SEQUENCE CLON ING AND EXPRESSION ANALYSIS OF RECOMBINATION ACT IVAT ING GENE 1 AND 2( rag1 AND rag2) IN GRASS CARP, CTENOPHARYNGODON IDELLUS

It is well known that the infectious diseases caused by the virus and bacterium frequently occur in the one-yearand two-year old grass carp but rarely in the three-year old grass carp,suggesting that perfect mi mune system might be establishedin three-year o ld grass carp. Bymediating V( D) J recom bination of the lymphocyte surface receptor gene exclusively,recom bination act ivat ing genes (RAGs) are the key genes play ing p ivotal role in regulating the developm ent of specific mi m un ity system in vertebrate. To understand the developm ental genetics of mi mune system in grass carp,w e hascloned grass carp rag1 and rag2 genesw ith polym erase chain reaction ( PCR) m ethod and then exam ined their expressionin various developm ental stages with method of reverse transcription-po lym erase cha in reaction ( RT-PCR). The totallength of the genomic sequence of the grass carp rag1 gene from the initiation codon to the stop codon is 4188bp,whichcomposed of three exons and two introns. T he length of entire open read ing frame (ORF) is 3192bp,encoding a prote inconta ining 1063 am ino ac ids. The grass carp rag2 gene has no intron and the length of open reading fram e is 1593bp encodinga prote in containing 530 amino acids. A lignm ent of RAGs am inoacids among various vertebrate species has shownthat the putative sequences in predictive function reg ions of grass carp shared high ident ity with other vertebrates. The expression of rag1 in grass carp embryos has been detected at 4 days post-fertilization (dpf),and higher expression level hasbeen detectedm ainta ining during the subsequent larva stage. rag1 expression has been detected in head kidney of one-yearold grass carp butnot in the three-year old grass carp. These results suggest that theV(D) J recom bination of the mimunoglobulin(Ig) and T-cell receptor (TCR) genes and the concom itant differentiation of lymphocyte start at hatching stageand arema intained in larva and one-year o ld grass carp; but in three-year old grass carp,the activation ofV(D) J recombination is greatly reduced meaning the libraries of diversified Ig and TCR and relat ive perfect mimune system are establishedat this stage.