灵芝长非编码RNA对三萜类化合物合成的调控作用和机理研究

Ganoderma lucidum is one of the most famous medicinal species. The lack of understanding of the biosynthesis of its active components triterpenoids has become a bottle neck in the research work aiming to improve the production of triterpenoid. Long non-coding RNA (lncRNA) is a class of RNA that has been recently found to play important roles in gene expression regulation. Based on literatures and our preliminary results, we hypothesize that lncRNAs regulate the biosynthesis of triterpenoid production through the regulation of key enzymes in the MVA pathway. The current study is designed to test this hypothesis. The proposed research can be divided into four parts: (1) systematically identifying lncRNA in Ganoderma lucidum using strand-specific RNA-Seq and Pacbio technologies, in combination with bioinformatic analyses; (2) identifying lncRNAs related to triterpenoid biosynthesis through data mining of a network constructed with the co-localization, co-expression of lncRNA and genes involved in triterpenoid biosynthesis, and the co-regulation of lncRNA expression and triterpenoid production; (3) validating the lncRNA's effects by over-production of lncRNA in Mycelia; (4) determining if the regulation is through an enhancer-like mechanism using a reporter system. The success of this project will not only reveal novel mechanisms involved in the biosynthesis of triterpenoids in Ganoderma lucidum, but also open up new avenues for research on the biosynthesis of triterpenoid in other medicinal species. In addition, it will lay the foundation for the application of genetic engineering and synthetic biology technologies for the improved production of triterpenoid in Ganoderma lucidum.

灵芝是最著名的药用物种之一，由于对灵芝三萜的合成调控机理尚不明确，导致目前有关提高灵芝三萜含量的研究存在盲目性，缺乏理论依据。长非编码RNA（lncRNA）是近来发现在基因表达调控中起重要作用的一类RNA。本项目建立假说"lncRNA通过调控相邻MVA关键酶基因的表达，调控三萜类化合物的合成"，旨在阐明lncRNA在灵芝三萜合成中的作用和机理。研究内容包括：（1）系统发现灵芝lncRNA；（2）通过构建lncRNA与三萜合成关键酶基因共定位、共表达，lncRNA表达和三萜化合物含量共变化的网络，筛选与三萜合成相关的lncRNA；（3）通过同源过量表达lncRNA，验证其调控作用；（4）利用同源报告基因表达系统，阐明其作用机理。本项目将揭示新的灵芝三萜合成调控机理，为探索三萜类化合物在其它物种中的合成与调控开辟新的道路，研究成果也将为应用遗传工程提高三萜产量奠定理论基础和提供技术保障。

因为本项目只获得一年资助，研究计划做了相应的修改.到目前为止，本研究已经完成了如下内容：1. 建立了lincRNA系统发现的数据分析流程；2..应用该流程，对灵芝RNA-seq数据进行了分析，共发现了402个lincRNA；3..建立了MRA方法，该方法是对3‘RACE方法和qPCR方法的改进，用来决定转录本的方向性并定量；4. 通过对lincRNA的相邻蛋白质编码基因（adjacent-protein-coding genes，apcGenes）的分析，发现了46个lincRNA的相邻的编码蛋白的基因可能和三萜的合成相关。这46个lincRNA基因中，37个lincRNA为单向转录，9个为双向转录。这37个单向转录的lincRNA中，16个与其相邻的基因表达高度正相关，而5个和相邻的蛋白质编码基因呈现高度负相关。5. 单个lincRNA基因的验证工作仍在进行之中。总之，本研究的结果支持lincRNA在灵芝酸合成中的潜在的调控作用。下一步的工作应该首先发现与灵芝酸合成相关的蛋白质编码基因，再来研究其调控。从成果角度，已发表与本项目直接相关的SCI论文两篇，本项目同时资助了另外3篇SCI论文的发表。