磷脂酶D介导的磷脂酸调节灵芝三萜合成的分子机理

Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids (TAs) are well recognized as the major bioactive constituents and important medicinal index of G. lingzhi. Our previous research has shown that Phospholipase D (PLD) mediated phosphatidic acid (PA) regulates TAs biosynthesis in G. lingzhi. However, the molecular mechanism of PA regulating TAs biosynthesis has not been elucidated. This project intends to further carry out the following work: 1. PA protein interactors are enriched by PA-Beads, and then the enriched proteins are identified by mass spectrometry. Pulldown technique is used to verify the specific PA protein interactors identified by mass spectrometry. 2. qRT-PCR and Western-blot are used to screen proteins involved in the regulation of TAs biosynthesis from PA protein interactors. The regulation of screened PA protein interactors on TAs biosynthesis is analyzed by gene silencing, knockout and overexpression techniques. This project aims to clarify the intracellular PA protein interactors of G. lingzhi and elucidate the regulatory mechanism of PA protein interactors on TA biosynthesis. The results of this study will further elucidate the molecular mechanism of PLD mediated PA regulating TAs biosynthesis in G. lingzhi, laying a theoretical foundation for efficient directional production of TAs. At the same time, the PA protein interactors identified in this study will also provide a reference for the study of PLD/PA signaling pathways in other species.

灵芝是名贵药用真菌，三萜是灵芝的关键药效成分。申请者前期研究发现，磷脂酶D（Phospholipase D，PLD）产生的磷脂酸（Phosphatidic acid，PA）可调控三萜合成，但PA调控三萜合成的分子机理尚未阐明。本项目开展以下工作：1、利用PA-Beads富集与PA互作的蛋白，采用质谱对富集到的蛋白进行鉴定，运用Pulldown对鉴定到的蛋白进行验证。2、通过qRT-PCR和Western-blot筛选PA互作蛋白中调控三萜合成的相关蛋白，运用基因沉默、敲除和过表达技术研究筛选出的蛋白对三萜合成的调控作用。以期揭示灵芝胞内PA互作蛋白，阐明PA互作蛋白对三萜合成的调控机制。研究结果将解析PLD介导的PA调控灵芝三萜合成的分子机理，为三萜的高效定向生产奠定理论基础，同时，本研究鉴定到的PA互作蛋白，也为其他物种的PLD/PA信号通路相关研究提供借鉴。

灵芝是名贵药用真菌，三萜是灵芝的关键药效成分。前期研究发现，磷脂酶D（Phospholipase D，PLD）产生的磷脂酸（Phosphatidic acid，PA）可调控三萜合成，为进一步阐明PA调控灵芝三萜合成的分子机制，本研究采用PA-beads富集结合LC-MS/MS技术，鉴定灵芝细胞中PA互作蛋白，结果共鉴定到了19个PA互作蛋白，主要包括细胞色素P450单加氧酶（GL22084）、特异性蛋白激酶MAPK（GL23765）、过氧化氢酶和细胞表面疏水性蛋白等。通过基因克隆、原核表达载体构建、蛋白诱导表达和分离纯化，获得了融合GST标签的GL22084和GL23765蛋白，采用GST-pull down实验，验证了灵芝GL22084和GL23765蛋白与PA互作。研究结果揭示了灵芝细胞中PA互作蛋白，为后续解析PLD介导的PA信号分子调控灵芝三萜合成的分子机理奠定了基础；同时，本研究鉴定到的PA互作蛋白，也为其他物种的PLD/PA信号通路相关研究提供借鉴。. 进一步从转录因子固醇调节元件结合蛋白SREBP（sterol regulatory element-binding protein）着手，采用DAP-seq技术，分析SREBP互作DNA，共获得2271个SREBP结合位点，SREBP最偏好的结合序列为5′-GRVGRVGRVGR-3′。进一步分析启动子结合位点附近的编码基因，共获得了1078个SREBP靶基因，功能注释显示，SREBP靶基因中有5个(g4989, g3847, g7601, g3941和g1941)编码萜类合成相关蛋白，6个基因(g4280, g4309, g1208, g5041, g6347和g805)编码脂质代谢相关蛋白。运用基因过表达技术构建SREBP过表达菌株，代谢组分析发现，与野生型菌株相比，SREBP过表达菌株中三萜合成中间代谢产物3,5-二羟基-3-甲基戊酸和羊毛甾醇以及17种三萜类化合物上调，并且多种磷脂、甘油脂以及鞘脂含量提高。本研究全基因组范围内揭示了SREBP调控灵芝三萜和脂质代谢的分子机制。