| 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。 |
附件 |
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| CONSTRUCTION OF A TWO-HYBRID LIBRARY OF PYROPIA YEZOENSIS AND SCREENING OF PYMAPK5 INTERACTION PROTEIN |
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DONG Dao-Ying, KONG Fan-Na, CUI Zheng-Cai, SUN Bin
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The Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, China
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| 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 |
