大豆GmMYB46基因的克隆、定位及表达分析

Cloning,localization and expression analysis of GmMYB46 in soybean

[目的]本文旨在研究MYB类转录因子基因GmMYB46的结构特征和定位,并阐述其对不同胁迫的响应,为明确其在逆境胁迫下的作用奠定基础。[方法]从大豆品种‘晋豆21’中克隆出GmMYB46的CDS序列,采用生物信息学方法对该基因及其编码蛋白进行分析,并利用PlantCARE软件分析GmMYB46基因启动子元件。通过洋葱表皮细胞瞬时表达系统对GmMYB46蛋白质全长及不同结构域M1(aa1～123)和M2(aa124～334)进行亚细胞定位分析。采用实时荧光定量PCR检测GmMYB46在不同逆境处理下的表达情况。[结果]GmMYB46 CDS序列全长为1 005 bp,编码334个氨基酸,蛋白质相对分子质量为82.53×10~3,氨基酸序列中含有2个高度保守的SANT结构域。进化树分析表明该基因编码的蛋白与野生大豆Gs MYB46亲缘关系最近。GmMYB46包含MBS、ABRE、GARE、TCA、LTR等逆境胁迫应答元件。亚细胞定位结果显示,p BIN-GmMYB46-GFP及p BIN-GmMYB46M1-GFP融合蛋白在细胞核中表达,p BIN-GmMYB46M2-GFP绿色荧光遍布整个细胞。实时荧光定量PCR分析表明,在干旱、盐(200 mmol·L^(-1)NaCl)、低温(4℃)、ABA(200μmol·L^(-1))、SA(500μmol·L^(-1))、GA(100μmol·L^(-1))处理下均能诱导GmMYB46基因在大豆根、茎、叶中的上调表达。[结论]GmMYB46基因的保守结构域对亚细胞定位起决定性作用,该基因可能参与大豆对非生物胁迫的响应。

[Objectives] In this study,the structural features and localization of the MYB transcription factor gene GmMYB 46 were analyzed.Expression patterns of GmMYB 46 under different stresses were tested,which can be applied to the further study of stress effect of soybean.[Methods] The CDS sequence of GmMYB 46 was cloned from the soybean variety‘Jindou 21’,and the gene and its encoding protein were analyzed by bioinformatics methods.The GmMYB 46 gene promoter motif element was analyzed by using the PlantCARE software.The subcellular localizations of the full-length and different domains of M1(aa1-123)and M2(aa124-334)of GmMYB46 protein were analyzed by the onion epidermal cell transient expression system.The expression of GmMYB 46 under different stress treatments were studied by RT-qPCR.[Results] Full-length CDS sequence of GmMYB 46 was 1 005 bp,encoding 334 amino acids,and the relative molecular mass of MYB46 protein was 82.53×10^3.Domain analysis showed that the amino acid sequence contained two highly conserved SANT domains.Phylogenetic tree analysis showed that GmMYB46 was highly homologous to GsMYB46 from Glycine soja.The promoter element analysis showed that GmMYB 46 contained stress responsive elements,such as MBS,ABRE,GARE,TCA and LTR.The subcellular localization results showed that the fusion protein of pBIN-GmMYB 46-GFP and pBIN-GmMYB 46M1-GFP were located in the nucleus,but the fusion protein of pBIN-GmMYB 46M2-GFP was expressed in the whole cell.Quantitative PCR(qPCR)analysis showed that the transcription level of GmMYB 46 in soybean root,stem and leaves was upregulated under the drought,salt(200 mmol · L^-1 NaCl),cold(4 ℃),ABA(200 μmol · L^-1),SA(500 μmol · L^-1)and GA(100 μmol · L^-1)treatments.[Conclusions] The conserved domain of GmMYB 46 played a crucial role in subcellular localization and the gene might be involved in stress response.