Functional Analysis Of Flavonoid B-Ring 3’-hydroxylase And 3’,5’-hydroxylases From Camellia Sinensis

Flavonoids are major polyphenol compounds in plant secondary metabolism.The hydroxylation pattern of the B-ring of flavonoids were determined by the flavonoid 3’-hydroxylase(F3’H)and flavonoid 3’,5’-hydroxylase(F3’5’H).In this paper,"Shuchazao" was used as the experimental material,and one CsF3’H and two CsF3’5’Hs(CsF3’5’Ha and CsF3’5’Hb)were isolated from the cDNA libraty.The functional analysis of CsF3’H,CsF3’5’Ha and CsF3’5’Hb were studied by the analysis of bioinformatics,tissue-specificity expression,heterogenous expression in yeast and site-directed mutation and some other skills.In the study,the functional differences between the structural genes F3’H and F3’5’H involved in the flavonoid metabolism pathway of tea trees were investigated,which were beneficial for the further understanding of the regulation mechanism in the synthesis and derivation of secondary metabolites.The main results are as following:1.One CsF3’H and two CsF3’5’Hs(CsF3’5’Ha and CsF3’5’Hb)genes were cloned and isolated from cDNA library by the End to End technique.These ORF frames of three genes were predicted by EXPASy website.The molecular weights of CsF3’H,CsF3’5’Ha and CsF3’5’Hb were 57.1 kDa,57.0 kDa and 57.0 kDa,respectively.The isoelectric points of CsF3’H,CsF3’5’Ha and CsF3’5’Hb were 6.82,8.83 and 6.98,respectively.The length of genes were 1557 bp,1533 bp and 1521 bp,respectively.2.F3’Hs and F3’5’Hs from various plants were analyzed by DN AM AN software.The consequences suggested that all of CsF3’H,CsF3’5’Ha and CsF3’5’Hb had four specific-conserved regions of cytochrome P450.The Phylogenetic trees were constructed,the results showed that F3’H and F3’5’Hs belonged to CYP75B and CYP75A,respectively.3.The PLANTCARE and SOGO website analyzed F3’H and CsF3’5’Hs gene promoters,and they were eventually linked into PZZ044 vector(the GUS gene carrier).The transfection technology of agrobacterium infected arabidopsis thaliana and the transgenic plants of CsF3’5’Hs gene promoter were acquired.The GUS staining technology of CsF3’5’Hs had the expression of specific-tissue.The CsF3’5’Ha promoter had the highest expression in the leaf stalks and the low expression of the blade;the promoter of CsF3’5’Hb gene was highly expressed around the leaf edge,but not expressed in the stem.4.The analysis of expression patterns and induced profiles of genes by qRT-PCR showed that the expression of CsF3’5’Ha and CsF3’5’Hb in the bud and 1st leaf were higher than other tissues.However,the CsF3’H had the highest expression in the 4th and mature leaf.The correlation analysis showed that the expression of CsF3’5’Hs was positively associated with the concentration of B-trihydroxylated catechins,and the expression of CsF3’H was positively associated with the accumulation of Q(quercetin)contentration.5.Heterologous expression of these genes in yeast showed that CsF3’H and CsF3’5’Ha could catalyze flavanones,flavonols and flavanonols to the corresponding 3’,4’or 3’,4’,5’-hydroxylated compounds,for which the optimum substrate was naringenin.The enzyme of CsF3’5’Hb could only catalyze flavonols(including K and Q)and flavanonols(DHK and DHQ),of which the highest activities in catalyzing were DHK.6.The key amino acid sites determining the functional differences of CsF3’H and CsF3’5’Ha were explored by the site directed technique.Interestingly,the experiment of site-directed mutagenesis suggested that two novel sites near the C-terminal were discovered impacting on the activity of the CsF3’5’Ha enzyme hydroxylation.