Effects Of DFR,ANS And UFGT Genes Encoding Proteins On Anthocyanin Synthesis In Zanthoxylum Bungeanum Pericarp

Zanthoxylum bungeanum,as an important spice plant in China,is deeply loved by the people as a condiment and medicine;Anthocyanin,as a flavonoid in plants,is an important pigment to produce plant color.It is also an important nutrient.It has the effects of anti-aging,anti-cancer and so on.However,the catalytic mechanism of anthocyanin synthesis pathway in Zanthoxylum bungeanum is still unclear.In this study,the flavonoids of Zanthoxylum bungeanum pericarp at different developmental stages were identified and their relative contents were analyzed;On this basis,ZbDFR,ZbANS and ZbUFGT genes of Zanthoxylum bungeanum were cloned and prokaryotic expressed;The mechanism of zbdfr catalyzing and regulating the production of anthocyanins was deeply studied.The following results were obtained:1.1.HPLC-Q/TOF-MS/MS technology was used to identify and relatively quantitatively analyze the flavonoids in the pericarp of Zanthoxylum bungeanum in green,semi red and red periods.A total of 37 flavonoids were identified,mainly quercetin,quercetin,isoquercetin and other glycosylated flavonoids derived from quercetin.Compared with green and half red pericarp,the anthocyanins and flavonoid glycosides in red pericarp increased significantly.As the substrate of ZbDFR,dihydroquercetin had the highest content in green pericarp,and dihydrokaempferol had the highest content in semi red pericarp.2.ZbDFR,ZbANS and ZbUFGT genes were cloned: the size of ZbDFR gene was about1014 bp,encoding 337 amino acids;The size of ZbANS gene is about 1074 bp and encodes357 amino acids;The size of ZbUFGT gene is about 1401 bp and encodes 466 amino acids.The prokaryotic expression vectors of p ET28a-ZbDFR,p ET28a-ZbANS,pmal-c5x-ZbANS and p ET28a-ZbUFGT were successfully constructed and purified.3.The catalytic and regulatory mechanisms of ZbDFR in Zanthoxylum bungeanum were analyzed.The in vitro enzyme activity of ZbDFR showed that the optimum temperature was 30 ℃,and the optimum p H was 6.5.The Km values of ZbDFR catalytic substrates DHK,DHQ and DHM were 491.29 μM,96.82 μM and 26.28 μM respectively,while the corresponding Kcat values were 0.06,0.04 and 0.011 respectively.The activity of ZbDFR was significantly inhibited by naringin,myricetin,quercetin and kaempferol,but not by epicatechin.Flavone glycosides(quercetin,isoquercetin,naringin,hypericin)did not inhibit the activity of ZbDFR,and quercetin and isoquercetin promoted the activity of ZbDFR to a certain extent,which indicated that the glycosylation of flavonoids would eliminate their inhibitory effect on DFR,and might regulate the activity of ZbDFR.Quercetin is a competitive inhibitor of ZbDFR at low concentration(5 μM),while at high concentration(200μM),it is a combination of competitive inhibition and allosteric inhibition.Site directed mutagenesis combined with enzyme activity test showed that S128,Y163 and K167 were the key catalytic sites of ZbDFR.The hydrogen transferred to the ketone group on the C ring of DHQ eventually came from solvent water,while the hydrogen transferred to C4 of DHQ came from the hydrogen donor site of NADPH.