在茶树中，儿茶素、花青素以及原花青素（PAs）都是通过类黄酮途径合成的，除了已知的主要结构基因外，许多转录因子基因比如MYB、bHLH、WD40等也都有参与类黄酮的合成。其中R2R3型MYB转录因子基因家族成员众多，在不同的物种中都具有调控类黄酮合成的作用。在本研究中，作者对茶树R2R3-MYB基因家族成员CsMYB2和CsMYB26进行了基因功能分析。

首先从‘龙井43’中克隆了这两个基因的全长编码区序列，然后通过系统进化分析发现二者与PAs合成相关的MYB聚在了一起；亚细胞定位表明CsMYB2位于细胞核；利用拟南芥的同源基因建立了CsMYB2和CsMYB26与类黄酮途径主要结构基因的互作网络；对CsMYB2和CsMYB26以及类黄酮途径主要结构基因启动子区域调控元件预测显示这些结构基因的表达可能受MYB基因的调控，而其他的MYB基因也可能调控CsMYB2和CsMYB26的表达；然后根据从启动子区域预测到许多光照响应元件以及ABA信号元件，作者接着分析了CsMYB2和CsMYB26以及类黄酮途径主要结构基因在ABA处理以及遮阴处理下的表达情况；紧接着以‘龙井43’和‘白叶1号’为材料分析了上述基因在两个品种不同叶片组织的表达情况，并分析了CsMYB2和CsMYB26的表达与主要结构基因表达模式的相关关系以及与儿茶素组分、花青素和原花青素等代谢物的相关关系，结果表明这些基因的表达情况与类黄酮的含量水平相关，但是基因间的表达相关关系因品种而异，CsMYB2可能调控CsF3'H的表达，而CsMYB26与CsLAR的表达正相关，并且CsMYB26可能是造成上述两个品种间EGC含量差异的调控基因。

Abstract

Background: Flavonoids are secondary metabolites that play important roles in the entire tea plant life cycle and have potential health-promoting properties. MYB transcription factors (TFs) are considered potentially important regulators of flavonoid biosynthesis in plants. However, the molecular mechanisms by which MYB TFs regulate the flavonoid pathway in tea plant remain unknown.

Results: In this study, two R2R3-MYB TFs (CsMYB2 and CsMYB26) involved in flavonoid biosynthesis in tea plant were investigated. The genes encoding CsMYB2 and CsMYB26 were cloned from the tea plant cultivar ‘Longjing 43’. Phylogenetic analysis showed that CsMYB2 and CsMYB26 were grouped into the proanthocyanidin biosynthesis-related MYB clade. Multiple sequence alignment revealed that conserved motif 1 in the two MYB factors was related to the bHLH TF. Subcellular localization assays suggested that CsMYB2 localized in the nucleus. Promoter analysis indicated that CsMYB2, CsMYB26 and the related structural genes contain MYB recognition elements. The expression levels of the CsMYB2 and CsMYB26 genes and the structural genes in the flavonoid biosynthesis pathway were determined in leaves from various sites in the two tea plant cultivars ‘Longjing 43’ and ‘Baiye 1 hao’.

Conclusions: The expression levels of these genes were correlated with the accumulated flavonoid content. The results demonstrated that the expression level of CsF3’H may be regulated by CsMYB2 and that CsMYB26 expression is positively correlated with CsLAR expression. The relative transcriptional level of CsMYB26 may be the main reason for the different epigallocatechin contents between the tea plant cultivars ‘Longjing 43’ and ‘Baiye 1 hao’. Our results will serve as a reference for the potential regulatory roles of CsMYB2 and CsMYB26 in flavonoid biosynthesis in tea plant and may also assist biologists in improving tea quality.