冬箭筈豌豆耐盐基因NHX1克隆及表达分析

doi:10.11733/j.issn.1007-0435.2024.05.010

草地学报 ›› 2024, Vol. 32 ›› Issue (5): 1401-1409.DOI: 10.11733/j.issn.1007-0435.2024.05.010

• 研究论文 • 上一篇

冬箭筈豌豆耐盐基因NHX1克隆及表达分析

姚娜¹, 云岚^1,2, 艾芊¹, 任晓敏¹, 周成美¹, 李博华¹

1. 内蒙古农业大学草原与资源环境学院, 内蒙古呼和浩特 010010;
2. 草地资源教育部重点实验室, 内蒙古呼和浩特 010010

收稿日期:2024-03-04 修回日期:2024-03-28 发布日期:2024-06-01
通讯作者: 云岚，E-mail:yunlan@imau.edu.cn
作者简介:姚娜(1998-)，女，汉族，内蒙古乌海人，硕士研究生，主要从事草种质资源与育种研究，E-mail:m18847319047_1@163.com
基金资助:
内蒙古自治区自然科学基金重点项目(2023ZD07)；内蒙古自治区种业创新重大示范工程项目(2022 JBGS00400303)资助

Cloning and Expression Analysis of Salt-tolerant Gene NHX1 in Vicia villosa Roth.

YAO Na¹, YUN Lan^1,2, AI Qian¹, REN Xiao-min¹, ZHOU Cheng-mei¹, LI Bo-hua¹

1. College of Grassland Resource and Environment, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010010, China；;
2. Key Laboratory of Grassland Resources of Education Ministry, Hohhot, Inner Mongolia 010010, China

Received:2024-03-04 Revised:2024-03-28 Published:2024-06-01

摘要/Abstract

摘要： 为了分析和挖掘更多豆科牧草耐盐基因，选育耐盐品种，本研究通过同源克隆PCR方法从冬箭筈豌豆(Vicia villosa Roth.)cDNA中克隆NHX1基因，并对其进行生物信息分析。结果表明：冬箭筈豌豆NHX1基因CDS序列长度为1 641 bp，编码546个氨基酸，为碱性疏水蛋白，与豌豆同源相似度最高；蛋白结构以α螺旋为主，包含10个跨膜结构域；相对定量分析表明NHX1在根、茎、叶组织中均有表达，盐胁迫下叶中表达量最高并在盐胁迫后48 h达到最大值；1300-cYFP-NHX1农杆菌转化烟草亚细胞定位显示其定位于细胞膜，并通过蛋白印迹法验证蛋白表达。冬箭筈豌豆NHX1基因参与盐胁迫响应，在耐盐性强的材料中相对高表达。本研究为豆科牧草耐盐性改良奠定基础。

关键词: 冬箭筈豌豆, NHX1, 基因克隆, 表达分析

Abstract: In order to analyze and excavate more salt-tolerant genes of legume forage and breed salt-tolerant varieties,the NHX1 gene was cloned from the cDNA of Vicia villosa Roth. by homologous cloning PCR method,and its biological information was analyzed. The CDS sequence length of the gene NHX1 of Vicia villosa Roth. is 1 641 bp,encoding 546 amino acids,and it is an alkaline hydrophobic protein with the highest homology similarity to pea. The protein structure is dominated by alpha helix and contains 10 transmembrane domains. Relative quantitative analysis showed that NHX1 was expressed in root,stem and leaf tissues. The highest expression was found in leaves and the maximum expression was found at 48 hours after salt stress treatment. Subcellular localization of Agrobacterium 1300-cYFP-NHX1 transformed tobacco showed that it was localized to the cell membrane,and the protein expression was verified by western blot. The NHX1 gene is involved in the salt stress response and is relatively highly expressed in salt-tolerant materials. This study laid a foundation for the improvement of salt tolerance of legume forage.

Key words: Vicia villosa Roth., NHX1, Gene cloning, Expression analysis

中图分类号:

S542

姚娜, 云岚, 艾芊, 任晓敏, 周成美, 李博华. 冬箭筈豌豆耐盐基因NHX1克隆及表达分析[J]. 草地学报, 2024, 32(5): 1401-1409.

YAO Na, YUN Lan, AI Qian, REN Xiao-min, ZHOU Cheng-mei, LI Bo-hua. Cloning and Expression Analysis of Salt-tolerant Gene NHX1 in Vicia villosa Roth.[J]. Acta Agrestia Sinica, 2024, 32(5): 1401-1409.

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参考文献

[1] 杨劲松,姚荣江,王相平,等. 中国盐渍土研究：历程、现状与展望[J]. 土壤学报,2022,59(1):10-27
[2] 李伟.盐胁迫下拟南芥与盐芥显微及超微结构的比较研究[D]. 哈尔滨:东北林业大学,2014:1-2
[3] 廖岩,彭友贵,陈桂珠. 植物耐盐性机理研究进展[J]. 生态学报,2007(5):2077-2089
[4] 刘云芬,彭华,王薇薇,等. 植物耐盐性生理与分子机制研究进展[J]. 江苏农业科学,2019,47(12):30-36
[5] 杨晓慧,蒋卫杰,魏珉,等. 植物对盐胁迫的反应及其抗盐机理研究进展[J]. 山东农业大学学报(自然科学版),2006(2):302-305,12308
[6] 李彦,张英鹏,孙明,等. 盐分胁迫对植物的影响及植物耐盐机理研究进展[J]. 中国农学通报,2008(1):258-265
[7] 王东明,贾媛,崔继哲. 盐胁迫对植物的影响及植物盐适应性研究进展[J]. 中国农学通报,2009,25(4):124-128
[8] MUNNS R,TESTER M. Mechanisms of salinity tolerance[J]. Annual Review of Plant Biology,2008,59:651-81
[9] 李静,刘明,孙晶,等. Na⁺/H⁺转运蛋白NHX基因的研究进展[J]. 大豆科学,2011,30(6):1035-1039
[10] ARIEL R,BENJAMIN J. Effect of K⁺,its Counter Anion,and pH on Sodium Efflux from Barley Root Tips[J]. Journal of Experimental Botany,1976,27(5):843-852
[11] FUKUDA A,NAKAMURA A,YOSHIYUKI T. Molecular cloning and ex-pression of the Na⁺/H⁺ exchanger gene in Oryza sativa[J]. Bio-chimica et Biophysica Acta,1999,1446(1-2):149-155
[12] FUKUDA A,CHIBA K,MAEDA M,et al. Effect of salt and osmotic stresses on the expression of genes for the vacuolar H⁺-pyrophosphatase,H⁺-ATPase subunit A,and Na+/H+ antiporter from barley[J]. Journal of Experimental Botany,2004,55(397):585-594
[13] BRINI F,GAXIOLA R A,BERKOWITZ G A,et al. Cloning and characterization of a wheat vacuolar cation /proton antiporter and pyrophosphatase proton pump[J]. Plant Physiology and Biochemistry,2005,43(4)：347-354
[14] ZORB C,NOLL A,KARL S,et al. Molecular characterization of Na⁺/H⁺antiporters(ZmNHX) of maize (Zea mays L.) and their expression under salt stress[J]. Journal of Plant Physiology,2005,162(1)：55-66
[15] 王建光. 牧草饲料作物栽培学[M]. 第二版.北京:中国农业出版社,2018:242-243
[16] 范龙,孙天杰,杨郡,等. 大豆GmNHX1基因克隆及其在酵母中的耐盐性分析[J]. 河北农业大学学报,2015,38(6):7-12,24
[17] 安宝燕,罗琰,李加瑞,等. 紫花苜蓿Na⁺/H⁺逆向转运蛋白基因在拟南芥中表达提高转基因植株的耐盐性[J]. 作物学报,2008(4):557-564
[18] KRISHNAMURTHY P,VIAHAL B,KHOO K,et al. Expression of AoNHX1 increases salt tolerance of rice and Arabidopsis,and bHLH transcription factors regulate AtNHX1 and AtNHX6 in Arabidopsis[J]. Plant Cell Reports,2019,38(10):1299-1315
[19] MISHRA M,TIWARI S,GOMES A V. Protein purification and analysis:next generation Western blotting techniques[J]. Expert Review of Proteomics,2017,14(11):1037-1053
[20] FAROOQ M,AHMAD R,SHAHZAD M,et al. Real-time expression and in silico characterization of pea genes involved in salt and water-deficit stress[J]. Molecular Biology Reports,2023,51(1):18
[21] MURER H,HOPFER U,KINNE R. Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney[J]. Biochemical Journal，1976,154(3):597-604
[22] TABATA S,KANEKO T,NAKAMURA Y,et al. Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana[J]. Nature,2000,408(6814):823-826
[23] ATSUNORI F,ATSUKO N,YOSHIYUKI T. Molecular cloning and expression of the Na⁺/H⁺exchanger gene in Oryza sativa[J]. Biochimica et Biophysica Acta-Gene Structure and Expression,1999,1446(1/2):149-155
[24] ERSHOV P V,VASEKINA A V,VOBLIKOVA V D. et al. Identification of K⁺/H⁺ antiporter homolog in barley:Expression in cultivars with different tolerance to NaCl[J]. Russ Journal of Plant Physiol,2007,54:16-24
[25] 李霞,孔丹宇,刘传鑫,等. 滨豇豆VmNHX基因克隆与表达分析[J/OL]. 分子植物育种,2024,22(2):402-413
[26] YAMAGUCHI T,APSE M P,SHI H,et al. Topological analysis of a plant vacuolar Na⁺/H⁺ antiporter reveais a luminal C terminus that regulates antiporter cation selectivity[J]. Proceedings of the National Academy of Sciences of the United States of America,2003,100(21):12510-12515
[27] 艾迪,魏永成,孟景祥,等. 木麻黄NHX基因家族的鉴定及盐胁迫下的表达分析[J]. 西北植物学报,2023,43(5):750-760
[28] 张业猛,朱丽丽,陈志国. 藜麦NHX基因家族鉴定及盐胁迫下表达分析[J]. 生物技术通报,2022,38(12):184-193
[29] ANTONELLA C,LORENZO C,ELISA P,et al. Can the transcriptional regulation of NHX1,SOS1 and HKT1 genes handle the response of two pomegranate cultivars to moderate salt stress?:Salt-tolerance of two pomegranate cultivars[J]. Scientia Horticulturae,2021,288:4238
[30] 武学霞. 蚕豆钠氢离子反向转运体NHX1参与干旱胁迫响应的分子机制研究[D]. 西宁:青海大学,2021:12-23
[31] HORIE T,KARAHARA I,KATSUHARA M. Salinity tolerance mechanisms in glycophytes:An overview with the central focus on rice plants[J]. Rice,2012,5(1):11
[32] DEZHI W,QIUFANG S,SHENGGUAN C,et al. Ionomic Responses and Correlations Between Elements and Metabolites Under Salt Stress in Wild and Cultivated Barley[J]. Plant and Cell Physiology,2013,54(12):1976-1988
[33] 黄小芳,石培礼,余成群,等. 非生物胁迫下牧草抗逆性研究进展[J]. 草地学报,2023,31(5):1293-1301
[34] ZHU J K. Plant salt tolerance[J]. Trends in Plant Science,2001,6(2):66-71
[35] WU D,CAI S,CHEN M,et al. Tissue metabolic responses to salt stress in wild and cultivated barley[J]. Public Library of Science One,2013,8(1):e55431
[36] 赵美荣,申玉华,李永春,等. 紫花苜蓿抗逆基因工程研究进展[J]. 草地学报,2014,22(2):243-248
[37] ZHU J K. Abiotic stress signaling and responses in plants[J]. Cell,2016,167(2):313-324
[38] HUAZHONG S,FRANCICO J,JOSE M,et al. The putative plasma membrane Na⁺/H⁺ antiporter SOS1 controls long-Distance Na⁺ transport in plants[J]. The Plant Cell,2002,14(2):465-477
[39] PU L,LIN R,ZOU T,et al. Genome-Wide Identification,Primary Functional Characterization of the NHX Gene Family in Canavalia rosea,and Their Possible Roles for Adaptation to Tropical Coral Reefs[J]. Genes-Basel, 2021,13(1):33

冬箭筈豌豆耐盐基因NHX1克隆及表达分析

Cloning and Expression Analysis of Salt-tolerant Gene NHX1 in Vicia villosa Roth.

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