大豆转录因子基因GmMYB111的克隆及功能分析  

Expression and Function Analysis of the Transcription Factor Gm MYB111 in Soybean

作　　者：许玲[1] 卫培培[1] 张大勇[1] 徐照龙[1] 何晓兰[1] 黄益洪[1] 马鸿翔[1] 邵宏波[1] 

机构地区：[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

出　　处：《中国农业科学》2015年第15期3079-3089,共11页Scientia Agricultura Sinica

基　　金：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)

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Gm MYB111;以盐胁迫处理的c DNA为模板,利用RT-PCR法分离克隆MYB基因c DNA编码序列;根据Gm MYB111蛋白序列进行同源性搜索,得到与Gm MYB111蛋白序列相似度较高的其他物种的蛋白序列;使用MEGA5.05对Gm MYB111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Gm MYB111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Gm MYB111,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Gm MYB76、Gm MYB12a以及苜蓿Mt MYB61的亲缘关系最近;Gm MYB111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,Gm MYB111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,Gm MYB111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,Gm MYB111在茎、叶和花中表达量最高,在根中表达量相对较低,在�【Objective】 A gene encoding MYB transcription factor, designated GmM YB111, was cloned, its basic biological functions and expression pattern were identified in soybean and yeast cells.【Method】A MYB transcription factor GmM YB111 was obtained from salt stress-related digital expression profiling（DGEP） data analysis. c DNA sequence of GmM YB111 was isolated and cloned using c DNA from soybean salt-treated roots by RT-PCR method. A homology search was performed using Gm MYB111protein sequence as a query, and protein sequences of high similarity with Gm MYB111 from other species were obtained. Using MEGA5.05, multiple sequence alignments between GmM YB111 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 GmM YB111 was analyzed using Arabidopsis protoplast transformation system, and its transcriptional activity and in vivo binding activity were determined by yeast hybrid system【.Result】GmM YB111 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 GmM YB76, GmM YB12 a, and Mt MYB61. The expression of GmM YB111 in soybean was induce

关 键 词：大豆 GmMYB111 表达分析 结合基序 转录激活活性 

分 类 号：S565.1[农业科学—作物学]