白木香AsWRKY4转录因子调控沉香倍半萜合成的分子机制

Agarwood has important medicinal value with sesquiterpene as one of the main efficacy components. Aquilaria sinensis (Lour.) Gilg is the only genuine plant resource for producing agarwood in China. Agarwood sesquiterpene can be induced after the A. sinensis was wounded. The mechanism of “defense-response induce agarwood formation” has been preliminary analyzed and the “whole-tree agarwood-inducing technique” has been established based on our previously research. But, because the molecular mechanism of wound-induced agarwood formation remains unclear, the efficiency of agarwood-inducing technique is sometimes low, and needs to be improved. In our previous studies, a typical wound-induced sesquiterpene synthase gene ASS1 has been cloned and functional analysis demonstrated ASS1 is a key enzyme that is responsible for the formation of agarwood sesquiterpenes. With yeast one-hybrid assay, we showed that AsWRKY4 binds the promoter of ASS1, and the interaction can be promoted by Methyl jasmonate at low concentrations; meanwhile, the transcript level of AsWRKY4 was up-regulated by wound and Methyl jasmonate treatment. Based on the previous study, in this project, we will confirm the combination of AsWRKY4 with ASS1 and clarify its regulation model through biochemical and genetic methods; screen the interacting protein with AsWRKY4 and carry out functional characterization; analyze their expression profiles and effect on the biosynthesis of sesquiterpene under wound stress. This study will clearly reveal the molecular network of wound-induced agarwood formation, providing a new theoretical basis for improving the efficient and stable agarwood-inducing technique.

沉香具有重要的药用价值，倍半萜是主要药效成分之一。白木香是国产沉香的唯一正品来源，沉香倍半萜是其受到伤害诱导形成的。课题组前期初步解析了“白木香防御反应形成沉香”的结香机理，发明了“通体结香技术”，但因分子机制未清晰揭示，结香技术还有的效率偏低，亟待改进。前期研究中，我们发现了1个显著受伤害诱导表达的倍半萜合酶基因ASS1。功能分析表明，ASS1是沉香倍半萜合成的关键酶。近期，通过酵母单杂交筛选到转录因子AsWRKY4能够与ASS1启动子结合，低浓度的MeJA促进二者互作；同时，AsWRKY4的表达受伤害和MeJA诱导。本项目拟在此基础上，通过生化和遗传学手段明确AsWRKY4对ASS1的调控模式；进一步筛选AsWRKY4的互作因子并进行功能鉴定；研究二者在伤害胁迫下的表达特性及对沉香倍半萜合成的影响，从而清晰揭示伤害诱导沉香倍半萜合成的分子网络，为高效稳定结香技术的改进提供新的理论基础。

珍稀濒危植物白木香是国产药用沉香的唯一正品来源。倍半萜类物质是其主要药效成分之一。项目组前期在白木香中发现了1个显著受伤害诱导表达的倍半萜合酶基因ASS1，并证实是沉香倍半萜合成的关键酶。为了研究ASS1的表达调控机制，对其启动子进行了克隆，并通过酵母单杂交筛选到两个与其启动子结合的转录因子AsWRKY44和AsMYC2。本项目在此基础上，进一步对启动子的关键调控区段及两个转录因子的功能进行研究，解析二者通过调控ASS1的表达参与沉香倍半萜合成的分子机制。研究发现，ASS1启动子是典型的伤害诱导表达启动子，其上游-973bp~-731bp区段（I段）为关键调控区段。健康状态下，AsWRKY44作为抑制子与ASS1启动子关键调控区段的W-box结合，抑制ASS1的启动子活性；而AsMYC2与抑制蛋白AsJAZ1结合，不与ASS1结合。当树体受到伤害后，随着内源JA升高，AsWRKY44通过泛素化途径降解，解除对ASS1的抑制作用，同时AsMYC2被释放，与启动子上游III段的G-box结合进一步促进ASS1表达。研究明确了转录因子AsWRKY44和AsMYC2的功能；揭示了ASS1健康状态下不表达，伤害后显著上调表达的原因，也是白木香伤害诱导结香的本质原因；为高效稳定结香技术的改进提供了新的理论基础；为全面揭示沉香属植物诱导结香的分子机制提供了新的切入点。本项目发表论文9篇，其中SCI论文4篇。申请专利2项，获得中医药科技进步二等奖1项。