| Citrus is the most important fruit plant in China.It plays an important role in the development of the agricultural economy.With the improvement of people’s living standards,the demand for improving the nutrition quality of citrus is increasing.Flavonoids are important components of secondary metabolites in citrus.They are not only involved in the resistances to biotic and abiotic stresses such as disease,insect,drought,and low temperature in citrus,but also as important components affecting the nutrition quality of citrus fruit.Therefore,study of genetic regulation mechanism of flavonoids biosynthesis in citrus has been received more and more attention.There have been many research reports on functional study of the structural genes in the main pathway of citrus flavonoid biosynthesis,but the transcriptional regulation of flavonoids biosynthesis were seldom studied.Mining transcription factors related to citrus flavonoid biosynthesis and identifying their functions is the basis for elucidating the transcriptional regulatory citrus flavonoids biosynthesis.In this study,different genotypes of citrus were used as the experimental materials,and the transcription factors related to the biosynthesis of flavonoids in citrus were identified and functionally studied with transcriptomic analysis,phylogenetic analysis,and transgenic methods.The main findings are as follows:(1)Identification of differential expressed genes related to secondary metabolismsTo identify the genes related to flavonoid biosynthesis in citrus,the transcriptomic analysis was conducted with the fruit peels collected at 100 days after flowering and 160 days after flowering from Brazil sour orange(Citrus aurantium L.)and Bendizao mandarin(Citrus reticulata Blanco).Comparation of the data of fruit peels between 160 days after flowering and 100 days after flowering,a total of 2897 and 2476 differentially expressed genes were detected in Brazil sour orange and Bendizao mandarin,respectively.Further analysis found 1290 co-differentially expressed genes among two citrus genotypes and fruit development stages.GO and KEGG functional annotation identified that these shared differentially expressed genes were mainly enriched in photosynthesis,amino acid metabolism,and phenylpropane biosynthesis pathways.This indicated that the large number of genes related to the biosynthesis of secondary metabolites,especially the biosynthesis of phenylpropane highly differentially expressed during fruit development.Many of them are related to the metabolism of flavonoid.(2)Identify transcription factors related to citrus flavonoid biosynthesisIn order to identify transcription factors related to flavonoid biosynthesis,the correlation between 1290 differently expressed genes from transcriptome and Cit CHS2,a known key structural gene related to flavonoids biosynthesis was analyzed.A total of 392 genes were identified highly correlated with the expression of Cit CHS2(|R| > 0.6,p < 0.05).These genes were enriched in MYB,Dof,ERF,and other transcription factor families.Many MYB transcription factor binding sites were found by analyzing the cis-acting elements of promoters from known structural gene in flavonoid biosynthesis pathway.Therefore,the MYB transcription factor highly related to the expression of Cit CHS2 was further analyzed.Five MYB transcription factors were identified.Among them,Cit DIV2(Ciclev10029205m)was positively correlated with Cit CHS2,and Cit MYB81(Ciclev10026107m),Cit MYB82(Ciclev10001979m),Cit MYB21(Ciclev10021699m),and Cit MYB33(Ciclev10012152m)were negatively correlated with Cit CHS2.Meanwhile,60 potential transcription factors were predicted using promoters of known structural genes of flavonoid biosynthetic pathway.Intersection analysis of the predicted 60 MYB genes and the above 5 MYB genes revealed 2 shared R2R3-MYB genes,Cit MYB21 and Cit MYB33.Phylogenetic analysis showed that Cit MYB33 belongs to the first subfamily of the R2R3-MYB gene family,while Cit MYB21 belongs to the second subfamily.(3)Correlation analysis between Cit MYB21 gene expression and flavonoids contentTo clarify the function of the Cit MYB21 gene on flavonoids biosynthesis,the correlation between Cit MYB21 expression and flavonoid content under different conditions was analyzed.The results of q RT-PCR analysis and flavonoids detection found that flavonoid content in the leaves under dark,low temperature,Me JA and SA treatments had no significant correlation with the expression of Cit MYB21 gene.Further analysis with different germplasm and tissues,the leaves and fruits in different developmental stages found that the expression of Cit MYB21 gene was not only significantly genotype specific but also obvious spatiotemporal specific.The expression of Cit MYB21 was the highest in the root among all the tissues.During the leaf and fruit development,expression of Cit MYB21 showed a trend of first increasing and then decreasing.The correlation analysis indicated that the expression level of the Cit MYB21 gene in different germplasms was significantly and negatively correlated with the content of flavonoids(R=-0.38,p < 0.001).There was also a significant and negative correlation between the expression level of Cit MYB21 and flavonoid content during fruit and leaf development(R =-0.88,p < 0.001;R =-0.66,p < 0.05;R =-0.49,p < 0.05,respectively).(4)Functional analysis of Cit MYB21 in citrusThe coding region sequence of Cit MYB21 gene was cloned from the leaves of Beibei 447 Jincheng orange(Citrus sinensis Osbeck).The CDS(coding sequence)of the gene is 804 bp in length,encoding 267 amino acids.To further analyze the biological function of Cit MYB21 gene,the overexpression vector and VIGS(Virus-induced gene silencing)were constructed for transient transformation of citrus.A total of 5 gene-silenced plants and 3 empty vector control plants were obtained.Compared with the empty control,the expression level of Cit MYB21 in the leaves of the silenced plants was significantly decreased by 67%、79%、52%、55% and 54%,respectively,and the flavonoid content was significantly increased by 11%、43%、29%、12%、34%,respectively.The expression level of Cit MYB21 gene in the transiently transformed overexpressed peel was average 1-fold higher than that of the control,and the flavonoid content decreased significantly.The results suggested that Cit MYB21 may negatively regulate the biosynthesis of flavonoids in citrus.To explore the mechanism of Cit MYB21 negative regulation of flavonoid synthesis,the expression of major structural genes in flavonoid biosynthesis pathway in different tissues and parts of plant,the leaves at different developmental stages,and transgenic plants were analyzed.Correlation analysis showed that the expressions of Cit CHS2 and Cit CHI were significantly and negatively correlated with the expression of Cit MYB21 in transgenic lines,but did not have significant and negative correlations in different tissues and parts of plant,and during leaf development.The rest of genes also did not appear to be significantly correlated with the expression of Cit MYB21.In conclusion,this study preliminarily identified the member of R2R3-MYB second subfamily gene Cit MYB21 as a novel transcription factor that negatively regulates flavonoid biosynthesis in citrus.The expression of Cit MYB21 demonstrated obvious spatiotemporal specificity,and was significantly and negatively correlated with flavonoid content.It should be a key genetic factor regulating the spatiotemporal distribution of flavonoids in citrus. |
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