甜果螨全线粒体基因组测序与分析

Sequencing and analysis of the complete mitochondrial genome of Carpoglyphus lactis(Acari:Carpoglyphidae)

【目的】测定和分析甜果螨Carpoglyphus lactis线粒体基因组全序列,并在线粒体基因组水平探讨其在真螨总目(Acariformes)中的系统发育地位,为真螨总目分类及果螨科线粒体基因组研究提供科学依据。【方法】挑取实验室饲养的甜果螨成螨,用传统的酚氯仿抽提法和试剂盒提取法提取甜果螨基因组DNA。然后采用节肢动物或螨类线粒体基因的通用引物PCR扩增出甜果螨线粒体基因cox1,cob,rrnS和nad 4-nad 5的部分序列;再设计种特异性引物进行Long-PCR扩增和步移法测序,测出甜果螨线粒体基因组全序列。应用SeqMan,SEQUIN 9.0和tRNAscan等生物信息学软件,对甜果螨线粒体基因组的基因结构等进行生物信息学分析。最后基于17种真螨总目螨类的蛋白质编码基因,采用最大似然法构建系统发育树。【结果】甜果螨线粒体全基因组总长为14060 bp(GenBank登录号:MN073839),为典型的闭合双链DNA分子,共由37个基因组成,包括13个蛋白质编码基因(PCGs)、22个tRNA基因和2个rRNA基因;甜果螨线粒体基因组还包括1个大的非编码区(large non-coding region,LNR)。系统发育分析结果显示,甜果螨Carpoglyphus lactis属于无气门亚目粉螨总科(Acaroidae),与椭圆食粉螨Aleuroglyphus ovatus构成一支。粉螨总科(Acaroidae)和薄口螨总科(Histiostomatoidae)聚成一簇,与痒螨股(Psoroptidia)构成姐妹群。【结论】本研究首次获得并分析了甜果螨线粒体基因组全序列。甜果螨与椭圆食粉螨的亲缘关系较近。

【Aim】To sequence and analyze the complete mitochondrial genome of Carpoglyphus lactis,and to explore the phylogenetic position of C.lactis in Acariformes based on the mitochondrial genome sequence,so as to provide a scientific basis for the classification of Acariformes and the study of mitochondrial genome of Carpoglyphidae.【Methods】Genomic DNA of C.lactis was initially extracted from adult mites of C.lactis bred in the laboratory using conventional phenol-chloroform and commercial DNA extraction kit.Partial sequences of mitochondrial genes including cox1,cob,rrnS and nad4-nad 5 were amplified by PCR using universal primers for arthropods or mites.Species-specific primers were designed for long-PCR amplification and primer-walking sequencing,and the complete mitochondrial genome of C.lactis was sequenced.Bioinformatic software SeqMan,SEQUIN 9.0 and tRNAscan were used to analyze the genetic structure of complete mitochondrial genome.Finally,the phylogenetic relationship was constructed by using maximum likelihood method(ML)based on the nucleotide sequences of concatenated protein-coding genes(PCGs)of 17 species of Acariformes.【Results】The complete mitochondrial genome of C.lactis(GenBank accession no.:MN073839)is 14060 bp in length.It is a classical closed circular double-stranded DNA molecule,and comprises 37 coding genes,including 13 protein-coding genes,2 ribosomal RNA genes,and 22 transfer RNA genes,and one large non-coding region(LNR).The results of phylogenetic analysis showed that C.lactis belongs to Acaroidea and clustered into one branch with Aleuroglyphus ovatus.Acaroidae and Histiostomatoidae clustered into a group,forming a sister group to Psoroptidia.【Conclusion】We successfully sequenced and analyzed the complete mitochondrial genome of C.lactis.The relationship between C.lactis and A.ovatus is close.