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目的:利用线粒体DNA(mtDNA)中278 bp的细胞色素氧化酶辅酶Ⅰ(cytochrome oxidaseⅠ,COⅠ)序列鉴定我国常见嗜尸性麻蝇,寻求快速、准确的嗜尸性麻蝇种类鉴定方法。方法:从12个省16个地区户外草地家兔尸体上采集麻蝇科3属4种共计19个样本。利用十二烷基硫酸钠-蛋白酶K法提取蝇类胸肌mtDNA;Eppendorf 5331型扩增仪对278 bp的COⅠ基因片段进行PCR扩增;检测扩增产物,PCR胶回收纯化,测序并上传GenBank;利用MEGA4.0软件按邻近法构建无根系统发育树,通过序列分析建立种内及种间进化分歧表。结果:邻近法构建的发育树中4种麻蝇各自聚群,与形态学鉴定结果一致,上述麻蝇按照不同属、种分别聚类,其种内分歧整体均数均小于或等于3%,种间进化分歧均数在8%~12%之间。结论:COⅠ中278 bp基因序列能有效地鉴定常见嗜尸性麻蝇种类,进一步完善我国嗜尸性麻蝇基因库,为今后在实际案例中应用分子标记鉴定嗜尸性麻蝇进而推断死亡时间奠定基础。
OBJECTIVE: To identify the common sarcophagous flies in China by using a 278 bp cytochrome oxidase Ⅰ (COⅠ) sequence in mitochondrial DNA (mtDNA), and to find a rapid and accurate method to identify the species of sarcophagous flies. Methods: Four specimens of 3 genera and 3 genera of Solenidae were collected from the carcass of outdoor grassland in 16 regions of 12 provinces. The mtDNA of pectoral muscle of flies was extracted by sodium dodecyl sulfate-proteinase K. The 278 bp COⅠ gene fragment was amplified by PCR using Eppendorf 5331. The amplification products were detected by PCR. The PCR products were recovered, sequenced and uploaded into GenBank. Using MEGA4.0 software to construct the root-free phylogenetic tree according to the neighbor-joining method, the intraspecific and interspecific divergence tables were established by sequence analysis. Results: Four species of flies were clustered in the adjacent trees. Consistent with the results of morphological identification, the flies were clustered according to different genera and species. The average number of species within the species was less than or equal to 3% Differences in evolution between 8% to 12%. CONCLUSION: The 278 bp gene sequence in COⅠ can effectively identify common species of Sarcophagous flies, and further improve the gene bank of Sarcocystis sibiricus in China. This will lay the foundation for future identification of Sarcophagous Flies by in vitro molecular markers and inference of death time.