天目地黄RcMYB4调控花青素合成的功能研究OA
Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii
以天目地黄为试验材料,从其花冠中克隆获得 1 个 R2R3-MYB 转录因子基因,命名为RcMYB4.该基因的开放阅读框(open reading frame,ORF)为 777 bp,编码 258 个氨基酸.实时荧光定量 PCR 分析发现其在花冠筒中高表达.蛋白多序列联配发现 RcMYB4 具有 S6 亚家族保守的结构域.以邻接法构建系统进化树,发现 RcMYB4 与天目地黄 RcMYB1 和地黄 RgMYB42 聚在一起.瞬时表达发现 RcMYB4 的异源表达激活了本氏烟草中的花青素合成,RcMYB4+RcANS 激活花青素合成的效果进一步增强.根癌农杆菌介导的地黄遗传转化表明,过量表达 RcMYB4 的地黄愈伤、组培苗叶中的花青苷含量均显著增加.营养土种植的地黄转化苗叶和叶柄的花青苷含量均显著增加;块根中仅有 OE7 株系的花青苷含量增加,其含量是野生型对照的 4.01 倍.RcMYB4 过量表达的地黄株系中花青素结构基因的表达量结果分析发现,在 3 个株系的叶中 RgANS、RgCHS、RgF3H 和 RgDFR 的表达量均有所增加,其中 OE7的增加幅度最大,而块根中这 4 个基因仅在 OE7 中上调表达.这些结果表明 RcMYB4 是天目地黄中激活花青素合成的正调控转录因子.
Using Rehmannia chingii as the experimental material,a novel R2R3-MYB transcription factor gene was cloned from its corolla,with the resulting gene being systematically named RcMYB4.The open reading frame(ORF)of the gene spans 777 bp,encoding 258 amino acids residues.Quantitative real-time PCR(qRT-PCR)analysis demonstrated predominant expression of RcMYB4 in the corolla tube.Multiple sequence alignment of protein homologs revealed that RcMYB4 contain conserved domains of the S6 subfamily.Phylogenetic tree construction using the neighbor-joining method revealed that RcMYB4 groups with RcMYB1 from R.chingii and RgMYB42 from R.glutinosa,exhibiting the closest phylogenetic relationship.Transient expression assays in Nicotiana benthamiana indicated that heterologous expression of RcMYB4 activated anthocyanin biosynthesis,with co-expression of RcMYB4+RcANS significantly enhanced anthocyanin accumulation compared to single transformation of RcMYB4.Agrobacterium tumefaciens-mediated genetic transformation in R.glutinosa revealed that transgenic lines overexpressing RcMYB4 exhibited significantly increased anthocyanin accumulation in both callus tissues and leaves of tissue-cultured seedlings compared to wild-type controls.In potted nutrient soil-grown transgenic R.glutinosa,both leaves and petioles exhibited significantly increased anthocyanin accumulation compared to wild-type controls.Notably,only the OE7 transgenic line showed enhanced anthocyanin content in tuberous roots,reaching 4.01 times that of the wild-type control.Expression analysis of anthocyanin biosynthetic structural genes in RcMYB4-overexpressing R.glutinosa lines revealed significant upregulation of RgANS,RgCHS,RgF3H,and RgDFR in leaves across three transgenic lines,with OE7 exhibiting the most pronounced increases.In tuberous roots,these four structural genes were exclusively upregulated in OE7.These results demonstrate that RcMYB4 is a positive regulatory transcription factor that activates anthocyanin biosynthesis in Rehmannia chingii.
杨雅贺;丁宁;郝秋雯;高俊鸽;张朋雨;胡靖翊;张重义;王丰青
河南农业大学农学院,郑州 450046河南农业大学农学院,郑州 450046河南农业大学农学院,郑州 450046河南农业大学农学院,郑州 450046河南农业大学农学院,郑州 450046河南农业大学农学院,郑州 450046福建农林大学农学院,福州 350002河南农业大学农学院,郑州 450046
生物科学
天目地黄RcMYB4地黄花青苷异源表达
Rehmannia chingiiRcMYB4Rehmannia glutinosaanthocyaninheterologous expression
《园艺学报》 2026 (3)
735-748,14
国家自然科学基金面上项目(82373985)中央本级重大增减支项目(2060302)河南省高等学校重点科研项目(25A360031)
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