人、猪鞭虫核糖体DNA及线粒体DNA遗传多样性的研究
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摘要
毛首鞭形线虫简称鞭虫,属于无尾感器纲(Aphasmidea)鞭尾目(Trichurata)鞭虫科(Trichuridae)鞭虫属(Trichuris),为人体和动物肠道常见的寄生线虫。据资料记载,猪鞭虫还可感染人体。随着生物种群的不断进化和发展,尤其对那些相似种或近缘种,寄生虫的形态学鉴定越来越显出它的局限性。尤其是近几年,PCR技术在寄生虫的分子分类学、分子遗传学、种(株)鉴定等方面的研究已取得了很大的成功,如以PCR扩增反应为基础的限制性片段长度多态性(PCR-RFLP)、随机扩增多态性DNA(RAPD)及单链构象多态性(SSCP)分析等。
本研究首次对来源于中国不同地方不同宿主鞭虫的间隔区序列(internal transcribed spacer,TS)以及线粒体(mtDNA)基因大亚基(lsrRNA)、NADH脱氢酶亚单位Ⅳ基因(nad4)进行PCR扩增、克隆及序列分析。结果,猪鞭虫ITS-1序列长为655-661bp,ITS-2序列长为530-538 bp;人鞭虫ITS-1序列长为600-627bp,ITS-2序列长为468-486 bp;二者的5.8S序列长均为154bp。其中,猪鞭虫与西班牙猪鞭虫ITS-1序列的差异碱基有14个,差异率为2.1%;ITS-2序列的差异碱基有18个,差异率为3.4%:5.8S序列的种(株)内差异碱基有1个,差异率为0.6%。人鞭虫ITS-1序列的种(株)内差异碱基有34个,差异率为5.4%;ITS-2序列的种(株)内差异碱基有30个,差异率为6.4%。猪鞭虫ITS-1序列的种(株)内差异碱基有15个,差异率为2.3%;ITS-2序列的种(株)内差异碱基有13个,差异率为2.4%。人鞭虫与猪鞭虫ITS-1序列的碱基差异率达45.1%,ITS-2序列的碱基差异率达74.7%,5.8S序列的差异碱基有6个差异率达3.9%。在猪鞭虫可见微卫星重复序列(ITS-1:AGC,CGC;ITS-2:GCT,CG,C),人鞭虫(ITS-1:CAG,GGC;ITS-2:CAG,CGA,CGG,GCG)。剔除ITS-1、ITS-2序列中的微卫星重复序列后,人鞭虫ITS-1序列的种(株)内差异碱基有10个,差异率为1.7%;ITS-2序列的种(株)内差异碱基有5个,差异率为1.1%。猪鞭虫ITS-1序列的种(株)内差异碱基有9个,差异率为1.4%;ITS-2序列的种(株)内差异碱基有9个,差异率为1.8%。
从GeneBank数据库下载Trichinella spiralis的ITS-2基因片段作为外群(accession number:AY851266),相关鞭虫的ITS-2基因片段(Trichuris.ovis,AJ238220;T.leporis,AJ251321;T.skrjabini,AJ489248;T.vulpis,AM234616;T.arvicolae,AJ310661;T.muris,AJ299407),采用PHYLIP(version3.67)的距离法(邻接法)以及PAUP(version 4.0b4a)的最大简约法,一齐进行进化树分析。
结果显示,rDNA的ITS-1序列的PCR-RFLP差异性分析以及ITS-2序列作为分子遗传标记的遗传进化树关系分析图显示,能有效区分鞭虫属内的各种(株),并且结果显示人鞭虫与猪鞭虫遗传距离最为接近。这些研究结果有助于鞭虫的生活史、传播途径、流行病学、种群遗传学的研究和其相关疾病的诊断,为进一步的分子遗传学和分子诊断学研究奠定基础。
Genus Trichuris(Aphasmidea:Trichurada:Trichuridae)as an intestinal infection parasitic disease that can be seen much more common on human and animal.According to records,Trichuris suis can also infect human.With constant evolution and development of the population,particularly those of similar or related species,the morphological identification of parasites increasingly shows its limitations.In particular,in recent years,the parasites PCR technology in the molecular taxonomy,molecular genetics,species(strains) identification of the research has achieved great success,such as the PCR amplification reaction-based restriction fragment length polymorphism(PCR-RFLP),random amplified polymorphic DNA(RAPD)and single strand conformation polymorphism(SSCP)analysis.
This study is the first-time to study Trichuris that come from different host and different parts of China.The internal transcribed spacer sequences(ITS)and mitochondria(mtDNA)such as large subunit gene(lsrRNA),NADHⅣdehydrogenase subunit gene(nad4)through PCR amplification,cloning and sequence analysis.The results:ITS-1 sequences length of T.suis are 655-661 bp,and ITS-2 sequence are 530-538 bp;ITS-1 sequences length of T.trichiura are 600-627 bp,and ITS-2 sequence are 468-486 bp; both the 5.8 S sequences are 154 bp.There are 14 different base pairs in ITS-1 sequence of Trichuris suis between China and Spain,the difference is 2.1%;18 different base pairs in ITS-2 sequence,the difference is 3.4%;1 different base pair in 5.8 S sequence,the difference is 0.6%.There are 34 different base pairs in ITS-1 sequence of T.trichiura,the difference is 5.4%;34 different base pairs in ITS-2 sequence,the difference is 6.4%.There are 15 different base pairs in ITS-1 sequence of T.suis,the difference was 2.3%;13 different base pairs in ITS-2 sequence,the difference was 2.4%.The difference in ITS-1 sequence between T.trichiura and T.suis up to 45.1%,and in ITS-2 sequence up to 74.7%.There are six different base pairs in 5.8 S sequence,difference is 3.9%.Microsatellite repeat sequences can be seen in T.suis (ITS-1:AGC,CGC;ITS-2:GCT,CG,C),in T.trichiura(ITS-1:CAG,GGC;ITS-2:CAG,CGA,CGG, GCG).Delete microsatellite repeat sequence in ITS-1 and ITS-2 sequence,there are 10 different base pairs in ITS-1 sequence of T.trichiura,the difference is 1.7%.In ITS-2 sequence,five different base pairs,the difference is 1.1%.There are 9 different base pairs in ITS-1 sequence of T.suis,the difference was 1.4%;in ITS-2 sequence,9 different bases,the difference is 1.8%.
Related GeneBank database were downloaded(accession number:AY851266;Trichuris.ovis, AJ238220;T.leporis,AJ251321;T.skrjabini,AJ489248;T.vulpis,AM234616;T.arvicolae,AJ310661;T. muris,AJ299407).ITS-2 gene fragments of the Trichinella spiralis as a out-group(accession number: AY851266),using Neighbor Joining method of PHYLIP(version 3.67),as well as maximum parsimony method of PAUP(version 4.0b4a).Confidence levels for each grouping were calculated with a bootstrap program(SEQBOOT)in the PHYLIP package,with a total of 1000 replicates.The trees were produced by application of the CONSENCE program of the PHYLIP package.
Even though the result of phylogenetic analysis showed a high degree of similarity between T. trichiura and T.suis.Ultimately,the resolution of the question as to whether the two taxa represent separate species requires reciprocal cross-mating experiments to be conducted,using appropriate controls, to determine the extent of mating and the reproductive capabilities of any "hybrid" individuals produced. Only if cross-mating experiments could provide an answer to the question as to whether they represent the same or different species.
本研究首次对来源于中国不同地方不同宿主鞭虫的间隔区序列(internal transcribed spacer,TS)以及线粒体(mtDNA)基因大亚基(lsrRNA)、NADH脱氢酶亚单位Ⅳ基因(nad4)进行PCR扩增、克隆及序列分析。结果,猪鞭虫ITS-1序列长为655-661bp,ITS-2序列长为530-538 bp;人鞭虫ITS-1序列长为600-627bp,ITS-2序列长为468-486 bp;二者的5.8S序列长均为154bp。其中,猪鞭虫与西班牙猪鞭虫ITS-1序列的差异碱基有14个,差异率为2.1%;ITS-2序列的差异碱基有18个,差异率为3.4%:5.8S序列的种(株)内差异碱基有1个,差异率为0.6%。人鞭虫ITS-1序列的种(株)内差异碱基有34个,差异率为5.4%;ITS-2序列的种(株)内差异碱基有30个,差异率为6.4%。猪鞭虫ITS-1序列的种(株)内差异碱基有15个,差异率为2.3%;ITS-2序列的种(株)内差异碱基有13个,差异率为2.4%。人鞭虫与猪鞭虫ITS-1序列的碱基差异率达45.1%,ITS-2序列的碱基差异率达74.7%,5.8S序列的差异碱基有6个差异率达3.9%。在猪鞭虫可见微卫星重复序列(ITS-1:AGC,CGC;ITS-2:GCT,CG,C),人鞭虫(ITS-1:CAG,GGC;ITS-2:CAG,CGA,CGG,GCG)。剔除ITS-1、ITS-2序列中的微卫星重复序列后,人鞭虫ITS-1序列的种(株)内差异碱基有10个,差异率为1.7%;ITS-2序列的种(株)内差异碱基有5个,差异率为1.1%。猪鞭虫ITS-1序列的种(株)内差异碱基有9个,差异率为1.4%;ITS-2序列的种(株)内差异碱基有9个,差异率为1.8%。
从GeneBank数据库下载Trichinella spiralis的ITS-2基因片段作为外群(accession number:AY851266),相关鞭虫的ITS-2基因片段(Trichuris.ovis,AJ238220;T.leporis,AJ251321;T.skrjabini,AJ489248;T.vulpis,AM234616;T.arvicolae,AJ310661;T.muris,AJ299407),采用PHYLIP(version3.67)的距离法(邻接法)以及PAUP(version 4.0b4a)的最大简约法,一齐进行进化树分析。
结果显示,rDNA的ITS-1序列的PCR-RFLP差异性分析以及ITS-2序列作为分子遗传标记的遗传进化树关系分析图显示,能有效区分鞭虫属内的各种(株),并且结果显示人鞭虫与猪鞭虫遗传距离最为接近。这些研究结果有助于鞭虫的生活史、传播途径、流行病学、种群遗传学的研究和其相关疾病的诊断,为进一步的分子遗传学和分子诊断学研究奠定基础。
Genus Trichuris(Aphasmidea:Trichurada:Trichuridae)as an intestinal infection parasitic disease that can be seen much more common on human and animal.According to records,Trichuris suis can also infect human.With constant evolution and development of the population,particularly those of similar or related species,the morphological identification of parasites increasingly shows its limitations.In particular,in recent years,the parasites PCR technology in the molecular taxonomy,molecular genetics,species(strains) identification of the research has achieved great success,such as the PCR amplification reaction-based restriction fragment length polymorphism(PCR-RFLP),random amplified polymorphic DNA(RAPD)and single strand conformation polymorphism(SSCP)analysis.
This study is the first-time to study Trichuris that come from different host and different parts of China.The internal transcribed spacer sequences(ITS)and mitochondria(mtDNA)such as large subunit gene(lsrRNA),NADHⅣdehydrogenase subunit gene(nad4)through PCR amplification,cloning and sequence analysis.The results:ITS-1 sequences length of T.suis are 655-661 bp,and ITS-2 sequence are 530-538 bp;ITS-1 sequences length of T.trichiura are 600-627 bp,and ITS-2 sequence are 468-486 bp; both the 5.8 S sequences are 154 bp.There are 14 different base pairs in ITS-1 sequence of Trichuris suis between China and Spain,the difference is 2.1%;18 different base pairs in ITS-2 sequence,the difference is 3.4%;1 different base pair in 5.8 S sequence,the difference is 0.6%.There are 34 different base pairs in ITS-1 sequence of T.trichiura,the difference is 5.4%;34 different base pairs in ITS-2 sequence,the difference is 6.4%.There are 15 different base pairs in ITS-1 sequence of T.suis,the difference was 2.3%;13 different base pairs in ITS-2 sequence,the difference was 2.4%.The difference in ITS-1 sequence between T.trichiura and T.suis up to 45.1%,and in ITS-2 sequence up to 74.7%.There are six different base pairs in 5.8 S sequence,difference is 3.9%.Microsatellite repeat sequences can be seen in T.suis (ITS-1:AGC,CGC;ITS-2:GCT,CG,C),in T.trichiura(ITS-1:CAG,GGC;ITS-2:CAG,CGA,CGG, GCG).Delete microsatellite repeat sequence in ITS-1 and ITS-2 sequence,there are 10 different base pairs in ITS-1 sequence of T.trichiura,the difference is 1.7%.In ITS-2 sequence,five different base pairs,the difference is 1.1%.There are 9 different base pairs in ITS-1 sequence of T.suis,the difference was 1.4%;in ITS-2 sequence,9 different bases,the difference is 1.8%.
Related GeneBank database were downloaded(accession number:AY851266;Trichuris.ovis, AJ238220;T.leporis,AJ251321;T.skrjabini,AJ489248;T.vulpis,AM234616;T.arvicolae,AJ310661;T. muris,AJ299407).ITS-2 gene fragments of the Trichinella spiralis as a out-group(accession number: AY851266),using Neighbor Joining method of PHYLIP(version 3.67),as well as maximum parsimony method of PAUP(version 4.0b4a).Confidence levels for each grouping were calculated with a bootstrap program(SEQBOOT)in the PHYLIP package,with a total of 1000 replicates.The trees were produced by application of the CONSENCE program of the PHYLIP package.
Even though the result of phylogenetic analysis showed a high degree of similarity between T. trichiura and T.suis.Ultimately,the resolution of the question as to whether the two taxa represent separate species requires reciprocal cross-mating experiments to be conducted,using appropriate controls, to determine the extent of mating and the reproductive capabilities of any "hybrid" individuals produced. Only if cross-mating experiments could provide an answer to the question as to whether they represent the same or different species.
引文
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