| Chinese dwarf cherry(Cerasus humilis(Bge.)Sok)is a unique fruit tree resource with high flavonoid content in China.The fruit tree is rich in germplasm resources and widely distributed in ecological groups.There are obvious differences in the content of flavonoid and the composition of metabolic substances among germplasm.In this study,the content and components of flavonoids in mature fruits of different germplasm resources firstly determined;then the dynamic changes of flavonoids content and components in fruits of several germplasm at their different fruit development stages were studied.The molecular regulation mechanism of flavonoid metabolites in C.humilis was studied by transcriptome and metabonomics analysis.The important structural genes of flavonoids metabolism in C.humilis fruit were explored to lay an important foundation for the study of the metabolic pathway and molecular regulation of flavonoids in C.humilis.The main results are as follows:1.The main characteristics of different C.humilis germplasms were determined,large variation was found in the single fruit weight and stone weight representing rich genetic diversity.The soluble solids content showed small variation indicating simple heredity.The fruit shape of C.humilis was basically oblate.Flavonoids content and antioxidant capacity of different germplasm differentiated significantly(P<0.01),with high variability and rich genetic diversity.Flavonoid contents in the same germplasm showed small variation in two years indicating that it is relatively stable.The flavonoid content of most Chinese Dwarf Cherry germplasm were in the medium range(9.57-15.23 mg/g FW),with a general normal distribution and quantitative genetic characters controlled by multiple genes.Among all the germplasms,the content of catechin was high,and the correlation between catechin and flavonoid content was the largest,which may be an important component affecting the flavonoid content in C.humilis fruit.There was no obvious correlation between the red peel color and the flavonoid content,however,it had a strong correlation with cyanidin-3-O-glucoside,suggesting that it is very important for peel color formation of red fruits.2.With the development period of C.humilis fruit,the variation trend of flavonoids content in different germplasms of C.humilis was slightly different,and the content of flavonoid in ‘Nongda5’showed a trend of continuous decline.There was a small increase in flavonoid content from the young fruit stage to the hard core stage,followed by a decreasing trend,and then to the lowest level in the mature stage.The trend of antioxidant capacity(ABTS,FRAP,DPPH)with the flavonoid content of C.humilis fruit was basically the same,and the correlation analysis results showed that the flavonoid content of C.humilis fruit was positively correlated with the antioxidant index(P< 0.01).Catechin,rutin and quercetin-7-O-glucose were detected in the six developmental stages of the four germplasms of C.humilis fruit,Among them,catechin was the highest component,accounting for about 10%.Myricetin and quercetin were generally detected only in the early stage of fruit development,but not in the later stage of fruit development.Correlation analysis showed that the flavonoid components with the content of flavonoids and antioxidant index of had an certain difference between different germplasms,overall rutin,catechins and liquiritigenin with the content of flavonoids and antioxidant index correlation is stronger,quercetin-7-O-β-D-glucopyranoside and epicatechin with the flavonoids content and antioxidant index correlation is weak.Cyanidin-3-O-glucoside was not detected until the beginning of coloration in two red C.humilis germplasms,‘Nongda 4’ and ‘DS-1’.3.A total of 171 kinds of flavonoids components were detected in the two germplasm of ‘Nongda 4’and ‘Nongda 5’,among which 170 were ‘Nongda 4’ and 145 were ‘Nongda 5’,After the classification of these components,they can be classified into isoflavones,flavones,flavonoids,flavanols,flavanones,flavanols and anthocyanins.There were 38 different metabolites in the two germplasms,which may be the reason for the difference in the total amount of flavonoids in the two germplasm fruits.After specific classification,12 different metabolites can be classified into anthocyanins,which may be the main reason for the formation of different epigenetic characteristics of C.humilis fruit.4.Through transcriptome sequencing technology,the ‘Nongda 4’ and ‘Nongda 5’ C.humilis fruit databases were constructed,and the effective data of each sample reached 6.08 gb,and the percentage of Q30 bases was 93.03% or above.Through KEGG metabolic pathway analysis,a total of 51 genes related to flavonoid synthesis involving 13 enzymes were obtained.After differential analysis,PAL,4CL,CHS(2),DFR,F3 H,F3’H,LDOX and LAR.In the fruit of ’Nongda 5’ was down regulated compared with ’Nongda 4’,of which CHS and LDOX were the most down regulated genes.It is speculated that these two genes may be related to the difference between the flavonoid content and the color of ‘Nongda 4’ and ‘Nongda 5’ C.humilis fruit.5.ChCHS and ChLDOX were homologous cloned from ‘Nongda 4’ C.humilis fruit,and their bioinformatics was analyzed.The ChCHS gene encodes 391 amino acids.The protein is a stable hydrophilic protein without signal peptides and is likely to be located in the cytoplasm.The secondary structure and tertiary structure of ChCHS were predicted to be mainly composed of Alpha helix,extended strand beta turn and random coil,among which the Alpha helix and random coil accounted for a relatively large proportion and had a typical CHS protein conserved domain.The ChLDOX gene encodes 357 amino acids,is an unstable hydrophilic protein without a signaling peptide and is likely to be located in the cytoplasm.The secondary structure and tertiary structure of ChLDOX were predicted to be mainly composed of Alpha helix,extended strand beta turn and random coil,among which the Alpha helix and random coil accounted for a relatively large proportion and had a typical LDOX protein conserved domain.6.By analyzing the dynamic changes and correlations of flavonoids content with CHS gene and LDOX gene expression levels in fruits of different C.humilis germplasm,the change regularity and amplitude of CHS gene were always highly consistent with the change regularity and amplitude of flavonoids content.The over expression vectors of pbin35sred2-CHS and pbin35sred2-LDOX genes were constructed,and 10 and 8 positive strains were obtained by Agrobacterium mediated method,The leaves of transgenic Arabidopsis thaliana with over expression of ChLDOX gene turn red,and the stem also turn red,which indicates that ChLDOX gene is the key gene for anthocyanin formation,plays an important role in red formation,and is the key gene for peel color formation of red fruits.The maximum and average content of flavonoids in Arabidopsis thaliana leaves were 1.05 mg/g and 0.66 mg/g respectively,which were469.24% and 259.45% higher than those in the control group.ChCHS is the gene closely related to the flavonoid content and plays an important role in the flavonoid content of C.humilis fruit. |
