摘要
为进一步研究条斑紫菜促分裂原活化激酶家族PyMAPK5的下游互作蛋白,理解其生物学功能,本研究通过酵母双杂交的方法进行其相互作用蛋白的筛选。提取不同温度和失水逆境胁迫下的RNA,利用Invitrogen体系构建条斑紫菜酵母双杂交cDNA文库,其库容为1.44×107CFU,重组率为91.8%。以pGBKT7-PyMAPK5为诱饵蛋白载体,利用共转化方法,从文库中筛选得到26个与PyMAPK5互作的候选蛋白。候选蛋白集中在光系统II相关蛋白、捕光蛋白、微管蛋白、ATP酶、GTP结合蛋白及假设蛋白等。微管蛋白、捕光蛋白、光系统II蛋白一对一验证结果为阳性,表明在酵母体内存在互作。本研究为阐明条斑紫菜PyMAPK5与其互作蛋白的关系及解析PyMAPK5下游作用机制奠定了基础。
To understand the downstream interacting proteins of mitogen-activated kinase family PyMAPK5 and its biological functions in Pyropia yezoensis,we screened the interacting proteins of PyMAPK5 by yeast two-hybrid.Total RNA of P.yezoensis under a different temperature and water stress was used to construct the yeast cDNA library with the Invitrogen system.The resulted storage capacity was 1.44×107 CFU,and the recombination rate was 91.8%.In addition,we screened the total 26 candidate proteins interacting with PyMAPK5 by co-transformation method.The candidate proteins were focused on photosystem II related proteins,light-harvesting proteins,tubulin,ATPase,GTP-binding proteins,and hypothetical proteins.At last,we one-to-one verified the reliabilities of three candidate proteins(including tubulin,light-harvesting protein,and photosystem II protein).This study may help clarify the relationship between PyMAPK5 and its interaction proteins and lay a foundation for the analysis of downstream mechanism of PyMAPK5.
作者
董道英
孔凡娜
崔正彩
孙斌
DONG Dao-Ying;KONG Fan-Na;CUI Zheng-Cai;SUN Bin(The Key Laboratory of Marine Genetics and Breeding,Ministry of Education,Ocean University of China,Qingdao 266003,China)
出处
《海洋与湖沼》
CAS
CSCD
北大核心
2019年第6期1271-1280,共10页
Oceanologia Et Limnologia Sinica
基金
国家自然科学基金项目,31672641号
