Transcriptome-based mining and functional characterization of class Ⅱ diterpene synthases from Isodon eriocalyx
Objective To identify and characterize new classⅡditerpene 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 classⅡditerpene 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 classⅡditerpene 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 classⅡditerpene 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.
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