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目的分析我国部分地区28种蚊虫的线粒体细胞色素c氧化酶亚基Ⅰ(mitochondrion cytochrome c oxidase subunitⅠ,COⅠ)基因序列,探讨其作为蚊虫分子标记的应用潜力。方法 2015年3月至2016年10月在我国海南、广东、广西、云南、福建、浙江、河南、山西、天津、内蒙古、吉林和黑龙江等12省(自治区、直辖市)的蚊虫孳生地采集幼虫,室内饲养至羽化成蚊,在蚊虫栖息地和蚊虫监测点,采用灯诱法和人诱法采集成蚊。所有成蚊经形态学鉴定后,提取单只蚊虫基因组DNA,采用国际双翅目通用引物PCR扩增并克隆线粒体COⅠ基因5′端片段,并进行测序。所得COⅠ基因序列上传Gen Bank获取登录号。序列通过Clustal1.83和MEGA5.05软件进行多序列比对,用DNAMAN9.0软件分析同源性。采用Mega5.05软件进行序列特征分析、计算遗传距离。每种蚊虫随机选择1~4条线粒体COⅠ基因序列,采用邻接法(NJ)构建系统发育进化树。结果共采集3亚科6属28种301只蚊虫,分别为伊蚊属(Aedes)的白纹伊蚊(Ae.albopictus)等5种、按蚊属(Anopheles)的中华按蚊(An.sinensis)等6种(亚种)、库蚊属(Culex)的二带喙库蚊(Cx.bitaeniorhynchus)等14种(亚种)、阿蚊属(Armigeres)的骚扰阿蚊(Ar.subalbatus)、曼蚊属(Mansonia)的常型曼蚊(Ma.uniformis)和巨蚊属(Toxorhynchites)的华丽巨蚊(Tx.splendens)。PCR扩增结果显示,28种蚊虫线粒体COⅠ基因的长度约711 bp,经多序列比对排齐后长度为651 bp。不同种别蚊虫的线粒体COⅠ序列同源性范围为97.85%~99.97%。种内K2P遗传距离为0.15%~2.89%,属内种间遗传距离为0.25%~14.50%。其中,按蚊属6种蚊虫COⅠ序列存在153个变异位点,131个简约信息位点,T+A平均含量为67.70%;编码的217个氨基酸残基中保守氨基酸206个(占94.9%);6种蚊虫的核苷酸同源性为98.31%~99.72%,种内K2P遗传距离为0.41%~1.56%,种间K2P遗传距离为1.07%~14.50%。28种蚊虫(60条)COⅠ序列的系统发育树分析结果显示,种团内近缘种(尖音库蚊、淡色库蚊和致倦库蚊)聚在一起,但近缘种之间未完全隔开;白跗按蚊(An.albitarsis)和迷走按蚊(An.vagus)聚在一起,其余23种蚊虫分别以种为单位聚在一起。结论基于线粒体COⅠ基因的分子标记可以区分捕获蚊虫的部分蚊种,但尚未能有效及准确地区分近缘种。
Objective To analyze the mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) gene sequences of 28 species of mosquitoes in some areas in China and explore their potential application as molecular markers for mosquitoes. Methods From March 2015 to October 2016, larvae were collected from mosquitoes in 12 provinces (autonomous regions and municipalities) in Hainan, Guangdong, Guangxi, Yunnan, Fujian, Zhejiang, Henan, Shanxi, Tianjin, Inner Mongolia, Jilin and Heilongjiang provinces, Indoor breeding to emergence of adult mosquitoes, mosquito habitat and mosquito monitoring point, the use of light-induced and human induced mosquito acquisition method. All adult mosquitoes were morphologically identified, single mosquito genomic DNA was extracted, the 5 ’end of mitochondrial COI gene was amplified by PCR using the international primer pairs of Diptera and sequenced. The resulting COI gene sequence is uploaded to Gen Bank to obtain the accession number. The sequences were aligned by multiple alignments using Clustal 1.83 and MEGA 5.05 software, and homology was analyzed using DNAMAN 9.0 software. Using Mega5.05 software to sequence characteristics analysis, calculate the genetic distance. One to four mitochondrial COI gene sequences were randomly selected for each species of mosquito, and the phylogenetic tree was constructed using the neighbor-joining (NJ) method. Results A total of 301 mosquitoes of 28 species belonging to 6 genera and 3 subfamilies were collected, including Aedes albopictus, Aedes albopictus, Anopheles sinensis, An.sinensis ), 14 species (subspecies) of Culex, Cx. Bitaeniorhynchus, Ar.subalbatus, Armigeres species, Mansonia, and M. sp., Of the genus Manunculaceae and Tx.splendens of the genus Toxorhynchites. The result of PCR amplification showed that the length of mitochondrial COI gene of 28 mosquitoes was about 711 bp, and the length of COI gene was 651 bp after aligned by multiple sequence alignment. The mitochondrial COⅠ sequence homology ranged from 97.85% to 99.97% for different species of mosquitoes. The intraspecific K2P genetic distance was 0.15% -2.89%, and the intraspecific genetic distance was 0.25% -14.50%. Among them, there were 153 mutation sites and 131 parsimony informative sites in COⅠ sequences of 6 species of mosquitoes, with an average content of T + A of 67.70%; 206 conserved amino acids (94.9%) of the 217 amino acid residues encoded, ; The nucleotide diversity of six mosquitoes was 98.31% -99.72%, the intraspecific K2P genetic distance was 0.41% ~ 1.56%, the interspecific K2P genetic distance was 1.07% ~ 14.50%. The phylogenetic tree analysis of COⅠ sequences of 28 species of mosquitoes showed that the relative species (Culex pipiens pallens, Culex pipiens pallens and Culex pipiens pallens) clustered together in the species, but the incomplete species An.albitarsis and An.vagus together, and the remaining 23 mosquitoes clustered together in species. Conclusion The molecular markers based on the mitochondrial COI gene can discriminate and capture some mosquito species of mosquitoes, but they have not been able to distinguish the relative species effectively and accurately.