苹果抗性相关的谷胱甘肽转移酶基因MdGSTU1的生物信息学和表达分析

doi:10.3864/j.issn.0578-1752.2014.24.010

摘要/Abstract

摘要： 【目的】克隆苹果（Malus domestica Borkh.）中抗苹果轮纹病相关的编码谷胱甘肽转移酶基因MdGSTU1，研究其在不同组织器官及不同逆境处理条件下的表达特性，为解析该基因的抗逆功能奠定基础。【方法】基于抗轮纹病相关的EST序列，通过在NCBI进行比对，得到一个谷胱甘肽转移酶（GST）基因相关的EST片段；然后在苹果基因组数据库中进行比对，获得该GST的编码框序列（coding sequence，CDS），同时，设计RACE引物，克隆该基因的UTR序列；利用MEGA5.0软件对该GST蛋白与拟南芥GST家族蛋白成员进行系统进化树分析，使用DNAMAN软件对该蛋白的分子量、等电点等进行预测，并对其氨基酸序列进行分析；采用荧光定量PCR检测该基因在不同组织中的表达以及该基因在盐、模拟干旱和苹果斑点落叶病原菌处理条件下的表达特性；克隆该基因的启动子，利用PlantCARE软件在线分析该基因启动子上的顺式作用元件。【结果】进化树分析显示该GST与拟南芥中GST家族的U亚家族成员亲缘关系最近，故被命名为MdGSTU1；MdGSTU1的CDS为666 bp，编码221个氨基酸残基，其蛋白分子量为25.41 kDa，等电点为5.28；RACE结果显示该基因3'UTR区域为135 bp；蛋白序列及结构分析显示，其与拟南芥GST家族蛋白相同，该蛋白也包含保守的谷胱甘肽-S-转移酶的N端及C端结构域；PCR结果显示，MdGSTU1在根、茎、叶、花、果各组织中均有表达，但根中的表达水平最高，同时，该基因能够被150 mmol·L^-1 NaCl和20%的PEG诱导表达，均在1 h时表达量达到最高值；另外，苹果斑点落叶病原菌也能诱导MdGSTU1的表达，表达量在病菌处理8 h时达到最大；启动子分析显示MdGSTU1启动子区域含有多个抗性相关的作用元件，其中包括ABA响应元件、厌氧响应元件、GA响应元件、SA响应元件、JA响应元件以及防御和逆境响应元件；此外，该启动子上还包含多个MYB转录因子结合位点。【结论】MdGSTU1属于植物tau亚家族（GSTU）成员，生物及非生物逆境胁迫均可诱导该基因的表达。

关键词: 苹果, 谷胱甘肽转移酶, 克隆, 序列分析, 表达分析

Abstract: 【Objective】MdGSTU1 gene was amplified from apple (Malus domestica Borkh) by RT-PCR. The transcriptional level of MdGSTU1 in different tissues and under various treatments were determined to identify the resistant function of MdGSTU1 .【Method】ESTs derived from suppression subtractive hybridization were blasted in NCBI and one EST fragment associated with apple ring rot was separated. This EST was found to be a GST gene by blasting in apple genome database. The primers were designed to amplify the ORF and 3'UTR of this gene; subsequently, the phylogenetic tree of the GST proteins, including MdGSTU1 and the GST family proteins from Arabidopsis, was constructed using the neighbor-joining (NJ) method of MEGA 5.0 software. In addition, the protein molecular weight and isoelectric point were predicted with DNAMAN software, and the full length amino acid sequences were analyzed between MdGSTU1 and the GSTU subfamily proteins of Arabidopsis. The expression level of MdGSTU1 gene was detected in different tissues and under NaCl, PEG and Alternaria mali Roberts treatments using RT-PCR. Finally, the promoter of MdGSTU1 gene was cloned and the cis-acting regulatory elements were analyzed through PlantCARE database. 【Result】An apple gene encoding a putative glutathione transferase was obtained from a resistance-related EST to apple ring rot. The amplified sequence of this gene contains a 666 bp coding sequence, which encodes a protein of 221 amino acid residues. The calculated molecular mass is 25.41 kDa, with the isoelectric point 5.28. The phylogenetic relationship of this gene is closer with Arabidopsis tau type GST family, so it was named MdGSTU1. Sequence analysis showed that MdGSTU1 protein contains conserved glutathione S-transferase N- and C-terminal domain. RT-PCR results showed that the MdGSTU1 gene was expressed in all tissues tested. However, its expression level varied with tissue type, and the highest transcriptional level occurred in root, compared with other tissues including stem, leaf, ?ower and fruit. Furthermore, the level of MdGSTU1 transcript was markedly induced by 150 mmol·L^-1 NaCl and 20% PEG, and the highest value was reached at 1 h. In addition, MdGSTU1 was also induced by Alternaria mali Roberts and the highest expression level of MdGSTU1 was appeared at 8 h after treatment. Promoter analysis revealed that there are multiple putative cis-acting elements that are involved in abscisic acid responsiveness, anaerobic induction, gibberellin- responsiveness, salicylic acid responsiveness, MeJA-responsiveness, and defense and stress responsiveness, and some MYB binding site were also found in the promoter of MdGSTU1 gene. 【Conclusion】MdGSTU1 belongs to a tau class GST family and both biotic and abiotic stresses could induce its expression.gene

Key words: apple, glutathione transferase, cloning, sequence analysis, expression analysis

安秀红，徐锴，厉恩茂，李壮，李敏，刘志，程存刚. 苹果抗性相关的谷胱甘肽转移酶基因MdGSTU1的生物信息学和表达分析[J]. 中国农业科学, 2014, 47(24): 4868-4877.

AN Xiu-hong1, XU Kai1, LI En-mao1, LI Zhuang1, LI Min1, LIU Zhi2, CHENG Cun-gang1. Bioinformatics and Expression Analysis of MdGSTU1 Gene Encoding a Resistance-Related Glutathione Transferase from Apple[J]. Scientia Agricultura Sinica, 2014, 47(24): 4868-4877.

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