Function And Expression Regulation Of DcDFR1 In Dracaena Cambodiana

Dragon’s blood is a kind of red resin secreted by the woody of Dracaena Cambodiana.It has the functions of activating blood circulation and removing blood stasis,relieving inflammation and pain,stopping bleeding and generating muscle.At present,there are many studies on the chemical constituents,pharmacological activities and clinical application of Dragon’s blood,but the formation mechanism of Dragon’s blood is not known.Dracaena Cambodiana is the main source plant of Dragon’s blood in China.Flavonoids are the main active substances in Dragon’s blood.dihydroflavonol-4-reductase(DFR)is one of the key enzymes in the flavonoid biosynthesis pathway of Dragon’s blood.In this study,a DFR gene DcDFR1 was cloned from Dracaena Cambodiana,and its expression regulation characteristics were studied,which laid a foundation for revealing the pathway of flavonoid biosynthesis and the mechanism of Dracaena Cambodiana.The open reading frame of DcDFR1 is 1005 bp,encoding 334 amino acid residues.The molecular formula is C1673H2619N439O485S23,the molecular weight is 37.38 kDa,and the predicted theoretical isoelectric point is 6.10.DcDFR1 protein has typical conserved NDPH binding domain and substrate binding domain of plant DFR and belongs to non-Asn/Asp type DFR.DcDFR1 has more than 70%homology with similar proteins in Anthurium andraeanum,Nelumbo lutea,Narcissus tazetta and Paeonia suffruticosa.Evolutionary tree analysis showed that DcDFRl was closely related to Narcissus tazetta and Anthurium andraeanum,but far from dicotyledon.Two fusion proteins,His-DcDFR1 and GST-DcDFR1,were obtained by prokaryotic expression.SDS-PAGE results showed that all fusion proteins of expected size were obtained.His-DcDFR1 fusion protein mainly existed in the form of inclusion bodies,while GST-DcDFR1 fusion protein existed in the form of soluble proteins and inclusion bodies.His-DcDFR1 fusion protein and GST-DcDFR1 fusion protein were purified separately.The activity of the fusion protein was detected by high performance liquid chromatography.It was found that the two fusion proteins had no activity on DHK,DHQ and DHM substrates.DcDFRl was the most expressed in flowers,followed by fruits,leaves,roots and stems.The expression of DcDFR1 was up-regulated by UV-B,NaCL,6-BA,ABA,JA and sucrose,Under UV-B,DcDFR1 expression was significantly up-regulated.A 1581 bp DcDFR1 promoter was cloned.The region has typical eukaryotic promoter structure,including hormone response elements such as jasmonic acid,ethylene and salicylic acid,and stress response elements such as light,drought and fungi.In addition,transcription factor binding elements such as MYB,bHLH,NAC and bZIP also exist in the promoter region.The results of yeast single hybridization showed that transcription factors DcTT2,DcMYB1,DcMYB3 and DcNAC1 could bind to DcDFR1 promoter.Subcellular localization analysis revealed that all four transcription factors were located in the nucleus.