家蚕追寄蝇线粒体DNA细胞色素b基因及其侧翼tRNA基因的克隆与序列结构和进化分析

Molecular Cloning,Structural and Evolutionary Analyses of Mitochondrial DNA cyto b Gene and its Flanking tRNA Gene from Exorista sorbillans

家蚕追寄蝇(Exorista sorbillans)为双翅目、寄蝇科、追寄蝇属昆虫,寄生家蚕后引起多化性蚕蛆病。研究家蚕追寄蝇的系统进化关系,有助于探讨其寄生机制。采用PCR技术扩增了家蚕追寄蝇的线粒体DNA(mtDNA)片段,序列分析结果表明:克隆的mtDNA片段大小约1.5 kb,包括完整细胞色素b基因(cyto b)及其侧翼tRNA-Leu基因序列,序列结构与其它21个昆虫物种的同源片段序列结构基本一致,说明线粒体DNA基因排列和基因结构的保守性;家蚕追寄蝇mtDNA的Cyto b蛋白和tRNA-Leu基因的二级结构符合功能大分子的空间结构特征,维持一定的保守进化;双翅目昆虫不同蝇类mtDNA中的cyto b基因、tRNA-Leu基因序列的同源性高,可保证构建蝇类系统发生树的准确性。基于包括家蚕追寄蝇在内的12个双翅目昆虫、5个鳞翅目昆虫、4个鞘翅目昆虫、1个直翅目昆虫的mtDNA cyto b基因编码氨基酸序列的进化分析表明:双翅目中蝇类的进化次序依次为狂蝇科、果蝇科、寄蝇科、蝇科、丽蝇科,寄蝇科与蝇科和果蝇科之间的进化距离较近;鳞翅目处于种系发生树分叉的起点,起源最早,双翅目处于种系发生树分叉的最末端,起源最晚。

Exorista sorbillans,insect of genus Exorista,family Tachinidae of Diptera order,leads to the multivoltine maggoty disease after parasitizing silkworms.Understanding phylogenetic relationships of Exorista sorbillans will facilitate exploitation to its parasitic mechanism.A piece of mitochondrial DNA （mtDNA） fragment of Exorista sorbillans was amplified by PCR technology.Sequence analysis showed that the cloned mtDNA fragment was of about 1.5 kb size,including a full-length cyto b gene and its flanking tRNA-Leu gene sequences.The structure of Exorista sorbillans mtDNA is generally consistent with the homologous fragments of 21 other insects,indicating conservatism on gene arrangement and gene structure of mtDNA.Besides,secondary struc-tures of Cyto b protein and tRNA-Leu gene had the typical spatial structure of functional macromolecules,having maintained certain conservatism during evolution.This high homology of mtDNA cyto b gene and tRNA-Leu gene in different flies of Diptera ensured the accuracy of the constructed phylogenetic tree for flies.Evolutionary analyses based on amino acids encoded by cyto b genes from 12 insect species in Diptera including Exorista sorbillans,5 species in Lepidoptera,4 species in Coleoptera,and 1 species in Orthoptera indicated that the evolutionary order in flies were from Oestridae to Drosophilidae,Tachinidae,Muscidae,and to Calliphoridae.Tachinidae had relatively closer evolutionary distance with Muscidae and Drosophilidae.Species of Lepidoptera formed the root clade,an indication of their earliest origin,while those of Diptera lied at the most distant clade,an indication of their latest origin.