The Cloning And Functional Analysis Of Dihydromyflavone 4-Reductase Gene (DFR) In Nitraria Tangutorum

Plants suffer a variety of abiotic stress in the process of growth and development,such as UV-B,drought,salt stress and so on.Plants can increase tolenrace to many kinds of abiotic stress by accumulateing a variety of small molecules of secondary metabolites,anthocyanins is one of the most important categories.Anthocyanin can reduce the oxidative damage caused by abiotic stress,because of it's good scavenging ability of reactive oxygen species(ROS).Dihydroflavonol 4-reductase(DFR)is a key enzyme in the downstream.pathway of anthocyanin synthesis,it determines which species of anthocyanin accumulation in plant.Nitraria tangutorum Bobr.grows in arid and semi-arid areas with poor natural environment,and has strong tolerance and resistance to various abiotic stresses under severe conditions.In this study,RT-PCR and RACE were used to clone the full-length cDNA of the dihydroflavonol 4-reductase(NtDFR)gene,which was 1392bp in length and the open reading frame(ORF)length is 1020bp encoding 339 amino acids.The amplified ORF fragment was ligated with pPZP221 to obtain pPZP221-NtDFR plant expression vector.We got first generation transgenic Arabidopsis by using inflorescence infiltration method,and screened it to T3 homozygote.The transgenic Arabidopsis thaliana of T3 was validated by PCR.The L2,L3 and L5 lines were selected for subsequent experiments.0h,2h,4h and 6h UV-B stress,0d,10d and 15d drought stress,0mM,75mM and 150mM NaCl stress were used for wide type(WT)and over expression(OE)Arabidopsis seedlings.The results showed that the content of anthocyanin in OE was higher than that in WT under three stress conditions,and it confirmed that overexpression of NtDFR could induce the accumulation of anthocyanins in Arabidopsis thaliana.Not only that,some other physiological and biochemical indexes were measured,including fresh weight,chlorophyll content,SOD,CAT activity and MDA,GSH content.The results showed that the accumulation of anthocyanin could improve the growth of Arabidopsis thaliana under stress conditions and regulate the antioxidant system.It revealed that the overexpression of NtDFR and accumulation of anthocyanins played an active role in Arabidopsis coping with the above abiotic stress.