中间锦鸡儿两个逆境相关R2R3-MYB基因的克隆及功能

Cloning and Functional Analysis of Two Stress Related R2R3-MYB Genes from Caragana intermedia

R2R3-MYB作为成员最多的MYB类转录因子,参与了植物发育、物质代谢、细胞分化以及胁迫应答等众多生物学过程。从中间锦鸡儿(Caragana intermedia.)干旱转录组数据库中筛选并克隆了2个MYB基因,命名为CiMYB74和CiMYB116。CiMYB74和CiMYB116基因g DNA长度分别为1 976和1 270 bp,均包含3个外显子和2个内含子;开放阅读框(ORF)长度分别为978和900 bp,分别编码326和300个氨基酸。采用染色体步移法克隆基因的启动子序列,克隆得到的CiMYB74启动子长516 bp,主要包含茉莉酸响应基序(CGTCA-motif和TGACGmotif)、ABA及病毒应答元件(CE3)、厌氧诱导响应元件(ARE)以及响应节律振荡元件(Circadian)等;克隆得到的CiMYB116基因的启动子长1 040 bp,主要包含热应答元件、水杨酸应答元件、防卫反应及胁迫应答元件。CiMYB74和CiMYB116基因的表达均受盐胁迫和干旱的诱导。在拟南芥中过表达CiMYB74后,转基因株系种子在萌发时期对盐胁迫更加敏感,预示该基因负调控种子萌发时期对于盐胁迫的耐受能力。

R2R3-MYB transcription factors family regulated numerous cellular and molecular mechanisms,such as plant development,primary and secondary metabolism,cell differentiation,and stress response. Two MYB genes were cloned from drought transcriptome of Caragana intermedia,and were named CiMYB74 and CiMYB116,respectively. The g DNA of CiMYB74 and CiMYB116 was 1 976 bp and 1 270 bp,respectively,consisted of three exons and two introns; the lengths of two genes ORF were 978 and 900 bp,encoding 326 and 300 amino acids,respectively. Promoter sequences were obtained by Genome Walking. Main predicted cis-elements of CiMYB74 promoter were CGTCA-motif,TGACG-motif,CE3,ARE,and Circadian. CiMYB116 promoter contained some stress responded elements,such as HSE( heat shock element),TCA-element,TC-rich repeat and circadian element. By real-time quantitative analysis,CiMYB74 and CiMYB116 were induced by Na Cl and drought treatments. CiMYB74 overexpression lines were more sensitivity to salt stress during seeds germination,indicating that CiMYB74 negatively regulated plants response to salt stress.