基于转录组测序的毛萼香茶菜Ⅱ型二萜合酶的挖掘与功能鉴定

Transcriptome-based mining and functional characterization of class Ⅱ diterpene synthases from Isodon eriocalyx

目的从毛萼香茶菜中挖掘Ⅱ型二萜合酶并进行功能鉴定。方法利用转录组测序和生物信息学分析技术从毛萼香茶菜的根、茎、叶、顶芽4种组织中挖掘Ⅱ型二萜合酶候选基因,通过RT-PCR方法获取候选基因cDNAs,运用无缝克隆技术构建携带候选基因的表达载体,并基于大肠杆菌异源表达体系及GC-MS测定分析技术进行二萜合酶基因的催化功能鉴定。结果分别从毛萼香茶菜的根、茎、叶、顶芽组织中提取总RNA,经过转录组测序和生物信息学分析获得4个Ⅱ型二萜合酶候选基因IeTPS1、IeTPS2、IeTPS3和IeTPS4。利用大肠杆菌异源表达体系对其中的IeTPS1和IeTPS4基因进行功能验证,发现IeTPS1能够催化底物香叶基香叶基焦磷酸(GGPP)生成对映-柯巴基焦磷酸(ent-CPP),命名为IeCPS4;IeTPS4能够催化底物GGPP生成柯巴基焦磷酸(normal-CPP),命名为IeCPS5。结论从毛萼香茶菜中筛选并鉴定了两个新的Ⅱ型二萜合酶基因IeCPS4和IeCPS5,其中IeCPS5是首个从毛萼香茶菜中发现的专一合成normal-CPP的二萜合酶基因,这不仅丰富了毛萼香茶菜的二萜合酶种类,而且为毛萼香茶菜二萜化合物生物合成途径的解析奠定了坚实基础。

Objective To identify and characterize new class Ⅱ diterpene synthases from Isodon eriocalyx,with the aim of elucidating the biosynthesis of its diterpenoids.Methods Transcriptome sequencing and bioinformatics analysis were employed to identify candidate genes encoding class Ⅱ diterpene synthases from various tissues of Isodon eriocalyx,including roots,stems,leaves,and buds.Subsequently,cDNAs of the candidate genes were obtained through RT-PCR,and expression vectors carrying these genes were constructed using seamless cloning technology.The biochemical functions of the candidate genes were then characterized using a well-established metabolic engineering system in Escherichia coli,with all compounds identified through GC-MS analysis.Results Total RNAs were extracted from the roots,stems,leaves,and buds of Isodon eriocalyx to generate transcriptome data.Four candidate genes encoding class Ⅱ diterpene synthases(IeTPS1,IeTPS2,IeTPS3,and IeTPS4)were identified through transcriptome-based bioinformatics analysis.Subsequent biochemical characterization in an engineered Escherichia coli system revealed that IeTPS1 converts GGPP into ent-CPP,while IeTPS4 produces normal-CPP from GGPP.As a result,these two TPSs were designated as IeCPS4 and IeCPS5,respectively.Conclusion Two novel class Ⅱ diterpene synthases,IeCPS4 and IeCPS5,have been discovered in Isodon eriocalyx,with IeCPS5 being the first CPS reported from this plant species.This study not only expands the repertoire of diterpene synthases but also provides a foundation for further exploration of key enzymes involved in the biosynthesis pathway of diterpenoids in Isodon eriocalyx.