天然产物研究与开发 ›› 2024, Vol. 36 ›› Issue (9): 1542-1553.doi: 10.16333/j.1001-6880.2024.9.010

• 研究简报 • 上一篇    下一篇

滇龙胆草中香叶醇10-羟化酶基因的克隆、蛋白结构及表达分析

王迎夏1,2，赵赛静1,2，田维圣1,2，崔晓雪1,2，黄文倩1,2，屠鹏飞1，史社坡1*，刘   晓1*


  

  1. 1北京中医药大学中药学院中药现代研究中心；2北京中医药大学中药学院，北京 100029
  • 出版日期:2024-09-25 发布日期:2024-09-25
  • 基金资助:
    国家自然科学基金（82173922，81402809）；中央高校基本科研业务费专项（2023-JYB-JBQN-054）；北京市自然科学基金（7192112）

Cloning,protein structure,and expression analysis of geraniol 10-hydroxylase gene from Gentiana rigescens

WANG Ying-xia1,2,ZHAO Sai-jing1,2,TIAN Wei-sheng1,2,CUI Xiao-xue1,2,HUANG Wen-qian1,2,TU Peng-fei1,SHI She-po1*,LIU Xiao1*   

  1. 1Modern Research Center for Traditional Chinese Medicine,Beijing Research Institute of Chinese Medicine,Beijing University of Chinese Medicine;2School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100029,China
  • Online:2024-09-25 Published:2024-09-25

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摘要：

本研究基于滇龙胆草转录组数据分析，使用RT-PCR技术克隆获得两条香叶醇10-羟化酶基因，命名为GrG10H1和GrG10H2。使用在线软件对其进行了生物信息学分析，两条基因全长分别为1 548 bp、1 482 bp。系统进化分析显示该基因与细胞色素P450家族中CYP76B亚家族亲缘关系最近。通过对该蛋白进行三维结构预测、同源模建和分子对接，分析了GrG10H蛋白活性口袋中可能参与催化功能发挥的关键氨基酸残基。荧光定量PCR表明GrG10H1基因在叶中表达水平最高，茎次之，根最低。而GrG10H2基因在根和叶中表达水平较高，在茎中几乎不表达。本研究还建立了能够生产龙胆苦苷的愈伤组织细胞培养体系，为进一步解析龙胆苦苷生物合成机制奠定了基础。

关键词: 滇龙胆草, 龙胆苦苷, 生物信息学分析, 香叶醇10-羟化酶, 基因克隆

Abstract:

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.

Key words: Gentiana rigescens, gentiopicroside, bioinformatics analysis, geraniol 10-hydroxylase, gene cloning

中图分类号:  R285

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