Serial Analysis of Gene Expression (SAGE) applied to the study of Brassica napus seed development

摘要

Serial analysis of gene expression (SAGE) is a high-throughput sequencing-based genomic technique that allows identification and quantification of tissue-specific gene expression based on the cloning of short sequence tags (13-26 bp) derived from expressed poly A+ transcripts. A modification of the original protocol, Robust-LongSAGE, which generates 21 bp tags, was used and optimised to enable efficient cloning and transcript identification from the large Brassica napus genome. Two libraries were produced and analysed from total RNA extracted from seeds harvested at 23 and 35 days after pollination (DAP). The tag-matching efficiency was restricted by the current limited availability of brassica genomic data and EST annotation quality,which, however, is expected to increase rapidly in the near future. Tags expressed differentially between 23 and 35 DAP that were successfully matched to genes present in databases include genes involved in storage protein accumulation, fatty acid and protein metabolism, photosynthesis, development and secondary compound metabolism. Differentiation between closely related target sequences was possible within the conserved napin storage protein gene family, which was highly up-regulated at 35 DAP. About 7% of the total counts of all tags derived from genes of the brassica napin gene family matched in antisense orientation within the napin gene coding regions, suggesting an involvement of poly A+ containing antisense RNAs in regulation of napin gene expression during B. napus seed development.