中国农业科学 ›› 2015, Vol. 48 ›› Issue (15): 3079-3089.doi: 10.3864/j.issn.0578-1752.2015.15.019

• 研究简报 • 上一篇    下一篇

大豆转录因子基因GmMYB111的克隆及功能分析

许玲,卫培培,张大勇,徐照龙,何晓兰,黄益洪,马鸿翔,邵宏波   

  1. 江苏省农业科学院农业生物技术研究所/江苏省农业生物学重点实验室,南京 210014
  • 收稿日期:2015-03-26 出版日期:2015-08-01 发布日期:2015-08-01
  • 通讯作者: 张大勇,Tel:025-84391105;E-mail:Cotton.z@126.com;邵宏波,Tel:025-84391921;E-mail:shaohongbochu@126.com E-mail:Cotton.z@126.com; shaohongbochu@126.com
  • 作者简介:许玲,Tel:025-84390351;E-mail:xuling@jaas.ac.cn
  • 基金资助:
    国家自然科学基金(31101166)、国家“十二五”科技支撑计划(2011BAD35B06-4-3)、江苏省农业科技自主创新资金项目(CX(15)1005)

Expression and Function Analysis of the Transcription Factor GmMYB111 in Soybean

XU Ling, WEI Pei-pei, ZHANG Da-yong, XU Zhao-long, HE Xiao-lan, HUANG Yi-hong, MA Hong-xiang, SHAO Hong-bo   

  1. Institute of Biotechnology, Jiangsu Academy of Agricultural Sciences/Provincial Key Laboratory of Agrobiology, Nanjing 210014
  • Received:2015-03-26 Online:2015-08-01 Published:2015-08-01

摘要: 【目的】克隆大豆MYB转录因子基因,进行序列分析和表达模式分析,并对其功能进行鉴定。【方法】通过对盐胁迫相关的数字表达谱(DGEP)数据分析,获得一个MYB转录因子GmMYB111;以盐胁迫处理的cDNA为模板,利用RT-PCR法分离克隆MYB基因cDNA编码序列;根据GmMYB111蛋白序列进行同源性搜索,得到与GmMYB111蛋白序列相似度较高的其他物种的蛋白序列;使用 MEGA5.05对GmMYB111蛋白序列及其同源序列进行多序列比对分析并构建同源物种间系统进化树;利用实时荧光定量PCR方法检测目的基因在大豆中受非生物胁迫诱导表达情况及组织特异性表达情况;利用拟南芥原生质体转化体系分析GmMYB111的亚细胞定位情况;通过酵母杂交系统检测其转录激活活性以及体外结合活性。【结果】根据前期江苏省农业科学院农业生物技术研究所盐土农业研究室盐胁迫相关的数字表达谱(DGEP)数据获得盐胁迫响应显著上调(27倍)的GmMYB111,利用RT-PCR方法从栽培大豆根组织中克隆该基因片段,序列比对发现其与已公布的Williams82基因组数据库序列一致,生物信息学分析表明,其编码的氨基酸序列具有MYB类转录因子的共同特征,其N端具有R2、R3两个MYB结构域,同时其C-端还存在一个富含酸性氨基酸的转录激活区;系统进化树分析表明,该基因编码的蛋白与GmMYB76、GmMYB12a以及苜蓿MtMYB61的亲缘关系最近; GmMYB111在大豆中的表达受高盐、干旱、冷害和ABA诱导表达,实时荧光定量PCR检测结果显示,在高盐和冷害胁迫下,GmMYB111呈上调表达,在干旱胁迫诱导后呈先上调后下调的表达模式,在ABA诱导下其表达量呈现波动式上调和下调表达;时空表达分析表明,GmMYB111为组成型表达,在大豆幼苗期和成熟期的表达量相对较强,成熟期的表达量相对较低,从不同组织来看,GmMYB111在茎、叶和花中表达量最高,在根中表达量相对较低,在豆荚中不表达;亚细胞定位结果显示GmMYB111定位于细胞核中,为典型的转录因子;酵母杂交系统检测表明,GmMYB111具有转录激活功能,并且能够与顺式作用元件TAACTG基序相结合。【结论】GmMYB111为典型的R2R3-MYB转录因子基因,具有转录激活活性及DNA结合活性,在大豆中的表达可能与大豆的非生物胁迫和ABA信号转导途径有关,推测其可能通过调节下游基因的表达来调控大豆对非生物胁迫的应答。

关键词: 大豆, GmMYB111, 表达分析, 结合基序, 转录激活活性

Abstract: 【Objective】 A gene encoding MYB transcription factor, designated GmMYB111, was cloned, its basic biological functions and expression pattern were identified in soybean and yeast cells. 【Method】 A MYB transcription factor GmMYB111 was obtained from salt stress-related digital expression profiling (DGEP) data analysis. cDNA sequence of GmMYB111 was isolated and cloned using cDNA from soybean salt-treated roots by RT-PCR method. A homology search was performed using GmMYB111 protein sequence as a query, and protein sequences of high similarity with GmMYB111 from other species were obtained. Using MEGA5.05, multiple sequence alignments between GmMYB111 protein and its homologous ones from other species were done and a phylogenetic tree of homologous species was constructed. The induced expression and tissue-specific expression profiles of target genes in soybean with abiotic stress were detected by real-time fluorescent quantitative PCR. The subcellular localization of GmMYB111 was analyzed using Arabidopsisprotoplast transformation system, and its transcriptional activity and in vivo binding activity were determined by yeast hybrid system. 【Result】GmMYB111 gene, a significantly upregulated gene (27 folds) in response to salt stress, was obtained based on the preliminary digital expression profiling (DGEP) data related to salt stress in authors laboratory. Using RT-PCR method, fragment of this gene was cloned from cultivated soybean root. Sequence alignment revealed that its sequence was consistent with that from the published Williams82 genome database. Bioinformatics analysis indicated that the deduced amino acids had common characteristics of MYB transcription factors with two MYB domains of R2 and R3 at the N-terminal and an acidic amino acid-rich transcriptional activation domain at the C-terminal. Phylogenetic tree analysis suggested that the encoded protein had the closest genetic relationship with GmMYB76, GmMYB12a, and MtMYB61. The expression of GmMYB111 in soybean was induced by high salt, drought, chilling, and ABA treatments, respectively. Real-time fluorescent quantitative PCR detection results showed that the induced GmMYB111 was upregulated by high salt and cold stress, and was first upregulated and followed by a down-regulation by drought stress. There was a wave-like up- and down-regulated expression of GmMYB111 inducted by ABA treatment. Analysis of temporal and spatial expression showed that GmMYB111 was nearly expressed in all detected tissues, and its expression level was relatively high at seedling and low at maturing stage. From the perspective of different tissues, GmMYB111 showed the highest expression in stem, leaf, and flower, relatively low in root, and no expression in pod. Subcellular localization results showed that GmMYB111 was located in the nucleus which belongs to a typical transcription factor, yeast hybrid assay indicated that GmMYB111 had transcriptional activation functions and could bind to the cis-acting element TAACTG motif. 【Conclusion】GmMYB111 is a typical R2R3-MYB transcription factor, with transcriptional activation function and DNA binding activity. Its expression in soybean may be related to the abiotic stress and ABA signal transduction pathway. GmMYB111 is speculated to regulate the soybean response to abiotic stresses by regulating the expression of downstream genes.

Key words: soybean, GmMYB111, expression analysis, binding motif, transcriptional activation activity

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