Sequence Cloning And Expression Analysis Of Rag Genes In Goldfish, Carassius Auratus

Recombination activating genes (Rag) are key genes in specific immunity system of vertebrate and also used as one of the molecular marker for analysis of vertebrate evolution. Goldfish, Carassius auratus, with strong adaptive and diseases-resistant abilities, is one of the important freshwater economic fish extensively cultivated in China. And it also appears to be an excellent model animal for the evolution study of fish genome for its varied ploidy and abounding genetic diversity. The goldfish Rag genes have not been cloned and the specificity of goldfish specific immunity system has not been studied yet. In the present study, goldfish Ragl and Rag 2 genes were cloned by polymerase chain reaction (PCR) methods, and the temporal and spatial expression pattern of RAG-1 were examined with methods of reverse transcription polymerase chain reaction(RT-PCR) and whole mount in situ hybridization. The total length of the genomic sequence of the goldfish Ragl gene from the initiation codon to the stop codon is 4188 bp, which composed of three exons and two introns. The length of exon1, 2 and 3 is 308bp, 1136bp, 1748bp respectively. The length of intronl and 2 is 105bp, 891bp respectively. The entire open reading frame (ORF) is 3192bp long, which predicated encoding a protein of 1063 amino acids. The goldfish Rag2 has no intron in its open reading frame. The length of the genomic sequence from the initiation codon to the stop codon is 1593bp, which was predicated encoding a protein of 530 amino acids. The putative structure of RAG protein in Carassius auratus are consisted of both an N-terminal domain and a core region. Comparative analysis on the sequences of ORFS and protein amino acid of Carcasses auratus, Danio rerio, Ctenopharyngodon idella, Cyprinus carpio and Oncorhynchus mykiss, revealed that both Rag1 and Rag2 are highly conserved in evolution. Phylogenetic tree analysis on these fishes based on Ragl ORF suggested that Carassius auratus is more closely related to Cyprinus carpio. Phylogenetic tree analysis on these fishes based on Rag2 ORF, however, indicated Carassius auratus is more closely related to Danio rerio. The second intron of the Ragl is also highly conserved during evolution. Transcription factor binding sites analysis on the conserved region of this intron suggested that there are some putative transcription factor binding sites in it. A common conserved area which is appeared to be the SRY and SOX5 transcription factor binding sites was observed in all the examined fishes, suggesting that the expression of ragl may have some relationship with the development of gonad. Tissue-specific expression analysis by RT-PCR methods revealed that Ragl expressed mainly in the spermary and head kidney. This observation suggested that the RAGs directed DNA recombination take place not only in immunity tissue but also in gonads. Ragl expression was first detected at day 5 post-fertilization by RT-PCR. Strong mRNA in situ hybridization signal was detected in the thymus primordium at day 9 post-fertilization, suggesting that this period might be an active DNA recombination stage of immunogenes in goldfish.