O-甲基化没食子儿茶素没食子酸酯是最先在日本的茶树品种‘Benifuuki’中发现报道的，因其显著的抗过敏效应而备受关注，而其中的3-O-甲基化没食子儿茶素没食子酸酯（EGCG3''Me）的功效最为显著。相比于EGCG，EGCG3''Me更容易被人体吸收。目前在茶树品种‘金牡丹’和‘金观音’中也发现了高积累的EGCG3''Me，但是对于EGCG3''Me合成的调控机制还不清楚。

bHLH（basic Helix-Loop-Helix）是一类转录因子家族，在茶树中有近120个成员。基于前期的RNA-seq分析，作者鉴定到了一个bHLH转录因子差异表达基因，它在EGCG3''Me高积累的茶树品种‘金牡丹’和‘金观音’中显著下调表达。在本研究中，作者分离了该转录因子的全长cDNA序列并将其命名为CsbHLH62；然后作者对其进行生物信息学分析，并检测了它在不同EGCG3''Me含量的茶树品种中的表达情况，表达结果同RNA-seq结果一致预示着CsbHLH62可能负向调控EGCG3''Me的积累；亚细胞定位证实其为核定位蛋白，双荧光素酶报告系统显示其为转录抑制子，进一步转录活性分析显示CsbHLH62能够通过结合 CCoAOMT、CsLAR 和 CsDFR 基因启动子区的E-box motif来抑制这些基因的转录表达，因为前期研究显示CCoAOMT、CsLAR 和 CsDFR的表达与EGCG3''Me的积累呈正相关，这也就预示着CsbHLH62能够抑制CCoAOMT、CsLAR 和 CsDFR的表达进而负向调控EGCG3''Me的合成。

Abstract

Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) in tea (Camellia sinensis (L.) O. Kuntze) is a major source of O-methylated catechin and renowned for a wide range of health effects. However, the transcriptional regulation mechanisms of EGCG3"Me biosynthesis remain unclear. In the present work, the basic Helix-Loop-Helix (bHLH) transcription factor, designated as CsbHLH62, belonging to GBOF group of bHLH families, was isolated and characterized from Camellia sinensis. CsbHLH62 contains an Open Reading Frame of 1662 bp and encodes a polypeptide of 553 amino acids. Subcellular location and transcriptional activity analysis showed it as a nucleus protein and possessed transcriptional inhibition activity. Furthermore, the expression of CsbHLH62 was decreased during EGCG3"Me accumulation. More importantly, E-box motifs (5'-CANNTG-3') were found in the promoters of CCoAOMT, CsLAR, and CsDFR, and further transient expression assays showed that CsbHLH62 repressed the transcription of CCoAOMT, CsLAR, and CsDFR. Collectively, these results suggest that CsbHLH62 acts as a transcriptional repressor that might be negatively affecting the accumulation of EGCG3"Me. These findings provide novel insights into the regulatory mechanism of EGCG3"Me biosynthesis, which might help to breed high EGCG3"Me-content tea plants.