棘球属绦虫线粒体基因组全序列生物信息学分析

Bioinformatic analyses on sequences of the complete mitochondrial genomes of Echinococcus genus

目的利用生物信息学技术分析棘球属绦虫线粒体基因组全序列碱基组成、基因的结构组成及排列、蛋白基因核苷酸序列变异位点、密码子使用情况及偏好性、系统发育等,为研究棘球绦虫系统起源、演化、分类及亲缘关系等提供理论基础。方法从GenBank数据库下载12种棘球属绦虫线mtDNA全序列,并以猪带绦虫作为外类群,构建系统发育树。利用OGDRAW在线分析网站及Vector NTI Express软件,分析mtDNA的组成及排列情况。用Clust-X软件对多个相似序列进行多重序列比对分析;使用Mega4.0软件选择邻接法构建进化树,并分析密码子使用情况;利用RNAfold在线预测网站使用minimum free energy (MFE) and partition function算法预测l-rRNA和s-rRNA二级结构。结果除Eg G1外,棘球绦虫属mtDNA有36个编码基因,包括22个转运RNA基因、12个蛋白基因、2个核糖体RNA基因;但Eg G1 mtDNA最小,只有30个编码基因,在起始编码区缺少6个编码转运RNA的基因,且第一个编码基因不是ND5,而是COX3;编码蛋白的基因核苷酸序列变异率为27.9%～42.7%,其中COX1最为保守,ND5变异率最大达到42.7%;棘球属绦虫线粒体蛋白质编码基因起始密码子除atg,也有一些蛋白质以gtg作为起始密码子,终止密码子以taa和tag常见,但也有以ttt作为终止密码子的;棘球绦虫属使用的密码子为密码表9,使用频率最高的密码子是UUG(2.72%),频率最低的是CUC(1%)、CGC(1%),编码亮氨酸、精氨酸的密码子最多达6个,编码甲硫氨酸、色氨酸最少只有一个,亮氨酸也是棘球属绦虫最偏好的氨基酸达到6%;棘球属绦虫核糖体基因有两个长度大小分别为977～985 bp、700～727 bp,两个基因的位置十分靠近中间只隔一个trnC基因;系统进化树中Ev、Eo单独为一枝,Em、Es及Eg G1、Ef形成姐妹枝,细粒棘球绦虫G4、G5、G6、G7、G8、G10亚型聚为一枝,进化距离较近。结论本研究将为棘球属绦虫mtDNA的研究、分子进化和分子诊断等方面提供诸多信息,并为种系鉴定起到一定的指导作用。

By means of bioinformatics,the whole sequence base composition,structure and arrangement of genes,nucleotide sequence variation sites of protein genes,codon usage and preference,phylogeny,etc.,were analyzed in Echinococcus.It will provide a theoretical basis for studying the origin,evolution,classification and phylogenetic relationship of Echinococcus genus.The whole mtDNA sequence in 12 Echinococcus genus was downloaded from GenBank database,and the phylogenetic tree was constructed with Taenia solium as an external group.The composition and arrangement of mtDNA were analyzed by OGDRAW online analysis website and Vector NTI Express software.Clust-X software was used to analyze multiple sequence alignment of similar sequences;Mega4.0 software selection adjacency method was used to construct evolutionary tree,and the use of codon was analyzed at last.The secondary structure of l-rRNA and s-rRNA was predicted by minimum free energy(MFE)and partition function algorithm using RNAfold online prediction website.Results showed that the size of mtDNA of Echinococcus genus was 13.5 kB-13.8 kB.The genome was rich in A and T,in which A content was up to about 48%,followed by T 20%,and the coding region accounted for about 73%of the whole genome sequence.Apart from Eg G1,the mtDNA of Echinococcus genus contains 36 coding genes which include 22 transporter RNA genes,12 protein genes and 2 ribosomal RNA genes.However,the genome of E.g.G1 mtDNA was the smallest with only 30 coding genes,lacking six tRNA genes in the initial coding region,and the first coding gene was COX3 instead of ND5.The nucleotide sequence variation rate of the gene encoding protein ranged from 27.9%to 42.7%,of which COX1 was the most conservative and ND5 had the highest variation rate of 42.7%.In addition to"atg",some proteins use"gtg"as the starting codon,while"taa"and"tag"were common for termination codons,but"ttt"was also used for termination codon.Codon Table 9 was used by Echinococcus genus,of which UUG(2.72%)was the most frequently used codon,while CUC(1%)and CGC(1%)were the lowest.There were six codons encoding leucine and arginine,only one codon encoding methionine and tryptophan.Leucine was also the most preferred amino acid of Echinococcus genus with 6%.The ribosomal genes of Echinococcus genus had two lengths ranging from 977-985 bp and 700-727 bp respectively,and the positions of the two genes were very close,separated by one trnC gene;in phylogenetic tree,Ev and Eo were single branch while Em,E.s and Eg G1,Ef form sister branches,and The G4,G5,G6,G7,G8,and G10 subtypes of Echinococcus granulosus were clustered into one branch with a relatively close evolutionary distance.In conclusion,this study will provide a lot of information for the research,molecular evolution and molecular diagnosis of mtDNA in Echinococcus genus,which plays a guiding role in phylogenetic identification,drug and vaccine development in the future.