SERIAL ANALYSIS OF GENE EXPRESSION (SAGE) DURING BRASSICA NAPUS SEED DEVELOPMENT

Abstract

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. About 18% of all EST-matched A. thaliana genes were matched by tags in antisense orientation, suggesting an involvement of poly A+ containing antisense RNAs in regulation of gene expression during B. napus seed development.