Cloning And Functional Analysis Of Glycosyltransferase Genes Related To Anthocyanin Biosynthesis In Rosa Rugosa

Rosa rugosa is a perennial deciduous shrub of genus Rosa in the Rosaceae family.It has beautiful flowers,fragrant smell,good shape and color,and has very high ornamental values.Although there are many kinds of R.rugosa,they are less in color,mainly pink and purplish red,less in white,and lack of yellow,deep red,orange and compound color.Therefore,how to innovate color of R.rugosa has become the main goal of breeders.Anthocyanin is closely related to plant flower color.Anthocyanin is a water-soluble natural pigment widely found in flowers,stems,leaves,fruits,seeds and other organs of natural plants.It is synthesized by the anthocyanin biosynthesis pathway of flavonoid synthesis pathway.It plays a dominant role in the color process of R.rugosa petals.Although many genes have been reported to regulate the formation of anthocyanin in anthocyanin biosynthesis pathway,there are few reports on downstream structural genes such as glycosyltransferase genes.The final formation of anthocyanin depends on the glycosylation of glycosyltransferases.Glycosylation plays an important role in increasing the stability and solubility of anthocyanin in plants.At present,there are few studies on the mechanism of R.rugosa flower color formation.Therefore,the study on the function and molecular mechanism of glycosyltransferase genes in anthocyanin biosynthesis is of great significance in elucidating the color mechanism of R.rugosa petals and further improving the R.rugosa flower color.In this study,R.rugosa'Zizhi'and R.davurica were used as the main experimental materials.Based on the transcriptional data of rose obtained in our laboratory,two full-length CDS sequences of glycosyltransferase genes?RrGT1 and RrGT2?were isolated from the cDNA of rose petals by RT-PCR method.The sequence structure,physicochemical properties and so on were identified and analyzed by bioinformatics.qRT-PCR technique was used to analyze the gene expression of two glycosyltransferase genes in the petals of R.rugosa'Zizhi'and R.davurica in different tissues and in different flowering stages.Two glycosyltransferase genes were silenced in R.rugosa'Zizhi'and R.davurica plants by virus induced gene silencing?VIGS?technique in the field,respectively,to verify their function in flower color formation.Two glycosyltransferase genes were stably transformed into tobacco by transgenic technique to verify their biological function in different species.After the transgenic tobacco plants were obtained,the R.rugosa glycosyltransferase genes were silenced by VIGS technique.The function of two glycosyltransferase genes in anthocyanin biosynthesis was further investigated and verified in positive and negative directions of gene expression level.The main results are as follows:1.The full-length CDS sequences of two glycosyltransferase genes were first cloned from the cDNA of R.rugosa'Zizhi'flower petals at budding stage.RrGT1,GeneBank accession Nos:MK034140,has an open reading frame?ORF?with length of 1161bp,and encoding 386 amino acids;RrGT2,GeneBank accession Nos:MK034141,has an ORF of1422 bp,and encoding 473 amino acids.2.Two glycosyltransferase genes both contained a typical PSPG domain composed of 44amino acid residues and belonged to the GTB superfamily.The formulas of proteins encoded by RrGT1 and RrGT2 were C₁₈₇₉H₂₉₆₄N₄₉₄O₅₅₆S₁₄ and C₂₃₃₄H₃₆₂₈N₆₀₂O₇₁₁S₁₈,respectively.The prediction results showed that there were mainly alpha-helix and Random coil in the secondary structures of the two glycosyltransferase proteins.The results of 3D structure prediction of protein show that both of the two glycosyltransferase proteins can construct 3D model using the existing 3D structure of glycosyltransferase as template.The prediction results of the basic physical and chemical properties of the protein showed that the two glycosyltransferase proteins were unstable acidic proteins.Both have glycosylation sites,many phosphorylation sites,no signal peptide and transmembrane domain,and belong to non-secretory protein.Phylogenetic tree analysis showed that the relationship between the two glycosyltransferase genes and most of the similar genes in other species was relatively distant,which suggested that the two genes had higher species specificity.3.RrGT1 and RrGT2 were both expressed in five flowering stages and seven tissues of R.rugosa'Zizhi'and R.davurica,but there were significant differences in expression levels.To sum up,the expression of RrGT1 and RrGT2 may be developmentally regulated and has a certain tissue specificity,which may be related to the biosynthesis of anthocyanin.In addition,because of their high expression level in leaves,it is assumed that they are also related to the glycosylation of other secondary metabolites in leaves.4.VIGS reduced the transcript abundance of the endogenous RrGT1 and RrGT2 gene in R.rugosa'Zizhi'and R.davurica.The petals in the control group and TRV-GFP group showed no definitive changes in color,but the petals in the gene silencing group were clearly lighter in color.The results of qRT-PCR analysis showed that the expression of endogenous RrGT1and RrGT2 in empty virus vector group and control group were basically the same,while the expression of endogenous RrGT1 and RrGT2 in gene silencing group were significantly decreased.The relative expressions of the six key structural genes?RrCHS,RrCHI,RrF3H,RrF3'H,RrDFR and RrANS?upstream of the RrGT1 and RrGT2 genes in the anthocyanin pathway in the petals of R.rugosa'Zizhi'and R.davurica were detected by qRT-PCR.The results showed that the relative expression of the six genes in the control group,empty virus vector group and gene silencing group of R.rugosa?Zizhi?and R.davurica did not clearly change.Therefore,the effects of these upstream structural genes can be excluded.It is inferred that the shallowness of flower color may be related to the posttranscriptional silencing of endogenous RrGT1 and RrGT2 genes.5.The kinds and contents of anthocyanin in the petals of R.rugosa'Zizhi'and R.davurica were determined qualitatively and quantitatively by HPLC.In?Zizhi?,six kinds of anthocyanins were detected:Cy3G5G,Pg3G5G,Cy3G,Pn3G5G,Pg3G,and Pn3G.Pn3G5G had the highest content,while Cy3G5G had the second highest;the contents of the other four anthocyanins were relatively low.Compared with those in the control group and TRV-GFP group,the contents of several anthocyanins in response to the VIGS treatment were clearly reduced.Pn3G5G exhibited the greatest decrease in content,followed by Cy3G5G;the content of Pg3G was no longer detectable.In R.davurica,the six anthocyanins listed above were also detected.However,Cy3G5G had the highest content,and Cy3G had the second highest content;the contents of the other four anthocyanins were relatively low.Compared with those in the control group and TRV-GFP group,the contents of the six anthocyanins in response to the VIGS treatment were clearly reduced.Cy3G5G exhibited the greatest decrease in content,followed by Cy3G;no detection of Pn3G was observed.And this was also the first time to find that the flower color of R.davurica is mainly controlled by Cy3G5G.6.Overexpression of the RrGT1 and RrGT2 genes increased the anthocyanin accumulation in tobacco.Compared with empty expression vector group and control group,the corolla color of transgenic tobacco lines with RrGT1 and RrGT2 genes was significantly deeper.It was found that the main anthocyanin detected in tobacco corolla was cyanidin glucoside,which was consistent with previous studies.Compared with empty expression vector group and control group,the anthocyanin contents of corolla of transgenic tobacco lines with RrGT1 and RrGT2 genes was significantly increased.It can be inferred that RrGT1and RrGT2 genes are related to anthocyanin biosynthesis in tobacco.7.VIGS reduced the anthocyanin accumulation in the corolla of transgenic tobacco.Transgenic tobacco lines with RrGT1 and RrGT2 genes were silenced by exogenous RrGT1and RrGT2 genes.Compared with control group and empty virus vector group,the color of corolla in gene silencing group was significantly lighter than that in control group and empty virus vector group.The expression of RrGT1 and RrGT2 genes in the gene silencing group decreased significantly,and the anthocyanin content in the tobacco corolla also decreased significantly in the gene silencing group.Therefore,it can be inferred that RrGT1 and RrGT2genes are the key enzyme genes directly affecting anthocyanin biosynthesis.