| Peel color, one of the main traits of citrus fruit appearance, has attracted increasing attention for citrus consumers. During Citrus ripening process, the external color transform from green to yellow along with the breakdown of chlorophyll and the accumulation of carotenoid. The commercial practice of degreening citrus fruit by exposure to ethylene to accelerate peel color development, it is very important for early varieties and for fruits that natural colour development is late and weak. But little is yet known about regulation of chlorophyll degradation on citrus peel.Stay-green is the phenomenon that chlorophyll does not degrade or slowly degrade in plant during ripening progress. Stay-green mutant is an ideal material to study the regulation of chlorophyll degradation. Citrus suavissima Tanaka is a native variety in wenzhou, zhejiang province called "Ougan" by people, and its spontaneous stay-green mutant called "Qing'ougan" whose chlorophyll degrade slowly. In this study, we used Qing'ougan and Ougan as our materials, analysed differences of the PAO pathway during postharvest and storage stages. The main findings are as follows:1. UPLC detected the content of chlorophyll and its metabolites of Qing'ougan and Ougan during different periods. During the color break stage, the chlorophyll content of Ougan rapidly reduced, while in Qing'ougan, the rate of chlorophyll degradation is slower. Total chlorophyll content in Qing'ougan was significantly higher than the Ougan, which including chlorophyll a, chlorophyll b, chlorophyllide a, pheophorbide a. This is the reason that Qing'ougan's flavedo stay green during maturation and storage process.2. The PAO homologous genes were cloned from Qing'ougan and Ougan respectively, and there is no difference between them after BLAST. The PAO's cDNA is 1798 bp in length. It contains a 1626 bp ORF which encodes a 541 amino-acid sequence, with a molecular mass of 60 kDa and isoelectric point of 6.41. According to the BLAST results in GenBank database, the amino-acid sequence of PAO shares 75% and 85% identity with Arabidopsis thaliana and Nicotiana tabacum respectively, and 79% identity with Ricinus communis and Vitis vinifera.3. Real-time fluorescent quantitative PCR was used to analyze the expression of the key genes in chlorophyll degradation pathway in Qing'ougan and Ougan. The results showed that relative expression of PAO, SGR, CAO, NYC1, CLH and PPH in Ougan were higher than Qing'ougan 210 days and 240 days after flowering, and the differences are significant. This may be the reason that the rate of chlorophyll degradation in Qing'ougan is slower.4. Western blot was used to analyze PAO protein expression of Qing'ougan and Ougan during development and storage stages. The level of PAO protein expression began to increase since the color change, Ougan stored about 50 days had the highest expression of PAO protein, and Qing'ougan bloomed after 170days had the lowest.5. About 2000 bp promoter fragment of PAO gene were cloned from Qing'ougan and Ougan respectively. From the results of sequence alignment, there are four kinds of promoters which have little difference. The main differences are:the first is the bite at 617 bp upstream the ATG, there is a missing of a 9 bp fragments (CCTCCGAAA); the second is the bite at 146 bp upstream of the ATG, there is a 4 bp sequence repeat (ACAG), some repeat for two times, some three times; others are the difference at single base pair.6. We predicted cis-acting elements of PAO gene promoter by on line software. Results showed that the sequence included a number of regulatory elements which could divide into two classes, the first class contained elements in response to plant hormones, and the other class contained elements in response to environmental signals. We used sweet orange genome data to get sequences 2000 bp upstream of SGR, CAO, NYC1, CLH, PPH, and predicted cis-acting elements of these genes. Finding that they all have ABA-responsive element, in addition to the SGR, they all have low temperature response element.7. Real time RT-PCR was used to study the expression of the transcription factor ABF family and DREB1/CBF family which binding to ABA-response element, and low-temperature-response element respectively. The results showed that the expression patterns of two ABF genes and two DREB1/CBF genes are similar with genes in chlorophyll degradation.8. Real time RT-PCR was used to study the expression of the relative genes and transcription factors in chlorophyll degradation pathway after ethylene and ABA treatment. SGR, NYC1, ABF2 were induced significantly after ethylene treatment, which were reached the hightest exprssion level at 48 hours after treatment, and Qing'ougan increased more than Ougan. There was no much differences in gene expression level between ABA treatment and control in Qing'ougan, the expression of PAO and ABF4 genes were inhibited compared with the control. |
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