| Flavonoids are a class of important secondary metabolites in plants.In plant cells,flavonoids biosynthesis occurs in the cytosol and then transported and stored in vacuole or other cellular compartments.The biosynthesis pathway of flavonoid were clear in tea plants.However,the process of transporting flavonoids into vacuole wasn't understood,in which,Glutathione S-transferase may play important roles in it.The recombinant CsGSTs proteins were obtained by prokaryotic expression system.Enzymatic assay showed three all rCsGSTs had the activities of glutathione acyl transferase.The fluorescence quenching experiments of tryptophan confirmed three rCsGSTs could bind the tested flavonoids,among them,three rCsGSTs dislayed the strongest affinitie for anthocyanin.Homology modeling and docking studies showed quercetin shared the same binding sites with GSH in CsGSTa,CsGSTb and CsGSTc.MVD value also indicated that rCsGSTs had the strongest affinitie for anthocyanins.The heterologous plant expression vectors infused with CsGSTs were constructed and transformed into Arabidopsis thaliana and tobacco,respectively.The results showed that CsGSTa complemented the deficient phenotype of mutant tt19 completely.CsGSTb and CsGSTc could complement the deficient phenotype of mutant tt19 paritially.The relative experiments confirmed CsGSTs participated in accumulation of anthocyanin and proanthocyanidin.The content of anthocyanins and flavonols were higher than those of wild type tobacco G28,especially the contents of quercetin-3-O-glucoside,kaempferol-3-O-glucoside,and cyanidin 3-rutinoside.The results of the gene overexpression in tobacco showed the CsGSTs involved in accumulation of anthocyanin and flavonol.It was found that the expression proteins of the CsGSTs gene located in cytoplasm and on cell membrane detected by N.benthamiana transient expression.The above results indicate that CsGSTs genes in tea plant probably involved in the transport of anthocyanin,flavonol and proanthocyanidin. |
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