茶树Na+/H+逆向转运蛋白基因CsNHX1、CsNHX2的克隆及表达分析

doi:10.13305/j.cnki.jts.2018.06.002

摘要/Abstract

摘要： Na⁺/H⁺逆向转运蛋白（Na⁺/H⁺ antiporter,NHX）在植物生长发育与逆境响应过程中扮演着重要角色。本研究以龙井长叶茶树品种为材料,克隆获得了茶树CsNHX1和CsNHX2基因cDNA全长序列,GeneBank登录号分别为：MG722977和MG515211。生物信息学分析结果显示,CsNHX1和CsNHX2的cDNA全长分别为1β691βbp和1β757βbp,均包含1个1β626βbp的开放阅读框,编码541个氨基酸,预测分子量为59.5βkD和59.7βkD,理论等电点为7.07和8.79;蛋白序列分析结果显示,CsNHX1和CsNHX2属于典型的跨膜蛋白,均含有保守的Na⁺/H⁺ Exchanger结构域;进化树分析显示,CsNHX1和CsNHX2均为IC类中定位于液泡膜上的Class I成员。qRT-PCR结果显示,干旱、低温和盐胁迫能够显著诱导CsNHX1和CsNHX2上调表达;外源ABA处理下,CsNHX1和CsNHX2表达水平整体变化趋势不显著;高温胁迫处理下,茶树CsNHX1表达水平显著降低,而CsNHX2表达水平逐渐增加,表明茶树CsNHX1和CsNHX2参与了茶树对多种环境胁迫的响应过程,但对于不同逆境胁迫的应答模式存在一定差异。

关键词: 茶树, NHX, 基因克隆, 表达分析

Abstract: The Na⁺/H⁺ antiporter (NHX) plays an important role in plant growth, development and stress response. In this study, the full-length cDNA sequences of CsNHX1 and CsNHX2 (GenBank: MG722977 and MG515211) were cloned from tea cultivar ‘Longjingchangye’. Bioinformatics analysis showed that the full-length cDNA of CsNHX1 and CsNHX2 were 1β691βbp and 1β757βbp, all containing a 1β626βbp open reading frame and encoding 541 amino acids. The molecular weights of CsNHX1 and CsNHX2 were 59.5βkD and 59.7βkD and pI were 7.07 and 8.79, respectively.The results of protein sequence analysis showed that CsNHX1 and CsNHX2 contained the conserved Na⁺/H⁺ exchange domain, and belong to the typical transmembrane proteins. Phylogenetic analysis of plant NHX revealed that CsNHX1 and CsNHX2 are the member of Class I that localized to the vacuolar membrane in IC subfamily. In addition, qRT-PCR results showed that the expressions of CsNHX1 and CsNHX2 were induced by drought, low-temperature and salt stress. In contrast, exogenous ABA could not induce the expressions of CsNHX1 and CsNHX2. In addition, the expression level of CsNHX1 in tea plant decreased significantly, but that of CsNHX2 increased gradually under heat stress, indicating that CsNHX1 and CsNHX2 were differently involved in tea plant responding to environmental stress, and possibly through different responding modes.

Key words: tea plant, NHX, gene cloning, expression analysis

中图分类号:

TS272.2
Q52

陈江飞, 余津铭, 杨建坤, 余有本, 肖斌, 杨亚军, 王伟东. 茶树Na⁺/H⁺逆向转运蛋白基因CsNHX1、CsNHX2的克隆及表达分析[J]. 茶叶科学, 2018, 38(6): 559-568. doi: 10.13305/j.cnki.jts.2018.06.002.

CHEN Jiangfei, YU Jinming, YANG Jiankun, YU Youben, XIAO Bin, YANG Yajun, WANG Weidong. Cloning and Expression Analysis of Na⁺/H⁺ Antiporter Gene CsNHX1 and CsNHX2 in Tea Plant (Camellia sinensis)[J]. Journal of Tea Science, 2018, 38(6): 559-568. doi: 10.13305/j.cnki.jts.2018.06.002.

参考文献 40

[1]	王新超, 杨亚军. 茶树抗性育种研究现状[J]. 茶叶科学, 2003, 23(2): 94-98.
[2]	Pardo J M, Cubero B, Leidi E O, et al.Alkali cation exchangers: roles in cellular homeostasis and stress tolerance[J]. Journal of Experimental Botany, 2006, 57(5): 1181-1199.
[3]	Brett C L, Donowitz M, Rao R.Evolutionary origins of eukaryotic sodium/proton exchangers[J]. American Journal of Physiology Cell Physiology, 2005, 288(2): C223-C239.
[4]	Apse M P, Sottosanto J B, Blumwald E.Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter[J]. Plant Journal for Cell & Molecular Biology, 2003, 36(2): 229-239.
[5]	Bassil E, Tajima H, Liang Y C, et al.The Arabidopsis Na+/H+Antiporters NHX1 and NHX2 control vacuolar pH and K+ homeostasis to regulate growth, flower development, and reproduction[J]. Plant Cell, 2011, 23(9): 3482-3497.
[6]	Yamaguchi T, Fukadatanaka S, Inagaki Y, et al.Genes encoding the vacuolar Na+/H+ exchanger and flower coloration[J]. Plant & Cell Physiology, 2001, 42(5): 451-461.
[7]	Rodríguezrosales M P, Francisco J G, Huertas R M, et al.Plant NHX cation/proton antiporters[J]. 2009, 4(4): 265-276.
[8]	Zhang H, Liu Y X, Xu Y, et al.A newly isolated Na+/H+ antiporter gene, DmNHX1, confers salt tolerance when expressed transiently in Nicotiana benthamiana or stably in Arabidopsis thaliana[J]. Plant Cell Tissue & Organ Culture, 2012, 110(2): 189-200.
[9]	Dacosta M.Research advances in mechanisms of turf grass tolerance to abiotic stresses: From Physiology to Molecular Biology[J]. Critical Reviews in Plant Sciences, 2014, 33(2/3): 141-189.
[10]	Quintero F J, Blatt M R, Pardo J M.Functional conservation between yeast and plant endosomal Na+/H+ antiporters[J]. Febs Letters, 2000, 471(2): 224-228.
[11]	Fukuda A, Nakamura A, Tagiri A, et al.Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice[J]. Plant & Cell Physiology, 2004, 45(2): 146-159.
[12]	Yokoi S, Quintero F J, Cubero B, et al.Differential expression and function of Arabidopsis thaliana NHX Na+/H+ antiporters in the salt stress response[J]. Plant Journal for Cell & Molecular Biology, 2010, 30(5): 529-539.
[13]	Venema K, Belver A, Marin-Manzano M C, et al. A novel intracellular K+/H+ antiporter related to Na+/H+ antiporters is important for K+ ion homeostasis in plants[J]. Journal of Biological Chemistry, 2003, 278(25): 22453-22459.
[14]	Porat R, Pavoncello D, Ben G.A heat treatment induced the expression of a Na+/H+ antiport gene (cNHX1) in citrus fruit[J]. Plant Science, 2002, 162(6): 957-963.
[15]	Li W Y, FRANCISCA L I, Wong F L, et al.Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells[J]. Plant Cell & Environment, 2010, 29(6): 1122-1137.
[16]	Wu C A, Yang G D, Meng Q W, et al.The cotton GhNHX1 gene encoding a novel putative tonoplast Na+/H+ antiporter plays an important role in salt stress[J]. Wiley, 2004, 45(5): 600-607.
[17]	卜华虎. 玉米Na+/H+质子泵ZmNHX1功能的初步研究[D]. 北京: 中央民族大学, 2011.
[18]	俞嘉宁. 小麦耐旱、耐盐相关基因的克隆、分析与功能研究[D]. 咸阳: 西北农林科技大学, 2003.
[19]	郭俊红, 王伟东, 谷星, 等. 茶树WRKY转录因子基因CsWRKY57的克隆及表达分析[J]. 茶叶科学, 2017, 37(4): 411-419.
[20]	王伟东. 高温和干旱胁迫下茶树转录组分析及Histone H1基因的功能鉴定[D]. 南京: 南京农业大学, 2016.
[21]	Hao X Y, Horvath D P, Chao W S, et al.Identification and evaluation of reliable reference genes for quantitative real-time PCR analysis in tea plant (Camellia sinensis (L.) O. Kuntze)[J]. International Journal of Molecular Sciences, 2014, 15(12): 22155-22172.
[22]	Kenneth J, Livak Thomas D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Method, 2001, 25(4): 402-408.
[23]	刘威, 李慧, 蔺经, 等. 杜梨PbNHX1基因的克隆、表达分析及功能验证[J]. 果树学报, 2018, 35(2): 137-146.
[24]	Hamada A, Shono M, Xia T, et al.Isolation and characterization of a Na+/H+ antiporter gene from the halophyte Atriplex gmelini[J]. Plant Molecular Biology. 2001, 46(1): 35-42.
[25]	Gaxiola R A, Rao R. Sherman A.et al.The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast[J]. Proceedings of the national academy of sciences of the United States of America, 1999, 96(4): 1480-1485.
[26]	Fukuda A, Yazaki Y, Ishikawa T, et al.Na+/H+ antiporter in tonoplast vesicles from rice roots[J]. Plant & Cell Physiology, 1998, 39(2): 196-201.
[27]	张雨良, 张智俊, 杨峰山, 等. 新疆盐生植物车前PmNHX1基因的克隆及生物信息学分析[J]. 中国生物工程杂志, 2009, 29(1): 27-33.
[28]	Bulle M, Yarra R, Abbagani S.Enhanced salinity stress tolerance in transgenic chilli pepper (Capsicum annuum L.) plants overexpressing the wheat antiporter (TaNHX2) gene[J]. Molecular Breeding. 2016, 36(4): 36.
[29]	Qiao W H, Zhao X Y, Li W, et al.Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants[J]. Plant Cell Reports. 2007, 26(9): 1663-1672.
[30]	Alhassan M, Daniso E, Boscaiu M, et al.Expression of the vacuolar Na+/H+ antiporter gene (NHX1) in three Plantago species differing in salt tolerance[J]. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Horticulture, 2015, 72(2): 441-442.
[31]	Brini F, Hanin M, Mezghani I, et al.Overexpression of wheat Na+/H+ antiporter TaNHX1 and H+-pyrophosphatase TVP1 improve salt and drought stress tolerance in Arabidopsis thaliana plants[J]. Journal of Experimental Botany, 2007, 58(2): 301-307.
[32]	Sun M H, Ma Q J, Liu X, et al.Molecular cloning and functional characterization of MdNHX1 reveals its involvement in salt tolerance in apple calli and Arabidopsis[J]. Scientia Horticulturae, 2017, 215(27): 126-133.
[33]	Wang L, Ma Y K, Li N N, et al.Isolation and characterization of a tonoplast Na+/H+ antiporter from the halophyte Nitraria sibirica[J]. Biologia Plantarum, 2016, 60(1): 1-10.
[34]	Munns R, Sharp R E.Involvement of abscisic acid in controlling plant growth in soil of low water potential[J]. Functional Plant Biology, 1993, 20(20): 425-437.
[35]	Fukuda A, Nakamura A, Hara N, et al.Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes[J]. Planta, 2011, 233(1): 175-188.
[36]	Liang M, Lin M, Lin Z, et al.Identification, functional characterization, and expression pattern of a NaCl inducible vacuolar Na+/H+ antiporter in chicory (Cichorium intybus L.)[J]. Plant Growth Regulation, 2015, 75(3): 605-614.
[37]	Adler G, Blumwald E, Bar-Zvi D.The sugar beet gene encoding the sodium/proton exchanger 1 (BvNHX1) is regulated by a MYB transcription factor[J]. Planta, 2010, 232(1): 187-195.
[38]	Afaq A M, Prasad S, Frans J M M. Improving crop salt tolerance: anion and cation transporters as genetic engineering targets[J]. Plant stress, 2009, 5(1): 64-70.
[39]	Munns R, Tester M.Mechanisms of salinity tolerance[J]. Annual Review of Plant Biology, 2008, 59: 651-681.
[40]	伍国强, 冯瑞军, 魏金魁, 等. 过量表达霸王ZxNHX和ZxVP1-1基因增强甜菜对渗透胁迫的耐受性[J]. 植物生理学报, 2017, 53(6): 1007-1014.