Serial analysis of gene expression (SAGE) bei Raps: Quantitative, differentielle Expressionsanalyse im sich entwickelnden Rapssamen zur Erzeugung von Transkriptionsmarkern für die Samenentwicklung
Zusammenfassung der Projektergebnisse
High-throughput global expression profiling was applied to Brassica napus seeds by using Serial Analysis of Gene Expression (SAGE) in order to evaluate the usefulness of this technique for plants with highly complex, not fully sequenced and annotated genomes, and to identify genes potentially associated with the synthesis of commercially valuable seed compounds. A LongSAGE protocol using 20 bp tags was successfully adapted and optimized to measure gene expression in B. napus seeds. A novel bioinformatics strategy for tag-to-gene assignment was developed, data processing scripts were adapted to the generated sequence data and MySQL databases were constructed for quality filtering and linking of observed tags and Brassica ESTs to Arabidopsis thaliana proteome annotation data. The efficiency of tag-to-gene assignment using 20 bp tags allowed reliable annotation of B. napus transcripts. However, specificity for resolving expression down to single transcripts derived from members of closely-related gene families was found to be limited in some cases. This was mainly due to a tag-to-gene assignment strategy that relies on annotation data from the A. thaliana proteome due to the unavailability of an annotated Brassica proteome. The most highly abundant transcripts at 23 days after pollination (DAP) were derived from genes coding for cysteine proteases (more than 4 % of all counts) and at 35 DAP from genes coding for storage proteins (more than 10 % of all counts). Differential expression and Gene Ontology (GO) enrichment analysis revealed a shift from cell organization and cytosolic metabolism with many diverse processes at 23 DAP to a metabolism more focussed on photosynthesis, oil biosynthesis, protein biosynthesis and plastid uptake at 35 DAP. A Realtime RT-PCR procedure and endogeneous control genes were developed, and comparison of expression profiles with LongSAGE profiles revealed a good correlation for all tested genes. Real-time RT-PCR was found to be more sensitive than LongSAGE in detecting lowabundance transcripts from B. napus seeds under the experimental conditions used for LongSAGE. These results indicate that resolution of LongSAGE could be further improved by increasing the total number of sequenced tags after filtering to more than 50,000 per timepoint. In this respect a combination of next-generation sequencing technologies with SAGE using the data processing techniques developed in this project has the potential to provide a highly-accurate global transcription profile. A proportion of 11.6 % antisense tags was detected among all tags most of them (96 %) matching in sense-antisense pairs to A. thaliana genes, and for a small number of genes the expression of sense-anti sense transcript pairs from seeds was validated and quantified by the development and application of a strand-specific Real-time RT-PCR procedure. This result suggests the involvement of antisense regulation in B. napus seed metabolic processes. After optimization of the LongSAGE expression profiling protocol and establishment of appropriate data processing pipelines we found this expression profiling technique to be a very useful method for global analysis of the seed transcriptome of B. napus. We have established a database with detailed expression data from the B. napus seed transcriptome, that represents a highly valuable resource for future research into the identification of genes and mechanisms involved in the synthesis and regulation of transcripts involved in development and deposition of commercially interesting compounds in seeds of A napus.
Projektbezogene Publikationen (Auswahl)
- 2006. Serial Analysis of Gene Expression (SAGE) - Globale Genexpression im Rapssamen. Gessellschaft zur Förderung der deutschen Pflanzenzüchtung, Sitzung der Abteilung Öl- und Eiweißpflanzen, Gatersleben. presentation
Obermeier C.
- 2007. SAGE analysis of Brassica seed. Seminar, Plant Biotechnology Institute (PBI-NRC), Saskatoon, Canada. presentation
Obermeier C. Hosseini B, Snowdon R.
- 2007. Serial Analysis of Gene Expression (SAGE) applied to the study of Brassica napus seed development. Proceedings 12th International Rapeseed Congress, Wuhan, China II: 120-123. presentation.
Obermeier C, Hosseini B, Snowdon R.
