关键词: 缺齿甲胁虱, 线粒体微环染色体, RNA基因, 蛋白质编码基因, 非编码区

Abstract:

Objective To conduct sequencing and analysis the mitochondrial (mt) genome of Hoplopleura edentula to understand the structure and variation of the mt genome of Hoplopleura. Methods We used stratified random sampling to capture the Eothenomys miletus in Mount Cangshan Geopark Dali, and collected all sucking lice on the surface of E. miletus by complete catching method and identified the species of the sucking lice. The DNA of each H. edentula was extracted using the Dneasy Tissue Kit. Universal primers were used to amplify the short fragment of small ribosomal submit RNA (rrnS) and large ribosomal submit RNA (rrnL) genes of H. edentula. Subsequently, the specific primers in the conserved regions of the short fragment were designed to amplify long fragment sequences of rrnS and rrnL genes, and the specific primers in the minichromosomes conserved region were designed to amplify all the coding regions of the minichromosomes. The successfully amplified PCR products were purified and sequenced by Illumina HiSeq X Ten platform high-throughput sequencing method. The structure and variation of the mt genome were analyzed by bioinformatics tools such as Geneious, tRNAscan, CodonW, BLAST, etc. Results A tatol of 6 812 606 bp sequence reads were obtained from the H. edentula mt genome and 24 mt genes (7 protein-coding genes, 15 tRNA genes and 2 rRNA genes) of common genes in arthropod mt genomes were identified after assembly. The mt genome of H. edentula fragmented into 8 minichromosomes (GenBank accession number: MW835203-MW835210). These genes were unevenly distributed on minichromosomes. The coding region of each minichromosome contains 1-4 genes, at least one protein-coding gene or rRNA gene. AT content of the coding region is 61.0%. All start codon of protein-coding genes were ATN, except for cox2, which start codon was TTG. All protein-coding genes use TAA and TAG as stop codons. The codon AUU is the most frequently used (RSCU: 1.53). The secondary structure of 15 tRNA genes is a typical clover like structure. There are 31 mismatches in tRNA genes, mainly G-U mismatch. The rrnS and M-L₁(tag)-rrnL-V minichromosomes have all the non-coding regions, and there are 2 types of tandem repetitive sequences, and they were 88.0%-90.0% identical. An AT-rich motif (50 bp, 64.0% A&T) is present in the non-coding region upstream of the 5′-end of the coding region, whereas a GC-rich motif (42 bp, 85.7% G&C) is present downstream of the 3′-end of the coding region. We also sequenced parts of the non-coding regions upstream and downstream of the coding regions of the other 6 H. edentula minichromosomes, and the identity reached to 86.5%-88.5%. Comparing the mt genomes of H. edentula, H. akanezumiand H. kitti showed that the mt genomes of all three species were fragmented. There were four minichromosomes that have the same gene composition and sequence: E-cob-S₁(tct)-S₂(tga), I-cox1, K-nad4 and rrnS. The H-nad5-F-T minichromosome of H. edentula was not found in the other two Hoplopleura species. trnT gene translocated frequently and distributed in the different minichromosomes of three Hoplopleura species. trnS₁(tct) of H. edentula has a typical clover-leaf structure, while trnS₁(tct) of the other two Hoplopleura species lacks D-arm. Conclusion H. edentula has 24 genes distributed unevenly on eight minichromosomes, each of them contains a coding region and a non-coding region, and is of AT rich. The number of base mismatches of tRNA is considerably high. The trnS₁(tct) of H. edentulais shows a typical clover like structure. The structure of Hoplopleura mt genome varies, and its particularity may be related to the genome cracking.

Key words: Hoplopleura edentula, Mitochondrial minichromosomes, RNA genes, Protein-coding genes, Non-coding regions