Tan Hong-Hu, Xia Meng, Su Ping, Huang Lu-Qi
School of Pharmacy, Nanjing University of Chinese Medicine Nanjing 210023, China 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 Beijing 100700, China.
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 Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2024 May;49(9):2410-2421. doi: 10.19540/j.cnki.cjcmm.20240214.102.
Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.
以牻牛儿基牻牛儿基焦磷酸（GGPP）为底物，内根-贝壳杉烯二磷酸合酶（CPS）和内根-贝壳杉烯合酶（KS）的顺序催化是植物启动赤霉素生物合成的关键步骤。本研究挖掘了狼毒大戟的转录组数据，并克隆了两个参与赤霉素途径的关键二萜合酶基因SchCPS和SchKS。这两个基因的完整开放阅读框分别为2595 bp和1701 bp，编码两种亲水性蛋白，分别由864和566个氨基酸残基组成，相对分子质量分别为97.9 kDa和64.6 kDa，理论等电点分别为5.61和6.12。序列比较和系统发育树显示，SchCPS包含CPS家族中保守的LHS、PNV和DxDD基序，属于TPS-c亚家族，而SchKS包含KS家族中保守的DDxxD、NSE/DTE和PIx基序，属于TPS-e亚家族。功能验证表明，SchCPS催化GGPP质子化并环化生成内根-贝壳杉烯焦磷酸（ent-CPP），而SchKS作用于ent-CPP去磷酸化并重新环化生成内根-贝壳杉烯。本研究首次克隆并功能验证了SchCPS和SchKS的全长序列，不仅丰富了现有的CPS和KS基因库，也为狼毒大戟中更多参与活性成分合成的基因克隆及生物合成途径分析奠定了基础。
