| Papaya(Carica papaya L.) fruit has a short shelf life due to fast ripening induced by ethylene,1-Methylcyclopropene (1-MCP) is used to slow down the fruit ripening. but little is known about the genetic control of ripening and attributes of fruit quality. Here we use papaya fruit after treated by ethylene (0.5 μL/L) and 1-MCP (1μL/L) to study differential Proteomics during ripening using iTRAQ technology and explore the function of UBI family gene. Studying the process of papaya fruit ripening treated by ethylene and 1-MCP, which can help us to learn general molecular mechanism about fruit ripening and also have theoretical significance of preservation research and development.(1) In this experiment, skin colour, firmness, malondialdehyde, peroxidase, soluble protein of papaya began to change between 12h and 24h after treated, but the total soluble solid did not have significant increase or decrease, which means that the total soluble solid has reached the peak at mature green stage.(2) Proteins extracted and evaluated by a phenol extraction and iTRAQ technology. 217907 pieces of total spectra,16957 pieces of spectra,8842 pieces of unique spectra, 6582 peptides,3853 unique peptides and 3048 papaya proteins were detected from three contrast groups. In ethrel/CK,49 proteins were up-regulated and 73 proteins were down-regulated, and in 1-MCP/CK, there were 30 up-regulated proteins and 40 down-regulated proteins.(3) Compared with CK, ethrel/CK and 1-MCP/CK were both enriched in the metabolic pathway and Glycolysis/Gluconeogenesis pathway. In the metabolic pathway, there were 55 proteins in ethrel/CK, which was 51.89% of the total significantly differentially expressed protein, in 1-MCP/CK, there were 38 proteins significantly differentially expressed, accounting for 62.3%. In Glycolysis/ Gluconeogenesis pathway, we found 14 proteins (13.21%) significantly differentially expressed in ethrel/CK, and 8 proteins (13.11%) in 1-MCP/CK.(4) We found some key proteins related with friut ripening using GO analysis and KEGG database, such as UDP-glycosyltransferases, (1-4)-beta-mannan endohydrolase, glyceraldehyde-3-phosphate dehydrogenase, papaya proteinase 4, alpha-L-arabinofuranosidase, cysteine proteinase, xyloglucan endo-transglycosylase, methionine synthase, alpha-amylase-like isoform 1, alpha-amylase-like isoform 2, protein disulfide-isomerase, aquaporin PIP2;2 and enolase.(5) Six different proteins were validated in mRNA, the results were consistent with mass spectrometry at the ratio of 66.7%, alpha-amylase-like isoform 1, methionine synthase, UDP-glycosyltransferases and cysteine proteinase were consistent with mass spectrometry, alpha-amylase-like isoform and xyloglucan endo-transglycosylase were opposite or disorder with mass spectrometry.(6) Ubiquitin proteins have been paid attention that they play important roles in responding to ripening and regulating certain developmental processes in plants. Therefore, UBI genes, encoding ubiquitin were isolated from papaya fruit and designated CpUBI1, CpUBI2, CpUBI3. The length of CpUBIl, CpUBI2, CpUBI3 cDNAs are 1485bp,1642bp,529bp encoding 306,308,156 amino acids. The sequence analysis showed that that CpUBI1 and CpUBI2 proteins contained four consecutive UBQ superfamily domain structures, which belonged to the polyubiquitin, While CpUBI3 was a S27a ubiquitin extension gene containing a Ribosomal S27a superfamily domain structure. RT-qPCR analysis showed that ethephon treatment significantly promoted the expression of CpUBIs compared with the 1-MCP treatment and control treatment, and reached the highest in 3 days after processing, CpUBI1 and CpUBI2 expression is significantly higher than CpUBI3. These results suggested that CpUBIl, CpUBI2 and CpUBI3 genes may play different roles in papaya fruit ripening and softening, speculating that the increase of ubiquitin gene expression was induced by ethylene. |
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