Recombinant Expression Of Tartary Buckwheat Dihydroflavonol 4-Reductase /Leucoanthocyanidin Reductase And Preparation Of Their Polyclonal Antibody

Fagopyrum tataricum(buckwheat), as a typical crop for food and medical functions, which contain abundant flavonoid discussed the future of the potential development and utilization of flavonoids. Flavonoid has been shown to exhibit anti-tumer, anti-oxidation and anti-aging, antibacterial and antivirus, immunological regulation and many health care and medicinal functions. DFR and LAR are key enzymes in flavonoid biosynthetic pathway. The research that express Ft DFR and FtLAR in E.coli on the basis of cloned FtDFR and Ft LAR genes, purify the recombinant protein and obtain the high titer polyclonal antiserum rasied against rabbit will lay the basis for the regulating mechanism of accumulation of secondary metabolites in tartary buckwheat. The following researches have been conducted:FtDFR and FtLAR coding genes was amplifies from the immature seed c DNA of tartary buckwheat by RT-PCR.To investigate the level of expression of FtDFR and Ft LAR genes in phenylpropanoid biosynthetic pathway in different organs of tartary buckwheat at seed-filling development period, the experiment showed FtDFR gene have relative higher expression in flowers and leaves, Ft LAR have relative higher expression in seeds.Nucleotide and conducted protein sequence of Ft DFR and Ft LAR were analyzed using bioinformatic approaches. The results revealed that FtDFR and Ft LAR were 1026 bp and1068 bp in length, which encoded 341 and 355 amino acids. The predicted molecular weight and pI of FtDFR were 38.53 kD and 5.78, that of FtLAR were 39.37 kD and 6.36. As analyzed by alignment of deduced anmino acid sequences of some DFR and LAR genes, the result revealed FtDFR possess a NADP binding site and a substrated specificity site, FtLAR possess RFLP、ICCN and THD motifs. The phylogenetic tree indicated that FtDFR and Ft LAR have a close relationship with them of Fagopyrum cymosum.FtDFR and FtLAR gene were amplified from the seed-filling period cDNA of tartary buckwheat by RT-PCR and ligated to expression vector pET47 b and pET28 a. The E.coli BL21(DE3) carrying the Ft DFR and FtLAR gene were obtained and induced IPTG. As analyzed by the optimized condition of expression, The results indicated that FtDFR had been expressed in E.coli in the form of soluble and insoluble and Ft LAR was highly expressed in the form inclusion bodies.The expression products of FtDFR and FtLAR were purified by cobalt chelating affinity chromatograph, the results indicated that very pure FtDFR fusion protein was obtained by the gradient elution method of imidazole( 0~20 mmol and 20~150 mmol) and pure FtLAR fusion protein was obtained by the solubilization and renaturation of inclusion body.Activity of FtDFR fusion protein in vitro detected by HPLC method. The result that it showed the DFR activity, which catalyzed DHQ to leucocyanidin and catalyzed DHM to leucodelphinidin.The high titer polyconal antiserum raised against rabbit was obtained using FtDFR and Ft LAR fusion protein by the technology of cutting SDS-PAGE gel and immune response. Western blotting analysis showed the raise antibody could specifically react with the antigen while native FtDFR and FtLAR existed in total protein of seed filling period of buckwheat.