广州管圆线虫线粒体COX1基因碱基置换饱和度分析

Substitution Saturation Analysis of Mitochondrial Cytochrome C Oxidase Subunit 1(COX1) Gene of Angiostrongylus cantonensis

目的检测广州管圆线虫线粒体COX1基因碱基置换饱和度。方法收集全国33个采样点的广州管圆线虫成虫130条,对其COX1基因全长进行PCR扩增和测序,拼接、比对后,利用DnaSP软件分析序列的碱基多样性、单倍型数量和突变位点,根据碱基颠换和碱基转换随遗传距离增加的变化趋势判断COX1基因饱和度。结果共获得130个COX1全长序列,长度均为1 577 bp,碱基突变位点为171个,占10.8%,核苷酸多态性为0.02841。突变的空间分布无明显聚集性,均匀分布于整条DNA链上。其中单倍型39个,多态性为0.8114。单倍型两两比较发现,随遗传距离增加,广州管圆线虫的COX1基因碱基转换突变和颠换突变均呈直线上升,且转换数量多于颠换数量,增加速率分别为0.76和0.16,碱基置换未饱和;不同氨基酸密码子位点碱基置换速率差异较大,速率最大的为第三位点,其次为第一位点,最小的为第二位点。后圆线虫总科(Metastrongyloidea)7种线虫的COX1基因饱和度分析显示,遗传距离约为0.15时,碱基颠换数量开始超过转换数量。将本研究所获COX1基因序列与后圆线虫总科COX1基因综合分析,结果表明,碱基转换的比例在6%时出现平台期,而碱基颠换仍直线增加。结论广州管圆线虫线粒体COX1基因碱基置换未饱和。

Objective To determine the substitution saturation of mitochondrial cytochrome c oxidase subunit I（COX1） gene of Angiostrongylus cantonensis. Methods One hundred and thirty A. cantonensis adult worms were collected from 33 sampling sites and used to amplify the complete sequence of COX1 gene. The nucleotide diversity,the number of haplotypes and the number of mutations were calculated by DnaSP software after sequence alignment. The distribution of base substitutions was characterized. The level of nucleotide substitution saturation was evaluated by plotting transitions and transversions against pairwise genetic distance. Results A total of 130 complete COX1 sequences of 1 577 bp were obtained. One hundred seventy-one nucleotides were found to be variable,resulting in 39 haplotypes with a diversity of 0.8114. The nucleotide diversity was 0.02841. Mutations were evenly distributed along the COX1 gene and did not show significant clustering. The analysis of COX1 gene showed that the amount of substitutions was increasing with the extension of genetic distance,and transitions outnumbered transversions;the increase rate was 0.76,and 0.16,respectively. The substitution rate was markedly different among the three codon positions：the maximum rate was found at the third codon position,followed by the first position,and the second position. The analysis of COX1 gene among 7 members of Metastrongyloidea showed that transversions outnumbered transitions when the genetic distance was more than 0.15. When the number of transitions exceeded 6%,a plateau was reached;while the transvertions increased linearly. Conclusion No substitution saturation is found in mitochondrial COX1 gene of A. cantonensis. Therefore, the substitutions at each codon position could be considered in phylogeny analysis.