The Mechanism Study On Flavonoid 3-O-glycosyltransferase Regulating The Formation Of Flower Color Of Rhododendron Delavayi

Rhododendron delavayi is an evergreen shrub or small tree which belongs to Ericales.It has high ornamental value because of the deep or bright red color in its flowers.Meanwhile,R.delavayi also have various medicinal values as its roots,stems,leaves and flowers can be used to clear away heat and detoxification,stop bleeding,cool blood,regulate menstruation and so on.Anthocyanins,a kind of flavonoids,are important secondary metabolites in plants which also regarded as one of the main substances that affect the formation of flower color.In anthocyanin pathway,flavonoid 3-O-glucosyltransferase(3GT)acts on the downstream and plays the pivotal role,because it can enhance the stability and water solubility of anthocyanin through glycosylating the anthocyanidins.Anthocyanin composition analysis of R.delavayi flowers showed that3-O-glycosylated anthocyanins were the most abundant in its flowers,which accounted for more than 93% of total anthocyanins.Thus,flavonoid 3-O-glycosyltransferasemay play a key role during the color formation of R.delavayi.Therefore,two flavonoid 3-O-glycotransferase genes(Rd3GT1 and Rd3GT6)related to anthocyanin synthesis were successfully screened in this study based on the correlation analysis between anthocyanin accumulation patterns and 3GT gene expression levels.Transcriptional analysis revealed that both Rd3GT1 and Rd3GT6 were expressed in all tested tissues.And during flowers developmental stages,Rd3GT1 expression showed a gradually increasing trend,while Rd3GT6 were opposite.Then,analyses of physical and chemical parameters,enzyme catalytic site of Rd3GT1 and Rd3GT6 were performed,and eukaryotic as well as prokaryotic expression vectors were successfully constructed.Genetic transformation of Arabidopsis thaliana and tobacco together with enzyme activity analyses confirmed the function of Rd3GT1 and Rd3GT6 in regulating the flower color formation of R.delavayi.Overall,the above results will provide a theoretical basis for the mechanism study on flower color formation of R.delavayi as well as the improvement of azalea flower color.The main results are as follows:1.Screening of the key 3GT gene for R.delavayi flower color formation.HPLC analysis showed that eight kinds of anthocyanins were detected in the flowers of R.delavayi,and these anthocyanins were then identified as delphinin galactoside,cyanidin galactoside,delphinin rhamnoside,cyanidin glucoside,cyanidin arabinoside as well as three cyanidin derivatives.And the contents of 3-O glycation anthocyanins were the most abundant all the times(accounting for 93.68～96.31% of total anthocyanins).Subsequently,two key flavonoid3-O-glycotransferase genes(Rd3GT1 and Rd3GT6)involved in flower color formation of R.delavayi were successfully screened according to the correlation analysis between anthocyanin accumulation patterns and expression levels of 3GT genes.2.Cloning and bioinformatics analysis of Rd3GT1 and Rd3GT6.Rd3GT1 and Rd3GT6 were successfully cloned by RT-PCR.The open reading frames of Rd3GT1 and Rd3GT6 were 1395 bp and 1365 bp,encoding 465 and 455 amino acids respectively.The predicted protein molecular weight of Rd3GT1 and Rd3GT6 were 50.459 and 49.556 KD,and both of them belong to the GT-B ultrasound family of glycosyltransferases which contain the PSPG conserved domain that unique to glycosyltransferases.Furthermore,the catalytic active site and amino acid residues that interact with sugar donors were also found in Rd3GT1 and Rd3GT6.Phylogenetic analysis displayed that Rd3GT1 was most closely related to the glycosyltransferase of Coffea arabica,while Rd3GT6 was most closely related to the glycosyltransferase of Vaccinium corymbosum.3.Eukaryotic expression vectors construction and genetic transformation of Rd3GT1 and Rd3GT6.The eukaryotic overexpression vectors p BI121-Rd3GT1 and p BI121-Rd3GT6 were successfully constructed,and then were introduced into Arabidopsis 3GT mutants.The results prove that Rd3GT1 and Rd3GT6 genes encode the flavonoid 3-O-glycotransferase genes which are fully functional in plants.Later,both Rd3GT1 and Rd3GT6 were overexpressed in tobacco to confirm their effection on flower color formation.The results showed that tobacco produced deeper pink flowers,and the kinds of anthocyanins in petals increased from 2 to 5,indicating that Rd3GT1 and Rd3GT6 fulfill the function on anthocyanins synthesis and could be used for the improvement of plant flower color.4.Prokaryotic expression vectors construction of Rd3GT1 and Rd3GT6 and enzyme activity detection in vitro.Prokaryotic expression vectors p ET32a(+)-Rd3GT1 and p ET32a(+)-Rd3GT6 were successfully constructed and inductively expressed in E.coli BL21(DE3).The enzymatic activity assays of the purified recombinant proteins were performed by using cyanidin as substrate and UDP-glucose as sugar donor.The results verified that the recombinant proteins of Rd3GT1 and Rd3GT6 could transfer UDP-glucose to the 3-position of cyanidin for the formation of cyanidin3-O-glucoside,suggesting they possess the activity of flavonoid3-O-glucosyltransferase.So the above results will lay a foundation for the further analysis of substrate specificity and glycogen donor specificity of Rd3GT1 and Rd3GT6.