海南蛾类及内共生菌沃尔巴克体的分子鉴定及检测研究

Molecular identification and phylogenetic analysis of moths of Hainan and detection of the endosymbiont Wolbachia

目的了解海南热带环境蛾类种群情况,并检测内共生菌沃尔巴克体(Wolbachia)感染情况。方法用Hot SHOT法提取海南蛾类基因组DNA,用PCR法扩增线粒体基因组细胞色素c氧化酶亚基I(COI)基因,对扩增产物进行基因测序,利用BLAST功能对测序序列进行比对分析确定蛾种类,构建系统发育树;同时扩增Wolbachia的wsp基因,明确海南蛾类内共生菌Wolbachia的感染情况。结果共鉴定23只蛾类样本,均隶属于鳞翅目。15只鉴定至种阶元,3只、1只及4只分别鉴别至属、科、目阶元。包括3只Hymenia perspectalis(13.03%)、2只Euproctis varians(8.69%)、1只Acontia thapsina、Apethistis uncinata、Cerynea trogobasis、Crypsiphona ocultaria、Epirrhoe rivata、Herpetogramma licarsisalis、Herpetogramma okamotoi、Hypena proboscidalis、Luceria striata、Zanclognatha lunalis、Herpetogramma sp.、Hypena sp.、Luceria sp.、Erebidae sp.,各占4.35%,4只Lepidoptera sp.,占17.38%。海南蛾COI基因序列长约500 bp,具有明显的A+T偏好性,A+T含量为67.78%~70.93%,与GenBank中蛾序列特征具有一致性,且系统发育关系相近的蛾类聚为一支。在海南屯昌蛾Cerynea trogobasis中检测出1株内共生菌沃尔巴克氏体,感染率为4.35%,该株沃尔巴克氏体与西班牙的雪松长足大蚜内共生菌沃尔巴克氏体系统发育关系最近。结论海南蛾类资源丰富、种类多。基于COI基因的DNA条形码技术可有效鉴定海南蛾类,揭示遗传特征,为探索热带环境蛾类多样性提供依据。

Objective To understand the moth populations in the tropical environment of Hainan Province,and to detect infection by the endosymbiont Wolbachia.Methods The genomic DNA of moths in Hainan Province was extracted by Hot SHOT method.The mitochondrial genome cytochrome c oxidase subunit I(COI)gene was amplified by PCR,the amplified product was genetically sequenced.The sequence was analyzed and aligned by using the BLAST tool to determine the moth species,and a phylogenetic tree was constructed.The wsp gene of Wolbachia was amplified to detect the infection of the endosymbiont Wolbachiain moths in Hainan Province.Results A total of 23 moth samples were identified,all belonging to the order Wolbachia in Lepidoptera.A total of 15 were identified to species,and 3,1,4 were identified to genus,family and order,respectively,including 3 of Hymenia perspectalis(13.03%),2 of Euproctis varians(8.69%),1 of Acontia thapsina,Apethistis uncinata,Cerynea trogobasis,Crypsiphona ocultaria,Epirrhoe rivata,Herpetogramma licarsisalis,Herpetogramma okamotoil,Hypena proboscidalis,Luceria striata,Zanclognatha lunalis,Herpetogramma sp.,Hypena sp.,Luceria sp.,Erebidae sp.,accounted for 4.35%,and 4 of Lepidoptera sp.accounted for 17.38%.The COI gene sequences of moths in Hainan Province were about 500 bp,with obvious A+T preference,A+T content of 67.78%to 70.93%,with the characteristics of the sequences of moths in GenBank.Moths with similar phylogenetic relationships were clustered into one branch.One strain of endosymbiont Wolbachia was detected in Cerynea trogobasis in Tunchang County,Hainan Province,with an infection rate of 4.35%.This strain of Wolbachia had the closest phylogenetic relationship with the endosymbiont Wolbachia of Cinara cedri in Spain.Conclusion Moths in Hainan Province were rich and diverse.The DNA barcoding technology based on COI geneeffectively identified moths in Hainan Province,and revealed the genetic characteristics,which provided a for exploring the diversity of moths in tropical environments.