Recombinant Expression Of FtFLS2 And FtCYP98A In Tartary Buckwheat And Preparation Of Their Polyclonal Antibody

Fagopyrum tataricum(L.) Gaertn, also known as tartary buckwheat, is a short season polygonaceae buckwheat. Tartary buckwheat is rich in nutrients, not only can be eaten, but also has effects of reduce blood sugar, lower blood lipid, anti-aging, antioxidant and other pharmacological effects. Flavonoids is the main bioactive component of tartary buckwheat,which involved in the resistance to UV-B radiation, cold, drought and many other physiological processes of tartary buckwheat. Flavonol is one kind of flavonoids, which exhibits antioxidant, anti-inflammatory activities and can protect plants from UV damage.FLS can catalyze dihydroflavonol into flavonol. As the key enzyme for the synthesis of flavonol, FLS is highly conserved in plants. Lignin, one of the important products of phenylpropanoid metabolic pathway, plays an important role in the normal growth and development of plants. CYP98 As are the members of cytochrome P450 family, can decide the carbon origin of lignin monomer, are greatly significant for regulating the biosynthesis of lignin.The gene FtFLS2 and FtCYP98 A from cultivar “Yu 6-21” of tartary buckwheat were obtained by homology cloning and analyzed by bioinformatic method. The amino acid sequence of FtFLS2 shares high similarity with tartary buckwheat, common buckwheat, grape FLSs, etc. The typical binding sites of Fe2+and 2-oxoglutaric acid are existed in the FtFLS2,besides the two conserved functional domains DIOX_N and 2OG-Fe_Oxy of 2-ODD family. The amino acid sequenc of Ft CYP98 A shares more than 70% similarity with the published CYP98 A and C3 H sequences. It suggests that we have cloned the complete coding sequence of FtFLS2 and partial sequence of the homologous gene FtCYP98 A of cytochrome CYP98 A family, and the recombinant expression vectors pET47b-Ft FLS2 and pET47b-Ft CYP98 A were constructed successfully. Results of Semi-quantitative RT-PCR showed that FtFLS2 gene was expressed at the highest level in immature seed, followed by flower, leaf and stem. While FtCYP98 A gene expressed the higher level in stem and immature seed, compared to leaf and flower. The recombinant expression vectors pET47b-Ft FLS2 and pET47b-Ft CYP98 A were induced to express the target proteinsefficiently in E.coli BL21 Star(DE3). After the target proteins purified with cobalt chelating affinity chromatography, the specific band was excised from the gel and the polyclonal antiserum was raised by immunizing rabbits. Western blotting analysis showed bright and single bands after the hybridization between total thallus proteins of prokaryotic expression and antiserum, indicating the antibodies had significant specificity and Ft FLS2 was mainly expressed in immature seed. The result of activity detection showed that FtFLS2 had the catalytic activity to convert dihydroquercetin to quercetin. The specific activity, Km and Vmax of FtFLS2 recombinase are 4.645×103IU /mg, 323 mol/L and 17.65 M/min.The obtained polyclonal antibodies laid the foundation for analysis the relationship between Ft CYP98 A and Lignin or Chlorogenic acid metabolic pathways in tartary Buckwheat on the protein expression level, as well as FtFLS2 and accumulation of flavonoids.