棉花E3泛素连接酶基因GhRING1-like的克隆及功能分析  

Cloning and functional analysis of cotton E3 ubiquitin ligase gene GhRING1-like

作　　者：夏朝阳 安晓晖 张中起 葛冬冬 刘康[1] XIA Zhaoyang;AN Xiaohui;ZHANG Zhongqi;GE Dongdong;LIU Kang(College of Agriculture/State Key Laboratory of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学农学院/作物遗传与种质创新国家重点实验室,江苏南京210095

出　　处：《南京农业大学学报》2019年第1期39-50,共12页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31371672)

摘　　要：[目的]RING(really interesting new gene) finger E3泛素连接酶在植物生长发育和抵御环境胁迫中具有重要作用。克隆棉花RING finger E3泛素连接酶基因GhRING1-like并分析其表达特征和功能,为该基因分子作用机制研究和育种应用奠定基础。[方法]根据棉花表达谱分析,选取并克隆干旱胁迫处理上调表达基因GhRING1-like,qRT-PCR分析GhRING1-like的组织表达特性及其对不同非生物胁迫和激素处理的响应模式;采用瞬时表达对GhRING1-like-GFP融合蛋白进行亚细胞定位分析;通过获得病毒介导的基因沉默(VIGS)棉花和过表达拟南芥植株,分析沉默或过表达GhRING1-like对植株生长发育及其抗旱性的影响。[结果]GhRING1-like基因序列全长996 bp,编码332个氨基酸,其C端含有RING-H_2(C3H_2C3)结构域。GhRING1-like定位于内质网上。GhRING1-like在叶片中优势表达; 200 g·L^(-1)PEG6000诱导处理1 h后瞬时显著上调表达12倍; GhRING1-like对Na Cl、ABA和SA的响应时间都在处理后3 h,上调表达均在5倍左右。干旱胁迫处理后,VIGS沉默GhRING1-like棉花叶片相对含水量和叶绿素荧光参数Fv/Fm分别较未沉默对照降低17.1%和30.6%,而电解质渗透率和丙二醛含量分别较对照提高70.8%和100%。过表达GhRING1-like显著提高了拟南芥种子萌发和幼苗期对于甘露醇引起的渗透胁迫的耐受性;可使营养生长中后期阶段的拟南芥在水分亏缺处理下的存活率提高2.4倍;转基因拟南芥叶片气孔开度降低51.2%,失水率明显降低。干旱胁迫下,过表达GhRING1-like使依赖于ABA诱导表达的At AREB1、At ERD15、AtRD29A上调表达,而对不依赖ABA的干旱胁迫诱导基因At DREB和脯氨酸合成基因At PLD没有影响。[结论]棉花GhRING1-like参与了植物非生物胁迫抗性的调控,主要通过依赖于ABA通路正向调控植物的抗旱反应。[Objectives] The RING(really interesting new gene)finger E3 ubiquitin ligase plays a crucial role in plant growth and response to various environmental stresses. Cloning and functional analysis of RING finger E3 ligase gene GhRING1 -like was conducted to understand its molecular mechanisms and lay the foundation for further application in cotton genetic improvement.[Methods] Based on transcriptome data analysis ,the GhRING1 -like gene up-regulated in drought stress was screened and molecularly cloned, the expression patterns in different tissues along with in response to different abiotic stress and phytohormone treatments were characterized by using quantitative real-time PCR;GhRING1-like-GFP fusion protein was transiently expressed for subcellular localization analysis;virus-induced gene silencing(VIGS)of GhRING1 -like cotton and transgenic Arabidopsis overexpressing GhRING1 -like were generated to determine the biological roles of GhRING1 -like in plant growth and drought resistance.[Results] GhRING1 -like consists of a 996 bp of open reading frame,which encodes a protein of 332 amino acids with a RING-H2(C3H2C3)domain at the C-terminus. GhRING1-like is located at the endoplasmic reticulum. qRT-PCR analysis indicated that GhRING1 -like transcript was preferentially accumulated in leaves, GhRING1 -like was markedly up-regulated by 12-fold at 1 h post treatment with 200 g · L^-1 PEG6000,approximately 5 times elevated expression of GhRING1 -like was observed after 3h treatment with NaCl,ABA and SA,respectively. Under water deficit,the relative water content and chlorophyll fluorescence parameter F v/ F m in the VIGS cotton leaves reduced by 17.1% and 30.6% than those in the control plants,respectively;while the electrolyte leakage and malondialdehyde content in the VIGS plants increased 70.8% and 100% than those in the control plants,respectively. Overexpression of GhRING1 -like significantly increased the tolerance of Arabidopsis seed germination and young seedlings to the osmotic stress derived from mannitol.

关 键 词：E3泛素连接酶 干旱胁迫 实时荧光定量PCR 病毒介导的基因沉默(VIGS) 转基因拟南芥 

分 类 号：S634.3[农业科学—蔬菜学]