瑞香狼毒贝壳杉烷型二萜类合成酶功能表征

Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme

ent-柯巴基焦磷酸合酶(ent-copalyl diphosphate synthase,CPS)与ent-贝壳杉烯合酶(ent-kaurene synthase,KS)的连续催化是植物以香叶基香叶基焦磷酸(geranylgeranyl pyrophosphate,GGPP)为底物开启赤霉素生物合成路径的关键步骤。该研究通过分析与挖掘瑞香狼毒的转录组数据,成功克隆得到赤霉素途径的2个关键二萜合酶基因SchCPS和SchKS。二者完整开放阅读框分别为2595、1701 bp,编码864、566个氨基酸残基,生物信息学分析预测其相对分子质量分别为97.9、64.6 kDa,理论等电点为5.61、6.12,均为亲水性蛋白。序列比对及系统发育树显示,SchCPS蛋白含有CPS基因家族保守的LHS、PNV及DxDD基序,归类于TPS-c亚家族;SchKS蛋白含有KS基因家族保守的DDxxD、NSE/DTE和PIx基序,归类于TPS-e亚家族。功能验证结果表明,SchCPS可催化GGPP质子化并环化成ent-CPP,SchKS则作用于ent-CPP去磷酸化并再次环化生成贝壳杉烯。该文首次从瑞香狼毒中克隆获得SchCPS与SchKS全长基因,并完成功能验证,不仅丰富了现有的CPS和KS基因库,也为瑞香狼毒中更多活性成分基因的克隆与生物合成途径解析奠定了基础。

Sequential catalysis by ent-copalyl diphosphate(CPS)and ent-kaurene synthase(KS)is a critical step for plants to ini-tiate 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 2595 bp and 1701 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 con-served in the CPS family and was categorized in the TPS-c subfamily,while SchKS contained DDxxD,NSE/DTE and PIx motifs con-served in the KS family and was categorized in the TPS-e subfamily.Functional validation showed that SchCPS catalyzed the protona-tion 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.