| To discuss the composition and metabolism mechanism of flavonoids in flowers of Narcissus tazetta var.chinensis Roem,petals and coronals at different developmental stages were used as materials,acid-hydrolysis method was employed on extracting flavonoids,and HPLC-MSn technology was applied on qualitative and quantitative analysis,combined with expression spectrum analysis and RT-PCR analysis,flavonoids synthesis pathway was studied.The main results are as follows.1 Extraction and identification technology of flavonoids in Narcissus tazetta var.chinensis flowers were establishedPetals from three years old Narcissus bulbs was used as materials.The advantages and disadvantages of two extraction methods including non-acid hydrolysis and acid hydrolysis method were compared by HPLC-MSn technology.The mobile phase composition of HPLC-MSn was also screened.The results showed that chlorogenic acid,quercetin and isorhamnetin obtained by acid hydrolysis were identified by HPLC-MSn mobile phase and gradient elution extraction conditions,and chlorogenic acid,rutin and narcissin obtained by non acid hydrolysis were identified.Quercetin and isorhamnetin are aglycon of rutin and narcissin respectively.Considering that the flavonoids extracted by non acid hydrolysis can provide more structure information,the non acid hydrolysis were chose as extraction method in this study.Retention time and peak type were compared under two mobile phase compositions including 1%acetic acid methanol and 1%acetic acid acetonitrile in the same extraction method and gradient elution.The result showed that 1%acetic acid acetonitrile was better than 1%acetic acid methanol.2 Changes of main components of flavonoids in Narcissus flowers during development process was determinedThe contents of chlorogenic acid,rutin and narcissin in corona and petals under different development stage were analyzed by HPLC-MSn technology.The results showed that the three components were not detected in buddha flame bud.With flower opening,the content of chlorogenic acid was highest in the green bud period of petals,then gradually decreased.The content of chlorogenic acid in petals were higher than that in crown.Rutin content was the highest in the green bud stage,and the content in corona was slightly higher than that in petals.Narcissin also showed a similar change trends,the content in green bud stage was the highest,the contents in crown was higher than that of petals.The content of narcissin in petals was higher than that in corona during full opening flower stage.In the same period,the content of narcissin was the highest at the same stage,and the eontent of chiorogenie was lowest.3 Construction of expression pattern and screening of differential expression genes in fiavonoid biosynthesisTo construct digital expression of Narcissus tazetta var.chinensis development spectrum,base quality values analysis results show that the average quality of base value distribution mostly in between 30?40(Q30?Q40),namely the sequence recognition error rate between 0.1%?0.01%.The statistics of the sequencing output:received a total of 68.79M reads,the total base number as 3.49G.in sequencing quality value statistical evaluation on the base of Q30,found in more than 85%.And compared to the reference genome,high efficiency comparison of 7 samples of reads with the reference genome,were more than 76%,showed that the selected reference gene sequencing and better meet the needs of subsequent analysis.Through DNA random testing,the distribution curve of Narcissus tazetta var.chinensis color development in each period of sample the relatively flat,indicated that all samples randomly obtained better overall sequencing.Two pathways of flavonoid metabolism in KEGG and related notes,respectively for flavonoid biosynthesis As group of the biosynthesis and ko00941),flavone and flavonol biosynthesis(flavone and flavonol biosynthesis,ko00944).Among the samples of different developmental stages of pairwise alignments that were differentially expressed genes in 13.Among them,in the flavonoid biosynthesis pathway 10,respectively:CHS?CCoAOMT?F3'H?C4H?HCT?C3'H.Flavone and flavonol biosynthetic pathway 4,respectively:F3-OMT?F3'H.Through BAST software of difference expression gene sequence and NR,Swiss prot,GO,COG,KEGG database for comparison,differences in the gene annotation information4 The expression patterns of differential expression genes during flower development were studied by RT-PCRIn the early period of the Buddha flame involucre(E0)expression level of each gene were compared in Narcissus tazetta var.chinensis petal flavonoids,flavone and flavonol differential gene expression in different ways in the period of F3'H,the expression of F3-OMT-1 decreased,F3-OMT-2,F3-OMT-3,C4H CHS,CCoAOMT-3 expression increased in the petals of Buddha flame involucre mid(E01-2)expression was the highest,then decreased,C3'H,CCoAOMT-1,CCoAOMT-2 expression level gradually increased.Compared Narcissus tazetta var.chinensis corona flavonoids,flavonoids and differential gene expression in different periods,F3-OMT-1,F3-OMT-2,F3-OMT-3 expression gradually decreased,F3'H,CHS expression increased in the green bud stage,the Deputy crown(E03)the highest expression level,then decreased,C3'H,CCoAOMT-1,CCoAOMT-2 expression gradually increased the expression of.CCoAOMT-3 in the overall upward trend in the green bud stage of corolla(E03)expression was the lowest.The Buddha flame involucre period Stamens(E01-1)expression of each gene were compared Chinese daffodil petals stamens,flavonoids,and the expression of genetic differences in the mid Huang Tonghe Buddha flame involucre flavonol pathway in F3'H,F3-OMT-1,F3-OMT-2,F3-OMT-3,C3'H,CCoAOMT-3,expression of C4H,stamens longer than petals,CHS,CCoAOMT-1,CCoAOMT-2 expression petals higher than stamens with green bracts.Petals during(E02)expression level of each gene were compared Narcissus tazetta var.chinensis petals,deputy crown flavonoids,flavone and flavonol differential gene expression in the green way,bud period of the F3'H,F3-OMT-1.F3-OMT-2,F3-OMT-3,CCoAOMT-1,CCoAOMT-2 expression was higher than that of petals corona,C4H the expression of CCoAOMT-3,CHS,.C3'H gene in the eorona above petals in two parts of the expression has no significant difference.The blooming petals(E04)expression of each gene were compared in Narcissus tazetta var.chinensis petals,corona flavonoids,flavonoids and Huang Tongchun Differential gene expression in flowering stage,F3'H,F3-OMT-3,CHS,CCoAOMT-1,expression was higher than the amount of petals,F3-OMT-1,F3-OMT-2t C3'H,C4H CCoAOMT-2,CCoAOMT-3 expression of the Deputy crown higher than the petals.CHS,F3'H,F3-OMT expression of the amount of changes in overall presentation in front of the florescence accumulation,flowering stage decreased trend,rutin and,narcissin in Narcissus tazetta var.chinensis development patterns of variation in the process of consistent,which F3-OMT may specific recognition using rutin as substrate methyl of narcissin formation and change raw material chemical activity and more stable in structure,the in front of the florescence of accumulation for the downstream anthocyanins and synthetic substrates may Narcissus tazetta var.chinensis failed to colored anthocyanins were found. |
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