比较蛋白质组学揭示茉莉酸甲酯诱导的雷公藤甲素生物合成  

Comparative Proteomics Reveals the Biosynthesis of Methyl Jasmonate-induced Triptolide

作　　者：张逸风[1] 赵瑜君 苏平[1] 吴晓毅[2] 夏梦 高伟[2] 黄璐琦[1] ZHANG Yi-feng;ZHAO Yu-jun;SU Ping;WU Xiao-yi;XIA Meng;GAO Wei;HUANG Lu-qi(State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijng 100700,China;School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China)

机构地区：[1]中国中医科学院中药资源中心道地药材品质保障与资源持续利用全国重点实验室,北京100700 [2]首都医科大学中医药学院,北京100069

出　　处：《中国现代中药》2024年第2期316-327,共12页Modern Chinese Medicine

基　　金：国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-D-202005);国家自然科学基金项目(82204547);国家重点研发计划项目(2020YFA0908000);中国中医科学院科技创新工程项目(CI2023E002)。

摘　　要：目的:探索雷公藤甲素生物合成途径,以提高其产量或用于合成生物学异源生产。方法:使用无标记(lable-free)比较蛋白质组学对茉莉酸甲酯(MeJA)诱导的雷公藤悬浮细胞进行测序,通过京都基因与基因组百科全书(KEGG)、基因本体(GO)分析等方法,结合基因转录表达分析和基因体内功能研究,逐步筛选参与雷公藤甲素生物合成的功能基因。结果:MeJA诱导不同时间的样品组鉴定得到376个差异蛋白质,包括8个细胞色素P450酶(CYP450)、3个转录因子(TF)和2-氧戊二酸依赖的双加氧酶(2ODD)。其中CYP71BE89、CYP82AQ2和Tw2ODD基因在MeJA诱导及植物不同组织部位中,均与已知参与雷公藤甲素母核环化的二萜合酶TwTPS7(v2)和TwTPS27(v2)基因具有相似的表达模式,表明其可能与雷公藤甲素生物合成相关。进一步利用植物细胞体内功能研究,验证了CYP71BE89、CYP82AQ2和Tw2ODD基因干扰使雷公藤甲素积累分别降低了24.1%、41.4%和71.4%,表明了3个基因与雷公藤甲素生物合成直接相关。结论:利用比较蛋白质组学技术揭示了几种与雷公藤甲素生物合成相关的蛋白,进一步表征了雷公藤甲素生物合成与外源激素刺激之间的关系,并为发掘中药活性成分生物合成途径提供了一种有效方法。Objective:To explore the biosynthetic pathway of triptolide to increase its yield or for heterologous production in synthetic biology.Methods:Suspended cells treated by methyl jasmonate(MeJA)-induced Tripterygium wilfordii were sequenced by label-free comparative proteomics.The functional genes involved in the biosynthesis of triptolide were screened step by step through Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)analysis,combined with gene transcriptional expression analysis and in vivo gene function studies.Results:MeJA-induced samples from different time points were identified to have 376 differentially expressed proteins,including eight cytochrome P450s(CYP450s),three transcription factors and a 2-oxoglutarate dependent dioxygenase(2ODD).Among them,CYP71BE89,CYP82AQ2 and Tw2ODD had similar expression patterns to the known diterpene synthases TwTPS7(v2)and TwTPS27(v2)involved in the cyclization of triptolide precursors,both under MeJA induction and in different plant tissues,suggesting that they may be related to triptolide biosynthesis.Further in vivo RNA-interference(RNAi)functional studies using plant cells verified that interference with the genes CYP71BE89,CYP82AQ2,and Tw2ODD reduced triptolide accumulation by 24.1%,41.4%,and 71.4%,respectively,indicating a direct correlation between the three genes and triptolide biosynthesis.Conclusion:The application of comparative proteomics technology reveals several candidate proteins associated with triptolide biosynthesis.Further characterization of the relationship between the biosynthesis of triptolide and exogenous hormone stimulation may provide an effective method for uncovering the biosynthetic pathways of active ingredients in traditional Chinese medicine.

关 键 词：雷公藤 比较蛋白质组学 雷公藤甲素 细胞色素P450酶 2-氧戊二酸依赖的双加氧酶 

分 类 号：R284[医药卫生—中药学] R285[医药卫生—中医学]