带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 Taenia species

通过利用生物生物信息学方法分析带属绦虫线粒体DNA（mtDNA）碱基组成、基因结构、密码子利用、基因变异及其在分子系统进化研究中的应用价值等,以期为带属虫种线粒体功能基因组学研究、分子分类学研究及其疾病诊断提供重要依据。从NCBI GenBank中下载已测序的6种带属绦虫线粒体基因组（mt genome）全序列,以细粒棘球绦虫mtDNA序列作为参考序列（外群）,用ClustalX软件（1.83）（http：//www2.ebi.ac.uk/clustal w/）进行多重序列比对,用DNAStar软件进行序列差异性分析,用MEGA4软件的邻接法（Neighbor-joining method）构建进化树,核苷酸进化距离算法用最大似然法（Maxi mumlikelihood method）,氨基酸进化距离算法用泊松校正法（Poissoncorrection method）,进化树检验用自展法（Bootstrap test）。tRNA和2个非编码区RNA二级结构预测分别使用软件ARWEN和RNAstructure Version 4.6。结果显示,带属绦虫mtDNA为双链闭环分子,为13.3~13.7 kb;4种碱基成分中,T含量最高（超过47%）,C含量最低（低于9%）;共有36个按照相同顺序排列的编码基因,其中蛋白质编码基因有12个,tRNA编码基因有22个（其中编码丝氨酸-tRNA和亮氨酸-tRNA的基因有2个,其余18种tRNA分子各有1个）,rRNA编码基因有2个（包括1个大亚基和1个小亚基）。基因组结构紧凑,基因间存在重叠区域,如nad4L和nad4基因;除广泛使用ATG作为起始密码子编码蛋氨酸,部分基因如多头绦虫nad6基因用GTG作为蛋白质翻译的起始密码子。线粒体tRNA长度为58~76 nt,二级结构多数呈典型的三叶草结构,少数呈D-loop结构;2个线粒体rRNA亚基基因rrnL和rrnS被trnC基因分隔开;线粒体基因组有2个大的非编码区,1个长非编码区（LNR）和1个短非编码区（SNR）;线粒体基因组中蛋白编码基因之间的差异为5.7%~28.9%,其中cox1和nad4L基因比较保守,而nad5和nad6则变异较大,进化较快。结果表明,根据线粒体基因组序列绘制的带属绦虫分子进化树表明,亚洲带绦虫（T.asiatica）和牛带绦虫（T.saginata）间的亲缘关系最近,成为姊妹种,而多头绦虫（T.multiceps）与前两者的亲缘关系比猪带绦虫（T.solium）与前两者的关系更近,但与虫种的生物学特征存在一定差异。

The current study is to analyze base composition,gene organization,codon usage and RNAs＇ structures of mitochondrial genomes of Taenia species,which provide a fundamental foundation for comparative mt genomics,systematic studies,molecular taxonomy and discrimination.Alignments were done by using the ClustalX（1.83）program.Percentage pairwise divergences of nucleotide and amino acid sequences were calculated using DNAStar software.The phylogenetic analyses were performed in MEGA4 program by the Neighbor-joining method with bootstrap and using Echinococcus granulosus as an outgroup.The evolutionary distances were computed using the Poisson correction method for amino acids and the Maximum Composite Likelihood method for nucleotides,respectively.The putative stem-loop structures of tRNAs and non-coding mitochondrial regions（LNR and SNR） were inferred using the ARWEN program and the RNAstructure program（Version 4.6）,respectively.The mt genomes of Taenia species were from 13.4 kb to 13.7 kb long,coding 12 proteins,two ribosomal RNAs（rRNAs,a small and a large subunit） and 22 transfer RNAs（tRNAs）,which were arranged in the same order.Overlapping regions are found among genes,such as between nad4L and nad4.The GTG initiation codon is used for some genes,for example,nad6 of T.multiceps.The size of the 12 protein-coding genes of the two Taenia tapeworms was conserved.The tRNA genes were 57-74 bp long,and although 18 predicted secondary structures have a typical clover-leaf shape with a paired dihydrouridine（DHU） arm,4 tRNAs have a D-loop structure without the DHU arm.The genes for the two mitochondrial rRNA subunit genes rrnL and rrnS in the two Taenia tapeworms are separated by trnC gene.The non-coding regions of the mt genomes of Taenia spp.consist of two regions： a short non-coding region（SNR） and a long non-coding region（LNR）,respectively.The mt DNAs of Taenia spp.have a high level of T（over 45%） and a low level of C（below 9%）.The overall nucleic sequence differences in the protein-coding genes of mt genomes among Taenia spp.were from 5.7% to 28.9%.Among the protein-coding genes,cox1 and nad4L genes were relatively conserved,while nad5 and nad6 varied much.Phylogenetic analysis of Taenia species showed that T.multiceps is more closely related to T.asiatica and T.saginata than T.solium using either the complete mitochondrial DNA or protein-coding or tRNAs＇ sequences,suggesting that T.asiatica,T.saginata and T.multiceps should be sister species.