甘薯块根cDNA酵母文库的构建及IbNCED3启动子互作蛋白的筛选鉴定  

Construction of a yeast cDNA library of sweet potato tuberous root and identification of IbNCED3 promoterinteracting proteins

作　　者：赵彩良 张洁 唐锐敏 贾小云[2] Zhao Cailiang;Zhang Jie;Tang Ruimin;Jia Xiaoyun(College of Agriculture,Shanxi Agricultural University,Jinzhong 030801,China;College of Life Sciences,Shanxi Agricultural University,Jinzhong 030801,China)

机构地区：[1]山西农业大学农学院,山西晋中030801 [2]山西农业大学生命科学学院,山西晋中030801

出　　处：《山西农业大学学报（自然科学版）》2022年第4期19-27,共9页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：山西省农业科学院应用基础研究计划项目(YGC2019FZ4);2020年度中央引导地方科技发展资金项目;山西省高等学校科技创新项目(2019L0388);吕梁市引进高层次科技人才重点研发项目(2021RC-2-21)。

摘　　要：[目的]橘心甘薯和黄心甘薯富含大量合成维生素A的前体物质⁃β⁃胡萝卜素。9⁃顺式环氧类胡萝卜素双加氧酶(9⁃Cis Epoxycarote⁃noid Dioxygenase,NCED)可降解植物中的类胡萝卜素,与脱落酸(Abscisic Acid,ABA)的合成和抗逆性相关。本研究通过构建甘薯块根cDNA酵母文库并利用酵母单杂交(Yeast One⁃Hybrid,Y1H)技术筛选与IbNCED3基因启动子互作的调控因子,为深入研究IbNCED3的功能和表达调控机制奠定基础。[方法]利用Gateway技术构建甘薯块根cDNA酵母文库,根据‘徐薯18’基因组数据克隆IbNCED3的启动子序列,并利用双酶切方法构建诱饵载体pAbAi⁃NCED3。利用同源重组的方法,将诱饵载体整合到Y1HGold菌株中。利用所构建的甘薯块根cD⁃NA酵母文库和诱饵载体pAbAi⁃NCED3筛选与IbNCED3启动子互作的蛋白,并通过酵母单杂交技术进行验证。[结果]成功构建了甘薯块根cDNA酵母文库,文库的容量达到1.1×10^(7)以上,重组率为100%,插入片段的大小为600~2500 bp。克隆得到的IbNCED3启动子区存在4个核心启动区域,选取其中的3和4(-311~-86)区域序列作为诱饵序列成功构建诱饵载体pAbAi⁃NCED3,利用该载体筛选并验证获得与IbNCED3启动子互作的3个蛋白,分别为胁迫相关的DnaJ蛋白、开花相关的FRIGIDA⁃Like蛋白和功能未知蛋白。[结论]构建的甘薯块根cDNA酵母文库质量较高,可用于通过酵母单杂交和酵母双杂交(Yeast Two⁃Hybrid,Y2H)筛选互作蛋白。以IbNCED3启动子为诱饵,在所构建的甘薯块根cDNA酵母文库中筛选到3个与其互作的蛋白。[Objective]Orange-fleshed sweet potatoes and yellow-fleshed sweet potatoes are rich in a large amount ofβ-carotene,which is a precursor of vitamin A.9Cis Epoxycarotenoid Dioxygenase(NCED)can degrade carotenoids in plants and is involved in Abscisic Acid(ABA)synthesis and stress resistance.In this study,a yeast cDNA library of sweet potato tuberous root was constructed and used to screen the regulatory factors interacting with IbNCED3 promoter by Yeast One-Hybrid(Y1H)technology.It lays a foundation for an in-depth study of the function and expression regulation mechanism of IbNCED3.[Methods]A yeast cDNA library of sweet potato tuberous root was constructed by Gateway technology.The promoter sequence of IbNCED3 was cloned according to the genome data of′Xushu 18′.A bait vector pAbAi-NCED3 was constructed by double enzyme digestion.The bait vector was integrated into Y1HGold strain by homologous recombination.The constructed yeast library and the bait vector pAbAi-NCED3 were used to screen the proteins interacting with the IbNCED3 promoter by Yeast One-Hybrid technique.[Results]The yeast cDNA library of sweet potato tuberous root was successfully constructed.The capacity of the library was over 1.1×10^(7),and the recombination rate was 100%,and the size of inserted fragment ranged from 600 to 2500 bp.The promoter sequence of IbNCED3 included four core promoter regions,and the sequences of the 3 and 4(−311~−86)core regions were selected as the bait sequences to construct the bait vector pAbAi-NCED3.Three proteins interacting with the IbNCED3 promoter were successfully screened and validated,which were a stress-related DnaJ protein,a flowering-related FRIGIDA-Like protein,and a unknown function protein.[Conclusion]The quality of the constructed cDNA yeast library was high,and can be used to screen interaction proteins by Yeast One-Hybrid and Yeast Two-Hybrid(Y2H).Using IbNCED3 promoter as a bait,three proteins interacting with the IbNCED3 promoter were screened from the constructed library.

关 键 词：甘薯 酵母cDNA文库 酵母单杂交 互作蛋白 IbNCED3 

分 类 号：S531[农业科学—作物学]