基于mtDNA-COⅠ基因的山东省蚊种群遗传多样性分析
|
摘要
目的探讨山东省不同地理株及实验室不同抗性品系淡色库蚊及5种常见蚊种的线粒体DNA细胞色素C氧化酶亚基Ⅰ(mt DNA-COⅠ)基因序列特征,分析其遗传多样性。方法采用山东省济南、济宁、青岛等地现场采集及实验室选育的淡色库蚊成蚊,以济宁市现场采集的5种蚊(淡色库蚊、三带喙库蚊、中华按蚊、白纹伊蚊、骚扰阿蚊)作为样本,PCR扩增mt DNA-COⅠ基因并测序,分析序列特征并构建系统进化树。结果 PCR特异扩增8个不同地理株及4个实验室抗性品系的淡色库蚊mt DNA-COⅠ区域,获得长度均为528 bp的扩增片段,A+T含量为67.4%,存在两个变异位点。不同地理株淡色库蚊mt DNA-COⅠ基因同源性为99.95%,不同抗性品系淡色库蚊基因序列相同,所有蚊虫COⅠ基因具有高度保守性。5种常见蚊虫的mt DNA-COⅠ基因片段长度均为528 bp,有408个保守位点,120个变异位点,42个简约信息位点,78个单态位点,A+T含量为65.7%~68.0%,同源性分析发现蚊种间基因同源性为86.17%~92.05%,所有蚊虫的COⅠ基因分子鉴定与其形态学相吻合。系统进化关系显示,同种和同属之间呈明显的聚集,但阿蚊属成单独的分支,与其他蚊种亲缘关系较远。结论线粒体COⅠ作为不同地理株及实验室不同品系淡色库蚊的遗传进化分子标记不够理想,但该基因具有种间和属间特异性,可用于蚊虫属和种的区分。
Objective To explore the characteristics of gene sequence of mt DNA-COⅠ of Culex pipiens pallens from different geographical regions in Shandong Province and different resistant strains from the lab and five common mosquito species,and analyze the genetic diversity of these mosquitoes. Methods Adult mosquitoes were collected from Jinan,Jining,Qingdao cities and other places in Shandong Province. The sensitive,dichlorvos-resistant,pyrethroid-resistant and propoxur-resistant strains were reared in the lab. Five species of mosquito(Cx. pipiens pallens,Cx. tritaeniorhynchus,Anopheles sinensis,Aedes albopictus,and Armigeres subalbatus)were collected from Jining City and identified in the lab. mt DNA-COⅠwas specifically amplified by PCR and sequenced. The gene sequences were compared and analyzed by the biological information systems,and the phylogenetic tree was constructed. Results The amplified mt DNA-COⅠfragments of Cx. pipiens pallens from eight different cities and four different resistant strains were 528 bp in length,with 67.4% A+T contents and two mutation sites. The nucleotide sequence homology among the different geographic strains was 99.95% and the gene sequences of the four resistant strains were the same,showing a high homogeny. The amplified mt DNA-COⅠfragments of the five species of mosquitoes were 528 bp with 408 conserved sites,120 variable sites,42 parsimony informative sites and 78 singleton sites. The A+T contents were between65.7% and 68.0%. The nucleotide sequence homology among the different mosquito species was between 86.17% and 92.05%,and the molecular identification was consistent with the traditional morphological identification. The molecular phylogenetic study showed that the different species were clustered at their own branch at the species and genus levels,while genera Armigeres was distantly related to the others. Conclusion mt DNA-COⅠcould not serve as the molecular marker to analyze the population genetic variation and phylogenesis of Cx. pipiens pallens from different geographical regions and different resistant strains,but it has species and genus specificities,which could be used for the identification of the mosquito species and genus.
Objective To explore the characteristics of gene sequence of mt DNA-COⅠ of Culex pipiens pallens from different geographical regions in Shandong Province and different resistant strains from the lab and five common mosquito species,and analyze the genetic diversity of these mosquitoes. Methods Adult mosquitoes were collected from Jinan,Jining,Qingdao cities and other places in Shandong Province. The sensitive,dichlorvos-resistant,pyrethroid-resistant and propoxur-resistant strains were reared in the lab. Five species of mosquito(Cx. pipiens pallens,Cx. tritaeniorhynchus,Anopheles sinensis,Aedes albopictus,and Armigeres subalbatus)were collected from Jining City and identified in the lab. mt DNA-COⅠwas specifically amplified by PCR and sequenced. The gene sequences were compared and analyzed by the biological information systems,and the phylogenetic tree was constructed. Results The amplified mt DNA-COⅠfragments of Cx. pipiens pallens from eight different cities and four different resistant strains were 528 bp in length,with 67.4% A+T contents and two mutation sites. The nucleotide sequence homology among the different geographic strains was 99.95% and the gene sequences of the four resistant strains were the same,showing a high homogeny. The amplified mt DNA-COⅠfragments of the five species of mosquitoes were 528 bp with 408 conserved sites,120 variable sites,42 parsimony informative sites and 78 singleton sites. The A+T contents were between65.7% and 68.0%. The nucleotide sequence homology among the different mosquito species was between 86.17% and 92.05%,and the molecular identification was consistent with the traditional morphological identification. The molecular phylogenetic study showed that the different species were clustered at their own branch at the species and genus levels,while genera Armigeres was distantly related to the others. Conclusion mt DNA-COⅠcould not serve as the molecular marker to analyze the population genetic variation and phylogenesis of Cx. pipiens pallens from different geographical regions and different resistant strains,but it has species and genus specificities,which could be used for the identification of the mosquito species and genus.
引文
[1]Gemperli A,Sogoba N,Fondjo E,et al.Mapping malaria transmission in West and Central Africa[J].Trop Med Int Health,2006,1(7):1032-1046.
[2]Onyango SA,Kitron U,Mungai P,et al.Monitoring malaria vector control interventions:effectiveness of five different adult mosquito sampling methods[J].J Med Entomol,2013,50(5):1140-1151.
[3]Zhu GD,Xia H,Zhou HY,et al.Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China[J].Parasite Vector,2013,6:176.
[4]Liu HM,Cheng P,Huang X,et al.Identification of TCT,a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na-channel of Culex pipiens pallens from Shandong Province,China[J].Mol Med Rep,2013,7(2):525-530.
[5]孙定炜,王善青,卓开仁,等.海南省中华按蚊对3种杀虫剂的抗药性研究[J].中国寄生虫学与寄生虫病杂志,2014,32(2):127-129.
[6]徐建敏,李成玲,梁雪莹,等.广州市白纹伊蚊幼虫对5种杀虫剂的抗药性调查[J].中华卫生杀虫药械,2016,22(2):127-129.
[7]Fang Y,Shi WQ,Zhang Y.Molecular phylogeny of Anopheles hyrcanus group(Diptera:Culicidae)based on mt DNA COI[J].Infect Dis Poverty,2017,6(1):61.
[8]Torres-Gutierrez C,Bergo ES,Emerson KJ,et al.Mitochondrial COI gene as a tool in the taxonomy of mosquitoes Culex subgenus Melanoconion[J].Acta Trop,2016,164:137-149.
[9]Wang X,Tu WC,Huang EJ,et al.Identification of disease-transmitting mosquitoes:development of species-specific probes for DNA Chip Assay using mitochondrial COI and ND2 genes and ribosomal internal transcribed spacer 2[J].J Med Entomol,2017,54(2):396-402.
[10]陆宝麟,陈汉彬,苏龙,等.中国动物志[M].北京:科学出版社,1997:1-884.
[11]Collins FH,Mendez MA,Rasmussen MO,et al.A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex[J].Am J Trop Med Hyg,1987,37(1):37-41.
[12]关申民,高邦.COI序列:影响动物分类学与生态学的DNA barcode[J].生态学杂志,2008,27(8):1406-1412.
[13]Bourke BP,Oliveira TP,Suesdek L,et al.A multi-locus approach to barcoding in the Anopheles strodei subgroup(Diptera:Culicidae)[J].Parasit Vectors,2013,6:111.
[14]Batovska J,Blacket MJ,Brown K,et al.Molecular identification of mosquitoes(Diptera:Culicidae)in southeastern Australia[J].Ecol Evol,2016,6(9):3001-3011.
[15]Foley DH,Bryan JH,Yeates D,et a1.Evolution and systematics of Anopheles:insights from a molecular phylogeny of Australasian mosquitoes[J].Mol Phylogenet Evol,1998,9(2):262-275.
[16]Araki AS,Maia DA,Gil-Santana HR,et al.Variation in mitochondrial cytochrome c oxidase I DNA can successfully identify Culex(Melanoconion)pedroi(Diptera:Culicidae)and Culex(Melanoconion)ribeirensis(Diptera:Culicidae)[J].J Med Entomol.2017,54(2):485-488.
[17]Dai YH,Wang HF,Cheng P,et al.Mosquito species(Diptera:Culicidae)reported from Shandong Province,China[J].J Entomal Sci,2011,46(3):247-255.
[18]Yuan Y,Li Q,Kong L,et al.The complete mitochondrial genome of the grand jackknife clam,Solen grandis(Bivalvia:Solenidae):a novel gene order and unusual non-coding region[J].Mol Biol Rep,2012,39(2):1287-1292.
[19]Oshaghi MA,Yaaghoobi F,Abaie MR.Pattern of mitochondrial DNA variation between and within Anopheles stephensi(Diptera:Culicidae)biological forms suggests extensive gene flow[J].Acta Trop,2006,99(2-3):226-233.
[20]常雪莲,钟代斌,李小聪,等.基于mt DNA-COⅠ基因序列分析我国中华按蚊种群的遗传结构[J].南方医科大学学报,2015,36(2):234-238.
[21]刘飞,郭秀霞,程鹏,等.山东省淡色库蚊核糖体DNA第二内转录间隔区基因多态性分析[J].中国媒介生物学及控制杂志,2015,26(6):550-554.
[22]师永霞,幸芦琴,洪烨,等.广东国境口岸五种蚊种核糖体基因第2内转录间隔区分子鉴定方法研究[J].中国国境卫生检疫杂志,2008,31(1):24-28.
[2]Onyango SA,Kitron U,Mungai P,et al.Monitoring malaria vector control interventions:effectiveness of five different adult mosquito sampling methods[J].J Med Entomol,2013,50(5):1140-1151.
[3]Zhu GD,Xia H,Zhou HY,et al.Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China[J].Parasite Vector,2013,6:176.
[4]Liu HM,Cheng P,Huang X,et al.Identification of TCT,a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na-channel of Culex pipiens pallens from Shandong Province,China[J].Mol Med Rep,2013,7(2):525-530.
[5]孙定炜,王善青,卓开仁,等.海南省中华按蚊对3种杀虫剂的抗药性研究[J].中国寄生虫学与寄生虫病杂志,2014,32(2):127-129.
[6]徐建敏,李成玲,梁雪莹,等.广州市白纹伊蚊幼虫对5种杀虫剂的抗药性调查[J].中华卫生杀虫药械,2016,22(2):127-129.
[7]Fang Y,Shi WQ,Zhang Y.Molecular phylogeny of Anopheles hyrcanus group(Diptera:Culicidae)based on mt DNA COI[J].Infect Dis Poverty,2017,6(1):61.
[8]Torres-Gutierrez C,Bergo ES,Emerson KJ,et al.Mitochondrial COI gene as a tool in the taxonomy of mosquitoes Culex subgenus Melanoconion[J].Acta Trop,2016,164:137-149.
[9]Wang X,Tu WC,Huang EJ,et al.Identification of disease-transmitting mosquitoes:development of species-specific probes for DNA Chip Assay using mitochondrial COI and ND2 genes and ribosomal internal transcribed spacer 2[J].J Med Entomol,2017,54(2):396-402.
[10]陆宝麟,陈汉彬,苏龙,等.中国动物志[M].北京:科学出版社,1997:1-884.
[11]Collins FH,Mendez MA,Rasmussen MO,et al.A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex[J].Am J Trop Med Hyg,1987,37(1):37-41.
[12]关申民,高邦.COI序列:影响动物分类学与生态学的DNA barcode[J].生态学杂志,2008,27(8):1406-1412.
[13]Bourke BP,Oliveira TP,Suesdek L,et al.A multi-locus approach to barcoding in the Anopheles strodei subgroup(Diptera:Culicidae)[J].Parasit Vectors,2013,6:111.
[14]Batovska J,Blacket MJ,Brown K,et al.Molecular identification of mosquitoes(Diptera:Culicidae)in southeastern Australia[J].Ecol Evol,2016,6(9):3001-3011.
[15]Foley DH,Bryan JH,Yeates D,et a1.Evolution and systematics of Anopheles:insights from a molecular phylogeny of Australasian mosquitoes[J].Mol Phylogenet Evol,1998,9(2):262-275.
[16]Araki AS,Maia DA,Gil-Santana HR,et al.Variation in mitochondrial cytochrome c oxidase I DNA can successfully identify Culex(Melanoconion)pedroi(Diptera:Culicidae)and Culex(Melanoconion)ribeirensis(Diptera:Culicidae)[J].J Med Entomol.2017,54(2):485-488.
[17]Dai YH,Wang HF,Cheng P,et al.Mosquito species(Diptera:Culicidae)reported from Shandong Province,China[J].J Entomal Sci,2011,46(3):247-255.
[18]Yuan Y,Li Q,Kong L,et al.The complete mitochondrial genome of the grand jackknife clam,Solen grandis(Bivalvia:Solenidae):a novel gene order and unusual non-coding region[J].Mol Biol Rep,2012,39(2):1287-1292.
[19]Oshaghi MA,Yaaghoobi F,Abaie MR.Pattern of mitochondrial DNA variation between and within Anopheles stephensi(Diptera:Culicidae)biological forms suggests extensive gene flow[J].Acta Trop,2006,99(2-3):226-233.
[20]常雪莲,钟代斌,李小聪,等.基于mt DNA-COⅠ基因序列分析我国中华按蚊种群的遗传结构[J].南方医科大学学报,2015,36(2):234-238.
[21]刘飞,郭秀霞,程鹏,等.山东省淡色库蚊核糖体DNA第二内转录间隔区基因多态性分析[J].中国媒介生物学及控制杂志,2015,26(6):550-554.
[22]师永霞,幸芦琴,洪烨,等.广东国境口岸五种蚊种核糖体基因第2内转录间隔区分子鉴定方法研究[J].中国国境卫生检疫杂志,2008,31(1):24-28.