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引用本文:董道英,孔凡娜,崔正彩,孙斌.条斑紫菜酵母双杂交文库的构建及PyMAPK5互作蛋白的筛选.海洋与湖沼,2019,50(6):1271-1280.
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条斑紫菜酵母双杂交文库的构建及PyMAPK5互作蛋白的筛选
董道英, 孔凡娜, 崔正彩, 孙斌
中国海洋大学海洋生命学院 海洋生物遗传学与育种教育部重点实验室 青岛 266003
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。
关键词:  PyMAPK5  条斑紫菜  酵母双杂交  互作蛋白  逆境胁迫
DOI:10.11693/hyhz20190400070
分类号:
基金项目:国家自然科学基金项目,31672641号。
CONSTRUCTION OF A TWO-HYBRID LIBRARY OF PYROPIA YEZOENSIS AND SCREENING OF PYMAPK5 INTERACTION PROTEIN
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
Abstract:
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 1.44×107CFU, 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.
Key words:  PyMAPK5  Pyropia yezoensis  yeast two-hybrid  interacting proteins  adversity stress
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