Proteomic Study On Bent Character Of Fruit In Cucumber (Cucumis Sativus L.)

Fruit shape is one of the important quality traits. Bending fruit has a serious impact on thequality of cucumber appearance, flavor and taste in cucumber. However, there has been littledetailed analysis of molecular mechanisms of this character. To better understand thepost-transcriptional changes of bent character, a proteomic approach was used to profile proteinchanges among the straight fruit, the abdomen and ridge of cucumber bending fruit at fruit settingstage, fruit development stage and fruit maturity stage, respectively. Two-dimensional gelelectrophoresis (2-DE) has been used to identify proteins that were differentially expressed in thestraight fruit, the abdomen and back of cucumber bending fruit, and image analysis has been usedto determine which proteins were up-or down-regulated by the experiments designed. Nextmatrix-Assisted Laser Ionization Time of Flight Mass Spectrometry (MALDI-TOF-TOF/MS) willbe used to determine the identity of these proteins. Searching the NCBI protein database with thecombination of gene ontology (GO) annotation database, Cluster analysis, PDB protein database,SWISS-MODEL pathway analysis and Chromosome location, it is possible to assign a scientificidentification of biological functions, regulatory functions and interaction network of these proteins.In addition, real-time quantitative RT-PCR will be used to assess the transcript levels of criticaldifferentially expressed proteins. Gene expression patterns correlate with or without proteinexpression identified by both proteomic and genomic approaches will be required to obtain adetailed understanding of the bending fruit. It is imperative to screen the proteome pattern ofcucumber bent character, in order to better understand the key pathway, the cells changes, and themolecular mechanism for regulating bending happened and development. Here we report thechanges in proteome of the straight fruit, the abdomen and ridge of cucumber bending fruit. Themain results were as followed：1）According to the stringent criteria of having more than one unique peptide per proteinpresent and a false discovery rate≤5%,2114、1929and2638proteins were identified at fruitsetting stage, fruit development stage and fruit maturity stage, respectively. A total of197difference proteins from different part of cucumber fruit were screened out, in which,61proteinspots were analyzed using MALDI-TOF-TOF MS. Differentially expressed proteins were screenedby2-sample t-test (p<0.05) and fold change (fold>1.5),11、7and43proteins were screened out at fruit setting stage, fruit development stage and fruit maturity stage, respectively.2）61protein spots were identified by MS. Most of them were up-regulated or down-regulatedbetween the straight and bending fruit, and few was up-regulated or down-regulated between theabdomen and ridge of cucumber bending fruit. During the three development period,3、4and19differential proteins were down-regulated in the straight fruit,5、2and14differential proteins wereup-regulated in the straight fruit,2、0and6differential proteins were down-regulated in theabdomen of bending fruit, all1differential proteins were down-regulated in the ridge, respectively.3）The identified proteins were involved in various metabolic processes and cellular responses,including proteolysis, proteasome regulatory particle assembly, cytoskeleton organization andbiogenesis, circadian rhythm, riboflavin biosynthetic process, biological-process, response to stress,response to dna damage stimulus, mRNA catabolic process, cellular protein metabolic, nucleotidemetabolic process, lipid metabolic process, metabolic process, nitrogen compound metabolicprocess, postreplication repair, translational elongation, regulation of dna replication, cell cycle,photosynthesis, light harvesting, oxidation-reduction process and transport according to the primarydatabases. Of them, oxidation-reduction process was the key process, and transport, cell cycle,photosynthesis were also important.4）Hierarchical cluster analysis of differentially expressed proteins. Two sub-class included20and41differentially expressed proteins respectively. HZ9412-HK4319differentially expressedproteins were down-regulated at fruit setting stage, HZ7802-HZ1314differentially expressedproteins were up-regulated at fruit setting stage. The abdomen and ridge of bending fruit at fruitdevelopment stage and fruit maturity stage were clustered the same group. Other parts of elsestages were clustered another group.5）60differentially expressed proteins were located in7chromosomes of cucumber. Thenumber of differentially expressed proteins in Ch6and Ch4were13and11respectively, the Ch5was only5. The differentially expressed proteins in Ch1, Ch3and Ch6included proteins from threedevelopment stage. The oxidation-reduction process proteins were located in Ch1, Ch2, Ch3, Ch4,Ch5and Ch6. The transport proteins were located in Ch5. The proteolysis proteins were located inCh4and photosynthesis in Ch6.6）We found65pathways during searching KEGG database, including39related pathway,such as glyoxylate and dicarboxylate metabolism, photosynthesis, oocyte meiosis, cell cyclepathway were presented enrichment and were related with fruit bending.7）Different proteins were validated in mRNA and protein levels of the straight fruit, theabdomen and ridge of cucumber bending fruit. Different proteins were riboflavin biosyntheticprocess, lipid metabolic process, nitrogen compound metabolic process, oxidation-reductionprocess and transport proteins. The results were consistent with mass spectrometry at the ratio of62.5%, HP8012, HZ5012, HZ5615, HZ7601and HZ1302were consistent with mass spectrometry.HZ7629and HZ4803were opposite with mass spectrometry. HP4412was disorder with massspectrometry.