金钗石斛法尼基焦磷酸合酶基因克隆及表达分析  

Clone and Expression Analysis of Farnesyl Pyrophosphatase Gene from Dendrobium nobile L.

作　　者：李丹丹 肖逸飞 李金玲[1,2] 黄明进[1,2] 罗春丽[1,2] 王华磊[1,2] Li Dandan;Xiao Yifei;Li Jinling;Huang Mingjin;Luo Chunli;Wang Hualei(College of Agriculture,Guizhou University,Guiyang,550025;Guizhou Key Laboratory of Propagation and Cultivation on Medicinal Plants,Guizhou University,Guiyang,550025)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州大学,贵州省药用植物繁育与种植重点实验室,贵阳550025

出　　处：《分子植物育种》2024年第20期6675-6682,共8页Molecular Plant Breeding

基　　金：贵州大学引进人才项目(2019#17);贵州大学本科生创新创业训练SRT项目(贵大SRT字[2019]320号);贵州省自然科学基金项目(黔科合基础[2017]1403);贵州省科技计划项目(黔科合支撑[2019]2962号);贵州省药用植物繁育与种植人才基地(黔人领发[2020]2号)共同资助。

摘　　要：为探究金钗石斛法尼基焦磷酸合酶基因(DnFPPS)是否参与倍半萜类生物碱、萜类合成等代谢过程,根据金钗石斛转录组信息,克隆金钗石斛DnFPPS基因全长cDNA序列,采用荧光定量PCR技术检测其在石斛不同生长年限茎和叶中的相对表达水平,构建pGEX-6t-1-DnFPPS原核表达载体并在大肠杆菌中表达,获得金钗石斛DnFPPS基因c DNA全长序列共1044 bp核苷酸。NCBI blastx结果显示,DnFPPS基因编码的蛋白与霍山石斛来源FPPS1具有最高相似性,氨基酸一致度达98%。DnFPPS具有异戊烯基转移酶基因的5个典型保守功能域。进化树分析结果显示,DnFPPS蛋白与霍山石斛的FPPS1具有较近的遗传距离。其推导出的蛋白质序列经预测其结构与穗状蒿(Artemisia spiciformis)中FPP/GFPP合成酶的嵌合体蛋白晶体结构相似性可达到73.16%。表达水平分析发现DnFPPS基因表达与石斛碱累积呈正相关。在大肠杆菌中表达后SDS-PAGE电泳观察得到72 kD左右的目的条带,与预测的蛋白分子量相符合。该研究为DnFPPS基因功能研究奠定重要基础,为阐明金钗石斛倍半萜及倍半萜类生物碱物质生物合成途径研究提供重要理论参考。To explore whether the farnesyl pyrophosphate synthase gene(DnFPPS)in Dendrobium nobile is involved in the metabolic processes such as sesquiterpene alkaloid and terpene synthesis,in this study,the full-length cDNA sequence of FPPS gene of Dendrobium nobile according to the reanalysis of transcriptome data published in NCBI for Dendrobium nobile was cloned;DnFPPS+PGEX-6t-1 prokaryotic expression vector was constructed and expressed in E.coli.A total of 1044 bp nucleotides of FPPS gene of Dendrobium nobile were obtained.The NCBI blastx showed that the protein encoded by DnFPPS gene was the highest homology with FPPS1 from Dendrobium huoshanense,and the consistency of amino acids was 98%.Five typical conserved domains of isopentenyl transferase gene existed in DnFPPS gene.Close genetic relationship was found between DnFPPS protein to DhFPPS1 by phylogenetic tree analysis.The predicted protein structure was 73.16%homologous with the crystal structure of FPP/GFPP synthetase in Artemisia spiciformis.RT-qPCR result revealed that DnFPPS gene expression was positively correlated with dendrobine accumulation.SDS-PAGE electrophoresis observed about 72 kD bands,which was consistent with the predicted protein molecular weight.This study laid a key foundation for the functional study of DnFPPS gene and provided an important theoretical reference for elucidating the biosynthesis pathway of sesquiterpenoids and sesquiterpenoid alkaloids in Dendrobium nobile.

关 键 词：法尼基焦磷酸合成酶 倍半萜类生物碱 基因克隆 生物合成途径 

分 类 号：S567.239[农业科学—中草药栽培]