香水柠檬RHF2A的克隆与互作蛋白的筛选  

Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon

作　　者：李雨泽 朱嘉伟 林蔚[1,2] 蓝茉莹 夏黎明 张艺粒 罗聪 黄桂香[1] 何新华[1] LI YuZe;ZHU JiaWei;LIN Wei;LAN MoYing;XIA LiMing;ZHANG YiLi;LUO Cong;HUANG Gui Xiang;HE XinHua(College of Agriculture,Guangxi University/State Key Laboratory of Subtropical Agricultural Biological Resources Protection and Utilization/National Experimental Teaching Demonstration Center of Plant Science,Nanning 530004;Institute of Subtropical Agriculture,Fujian Academy of Agricultural Sciences,Zhangzhou 363005,Fujian)

机构地区：[1]广西大学农学院/亚热带农业生物资源保护与利用国家重点实验室/植物科学国家级实验教学示范中心,南宁530004 [2]福建省农业科学院亚热带农业研究所,福建漳州363005

出　　处：《中国农业科学》2022年第24期4912-4926,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31960585);亚热带农业生物资源保护与利用国家重点实验室自主课题(SKLCUSA-a201920);亚热带农业生物资源保护与利用国家重点实验室科教结合创新基地课题(SKLCUSA-c201901)。

摘　　要：【目的】以香水柠檬(Citrus limon (L.) Burm. F.)为研究对象,分析两个RHF2A的时空表达规律,利用酵母双杂交技术和双分子荧光互补试验(BiFC)筛选、验证其互作蛋白,为深入研究RHF2A在香水柠檬自交不亲和过程中的分子作用机制奠定基础。【方法】从前期转录组和泛素修饰组(未公开)筛选获得2个E3泛素连接酶RHF2A(RING-H2 Zinc Finger2A)基因RHF2A-1、RHF2A-2,并克隆其全长序列,通过生物信息学分析2个RHF2A的序列和蛋白质结构,预测其启动子顺式作用元件,构建35S-RHF2A-GFP融合蛋白表达载体用于亚细胞定位分析,采用实时荧光定量PCR分析两个RHF2A的时空表达模式,构建酵母双杂诱饵载体从香水柠檬酵母文库筛选互作蛋白。构建BiFC载体,在洋葱活体细胞内对目标蛋白进行互作验证。【结果】从香水柠檬克隆获得RHF2A-1、RHF2A-2,ORF全长分别为1 161和1 134 bp;NCBI结构域预测发现其具有Ring/U-box结构域。启动子分析发现其具有花粉特异性表达相关元件POLLEN1LELAT52、GTGANTG10。组织表达分析发现RHF2A-1在花粉特异性表达,RHF2A-2在叶中特异性表达;时空表达分析结果显示,RHF2A-1在自交柱头表达量从第1天开始升高,第3天达到峰值,是杂交柱头表达量的5倍以上。亚细胞定位显示RHF2A-1和RHF2A-2定位在细胞核。经过Uniprot网站预测其互作蛋白显示RHF2A能与KRP6、AT3G57370、UBA1、FBL17、SK11蛋白相互作用,推测其参与自交不亲和泛素化反应途径、配子体发育调控、花粉的生长发育等生物学过程。通过酵母双杂交技术筛选出72个克隆,对其测序并进行Blast比对后排除重复克隆,最终得到ABCF3等20个候选互作蛋白。经过一对一互作验证、双分子荧光互补实验确定RHF2A-1与ABCF3-2存在互作关系。【结论】RHF2A-1的时空表达规律与自交不亲和过程中花粉在雌蕊上的萌发规律相吻合;筛选得到柠檬授粉过程中直接影响花粉生长发�【Objective】Xiangshui lemon(Citrus limon(L.) Burm. F.) was used to study the expression of two RHF2A genes, and to screen and verify their interaction proteins by yeast two-hybrid technology and BiFC, so as to lay a foundation for further studying the molecular mechanism of RHF2A in the process of lemon self-incompatibility. 【Method】 Two E3 ubiquitin ligase RHF2A(RING-H2 Zinc Finger2A) genes including RHF2A-1 and RHF2A-2 of Xiangshui lemon were screened from the transcriptome and ubiquitin modification group, and their full-length sequences were cloned. The sequence and protein structure of two RHF2A genes were analyzed by bioinformatics to predict the cis acting elements of their promoters. 35S-RHF2A-GFP fusion protein expression vector was constructed for subcellular localization analysis. The temporal and spatial expression patterns of two RHF2A were analyzed by real-time fluorescence quantitative PCR. The yeast two-hybrid bait vector was constructed to screen the interaction proteins from the lemon yeast library. The BiFC vector was constructed to verify the interaction of the target protein in onion living cells.【Result】The RHF2A-1 and RHF2A-2 genes were obtained from ‘Xiangshui’ lemon, and the total length of ORF was 1 161 and 1 134 bp, respectively. NCBI domain prediction found that it had a Ring/U-box domain. Promoter analysis showed that there were POLLEN1LELAT52 and GTGANTG10 related to pollen specific expression elements. Tissue expression analysis showed that RHF2A-1 gene was specifically expressed in pollen and RHF2A-2 was specifically expressed in leaves;the results of temporal and spatial expression analysis showed that the expressed of RHF2A-1 in self-stigma tended to increase from the first day and reached the peak on the third day, which was more than 5 times that of hybrid stigma. Subcellular localization showed that RHF2A-1and RHF2A-2 were localized in the nucleus. The interaction protein predicted by Uniprot website showed that RHF2A could interact with KRP6, AT3G57370, UBA1, F

关 键 词：香水柠檬 RHF2A 基因克隆 时空表达 酵母双杂交技术 

分 类 号：S666.5[农业科学—果树学]