滁菊CmWRKY7调控花香成分萜类物质合成的分子机制

Chrysanthemum morifolium 'Chuju' is one of the important medicine and tea in China. The floral scent is an important horticultural trait of it. The terpenoids and its derivatives were considered as the main components of fragrance volatiles in chrysanthemum, but the molecular mechanism of regulation pathway is still not clear. Our previous study found that CmWRKY7 was orthologue of Artemisia annua AaWRKY1, which can regulate the expression of amorpha-4,11-diene synthase gene. We also found that CmWRKY7 can bind to the W-box region of the CmHMGR1 and CmFPPS1 promoters. By generating CmWRKY7 over-expression/chimeric repressor gene-silencing transgenic chrysanthemum, we try to explore the function of CmWRKY7 played in floral scent biosynthesis. We also use the yeast-one-hybrid assay and EMSA to validate whether CmWRKY7 binds to their promoters directly or not. The yeast-two-hybrid assay will be applied to screen CmWRKY7 interacted proteins, then using the BiFC and GST Pull-down to validate the interaction both in vivo and in vitro. Additionally, functional analysis of CmWRKY7 interaction protein via transgene will reveal the post-translational regulation mechanism of CmWRKY7 involved in terpenoids biosynthesis. Finally, we will clarify molecular mechanism of CmWRKY7 involved in the regulation of floral scent biosynthesis. Our proposal can provide a theoretical basis and the elite gene pool for molecular assisted breeding of floral scent.

滁菊是我国重要的药、茶两用菊花品种之一。花香成分是影响滁菊品质的关键性状，其主要成分为萜类及其衍生物，但滁菊花香合成的调控机制仍不明确。项目组从滁菊中克隆了青蒿萜烯合酶基因AaWRKY1的同源基因CmWRKY7，其表达特性与滁菊花朵香气含量相关，且发现其可结合滁菊萜类合成关键酶基因CmHMGR1和CmFPPS1启动子的W-box区域。为此，本项目拟利用超表达和融合抑制技术获得CmWRKY7转基因菊花，鉴定CmWRKY7调控萜烯合成的功能；利用酵母单杂检测CmWRKY7与萜类合成关键酶基因CmHMGR1、CmFPPS1启动子的结合，并采用EMSA进行验证；构建菊花花朵不同阶段酵母双杂文库筛选CmWRKY7互作蛋白，进行BiFC等验证；并对互作蛋白基因进行转基因功能鉴定；阐明CmWRKY7在翻译后水平调控滁菊萜烯合成的分子机制，为滁菊花朵香气育种提供理论依据和基因储备。

菊花（Chrysanthemum morifolium）是我国十大传统名花和世界四大切花之一，是菊科（Compositae）菊属（Chrysanthemum）多年生宿根花卉，具有观赏、食用以及药用等多种价值。萜类化合物是菊花香气的重要组成成分，影响着菊花观赏品质和功效，同时也在吸引传粉者、防御植食性昆虫以及植株间信号传导等生命活动中发挥着重要作用。WRKY转录因子家族是植物中最大的转录因子家族之一，参与了植物抗逆响应、生长发育以及次生代谢等多种生命活动。在本研究中，我们克隆获得了WRKY转录因子家族成员CmWRKY41，对该转录因子调控萜类化合物生物合成的功能进行研究。研究发现CmWRKY41通过特异结合以GTGACA和CTGACG为序列的W-box直接激活CmHMGR2和CmFPPS2表达，调控菊花倍半萜类物质生物合成；发现了CmMPK3.1能与CmWRKY41发生互作，且CmMPK3.1磷酸化CmWRKY41，并参与菊花倍半萜类物质生物合成的调控。项目实施期间已投稿论文4篇，发表论文3篇；培养研究生3名，其中博士2人，硕士1人。