The Cloning And Characterization Of Glucosyltransferase Genes In Tartary Buckwheat And Their Expression After Cold Stress

Tartary buckwheat is a medicinal and edible grain crop.It has become an important functional food material for exploiting and utilizing.As a new kind of health food,tartary buckwheat sprouts have become increasingly popular by vegetarians because of high anthocyanin content.Proper eating anthocyanin can reduce the probability of suffering miocardial infarction,and prevent cardiovascular and cerebrovascular diseases,and promote the resynthesis of erythrolabe.Increasing anthocyanin content is key to improve the quality and economic value of tartary buckwheat sprouts.Isolation and identification of key enzyme in anthocyanin synthesis has important theoretical significance and application value.UDP-Glucose:flavonoid 3-O-glucosyltransferase(UFGT)is the key enzyme of anthocyanin biosynthetic pathway.Glycosylation can alter the solubility,stability and color of anthocyanin.Glycosylation products play significant roles in many physiological and biochemical processes of plant.In this study,tartary buckwheat('Xi Qiao No.2')was used as material.UFGT,a key enzyme gene in anthocyanin biosynthetic pathway,was cloned.The structure,function and expression of UFGT were studied for understanding molecular mechanism of anthocyanin synthesis and for providing a theoretical basis in artificial cultivation of high anthocyanin in buckwheat sprouts.The main results are as follows:1.The total RNA of buckwheat flower was used as material.According to tartary buckwheat flower transcriptome database,three UFGT-like cDNA sequences were obtained and designated FtUFGTl,FtUFGT2 and FtUFGT3.Sequence analyses showed that the ORF of FtUFGTl,FtUFGT2 and FtUFGT3 were 1341,1500,1413 bp,encoding 446,499,470 amine acid resides,respectively.Molecular docking analyses showed that FtUFGT1-3 can be successfully docked with UDP and cyanidin.Their structures are mainly composed of N-and C-terminal domains.The sugar acceptor interacts with the N-terminal domain,whereas the sugar donor interacts with the C-terminal domain.Phylogenetic tree analyses indicated that FtUFGT1-3 belong to the UF3GT family.2.Anthocyanin content determination showed that anthocyanins mainly distributed in flowers,followed by leaves and roots,and the lowest is in stems.qPCR showed that the expression of FtUFGT1-3 have tissue-specificity and can be detected in all tissues,with the highest expression level in the flower.In addition,the expression levels of FtUFGT3 were much higher than those of FtUFGT1-2.FtUFGT3 may be major gene in anthocyanin glycosylation.3.To analyze the gene expression of FtUFGT1-3 and anthocyanin synthesis at seedling stage under cold stress,qPCR and spectrophotometry was carried out.The results showed that FtUFGT1-3 were up-regulated after cold treatment.The highest level was FtUFGT1,followed by FtUFGT2 and FtUFGT3.Anthocyanin content of tartary buckwheat sprouts quickly increased after cold treatment and peaked at 6 h,then stabilized at 12-24 h.The peak level of anthocyanin in the cold-treated sprouts was about 2.3-fold higher than that of the control.4.The ORF of FtUFGT1-3 were constructed into prokaryotic expression vector pGEX 4T-1.Each construct was transformed into E.coli BL21(DE3)cells.The culture was collected under a final concentration of 0.2 mM,incubating at 25 ? for 10 h.SDS-PAGE showed that FtUFGTl,FtUFGT2,and FtUFGT3 GST-fusion proteins achieve soluble expression,and their molecular masses were estimated to be 74,80,and 76 kDa.5.After being purified by glutathione-agarose affinity chromatography,the recombinant proteins were cleaved using thrombin to remove GST tag,obtaining high purity protein of FtUFGTl-3.The activities of FtUFGT1-3 were assayed by HPLC-MS/MS.The results showed that cyanidin 3-O-glucoside was generated in the reaction products.FtUFGTl,FtUFGT2 and FtUFGT3 all have catalytic activity of glucosyltransferase.