茶树CsbHLHs调控花青素合成关键基因CsDFR1在花青素积累中的功能解析

二氢黄酮醇4-还原酶（DFR）是花青素合成途径中的重要酶。申请者通过遮阴茶叶及转录组分析发现，CsDFR1是茶树花青素积累的关键基因，WGCNA分析表明20多个光响应bHLH与CsDFR1共表达，但其如何调控CsDFR1并影响花青素合成与积累的机制尚不清楚。对CsDFR1启动子克隆与分析发现，该启动子具有G-box等多个光响应元件。由此我们提出具有光响应结合位点的CsDFR1，在光胁迫条件下受bHLH调控从而引起茶叶中花青素的积累的设想。申请者进一步通过构建来自CsDFR1启动子的G-box为杂交诱饵，采用酵母单杂交方法已经筛选出CsbHLH2等3个bHLH类转录因子。本项目拟进一步通过设计ACE、L-box杂交诱饵和酵母单杂交筛选bHLH类转录因子。并采用EMSA、双荧光素酶报告基因检测、烟草异源表达等方法来证明这一设想，以阐明CsbHLH转录调控CsDFR1并引起茶树花青素积累的机制。

DFR(Dihydroflavonol 4-Reductase)is a key reductase involved in anthyocynin pathway in tea（camellia sinensis).In our previous study, we have found that the important transcription factor bHLHs, which had a correlation with the expression of CsDFR1 during light stress in purple tea using metabolome, transcriptome sequencing analysis and WGCNA. But the molecular mechanisms of CsbHLHs regulate CsDFR1 involved in anthocyanin synthesis in tea remains unclear. Our early research showed that there were G-Box, ACE, Box-I, CATT-motif,GT1-motif, LAMP-element and TCT-motif light response cis-element motifs in the promoter of CsDFR1 by plantcare online analysis. We have cloned and verified the sequences of promoter of CsDFR1. Bait yeast strain was constructed by synthesizing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast. One-hybrid cDNA library was simultaneously constructed and screened directly in yeast as a result of in vivo plasmid recombination. cDNA inserts in positive clones was amplified by yeast colony PCR and analyzed through NCBI Blast after sequencing. The results showed that the construction of yeast one-hybrid library was successful, there were 3 bHLHs candidate proteins for G-box binding proteins were acquired by preliminary library screening. Our RNA-seq research and yeast One-hybrid screening results showed that the expression level of CsbHLHs had directly relationship with CsDFR1 when tea tree was subjected to the light stress. So, we supposed the molecular mechanisms of anathycynins accumulation in tea, the activity of bHLHs can effect on CsDFR1 directly. In order to validate the assumption, two bait yeast strain was constructed by synthesizing oligonucleotides containing ACE and L-box cis-acting element sequence for CsDFR1 promoter, and screen bHLHs by yeast One-hybrid. And then we will focus on the investigating the relationship of all CsbHLHs and CsDFR1 by dual-luciferase, yeast one-hybrid analysis. Our research may reveal CsbHLHs could physically bind and directly trans-activate the CsDFR1 promoter in vivo. we will try to validate CsbHLH2 can activate transcription of CsDFR1 as well as anathycynins accumulation in transgenic tobacco. Our results may enrich the molecular mechanism of anathycynin biosynthesis regulation and accumulation in tea.