白木香microRNA在伤害诱导沉香形成中的作用机制研究

Agarwood is in great demand for its high value in medicine, incense, and perfume across Asia, Middle East, and Europe. As agarwood is formed only when the Aquilaria trees are wounded or infected by some microbes, overharvesting and habitat loss are threatening some populations of agarwood-producing species. Aquilaria sinensis is the only agarwood producing species in China. To promote the production efficiency and protect the resource of A. sinensis as well as other agarwood producing species, it would be critical to reveal the regulation mechanisms of stress-induced agarwood formation. MicroRNAs (miRNAs), an important gene expression regulator involved in various plant stress responses and metabolic processes, might have function in agarwood formation. Our previous work identified miRNAs from A. sinensis by small RNA sequencing and EST library, and indicated that the miRNAs might participate in a series steps of the whole progress from the wound response, signal transduction to the biosynthesis of secondary metabolism. Depend on these results, this study will thoroughly identify miRNAs and their targets using our small RNA library and the genome sequences obtained recently. Then select the key miRNAs in wound induced agarwood formation by their expression pattern and target prediction. The relationship of these key miRNAs and targets will further be verified by 5' RACE and transient expression. And the character, function and mechanism of these targets will also be studied. This study will help to develop the production method, especially in the future industrial production. The result will also help for studies of stress-induced production of useful secondary metabolites in many plants.

名贵芳香类药材沉香的野生资源已近枯竭，人工生产迫在眉睫。健康的沉香属植物只有受到外界伤害时，才在伤口周围积累倍半萜、色酮等，形成沉香药材。miRNA 是一种重要的转录后调控因子，参与多种生物、非生物胁迫应答反应。前期我们采用高通量测序技术构建了白木香的EST 库及小RNA 库，筛选鉴定出一批伤害诱导的关键miRNA，并发现miRNA可能参与了白木香从伤害应答到信号转导到次生代谢物合成的全过程，但由于基因组信息的匮乏，制约了关键miRNA功能的阐释。本研究将利用业已完成的白木香全基因组测序工作，通过生物信息学手段全面预测白木香miRNA及其靶基因，结合前期小RNA表达谱研究，补充筛选伤害诱导关键miRNA；利用多种实验手段验证其靶基因，揭示其作用靶点，并进一步深入挖掘miRNA及靶基因的功能和作用机制。本研究将为高效结香技术建立奠定基础，为诱导型药用次生代谢产物形成的分子机制制阐述提供参考。

名贵芳香类药材沉香的野生资源已近枯竭，人工生产迫在眉睫。健康的沉香属植物只有受到外界伤害时，才在伤口周围积累倍半萜、色酮等，形成沉香药材。miRNA 是一种重要的转录后调控因子，参与多种生物、非生物胁迫应答反应。本研究利用白木香全基因组测序工作，通过生物信息学手段全面预测白木香miRNA及其靶基因，结合前期小RNA表达谱研究，补充筛选伤害诱导关键miRNA。在靶基因中发现了萜类合成通路中的3个TPS基因和2个DXS基因。通过转录因子与TPS表达相关性分析筛选关键转录因子家族，进一步找出9个靶基因属于转录因子WRKY家族，15个基因属于MYB家族，2个基因属于HSF家族，3个基因属于AP2/ERF家族。后续将以这些靶基因的miRNA为主要研究对象。研究miRNA对这些转录因子的调控作用以及这些转录因子对沉香形成的调控作用。经过严格筛选，共预测得到38个可能参与伤害诱导沉香形成信号通路的关键miRNA-靶基因对。29个可能参与伤害诱导沉香形成的转录因子调控的关键miRNA-靶基因对。为后续深入研究各个miRNA-靶基因对的调控机制提供了可靠的研究标的。进一步通过5’RACE明确了miRNA-靶基因对的剪切关系，并通过烟草叶片瞬时转化明确了miRNA对靶基因表达的抑制作用。此外，研究发现白木香聚酮合酶PKS家族中KS3参与色酮合成，明确了白木香色酮合成通路为苯丙烷通路，填补了沉香色酮合成研究的空白，在靶基因中发现该通路中的1个PKS基因。木蠹蛾蛀食虫漏形成机理研究发现：在虫蛀的早期与晚期特有的植物激素信号通路与信号因子，发现了虫漏香特有香味的可能的主要相关基因，亦为miRNA靶基因筛选进一步提供参考。本研究为高效结香技术建立奠定了基础，为诱导型药用次生代谢产物形成的分子机制阐述提供重要参考。