葡萄钾离子通道基因VviSKOR的克隆、表达及电生理功能  

Cloning,Expression and Electrophysiological Function Analysis of Potassium Channel Gene VviSKOR in Grape

作　　者：沈静沅 唐美玲 杨庆山 高雅超[1] 刘万好 程杰山 张洪霞 宋志忠 SHEN JingYuan;TANG MeiLing;YANG QingShan;GAO YaChao;LIU WanHao;CHENG JieShan;ZHANG HongXia;SONG ZhiZhong(College of Agriculture,Ludong University/Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong,Yantai 264025,Shandong,China;Institute of Grape,Yantai Academy of Agricultural Science,Yantai 264000,Shandong,China;Shandong Academy of Forestry,Jinan 250014,China;Department of Plant Science,University of Cambridge,Cambridge UK CB23EA)

机构地区：[1]鲁东大学农学院/山东省高等学校重点实验室“作物高产抗逆分子模块育种实验室”,中国山东烟台264000 [2]烟台市农业科学院葡萄研究所,中国山东烟台264000 [3]山东省林业科学研究院,中国济南250014 [4]剑桥大学植物系,英国剑桥CB23EA

出　　处：《中国农业科学》2020年第15期3158-3168,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2019YFD1000500);山东省农业良种工程基金(2019LZGC009-2);山东省重点研发计划(2018JH2006);国家现代农业产业技术体系专项资金(CARS-29-16)。

摘　　要：【目的】从葡萄中克隆并鉴定钾离子通道基因VviSKOR,在转录水平分析其组织特异性表达特征及对缺钾、氯化钠(NaCl)与脱落酸(ABA)等胁迫的响应情况,通过膜片钳电生理技术研究其生理学功能。【方法】通过同源克隆法,在葡萄基因组中筛选并鉴定钾离子通道基因VviSKOR;借助多种生物信息学软件分析葡萄SKOR及其编码蛋白的特征;利用MEGA 7.0软件建立葡萄、拟南芥、水稻、玉米、高粱、短柄草、大豆、番茄、黄瓜、白杨、桃、梨、草莓、苹果、木瓜、柑橘、香蕉、凤梨等18种不同科属植物SKOR同源蛋白成员的系统进化树;利用实时荧光定量PCR分析VviSKOR在葡萄不同组织部位的表达模式及对缺钾、NaCl、ABA与sorbitol等胁迫的响应情况;利用膜片钳电生理系统分析葡萄VviSKOR的生理学功能。【结果】在葡萄基因组中克隆获得一个钾离子通道基因VviSKOR,其编码蛋白含有环核苷酸结合域、离子通道跨膜域、Ankyrin repeats和KHA功能结构域,属于典型的Shaker类钾离子通道;18种不同科属植物SKOR蛋白在氨基酸水平具有58.92%的一致性,系统进化树表明葡萄VviSKOR与黄瓜CsaSKOR紧密聚在一起,其遗传进化关系上较近,4种禾本科植物(玉米、水稻、短柄草和高粱)SKOR家族成员在系统进化树上更倾向于聚在一起,而同属蔷薇科植物(草莓、苹果、梨和桃)SKOR在进化关系上紧密聚在一起;亚细胞定位预测表明葡萄VviSKOR蛋白主要定位于细胞质膜,且含有6个跨膜区,其等电点PI为6.24,表明该蛋白含有偏酸性氨基酸残基较多;在VviSKOR启动子区域预测到12种顺式作用元件,主要包括胁迫响应、激素响应和细胞周期调控等不同生命活动相关的调控元件;数据库表达谱分析结果表明葡萄VviSKOR在多种组织或器官中均有表达,在葡萄树根中的表达水平最高,其次是叶和木质部;实时荧光定量PCR分析表明VviSKOR在7年生‘马�【Objective】The aim of this study was to isolate and characterize potassium(K+)channel gene SKOR(stelar K+outward rectifier)from grape genome,based on the analysis of the tissue-specific expression patterns of VviSKOR gene and response to K+depletion,K+excess,NaCl and ABA treatments,as well as function study by patch clamping electrophysiological technology.【Method】By carrying out homology-based cloning,a putative K+channel gene VviSKOR was screened and isolated from grape genome.The details of VviSKOR gene were analyzed,and the protein encoded via utilizing bioinformatical analysis software.With the help of MEGA7.0 software,a phylogenetic tree was constructed by multiple alignments of SKOR proteins from grape,Arabidopsis,rice,maize,sorghum,slender false brome,soybean,tomato,cucumber,poplar,peach,pear,strawberry,apple,papaya,citrus,banana and pineapple.Using quantitative real-time PCR(qRT-PCR),the expression profiles of VviSKOR gene and its response to K+depletion,NaCl and ABA treatments were analyzed.Preliminary analized the physiological function of VviSKOR gene by using patch clamping.【Result】VviSKOR was isolated and determined from grape genome.VviSKOR contained the functional domains of cyclic nucleotide-binding domain,ion channel transmembrane,ankyrin repeats and KHA domain,which belonged to the classic plant potassium channels.The amino acid sequences of SKOR protein from 18 plants shared an overall identity of 58.92%.Phylogenetic tree analysis showed that VviSKOR was closely clustered with homolog of CsaSKOR from cucumber.SKOR members from 4 grass family plants(maize,rice,slender false brome and sorghum)were prone to be clustered together,while SKOR members from Rosaceae,including strawberry,apple,pear and peach,were prone to be clustered together.VviSKOR was mainly localized in plasma membrane,which contained 6 transmembrane domains(TMs),and the theoretical isoelectric point(PI)was 6.24.Twelve kinds of cis-acting regulatory elements,including stress response,hormone response and cell cycle reg

关 键 词：葡萄 Shaker类钾离子通道 SKOR 基因克隆与表达 膜片钳技术 

分 类 号：S663.1[农业科学—果树学]