Genes Cloning And Optimal Substrate Analysis Of Flavonols Synthase Homologuous Proteins FLS2 And FLS3 From Fagopyrum Tataticum

As the traditional medicine and food plants in nature, tartary buckwheat (Fagopyrum tatarium) contains not only various kinds of nutrient, but also rich flavonols compounds such as rutin. Flavonols synthesis is one branch of phenylpropanoid metabolic pathway in plant. With the catalysis of flavonols synthase (FLS), dihydroquercetin (DHQ), dihydrokaempferol (DHK) and dihydromyricetin (DHM) are transformed into quercetin, kaempferol and myricetin, respectivley. Quercetin would be further glycosylated into rutin. Many recent researches indicate FtFLS multicopy exsits in tartary buckwheat chromosomes, which implies that the different FtFLS homologuous proteins are involved in rutin biosynthesis probably. Based on transcriptome data of tartary buckwheat at flowering phase, FtFLS2 and FtFLS3 genes were cloned, analyzed and expressed in Escherichia coli. Furthermore, optimal substrate was analyzed after kinetics parameters to three substrates were compared along three recombination FtFLSs. The main results are as follows:1. Based on homology cloning and RACE technology, two flavonols synthase genes FtFLS2 and FtFLS3 were obtained from tartary buckwheat. Sequences analysis indicated that FtFLS2 and FtFLS3 genes ORFs are 996b pand 1008 in full-length, they code 331 and 335 amino acid residues, molecular formula are C1771H2651N451O502S10 and C1723H2689N449O509S10, their relative molecular masses are about 37.8KD and 38.1KD, pis are 5.52 and 5.74, respectively. BLAST result shows FLS2 and FLS3 has 97%and 77% identity with FtFLS 1 (GenBank ID:JX401285.1). Multiple sequence alignment indicates that FtFLS2 and FtFLS3 contain conserved region Conserved domain A, Conserved domain B, Fe2+ binding site and 2-ketoglutaric acid binding site of 2-ODD protein family. Hence, they belong to the typical 2-ketoglutaric acid-positive dioxygenase. Homologous evolutionary tree shows that FtFLS2 and grape (Vitis viniferd) VvFLS (GenBank ID: AB213566) are gathered into cluster III, when FtFLS3, FtFLS1 and commom buckwheat (Fagopyrum esculentum) FeFLS are assembled into cluster I. So, there are different catalytic characteristics between FtFLS2 and FtFLS3 probably.2. Two recombinant plasmids named pET30b-F/FLS2 and pET30b-FtFLS3 were constructed and expressed in E. coli. SDS-PAGE shows their molecular weights are 37.8KDa and 38.1KDa, respectively. The E. coli strains with pET30-FtFLS2, pET30-FtFLS3 and pET30-FtFLSl were induced in lmmol/L IPTG,25℃ for 10h. The cell suspensions were broken by ultrasonication and purified by Ni-affinity chromatography for electrophoresis-pure FtFLSs proteins. Thin layer chromatography (TLC) results indicate three FtFLSs could catalyst DHQ into quercetin. In 100μmol/L DHQ reaction system with 12.5ug FtFLS proteins, a linear correlation is presented between quercetin production and reaction time under presented in 15min. Enzymatic kinetic parameters of three recombinant FtFLS to DHQ, DHK and DHM were measured through Lineweaven Burk method, the results are as follows:For FtFLS 1,KmDHQ=594.17 μmol/L (VmaxDHQ=42.55 μmol/L·in), KmDHK=1257.18 μmol/L (VmaxXDHQ= 25.53 μmol/L·min) and KmDHM=1516.36μmol/L (VmaxDHM=17.76 μmol/L·min), respectively; For FtFLS2, KmDHQ=8411.27μmol/L (VwaxDHQ=10.25 μmol/L·min), KmDHK= 23646.74 μmol/L (VmaxDHQ=13.59μmol/L·min) and DHM=21583.23 μmol/L (VmaxDHM= 12.15μmol/L·min), respectively; For FtFLS3, KmDHQ=6478.21 umol/L (μmaxDHQ=13.97 umol/L·min),KmDHK=717.93 umol/L (VmaxDHQ=24.04 μmol/L·min) and KmDHM=1609.36 μmol/L (VmaxDHM=17.83 μmol/L·min), respectively. Seen from the above, the optimal substrates of FLS1, FLS2 and FLS3 are DHQ, DHQ and DHK, respectively. In this study, the flavonols biosynthesis branch in tartary buckwheat is understood about metabolic pathway and metabolic flux in detail.