利用酵母双杂交系统筛选水稻中与OsCRK5互作蛋白  

Screening of OsCRK5-interacted Proteins in Rice Using Yeast Twohybrid System

作　　者：王子颖 龙晨洁 范兆宇 张蕾 WANG Zi-ying;LONG Chen-jie;FAN Zhao-yu;ZHANG Lei(College of Life Sciences,Wuhan University,State Key Laboratory of Hybrid Rice,Wuhan 430072)

机构地区：[1]武汉大学生命科学学院杂交水稻国家重点实验室,武汉430072

出　　处：《生物技术通报》2023年第9期117-125,共9页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31570288)。

摘　　要：水稻是重要的粮食作物,研究水稻生长发育调控机制可为水稻品种改良奠定理论基础。钙依赖蛋白激酶(calcium dependent protein kinases,CDPKs)是植物中重要的蛋白激酶,参与植物生长发育以及对环境反应的应答。水稻中的CRK5(CDPKrelated kinase 5)在蛋白序列和结构上与CDPK高度同源。为进一步研究OsCRK5在水稻干旱反应中的功能,本研究利用酵母双杂交筛库技术筛选了OsCRK5的互作蛋白。首先将OsCRK5的1-1332 bp片段克隆至pGBKT7载体中,获得诱饵载体pGBKT7-OsCRK5,经测序无误后,转化至酵母菌株Y2H Gold中。在营养缺陷培养基中观察到重组蛋白不具有毒性作用及自激活活性,同时利用Western blot分析重组蛋白的表达。进一步利用水稻cDNA文库筛选OsCRK5的互作蛋白,共得到77个阳性克隆。功能预测结果显示,互作蛋白涉及蛋白质合成、贮存和降解过程、转录调控、植物细胞生长和分裂过程、能量代谢和细胞代谢等方面的功能。最后,从阳性克隆中选取参与植物干旱胁迫应答的两个蛋白OsWR1和OsDi19-1,利用酵母双杂交和双分子荧光互补的方法验证其与OsCRK5的相互作用。研究结果为水稻抗旱的遗传改良奠定了基础。Rice(Oryza sativa L.)is an important food crop,research on the regulation mechanism of rice growth and development can lay a theoretical foundation for the improvement of rice varieties.Calcium dependent protein kinases(CDPKs)are important protein kinases in plants which participate in plant growth and development,as well as in response to environmental reactions.CRK5(CDPK related kinase 5)in rice is highly homologous to CDPK in protein sequence and structure.Yeast two-hybrid screening library technology was used to screen OsCRK5 interacting proteins related to plant drought resistance in order to explore the molecular mechanism of OsCRK5 participating in drought response in rice plants.Firstly,the 1-1332 bp fragment of OsCRK5 was cloned into the plastmid pGBKT7,and the bait vector pGBKT7-OsCRK5 was obtained.After verification by sequencing,the bait vector was transformed into the yeast strain Y2H Gold.It was observed that the recombinant protein didn't demonstrate the toxic effects and self-activation in selective nutrition medium,and the expression of the recombinant protein was analyzed by Western blot.Furthermore,the interacting proteins of OsCRK5 were screened from the rice cDNA library and 77 positive clones were obtained.The functional prediction showed that the interacting proteins were involved in protein synthesis,degradation and storage process,transcriptional regulation,cell growth and division process,energy metabolism and cell metabolic processes.Finally,OsWR1 and OsDi19-1 related to plant drought stress response were selected from positive clones.The interaction between OsCRK5 and OsWR1 and OsDi19-1 was verified by yeast two-hybrid and biomolecular fluorescence complementation experiments.The results lay a foundation for the genetic improvement of drought tolerance in rice.

关 键 词：水稻 OsCRK5 酵母双杂交 CDNA文库 干旱胁迫 

分 类 号：S511[农业科学—作物学]