Proteomics Profiling Of Ethylene-induced Tomato Flower Pedicel Abscission And Verification Of Key Protein S1CDPK5

Organ abscissionis a natural phenomenon commonly occurring plant kingdom, but it is one of the important factors that affect the yield and quality of crops. It hasbeen demonstrated that ethylene can promote the role of tomato flower pedicel abscission, However, only few large-scale proteomic studies focused on tomato pedicel abscission, during the process of abscission phosphoproteomics research is no report.Therefore, by iTRAQ technology this test large-scale analysis ethylene and 1-MCP-induced protein and phosphoproteinduring tomato flower pedicel abscission. By protein function analysis, bioinformatics analysis, RT-PCR and Western Blot verification role of transcription and protein phosphorylation.This research attempts to study the ethylene-induced molecular mechanisms of tomato flower pedicel abscission from proteomic aspects. The main results are as follows:1. Analysis of the tomato stalk abscission zone relative protein expression levels using iTRAQ. Among the 1429 quantified proteins,383 unique peptides corresponding to 166 proteins showed higher than 1.5-fold change in abundance. By clustering analysis on expression of these proteins, they are divided into eight categories. A lot of proteins were increased by ethylene treatment.2 A total of 450 phosphopeptides were detected, Only those phosphopeptides that met the following restrictions were regarded as having undergone a significant change at the phosphorylation abundance:（1） increasing or decreasing 1.5-fold in phosphorylation activity; （2） the log2 （ethylene/CK,1-MCP/CK,1-MCP/ethylene） value≥0.6 or≤-0.6; （3） Mascot Score≥20; and （4） pRS Score≥50.138 phosphorylated sites corresponding to 85 phosphopeptides with the phosphorylation level significantly changed were screened out.3. The use of GO annotation analysis, proteomics and phosphoproteomics of proteins conducted Leve₂ level of biological process, molecular function, and cellular component classification. A total of 166 proteins comment to the 1833 GO annotation terms:994 biological processes,284 molecular function,555 cellular components. A total of 73 phosphoproteins comment to the 955 GO annotation terms:557 biological processes,135 molecular function,263 cellular components. Proteins and phosphoproteins were matched to the 79 and 24 KEGG pathway respectively. Proteomics and phosphoproteomics of have 14 and 4 proteins involved in the group starch and sucrose metabolism pathway. Ethylene-induced abscission is closely related to starch and sucrose metabolism.4. Some protein and phosphorylated proteins were analyzed transcription levels. In proteomics, most inconsistent level of transcription and translation of genes, in phosphoproteomics, most consistent level of transcription and modification of genes. S-adenosylmethionine synthase, CELLULASE 4, Expansin, osmotin-like protein, Auxin-regulated protein may be regulated by ethylene. Combined with other studies we found no correlation between transcription and translation, Consistency of transcription and modification is greater than the consistency of transcription and translation. There 25 proteins were identified in the two trials, of which 20 proteins were analyzed transcription levels. This research found that the role of phosphorylation changes are not due to changes in transcription or translation levels caused. Phosphoproteomics data show effective and reliable.5. Established for the Trans-Blot（?）urbo TM transmembrane system, using a suitable transfer buffer for the different molecular-weight proteins. The antibody was diluted into different multiple, we found optimal dilution of 1:1000 after immunization. Using western blot verified SRL3S329, CDPK5S523, CDPK5S527 phosphorylation consistent with iTRAQ.6. In the 138 iTRAQ identified significant differences in phosphorylation sites, serine, threonine, tyrosine, respectively, accounted for:68%,29%,3%. Motif-X analysis found that these modifications phosphorylation sites have six kinds:[sP], [Axxxxxs], [txxxxxxxxxT], [txxT], [Txt], [Kxxxxxt].7 Obtain SICDPK5 full-length cDNA, and successfully connected to the T vector. Use of Takara MutanBEST Kit Kit successfully CDPK5 of S （AGC） was mutated to A （GCC）. Use of Gateway technology successfully SlCDPK5and SICDPK5S527A were turned into the pB7YWG2 vector, and transformed into Agrobacterium.8. TMpred predicts SlCDPK5 have a transmembrane domain at 255-273-bit, TargetP 1.1 Server for CDPK5 cell localization prediction:SlCDPK5 mainly in the cytoplasmic and membrane. Tobacco transient expression proved SlCDPK5 expression in higher amounts near the cell membrane, and SlCDPK5S527A only uniformly expressed in the cell. This shows phosphorylation of SlCDPK5 can change its subcellular localization.