Identification And Function Analysis Of Glycosyltransferase Genes Related To Flavonoids And Anthocyanins Metabolism In Rice

Flavonoids are a kind of natural secondary metabolites which are ubiquitous in plants and play an important role in plants.Studies have found that the flavonoids have anti-cancer,antioxidant,antibacterial,and other functions,so flavonoids have important health care function to human beings.At the same time,the flavonoids can obviously enhance the adversity stress resistance of plants.Flavonoids exist in plants mainly in the binding state or free state.Glycosyltransferases are enzymes that add sugars to the receptor and catalyze the formation of glycosides.Glycosyltransferases modify the activity,stability and solubility of biomolec?Les.The glycosyltransferases in rice have many members,but the functions of most members are not clear.This paper mainly focused on the glycosylation modification of flavonoids and anthocyanins in rice.We identified the relevant glycosyltransferase genes and analyzed their functions,and thus laying a theoretical foundation for the improvement of nutritional quality and stress-resistant breeding of rice.The main research contents and results of this paper are as follows:1.Identification and function analysis of glycosyltransferase gene related to flavonoids in riceAccording to the published gene chip data,a batch of candidate genes of rice glycosyltransferases were selected,cloned,expressed and purified.Biochemical analysis revealed that the coding product of UGT40 glycosyltransferase gene can glycosylate flavonoids including quercetin,kaempferol,naringin and so on.In order to further investigate the role of UGT40 in vivo,we transferred this gene into Arabidopsis thaliana and obtained overexpression lines.First,we detected the content of flavonoids in transgenic Arabidopsis thaliana and found that UGT40 overexpressing plants could accumulate much more flavonoids than wild plants.At the same time,the expression of flavonoids synthesis related genes(CHS,CHI,FLS1,F3H)in transgenic plants was analyzed,and these genes were significantly up-regulated in UGT40 overexpressing plants compared with the wild type.It may be that the transfer of UGT40 affects the metabolic pathway of flavonoids,which leads to the accumulation of flavonoids.In addition,the stress resistance of transgenic Arabidopsis thaliana were analyzed.It was found that UGT40 overexpressing lines exhibited enhanced stress resistance in seed germination rate,cotyledon greening,root length and post-germination growth under osmotic or drought stress conditions compared with wild type.Some physiological changes of transgenic Arabidopsis thaliana under stress conditions were further determined.It was found that the contents of soluble sugar and proline were higher than that of the wild type,and the in vitro water loss rate was significantly lower than that of the wild type.At the same time,NBT and Evans Blue staining demonstrated the enhanced ability of scavenging reactive oxygen species(ROS)after UGT40 overexpression.Finally,the induced expression of stress resistance genes in plants under drought conditions was detected,including DREB2A,AIL1,COR47 and CAT1.The expression levels of these genes in UGT40 overexpression lines were significantly higher than that of the wild type.Therefore,we speculated that under stress conditions,the up-regulation of UGT40 expression might lead to the increase of flavonoids in plants.On the one hand,flavonoids,as scavengers of ROS,remove excessive reactive oxygen species,thus improving drought tolerance of plants.2.C loning and substrate identification of the anthocyanins related glycosyltransferase gene of purple riceBased on the predicted anthocyanin glycosyltransferase gene UGT33 sequence in the common rice database,a homologous gene of glycosyltransferase was obtained from purple rice by primer design and PCR amplification,and named PUGT33.It was found that the PUGT33 gene had a base substitution compared with the homologous gene UGT33 in ordinary rice.Comparing the enzyme activities of encoded proteins by these two homologous genes,it was found that the glycosylation activity of PUGT33 on anthocyanins was higher than that of UGT33.Furthermore,the transgenic Arabidopsis thaliana of PUGT33 was obtained.It was found that plants with PUGT33 overexpression exhibited significantly increased anthocyanin accumulation phenotype in the hypocotyl and other parts compared with the wild type.The anthocyanin content of transgenic lines was detected,and the results showed that anthocyanin content of transgenic lines was higher than that of wild type.In addition,the expression levels of genes related to anthocyanin synthesis pathway were also detected,including CHS,F3H,DFR,ANS and other genes.We found that the expression levels of these genes in transgenic Arabidopsis thaliana were significantly higher than that in wild type.The above research results on enzyme activity and heterogenic expression suggest that PUGT33 may be a glycosyltransferase gene responsible for anthocyanin glycosylation in purple rice.This gene may regulate anthocyanin synthesis in purple rice by affecting anthocyanin metabolic pathway and related gene expression.