天目地黄RcMYB4调控花青素合成的功能研究

doi:10.16420/j.issn.0513-353x.2025-0309

摘要/Abstract

摘要：

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。

关键词: 天目地黄, RcMYB4, 地黄, 花青苷, 异源表达

Abstract:

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.

Key words: Rehmannia chingii, RcMYB4, Rehmannia glutinosa, anthocyanin, heterologous expression

杨雅贺, 丁宁, 郝秋雯, 高俊鸽, 张朋雨, 胡靖翊, 张重义, 王丰青. 天目地黄RcMYB4调控花青素合成的功能研究[J]. 园艺学报, 2026, 53(3): 735-748.

YANG Yahe, DING Ning, HAO Qiuwen, GAO Junge, ZHANG Pengyu, HU Jingyi, ZHANG Zhongyi, WANG Fengqing. Functional Study of RcMYB4 in Regulating Anthocyanin Biosynthesis in Rehmannia chingii[J]. Acta Horticulturae Sinica, 2026, 53(3): 735-748.

图/表 8

图1 天目地黄RcMYB4的PCR扩增（A）及其在花不同组织中的表达（B）

Fig. 1 PCR amplification of RcMYB4（A）and its expression in different tissues of Rehmannia chingii（B）

图2 RcMYB4与其同源蛋白的多序列联配及结构域 Rh：湖北地黄；Rg：地黄；Rc：天目地黄；Rp：裂叶地黄；At：拟南芥

Fig. 2 Multiplesequence alignmentand domains of RcMYB4 withits homologous proteins Rh：Rehmannia henryi；Rg：Rehmannia glutinosa；Rc：Rehmannia chingii；Rp：Rehmannia piasezkii；At：Arabidopsis thaliana

图3 RcMYB4与其同源蛋白的系统进化树

Fig. 3 Phylogenetic tree of RcMYB4 and its homologous proteins

图4 本氏烟草瞬时过量表达RcMYB4的表型分析 A：RcMYB4及RcMYB4 + RcANS瞬时表达的本氏烟草叶片表型；B：烟草叶中总花青苷含量检测。** P < 0.01，下同

Fig. 4 Phenotypic analysis of transient overexpression of RcMYB4 in Nicotiana benthamiana A：Transient expression of RcMYB4 and RcMYB4 + RcANS in Nicotiana benthamiana leaves phenotype；B：Detection of total anthocyanin content in Nicotiana benthamiana leaves. ** P < 0.01. The same below

图5 RcMYB4过量表达的地黄愈伤诱导及形态（A）和花青苷含量分析（B） WT：野生型；OE：过表达株系

Fig. 5 Overexpression of RcMYB4 in callus induction and morphology（A）and anthocyanin content analysis（B）of Rehmannia glutinosa WT：Wild type；OE：Overexpression lines

图6 过量表达RcMYB4的地黄组培苗表型（A）与花青苷含量分析（B）

Fig. 6 Phenotype（A）and anthocyanin content analysis（B）of RcMYB4-overexpressing Rehmannia glutinosa tissue-cultured seedlings

图7 过量表达RcMYB4的地黄植株表型与花青苷含量分析 A：过量表达RcMYB4的地黄植株表型；B：过量表达RcMYB4的地黄叶、叶肉和叶柄横切面形态；C：过量表达RcMYB4的地黄块根及其横切面形态；D ~ F：不同转化株系叶、叶柄和块根中花青苷含量

Fig. 7 Phenotype and anthocyanin content analysis of RcMYB4-overexpressing Rehmannia glutinosa plants A：The phenotype of Rehmannia glutinosa plants overexpressing RcMYB4；B：Cross-sectional morphology of leaves，mesophyll，and petioles in RcMYB4-overexpressing Rehmannia glutinosa plants；C：The tuberous roots and their cross-sectional morphology of RcMYB4-overexpressing Rehmannia glutinosa；D-F：Anthocyanin content in leaves，petioles，and tuberous roots of different transgenic lines

图8 稳定表达RcMYB4的地黄叶（A）和块根（B）中花青素合成途径结构基因的表达量

Fig. 8 Expression levels of structural genes in the anthocyanin biosynthesis pathway in leaves（A）and tuberous roots（B）of Rehmannia glutinosa stably expressing RcMYB4

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