藜麦Cq6GT基因的克隆与表达分析  

Cloning and expression analysis of Cq6GT gene from Chenopodium quinoa

作　　者：丰扬 郭凤根[1] 王仕玉[2] 刘正杰[1] 龙雯虹 FENG Yang;GUO Fenggen;WANG Shiyi;LIU Zhengjie;LONG Wenhong(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming 650201,China;College of Horticulture and Landscape,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学农学与生物技术学院,昆明650201 [2]云南农业大学园林园艺学院,昆明650201

出　　处：《植物生理学报》2022年第10期2017-2024,共8页Plant Physiology Journal

基　　金：国家自然科学基金(31960417)。

摘　　要：甜菜苷配基-6-O-葡萄糖基转移酶(6GT)是石竹目植物中合成甜菜红素的关键酶,具有催化甜菜红苷元糖基化生成甜菜红素的功能。本研究以藜麦为材料,通过近缘物种6GT序列比对分析,使用RT-PCR扩增获得了藜麦6GT基因的全长c DNA序列,并对其进行生物信息学、表达特异性及原核表达分析。结果表明,Cq6GT基因ORF全长1452 bp,编码483个氨基酸,Cq6GT蛋白相对分子量为53.98 kDa。该蛋白无信号肽,疏水系数为–0.205,不稳定指数为44.6,属于不稳定亲水蛋白。多序列比对及进化树分析显示Cq6GT蛋白与三色苋6GT和彩虹菊6GT蛋白氨基酸序列高度相似,具有共同的糖基转移酶家族PLN02554功能保守结构域。荧光定量PCR显示Cq6GT基因在红色、红条纹、黄条纹和绿色的藜麦茎中均有表达,其中红色茎表达量最高,绿色茎表达量最低,红色藜麦中茎和叶的Cq6GT表达量上显著高于根。将Cq6GT构建到pET28a原核表达载体,转化至大肠杆菌BL21(DE3)诱导表达,SDS-PAGE电泳显示在53.98 kDa大小处有特异性条带,与预测的目的蛋白分子量大小一致。本研究结果为进一步探究Cq6GT基因功能奠定了基础。Betanidin-6-O-glucosyltransferase is a key enzyme in the biosynthesis of betacyanin in Caryophyllales,which catalyzes the glycosylation of betanidin to betacyanin.The full length of Cq6GT gene was obtained by RT-PCR and its bioinformatics,expression specificity and prokaryotic expression were analyzed.The results showed that the ORF of Cq6GT was 1452 bp,encoding 483 amino acids and a protein with molecular mass of 53.98 kDa.Cq6GT protein had no signal peptide,its average hydrophobicity was–0.205 and the instability index was 44.6,indicating Cq6GT was an unstable hydrophilic protein.Multiple sequence alignment and phylogenetic analysis indicated that Cq6GT protein shared high identity with those of Amaranthus tricolor and Cleretum bellidiforme,and shared the common PLN02554 conserved domain of glucosyltransferase family.qRT-PCR showed that Cq6GT was expressed in quinoa stems of red,red stripe,yellow stripe and green,with the highest expression level in red stems and the lowest expression level in green stems.In red quinoa the expression of Cq6GT in stems and leaves was significantly higher than that in roots.Cq6GT was constructed into the prokaryotic expression vector pET28a and transformed into E.coli BL21(DE3)for inducing expression.SDS-PAGE electrophoresis showed that the expressed protein was 53.98 kDa and accorded with our forecast.This study provides a theoretical foundation for further functional studies of Cq6GT.

关 键 词：藜麦 甜菜红素 甜菜苷配基-6-O-葡萄糖基转移酶 荧光定量PCR 原核表达 

分 类 号：S519[农业科学—作物学]