Cloning And Functional Identification Of Dihydroflavonol-4-reductase Gene In Strawberry

Strawberry(Fragaria ×ananassa)is herbaceous plant of Rosaceae Fragaria.It is loved by most customers due to the high concentration of flavonids in fruit,which make the fruit colorful and help people to keep healthy.Anthocyanins and proanthocyanidins(PAs)are two different kinds of important flavonoids.They have strong antioxidant capacity which can protect people from aging,cancer and cardiovascular disease.Anthocyanins are nature water soluble pigment,the color of flowers and fruits for most angiosperm are decided by anthocyanins.Dihydroflavonol-4-reductase is a key enzyme in Anthocyanins and PAs synthetic pathway.It can catalyze 3 kinds of dihydroflavonol and 2 kinds of flavanone and produce 5 different anthocyanins precursors,these precursors are crucial prerequisite for colorful organ and tissue formation in plant.PAs is haploid or polymer of one kind restored anthocyanin(cyanidin).Thus DFR is important for both anthocyanin and PAs synthesis.In this study,we found a DFR gene from the transcriptome database,and the expression level of this gene is significant different under blue and white light treatments.The content of anthocyanin also different in two treatments.The sequence of this gene is very different from other strawberry DFR genes found in NCBI database.Reaserch on this gene can help us to understand the function of DFR gene family.Coding sequence(CDS)and promoter of this DFR was isolated,qRT-PCR was performed to evaluate the spatial and temporal expression level of this gene in strawberry,prokaryotic expression and over-expression this DFR gene in Arabidopsis thaliana were also performed to characterize the function of this gene.Several significant results are shown below.(1)A 1065 bp CDS was isolated from strawberry based on the transcriptome database and named FaDFRL.The identity of this protein and other DFR protein was only 35%.But the NADPH binding site and substrate binding site were putative to include in FaDFRL as other DFR.The promoter of FaDFRL gene was predicted to have several light and hormone response element.(2)The expression level of FaDFRL was significantly higher in strawberry fruit when supplement with blue light on 14 d after bloom,and the expression level also higher under red light treatment on 28 d after bloom.Indicating that blue light can induce FaDFRL in the degreening stage,and this gene also can be induced by red light in the mature fruit.(3)FaDFRL recombinant protein was obtained using prokaryotic expression.HPLC was performed to evaluate the function of FaDFRL.The results showed that FaDFRL can not catalyze the two common substrate of DFR in strawberry.The T3 homozygosis Arabidopsis which contain FaDFRL over-expression vector were acquired,the expression of FaDFRL was increased as compared to wild type plants by qRT-PCR detection.But the content of anthocyanins and PAs has no promotion.As the content of anthocyanins and PAs in Arabidopsis is extreme low,Arabidopsis is not very suitable for anthocyanins research,transform FaDFRL gene to strawberry would be better to verify the function of FaDFRL.In addition,the expression level of FaDFRL in transgene Arabidopsis was inhibited under blue light treatment,we speculate that the regulation of FaDFRL by blue light was post-transcriptional control,and FaDFRL takes part in stress resistance based on other reasearch.