草鱼RAGs的克隆及不同发育阶段的表达分析
SEQUENCE CLON ING AND EXPRESSION ANALYSIS OF RECOMBINATION ACT IVAT ING GENE 1 AND 2( rag1 AND rag2) IN GRASS CARP, CTENOPHARYNGODON IDELLUS
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摘要: 草鱼(Ctenopharyngodon idellus)在1、2龄阶段的抗病能力很差,但到3龄阶段后又具有了很强的抗病能力,提示草鱼的免疫系统可能要到3龄发育阶段才会完善。重组激活基因(Recombination activating genes,RAGs)编码的蛋白质专一性介导淋巴细胞抗原识别受体基因的V(D)J重组,在脊椎动物淋巴细胞正常发育成熟过程中起关键作用。为了进一步研究草鱼免疫系统建立和完善的发育遗传学因素,通过PCR方法克隆了草鱼重组激活基因rag1和rag2,分析了它们在不同发育阶段的表达。草鱼rag1基因从起始密码到终止密码总长4188 bp,由三个外显子和两个内含子组成,其开放阅读框长3192 bp,编码1063个氨基酸。草鱼rag2基因从起始密码到终止密码总长1593bp,没有内含子,编码530个氨基酸。氨基酸序列比对分析表明,预测的草鱼RAG1和RAG2蛋白的功能区域与其他脊椎动物的相应功能区具有很高的同源性。从受精后第4天开始,RT-PCR即可检测到rag1基因的表达,在随后的幼鱼阶段rag1基因维持了高水平的表达。在1龄草鱼的头肾中仍可以检测到rag1基因的表达,但在3龄草鱼头肾中没有检测到rag1基因的表达。这些结果表明草鱼从胚胎的孵化期开始到1龄幼鱼阶段是免疫球蛋白(Ig)和T细胞受体(TCR)V(D)J重组的活跃时期,在3龄发育阶段其淋巴细胞系中V(D)J重组活动已经很少,免疫系统中Ig和TCR库的多样化过程趋于完成,其免疫系统已经比较完善。Abstract: 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.