灵芝细胞中磷脂酸互作蛋白鉴定  

Identification of phosphatidic acid interacting proteins in Ganoderma lingzhi

作　　者：刘勇男 尹媛媛 郝宏伟 王睿[1,2] 何哲 田人元 刘高强[1,2] Yongnan Liu;Yuanyuan Yin;Hongwei Hao;Rui Wang;Zhe He;Renyuan Tian;Gaoqiang Liu(Hunan Provincial Key Laboratory of Forestry Biotechnology,Central South University of Forestry&Technology,Changsha 410004,Hunan,China;International Cooperation Base of Science and Technology Innovation on Forest Resource Biotechnology of Hunan Province,Central South University of Forestry&Technology,Changsha 410004,Hunan,China)

机构地区：[1]中南林业科技大学林业生物技术湖南省重点实验室,湖南长沙410004 [2]中南林业科技大学森林资源生物技术湖南省国际科技创新合作基地,湖南长沙410004

出　　处：《生物工程学报》2021年第9期3293-3299,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.31900027,31772374,32071673);中国博士后科学基金(No.2020M682601);湖南省自然科学基金(No.2020JJ5972);湖南省科技创新计划(No.2020RC2059);湖南省教育厅科学研究项目(No.18B167);中南林业科技大学研究生科技创新基金(No.CX20202014)资助。

摘　　要：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,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,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。Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids(TAs) are the key bioactive medicinal components of G. lingzhi. Our previous studies have shown that phospholipid acid(PA) produced by phospholipase D(PLD) plays a regulatory role in TA synthesis. In order to further elucidate the molecular mechanism how PA regulates TA synthesis in G. lingzhi, PA beads enrichment combined with LC-MS/MS technology was used to identify PA interacting proteins in G. lingzhi. A total of 19 PA interacting proteins were identified, including cytochrome P450 monooxygenase(GL22084), specific protein kinase MAPK(GL23765), catalase and cell surface hydrophobicity-associated protein. GST tagged GL22084 and GL23765 proteins were obtained through gene cloning, heterologous expression, and purification. The interactions between GL22084/GL23765 and PA were verified by GST pull down assay. The identification of PA interacting proteins provides a basis for further understanding the molecular mechanism how PLD-mediated PA signaling molecules regulates the TA synthesis in G. lingzhi. Moreover, the PA interacting proteins identified in this study can also provide clues for the research of PLD/PA signaling pathway in other species.

关 键 词：磷脂酸互作蛋白 灵芝 三萜合成 蛋白鉴定 谷胱甘肽-S-转移酶蛋白下拉实验 

分 类 号：S567.31[农业科学—中草药栽培]