2023/5/17 9:28:24 阅读：128 发布者：
4月25日，河南农业大学王丰青教授团队在国际植物学知名期刊Physiologia Plantarum（2021年影响因子/JCR分区：5.081/Q1，中科院JCR分区植物科学2区）在线发表了题为“Purple Rehmannnia : investigation of the activation of R2R3-MYB transcription factors involved in anthocyanin biosynthesis”的研究论文。该研究揭示了地黄、天目地黄和湖北地黄3个物种花色差异的物质基础和分子调控机制，挖掘了6个调控花青素合成的MYB转录因子，并在体外和体内验证了它们调控花青素合成的功能，创制了高花青素含量的地黄新种质。
花青素是植物中广泛存在的一种水溶性天然色素，不仅能够赋予植物丰富的颜色产生一定的保护作用，同时还作为一种强氧化剂在保护人类健康方面发挥重要作用。地黄属有6种植物，其中地黄（Rehmannia glutinosa）是常用大宗药材，始载于《神农本草经》，列为上品，疗效确切，在我国具有悠久的用药历史。地黄花与地黄块根的功效类似，《本草纲目》中记载 ：“为末服食，功同地黄，肾虚腰脊痛，为末，酒服方寸匕，日三”。天目地黄（R. chingii）和湖北地黄（R. henryi）是地黄的近缘种，为传统的民间用药。地黄、天目地黄和湖北地黄的花色有很大差异（图1A），是辨别3个物种的典型器官特征。然而花色差异的原因及其调控机制还未见报道。
通过广泛靶向代谢组学和花青素靶向代谢组学分析，发现花青素含量差异是地黄、天目地黄和湖北地黄花色差异的物质基础，矢车菊素、天竺葵素、芍药素、飞燕草素和矮牵牛素是3种植物花的主要花青素（图1B）。3个物种花冠中含量最高的花青素成分均为矢车菊素-3-O-芸香糖苷，而其在天目地黄中的含量最高，其次为地黄，湖北地黄含量最低（图1C）。
Figure 1. Corolla phenotype and metabolite analysis of R. glutinosa, R. chingii and R. henryi. (A) Corolla phenotypic characteristics of R. glutinosa, R. chingii and R. henryi. (B) Clustering heat maps of anthocyanin-targeted metabolites. (C) The content of anthocyanin compounds in corollas of R. glutinosa, R. chingii and R. henryi. Data represent the mean ± standard (n = 3 biological replicate, each being a pool of 6 corollas from an individual plant). The capital letters indicate the significant difference at 0.01 level, and lower case letters indicate at 0.05 level; Student’s t-test.
利用转录组测序和实时荧光定量PCR分析，从地黄、天目地黄和湖北地黄中鉴定出6个参与花青素合成的候选MYB基因（RgMYB41、RgMYB42、RgMYB43、RcMYB1、RcMYB3和RhMYB1）。进化分析发现这6个转录因子属于S6亚家族（图2A），具有典型的R2R3 MYB结构域（图2B）。亚细胞定位显示RgMYB41、RgMYB42、RgMYB43、RcMYB1、RhMYB1和RcMYB3主要定位于细胞核（图2D）。
Figure 2. Identification and analysis of R2R3-MYB members in R. glutinosa, R. chingii and R. henryi. (A) Phylogenetic analysis of MYB proteins. R. glutinosa, R. chingii, R. henryi and Arabidopsis MYBs are indicated with red circles, green square boxes, blue diamonds and black triangles, respectively. At, Arabidopsis thaliana; Dc, Daucus carota; Si, Sesamum indicum; Ac, Actinidia chinensis; Pyp, Pyrus pyrifolia; Pav, Prunus avium; Pc, Prunus cerasifera; Md, Malus domestica; Pt, Populus tomentosa. (B) Amino acid sequence alignment of proteins in subgroup 6. Highly conserved sequences are highlighted in black, partially conserved in grey. R2, R3 domains are highlighted with blue underline. The bHLH interaction motif (DLX2RX3LX6LX3R) is indicated in green box. The conserved motifs of dicot anthocyanin-promoting MYBs motif ([A/S/G]NDV) and the anthocyanin-regulating MYBs motif ([R/K]Px[P/A/R]xx[F/Y]) are shown in red boxes. (C) Relative expression analysisof RgMYB41, RgMYB42, RgMYB43, RcMYB1, RcMYB3 and RhMYB1 genes. C1, C2, C3, F1 and F2 represent the five developmental stages of the corolla, and L represents the leaf. (D) RgMYB41-GFP, RgMYB42-GFP, RgMYB43-GFP, RcMYB1-GFP, RcMYB3-GFP and RhMYB1-GFP expressed in N. benthamiana leaf cells. Bar=20 μm. Data represent the mean ± standard.
基于转录组因子-代谢产物-基因共表达调控网络分析，明确了6个MYB基因与花青素积累、花青素结构基因表达的相关性。利用双荧光素酶报告研究揭示了6个MYB转录因子均能够激活烟草和地黄属植物花青素合成关键催化酶基因启动子的表达（图3C-F），但不同MYB转录因子的调控强度有差异，其中RcMYB3的激活效应最大。体外实验证实了6个MYB转录因子均可以正向调控花青素合成途径结构基因的表达。
Figure 3. Regulation analysis of the six MYB proteins on the promoters of structural genes involved in anthocyanin biosynthesis. (A) RcMYB3-structural gene-anthocyanin correlation network analysis. The gray dotted line represents no significant correlation, the orange and red solid lines represent the significant and very significant positive correlation, the light green and green solid lines represent the significant and very significant negative correlation. (B) Diagrams of reporter and effector vector construction in dual luciferase assays (REN, Renilla luciferase; LUC, firefly luciferase). (C) Effects of six MYB proteins on NtDFR and NtANS promoter activities in N. benthamiana cells. (D) Effects of RgMYB41, RgMYB42 and RgMYB43 on RgANS promoter activity. (E) Effects of RcMYB1 and RcMYB3 on RcCHS and RcANS promoter activities. (F) Effects of RhMYB1 on RhANS promoter activity. Data are shown as mean ± standard deviation. *p<0.05, **p<0.01; Student’s t-test.
进一步利用烟草异源稳定过量表达6个MYB基因，发现转基因烟草的叶片和花器官中花青素的积累显著增加，花青素途径结构基因的表达量明显提高。同时，为了分析MYB基因在地黄属植物中的分子功能，把RgMYB41、RgMYB42、RgMYB43、RcMYB1、RcMYB3和RhMYB1分别在地黄、天目地黄和湖北地黄中过量表达，转化体的叶片和块根中均积累了丰富的花青素（图4A-E），花青素合成途径的结构基因也均显著上调表达（图4F-H）。其中RcMYB3过量表达的天目地黄叶和根中积累的最多，说明RcMYB3激活花青素积累的功能最强。体内实验进一步证实克隆的6个MYB是地黄属植物花青素合成的正调控转录因子。
Figure 4. Coloration characterizations of different tissues of R. glutinosa, R. chingii and R. henryi with overexpressed the six MYB genes. (A–B) Seedling, leaf, vein, and root/tuberous root coloration phenotypes of R. glutinosa, R. chingii and R. henryi. (A) Seedling, leaf and vein; (B) tuberous root or root. Bar=1.0 cm. (C-E) Total anthocyanin content in the leaves(C), veins (D) and roots/tuberous roots (E). (F-H) qRT-PCR analysis of genes involved in anthocyanin biosynthesis pathway in leaves of R. glutinosa (F), R. chingii (G) and R. henryi (H). Data are shown as mean ± standard deviation. **p<0.01; Student’s t-test.
进一步把RcMYB3在地黄中过量异源表达，创制了高花青素含量的“紫地黄”（图5A），其花冠、叶和块根中总花青素的含量分别达到18.43、10.36和2.49 mg/g·FW（图5B）。
Figure 5. Effect of RcMYB3 overexpression on anthocyanin accumulation in different tissues of R. glutinosa. (A) Pigmentation phenotypes in seedlings and leaves, tuberous roots, corollas, calyxes, pistils and stamens of R. glutinosa overexpressing RcMYB3 gene. Wild type R. glutinosa was used as a control. Bar=1.0 cm. (B) Total anthocyanin content in different tissues of R. glutinosa overexpressing RcMYB3 gene. (C) Cyanidin-3-O-glucoside content in leaves and tuberous roots in RcMYB3-overexpressing R. glutinosa. Data are shown as mean ± standard deviation. **p<0.01; Student’s t-test.
天目地黄的花冠裂片紫红色，明显区别于其它物种。利用CRISPR/Cas9基因编辑技术对天目地黄的RcMYB3基因进行靶向敲除，获得了1株花色有显著差异的突变体，其花冠裂片由野生型的紫红色变为白色（图6B），花冠花青素含量降低了41.4%（图6C），表达量降低了73.4%（图6D）。说明RcMYB3基因的正常表达对于天目地黄花冠裂片花青素的积累是不可缺少的，推测是地黄属植物物种分化中受到选择的关键基因之一。
Figure 6. CRISPR/Cas9-induced mutation in the RcMYB3 gene of R. chingii. (A) The gene structure of RcMYB3 along with the target site for mutation. (B) Phenotypes of R. chingii with RcMYB1-edited. Bar=1.0 cm. (C) Total anthocyanin content in corollas, leaves, roots, calyxes, stamens and pistils of RcMYB3-edited R. chingii mutant. (D) Relative expression levels of RcMYB3 gene in the corolla of RcMYB3-edited R. chingii mutant. Data are shown as mean ± standard deviation. **p<0.01; Student’s t-test. (E) Expression levels of anthocyanin biosynthesis-related genes in the corolla of RcMYB3 mutant. (F) Sequencing results of RcMYB3 target site mutation pattern. The PAM motif (NGG) is underlined, dashes indicate deletions, green letters indicate insertions, and purple letters indicate substitutions. The numbers at the right of sequence indicate the number of deletion (-), insertion (+) or substation (S) bases for the target site.
河南农业大学农学院已毕业硕士研究生左鑫（现为北京中医药大学博士研究生）为本论文的第一作者，王丰青教授为本论文的通讯作者。河南农业大学硕士生苗春妍、李铭铭、杨旭、宋词、杜家方博士、刘向阳副教授、孙红正副教授、李连珍教授、福建农林大学张重义教授、古力副教授、李明杰副教授、河南中医药大学谢彩侠教授等也参与了该项工作。中国中医科学院中药资源中心袁媛研究员为该论文提供了宝贵的指导意见。本研究得到中央本级重大增减支项目（2060302）、国家自然科学基金（81872950）和河南省教育厅重点项目（22A360009）等项目的资助。
论文链接：
https://onlinelibrary.wiley.com/doi/10.1111/ppl.13920
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