Analysis On Sequence Polymorphism And Activity Of Citrus Flavonoid 3’-Hydroxylase Gene Promoter

Flavonoids are a class of secondary polyphenols metabolites with strong biological activity.They are widely distributed in various tissues of citrus,determining fruit flavor and color,as well as plant resistance.Flavonoid 3’-hydroxylase(F3’H),a multifunctional enzyme in the flavonoid biosynthesis pathway,can catalyze the hydroxylation of the 3’position on the B-ring of the flavonoid skeleton and plays an important role in the anthocyanin biosynthesis.In this study,we cloned and analyzed the differences cis-acting elements of the F3’H promoter in different citrus germplasms.To determine the distinct promoter activities,the promoters was transiently over-expressed in tobacco leaves and Newhall callus.Additionally,the effect of F3’H promoter polymorphism on the flavonoid metabolic pathway was analyzed.The results were shown as blow:1.Cloned and analyzed the F3’H promoter sequence: There were three types of promoters in different citrus germplasms.The A2 promoter had a 44 bp sequence insertion at-288 bp upstream of the start codon compared with the A1 promoter.While compared with the A1 promoter,A3 promoter type had 162 bp and 44 bp sequences insertion at-227 bp and-288 bp upstream of the start codon,respectively.The promoters of Hua zhou pummelo,such as zheng mao,Fu mao and Jia xi yang,were A2A1.Among the Poncirus and its’ hybrids,the promoter type of P.trafoliata and C.sinensis × P.trafoliata were A3A3,and the C.grandis × P.trafoliata was A3A1.The promoter type of common pommelos,loose-skin citrus,sweet orange and ichangensis were A1A1.2.Analyzed the insertion sequence in F3’H promoter: The 44 bp and 162 bp insertion sequences in the A2 and A3 promoters were characterised as the plant micro-inverted transposable element(MITE),named ’MITE1’ and ’MITE2’,respectively.The ciselements on the ’MITE1’ and ’MITE2’ sequences were predicted to contain one and several binding domains of transcription factors,respectively.In addition,compared with the A1 promoter,the A2 and A3 promoters had multiple single nucleotide difference(SNP)sites,resulting in different cis-elements.3.Analyzed the activity of F3’H promoter: The A1 to A3 promoters were overexpressed in N.benthamiana leaves and Newhall callus.The results indicated that the activity of A2 and A3 promoters was significantly higher than that of the A1.Interestingly,the promoter activity of the A2 and A3 promoters without MITEs insertion sequence were decresed.4.Analyzed the full coding sequence of F3’H: The length of F3’H gene was 4031 bp,containing 4 exons and 3 introns.The SNP site linked to the MITEs insertion was not detected in first to third exon regions of zheng mao F3’H gene.Taken together,the differences in the promoter sequence of F3’H gene might affect on the metabolic of flavonoids,and it had certain significance for the study of citrus evolution.