Identification Of Genes Regulating Carotenoids Metabolism In Citrus

Carotenoids,as the precursor of vitamin A,play an important role in human health.Citrus,as one of the most productive fruits in the world,are rich in carotenoids.The content and composition of carotenoids in citrus fruits determine their appearance quality,nutritional value and economic benefits.Therefore,it is of great significance to carry on researches about the carotenoid in citrus.In this study,based on the research progress of citrus carotenoids metabolism and the analysis and screening of color-related omics data,combined with quantitative expression,sequence analysis and transgenic experiments,the genes regulating the carotenoid metabolism of citrus were discovered and verified.The main findings are as follows:1.Several omics data were analyzed,and candidate genes Cgb HLH28 and Cg PK1(Protein Kinase)related to the regulation of carotenoids were screened.By analyzing the transcriptome of low-temperature treated blood orange and bagging treated purple-skinned pummelo with the published transcriptome of tomato overexpression OR(ORANGE Protein),the candidate gene Cgb HLH28 related to the regulation of carotenoids was screened.Through comparative analysis of the whole genome resequencing of ‘Guanximiyou’ pummelo and its color mutants,the candidate gene Cg PK1 related to the regulation of carotenoids was selected.2.The expression levels of candidate genes Cgb HLH28 and Cg PK1 were verified,confirmed that the expression levels of candidate genes were positively correlated with the carotenoid content in plants.Through the quantitative PCR analysis of candidate genes in ‘Guanximiyou’ pummelo and its color mutants and multi-color callus materials,it was found that the candidate genes have higher expression levels in materials with high carotenoids content,speculating that the difference of carotenoid content in different materials is related to the difference of expression level of candidate genes.3.The promoter sequences of the candidate genes Cgb HLH28 and Cg PK1 were cloned,and their elemental differences of the promoters in different materials were identified.Through cloning and comparison of promoter sequences and protein coding sequences of candidate genes in different-color materials,it was found that the promotersequences of candidate genes have differences in key elements between different-color materials,speculating that the difference in gene expression of candidate genes is related to the difference in promoter elements of candidate genes.4.The overexpression vectors of candidate gene Cgb HLH28 were constructed and three kinds of transgenic positive materials of candidate gene Cgb HLH28 overexpression were created.By constructing overexpression vectors for candidate gene Cgb HLH28,it was transferred into Arabidopsis thaliana,citrus callus and apple callus by Agrobacterium-mediated method for overexpression.After cultivating and screening the transgenic materials,the positive line materials successfully transformed were obtained.