中国农业科学

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最新录用:香水柠檬RHF2A基因的克隆与互作蛋白的筛选

李雨泽1,朱嘉伟1,林蔚1,2,蓝茉莹1,夏黎明1,张艺粒1,罗聪1,黄桂香1,何新华1*
  

  1. 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
  • 发布日期:2022-06-26

Cloning and Interaction Protein Screening of RHF2A Gene from ‘Xiangshui’ Lemon

LI YuZe1, ZHU JiaWei1, LIN Wei1,2, LAN MoYing1, XIA LiMing1, ZHANG YiLi1, LUO Cong1, HUANG GuiXiang1, HE XinHua1* #br#   

  1. 1 College of agriculture, State Key Laboratory of subtropical agricultural biological resources protection and utilization, national experimental teaching demonstration center of plant science, Guangxi University, Nanning 530004;2 Institute of Subtropical Agriculture, Fujian Academy of Agricultural Sciences, Zhangzhou 363005, Fujian
  • Online:2022-06-26

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实验(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2E3泛素连接酶 RHF2ARING-H2 Zinc Finger2A)基因RHF2A-1RHF2A-2并克隆其全长序列,通过生物信息学分析2RHF2A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的时空表达模式分析,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-1RHF2A-2ORF全长分别为1 1611 134 bpNCBI结构域预测发现其具有Ring/U-box结构域。启动子分析发现具有花粉特异性表达相关元件POLLEN1LELAT52GTGANTG10。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一天开始升高,第三天达到峰值,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、UBA1FBL17SK11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ABCF320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-1ABCF3-2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育的互作候选蛋白,初步证明RHF2A-1在自交不亲和过程中具有重要作用。


关键词: 香水柠檬, RHF2A, 基因克隆, 时空表达, 酵母双杂交技术

Abstract:

Objective】‘Xiangshuilemon (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, and lay a foundation for further studying the molecular mechanism of RHF2A in the process of lemon selfincompatibility. MethodTwo E3 ubiquitin ligase RHF2A(RING-H2 Zinc Finger2A) genes 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 proreinfrom the lemon yeast library. The BiFC vector was constructed to verify the interaction of the target protein in onion living cells.ResultThe RHF2A-1 and RHF2A-2 genes were obtained from ‘Xiangshui’ lemon, and the total length of ORF was 1161 and 1134 bp respectively. NCBI domain prediction found that it had a Ring/U-box domain. Promoter analysis showed that there are 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 shows that RHF2A can interact with KRP6, AT3G57370, UBA1, FBL17 and SK11proteins, and that RHF2A gene is involved in biological processes such as self incompatibility ubiquitination reaction pathway, gametophyte development regulation and pollen growth and development. 72 clones were screened by yeast two hybrid technology. After sequencing and blast comparison, repetitive clones were excluded. Finally, 20 candidate interaction proteins such as ABCF3 were obtained. Through one-to-one interaction verification and BiFC, it was determined that there is an interaction relationship between RHF2A-1 and ABCF3-2. ConclusionThe temporal and spatial expression of RHF2A-1 gene was consistent with the germination of pollen on pistil in the process of self incompatibility; The interaction candidate proteins directly affecting pollen growth and development during the pollination were screened, which preliminarily proved that RHF2A-1 gene played an important role in the process of self incompatibility.

Key words: lemon, RHF2A, gene cloning, spatiotemporal expression, yeast two hybrid technology