缺齿甲胁虱线粒体基因组测序与分析

doi:10.12140/j.issn.1000-7423.2022.02.010

摘要/Abstract

摘要：

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。

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

中图分类号:

R384.3

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.

SUN Jia-ning, CHEN Ting, DONG Wen-ge. Sequencing and analysis of the mitochondrial genome of Hoplopleura edentula[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(2): 194-203.

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微环染色体 Minichromosome	编码区大小/bp Size of coding region/bp	序列读数/bp No. sequence read/bp
E-cob-S₁(tct)-S₂(tga)	1 303	456 433
I-cox1	1 599	206 917
D-Y-cox2	791	822 133
Q-nad1-G-nad3	1 446	712 883
K-nad4	1 322	960 072
H-nad5-F-T	1 889	441 440
M-L₁_(tag)-rrnL-V	1 311	1 681 291
rrnS	874	1 290 852
合计Total	10 535	6 572 021

特征 Feature	缺齿甲胁虱 H. edentula	克氏甲胁虱 H. kitti	红姬甲胁虱 H. akanezumi
微环染色体组成Composition of minichromosome	-	atp8-atp6-N	atp8-atp6-N
	E-cob-S₁(tct)-S₂(tga)	E-cob-S₁(tct)-S₂(tga)	E-cob-S₁(tct)-S₂(tga)
	I-cox1	I-cox1	I-cox1
	D-Y-cox2	D-Y-cox2-	D-Y-cox2
	-	R-nad4L-P-cox3-A	R-nad4L-P-cox3-A-
	Q-nad1-G-nad3	Q-nad1-G-nad3	-
	-	nad2	nad2
	K-nad4	K-nad4	K-nad4
	H-nad5-F-	-	-
	-	C-nad6-W-L₂(taa)	C-nad6-W-L₂(taa)
	M-L₁(tag)-rrnL-V	M-L₁(tag)-rrnL-V	-rrnL-V
	rrnS	rrnS	rrnS
微环染色体数No. minichromosome	8	11	11
基因数No. gene	24	34	28

基因序列 Gene sequence	长度/bp Length/bp	占比/% Proportion/%					AT偏倚值 AT-skew	GC偏倚值 GC-skew
基因序列 Gene sequence	长度/bp Length/bp	A	T	G	C	A + T	AT偏倚值 AT-skew	GC偏倚值 GC-skew
编码区Coding region	10 545	26.9	34.1	20.7	18.3	61.0	-0.118	0.062
蛋白质编码基因 Protein-coding gene	7 563	25.0	34.8	19.1	21.1	59.8	-0.164	-0.050
密码子第1位点 The 1st codon position	2 521	28.2	28.4	25.6	17.8	56.6	-0.004	0.180
密码子第2位点 The 2nd codon position	2 521	16.9	43.5	18.0	21.6	60.4	-0.440	-0.091
密码子第3位点 The 3rd codon position	2 521	26.8	35.7	19.5	18.0	62.5	-0.426	0.040
tRNAs	995	31.5	32.8	19.6	16.1	64.3	-0.020	0.098
rrnL	1 117	31.5	32.2	21.1	15.2	63.7	-0.011	0.163
rrnS	874	31.2	31.5	19.2	18.1	62.7	-0.005	0.029

氨基酸 Amino acid	密码子 Codon	使用次数 No. use	相对同义密码子使用频率 Relative synonymous codon usage	氨基酸 Amino acid	密码子 Codon	使用次数 No. use	相对同义密码子使用频率 Relative synonymous codon usage
丙氨酸 Ala	GCU	39	1.65	脯氨酸 Pro	CCU	63	2.23
	GCC	64	1.01		CCC	23	0.81
	GCA	32	0.83		CCA	16	0.57
	GCG	20	0.52		CCG	11	0.39
半胱氨酸 Cys	UGU	21	1.08	谷氨酰胺 Gln	CAA	15	0.77
	UGC	18	0.92		CAG	24	1.23
天冬氨酸 Asp	GAU	31	1.24	精氨酸 Arg	CGU	6	0.27
	GAC	19	0.76		CGC	10	0.45
谷氨酸 Glu	GAA	41	1.32		CGA	10	0.45
	GAG	21	0.68		CGG	12	0.54
苯丙氨酸 Phe	UUU	129	1.50		AGA	43	1.93
	UUC	43	0.50		AGG	53	2.37
甘氨酸 Gly	GGU	24	0.57	丝氨酸 Ser	UCU	77	2.23
	GGC	25	0.60		UCC	40	1.16
	GGA	50	1.20		UCA	40	1.16
	GGG	68	1.63		UCG	12	0.35
组氨酸 His	CAU	24	0.92		AGU	21	0.61
	CAC	28	1.08		AGC	17	0.49
异亮氨酸 Ile	AUU	137	1.53	苏氨酸 Thr	ACU	46	1.72
	AUC	38	0.43		ACC	26	0.97
	AUA	93	1.04		ACA	30	1.12
赖氨酸 Lys	AAA	25	0.82		ACG	5	0.19
	AAG	36	1.18	缬氨酸 Val	GUU	75	1.42
亮氨酸 Leu	UUA	124	1.97		GUC	32	0.60
	UUG	46	0.73		GUA	65	1.23
	CUU	91	1.45		GUG	40	0.75
	CUC	32	0.51	色氨酸 Trp	UGG	32	1.00
	CUA	41	0.65		UGA	45	2.60
	CUG	43	0.68	酪氨酸 Tyr	UAU	46	1.12
蛋氨酸 Met	AUG	68	1.00		UAC	36	0.88
天冬酰胺 Asn	AAU	45	1.25	终止密码子* Stop codon*	UAA	6	0.35
	AAC	27	0.75	终止密码子* Stop codon*	UAG	1	0.06