| Rosa rugosa originated in China,it is an important garden ornamental plant whichbelongs to the genus Rosa of the family Rosaceae with graceful shape and sweet smell.As one of its most important ornamental properties,coloration directly affects its ornamental value.Wild and cultivated petals of R.rugosa are mostly purple,pink,a small amount of white,but lack of yellow.Although people had bred new varieties of R.rugosa before,they had never got a variety with yellow flowers.Therefore,the application of R.rugosa in garden was restricted until now.We used resources of R.rugosa for distant hybridization and produce four new varieties of R.rugosa（R.rugosa‘Miaoyu’,R.rugosa‘Rudiepianpian’,R.rugosa‘Jiaomeisanbian’and R.rugosa‘Sijihuangmei’）.And these cultivars’petals appear yellowcolor or at least have a small amount of yellow color.It is an important research topic thatwhat the internal factors cause these new varieties to be different from the original varieties of R.rugosa and appear yellow.In this study,the petals of Rosa xanthina,wild R.rugosa‘Hunchun’and their four new hybrid varieties at initial open stage were used as plant materials,six kinds of anthocyanins including cyanidin 3,5-di-O-glucoside（Cy3G5G）,pelargonidin 3,5-di-O-glucoside（Pg3G5G）,peonidin 3,5-di-O-glucoside（Pn3G5G）,cyanidin 3-O-glucoside（Cy3G）,pelargonidin3-O-glucoside（Pg3G）,peonidin 3-O-glucoside（Pn3G）and chalcone were analyzed in their petals.In addition,the petals of wild R.rugosa‘Hunchun’were used as plant materials,two key structural genes RrFLS and Rr F3’H in the flavonoid pathway were cloned according to the transcriptome data,and the bioinformatics analysis was carried out.Moreover,the petals of Rosa xanthina,wild R.rugosa‘Hunchun’and their four new hybrid varieties at initial open stage were used as plant materials,the expression level of seven structural genes（RrCHS,RrCHI,RrF3H,RrFLS,RrF3’H,RrDFR and RrANS）in the biosynthesis pathway of flavonoids had been quantitatively analyzed in this experiment by using qRT-PCR.The main results are as follows:1.It is the first time to clone the two key genes of RrFLS and RrF3’H in the flavonoid pathways used the petals of wild R.rugosa‘Hunchun’at initial opening stage as an experimental material.RrFLS（GeneBank accession Nos:MG735185）,has an open reading frame（ORF）with length of 1005 bp,and encoding 335 amino acids;RrF3’H（GeneBank accession Nos:MG735186）,has an ORF of 1530 bp,and encoding 510 amino acids.2.The two structural genes of RrFLS and RrF3’H are very stable and conservative.RrFLS belongs to the 2OG-Fe（II）oxy superfamily,and RrF3’H belongs to the P450superfamily,with corresponding conservative structure domains.By comparison,the encoding amino acid of RrFLS and RrF3’H have a high similarity with the the encoding amino acid in Prunus persica,Prunus avium and Prunus mume,the similarity can reach over80%.3.The formulas of proteins encoded by RrFLS and RrF3’H were C1731H2681N443O502S9and C2666H4149N699O734S24 respectively.The secondary structures of the two proteins are formed by alpha helix,random coil,beta turn and extended strand,inaddition,alpha-helix and random coil are the main composition.O-glycosylation sites and numerous phosphorylation sites are existed in the two proteins.The protein encoded by RrFLS is stable hydrophilic protein,no signal peptide,no transmembrane construction.The protein encoded by RrF3’H is unstable hydrophilic protein,and has a signal peptide and a transmembrane structure.4.Cy3G,Cy3G5G,Pg3G,Pg3G5G,Pn3G and Pn3G5G the six anthocyanins were all detected in‘Hunchun’.A small amount of Cy3G5G was detected in the‘Miaoyu’and‘Jiaomeisanbian’.None of these six anthocyanins were detected in Rosa xanthina and other varieties.Therefore,we concluded that except for‘Hunchun’,only one anthocyanin synthesis pathway exist in the petals of other plant materials,which synthetic cyanidin.According to the results of HPLC,the content of chalcone is very low in all plant materials,so chalcone is not the substance that makes the petals appear yellow.5.The petals of Rosa xanthina,wild R.rugosa‘Hunchun’and their four hybrid varieties（R.rugosa‘Miaoyu’,‘Rudiepianpian’,‘Jiaomeisanbian’and‘Sijihuangmei’）at initial opening stage were used as plant materials.The expression level of seven structural genes（Rr CHS、RrCHI、RrF3H、RrF3’H、RrFLS、RrDFR and RrANS）had been quantitatively analyzed in this experiment by using qRT-PCR.We found that RrFLS,RrDFR and RrF3’H were the extremely important genes that make their petals appeared different colors.6.Dihydroflavonol 4-reductase（DFR）was specific to substrate binding in Rosa xanthina and three hybrid culvars.The content of flavonol and anthocyanin would be different because of the enzymes（FLS and DFR）have different abilities to compete for the substrate of dihydrokaempferol in‘Rudiepianpian’and‘Miaoyu’,which lead to the color change of petals. |
