水稻酵母双杂交文库构建及PID2胞内结构域互作蛋白的筛选  

Construction of a Yeast Two Hybrid Library of Rice and the Validation of a PID2-Intracellular Domain Interaction Protein

作　　者：孙一丁[1] 杨奕[1] 刘畅媛 马继琼[1] 许明辉[1] Sun Yiding;Yang Yi;Liu Changyuan;Ma Jiqiong;Xu Minghui(Key Lab of Southwestern Crop Gene Resources and Germplasm Innovation of Ministry of Agriculture and Rural Affairs of the People's Republic of China,The Key Laboratory of Biotechnology Research of Yunnan Province,Institute of Biotechnology and Genetic Resources,Yunnan Academy of Agricultural Sciences,Kunming,650223;College of Plant Protection,Yunnan Agricultural University,Kunming,650201)

机构地区：[1]云南省农业科学院生物技术与种质资源研究所,云南省农业生物技术重点实验室,农业农村部西南作物基因资源与种质创制重点实验室,昆明650223 [2]云南农业大学植保学院,昆明650201

出　　处：《分子植物育种》2022年第12期3931-3937,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31860369);云南省科技计划面上项目(2018FB063)共同资助。

摘　　要：为了进一步挖掘水稻PID2胞内互作蛋白质,阐明Pid2介导的稻瘟病抗性机制。本研究利用SMART技术,构建了受稻瘟病诱导后不同时间段的含抗性基因Pid2水稻材料的cDNA文库;通过PCR扩增获得PID2胞内结构域编码片段Pid2-JM,构建了诱饵表达载体pGBK-Pid2-JM,并进一步利用酵母双杂交技术筛选Pid2-JM的互作蛋白。结果表明,构建的cDNA酵母文库细胞密度为9.0×10;Cells/mL,文库插入片段在500~2000 bp之间,重组率为100%,诱饵载体无自激活活性;利用诱饵载体经筛选文库后,新发现一个能与Pid2-JM互作的U-box类E3泛素连接酶PBP1,经回转试验验证了PBP1与Pid2-JM可在烟草细胞内互作。综上,本研究成功构建了稻瘟病诱导下的水稻酵母双杂交文库,并筛选到一个能与PID2胞内结构域互作的U-box类E3泛素连接酶,该结果为进一步揭示水稻抗稻瘟病信号转导机制提供参考。In order to further elucidate the mechanism of Pid2-mediated resistance to rice blast and explore the intracellular interaction protein of Os P ID 2 in rice,a yeast two-hybrid cDNA library of rice cultivar having Pid2-mediated resistant was constructed in this study.PCR amplification was performed to obtain the encoding fragment of intracellular domain of PID2 and then the the bait expression vector pGBK-PID2-JM was constructed.The interaction proteins of Pid2-JM was further screened by yeast double hybridization.The results showed the library titer was 9.0×107Cells/mL,the size of insert fragments was 500~2000 bp,and the recombination rate was100%.The bait expression vector had no auto-activating activity.PBP1,a E3 ubiquitin ligase interacting with PID2-JM protein,was obtained by screening the library.In summary,the present study successfully constructed a yeast-two-hybrid library of rice induced by rice blast,and an E3 ubiquitin ligase that interacted with intracellular domain of Pid2 was found.The results provide a reference for further revealing the signal transduction mechanism of rice blast resistance.

关 键 词：稻瘟病 酵母双杂交 Pid2 互作蛋白 E3泛素化连接酶 

分 类 号：S511[农业科学—作物学]