金线莲磷酸甘露糖变位酶基因的克隆与表达分析  

Cloning and Expression Analysis of Phosphomannomutase Gene in Anoectochilus roxburghii

作　　者：李和平 林江波 黄惠明 邹晖 戴艺民 LI He-Ping;LIN Jiang-Bo;HUANG Hui-Ming;ZOU Hui;DAI Yi-Min(Institute of Subtropical Agriculture,Fujian Academy of Agricultural Sciences,Zhangzhou 363005,China)

机构地区：[1]福建省农业科学院亚热带农业研究所,漳州363005

出　　处：《农业生物技术学报》2023年第11期2284-2293,共10页Journal of Agricultural Biotechnology

基　　金：福建省省属公益类科研院所基本科研专项(2020R1030003,2021R1030001);福建省农业科学院南药植物科技创新团队(CXTD2021001-2)。

摘　　要：金线莲多糖是金线莲(Anoectochilus roxburghii)的主要药效成分之一。为了深入了解金线莲多糖的生物合成途径,本研究对该途径中的磷酸甘露糖变位酶(phosphomannomutase,PMM)基因进行克隆和原核表达,并进行理化性质、结构特点、亚细胞定位和基因表达调控分析。结果显示,ArPMM基因ORF区序列长759 bp,共编码252个氨基酸,原核表达的融合蛋白分子量为30.75 kD。生物信息学预测该蛋白属于卤酸脱卤酶(haloacid dehalogenase,HAD)超家族成员,是一种稳定的、不具有信号肽和跨膜结构的亲水蛋白。PMM蛋白在前人研究中均定位于细胞质,而本研究通过植物瞬时表达的亚细胞定位分析发现,ArPMM蛋白定位于细胞核。系统进化树分析发现,ArPMM蛋白与铁皮石斛(Dendrobium officinale)、霍山石斛(D.huoshanense)、蝴蝶兰(Phalaenopsis equestris)等兰科植物亲缘关系较近。基因表达分析发现,ArPMM基因在金线莲不同组织中均有表达,在花中的相对表达量最高,茎中最低;ArPMM基因受盐胁迫影响上调表达,推测其可能催化甘露糖-1-磷酸向甘露糖-6-磷酸转化;同时,温度对该基因也有调控作用,高温和低温均抑制其表达,且对高温处理反应灵敏。本研究为深入开展PMM基因参与甘露糖代谢途径的相关研究提供基础资料。Polysaccharide is one of the main medicinal components in Anoectochilus roxburghii.To further understand the biosynthetic pathway of A.roxburghii polysaccharides,the phosphomannomutase(PMM)gene was cloned and prokaryotic expressed in this study.In addition,the bioinformatics,subcellular localization,and expression regulation were analyzed.The results showed that the ORF region of ArPMM gene was 759 bp in length,encoding 252 amino acids,and the molecular weight of fusion protein expressed by prokaryotes was 30.75 kD.Bioinformatics prediction showed that the PMM protein belonged to the haloacid dehalogenase(HAD)superfamily,and was a stable hydrophilic protein without a signal peptide and transmembrane structure.The ArPMM protein was found,for the first time,located in the nucleus by transient expression method,although the PMM was believed located in the cytoplasm in previous reports.Phylogenetic tree analysis showed that ArPMM protein was closely related to Dendrobium officinale,D.huoshanense,and Phalaenopsis equestris.The result of gene expression experiment revealed that the ArPMM gene was expressed in multiple tissues,and the highest relative expression level was detected in flowers while the lowest expression was found in stems.The ArPMM gene was up-regulated by salt stress,and it was speculated that ArPMM might catalyzing the transformation of mannose-1-phosphate to mannose-6-phosphate.Meanwhile,the ArPMM gene was also regulated by temperature,both high and low temperatures inhibited its expression,and ArPMM gene was sensitive to high-temperature treatment.This study provides basic materials for further research of ArPMM gene participating in the mannose metabolism pathway of A.roxburghii.

关 键 词：金线莲 磷酸甘露糖变位酶(PMM) 基因克隆 亚细胞定位 表达调控 

分 类 号：S567.2[农业科学—中草药栽培]