In this study,two geraniol 10-hydroxylase (G10H) genes,namely GrG10H1 and GrG10H2,were cloned from Gentiana rigescens through RT-PCR based on transcriptome data analysis.In silico bioinformatics analysis was performed towards these two genes whose open reading frame were 1 548 bp and 1 482 bp in length,respectively.Phylogenetic analysis revealed that the two genes are phylogenetically closed to the CYP76B subfamily of the cytochrome P450 enzymes.Homology modeling was employed to construct the three-dimensional structures of GrG10H1 and GrG10H2.Combined with molecular docking,the key residues in the substrate binding pocket of GrG10H1 and GrG10H2 protein were analyzed.Quantitative PCR demonstrated that the expression level of the GrG10H1 gene was highest in leaves,followed by stems and lowest in roots.However,the expression level of the GrG10H2 gene was high in roots and leaves but almost absent in stems.This study also established a callus cell culture system for G. rigescens to achieve stable production of gentiopicroside,which provides a foundation for further investigation into gentiopicroside biosynthesis mechanism.