霍山石斛UDP-鼠李糖合成酶的克隆及功能分析  

Cloning and function characterization of a UDP-rhamnose synthase from Dendrobium huoshanense

作　　者：黄晓 陈创博 郑志玉 王广林[2] 宋程 戴军[2] 韩邦兴 易善勇[2] HUANG Xiao;CHEN Chuang-bo;ZHENG Zhi-yu;WANG Guang-lin;SONG Cheng;DAI Jun;HAN Bang-xing;YI Shan-yong(College of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;Analytical and Testing Center,Anhui Dabieshan Chinese Medicine Research Institute,Anhui Engineering Research Center for Eco-agriculture of Traditional Chinese Medicine,West Anhui University,Lu′an 237012,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]皖西学院安徽省中药生态农业工程研究中心安徽省大别山中医药研究院分析测试中心,安徽六安237012

出　　处：《中国中药杂志》2024年第19期5147-5157,共11页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2023YFC3503804);国家自然科学基金项目(82304679);安徽高校协同创新项目(GXXT-2023-070)。

摘　　要：鼠李糖合成酶(rhamnose synthase,RHM)催化合成的UDP-鼠李糖,是植物体内合成鼠李糖糖苷类次生代谢产物的重要糖供体。该研究基于已报道的鼠李糖合成酶,通过序列比对从霍山石斛本地化基因数据库中筛选出1条RHM基因(DhuRHM)。克隆该基因并进行生物信息学分析及体外功能验证,结果显示DhuRHM的开放阅读框为2016 bp,编码蛋白的氨基酸数目为671,预测其大小为75.43 kDa,理论等电点为6.12。亚细胞定位分析表明DhuRHM定位于细胞膜,多序列比对及系统进化树显示,DhuRHM有植物RHM家族的特征结构域(GxxGxxG/A和YxxxK),且与其他物种的RHM具有同源性。体外酶活实验表明DhuRHM可体外催化UDP-葡萄糖生成UDP-鼠李糖。此外,RT-qPCR检测发现DhuRHM在茎的表达量高于叶和根,4种非生物胁迫(高温、低温、干旱、紫外)及MeJA处理下,紫外、低温及高温胁迫上调DhuRHM表达较为显著。该研究首次从霍山石斛中获得鼠李糖合成酶并完成其功能鉴定,进一步丰富了植物RHM家族的研究,为霍山石斛UDP-鼠李糖生物合成途径以及相关代谢产物应对环境胁迫等研究提供参考。UDP-rhamnose,which is synthesized under the catalysis by the rhamnose synthase(RHM),is an essential sugar donor for the synthesis of rhamnoside in plants.Based on the reported rhamnose synthase,this study screened one RHM gene(DhuRHM)from the localized gene database of Dendrobium huoshanense by sequence alignment.This gene was cloned,and then bioinformatics analysis and in vitro functional verification were carried out.The results revealed that DhuRHM had an open reading frame of 2016 bp,encoding a protein with 671 residues,a molecular weight of 75.43 kDa,and a theoretical isoelectric point of 6.12.Subcellular locali-zation analysis indicated that DhuRHM was located on the cell membrane.Multiple sequence alignments and the phylogenetic tree showed that DhuRHM possessed the characteristic domains(GxxGxxG/A and YxxxK)of the plant RHM family and presented high ho-mology with the RHMs of other species.DhuRHM had the activity to catalyze the transformation of UDP-glucose into UDP-rhamnose.Furthermore,RT-qPCR results showcased that the expression level of DhuRHM in stems was higher than those in leaves and roots.Among four abiotic stress conditions(high temperature,low temperature,drought,and ultraviolet radiation)and MeJA treatment,ultraviolet radiation,low temperature,and high temperature significantly up-regulated the expression of DhuRHM.This study is the first to obtain a rhamnose synthase from D.huoshanense and identify its functions.The findings enrich the knowledge about the plant RHM family and provide a reference for the research on the UDP-rhamnose biosynthetic pathway in D.huoshanense and the responses of related metabolites to environmental stress.

关 键 词：霍山石斛 鼠李糖合成酶 黄酮苷 功能鉴定 

分 类 号：S567.239[农业科学—中草药栽培]