收稿日期: 2021-09-22
修回日期: 2021-11-18
网络出版日期: 2022-04-12
基金资助
国家自然科学基金(31660314);国家自然科学基金(32060143)
Sequencing and analysis of the mitochondrial genome of Hoplopleura edentula
Received date: 2021-09-22
Revised date: 2021-11-18
Online published: 2022-04-12
Supported by
National Natural Science Foundation of China(31660314);National Natural Science Foundation of China(32060143)
目的 对缺齿甲胁虱线粒体基因组序列进行测定与分析,了解甲胁虱属线粒体基因组的结构特征和变异情况。 方法 在大理苍山世界地质公园捕获大绒鼠,全捕法采集大绒鼠体表吸虱,鉴定后选取缺齿甲胁虱用组织DNA提取试剂盒提取单只缺齿甲胁虱DNA。用通用引物扩增缺齿甲胁虱的rrnS和rrnL基因的短片段序列,测序后在短片段序列的保守区设计特异引物,PCR扩增包含rrnS和rrnL基因的全长或近乎全长的微环染色体,微环染色体组装成功后在其保守区设计一对微环染色体编码区特异性引物,PCR扩增出全部微环的编码区。扩增产物纯化后进行高通量测序法测序。利用Geneious、tRNAscan、CodonW、BLAST等生物信息学工具分析其线粒体基因结构特征与变异情况。 结果 共获得缺齿甲胁虱优质序列读数6 812 606 bp。组装后共找到节肢动物线粒体基因组常见基因24个,包括7个蛋白质编码基因（PCG）、15个tRNA基因和2个rRNA基因。缺齿甲胁虱线粒体基因组裂化为8个微环染色体（GenBank登录号：MW835203~MW835210）,这些基因不均匀地分布在微环染色体上,每个微环染色体编码区包含1~4个基因,至少有1个PCG或rRNA基因。编码区的AT含量为61.0%。除cox2基因以TTG为起始密码子,其余PCG均以ATN为起始密码子,以典型的TAA和TAG为终止密码子。密码子AUU使用频率最高（RSCU：1.53）。15个tRNA基因的二级结构均为典型的三叶草结构,存在31处错配,主要为G-U错配。rrnS和M-L1(tag)-rrnL-V微环染色体获得了全部非编码区,存在2种串联重复序列模块,相似度达88.0%~90.0%,在编码区5′端上游的非编码区存在一处AT富集区（50 bp,64.0% A&T）,在编码区3′端下游存在一处GC富集区（42 bp,85.7% G&C）。其余6个微环染色体仅获得部分非编码区,相似度达86.5%~88.5%。比较缺齿甲胁虱,克氏甲胁虱和红姬甲胁虱线粒体基因组发现：3种甲胁虱的线粒体基因组均裂化;E-cob-S1(tct)-S2(tga)、I-cox1、K-nad4和rrnS等4个微环的基因组成以及基因排序完全相同;缺齿甲胁虱的H-nad5-F-T微环,在其他2种甲胁虱没有发现;trnT移位频繁,分布在3种甲胁虱不同的微环上;缺齿甲胁虱trnS1(tct)二级结构为典型的三叶草结构,而其他2种甲胁虱trnS1(tct)缺少D臂。 结论 缺齿甲胁虱的24个基因不均匀地分布于8个微环染色体上,每个微环染色体上均含有1个编码区和1个非编码区。缺齿甲胁虱的24个基因虽AT含量较高,tRNA碱基错配次数较多,缺齿甲胁虱trnS1(tct)为典型的三叶草结构。甲胁虱属线粒体基因组的结构有差异,缺齿甲胁虱线粒体基因组结构的特殊性可能与线粒体基因组的裂化有关。
孙佳宁 , 陈婷 , 董文鸽 . 缺齿甲胁虱线粒体基因组测序与分析[J]. 中国寄生虫学与寄生虫病杂志, 2022 , 40(2) : 194 -203 . DOI: 10.12140/j.issn.1000-7423.2022.02.010
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.
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