江南卷柏miRNA和circRNA的识别及其在萜类合成中的调控网络解析

Terpene synthase(TPS) is the key enzyme for the biosynthesis of terpenoid. Except for a seed plant type of terpene synthase, we also discovered a microbial TPS-like type of terpene synthase only in nonseed plant. microRNA (miRNA) is a novel regulator of plant secondary metabolism, whose regulation of the target can be inhibited by circular RNA (circRNA). In seed plant, miRNA can target TPS gene and transcriptional factor (TFtps) gene which controls the transcription of the TPS gene in order to regulate the biosynthesis of terpenoid. However, now it is unclear that what is the roles of miRNA in nonseed plant..Selaginella moellendorffii as an object, firstly miRNA, circRNA and TFtps were identified based on bioinformatics methods and experimental validation by integrating data from known transcriptome and new data from Selaginella moellendorffii. Then the miRNA-circRNA-TPS/TFtps gene network was constructed and analyzed to screen candidate miRNA and circRNA involved in terpenoid biosynthesis. Finally, RT-PCR, GC-MS, RNAi and CRISPR were used to verify the regulatory role of miRNA and circRNA in terpenoid synthesis.The results of the study laid the foundation for the further study on the mechanism of terpene regulation.

萜类合成酶(TPS)是萜类合成的关键酶。除植物型TPS外，我们还发现只存在于非种子植物中的微生物型TPS。微小RNA（miRNA）是植物次生代谢的新型调控分子，其对靶标的调控作用可受环状RNA（circRNA）的抑制。种子植物中miRNA以靶向控制植物TPS基因转录的转录因子（TFtps）基因和TPS基因来调控萜类的合成，但对非种子植物，其调控作用尚不清楚。.本项目以非种子植物江南卷柏为对象，首先整合现有的和自建的转录组数据，进行miRNA、circRNA和TFtps基因的生物信息学分析和实验鉴定。然后构建和分析miRNA-circRNA-TPS/TFtps基因网络，筛选参与萜类合成调控的候选miRNA和circRNA。最后利用RT-PCR、GC-MS、RNAi和CRISPR等方法，验证代表性miRNA和circRNA在萜类合成中的调控作用。项目的结果为今后研究萜类调控的分子机制奠定基础。

萜类合成酶(TPS)是萜类合成的关键酶。除植物型TPS外，我们还发现只存在于非种子植物中的微生物型TPS（MTPSL）。微小RNA（miRNA）和环状RNA（circRNA）是植物次生代谢的新型调控分子，目前尚不清楚这些非编码RNA在非种子植物TPS中的调控作用。本项目我们首先在江南卷柏中克隆并鉴定了8个MTPSL基因，并对它们的生化和生物学功能进行了研究；然后建立了植物circRNA的识别工具Pcirc以及可翻译circRNA的识别工具CircCode；接着建立在卷柏转录组数据的基础上，识别出236个circRNA；最后通过circRNA-miRNA-mRNA网络分析与实验相结合的方法，鉴定出两个与萜类合成酶相关的circRNA。项目的结果为今后深入研究circRNA和miRNA在萜类调控中的分子机制奠定了坚实的基础。