Molecular Cloning And Characterization Of Flavonoid 3-O-glycosyltransferase Gene In Freesia Hybrida

Freesia hybrida is a kind of ornamental horticulture flower that belongs to freesia genus of Iridaceae family,it could be considered as an ideal material to investigate the biosynthesis of flavonoids,which are determinant to flower colors.Flavonoid 3-O-glucosyltransferase(3GT)is a key enzyme at the downstream steps of flavonoid biosynthetic pathway.It could make flavonoids more stable and water-soluble through glycosylation modification of the flavonoids aglycon.Numerous studies have shown that 3GT played very important roles in the catalysis of anthocyanins and flavonols.In this study,using Red River~?,one representative cultivar of Freesia hybrida,as experimental materials,we successfully cloned the Fh3GT2 gene and analyzed its function through a series of methods including bioinformatics prediction,spatio-temporal expression pattern analysis,biochemical analysis and plant protoplast transient tranfection analysis.In contrast to the results of Fh3GT1,which was isolated previously in our laboratory,the functional divergences between them was summarized.This study would lay a good foundation to reveal the evolution of flavonoid 3-O-glucosyltransferase family members,and provide a theoretical basis for the modifying of flower color by genetic engineering.The main research methods and conclusions were presented as follows:1.Fh3GT2 gene cloning and phylogenetic analysis.According on transcriptome sequencing results of flowers organs,the c DNA sequence of Fh3GT2 gene was isolated by 5’RACE techniques.Sequence analysis showed that the cloned gene sequence is 1362 bp long,encoding for a 453 amino acid protein with a calculated molecular weight of 48.5 k Da.Multiple sequence alignments showed that Fh3GT2 has the conserved catalytically active sites and residues interacting with glycosyl donors.Phylogenetic analysis showed that Fh3GT2 had the highest similarity with Zm Fl GT and Hv Fl3GT,and was clustered in a different branch with Fh3GT1,indicating that Fh3GT2might have differential catalytic properties compared with Fh3GT1.2.Expression patterns of Fh3GT2.Expression levels of Fh3GT2 in different stages of Freesia flower development were determined by q RT-PCR.The results showed that the expression of Fh3GT2 was gradually decreased along with the development of Freesia flowers,which was positively related to the accumulation pattern of flavonols.In additon,Fh3GT2 was also found to be expressed broadly in different vegetative tissues except flower tissues,which is also consistent with the accumulation of flavonols in plants.Thus,it is reasonable to deduce that Fh3GT2 might be mainly involved in the glycosylation of flavonols in Freesia per se.3.Preparation of the recombinant protein and enzymatic activity detection in vitro.Firstly,the ORF sequence of Fh3GT2 gene was connected into p ET32a(+)plasmid to construct recombinant prokaryotic expression vector p ET32a-Fh3GT2,which was subsquently introduced into Escherichia coli BL21(DE3),the recombinant protein was induced at low temperature and purified through Nickel column affinity chromatography purification method.In vitro enzymatic reaction system was established using the purified proteins,and the biochemical analysis showed that anthocyanin and flavonol glycosides was generated using flavonol and anthocyanidin as substrates,respectively,demonstrating that Fh3GT2 is the bona fide catalysts having flavonoid 3-O-glucosyltransferase enzymatic activity.Substrate specificity studies showed that the catalytic efficiency of Fh3GT2 on flavonols was greater than that of anthocyanins.The analysis of enzyme kinetics parameters showed that Fh3GT2 had the highest affinity for kaempferol,suggesting that kaempferol might be the physiological substrate of Fh3GT2 in vivo.4.Promoter activation verification of Fh3GT2.Several constructed plasmids were used in Arabidopsis protoplast transfection system.The results showed that Fh MYB12L1,a transcription factor that potentially regulates flavonol synthesis in Freesia,could activate the expression of Fh3GT2 but not Fh3GT1,further suggesting that Fh3GT2 could be involved in the glycosylation of flavonols in Freesia.5.UV-B radiation strengthened the expression of Fh3GT2 gene.The expression of Fh3GTs genes in Fressia flowers subjected to UV-B radiation for 24hours was investigated.The results showed that the expression of Fh3GT2 was significantly increased after 24 hours of UV induction,implying its roles in UV-B resistance through regulating the biosyntheis of flavonols.