假地豆碳糖基转移酶基因的克隆、生物信息分析及原核表达  

Cloning,Bioinformatics Analysis and Prokaryotic Expression of C-glycosyl Transferases Gene of Desmodium heterocarpon

作　　者：袁慧娟 向贵生 郝冰 杨生超 Yuan Huijuan;Xiang Guisheng;Hao Bing;Yang Shengchao(National-Local Joint Engineering Research Center on Germplasm Innovation&Utilization of Chinese Medicinal Materials in Southwestern China,Yunnan Agricultural University,Kunming,650201;The Key Laboratory of Medicinal Plant Biology of Yunnan Province,Yunnan Agricultural University,Kunming,650201;College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming,650201)

机构地区：[1]云南农业大学,西南中药材种质创新与利用国家地方联合工程研究中心,昆明650201 [2]云南农业大学,云南省药用植物生物学重点实验室,昆明650201 [3]云南农业大学农学与生物技术学院,昆明650201

出　　处：《分子植物育种》2022年第17期5569-5577,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31701854);云南省应用基础研究计划项目[2017FG001(-076)];云南省科技厅重大科技专项(2019ZF011-1)共同资助。

摘　　要：碳糖基转移酶(CGTs)是植物中黄酮碳苷生物合成途径中关键酶,具有催化黄酮类底物生成黄酮苷类化合物的功能。本研究对药用植物假地豆(Desmodium heterocarpon(L.)DC.)中黄酮碳糖基转移酶基因(DhCGT1)进行克隆、生物信息学分析、异源表达和蛋白纯化。结果表明DhCGT1基因全长1446 bp,含481个氨基酸,DhCGT1蛋白的相对分子质量为53.55 kD,含47个极性氨基酸残基,51个非极性氨基酸残基,等电点(pI)为6.44,分子式CHNOS,脂溶系数为88.38,平均疏水性为0.822,半衰期为30 h。不稳定指数为47.80,属于不稳亲水蛋白。DhCGT1蛋白是非分泌蛋白,没有信号肽识别功能,并且该蛋白不存在跨膜区,亚细胞定位预测分析结果显示DhCGT1蛋白位于质膜上。经多序列比对和亲缘关系分析显示,DhCGT1基因编码的氨基酸序列与大豆(Glycine max)和豇豆(Vigna unguiculata)序列相似度高达67.38%。作为黄酮碳苷的生物合成过程中的关键酶,CGTs催化并生成了多种具有重要活性的黄酮碳苷类化合物。本研究首次成功克隆、异源表达并纯化了山蚂蝗属假地豆植物中的DhCGT1基因,这为进一步研究DhCGT1基因功能及假地豆中黄酮碳苷合成途径的分子机制提供基础。C-glycosyl-transferases are key enzymes in the biosynthesis of C-glycosylflavonoid in plants which catalyze flavonoid substrates into C-glycosyl-flavonoid.In this study,the C-glycosyl-transferase gene(DhCGT1)from Desmodium heterocarpon(L.)DC was cloned,Bioinformatics analyzed,heterologous expressed and the protein was purified.The results showed that the full length of DhCGT1 gene was 1446 bp,encoding 481 amino acids.The relative molecular weight of DhCGT1 protein was 53.55 kD,containing 47 polar amino acid residues and 51 non-polar amino acid residues with an isoelectric point of 6.44.An average hydrophobicity and aliposolubility coefficient of Dh CGT1 were 0.822 and 88.38 respectively.The molecular formula of DhCGT1 was C_(2399)H_(3761)N_(645)O_(702)S_(21)and a half-life period was 30 h with the instability index of 47.80,indicated DhCGT1 unstable hydrophilic protein.DhCGT1 was a non-secreted protein with no signal peptide recognition function and transmembrane region.Subcellular localization prediction analysis indicated the amino acid sequence encoded by DhCGT1 was 67.38%similar to Glycine max and Vigna unguiculata.As a key enzyme in the biosynthesis of flavonoid glycosides,C-glycosyl-transferase catalyze and produce a variety of flavonoid glycosides with important activities.In this study,the DhCGT1 was cloned,heterologously expressed and purified from Chinese herb D.heterocarpon for the first time,which laid a foundation for further study on the function of CGTs and the molecular mechanism of C-glucolyflavones synthesis pathway in D.heterocarpon.

关 键 词：假地豆 碳糖基转移酶 生物信息学分析 原核表达 

分 类 号：S567.19[农业科学—中草药栽培]