花生AhMYB113异位表达促进烟草花青素积累

Ectopic expression of peanut AhMYB113 gene enhances anthocyanin accumulation in tobacco

为进一步探索MYB转录因子在花生花青素积累过程中的调控机理,以花生常规品种鲁花11(绿色叶片)和紫色叶片花生种质056杂交后代为材料,基于绿色和紫色杂交群体数字基因表达谱克隆了AhMYB113基因,利用生物信息学手段进行保守结构域、系统进化树等分析,通过实时荧光定量PCR技术检测在花生中的表达模式,在转基因烟草中进行功能鉴定。结果表明,花生AhMYB113基因的开放阅读框全长864 bp,编码287个氨基酸残基;编码蛋白的N端包含2个高度保守的MYB结构域,属于R2R3-MYB转录因子;与拟南芥、金鱼草、矮牵牛、苹果、葡萄中调控花青素积累的R2R3-MYB聚成一大类;通过荧光定量PCR分析发现,AhMYB113在花生紫色杂交后代(PH、PS)中的表达水平要显著高于绿色杂交后代(GH、GS);在烟草NC89中异位表达AhMYB113,能够促进花青素积累,使得转基因烟草叶片转变为紫色,随着叶龄增长叶片紫色逐渐加深,花瓣、雄蕊(花丝、花药)、雌蕊(柱头、花柱)、萼片等组织也分别呈现为紫红色或紫黑色。

To better understand the regulatory mechanism of MYB transcription factor in peanut anthocyanin accumulation,AhMYB113 gene was cloned from the hybrid of Luhua 11(green leaves)and 056(purple leaves).Conserved domains,phylogenetic tree,expression patterns of the gene were analized using bioinformatics method and quantitative real-time PCR.In addition,AhMYB113 was genetically transformed into tobacco for ectopic expression.Result showed that ORF of AhMYB113 was 864 bp in length and encoded 287 amino acids,with 2 highly conserved MYB domains at N-terminal,indicating that AhMYB113 belonged to R2R3-MYB transcription factor.AhMYB113 showed high homologous with the same conserved motif of R2R3-MYB in Arabidopsis,Antirrhinum,petunia,apple and grape.Its expression was significantly higher in purple hybrid plants(PH,PS)than in green hybrids(GH,GS).Phenotype analysis result showed that,AhMYB113 ectopic expression could promote anthocyanin accumulation in transgenic tobacco NC89.Tobacco leaves changed from green to purple,and was deepened gradually with the growth.Petals,stamens(anthers and filaments),pistil(stigmas and style)and sepals were maroon or purple-black,respectively.