摘要
目的 尝试用简并引物扩增序列未知的channelcatfish基因组DNA中rag1基因的片段并测序 ,为rag1基因的早期进化模式和遗传学变异情况的研究提供直接的证据。方法 基于rag1基因的高度保守设计简并引物 ,PCR扩增channelcatfish基因组DNA中rag1基因的片段 ,TA克隆并双向测序。将所得序列资料拼接 ,用多种生物信息学方法分析其ORF、内含子、与其它物种rag1基因及Rag1蛋白的相似性 ,并做多序列比较和系统进化树分析。结果 扩增出 3条chan nelcatfishrag1基因的DNA片段。从拼接后的序列中找到一 2 2 3 5bp的ORF和一 2 93bp的内含子 ,所得序列与其它物种rag1基因的相似程度高 (多大于 70 %) ,去除内含子后的channelcatfishrag1基因序列与zebrafish、rainbowtrout、bullshark的rag1基因cDNA全序列碱基一致的位点占 43 9%,自第 13 5 2位碱基至 2 92 5位碱基为 5 3 1%,而自第 1位碱基至 13 5 1位碱基为 3 3 2 %,有非常显著的差异 (P <0 .0 1)。结论 用简并引物成功扩增出channelcatfishrag1基因的DNA片段。序列在GenBank中的登记号是 :AY42 3 85 8(去除内含子序列 ) ,AY42 3 85 9。rag1基因进化缓慢 ,高度保守 ,不同物种rag1基因的变异相对集中在氨基末端的区域。系统进化树分析显示了 13种硬骨鱼在进化上关?
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.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2004年第8期700-703,共4页
Journal of Third Military Medical University
关键词
重组激活基因
V(D)J重排
聚合酶链反应
简并引物
生物信息学
recombination activating gene
V(D)J rearrangement
polymerase chain reaction
degenerate primer
bioinformatics
