摘要
闹羊花是我国传统药材,来源于杜鹃花科植物羊踯躅Rhododendron molle,其主要活性成分为二萜类化合物。二萜合酶是二萜类成分生物合成途径的关键酶,该研究通过分析羊踯躅转录组数据,发现2条ent-柯巴基焦磷酸合酶(ent-copalyl diphosphate synthase,CPS)基因RmTPS33和RmTPS64以及1条ent-贝壳杉烯合酶(ent-kaurene synthase,KS)基因RmTPS65,通过对这3条基因进行全长克隆、生物信息学分析及功能验证结果发现:①RmTPS33、RmTPS64、RmTPS65开放阅读框分别为2280、2476、2370 bp,分别编码759、819、789个氨基酸;②系统进化分析及多重序列比对显示:RmTPS33及RmTPS64均有CPS基因家族保守的LHS、PNV、DxDD基序,归类于TPS-c亚家族;RmTPS65包含Ⅰ类二萜合酶α结构域中保守的DDxxD和NSE/DTE基序,归类于TPS-e/f亚家族;③功能验证结果表明,RmTPS33及RmTPS64可催化GGPP质子化并环化形成ent-CPP,且RmTPS33催化活性高于RmTPS64;RmTPS65可催化ent-CPP二磷酸基团脱离,并形成单一产物16α-hydroxy-ent-kau-rane。此外,由于RmTPS33和RmTPS64存在一定的活性差异,对其进行蛋白改造研究,结果揭示了其活性差异可能源于功能结构域中的差异氨基酸。该研究获得3条二萜合酶基因RmTPS33、RmTPS64、RmTPS65,丰富了现有二萜合酶基因库,也为羊踯躅中活性成分生物合成途径相关基因研究奠定基础。
Rhododendri Mollis Flos is a Chinese herbal medicine derived from Rhododendron molle(Ericaceae),with diterpenoids being its primary bioactive constituents.Diterpene synthases are key enzymes in the biosynthetic pathway of diterpenoids.Transcriptome analysis of R.molle identified two ent-copalyl diphosphate synthase(CPS)genes(RmTPS33 and RmTPS64)and one ent-kaurene synthase(KS)gene(RmTPS65).Full-length cloning,bioinformatics analysis,and functional characterization of these three genes yielded the following results.①The open reading frames of RmTPS33,RmTPS64,and RmTPS65 were 2280,2476,and 2370 bp,encoding 759,819,and 789 amino acid residues,respectively.②Multiple sequence alignment and phylogenetic analysis demonstrated that both RmTPS33 and RmTPS64 contained conserved CPS family motifs(LHS,PNV,and DxDD)and belonged to the TPS-c subfamily.RmTPS65 possessed conservedα-domain motifs(DDxxD and NSE/DTE)of classⅠditerpene synthases and was classified into the TPS-e/f subfamily.③Functional assays confirmed that both RmTPS33 and RmTPS64 catalyzed the protonation-induced cyclization of GGPP to form ent-CPP,with RmTPS33 exhibiting higher catalytic activity than RmTPS64.RmTPS65 catalyzed the diphosphate elimination of ent-CPP to produce a single product,16α-hydroxy-ent-kaurane.Furthermore,protein engineering of RmTPS33 and RmTPS64 suggested that their activity divergence stemmed from variant amino acids within functional domains.This study successfully cloned three diterpene synthases gene(RmTPS33,RmTPS64,and RmTPS65),expanding the current diterpene synthase gene repository and establishing a foundation for investigating biosynthetic pathway genes of active compounds in R.molle.
作者
刘政
王鑫梦
王荣凤
高伟
王家典
胡雅婷
黄璐琦
LIU Zheng;WANG Xin-meng;WANG Rong-feng;GAO Wei;WANG Jia-dian;HU Ya-ting;HUANG Lu-qi(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China;Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China;National 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,Beijing 100700,China)
出处
《中国中药杂志》
北大核心
2025年第22期6328-6337,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金青年科学基金项目(82304653)
国家重点研发计划项目(2020YFA0908000)
中央本级重大增减支项目(2060302-1806-03)。
关键词
羊踯躅
二萜合酶
生物合成
功能鉴定
Rhododendron molle
diterpene synthases
biosynthesis
functional identification
