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目的分析国境口岸13蚊种的细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列,为建立蚊种分子鉴定方法奠定基础。方法设计1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、中华按蚊等成蚊进行PCR扩增和序列测定,分析COⅠ核苷酸的系统进化关系。结果 13种蚊种的COⅠ基因扩增片段长度均约为400 bp,A+T含量为69.64%~72.78%。同源性比较表明,同一蚊种间核苷酸序列同源性>95.6%;不同蚊种间COⅠ片段核苷酸序列同源性为79.1%~93.4%。COⅠ核苷酸系统进化关系显示,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,均与形态学鉴定结果相一致。结论建立的COⅠ核苷酸鉴别技术可应用于国境口岸范围内成蚊种的区分,基本上可正确反映蚊虫的系统发育关系。这可以弥补蚊虫形态特征信息量的不足等传统分类系统的缺点,为国境口岸范围内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 analyze the sequence of cytochrome C oxidase subunit Ⅰ (COⅠ) gene in 13 mosquito species at the border crossing, and lay the foundation for the establishment of the method for molecular identification of mosquito species. Methods A pair of PCR primers were designed to amplify COⅠ partial coding region and PCR-amplified and sequenced the adult mosquitoes of Culex quinquefasciatus, Aedes albopictus and Anopheles sinensis collected in the border ports of Guangdong, Hainan and Yunnan. Phylogenetic relationship of CO Ⅰ nucleotides. Results The lengths of COⅠ gene amplified from 13 species of mosquitoes were all about 400 bp and A + T contents ranged from 69.64% to 72.78%. Homology comparison showed that the homology of the nucleotide sequences of the same mosquito species was> 95.6%. The nucleotide sequence homology of the COI fragments between different mosquito species was 79.1% -93.4%. The phylogenetic relationship of COⅠ nucleotide system showed that all the above mentioned mosquito species such as Aedes albopictus, Culex pipiens quinquefasciatus and Anopheles sinensis clustered into clusters from the species level Consistent with morphological identification results. Conclusion The established COⅠ nucleotide discrimination technique can be applied to the distinction of adult mosquito species within the border crossings of the border areas and can basically correctly reflect the phylogenetic relationship of the mosquitoes. This can make up for the shortcomings of the traditional taxonomic system such as lack of information on the morphological characteristics of mosquitoes and provide a molecular basis for the identification of exotic or newly discovered mosquito species within the border crossings.