CsLAC介导木质素合成参与调控茶树新稍嫩度的分子机制研究

Tenderness is an important factor in evaluating the quality of tea as well as in the tenderness-keeping ability of Camellia sinensis. The research on the shoot tenderness of C. sinensis is only confined to physiological and biochemical analysis, while there are almost no available reports on the molecular mechanism. Based on lignin affecting C. sinensis shoot tenderness and CsLAC mediating lignin synthesis, this project is designed to research the following works: Detecting the lignin by histochemical staining, content and monomer composition analysis to explore the role of lignin in regulating C. sinensis shoot tenderness; Identifying the key candidate genes of CsLAC family in the above process by bioinformatics, gene expression level and enzyme analysis, and then verifying the function of key CsLAC genes by over-expressing in Arabidopsis and inhibit-expressing in C. sinensis to reveal the effects of lignin synthesis in regulating C. sinensis shoot tenderness; Obtaining the miRNA of key CsLAC gene to analyze the molecular regulatory mechanism that dependent on upstream miRNA in the process of CsLAC-affected lignin synthesis in regulating C. sinensis shoot tenderness. The results of this project will reveal the underlying molecular mechanism of CsLAC-mediated lignin synthesis involved in regulating the shoot tenderness in C. sinensis, which will be highly valuable for promoting the applications of molecular biology in the research of tenderness and the tenderness-keeping ability of C. sinensis.

嫩度是茶叶品质鉴定的重要指标，亦是衡量茶树‘持嫩性’的关键因子，但关于茶树新稍嫩度的研究仅局限在简单的生理生化分析，分子机制研究鲜有报道。本项目拟以木质素对茶树新稍嫩度的影响为切入点，以CsLAC介导木质素的合成为理论基础开展以下工作：通过组织化学染色、含量和单体组成分析探究木质素在调控茶树新稍嫩度变化过程中的作用；结合生物信息学、基因表达水平和酶学分析筛选此过程的关键CsLAC家族基因，通过过表达拟南芥和瞬时抑制表达茶树探究其影响木质素合成调控茶树新稍嫩度的功能机制；获得关键CsLAC家族基因的miRNA，利用qRT-PCR、RLM-RACE和共表达技术探讨关键家族基因介导木质素合成调控茶树新稍嫩度过程中依赖miRNA的分子调控机制。本项目旨在探究CsLAC介导木质素合成参与调控茶树新稍嫩度的分子机制，对推动分子生物学在茶树新稍嫩度研究中的应用，开展茶树‘持嫩性’的研究具有重要理论意义。

茶树新梢嫩度是影响茶叶产量和品质的重要因素，但有关新稍嫩度变化的分子调控机制鲜有报道。本项目系统分析了茶树新梢嫩度变化过程中木质素含量变化趋势，明确了木质素在调控茶树新稍嫩度变化中的作用，确定了木质素积累关键时期；共鉴定了45个CsLAC家族基因，其中12个被确定为参与木质素生物合成的关键基因；构建了CsLAC2、3、4、5、6真核表达载体，亚细胞定位分析显示其均定位与细胞壁，同时获得了转基因拟南芥植株用以分析在木质素生物合成中的功能；通过异源和同源转化体系证实CsmiR397和CsmiR408a/b对多个关键CsLAC基因具有调控作用，获得了过表达CsmiR397、CsmiR408a、CsmiR408b的转基因拟南芥，用于进一步生物学功能验证；另外，对茶树新梢木质素积累关键时期进行了转录组学分析，全面探究了新梢嫩度变化过程中木质素生物合成的分子调控机制。项目研究结果有助于解析CsLAC介导木质素合成参与调控茶树新稍嫩度的分子机制，为茶树新梢嫩度变化调控机制研究以及高‘持嫩性’品种选育提供理论依据。