Identification of Growth-Dependent Genes in Brook Charr (Salvelinus fontinalis)
Québec, Université du Québec, Institut National de la Recherche Scientifique, Doctorat en biologie, 192 p.
Fish growth is important for fisheries management and aquaculture. The growth pattern of fish is a complex process which is influenced by environmental factors, hormones and genetic composition. Thyroid hormones (THs) play an important role in regulating fish growth. The identification of TH-dependant genes in fish could provide valuable information for predicting growth. Brook charr is an important species in aquaculture and therefore represents a good model to study fish growth. The objectives of this study were: (1) to identify TH-regulated genes in brook charr, (2) to identify growth responsive genes in brook charr and assess whether or not the expression of TH dependent genes are altered in fast and slow growing fish using a genomics approach.
To identify TH-dependant genes, three experimental groups of immature charr were used: a euthyroid control group (n=lO), a hyperthyroid group (n=lO) in which brook charr were injected (i.p.) with 3, 5, 3'-triiodo-L-thyronine (T3; 10 ug/kg body weight), and a hypothyroid group (n=lO) in which fish were injected with sodium ipodate (50 mg/kg body weight) a TH inhibitor. Fish were injected on days 1 and 4 of the experiment and sampled on day 7. Serum T3 levels were measured by radioimmunoassay. Results indicate that serum T3 concentrations decreased from 2.20 ng/ml in controls to 0.85 ng/ml in the ipodate treated group. T3 levels in the hyperthyroid group were 263.4 ng/ml. THs induced hepatic gene expression as measured by differentiai display PCR. The results show that over 100 transcripts were differentially regulated by THs. The expression of 18 of these genes was further confrrmed by reverse dot blot. These were then cloned and sequenced. Sequence homology comparisons revealed seven known genes: trypomyosin (3 isoforms), leukocyte-derived chemotaxin, mitochondrial NADH dehydrogenase subunit 5, basic transcription factor 3 and crystalline. Eleven genes had no significant homology to known gene and were therefore considered novel. Tissue distribution of the transcripts, as determined by Northem blot analysis, varied for each gene. While ali genes were expressed in liver, clearly the functions of these genes were not restricted to hepatic function as most of these were expressed in multiple tissues.
Since THs are known to be important regulators of growth, we wanted to determine whether or not the expression of TH-dependent genes differed between animais exhibiting different growth rate. To characterize growth-dependent gene expression profiles, eDNA arrays (GRASP project, University of Victoria) containing
16,000 genes were used. Two families of immature brook charr that were known to exhibit different growth characteristics were compared. These fish bad been raised under similar rearing conditions from egg incubation to the time of sampling (April, 2003). One family bad fast growing fish (n=8; average weight 54.73 ± 12.14 g) while the second bad slow growing fish (n=8; average weight 8.43 ± 1.59 g). Total RNA isolated from livers of fast and slow growing fish were used to prepare eDNA probes. The eDNA probes were labeled and hybridized to the eDNA microarrays. Microarrays were scanned and analyzed using GeneSpring software according to MIAME standards. A total of 94 genes were found to be growth-dependent exhibiting at least a 2-fold change in expression. There were 69 genes that were up-regulated and 25 genes were down-regulated in fast growing fish. Out of the 94 differentially expressed genes, 39 of these are known genes that have been implicated in regulating carbohydrate, fatty acid and protein metabolism, as weil as mitochondrial function and signaling pathways. Twenty of these have been shawn in the literature to be TH-dependent. The remaining 55 genes have unknown functions. Only two of the genes (basic transcription factors and leukocyte-derived chemotaxin) identified in the first experiment were present on the microarrays. However, the microarray results did not indicate different expression for these two between our fast and slow growing fish. These results indicate that TH-dependent genes may be associated with growth.
In conclusion, we have identified 18 TH-dependent genes in brook charr. This bas allowed us to show that THs are implicated in a wide variety of physiological processes as indicated by their function and tissue expression pattern. The genomic analysis of hepatic gene expression in fast and slow growing fish revealed that 20 genes that are known to be TH-dependent and regulated by growth. This supports previous reports that THs may be involved in the regulation of growth.
|Type de document:
|Directeur de mémoire/thèse:
||Cyr, Daniel G.
|Co-directeurs de mémoire/thèse:
||Audet, Céline (ISMER-UQAR)
||omble ; fontaine ; salvelinus ; fontalis ; hormone ; thyroïde ; gene ; croissance
||Centre INRS-Institut Armand Frappier
|Date de dépôt:
||27 sept. 2013 15:03
||18 déc. 2015 16:38
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