成都地区四种食尸性蝇类mtDNA中COⅠ基因序列检测

Sequencing of mitochondrial DNA cytochrome oxidase subunitⅠfor identification of sarcosaphagous flies (Diptera) in Chengdu

通过检测食尸性苍蝇线粒体DNA(mtDNA)上细胞色素氧化酶亚基Ⅰ (COⅠ )中 2 78bp基因序列 ,鉴定食尸性苍蝇的种类 ,解决依据形态学方法不能鉴定苍蝇卵的种类、很难鉴定幼虫种类的难题 ,作为法医鉴别食尸性苍蝇及其幼虫、卵种类依据。随机采集放置在成都地区室外草地兔尸体上的 4种 15个食尸性苍蝇。利用改进的小型昆虫DNA匀浆方法提取上述苍蝇mtDNA ;通过Perkin Elmer 96 0 0扩增仪进行PCR扩增 ;聚丙烯酰胺非变性凝胶连续缓冲体系垂直电泳和银染显色技术进行扩增结果检测 ;PCR胶回收试剂盒纯化 ;ABI 377测序仪测序 ;MEGA2 1软件包进行序列分析和构建系统发育树。在双翅目食尸性苍蝇的种内进化分歧均数小于 1% ,种间进化分歧均数大于 7%。mtDNA上COⅠ序列分析能有效地对主要的食尸性苍蝇进行种类鉴定。该检测方法快速、简便和精确 ,能作为法医鉴别食尸性苍蝇种类的可靠依据。

To solve the problems of identification of sarcosaphagous flies and their larvae and eggs, for which all of eggs and most of larvae could not be identified using their morphological features only, the feasibility of species identification based on a 278 bp region of the gene for cytochrome oxidase subunitⅠ(COⅠ) encoding region of mtDNA was evaluated. Samples were collected on the corpses of rabbits on the grassland in the Chengdu district. The mtDNA of flies was extracted using the improved technique in grinding tissue during extracting DNA from small insects. Reactions were conducted on a Perkin_Elmer 9600 thermal cycler, followed by vertical non_denaturing polyacrylamide electrophoresis. PCR products were purified using the Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequence of the double_stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. A neighbour_joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2.1 package. The analysis of a 278 base pairs region of the gene for COⅠencoding region of mtDNA of sarcosaphagous flies collected from Chengdu district showed less than 1% sequence divergence within species and about 10% divergence between species. So it is concluded that this region of the gene for COⅠencoding region of mtDNA of sarcosaphagous flies can be effectively used for identification of them to species level. Owing to the speed, easiness and accuracy of current nucleotide sequencing technology, it is likely to enable the reliable identification of sarcosaphagous flies.