昆虫学报2023，Vol.66Issue(11)：1451-1458,8.DOI:10.16380/j.kcxb.2023.11.004
家蝇酵母双杂交cDNA文库构建和MD14-3-3互作蛋白筛选
Construction of yeast two-hybrid cDNA library of Musca domestica(Diptera:Muscidae)and screening of MD14-3-3 interacting proteins
摘要
Abstract
[Aim]To construct a yeast two-hybrid(Y2H)cDNA library of Musca domestica and investigate the proteins that could interact with the MD14-3-3.[Methods]Taking M.domestica as the research object,the mRNA was purified,the primary cDNA library was constructed,the secondary library was constructed by the recombinant pGADT7-DEST vector,so as to construct the Y2H.Then,the plasmid pGBKT7-MD14-3-3 were constructed as bait and used for screening the proteins interacting with MD14-3-3.Finally,the transformation and validation were verified.[Results]A Y2H cDNA library of M.domestica was successfully constructed,with the library storage capacity of 1.6 × 107 CFU and the recombination rate of 100％.Two target proteins were obtained by Y2H screening.The results of transformation and validation showed that mucin-like protein HKR1 and antibacterial peptide ctenidin-1 interacted with MD14-3-3.[Conclusion]Two proteins that interact with MD14-3-3 were identified from the successfully constructed Y2H cDNA library of M.domestica.Our results provide a basis for investigating the mechanism of immune response of M.domestica.关键词
家蝇/血细胞/MD14-3-3/酵母双杂交/互作蛋白Key words
Musca domestica/hemocyte/MD14-3-3/yeast two-hybrid/interaction protein分类
生物科学引用本文复制引用
陈明明,焦振龙,赵文静,国果..家蝇酵母双杂交cDNA文库构建和MD14-3-3互作蛋白筛选[J].昆虫学报,2023,66(11):1451-1458,8.基金项目
国家自然科学基金项目(82060381,32360702) （82060381,32360702）
贵州省科技计划项目(黔科合基础-ZK[2022]一般345,黔科合基础-ZK[2023]一般329) （黔科合基础-ZK[2022]一般345,黔科合基础-ZK[2023]一般329）
贵州省卫生健康委员会基金项目(gzwkj-2023-569) （gzwkj-2023-569）
