大豆耐盐相关基因GmNcl1的序列单倍型及表达分析

doi:10.3864/j.issn.0578-1752.2014.03.001

摘要/Abstract

摘要： 【目的】鉴定大豆基因GmNcl1的序列多态性，探求逆境下该基因的表达模式。【方法】通过BLASTP比对GmNCl1的氨基酸序列，寻找同源基因。测定50个大豆品种中GmNcl1的启动子和基因的DNA序列差异以及这些品种的耐盐性差异，利用MAGE软件进行单倍型聚类分析和进化树分析，寻找品种耐盐性和序列间的相关性。以耐盐品种铁丰8号和盐敏感品种85-140的幼苗根为材料，利用实时荧光定量PCR分析GmNcl1在多种处理下的表达模式，处理条件包括蛋白抑制剂阿米洛利（Na+/H+逆转运蛋白抑制剂）和钒酸钠（Ca2+-ATPase抑制剂）、pH4.0 和pH5.7、ABA处理及多浓度盐、碱、旱逆境胁迫。【结果】GmNcl1与拟南芥的Na+(K+)/H+交换蛋白CHX同源。GmNcl1序列有单核苷酸序列多态性位点16个和插入缺失位点2个，其中，包括一个位于外显子3的G/A（敏/耐）碱基改变，引起丙氨酸到苏氨酸的非同义突变。18个变异位点共组成14个单倍型，其中，耐盐品种有3种单倍型，盐敏感品种有11种单倍型。由6个位点组成的单倍型GAGATATTC（耐）/TTT----CT（敏）能高效区分耐盐和盐敏感品种。大豆幼苗根中的GmNcl1在阿米洛利处理下表达量增加，在钒酸钠处理下表达量不变。GmNcl1在碱性pH处理下表达量增加，在酸性pH处理下呈现上调和下调波动。GmNcl1受ABA诱导上调表达。在碱和旱胁迫下表达强度增大，在盐胁迫下上调表达最为明显，3种逆境胁迫强度越大，GmNcl1表达量上调幅度越大。耐盐品种铁丰8和盐敏感品种85-140的GmNcl1在盐处理下有近似的上调表达趋势，但85-140中GmNcl1的上调幅度大于铁丰8。【结论】GmNcl1与Na+(K+)/H+交换蛋白高度相似，该基因的序列多态性与耐盐相关，GmNcl1参与调节pH平衡，受ABA强烈诱导，响应盐、碱、旱胁迫。

关键词: 大豆 , 离子交换蛋白 , 耐逆 , 表达分析

Abstract: 【Objective】The objective of the study is to confirm sequence polymorphisms of GmNcl1 gene and its differential pattern in stress. 【Method】Amino acid sequence of GmNCl1 was compared by BLASTP to find homologous genes. Fifty soybean varieties were used for the sequencing of GmNcl1 promoter and gene, and indicating of salt tolerance, and haplotype cluster and phylogenetic tree were analyzed by MAGE, looking for the correlation between salt-tolerance and sequence of GmNcl1. The expression pattern of GmNcl1 by real time PCR analysis in roots of salt-tolerant cultivar Tiefeng 8 and salt-sensitive cultivar 85-140 under a series of treatments including the protein inhibitors, pH, ABA treatments and salt, alkali and drought stress. 【Result】 GmNcl1 was homologous with Na +(K+)/H+ exchanger CHX. Sixteen single nucleotide polymorphism loci and two InDel loci were found in GmNcl1 gene sequence, including one G/A (salt-sensitive/salt-tolerant) base change in exon 3, which caused the nonsynonymous mutation from alanine to threonine. Eighteen polymorphism loci were composed into fourteen haplotypes, of which there were three salt-tolerant haplotypes and eleven salt-sensitive haplotypes. The haplotypes, GAGATATTC (salt-tolerant)/TTT ---- CT (salt-sensitive) composed of six loci were high-effectively distinguished between the salt tolerance and sensitivity. In the roots of soybean seedling, the expression of GmNcl1 gene was increased under the Na+/H+ antiporter inhibitor, amiloride, and unchanged under Ca2+-ATPase inhibitor, sodium vanadate. The expression of GmNcl1 was increased under alkaline pH, and showed fluctuations under acidic pH. The expression of GmNcl1 was also induced by ABA, and up-regulated by salt, alkali and drought stress. The greater stress, the more up-regulated expression of GmNcl1. Expression of GmNcl1 showed a similar trend in Tiefeng 8 and 85-140 under salt stress, but there was the greater increase in 85-140 than Tiefeng 8.【Conclusion】GmNcl1 was highly similar with Na+(K+)/H+ exchanger, its sequence polymorphism was related to salt-tolerance and sensitivity. GmNcl1 was inducted by ABA and GmNcl1 might be involved in the balance of the organism internal environment, and be a key factor in the stress response to salt, alkali and drought stress in soybean.

Key words: soybean , cation/proton exchanger , stress-tolerance , expression analysis

赫卫1, 刘林2, 关荣霞2, 邱丽娟2. 大豆耐盐相关基因GmNcl1的序列单倍型及表达分析[J]. 中国农业科学, 2014, 47(3): 411-421.

HE Wei-1, LIU Lin-2, GUAN Rong-Xia-2, QIU Li-Juan-2. Haplotypes and Expression Analysis of Salt-Tolerant Gene GmNcl1 in Soybean[J]. Scientia Agricultura Sinica, 2014, 47(3): 411-421.

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