灵芝三萜MVA途径关键酶基因的转录因子的筛选及功能验证

Triterpenoids are the major medicinal effective ingredients in Ganoderma spp. and influence the quality of it. Although there have many studies focus on the optimization of cultivation and fermentation conditions to improve the triterpenes yield of Ganoderma spp., but there have few reports about the mechanism of transcriptional regulation of triterpenoids biosynthesis pathway. At present, whole genome sequencing of G. lucidum has been completed, providing mass data for the study of the biology of Ganoderma spp.. Therefore, we will study five key enzyme genes including Hmgr、Fps、Sqs, Se and Las in MVA pathway of G. lucidum, cloning the promoter sequences of the 5 genes and analysis their cis elements; using promoter sequences and their 5 ' terminal deletion fragments as baits to construct yeast one hybrid library and separating the transcription factors which interact with cis elements of key enzyme genes; using heterologous expression and MJ induction experiments, qPCR and HPLC-MS technology to screen out the transcription factors which participate in triterpenoids synthesis and regulation network of G. lucidum. In this project, we study the transcription factors of 5 key enzyme genes to provide the basis for the study of expression and regulation mechanism of related genes and the synthesis pathway. In addition, this study is important to promote the triterpenoids industrialization of Ganoderma spp..

三萜类成分是灵芝的主要药效成分之一，影响着灵芝品质的优劣。虽然已有许多研究集中在优化栽培和发酵条件上以提高灵芝三萜产量，但关于灵芝三萜生物合成途径的转录调控机制还鲜有报道。目前，赤芝全基因组测序工作已经完成，为灵芝生物学的研究提供了海量的数据。因此，本项目在前期测序工作的基础上以灵芝三萜生物合成MVA途径中的关键酶基因Hmgr、Fps、Sqs、Se和Las为目标，克隆这5个关键酶基因相应的启动子序列，分析其中可能存在的顺式元件；用启动子全长及其5′端缺失片段为诱饵，构建酵母单杂交文库，分离与关键酶基因顺式元件作用的转录因子；用酵母异源表达及MJ诱导实验，通过定量PCR及HPLC-MS化学检测技术，筛选出参与灵芝三萜合成与调控网络的转录因子。本项目通过对这5个关键酶基因转录因子的研究，为相关基因及整个合成途径的表达调控提供研究基础，对推进灵芝三萜的产业化生产具有重要意义。

灵芝三萜的合成与调控机制研究尚处于初步阶段，而对灵芝三萜关键酶基因转录调控的研究是阐明其生物合成分子机制的重要内容。现有研究表明转录因子对次生代谢调控起着重要作用，能够调节特定次生代谢产物的合成。本项目利用酵母单杂交技术，筛选了LS和FPS基因上游的候选调控因子；利用MJ诱导和基因过表达实验，筛选了参与灵芝三萜合成的转录因子；利用全基因组重测序方法，筛选了可能参与到灵芝三萜合成及菌丝生长过程中的转录因子。结果表明TFIIB为调控LS的候选转录因子，Zn2Cys6、SNF2和MHR为调控FPS基因的候选转录因子；MJ诱导实验筛选出104个可能参与到灵芝三萜合成过程中，其中TFIIB使得灵芝三萜的含量提到2.87倍；全基因组重测序数据分析得到GlSwi6和GlPacC与灵芝菌丝生长及灵芝三萜含量相关。本项目的实施，为灵芝三萜合成的调控机制研究提供了重要的理论数据。