| Chinese narcissus(Narcissus tazetta var.Chinensis)is a kind of bulb flower,belongs to Narcissus spp,Amaryllidaceae.It is one of the ten traditional flowers in China because of its beautiful patterns and sweet elegant.Nowadays the wild germplasm resources of narcissus are deficient and the flower colors of the cultivated varieties are single,which severely restrict the development of narcissus industry,therefore,the color improvement of Chinese narcissus has being one of the hotspots of many researches.To conquer this plight,the use of genetic engineering to modify narcissus flower color seems to be a feasible way to create new varieties.Unfortunately,for lack of systemically understanding of the flower pigmentation mechanism of Chinese narcissus,the utility of genetic engineering for the above purpose was frustrated.In order to change this situation,we should study the mechanism of flower tepal pigmentation of Chinese narcissus first."Jinzhanyintai" was used as materials in this research and it can be divided into four stages based on the development of tepals color.Tepals from four stages were used for transcriptome sequencing,and the color-related metabolic pathways were obtained through enrichment significantly differentially expressed genes from sequencing data.Then the expression pattern of structural genes from metabolic pathways color-related was analysis by semi-quantitative PCR and the pigments metabolites were measured by HPLC with traditional ethanol extraction.Based on the analysis above all,metabolic pathways of flower pigmentation of"Jinzhanyintai" were elucidated.The main results are described as follows:1.RNA-Seq analysis was performed on Chinese narcissus.A total of 63,141 unigenes were obtained,204 of which were putative homologs to color-related genes based on 29,730 unigenes of functional annotation.2.The expression patterns analysis of color-related genes in Chinese narcissus were conducted by semi-quantitative PCR with NtGAPDH as reference gene.The expression patterns of most genes were corresponding to the data from RNA-Seq.Many genes related to flower pigmentation synthesis showed up-regulated expression and genes related to flower pigmentation degradation showed down-regulated expression.3.The contents of seven kinds of flavonoids and four kinds of carotenoids in tepal and corona of Chinese narcissus were measured using HPLC.The results indicated that there were a large number of various pigmentations accumulated in tepal and corona.Main flavonoids were naringin and rutin and main carotenoids was lutein in petal.In addition,a small amount of beta carotene and zeaxanthin were also detected in petal,and zeaxanthin was detected in Chinese narcissus for the first time.The dynamics of their contents were consistent during four stages,showing that the pigments increased up to maximum at T2 stage then decreased gradually.Main flavonoids were naringin and rutin in corona,moreover,a small amount of taxifolin hydrate,quercetin and kaempferol were also tested in corona.And there are no significant differences in the kinds of carotenoids between corona and tepal.The change of naringin and rutin showed the same trend between corona and tepal.The Content of taxifolin hydrate has declined gradually since T1 stage and undetected at T4 stage.Quercetin and kaempferol were detected only at T1 stage.The changes of all carotenoids content in corona showed a different pattern,which was gradually increased along with the development of flower and up to the maximum in T3 stage,and then decreased gradually.All the analysis results showed that the lutein played a key role for the formation of yellow in corona of Chinese narcissus.4.The content of chlorophyll in tepal and corona was detected by ethanol extraction and the research with chlorophyll as flower pigmentation in Chinese narcissus was the first time.The results showed that the color differences in tepals of Chinese narcissus may be affected mainly by the chlorophyll content.Chlorophyll and carotenoids played an important role in the color of corona of Chinese narcissus.And flavonoids with high content may be important supplementary factors for pigmentation.5.Three major metabolic pathways related to tepal pigmentation of Chinese narcissus were deduced through a combination of bioinformatics from transcriptome sequencing data,gene color-related expression pattern by semi-quantitative PCR and pigments composition and change by HPLC with traditional ethanol extraction,providing a certain theoretical basis for the improvement of colors in Chinese narcissus by molecular breeding technology in the future. |
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