褐飞虱酵母双杂交cDNA文库的构建及PIGM互作蛋白的筛选  

Construction of yeast two-hybrid cDNA library of brown planthopper andscreening of proteins interacted with PIGM

作　　者：郑荣儿 吴佳敏 张瑞娟 姬金亮 李雅彬 俞晓平 许益鹏 ZHENG Ronger;WU Jiamin;ZHANG Ruijuan;JI Jinliang;LI Yabin;YU Xiaoping;XU Yipeng(Zhejiang Provincial Key Laboratory of Biometrology,Inspection and Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China)

机构地区：[1]中国计量大学生命科学学院浙江省生物计量及检验检疫技术重点实验室,浙江杭州310018

出　　处：《中国计量大学学报》2021年第3期421-428,共8页Journal of China University of Metrology

基　　金：国家自然科学基金项目(No.31871961,31501632);浙江省重点研发计划项目(No.2019C02015);浙江省属高校基本科研业务费专项项目(No.2020YW14)。

摘　　要：目的:构建用于酵母双杂交的褐飞虱cDNA文库以及pGBKT7-NlPIGM诱饵载体,筛选与NlPIGM具有相互作用的蛋白。方法:以褐飞虱全虫mRNA为起始模板,采用CloneMiner构建褐飞虱cDNA文库并通过涂布鉴定文库库容量和滴度,通过两步法构建pGBKT7-NlPIGM诱饵载体并鉴定重组诱饵载体的自激活活性及毒性,然后通过mating法筛选与NlPIGM具有相互作用的蛋白。结果:构建的初级cDNA文库总库容为1.04×10^(8) CFU,次级cDNA文库滴度为3.4×10^(7) CFU/mL,重组率为100%,插入片段大小集中在750~2000 bp之间;pGBKT7-NlPIGM载体无自激活活性,对Y2H Gold酵母菌株无毒性。并筛选到9个能与NlPIGM互作的蛋白。结论:成功构建了可用于酵母双杂交的高质量褐飞虱cDNA文库及褐飞虱PIGM诱饵载体,并筛选到了能与NlPIGM互作的蛋白。Aims:This paper aims to construct the yeast two-hybrid cDNA library of brown planthopper and pGBKT7-NlPIGM(Nilaparvata lugens PIGM)bait vector and to screen the proteins interacted with NlPIGM.Methods:Using the mRNA of N.lugens whole body as the initial template,we constructed a yeast two-hybrid cDNA library which was constructed with the CloneMiner^(TM) cDNA Library Construction Kit and identified the capacity and titer of cDNA library with plate coating.The pGBKT7-NlPIGM bait vector was constructed by the two-step method and then the self-activating activity and the toxicity of the bait vector were checked;and then the proteins interacted with NlPIGM were screened out.Results:The unamplified library consisted of 1.04×10^(8) CFU.The titer of amplified cDNA library was 3.4×10^(7) CFU/mL,and the recombinant rate was 100%.The length of most cDNA inserts was 750~2000 bp.The pGBKT7-NlPIGM vector had no self-activation activity and toxicity to the Y2H Gold yeast strain.We screened out 9 proteins interacted with NlPIGM.Conclusions:A high quality cDNA library of the brown planthopper and the PIGM bait vector was constructed successfully for yeast two hybrid assay;and the proteins interacted with PIGM were screened out.

关 键 词：酵母双杂交 CDNA文库 诱饵载体 PIGM 

分 类 号：Q933[生物学—微生物学]