山西晋中030801 [2]马铃薯遗传改良与种质创新山西省重点实验室,山西大同037008 [3]山西农业大学植物保护学院,山西晋中030801 [4]山西农业大学农学院,山西晋中030801 [5]山西农业大学高寒区作物研究所,山西大同037008

出　　处：《植物生理学报》2024年第12期1747-1758,共12页Plant Physiology Journal

基　　金：山西省回国留学人员科研资助项目(2022-094);山西省育种联合攻关项目课题(YZGG-05-01);山西省重点研发计划课题(202102140601004-01)。

摘　　要：马铃薯(Solanum tuberosum)在生长发育过程中容易受到各种生物及非生物胁迫,从而严重影响其营养物质的积累,造成品质与产量的下降。细胞分裂素(CK)是一类调控胞质分裂、芽的形成和分化的植物激素,在植物抵御外界胁迫方面发挥重要作用。异戊烯基转移酶(IPT)是CK合成途径中的第一个限速酶,能够有效调控植物内源CK合成。为了研究IPT在马铃薯中的功能和作用机制,本研究成功构建了马铃薯酵母双杂交cDNA文库,检测文库容量为1.52×10^(7)cfu(菌落形成单位),插入片段长度750~2500 bp,重组率为100%。进而克隆出StIPT基因并将其与诱饵载体pGBKT7连接,通过杂交法(mating法)从文库中筛选到32个与StIPT互作的候选蛋白。通过酵母回转验证,确定了StIPT可与StVOZ1、StHXK4发生体内互作。本结果为深入解析StIPT在马铃薯生长发育和逆境响应中的作用提供理论基础,并为马铃薯抗逆新种质的选育提供基因资源。Potato(Solanum tuberosum)is susceptible to various biotic and abiotic stresses during its growth and development,which seriously affect the accumulation of nutrients and result in a decrease in quality and yield.Cytokinin(CK)is a crucial plant hormone that regulates cytoplasmic division,bud formation and differentiation,and plays an important role in plant resistance to external stresses.As the first rate-limiting enzyme in the CK synthesis pathway,isopentenyltransferases(IPT)can effectively regulate the production of endogenous CK in plants.In order to investigate the function and underlying mechanism of IPT in potato,the yeast two-hybrid cDNA library was successfully constructed in this study.The detected library had a capacity of 1.52×10^(7)cfu,with an inserted fragment length of 750-2500 bp and a recombination rate of 100%.Then the StIPT gene was cloned and connected with the bait vector pGBKT7.In total,32 candidate proteins interacting with StIPT were selected by mating method.Further validation of the interaction of StIPT with StVOZ1 and StHXK4 was conducted by yeast two-hybrid system(Y2H)rotation test.These findings provide a theoretical basis for further investigation of the role of StIPT in potato growth,development and stress response,and furnish valuable gene resources for the breeding of new potato varieties with enhanced resistance.

关 键 词：马铃薯 细胞分裂素 异戊烯基转移酶 互作蛋白 

分 类 号：S532[农业科学—作物学]