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

Abstract

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

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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