Journal of Nuclear Agricultural Sciences ›› 2020, Vol. 34 ›› Issue (12): 2638-2646.DOI: 10.11869/j.issn.100-8551.2020.12.2638

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Cloning and Expression Analysis of A Na+/H+Antiporter Gene CmaSOS1 in Cucurbita maxima

SHI Pibiao1, HONG Lizhou1, WANG Jun1, FEI Yueyue1, WANG Weiyi1, LYU Yuanda2, GU Minfeng1,*   

  1. 1Xinyang Agricultural Experiment Station of Yancheng City, Yancheng, Jiangsu 224049;
    2Institute of Crop Germplasm and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014
  • Received:2019-07-08 Online:2020-12-10 Published:2020-10-13

印度南瓜Na+/H+逆向转运蛋白基因CmaSOS1的克隆与表达分析

时丕彪1, 洪立洲1, 王军1, 费月跃1, 王伟义1, 吕远大2, 顾闽峰1,*   

  1. 1盐城市新洋农业试验站,江苏 盐城 224049;
    2江苏省农业科学院种质资源与生物技术研究所,江苏 南京 210014
  • 通讯作者: *顾闽峰,男,研究员,主要从事农作物遗传育种研究。E-mail:ycgmf@126.com
  • 作者简介:时丕彪,男,助理研究员,主要从事农作物新品种选育及分子育种研究。E-mail:1032175660@qq.com
  • 基金资助:
    江苏现代农业(蔬菜)产业技术体系(盐城)推广示范基地项目(JATS(2018)137),江苏省自然科学基金项目(BK20160582),江苏省农业科学院探索性颠覆性创新计划项目(ZX(17)2015),江苏沿海地区农业科学研究所科研基金项目(YHS201706)

Abstract: Salt overly sensitive 1 (SOS1) gene, encoding a Na+/H+ antiporter protein, is an essential gene for plant salt tolerance and plays an important role in biological processes of plants against abiotic stress. Bioinformatics and molecular biology methods were used to study the sequence characteristics and functions of SOS1 gene in Cucurbita maxima. In this study, the full-length cDNA sequence of Na+/H+antiporter gene of Cucurbita maxima was cloned, named as CmaSOS1 and the GenBank accession number is NW_019272028. Sequence analysis showed that the cDNA of CmaSOS1 gene is 3 940 bp in length and contains a 3 429 bp open reading frame (ORF, 162 to 3 590 bp), encoding 1 142 amino acid residues of CmaSOS1 protein. CmaSOS1 has 23 exons and 22 introns with a total length of 46 314 bp at the DNA level. Bioinformatics analysis indicated that the estimated molecular weight and theoretical isoelectric point (pI) of CmaSOS1 protein are 126.7 kDa and 5.92, respectively. The putative protein CmaSOS1 belongs to Na_H_Exchanger superfamily and CAP_ED superfamily, containing 12 transmembrane domains. CmaSOS1 belongs to hydrophobic stable protein, the mainly secondary structure elements are random coil and alpha helix. By multiple sequence alignment and phylogenetic tree analysis, CmaSOS1 has a high homology with Na+/H+ antiporter from Cucurbita moschata, Cucurbita pepo, Cucumis melo, Cucumis sativus and Momordica charantia in the same family of cucurbitaceae with the amino acid sequence identities of 98%, 98%, 90%, 89% and 89%, respectively. Real-time quantitative PCR (RT-qPCR) analysis revealed that the expression of CmaSOS1 gene was higher in roots and leaves, but lower in stems, flowers and fruits. Additionally, the expression of CmaSOS1 was up-regulated by NaCl and PEG treatments, suggesting that this gene might play an important role in the resistance to salt stress and drought stress of Cucurbita maxima. This study provided a foundation for further revealing the function of CmaSOS1 under abiotic stress.

Key words: Cucurbita maxima, CmaSOS1 gene, gene cloning, sequence analysis, expression analysis

摘要: Na+/H+逆向转运蛋白(SOS1)是植物耐盐的关键因子之一,在植物响应非生物胁迫过程中发挥着重要作用。为解析印度南瓜SOS1基因的序列特征和功能,利用生物信息学和分子生物学方法对其进行研究。结果表明,克隆获得印度南瓜SOS1基因cDNA全长序列,命名为CmaSOS1,GenBank登录号:NW_019272028。序列分析表明,CmaSOS1基因的cDNA全长3 940 bp,包含一个3 429 bp的开放阅读框架,编码1 142个氨基酸。CmaSOS1基因含有23个外显子和22个内含子,全长46 314 bp。CmaSOS1蛋白的分子量为126.7 kDa,理论等电点为5.92,包含12个跨膜结构区域,具有一个Na_H_Exchanger superfamily结构域和一个CAP_ED superfamily结构域;CmaSOS1蛋白属于疏水性稳定蛋白,二级结构元件多为无规卷曲和α-螺旋。CmaSOS1蛋白与葫芦科的中国南瓜、西葫芦、甜瓜、黄瓜和苦瓜Na+/H+逆向转运蛋白的同源性较高,序列一致性分别为98%、98%、90%、89%和89%。实时荧光定量PCR分析表明,CmaSOS1基因在印度南瓜的根和叶中表达量较高,在茎、花、果实中的表达量较低;该基因受NaCl和聚乙二醇(PEG)诱导后均呈上调表达,推测CmaSOS1基因可能在印度南瓜抵御盐分胁迫和干旱胁迫过程中发挥重要作用。本研究为进一步揭示CmSOS1在非生物胁迫下的功能奠定了基础。

关键词: 印度南瓜, CmaSOS1基因, 基因克隆, 序列分析, 表达分析