| Citrus(Citrus)is one of the most widely planted and highest-yielding fruit trees in the world.Citrus fruit is rich in a variety of nutrients and is thus well loved by consumers.At present,there is a high content of organic acids in early-maturing and extra-precocious citrus varieties,so reducing the content of organic acids after fruit ripening is of great significance to the industrial structure of citrus.In this study,DF4 and WZ were used to determine the quality characters of two varieties in six different developmental stages,and the changes of quality characters in different developmental stages were studied.Transcriptome sequencing and WGCNA analysis were used to screen differentially expressed genes in six different developmental stages.In the tricarboxylic acid cycle,which is the key pathway of citric acid metabolism,we have enriched 19 candidate genes.In this study,the candidate gene Ciclev10031423 m.1 enriched in citrate synthase(CS)and the candidate gene Ciclev10031633 m.1enriched in ATP-citrate lyase(ACL)were studied as key genes.Based on the expression of two key genes at the transcriptional level and the results of RT-q PCR,we have further confirmed the two key genes.In order to explore the interaction between the regulation of citric acid by key genes,the functional verification of the cloned key genes was carried out in this study.Through the verification of transient overexpression in tomato fruit and VIGS gene silencing in citrus fruit,it was found that the two key genes had a great influence on the regulation of citric acid metabolism and influenced each other.The main results of this study are as follows:(1)The results showed that there was no significant difference in soluble sugar content between low acid variety DF4 and high acid variety WZ,but there was significant difference in fruit citric acid content.The citric acid content of two varieties of 94 DAF reached the minimum,and the citric acid content of low acid variety DF4 remained at a low level after 94 DAF.The citric acid content of the high acid variety WZ increased significantly after 94 DAF and maintained at a high level.The citric acid content of the low acid variety DF4 was significantly higher than that of the high acid variety WZ in the early stage of development,while the citric acid content of the high acid variety WZ was significantly lower than that of the high acid variety WZ at the late stage of development.This shows that soluble sugar content has little effect on fruit quality traits,and fruit citric acid content is the main factor affecting fruit quality traits.(2)In this study,transcriptome sequencing combined with weighted gene co-expression network analysis(WGCNA)was used to screen differentially expressed genes in six different developmental stages of two citrus fruits.The results showed that after pairwise comparison of different varieties in the same period and different periods of the same variety,a total of 7105 differentially expressed genes were screened.In order to further screen candidate genes,the 7105 differentially expressed genes and phenotypic data were analyzed by WGCNA.In the analysis results,we will find that the correlation between the weight genes in the MEtan、MEbrown module and citric acid content is the highest,and then the weight genes in the MEtan、MEbrown module are analyzed by GO and KEGG enrichment analysis.Through enrichment analysis,we found that a total of 19 candidate genes were enriched in the tricarboxylic acid cycle pathway related to citric acid metabolism.Through the further functional enrichment of the tricarboxylic acid cycle pathway of these 19 candidate genes,we found that the candidate genes Ciclev10031423 m.1 and Ciclev10031633 m.1 were simultaneously enriched in the physiological process of the key enzymes regulating citrate metabolism,and then we analyzed the two candidate genes at different developmental stages and among different varieties.The results showed that the two candidate genes showed the opposite trend with the change of developmental stage,but the change trend was the same between the two varieties.We calculated the Pearson correlation coefficient(PCC)to evaluate the correlation between the relative expression of candidate genes Ciclev10031423 m.1,Ciclev10031633 m.1 and FPKM.The results showed that the PCC values between the relative expression of the two candidate genes and FPKM were higher than 0.7 in all samples,indicating that the q PCR results of the two candidate genes were basically consistent with the expression of the two genes obtained by RNA-seq analysis.Then the two genes Ciclev10031423 m.1and Ciclev10031633 m.1 were used as key genes for further analysis.(3)In this study,we have a preliminary understanding of the biological function of the key genes through the analysis of the functional structure of the two key genes.The expression pattern of key genes was preliminarily predicted by analyzing the correlation between gene expression and citric acid content in different developmental stages and among varieties.It is suggested that the regulation of citric acid by Ciclev10031423 m.1 may be negative,which is more active in the later stage of development,and the gene expression is significantly higher than that in the early stage of development.The regulation of citric acid by Ciclev10031633 m.1 may be positive regulation,which is more active in the early stage of development.The gene expression in the late stage of development is significantly lower than that in the early stage of development,and remains at the same level.In addition,there may be an expression pattern of reverse regulation between the two key genes.(4)In this study,the cloning and preliminary verification of key candidate genes were completed,and the overexpression vectors of key genes,gene silencing vectors mediated by TRV virus and gene editing vectors based on CRISPR/Cas9 technology were successfully constructed.The effect of key genes on citric acid content was verified by transient overexpression of key genes in tomato fruit and silencing key genes in citrus fruit,and the expression pattern of key genes was further verified.The analysis of the expression of key genes,titratable acid content and citric acid content in positive fruits showed that in DF4 and WZ varieties,the key gene Ciclev10031423 m.1 had negative regulation on citric acid content,while the key gene Ciclev10031633 m.1 had positive regulation on citric acid content,and they were inversely regulated each other. |
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