大豆水杨酸结合蛋白基因GmSABP2的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2015.18.002

摘要/Abstract

摘要： 【目的】对大豆（Glycine max）水杨酸结合蛋白基因GmSABP2进行克隆与表达分析，并转化拟南芥进行耐盐、耐干旱分析，进一步了解该基因耐盐、耐干旱的分子机制。【方法】以拟南芥SABP2为探针，搜索大豆基因组数据库，从中挑选出同源性最高的序列，将其命名为GmSABP2。利用电子克隆技术，从大豆叶子中克隆得到大豆水杨酸结合蛋白基因GmSABP2。通过DNAMAN程序进行氨基酸的多序列比对，利用NCBI的CD-search进行氨基酸的保守结构域分析，应用MEGA程序进行系统进化树分析。对大豆幼苗进行盐和干旱胁迫处理来分析其在胁迫下的表型变化。通过Real time-PCR分析大豆幼苗在盐和干旱处理条件下GmSABP2的表达特性。利用Gateway技术构建植物表达载体pEarleyGate103-GmSABP2，转入根癌农杆菌EHA105，利用蘸花法侵染拟南芥，经抗性筛选得到转基因株系。对野生型植株和转基因植株进行盐和干旱胁迫处理，并统计在胁迫条件下两者的种子萌发率、主根长和成熟植株的存活率。【结果】克隆得到GmSABP2的cDNA序列，序列全长1 235 bp，其开放阅读框为786 bp，编码261个氨基酸，分子量为29.15 kD，等电点为5.58。氨基酸序列比对发现大豆和毛白杨、可可以及烟草相似度较高。利用NCBI的CD-search发现大豆SABP2序列中存在一个Abhydrolase_6（pfam：12697）水解酶保守结构域。大豆SABP2蛋白属于α/β水解酶超家族。应用MEGA程序构建多物种的系统发生树，发现大豆和毛白杨及可可亲缘关系较近，而与拟南芥亲缘关系较远。对大豆幼苗胁迫后的表型分析发现，在盐和干旱条件下大豆幼苗均受到明显的胁迫效应。Real time-PCR分析表明大豆幼苗叶子中的GmSABP2在盐和干旱处理条件下均上调表达。拟南芥耐逆性分析发现，在正常培养条件下，野生型植株和转基因株系均能正常萌发、生长。在高盐（150 mmol·L^-1 NaCl）处理条件下，野生型植株的种子萌发率为38%，12 d大小幼苗主根长为0.4 cm，成熟植株的存活率为49%；转基因株系的种子萌发率为67%，12 d大小幼苗主根长为1.1 cm，成熟植株的存活率为78%。在模拟干旱（20% PEG6000）处理条件下，野生型植株的种子萌发率为31%，12 d大小幼苗主根长为0.5 cm，成熟植株的存活率为36%；转基因株系的种子萌发率为57%，12 d大小幼苗主根长为1.0 cm，成熟植株的存活率为66%。【结论】GmSABP2在拟南芥植株对盐和干旱的抗性中有一定的作用。

关键词: 大豆, GmSABP2, 组织表达分析, 转基因拟南芥, 功能分析

Abstract: 【Objective】The aim of this study is to clone and analyze soybean protein gene GmSABP2, which is binded with salicylic acid, and transform Arabidopsis for analyzing salt tolerance and drought tolerance, and further understand the molecular mechanism of salt tolerance and drought-resistance of the gene. 【Method】 Using SABP2 as a probe, the soybean genome database was searched, and the highest sequence homology was picked out and named as GmSABP2. The gene GmSABP2 was cloned by using electronic cloning technology. The DNAMAN program was used to analyze the amino acid sequence alignment and the conserved domain amino acid by the CD-search conducted NCBI. The MEGA program was applied to make the phylogenetic analysis. The phenotypic variation of soybean seedlings under salt and drought stress was analyzed. The expression of the characteristics of GmSABP2 under salt and drought conditions was analyzed by Real time-PCR of soybean seedlings. Gateway technology was used to build plant expression vector pEarleyGate103-GmSABP2, shifted into Agrobacterium tumefaciens EHA105, infected Arabidopsis by utilizing flower dip method, then the homozygous transgenic plants were obtained by resistance screening and finally the salt and drought tolerance was analyzed. The wild-type plants and transgenic plants were treated under salt and drought stresses, and both the seed germination, root length and mature plants were counted under stress conditions. 【Result】The cDNA sequence of GmSABP2 was obtained and the open reading frame is 786 bp and total length of the sequence is 1 235 bp, encoding 261 amino acids. And molecular weight is 29.15 kD, an isoelectric point is 5.58. The amino acid sequence alignment and phylogenetic analysis showed that GmSABP2 and tobacco SABP2, Rauvolfia serpentina PNAE had the highest similarity. Using the CD-search of NCBI, it was found that the Abhydrolase_6 (pfam: 12697) as conserved domain hydrolases. Soybean SABP2 protein belongs to SABP2 α/β hydrolase superfamily. Using MEGA program to build a system of species multiple phylogenetic tree, it was found that SABP2 of soybeans, Theobroma cacao SABP2 and Solanum lycopersicum SABP2 have a close genetic relationship, but has a distant genetic relationship with Arabidopsis SABP2. The phenotype of soybean seedlings under salt and drought conditions was analyzed and it was found that there were significant stress effects. Real time-PCR analysis showed that GmSABP2 under salt and drought conditions were upregulated expression. Arabidopsis thaliana resistance analysis showed that under normal culture conditions, the wild-type plants and transgenic plants could germinate and grow. Under 150 mmol·L^-1 NaCl treatment conditions, seed germination rate of wild-type plants was 38%, seedling root length after 12 days was 0.4 cm and the survival rate of mature plants was 49%; The seed germination rate of transgenic lines was 67%, seedling root length after 12 days was 1.1 cm and mature plants survival was 78%. Under 20% PEG6000 treatment conditions, the seed germination rate of wild-type plants was 31%, seedling root length after 12 days was 0.5 cm and mature plants survival rate was 36%; The seed germination rate of transgenic lines was 57%, seedling root length after 12 days was 1.0 cm and mature plants survival rate was 66%.【Conclusion】 The GmSABP2 gene increases the resistance of Arabidopsis plants under salt and drought conditions.

Key words: soybean, GmSABP2, tissue expression analysis, transgenic Arabidopsis, functional analysis

贾亚军，王晓婷，许娜，郭娜，邢邯. 大豆水杨酸结合蛋白基因GmSABP2的克隆及功能分析[J]. 中国农业科学, 2015, 48(18): 3580-3588.

JIA Ya-jun, WANG Xiao-ting, XU Na, GUO Na, XING Han. Cloning and Function Analysis of Salicylic Acid Binding Protein Gene GmSABP2 from Soybean[J]. Scientia Agricultura Sinica, 2015, 48(18): 3580-3588.

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