Comparison of intestinal gene expression in Atlantic cod (Gadus morhua) fed standard fish meal or soybean meal by means of suppression subtractive hybridization and real-time PCR

Abstract

Gene expression was studied in Atlantic cod fed two different diets, fish meal (FM) and dehulled and extracted soybean meal (SBM). RNA was isolated from the distal part of the mid-intestine of Atlantic cod and suppression subtractive hybridization (SSH) was employed to screen for genes that showed changes in expression in response to the two dietary treatments. We made a cDNA subtracted library, isolated and sequenced 192 clones. Identification of 157 clones was predicted by BLAST. Most of the clones were previously unidentified in cod. Expression of 12 selected clones was further studied by quantitative PCR. Expression of four clones showing similarity to aminopeptidase N, transcobalamin I precursor, cytochrome P450 3A40, and ras-related nuclear protein was significantly up regulated in intestine of cod fed SBM compared to cod fed FM. A trend towards up regulation of a clone with similarity to fatty acid binding protein in SBM-fed cod was also observed. No significant differences in expression were observed for: transmembrane 4 superfamily protein member, polypeptide N-acetylgalactosaminyltransferase, glutathione peroxidase, peroxiredoxin 4, SEC61, F-BOX, and 14-3-3.

Keywords: Atlantic cod; Expression; Intestine; Real-time PCR; SSH; Soybean meal.

Fig. 1

Distribution of clones from the SSH from Atlantic cod intestine, grouped by protein function. Clones with no or low (E value > 8e− 04) similarity to other proteins are shown as unidentified/low similarities. Enzymes not belonging to any of the other groups are shown as enzymes.

Fig. 2

Relative gene expression of selected clones. Expression of target mRNAs normalized to both 18S rRNA and ELF1a in intestinal tissue of Atlantic cod fed either FM or SBM diets. A representative template from a FM fed fish was chosen as the calibrator. Reverse transcriptase (RT) was performed in duplicates for each individual sample, and template from each RT reaction was run in duplicates in quantitative PCR. An average normalized calibrated ratio for each individual was calculated and is presented as an individual point in the plot. Mean normalized calibrated ratios are presented as the mid-line of each diamond. The vertical range of the diamonds represents 95% confidence intervals. Asterisk ^⁎ denotes genes that show significantly different expression between dietary treatments (P < 0.05), whereas † indicates a trend (P < 0.10). APN, aminopeptidase M (Alanyl aminopeptidase)(CD13)/Aminopeptidase N; FABP, fatty acid binding protein; TCI, transcobalamin I precursor; CYP3A40, cytochrome P450 3A40; SEC61, sec61-alpha; F-BOX, F-box protein 44; TM4, putative transmembrane 4 superfamily member protein; RAN, ras-related nuclear protein; ppGaNTase, probable polypeptide N-acetylgalactosaminyltransferase 8; 14-3-3, 14-3-3B2 protein mRNA; GPx, glutathione peroxidise; Prx4, peroxiredoxin 4.

Fig. 2

Relative gene expression of selected clones. Expression of target mRNAs normalized to both 18S rRNA and ELF1a in intestinal tissue of Atlantic cod fed either FM or SBM diets. A representative template from a FM fed fish was chosen as the calibrator. Reverse transcriptase (RT) was performed in duplicates for each individual sample, and template from each RT reaction was run in duplicates in quantitative PCR. An average normalized calibrated ratio for each individual was calculated and is presented as an individual point in the plot. Mean normalized calibrated ratios are presented as the mid-line of each diamond. The vertical range of the diamonds represents 95% confidence intervals. Asterisk ^⁎ denotes genes that show significantly different expression between dietary treatments (P < 0.05), whereas † indicates a trend (P < 0.10). APN, aminopeptidase M (Alanyl aminopeptidase)(CD13)/Aminopeptidase N; FABP, fatty acid binding protein; TCI, transcobalamin I precursor; CYP3A40, cytochrome P450 3A40; SEC61, sec61-alpha; F-BOX, F-box protein 44; TM4, putative transmembrane 4 superfamily member protein; RAN, ras-related nuclear protein; ppGaNTase, probable polypeptide N-acetylgalactosaminyltransferase 8; 14-3-3, 14-3-3B2 protein mRNA; GPx, glutathione peroxidise; Prx4, peroxiredoxin 4.