马尾松毛虫线粒体全基因组的测定与分析

Sequencing and Analysis of the Complete Mitochondrial Genome of Dendrolimus punctatus(Lepidoptera: Lasiocampidae)

【目的】 测定和分析马尾松毛虫线粒体基因组的特征,从线粒体基因组水平探究蛾类昆虫高级阶元的系统发育关系。【方法】 采用HiSeq X Ten测序仪测定马尾松毛虫线粒体的全基因组序列,参考鳞翅目昆虫已知线粒体基因组的全序列对其各基因进行定位和注释。采用tRNA Scan-SE 2.0软件预测tRNA基因的二级结构。基于线粒体全基因组的核苷酸序列构建鳞翅目蛾类13个科50种昆虫的系统发育关系。【结果】 马尾松毛虫线粒体基因组全长15 417 bp,A+T含量为79.6%,包括13个蛋白质编码基因、22个tRNA基因、2个rRNA基因和一段长度为320 bp的A+T富含区,无基因重排。13个蛋白质编码基因中,氨基酸使用较频繁的依次包括Phe、Leu、Ile、Tyr和Asn(Count>290),而Ala和Arg的使用相对较少(Count<50);在其密码子使用中,除COI以CGA为起始密码子外,其余基因的起始密码子均为昆虫典型的ATN(ATA、ATT、ATG);终止密码子除COI、COII和ND4基因为T外,其他基因的均为TAA。在所测得22个tRNA基因中,tRNA^Ser(AGN)缺少DHU臂,其余tRNA均为典型的三叶草结构。A+T富含区存在1段由ATAGAA引导保守长度为14 bp的多聚T结构和2段长度为13 bp的重复单元以及1个(AT)7结构。马尾松毛虫与其他松毛虫属的线粒体基因组全序列及13个蛋白质编码基因的核苷酸序列进行比对分析,结果表明,马尾松毛虫与文山松毛虫和油松毛虫三者间具有较高的同源性。系统发育树的结果显示: 马尾松毛虫与油松毛虫亲缘关系最近,思茅松毛虫和赤松毛虫亲缘关系最近。【结论】 马尾松毛虫线粒体基因结构稳定,基于线粒体基因组构建的鳞翅目蛾类昆虫的系统发育关系与传统的形态分类学结果基本一致。

【Objective】 In this study, we sequenced the mitochondrial genome of Dendrolimus punctatus, and analyzed the mitochondrial genome characteristics and the higher-level phylogenetic relationships in moths of Lepidoptera. 【Method】 The complete genome sequence of the mitochondrial genome of D. punctatus was determined by Hiseq X Ten sequencer. The genes were located and annotated based on the known complete mitochondrial genomes of Lepidopteran species. The tRNA Scan-SE 2.0 was used to predict the secondary structure of the tRNA genes online. Based on the whole nucleotide sequence of mitochondrial genome, the phylogenetic relationships of 50 insect species from 11 families of Lepidoptera were reconstructed. 【Result】 The results showed that the complete mitochondrial genome of D. punctatus was a circular molecule of 15 417 bp, including 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and one control region without gene rearrangement. Among the 13 protein-coding genes, amino acids of Phe, Leu, Ile, Tyr, and Asn (Count>290) were used more frequently, while Ala and Arg were uesd less frequently (Count<50). In the use of its codon, except CGA as the starting codon of COI gene, the starting codons of the other genes were all typical ATN (ATA, ATT, ATG). The stop codon of COI、COII and ND4 was T, while that of the other genes was TAA. All tRNAs showed the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNA^Ser(AGN) formed a simple loop. The (A+T)-rich region contained some conserved structure including the motif ATAGAA, 14 bp ploy (T) stretch and two repeating units with a length of 13 bp and one (AT)7 structure. The complete mitochondrial genome sequence and the nucleotide sequence of 13 protein-coding genes of D. punctatus were compared with other species of Dendrolimus genus, and the results showed that there was high homology between D.punctatus, D.wenshanensis, and D.tabulaeformis. Phylogenetic tree results show that D. punctatus is closely related with D. tabulaeformis, and D. spectabilis is closely related with D. kikuchii. 【Conclusion】 The mitochondrial genes structure of D. punctatus are stable, the phylogenetic relationship of some species from Lepidoptera based on mitochondrial genome is consistent with the traditional morphological classification.