甜荞花青素糖基转移酶基因<i>FeUFGT1</i>的克隆、遗传转化及生物信息学分析

doi:10.7668/hbnxb.20194527

摘要/Abstract

摘要：

为探究花青素糖基转移酶基因(UFGT)在甜荞花色苷生物合成中的作用,以甜荞贵红1号为材料,基于同源比对分析,筛选并克隆得到1条UFGT基因序列,命名为FeUFGT1。对FeUFGT1进行生物信息学分析,并构建过表达载体转入拟南芥花青素3-O-糖基转移酶突变体(atugt78d2)中分析其功能。结果表明,FeUFGT1开放阅读框为1 404 bp,编码467个氨基酸残基,推测其分子质量51.46 ku,理论等电点为4.97。FeUFGT1蛋白不存在信号肽和跨膜区,含有植物糖基转移酶家族典型结构域(GT-B型),并且在C末端具有植物UGT家族成员特有的PSPG-box。进化分析表明,FeUFGT1蛋白与罗汉果的3-O-葡萄糖基转移酶UGT74AC1亲缘关系最近。在白花品种丰甜1号和红花品种贵红1号2个品种盛花时期的花瓣中,红花中FeUFGT1的表达量约为白花中的3.7倍,差异显著。突变体恢复试验表明,FeUFGT1能够恢复拟南芥突变体atugt78d2缺乏花色苷积累的表型,具有催化花色苷生成的功能。

关键词: 甜荞, FeUFGT1, 基因克隆, 花色苷, 3-O-葡萄糖基转移酶

Abstract:

This study aimed to investigate the function of the anthocyanidin glycosyltransferase gene(UFGT)in the anthocyanin biosynthesis in common buckwheat.Guihong 1 was used as the experimental material.A UFGT gene,named FeUFGT1,was obtained by homologous alignment analysis,and cloned followed by performing on bioinformatics analysis.Overexpression vector of this gene was constructed,then transferred into Arabidopsis thaliana anthocyanin 3-O-glycosyltransferase mutant atugt78d2 to assess its function.The results revealed that the open reading frame of FeUFGT1 was 1 404 bp and encoded 467 amino acid residues with speculated molecular weight of 51.46 ku and theoretical isoelectric point of 4.97.FeUFGT1 protein contained GT-B type glycosyltransferase family domain and a PSPG-box,specific one of the plant UGT family at the C-terminus.The phylogenetic analysis showed that FeUFGT1 protein was closely related to the 3-O-glucosyltransferase of Siraitia grosvenorii UGT74AC1.The expression level of FeUFGT1 in the white-flower petals of Fengtian 1 was 3.7-fold as high as that in the red-flower petals of Guihong 1 with a significant difference.Mutant recovery experiment showed that FeUFGT1 could restore the phenotype of the Arabidopsis mutant which lacked anthocyanin accumulation.

Key words: Buckwheat, FeUFGT1, Gene cloning, Anthocyanins, 3-O-glucosyltransferase

张兰, 杨铝, 杨朝结, 陈红, 黄娟, 朱丽伟, 陈庆富, 邓娇. 甜荞花青素糖基转移酶基因FeUFGT1的克隆、遗传转化及生物信息学分析[J]. 华北农学报, 2024, 39(3): 43-50. doi: 10.7668/hbnxb.20194527.

ZHANG Lan, YANG Lü, YANG Chaojie, CHEN Hong, HUANG Juan, ZHU Liwei, CHEN Qingfu, DENG Jiao. Cloning,Genetic Transformation and Bioinformatics Analysis of the Glycosyltransferase Gene FeUFGT1 of Common Buckwheat[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 43-50. doi: 10.7668/hbnxb.20194527.

http://www.hbnxb.net/CN/Y2024/V39/I3/43

图/表 8

表1 引物序列

Tab.1 Primer sequences

引物名称 Primer name	引物序列(5'—3') Primer sequences (5'—3')	用途 Purpose
FeUFGT1-1	F:ATGGCGATAAGCAACGGAAACT	基因克隆
	R:TTAACTAATCCTTGCTATGAAC
FeUFGT1-2	F:AAGGCGACAGAGGAGGAC	RT-qPCR
	R:CAACTGAGGGCACCAACC
FeUFGT1-3	F:agaggacctcgactctagaactagtATGGCGATAAGCAACGGAAACT	构建过表达载体
	R:gccccccctcgaggcgcgccACTAATCCTTGCTATGAACT

图1 甜荞FeUFGT1基因CDS序列扩增 M.DL2000 Marker;A:1.FeUFGT1基因扩增条带;B :1—8.pMD19T-FeUFGT1的菌落PCR。

Fig.1 CDS sequence amplication of FeUFGT1 gene in Fagopyrum esculentum M.DL2000 Marker;A:1.The PCR amplication target band of FeUFGT1 gene;B:1—8.Colony PCR of pMD19T-FeUFGT1.

图2 FeUFGT1基因编码的蛋白质特征 A.FeUFGT1蛋白氨基酸亲、疏水预测;B.保守结构域;C.二级结构:蓝色.α-螺旋,红色.延伸链,绿色.β-转角,紫色.无规则卷曲;D.三级结构。

Fig.2 Characteristics of proteins encoded by the FeUFGT1 gene A.FeUFGT1 protein amino acid hydrophilicity and hydrophobic prediction;B.Conserved domains;C.Secondary structure:Blue.α-helix,Red.Extended strand,Green.β-turn,Purple.Random coil;D.Tertiary structure.

图3 FeUFGT1与其他物种的UFGT的系统进化树及保守基序分析 VIUF3GT.美洲葡萄(ABR24135.1);FaUF3GT.草莓(AAU12367.1);At5g17050.拟南芥(AAM91139.1);PhUF3GT.碧冬茄(BAA89008.1);PfUF3GT.紫苏(BAA19659.1);CaUF3GT.银子葫芦(KAG7023401.1);FtUF3GT.苦荞(AOS85164.1);ZmUF3GT.玉米(NP_001105886.2);RpUF3GT.掌叶大黄(QOI79387.1);SgUGT74AC1.罗汉果(K7NBW3.1)。

Fig.3 Phylogenetic tree and conserved motif analysis of FeUFGT1 with other species of UFGT VIUF3GT.Vitis labrusca (ABR24135.1);FaUF3GT.Fragaria ananassa(AAU12367.1); At5g17050.Arabidopsis thaliana(AAM91139.1); PhUF3GT.Petunia hybrida(BAA89008.1); PfUF3GT.Perilla frutescens(BAA19659.1); CaUF3GT.Cucurbita argyrosperma(KAG7023401.1);FtUF3GT.Fagopyrum tataricum(AOS85164.1); ZmUF3GT.Zea mays(NP_001105886.2);RpUF3GT.Rheum palmatum(QOI79387.1);sgUGT74AC1.Siraitia grosvenorii (K7NBW3.1).

图4 FeUFGT1与其他物种的类黄酮3-O-糖基转移酶多重对比黑色框部分的序列为PSPG-box。

Fig.4 Multiplex alignment between FeUFGT1 and flavonoid 3-O-glycosyltransferases from other species The sequence of the black box part is PSPG-box.

图5 FeUFGT1启动子作用顺式作用元件分析

Fig.5 Analysis of cis-acting elements of the FeUFGT1 promoter

图6 甜荞红、白花FeUFGT1相对表达量和总花色苷含量分析数值代表3个重复的平均值±s,并且不同的小写字母代表差异显著(P<0.05)。图7同。

Fig.6 Relative expression of FeUFGT1 and total anthocyanin contentanalysis of Fagopyrum esculentum red and white flowers The values represent 3 replicate averages ±s and the different lowercase represent significant differences(P<0.05).The same as Fig.7.

图7 FeUFGT1转拟南芥atugt78d2突变体植株的幼苗分析 A.WT、atugt78d2突变体、35S∶FeUFGT1系的幼苗表型观察;B.总花色苷含量测定。

Fig.7 Seedling analysis of FeUFGT1 transgenic Arabidopsis thaliana atugt78d2 mutant A.Phenotypic observation of seedlings of WT,atugt78d2 mutant,and 35S∶FeUFGT1;B.Total anthocyanins content determination.

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甜荞花青素糖基转移酶基因FeUFGT1的克隆、遗传转化及生物信息学分析

Cloning,Genetic Transformation and Bioinformatics Analysis of the Glycosyltransferase Gene FeUFGT1 of Common Buckwheat

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甜荞花青素糖基转移酶基因FeUFGT1的克隆、遗传转化及生物信息学分析

Cloning,Genetic Transformation and Bioinformatics Analysis of the Glycosyltransferase Gene FeUFGT1 of Common Buckwheat

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