白木香倍半萜合酶基因ASS1启动子结合抑制子导致ASS1沉默及伤害解除机制研究

Agarwood is a kind of precious and rare traditional Chinese medicinal materials and high valued world-wide resources. It is resinous heartwood produced from wound-induced Aquilaria spp. trees, such as A. sinensis. The major components of agarwood are sesquiterpenes and phenylethyl chromones. Sesquiterpene synthases (SS) are the most important catalyzing enzymes of sesquiterpene biosynthesis. Our previous studies found that sesquiterpene synthase gene ASS1 doesn’t express under health status, and the promoter is an inducible one which initiates transcription only in the presence of wound signals. The 242bp segment of the promoter between -973 to -731 is considered as the key sequence, which negatively regulates the transcription of ASS1. And the transcriptional inhibition can be released by wounding or deleting this fragment. Meanwhile, methylated sites don’t exist in the promoter. So we predict that there is a repressor binding to the 242bp segment. In this work, we will select the transcriptional repressor, analyze the suppression reason and figure out the releasing mechanism. Yeast one-hybrid method isused to get the protein AsR-X that binds to the 242bp segment of ASS1 promoter. Moreover, transient expression and transgenic technology are used to clear the function protein AsR-X. Furthermore, Yeast two-hybird and coimmunoprecipitation assays are going to be utilized to demonstrate that protein AsRx-Y directly interact with AsR-X, and clarify the regulation mechanism how wound signal release the inhibition. This study will clearly reveal the most essential molecular mechanism of agarwood sesquiterpene biosynthesis, completely solve the mysteries of induced agarwood formation, providing a new theoretical basis for improving the efficient and stable agarwood-inducing technique. At the same time, it also has a great reference value for studying the molecular mechanism of secondary metabolites produced by wound-induced plant defense response.

沉香为珍稀濒危南药和高价值世界性资源，是沉香属白木香等物种受伤害后诱导产生的含树脂木材。倍半萜合酶是沉香主要成分倍半萜生物合成最重要的催化酶。申请者团队发现，白木香倍半萜合酶基因ASS1在健康白木香树中不表达，其启动子为诱导型；启动子5’端242bp具有负调控作用，但伤害信号或删除该区段可解除抑制,启动ASS1表达，且启动子区无甲基化位点。推测该区段结合了某种抑制子。本课题以寻找与ASS1启动子5’端242bp序列结合的抑制蛋白为切入点，通过瞬时表达及转基因技术明确该抑制蛋白功能；通过免疫共沉淀和酵母双杂交筛选该抑制蛋白的互作蛋白，探析伤害信号解除抑制作用的机制。研究将揭示伤害诱导ASS1基因启动表达合成倍半萜最本质的分子机制，为全面阐述健康白木香树不形成沉香、伤害后可诱导产生沉香倍半萜的关键分子调控机制奠定基础。研究对其他植物伤害诱导产生防御性次生代谢产物机理解析及理论构建也有重要价值。

沉香为珍稀濒危南药和高价值世界性资源。在中国，白木香(Aquilaria sinensis (Lour.) Gilg)为其药用沉香唯一法定来源物种。白木香只有在受到外界伤害后才能诱导形成沉香。倍半萜合酶是沉香倍半萜类化合物生物合成最重要的催化酶。倍半萜合酶基因ASS1在健康白木香树中不表达，受到伤害诱导后才能强烈表达。前期研究发现白木香倍半萜合酶基因ASS1的启动子为诱导型启动子，其 5’端 242 bp (-973~-731 bp)区域存在着某种抑制作用，抑制了正常情况下ASS1全长基因的转录表达。在本项目中，我们以ASS1的启动子上游242bp区段为研究对象，通过酵母单杂交实验和染色体免疫共沉淀实验，筛选并确定了转录因子AsWRKY44与该区段的W-box特异性相结合，过表达的 AsWRKY44在转基因烟草中抑制了ASS1的表达；RNAi-AsWRKY44 转基因白木香体系中，AsWRKY44的表达量下降，而ASS1的表达量明显上调；此外，茉莉酸甲酯(MeJA)处理导致 AsWRKY44蛋白的降解，ASS1基因表达量的显著上调。因此，我们推测AsWRKY44是伤害诱导ASS1基因的关键的负调控蛋白。IP实验发现，AsWRKY44和泛素分子相互作用，暗示MeJA处理后AsWRKY44被泛素化；蛋白酶抑制剂MG132可以抑制AsWRKY44的降解，证明AsWRKY44蛋白通过泛素-蛋白酶体途径被降解。本项目清晰揭示了 ASS1健康状态下沉默、伤害诱导后显著上调表达最核心的分子机制，本项目为全面解析伤害诱导白木香防御反应形成沉香的调控机制提供了分子依据，为高效结香技术的改良提供了理论指导，同时对其他植物伤害诱导防御反应产生次生代谢产物的分子机制揭示也有重要参考价值。