水稻抽穗期基因OsFKF1的克隆和互作蛋白筛选  

Cloning of rice heading date gene OsFKF1 and screening of interacting proteins

作　　者：闫晓峰 胡渊 黄晓龙 金涛 李海申 田云录[1] 江玲[1] 周时荣[1] 万建民[1,2] YAN Xiaofeng;HU Yuan;HUANG Xiaolong;JIN Tao;LI Haishen;TIAN Yunlu;JIANG Ling;ZHOU Shirong;WAN Jianmin(National Key Laboratory of Crop Genetics&Germplasm Enhancement and Utilization,Nanjing Agricultural University,Nanjing 210095,China;Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]南京农业大学作物遗传与种质创新利用全国重点实验室,江苏南京210095 [2]中国农业科学院作物科学研究所,北京100081

出　　处：《南京农业大学学报》2023年第3期429-437,共9页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31971910);江苏省重点研发项目(BE2021359);江苏省农业自主创新资金课题[SCX(19)1079]。

摘　　要：[目的]本文旨在探究水稻抽穗期基因OsFKF1(Rice FLAVIN-BINDING,KELCH REPEAT,F-BOX 1)感受蓝光并调控抽穗期的分子机制。[方法]以‘宁粳2号’及其晚抽穗突变体lhn2和lhn3为材料,分析野生型和突变体的农艺性状。利用MutMap的方法定位并克隆导致lhn2和lhn3突变的基因,研究候选基因OsFKF1的功能及水稻原生质体中OsFKF1的亚细胞定位。利用不同波长光源处理检测OsFKF1对Ehd1表达的影响。通过酵母文库筛选试验寻找与OsFKF1互作的蛋白,并利用荧光素酶互补等试验进行验证。[结果]在南京自然长日照条件下lhn2和lhn3突变体抽穗期较野生型均延迟26、27 d,株高、粒长、粒宽、千粒重均显著下降。MutMap分析结果表明导致lhn2和lhn3突变的基因均为水稻抽穗期基因OsFKF1。亚细胞定位结果显示OsFKF1在水稻原生质体中定位于细胞质和细胞核内。OsFKF1仅在蓝光下诱导Ehd1表达,且蓝光处理3 h达到峰值。利用酵母文库筛选试验筛到与OsFKF1互作的一个含有泛素结合相关结构域的蛋白HGW(HEADING AND GRAIN WEIGHT),荧光素酶互补试验结果证实OsFKF1与HGW互作。[结论]蓝光诱导Ehd1的表达部分依赖于OsFKF1。推测OsFKF1和HGW可能形成复合体,通过泛素化降解蛋白途径调控水稻抽穗。[Objectives]This study aimed to elucidate the molecular mechanism of the rice FLAVIN-BINDING,KELCH REPEAT,F-BOX 1(OsFKF1)gene on sensing blue light and regulating heading date.[Methods]The agronomic traits of the japonica rice variety‘Ningjing 2’(the wild type)and the late flowering mutants lhn2 and lhn3 were analyzed.The genes causing lhn2 and lhn3 mutations were cloned by MutMap method.To analyze the function of the candidate gene OsFKF1,the subcellular localization of OsFKF1 in rice protoplasts was observed.The expression of Ehd1 under different light treatments was analyzed by quantitative real-time PCR.The proteins interacting with OsFKF1 were identified through yeast library screening experiment,and verified by luciferase complementation experiments.[Results]The heading date of lhn2 and lhn3 mutants was delayed by 26 and 27 days compared with the wild type,and the plant height,grain length,grain width and 1000-grain weight significantly decreased under Nanjing long-day conditions.OsFKF1 induced Ehd1 expression only under blue light,and reached a peak at 3 h after blue light treatment.Subcellular localization results showed that OsFKF1 was localized in the cytoplasm and nucleus in rice protoplasts.A ubiquitin-binding-related protein HEADING AND GRAIN WEIGHT(HGW),which interacted with OsFKF1,was screened by yeast screening library experiments,and the interaction between OsFKF1 and HGW was verified by luciferase complementation experiments.[Conclusions]The expression of Ehd1 gene induced by blue light was partially dependent on OsFKF1.It was speculated that OsFKF1 and HGW might form a complex to regulate rice heading through the ubiquitination degradation protein pathway.

关 键 词：水稻 抽穗期 蓝光 OsFKF1 HGW 

分 类 号：S511[农业科学—作物学]