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Cloning and identification of the rag1 gene in channel catfish

Objective To amplify and sequence the fragments of recombination activating gene 1 ( rag1 ) from the channel catfish genomic DNA so as to provide new insights into the early evolutionary patterns and the mechanisms of genetic diversity of rag1 gene. Methods The degenerate primers, designed based on the high conserved characteristics of rag1 gene, were used to amplify the fragments of rag1 gene from the channel catfish genomic DNA by PCR. The fragments were cloned into a T/A plasmid. Clones were randomly chosen for sequencing and all sequences were obtained on both strands. Several kinds of bioinformatic methods were used to analyze the open reading frames, introns, and identity percentage with those of other species. Selected sequences were also analyzed using the program “CLUSTAL W” for the multiple alignment and unrooted phylogenetic tree. The obtained sequences were submitted to GenBank using the program “Bankit” to apply for the sequence numbers. Results Three fragments of rag1 gene were obtained with a total length of 3 218 bp. An ORF of 2 235 bp and an intron of 293 bp within the sequence were identified. The percentage identity of the sequence with rag1 gene of other species was more than 70%. The multiple alignment of the cDNA sequence with rag1 complete cDNA sequences in zebrafish, rainbow trout, and bull shark indicted that the proportion of identical base sites was 43.9%. The proportion of identical base sites from the 1st to the 1 351st was 33.2%, and the proportion of identical base sites from the 1 352nd to the 2 925 th was 53.1%. There was a significant diversity between these two regions ( P 0.01). Conclusion The fragments of channel catfish rag1 gene are obtained by PCR using degenerate primers. The registered numbers of those sequences are AY423858, AY423859, and AY423860. The rag1 gene is highly conserved in different species. The dissimilarities in rag1 gene in different species are concentrated to the amino terminal region. The phylogenetic tree indicates the relationships of 13 kinds of bony fishes in the course of evolution.