大豆水杨酸结合蛋白基因GmSABP2的克隆及功能分析  

Cloning and Function Analysis of Salicylic Acid Binding Protein Gene GmSABP2 from Soybean

作　　者：贾亚军[1] 王晓婷[1] 许娜[1] 郭娜[1] 邢邯[1] 

机构地区：[1]南京农业大学大豆研究所/国家大豆改良中心/作物遗传与种质创新国家重点实验室,南京210095

出　　处：《中国农业科学》2015年第18期3580-3588,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31301340);国家转基因生物新品种培育科技重大专项(2014ZX08004);国家公益性行业(农业)科研专项(nycytx-004);长江学者和创新团队发展计划(PCSIRT13073)

摘　　要：【目的】对大豆(Glycine max)水杨酸结合蛋白基因Gm SABP2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Gm SABP2。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Gm SABP2。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Gm SABP2的表达特性。利用Gateway技术构建植物表达载体p Earley Gate103-Gm SABP2,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Gm SABP2的c DNA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 k D,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Gm SABP2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-1Na Cl)处理条件下,野生型植�[ 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 in A rabidopsis thaliana as a probe, thesoybean 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 pEarleyGate 103-GmSABP2, shifted into Agrobacterium tumefaciens EHA 105, 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 eDNA 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 o./fl hydrolase superfamily. U

关 键 词：大豆 GmSABP2 组织表达分析 转基因拟南芥 功能分析 

分 类 号：S565.1[农业科学—作物学]