| Citrus in our country are mainly mid-varieties and the mature period is too concentrated. Therefore, breeding different varieties and lengthening fresh fruit supplying period can largely improve the competitiveness of citrus industry. Fruit ripening is a complicated process, subject to a number of factors, such as light, temperature, hormones and regulation of key genes. Citrus belongs to non-climacteric fruit, but fruit ripening molecular mechanisms are still not clear, so the revelation of citrus fruit ripening regulation mechanisms has important significance for adapting existed citrus varieties and promoting the development of citrus industry. In this study, ’Fengjie 72-1’ and the late-maturing mutant of ’Fengwan’ are as the materials. We measured the physiological qualities and analyzed the difference of the citric acid content, and also screened three transcription factors related in fruit development for functional verification. The main findings are as follow:1. Fruit color: four different stages fruits of ’Fengjie 72-1’ and ’Fengwan’ orange, that is 190, 208, 223 and 248 DAF(Days after flowering) were measured, we found that during 190- 223 DAF, the peel H value of ’Fengwan’ orange was significantly higher than that of ’Fengjie 72-1’, however, the pulp H value was just higher than ’Fengjie 72-1’ at 190 DAF and there was no difference between each other during 208- 248 DAF. In additon, soluble solids content of ’Fengjie 72-1’ was higher than ‘Fengwan’ during 190- 208 DAF, but the titratable acid content was significantly lower than ’Fengwan’ at four different stages.2. Citric acid metabolism: The difference of citric acid content in ’Fengjie 72-1’ and ’Fengwan’ orange was analyzed, and then we used qRT-PCR to measure the expression of citrate metabolic related genes during fruit development stages. The results revealed that citric acid content was regulated by multiple genes. Genes involved in synthesis(CsCS4, CsME1、CsME4、CsPEPC1) and degradation metabolic pathways(CsACO1, CsGAD4 and CsGS1) coordinately contribute to the different content of citric acid. Correlation analysis revealed that CsME1 and CsME4 may be key genes affecting the different content of citric acid between ’Fengwan’ orange and its wild type.3. Bioinformatics analysis: protein size, isoelectric point and conserved domains of three key transcription factors were predicted. Futhermore, promoter region were cloned and analyzed combined with cis-acting elements. The results showed that three transcription factors contain more hormone response elements. It may explain three transcription factors can affect fruit development and maturation by participating in a variety of hormones signaling pathways. In addition, they also contain a large number of light-related response element binding sites, indicating that three transcription factors may involve in photosynthesis or affect the interaction of optical signal and its receptor genes, and thus participate in the regulation of varieties of plant physiological processes.4. Transcription factor function verification: CDS region of three transcription factors were cloned and then we constructed subcellular localization and overexpression vectors. Results showed that three transcription factors were localized in the nucleus, which meaned they were nucleus proteins. Using agrobacterium transformation to construct overexpression transgenic in tomato, we obtained three 35S-DREB26, two 35S-ESE3 and one 35S-bZIP9 transgenic plant by positive identification. Overexpression of CsDREB26 gene can delay the time to breaker from fruit setting and reduce the degree of fruit coloring. Compared with the wild type, transgenic plants of CsESE3 can significantly inhibit fruit coloring, although the time to breaker from fruit setting is closely. The results reveal that CsDREB26 and CsESE3 may negatively regulate fruit ripening. |
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