Study On Flower Color Formation Of Rosa Rugosa Thunb.

Rosa rugosa Thunb., origin in China, has a long history of cultivation, including aromatic genes, winter hardiness and drought resistance, it is the excellent materials in landscaping and water conservation. Most of the varieties bloom purple flowers, only a few varieties bloom pink and white, yellow flowers, lack of orange and redflowers. Compared with chinese rose,flower colors of Rosa.r is very single which seriously limitting its application in landscaping.Therefore, it is important to cultivate new colors cultivars for expanding the scope of application in landscaping;however research of color mechanism and anthocyanin metabolic pathwayof Rosa.r is rare, so it is important to definite the substance of color reaction, reveal color mechanism, discussion reason of rare color of Rosa rugosa Thunb. which have the great theoretical and practical significance to propels the development of landscaping and development of ornamental industry.In this study, we used the petals of purple color varieties Rosa.R ‘Zi zhi’ and its bud sported varieties Rosa.R ‘Bai zizhi’ and Rosa.R ‘ Fen zizhi’ as the test materials, the anthocyanin, flavonoid and carotenoid related compounds of five blossom periods of three varieties were quantitatively and qualitatively, using high performance liquid chromatography(HPLC) and liquid chromatography-mass spectrometry(HPLC-ESI-MS) technique of presumption of anthocyanin and flavonoids mainly, using a high-throughput transcriptome sequencing and quantitative fluorescence methods, analysis the expression of anthocyanin biosynthesis related genes during blossom periods among different varieties, analysis rose anthocyanin metabolic pathway and rose color mechanism, aim to provide reference for the breeding of new flower color rose culticators.The main results of this study are followed:1. Petals of three varieties all contain flavonoids compounds, not including chalcone and aurones, ‘Zi zhi’ and ‘Fen zizhi’ petals also contain anthocyanin; HPLC assay showed that,during the period of five blossom three varieties were detected in trace beta carotene(beta-Carotene), lutein were not detected.2. The carotenoid content has little difference between the early stage of different varieties of roses; the total chlorophyll content of ‘Bai zizhi’ >’Fen zizhi’ > ‘Zi zhi’; three strains of carotenoid content and total chlorophyll content and change trend of difference,from the budding to decline sharply, the largest a decrease of more than 80%; the content of total flavonoids ‘Fen zizhi’ > ‘Bai zizhi’ >‘Zi zhi’, total flavonoids content of three varieties is relatively stable in the blossom periods.3. The structures of the presumption of anthocyanin biosynthesis of flavonoids.The results showed that: 20 peaks were detected in ‘Bai zihi’ rose petals, there were 13 kinds of them can be infer the structure, respectively are: quercetin-3-gentiobioside 、Quercetin 、 quercetin-3-sophoroside 、 kaempferol-3-gentiobioside 、 Lutin 、Kaempferol-3-sophoroside 、 Quercetin-3-glucoside 、 kaempferol-3-glucoside 、kaempferol-3-(ρ-coumaroyl)- rhamnoside-7-(acetyl)- rhamnoside 、kaempferol-3-(feruloyl)-glucoside-7-glucoside 、 Quercetin-3-galactoside 、kaempferol-3-feruloyl-glucosidkeaempferol-3-ρ-coumaroyl-glucoside-7-glucoside.There are 11 kinds of them can be infer the structure in ‘Fen zizhi’, respectively is:Quercetin-3-arabinose-glucoside、Lutin、Quercetin-3-sophoroside、kaempferol、Kaempferol-3-sophoroside、kaempferol-3-glucoside、kaempferol-3-ρ-coumaroyl- rhamnoside-7-acetylrhamnosid、kaempferol-3-(ρ-coumaroyl)-glucoside、keaempferol-3-ρ-coumaroyl-glucoside-7-glucoside、Quercetin-3-gentiobioside、Kaempferol-3-gentiobioside.There are 18 kinds of them can be infer the structure in ‘Zi zhi’, respectively is:Quercetin-3-gentiobioside、Quercetin-3-sophoroside、kaempferol-3-gentiobioside、Quercetin-3-arabinose-glucoside、Quercetin-3-galactoside、kaempferol-3-glucosid、Isorhamnetin-3-sophoroside、kaempferol-3-(ρ-coumaroyl)-rhamnoside-7-(acetyl)- rhamnoside、kaempferol-3-(feruloyl)-glucoside-7-glucoside、Quercetin-3-glucoside、kaempferol、Quercetin、Lutin、kaempferol-3-sophoroside、kaempferol-3-(feruloyl)-glucoside、kaempferol-3-(ρ-coumaroyl)-glucoside-7-glucoside、Isorhamnetin-3-glucoside、kaempferol-3-(ρ-coumaroyl)-glucoside,flavonoid compounds identified in this study are reported in previous studies.4. There are only Cyanidin-3,5-diglucoside and onidin-3-galactoside-5-glucoside can be detected in ‘Fen zizhi’; there are 8 glycosides can be detected in ‘Zi zhi’ rose petals respectively are:Cyanidin-3,5-diglucoside、Peonidin 3,5-diglucoside、Peonidin-3-galactoside-5-glucoside、Delphinidin 3,5-diglucoside、Delphinidin-3-glucoside、Peonidin-3-cis-(ρ-coumaroyl)-glucoside-5-glucoside、Pelargonidin-3,5-diglucoside、peonidin-3-trans-(coumaroyl)-glucoside-5-glucoside; ’white Zizhi’ rose petals have not anthocyanin.5. The relative quantitative analysis results of the ‘Zi zhi’ rosocyanine showed that: the relative content of 8 kinds of anthocyanin were the highest in flower budding period and half bloomming periods, and the relative content of cyanidin glycosides, paeoniflorin and pelargonin with the flower opening gradually decreased, and the relative content of two kinds of pigment delphinidin glycosides in blossom process changes little.6. There are 16 flavonoids derivatives, kaempferol and quercetin and kaempferol derivatives of acyl derivatives as the main component; content of quercetin and kaempferol related components stabily of the entire blossom periods; content of acylation material kaempferol decreased with the flower blooming in the flowering stage, content of paeoniflorin is the lowest, and the same trend; amounts of flavonoids also contains trace methylated derivatives of quercetin and isorhamnetin derivatives, its content with the flowers reduced.7. The components of anthocyaninin in ‘Zi zhi’ rose are paeoniflorin, anthocyanin accounted for more than 80%, followed by delphinin, accounting for about 10%; centaurin,accounted for about 5%; few pelargonin content, accounted for about 0.3%. Change of content of delphinin and pelargonin during the blossom periods is small; little change of paeoniflorin in four blossom periods, but in the end of flowering compared withe bud degradation significantly, relative reduction of nearly 50%; in the five blossom periods, tre atio paeoniflorin to delphinin has obvious change, reduce more than 50% of flowering compared with late bud stage.8.The flavonoids components in ‘Zi zhi’ rose are mainly flavonoids, quercetin,kaempferol glycoside, acylation and methylated derivatives, content of basic substances of quercetin and kaempferol is very low, the proportion is very small; in accordance with modification of structure analysis, the vast majority of glycosides and content of acylated derivatives of kaempferol and quercetin, flavonoids were very low.9. There may be two different methylation pathway ways in ‘Zi zhi’ rosocyanine,methylation of different catalytic enzyme substrate could have more than one, related with not reported in previous studies.10. Three varieties of rose petals were used: a summary of the results of the transcriptome‘Fen zizhi’, ‘Bai zizhi’ and ‘Zi zhi’ rose petals with total RNA transcripts sequenced, there were 71415 unigenes, a note to the NR database, a total of 43564 comments to the NT database, a total of 46918, a note to the SWISS-PROT database, a total of 27336 comments to the KEGG database, a total of 24765 comments to the COG database, a total of 15570 comments to the GO database, a total of 30275.11. Fluorescent quantitative analysis of key genes in the biosynthesis of anthocyaninBy using the methods of fluorescence quantitative PCR of 16 key genes of anthocyanin biosynthesis in the three kinds of color expression pattern in the rose varieties, 14 quantitative results and transcriptome data. Part of the structure of FLS gene fragment F3 H, F3, H, UA3 ’,the expression of 5GT in the’ Purple Branch ’in a rose was significantly higher than that in the expression ‘Zi zhi’ and ‘Bai zizhi’, play an important role that these gene structure formed in the ‘Zi zhi’ flower color.Four class MYB genes expression: the expression of ‘Zi zhi’ was significantly higher than that of ‘Bai zizhi’ and ‘Fen zizhi’. We believe that the expression of MYB gene in ‘Zi zhi’played an important role. This study from the transcriptome data to search for homologous gene methylation enzyme.