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

doi:10.7668/hbnxb.20194527

Abstract

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

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.

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Figures/Tables 8

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

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.

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.

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).

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

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

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.

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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