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目的通过研究国境口岸常见11种蚊虫的细胞色素C氧化酶亚基I(COI)基因序列差异性,并分析其系统进化关系,建立口岸常见蚊种的分子鉴定方法。方法设计l对扩增COI部分编码区的PCR引物,对广东、海南和云南等国境口岸采集的致倦库蚊、白纹伊蚊、中华按蚊等常见11种成蚊进行PCR扩增和序列测定,依据COI核苷酸序列进行系统进化分析。结果 11种蚊虫的COI基因扩增片断长度均为650 bp左右,A+T含量为66.51%~69.97%。同源性比较表明,同一蚊种间核苷酸序列同源性为94.8%~100.0%;不同蚊种间核苷酸序列同源性为77.7%~91.8%。系统进化关系显示,从种的水平上看,白纹伊蚊、致倦库蚊、中华按蚊等上述所有蚊种均聚集成簇,即同种之间呈明显的聚集,与形态学鉴定结果相一致。在属的水平上,库蚊属、阿蚊属、伊蚊属等均聚集成簇,形成各自的单独一支;各属间,阿蚊属与伊蚊属先聚类,再分别与库蚊属、曼蚊属聚类为库蚊亚科,按蚊属与上述蚊种亲缘关系较远。结论本次研究中的COI基因差异可作为国境口岸常见11种蚊虫分类鉴定的分子标记,为口岸常见蚊种的分子鉴定提供帮助,也可为口岸外来的或新发现的蚊种的鉴别提供分子水平的技术依据。
OBJECTIVE: To study the sequence divergence of cytochrome C oxidase subunit I (COI) genes in 11 common mosquitoes at the border crossings and to analyze their phylogenetic relationships and to establish a molecular identification method for common mosquito species at the port. Methods Design PCR primers for amplifying COI coding region were used to amplify and sequence 11 common adult mosquitoes, Culex pipiens quinquefasciatus, Aedes albopictus and Anopheles sinensis collected at the cross-border ports of Guangdong, Hainan and Yunnan The phylogenetic analysis was based on the COI nucleotide sequence. Results The amplified fragments of COI gene of 11 species of mosquitoes were about 650 bp in length with A + T content of 66.51% -69.97%. Homology comparison showed that the homology of the nucleotide sequences of the same mosquito was 94.8% -100.0%. The nucleotide sequences of different mosquito species were 77.7% -91.8%. Phylogenetic relationships showed that all of the above mentioned mosquito species such as Aedes albopictus, Culex pipiens quinquefasciatus and Anopheles sinensis clustered into clusters at the species level, that is, significant aggregation was found among the same species, and the results of morphological identification Consistent. On the genus level, Culex, A mosquitoes, Aedes mosquito and so on are clustered into clusters, forming a separate one; each genus, A mosquito and Aedes genus cluster first, and then respectively Culex mosquito Genus, Manchus is a cluster of Culex mosquito subfamily, Anopheles and the mosquito species distantly related. Conclusion The COI gene differences in this study can be used as molecular markers for the classification and identification of 11 common mosquitoes at the border crossings, which can help to identify the common mosquito species in ports. It can also provide molecular identification for exotic or newly discovered mosquito species Horizontal technical basis.