基于COⅠ、12S rDNA和ITS-2基因序列的犬恶丝虫分子系统发生研究
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摘要
犬恶丝虫(Dirofiliaria immitis)又名心丝虫(heartworm),为丝虫的一种,在传统形态学分类中被划分于丝虫总科,盘尾丝虫科,恶丝虫属。犬恶丝虫病为人兽共患寄生虫病,呈世界性分布,严重影响多种犬科和猫科动物以及部分野生肉食动物的健康,给社会经济、生态环境带来巨大损失和危害。寄生虫的系统发生研究是寄生虫病防治的理论基础,进行犬恶丝虫的系统发生研究对于全面认识犬恶丝虫及犬恶丝虫病的防治具有重要意义。
论文以线粒体基因细胞色素氧化酶亚单位Ⅰ(cytochrome coxidase subunitⅠ,COI)和小亚单位核糖体核糖核酸(small subunit ribosomal RNA,12S rDNA)及核糖体基因内部转录区2(internal transcribed spacer 2,ITS-2)为靶序列,通过对相关序列进行分析,运用不同的进化树构建方法,包括最小进化法、邻接法和最大似然法,从距离距阵和遗传性状两个方面构建系统树,对小熊猫恶丝虫(重庆株、成都株)和犬恶丝虫(雅安株)的分子分类及两者与其它相关丝虫的种系发生关系进行研究。
通过分析不同虫种COI、12S rDNA和ITS-2基因序列间的差异和构建系统进化树,结果显示,不同虫种COI基因序列的同源性高于82.8%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了99.8%;各序列间的趋异值在0～19.6之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于0.2。不同虫种12S rDNA基因序列的同源性高于78.0%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了98.4%;各序列间的趋异值在0.2～26.1之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于1.6。不同虫种ITS-2基因序列的同源性高于71.20%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了98.5%;各序列间的趋异值在0.4～36.3之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于1.6。序列分析的结果表明小熊猫恶丝虫和犬恶丝虫具有高度的同源性,应为同一虫种,小熊猫恶丝虫为犬恶丝虫的同种异名。
在系统进化树中,包括基于COI基因构建的ME树和MP树,基于12S rDNA基因构建的ME树和MP树以及基于ITS-2基因构建的NJ树和MP树,小熊猫恶丝虫和犬恶丝虫均被聚合到一起,形成一个深的分支树,其置信值高于99%;盘尾丝虫科聚合成为3个大的分支树,即盘尾丝虫属,恶丝虫属及布鲁丝虫属+吴策丝虫属。这一结果表明基于形态学和分子生物学特征的盘尾丝虫超科分类结果存在一定差异,从一定程度上来说,应对盘尾丝虫超科的虫种分类结果进行部分的修订。
Comparing with high speed development of molecular biology,molecular systematization has been used in all areas of species classified as a branch,included in parasitic species taxonomy.Heartworm,as a filarial nematode,was placed in genus Dirofilaria under the family Onchocercidae based on morphological characters,which belongs to the Filarioidea superfamily(order Spirurida).Heartworm disease is a worldwide parasitic zoonosis,which mainly infected husbandry animal(dogs and cats) but could infect some wildlife,such as wolves,foxes,lions,ferrets,Raccoon dogs,Saki monkeys, Alopexs,Wolverines,tigers,bears,Cougars,Zalophuses,Neofelis nebulosas,harbor seals, castor fibers,otters,Raccoons,muskrats,Gorillas,chimpanzees and so on.To learn this disease has an important effect on developing of animal husbandry-raising and protecting human health.
In this paper,mtDNA gene included cytochrome coxidase subunitⅠ(COI) and small subunit ribosomal RNA(12S rDNA),and ribosomal gene internal transcribed spacer 2 (ITS-2) as targets were used to analyse phylogentic relation about heartworm from red panda and dog with other filariae.Sequence alignments were used to analyse the difference of sequences by DNAStar and Clustal X program.Different evolutional approaches include minimal evolution(ME),neighbour-joiner(NJ) and maximum parsimony(MP), from distance matrix and character state,were used to construct the phylogenetic tree by MEGA 3.1 program.The result of sequences alignment and phylogenetic tree were used to evaluate the evolutional relation between Dirofilaria spp and other species included in the family Onchocercidae.
The COI gene sequences alignment included 18 taxa(17 ingroup and 1 outgroup) and 652bp.The pairwise distances of different COI gene sequences were displayed by percent identity and divergence value.The percent identities were larger than 82.8%with all sequences,species Dirofilaria ailure and Drofilaria immitis had higher percent identity, which exceed 99.8%.The divergences value were 0～19.6 in all COI gene sequences,the value between the specie D.ailure and D.immitis was less than 0.2.The 12S rDNA gene sequences alignment included 16 taxa(15 ingroup and 1 outgroup) and 465bp.The percent identities were larger than 78.0%with all sequences,species D.ailure and D.immitis had higher percent identity,which exceed 98.4%.The divergence values were 0.2～26.1 in all 12S rDNA gene sequences,the value between the specie D.ailure and D.immitis was less than 1.6.The ITS-2 gene sequences aligenment included 7 taxa(6 ingroup and 1 outgroup) and 521bp.The percent identities were larger than 71.2,species D.ailure and D.immitis had higher percent identity,which was exceed 98.5%.The divergence value were 0.4～36.3 in all ITS-2 gene sequence.The value between the specie D.ailure and D.immitis was less than1.6.
These different phylogenetic trees,included ME and MP trees based on COI gene sequences,ME and MP trees based on 12S rDNA gene sequences,NJ and MP trees based on ITS-2 gene sequences,showed a definitive conclusion,that D.ailure and D.immitis were put together as a depth branch tree with a high confidence level(99%or 100%). Meanwhile,the family Onchocercidae was assembled to three major groupings of species/genera,including Onchocerca spp.;Dirofilaria spp.;Brugia+Wuchereria.
Our investigation results implied that D.ailure and D.immitis have a high homology, D.ailure was the synonym of D.immitis.Systematization based on morphological characters was not congruent with molecular characters in the family Onchocercidae.From a molecule phylogenetics visual angle,systematization should be evaluated again in the Filarioidea superfamily.
论文以线粒体基因细胞色素氧化酶亚单位Ⅰ(cytochrome coxidase subunitⅠ,COI)和小亚单位核糖体核糖核酸(small subunit ribosomal RNA,12S rDNA)及核糖体基因内部转录区2(internal transcribed spacer 2,ITS-2)为靶序列,通过对相关序列进行分析,运用不同的进化树构建方法,包括最小进化法、邻接法和最大似然法,从距离距阵和遗传性状两个方面构建系统树,对小熊猫恶丝虫(重庆株、成都株)和犬恶丝虫(雅安株)的分子分类及两者与其它相关丝虫的种系发生关系进行研究。
通过分析不同虫种COI、12S rDNA和ITS-2基因序列间的差异和构建系统进化树,结果显示,不同虫种COI基因序列的同源性高于82.8%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了99.8%;各序列间的趋异值在0～19.6之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于0.2。不同虫种12S rDNA基因序列的同源性高于78.0%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了98.4%;各序列间的趋异值在0.2～26.1之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于1.6。不同虫种ITS-2基因序列的同源性高于71.20%,小熊猫恶丝虫和犬恶丝虫之间的序列同源性超过了98.5%;各序列间的趋异值在0.4～36.3之间,小熊猫恶丝虫和犬恶丝虫之间的序列趋异值小于1.6。序列分析的结果表明小熊猫恶丝虫和犬恶丝虫具有高度的同源性,应为同一虫种,小熊猫恶丝虫为犬恶丝虫的同种异名。
在系统进化树中,包括基于COI基因构建的ME树和MP树,基于12S rDNA基因构建的ME树和MP树以及基于ITS-2基因构建的NJ树和MP树,小熊猫恶丝虫和犬恶丝虫均被聚合到一起,形成一个深的分支树,其置信值高于99%;盘尾丝虫科聚合成为3个大的分支树,即盘尾丝虫属,恶丝虫属及布鲁丝虫属+吴策丝虫属。这一结果表明基于形态学和分子生物学特征的盘尾丝虫超科分类结果存在一定差异,从一定程度上来说,应对盘尾丝虫超科的虫种分类结果进行部分的修订。
Comparing with high speed development of molecular biology,molecular systematization has been used in all areas of species classified as a branch,included in parasitic species taxonomy.Heartworm,as a filarial nematode,was placed in genus Dirofilaria under the family Onchocercidae based on morphological characters,which belongs to the Filarioidea superfamily(order Spirurida).Heartworm disease is a worldwide parasitic zoonosis,which mainly infected husbandry animal(dogs and cats) but could infect some wildlife,such as wolves,foxes,lions,ferrets,Raccoon dogs,Saki monkeys, Alopexs,Wolverines,tigers,bears,Cougars,Zalophuses,Neofelis nebulosas,harbor seals, castor fibers,otters,Raccoons,muskrats,Gorillas,chimpanzees and so on.To learn this disease has an important effect on developing of animal husbandry-raising and protecting human health.
In this paper,mtDNA gene included cytochrome coxidase subunitⅠ(COI) and small subunit ribosomal RNA(12S rDNA),and ribosomal gene internal transcribed spacer 2 (ITS-2) as targets were used to analyse phylogentic relation about heartworm from red panda and dog with other filariae.Sequence alignments were used to analyse the difference of sequences by DNAStar and Clustal X program.Different evolutional approaches include minimal evolution(ME),neighbour-joiner(NJ) and maximum parsimony(MP), from distance matrix and character state,were used to construct the phylogenetic tree by MEGA 3.1 program.The result of sequences alignment and phylogenetic tree were used to evaluate the evolutional relation between Dirofilaria spp and other species included in the family Onchocercidae.
The COI gene sequences alignment included 18 taxa(17 ingroup and 1 outgroup) and 652bp.The pairwise distances of different COI gene sequences were displayed by percent identity and divergence value.The percent identities were larger than 82.8%with all sequences,species Dirofilaria ailure and Drofilaria immitis had higher percent identity, which exceed 99.8%.The divergences value were 0～19.6 in all COI gene sequences,the value between the specie D.ailure and D.immitis was less than 0.2.The 12S rDNA gene sequences alignment included 16 taxa(15 ingroup and 1 outgroup) and 465bp.The percent identities were larger than 78.0%with all sequences,species D.ailure and D.immitis had higher percent identity,which exceed 98.4%.The divergence values were 0.2～26.1 in all 12S rDNA gene sequences,the value between the specie D.ailure and D.immitis was less than 1.6.The ITS-2 gene sequences aligenment included 7 taxa(6 ingroup and 1 outgroup) and 521bp.The percent identities were larger than 71.2,species D.ailure and D.immitis had higher percent identity,which was exceed 98.5%.The divergence value were 0.4～36.3 in all ITS-2 gene sequence.The value between the specie D.ailure and D.immitis was less than1.6.
These different phylogenetic trees,included ME and MP trees based on COI gene sequences,ME and MP trees based on 12S rDNA gene sequences,NJ and MP trees based on ITS-2 gene sequences,showed a definitive conclusion,that D.ailure and D.immitis were put together as a depth branch tree with a high confidence level(99%or 100%). Meanwhile,the family Onchocercidae was assembled to three major groupings of species/genera,including Onchocerca spp.;Dirofilaria spp.;Brugia+Wuchereria.
Our investigation results implied that D.ailure and D.immitis have a high homology, D.ailure was the synonym of D.immitis.Systematization based on morphological characters was not congruent with molecular characters in the family Onchocercidae.From a molecule phylogenetics visual angle,systematization should be evaluated again in the Filarioidea superfamily.
引文
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