Recombinant Expression And Enzymatic Properties Of Flavonoid Glycosyltransferase From Tartary Buckwheat

Flavonoids are plant secondary metabolities containing 2-phenylchromone structure,which usually present in the form of glycosides and perform a variety of biological functions.The glycosylation modification of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferase?UGT?,which could transform multiple sugar-moieties?such as glucose,galactose,rhamnose,arabinose,etc.?from UDP-sugar to the flavonoid aglycone.Glycosylation could change the stability,solubility and biological activity of flavonoids,affecting the growth and development,secondary metabolism and stress response processes of plants.This study selected and cloned four UDP-glycose:flavonoid glycosyltransferase gene?FtUFGT1,FtUFGT5,FtUFGT6,FtUFGT7?from tartary buckwheat.Then,a recombinant expression and purification system was constructed to obtain four target protein.This study designed to analysize the enzymatic properties of FtUFGT and attemped to lay a foundation for vitro flavonoid derivatives synthesis.In addition,the study intended to solve the UDP-rha deficiency in the glycosyltransferase activity analysis by expressing UDP-rhamnose synthase?RHM?.Otherwise,in order to understand how the FtUFGTs regulated secondary metabolite accumulation in tartary buckwheat,four FtUFGTs would be overexpressed in SR-1 tobacco?Nicotiana tabacum cv.Petit Havana SR-1?based on the pCAMBIA3301-eGFP plant expression vector.The mainly conclusion of this study are:.:?1?This study had cloned four flavonoid glycosyltransferase gene?FtUFGT1,FtUFGT5,FtUFGT6 and FtUFGT7?from tartary buckwheat.Multiple Multiple sequence alignment displayed that four FtUFGTs showed high identity with UFGTs from other plants.We predicted that FtUFGT6 and FtUFGT5 were UDP-rhamnose:flavonol 3-O-glucoside?1->6?rhamnosyltransferase,FtUFGT1 was flavonoid O-glycosyltransferase and FtUFGT7was flavonoid C-glycosyltransferase.We analyzed the expression level of four FtUFGTs in tartary buckwheat during the vigorous growth period by qRT-PCR.The results indicated that FtUFGT1 and FtUFGT7 both had the highest expression level in flowers;FtUFGT5and FtUFGT6 had highest-level in seeds.And they all expressed lowest in stem.The expression patterns of each gene in the buckwheat tissue were significantly different,which may be due to the different functions they performe in the secondary metabolic pathway.?2?This study gained each FtUFGT gene by RT-PCR and subcloned them into the pGEX-6p-1 vector by homologous recombination.Four target protein were expressed in Rosetta?DE3?in soluble form and were obtained by GST affinity chromatography.We used quercetin as substrate and assayed the enzymatic properties of FtUFGT1 by TLC and HPLC.The result showed that FtUFGT1 has flavonol 3-O-glucosyltransferase activity and could convert quercetin to isoquercetin efficiently.This enzyme has highest activity under30°C and pH 6-8 condition.The enzyme activity and substrate concentration showed good linear relationship when the concentrations of FtUFGT1,quercetin and UDPG were in the range of 1?mol/L,1.526×10³².287×10³?mol/L and 5.084×10⁴¹6.980×10⁴?mol/L,respectively.Otherwise,we constructed a mutation named F21YR_F23Y_F127Y of FtUFGT1 and it lost the glycosylation activity.?3?This study choned a UDP-rhamnose synthase from tartary buckwheat and constructed the pMAL-c4x-FtRHM vector.Then the FtRHM protein was purified by amylose-sepharose affinity chromatography.We used UDPG as substrate to analyzed its rhamnose synthesis activity but failed to find a effective method.?4?This study constructed four plant expression vector of FtUFGT based on the pCAMBIA3301-eGFP vector and transformed GV3101 Agrobacterium.Then we infested SR-1 tobacco by leaf disc method.Finally,77 FtUFGT1 overexpressed tobacco,36 UFGT6overexpressed tobacco and 2 UFGT7 overexpressed tobacco were selected.The content of total flavonoids in some UFGT1 overexpressed T₀ tobaccos were assayed by spectrophotometry.However,the total flavonoids in each strain was greatly different and we couldn't find any regularity.