Integrated Proteome/Transcriptome Profiling of Tomato Fruit
We are integrating a tomato fruit transcript expression profiling initiative with the cell wall proteome analysis, in both wild type ripening fruit and those of the ripening impaired mutants ripening inhibitor (rin), non-ripening (Nor) and never ripe (Nr). The transcriptome data is being generated with the 8,700 unigene TOM1 cDNA array, the long oligonucleotide 12,000 unigene (TOM2) microarrays and two RNASeq platforms (454 and Illumina).
Following the identification of genes and their cognate proteins through comparison of MS-derived protein sequence analysis with the complement of the tomato microarray, microarray and proteomics data of this gene set are directly compared to identify: (a) genes showing significant expression changes by proteomic analysis but not by microarray analysis, or vice versa, through fruit development, or in comparison with mutant fruits; (b) genes showing significant differences between changes in transcript and cognate protein levels.
- Compare the protein and transcriptome profile of each gene in the groups above using correlation analysis and define three different categories: those showing positive, negative or no significant correlation between microarray and proteomic analysis.
- If data sets of sufficient size result, the number of genes represented in each group will be used to derive estimates of secretome genes under transcriptional and/or post-transcriptional control.
- Classify the genes into different functional categories to determine whether certain classes of secretome genes are primarily under transcriptional and/or post-transcriptional control during ripening.
Tissue specific analysis of the tomato pericarp tissues transcriptome: an approach to increase specificity in secretome studies.
Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes (transcripts, proteins or metabolites) are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection.
We are using laser capture microdissection (LMD), coupled with transcript profiling using RNAseq to identify tissue type specific transcripts and molecular pathways, in to gain new insights into aspects of tissue-specific gene expression, and consequently tissue and organ physiology. In this regard, we are particularly interested in defining tissue-specific secretomes. In addition, this deeper mining of the transcriptome is extremely valuable for tomato gene annotation; for example, revealing substantial alternative splicing, which in turn is critical for enhancing the proteome analyses.
- Construct and sequence tissue specific transcript libraries for each tissue in tomato fruit pericarp, using 454 and Illumina technologies , at various developmental stages
- Characterize the predicted secretomes of each tissue
- Use the deep coverage of the transcripts to identify enhance gene space definition, and therefore peptide matching for the proteomic studies
To date, a total of 1,456,024 high quality sequences have been generated, distributed among the tissue libraries. Following sequence assembly, 20,976 tomato unigenes (assembled from at least five reads) were associated with one or more of the tissues.
- File 1: 454 GS FLX reads from library AC1001
- File 2: 454 GS FLX reads from library AC1002
- File 3: 454 GS FLX reads from library AC1003
- File 4: 454 GS FLX reads from library AC1004
- File 5: 454 GS FLX reads from library AC1005
- File 6: Assembled sequences of the tomato transcripts (fasta)
- File 7: Solexa/Illumina reads from library AC1001
- File 8: Solexa/Illumina reads from library AC1002
- File 9: Solexa/Illumina reads from library AC1003
- File 10: Solexa/Illumina reads from library AC1004
- File 11: Solexa/Illumina reads from library AC1005
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