三种蚊虫COⅡ基因的克隆与序列分析
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摘要
蚊虫属双翅目(Diptera)长角亚目(Nematocera)蚊科(Culicidae)，是危害人畜的重要医学昆虫。中华按蚊(Anopheles sinensis)、白纹伊蚊(Aedes albopictus)、致倦库蚊(Culex quinquefasciatus)分别是按蚊属、伊蚊属和库蚊属的代表蚊种，均为最常见的蚊虫，在我国都有较广泛的分布。本文运用T-A克隆技术分别获得三种蚊虫的细胞色素C氧化酶亚基Ⅱ(COⅡ)基因重组质粒。测定和比较了中华按蚊、白纹伊蚊和致倦库蚊的COⅡ基因全序列，分析了三种蚊虫之间及与其它几种蚊虫的COⅡ基因序列间的差异及进化关系。并根据COⅡ基因序列构建了分子系统树，对这些蚊虫的进化年代及亲缘关系进行了讨论。结果显示：三种蚊虫COⅡ基因的核苷酸序列同源性为84.1％～87.9％，氨基酸序列的同源性为85.1％～89.5％；C+G含量为23.2％～24.9％，颠换频率高于转换频率。这三种蚊虫在COⅡ分子结构上，白纹伊蚊与致倦库蚊具有更近的亲缘关系；COⅡ基因序列用于蚊类种间系统发育的研究非常有效。
Mosquitoes (Diptera: Culicidae), by nature of its association with problems of human and animal health, are medically important insects. Anopheles sinensis^ Aedes albopictus and Culex quinquefasciatus are all the most common mosquitoes in China. In this article, the cytochrome c oxidase subunit II gene (COII) in the mitochondrial DNA of the three mosquitoes was amplified by the polymerase chain reaction (PCR) technique, then was cloned into the pUCIS T-vector. The complete sequences of the three COII genes from Anopheles sinensis^ Aedes albopictus and Culex quinquefasciatus were sequenced and analyzed. The homology of the COII gene nucleotide and the deduced amino acid sequences of COII among three mosquitoes varied from 84.1% to 87.9% and from 85.1% to 89.5%, respectively. The C+G content of COII gene is about 23.2%~24.9%, and the frequency of transversion is higher than that of transition. The three mosquitoes were compared with others according to the COII gene, molecular phylogenetic tree was constructed base on the sequences of COII gene. The evolution history and relationships among those mosquitoes were discussed. Aedes albopictus and Culex quinquefasciatus are closely related species for their COII molecular level according to COII gene. The COII gene shows promise for resolving evolution and systematics of mosquitoes.
Mosquitoes (Diptera: Culicidae), by nature of its association with problems of human and animal health, are medically important insects. Anopheles sinensis^ Aedes albopictus and Culex quinquefasciatus are all the most common mosquitoes in China. In this article, the cytochrome c oxidase subunit II gene (COII) in the mitochondrial DNA of the three mosquitoes was amplified by the polymerase chain reaction (PCR) technique, then was cloned into the pUCIS T-vector. The complete sequences of the three COII genes from Anopheles sinensis^ Aedes albopictus and Culex quinquefasciatus were sequenced and analyzed. The homology of the COII gene nucleotide and the deduced amino acid sequences of COII among three mosquitoes varied from 84.1% to 87.9% and from 85.1% to 89.5%, respectively. The C+G content of COII gene is about 23.2%~24.9%, and the frequency of transversion is higher than that of transition. The three mosquitoes were compared with others according to the COII gene, molecular phylogenetic tree was constructed base on the sequences of COII gene. The evolution history and relationships among those mosquitoes were discussed. Aedes albopictus and Culex quinquefasciatus are closely related species for their COII molecular level according to COII gene. The COII gene shows promise for resolving evolution and systematics of mosquitoes.
引文
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7．李凤舞，牛春，叶柄辉，等．套式PCR检测蚊体内疟原虫的敏感性和特异性．中国寄生虫学与寄生虫病杂志，1998，16：164～167
8．李本文，卢惠霖，姚开泰，等．赫坎按蚊种团内各亲缘种间基因组重复DNA的限制酶切长度差异．中国寄生虫学与寄生虫病杂志，1991，9：8～10
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2．马雅军，瞿逢伊，曹毓存，等．我国雷氏按蚊和嗜人按蚊的分子鉴别和分类地位的探讨．中国寄生虫学与寄生虫病杂志，2000，18(6)：325～328
3．牛玲玲，袁征，蔡国英．DNA探针用以中华按蚊和嗜人按蚊的分类研究．中国寄生虫学与寄生虫病杂志，1992，10：267～270
4．王文，施立明．一种改进的动物线粒体DNA提取方法．动物学研究，1993，14(2)：197～198
5．刘忠湘，薛采芳，刘斌，等．中华按蚊不同地理株基因组DNA的多态性．第四军医大学学报，1998，19(2)：175～177
6．许晓春，徐建农，瞿逢伊．用聚合酶反应区分我国大劣按蚊复合体A和D种．中国寄生虫学与寄生虫病杂志，1998，16(3)：172～175
7．李凤舞，牛春，叶柄辉，等．套式PCR检测蚊体内疟原虫的敏感性和特异性．中国寄生虫学与寄生虫病杂志，1998，16：164～167
8．李本文，卢惠霖，姚开泰，等．赫坎按蚊种团内各亲缘种间基因组重复DNA的限制酶切长度差异．中国寄生虫学与寄生虫病杂志，1991，9：8～10
9．李新波，赵小步，田德志，等．一种PCR产物克隆的新方法—T-A克隆．生物化学与生物物理进展，1999，26(2)187～189
10．邵红光，张亚平，柯欣，等．南极隐虫兆COII基因序列及无翅类昆虫分子进化．中国科学(C辑)，2000，30(3)：330～336
11．陈永久，王学忠，王丕玉，等．云南微小按蚊随机扩增多态DNA及遗传分化关系．动物学研究，1998，19(3)：184～189
12．陈汉彬．中国蚊科亚科的支序分类(双翅目：蚊科)．四川动物，1987，6(1)：10～14
13．胡维，向华，周艳，等．用PCR法直接快速筛查重组阳性克隆．生物技术通报，1999，148：39～40
14．赵彤言，陆宝麟．尖音库蚊复合组RAPD-PCR分子分类的研究．中国昆虫学会第四届全国医学昆虫学学术讨论会论文摘要集，1995
15．徐建农，瞿逢伊，董学书．不同蚊种核糖体DNA第二内转录间隔区的PCR扩增．第二军医大学学报，1996，17：180～181
16. Audtho M, Tassanakjon A, Boonsaeng JM, et al. Simple nonradioactive DNA hybridization method of identification of sibling species of Anopheles dirus (Diptera: Culicidae) complex. J Med Entomol., 1995, 32:107～112
17. Ballinger-Crabtree ME, Black WCIV, Miller BB. Use of genetic polymorphisms detected by the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) for differentiation and identification of Aedes aegypti subspecies and populations. Am J Trop Med Hyg., 1992, 47:893～901
18. Beard CB, Mills Hamn D and Collins FH. The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects. Insect Mol Biol., 1993, 2(2):103～124.
19. Beatriz DSG, Panzetta DDG, Noemi GC. Genetic structure of Aedes albifasciatus (Diptera: Culicidae) populations in central Argentina determined by random amplified polymorphic DNA～polymerase chain reaction markers. J Med Entomol.,
1999,36(3) : 400-404
20． Beckenbach AT,Wei YW,and Liu H. Relationships in the Drosophila obscura species group,inferred from mitochondrial cytochrome oxidase II sequences. Mol Biol Evol.,1993,10: 619-634
21． Boore JL. Survey and summary: animal mitochondrial genomes. Nucl Acid Res.,1999,27(8) : 1767-1780
22． Brown WN. Polymorphism mitochondrial DNA of human as revealed by restriction endonuclease analysis. Proc Natl Acad Sci USA.,1980,77: 3605-3609
23． Brust RA,Ballard JWO,Driver F,et al. Molecular systematics,morphological analysis,and hybrid crossing identify a third taxon,Aedes (Halaedes) wardangensis sp.nov.,of the Aedes (Halaedes) australis species-group (Diptera: Culicidae). Canadian Journal of Zoology,1998,76(7) : 1236-1246
24． Busse G,Steffens GJ,Steffens GCM. Studies on cytochrome c oxidase III. Relationship of cytochrome oxidase subunits to electron carriers of photophosphorylation. Hoppe-Seyler'Z Physiol Chem.,1978,359: 1011-1013
25． Caccone A,Garcia BA and JR Powell. Evolution of the mitochondrial DNA control region in the Anopheles gambiae complex. Insect Mol Biol.,1996,5(1) : 51-59
26． Chuang SE,Wang KC,Cheng AL. Single-step direct cloning of PCR products. Trend in Genetics,1995,11(1) : 7
27． Clarke JM. Novel non-templated nucleotide addition reaction catalyzed by procaryotic and eucaryotic DNA polymeraese. Nucl Acid Res.,1988,14: 9677-9686
28． Cockburn AF and Seawright JA. Techniques for mitochondrial and ribosomal DNA analysis of Anopheline mosquitoes. J Am Mosq Control Assoc.,1988,4: 261-265
29． Colless DH. The Australian Chaoboridae (Diptera). Aust J
Zool.,1986,124: 1-66
30． Collins FH,Mendez MA,Rasmussen MO,et al. A ribosomal RNA probe differentiates member species of the Anopheles gambiae complex. Am J Trop Med Hyp.,1987,37: 37~41
31． Cornel AJ,Porter CH,Collins FH. Polymerase chain reaction species diagnostic assay for Anopheles quadrimaculatus cryptic species (Diptera: Culicidae) based on ribosomal DNA ITS2 sequences. J Med Entomol,1996,33: 109-116
32． Favia G,Dimopoulos G,Delia Torre A,et al. Analysis of the Anopheles gambiae genome using RAPD markers. Insect Mol Biol.,1994,3: 149-157
33． Felsentein J. Phylogenies from molecular sequences: inference and reliability. Annu Rev Genet.,1988,22: 521-565
34． Fischer SG and Lerman LS. DNA fragments differing by single base pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Pro Natl Acad Sci USA.,1983,80: 1579-1583
35． Foley DH,Bryan JH,Yeates D,et al. Evolution and systematics of Anopheles: insights from a molecular phylogeny of Australasian mosquitoes. Mol Phylogenet Evol.,1998,9(2) : 262-275
36． Fritz GN,Narang SK,Kline DL,et al. Diagnostic characterization of Anopheles freeborni and An.hermsi by hybrid crosses,frequencies of polytene X chromosomes and rDNA restriction enzyme fragments. J Am Mosq Control Assoc.,1991,7: 198-206
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