Development of expressed sequence tags (ESTS) and cDNA microarrays for analysis of differentially expressed genes in the channel catfish (Ictalurus punctatus) brain

The objectives of this research were to develop expressed sequence tags from the channel catfish (Ictalurus punctatus) brain and to develop microarray technologies for analysis of differentially expressed genes in the brain in response to cold acclimation. Expressed sequence tag (EST) analysis was conducted using a cDNA library made from the brain mRNA of channel catfish. ESTs were used to develop microarray technology to determine differentially expressed genes to reveal molecular mechanisms of cold acclimation. A total 1,201 brain cDNA clones that represented 660 unique genes were analyzed for transcriptome analysis in catfish to develop molecular reagents for cDNA microarray analysis to reveal molecular mechanisms of cold acclimation. Of the 660 unique genes, 330 clones were identified as known genes by BLAST searches and 330 clones as unknown genes.; Among 660 analyzed genes, 62 were differentially expressed at 12°C as compared to 24°C. The gene induction upon temperature shift was rapid within two hours. The major categories of differentially expressed genes include: (1) chaperones such as Hsp70 and Hsp70/Hsp90 organizing protein, (2) transcription factors and genes involved in signal transduction pathways such as zinc finger proteins, calmodulin kinase inhibitor, the nuclear autoantigen SG2NA, interferon regulatory factor 3, and inorganic pyrophosphatase, (3) genes involved in lipid metabolism such as TB2 and acyl CoA binding protein, and (4) genes involved in the translational machinery such as ribosomal proteins. Some genes were induced transiently, whereas others were induced in a durable fashion. Several genes were down regulated among which were mainly ribosomal protein genes, indicating reduced metabolic activity with extended incubation at the low temperature. This research indicated that catfish respond to low temperature by adjusting expression of a large number of genes. The rapid induction of proteins involved in signal transductions and chaperones suggest that both de novo synthesis of cold-induced proteins and modification of existing proteins are required in adaptation and tolerance of fish to low temperature environment.