草鱼RAGs的克隆及不同发育阶段的表达分析

SEQUENCE CLONING AND EXPRESSION ANALYSIS OF RECOMBINATION ACTIVATING GENE 1 AND 2(rag1 AND rag2) IN GRASS CARP,CTENOPHARYNGODON IDELLUS

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。

It is welt known that the infectious diseases caused by the virus and bacterium frequently occur in the one-year and two-year old grass carp but rarely in the three-year old grass carp, suggesting that perfect immune system might be established in three-year old grass carp. By mediating V （D） J recombination of the lymphocyte surface receptor gene exclusively, recombination activating genes （RAGs） are the key genes playing pivotal role in regulating the development of specific immunity system in vertebrate. To understand the developmental genetics of immune system in grass carp, we has cloned grass carp rag1 and rag2 genes with polymerase chain reaction （PCR） method and then examined their expression in various developmental stages with method of reverse transcription-polymerase chain reaction （RT-PCR）. The total length of the genomic sequence of the grass carp rag1 gene from the initiation codon to the stop codon is 4188bp, which composed of three exons and two introns. The length of entire open reading frame （ORF） is 3192bp, encoding a protein containing 1063 amino acids. The grass carp rag2 gene has no intron and the length of open reading frame is 1593bp encoding a protein containing 530 amino acids. Alignment of RAGs amino acids among various vertebrate species has shown that the putative sequences in predictive function regions of grass carp shared high identity with other vertebrates. The expression of rag1 in grass carp embryos has been detected at 4 days post-fertilization （dpf） , and higher expression level has been detected maintaining during the subsequent larva stage, rag1 expression has been detected in head kidney of one-year old grass carp but not in the three-year old grass carp. These results suggest that the V （D） J recombination of the immunoglobulin （Ig） and T-cell receptor （TCR） genes and the concomitant differentiation of lymphocyte start at hatching stage and are maintained in larva and one-year old grass carp ; but in three-year old grass carp, the activation of V （D） J recombination is greatly reduced meaning the libraries of diversified Ig and TCR and relative perfect immune system are established at this stage.