浣熊贝氏蛔虫核糖体基因和线粒体基因分析

doi:10.12140/j.issn.1000-7423.2022.05.005

中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (5): 594-602.doi: 10.12140/j.issn.1000-7423.2022.05.005

浣熊贝氏蛔虫核糖体基因和线粒体基因分析

韦开文¹(), 曾红霞¹, 何牧², 胡俊杰³^,^*()

1.云南大学生命科学学院,昆明 650000
2.昆明动物园,昆明650000
3.云南大学生态与环境学院,云南省高原山地生态与退化环境修复重点实验室,昆明 650000

收稿日期:2022-01-05 修回日期:2022-03-28 出版日期:2022-10-30 发布日期:2022-10-17
通讯作者: 胡俊杰
作者简介:韦开文（1997- ）,男,硕士研究生,从事动物寄生虫学研究。E-mail： 2654464562@qq.com
基金资助:
云南省教育厅科学研究基金(2021Y044)

Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis

WEI Kai-wen¹(), ZENG Hong-xia¹, HE Mu², HU Jun-jie³^,^*()

1. School of Biological Sciences, Yunnan University, Kunming 650000, China
2. Kunming Zoological Garden, Kunming 650000, China
3. School of Ecology and Environmental Sciences and Yunnan Key Laboratory or Plateau Mountain Ecology and Restoration of Degraded Environments, Yunnan University, Kunming 650000, China

Received:2022-01-05 Revised:2022-03-28 Online:2022-10-30 Published:2022-10-17
Contact: HU Jun-jie
Supported by:
Scientific Fund of Education Department of Yunnan Province(2021Y044)

摘要/Abstract

摘要：

目的探讨浣熊贝氏蛔虫（Baylisascaris procyonis）核糖体基因和线粒体基因的特征及其与其他贝氏蛔虫的系统进化关系。方法从昆明动物园1头病死的浣熊肠道收集浣熊贝氏蛔虫成虫,形态学初步鉴定后挑取20条（雌虫、雄虫各10条）进行形态特征测量和相关分子生物学研究。用DNA提取试剂盒提取浣熊贝氏蛔虫DNA,PCR扩增核糖体内转录间隔区（ITS）、细胞色素c氧化酶亚基1（cox1）、cox2、cox3、细胞色素氧化酶b（cytb）、烟酰胺腺嘌呤二核苷酸脱氢酶亚基1（nad1）、tRNA-nad2-tRNA和nad5,测序后与GenBank中的序列进行BLAST比对,使用MEGA X软件进行单核苷酸多态性（SNP）位点分析,采用最大似然法分别构建基于ITS、cox1和cox2序列的系统进化树。结果浣熊贝氏蛔虫成虫呈乳白色,雌、雄虫大小分别为（9.15~14.50）cm ×（0.20~0.32）cm和（6.45~8.70）cm ×（0.15~0.20）cm,雄虫尾端有交合刺。浣熊贝氏蛔虫的8个遗传标志均被成功测序并提交至GenBank,分别为ITS（MZ092850~MZ092855）、cox1（MZ164969）、cox2（MZ172987）、cox3（MZ172988）、cytb（MZ172982~MZ172986）、nad1（MZ172989）、nad5（MZ172981）和tRNA-nad2-tRNA（MZ172980）序列与GenBank中已有的浣熊贝氏蛔虫序列的相似性分别为98.6%~100%、98.5%~99.5%、99.4%~99.7%、98.3%、98.8%~99.0%、98.2%、97.8%和99.2%;与GenBank中已有的柱状贝氏蛔虫ITS、cox1、cox2和tRNA-nad2-tRNA序列的相似度分别为98.6%~100%、99.2%~100%、99.4%~100%和99.6%~99.9%。SNP位点分析结果发现,ITS、cox1、cox2和tRNA-nad2-tRNA与GenBank中已有的浣熊贝氏蛔虫和柱状贝氏蛔虫相应序列分别有8、28、7和12个SNP位点,但这些位点并不能对这2个物种进行有效区分;新测序的ITS序列中包含有7~9个GA串联重复序列,其中7和8个GA串联重复为本研究发现。在基于ITS、cox1和cox2序列构建的系统进化树上,浣熊贝氏蛔虫和柱状贝氏蛔虫均形成一个独立的分支,该分支中不同地理来源的浣熊贝氏蛔虫和柱状贝氏蛔虫完全聚合在一起,且该分支与德氏贝氏蛔虫聚合在一起。结论浣熊贝氏蛔虫的标记序列、SNP位点特征与柱状贝氏蛔虫均高度相似,基于系统进化分析不能有效区分浣熊贝氏蛔虫和柱状贝氏蛔虫。

关键词: 浣熊, 浣熊贝氏蛔虫, 核糖体和线粒体基因, 单核苷酸多态性, 系统发育

Abstract:

Objective To characterize the ribosomal and mitochondrial genes of Baylisascaris procyonis, and their phylogenetic relationship with other Baylisascaris spp. Methods Adult worms of B. procyonis were collected from the intestine of a deceased raccoon at the Kunming Zoo in Kunming, China. After morphological identification, 20 adult worms comprising 10 males and 10 females were selected for morphological measurement and related molecularbiological study. The DNA was extracted from the tissue sample of the parasite using a DNA extraction kit. For amplification of ribosome internal transcribed spacer (ITS), cytochrome c oxidase 1 (cox1), cox2, cox3, cytochrome b (cytb), nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1), nad5 and tRNA-nad2-tRNA by PCR, which were sequenced and aligned with the database deposited in GenBank using BLAST. Single nucleotide polymorphisms (SNP) were analyzed using MEGA X software, and phylogenetic trees based on ITS, cox1 and cox2 sequences were constructed respectively, using the maximum likelihood (ML) method. Results The adult worms were in ivory color, at a size of (9.15-14.50) cm × (0.20-0.32) cm for females and (6.45-8.70) cm × (0.15-0.20) cm for males. Adult male has copulatory spicule at the caudal ends. The eight genetic markers of B. procyonis were successfully sequenced, and all sequences were deposited in GenBank under the accession numbers MZ092850-MZ092855, MZ164969, MZ172987, MZ172988, MZ172982-MZ172986, MZ172989, MZ172981, and MZ172980 for ITS, cox1, cox2, cox3, cytb, nad1, nad5, and tRNA-nad2-tRNA, respectively. Of the eight loci, the newly obtained sequence shared identities of 98.6%-100%, 98.5%-99.5%, 99.4%-99.7%, 98.3%, 98.8%-99.0%, 98.2%, 97.8% and 99.2%, respectively, with those of B. procyonis previously deposited in GenBank. Additionally, the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had identities of 98.6%-100%, 99.2%-100%, 99.4%-100% and 99.6%-99.9%, respectively, with those of B. columnaris previously deposited in GenBank. SNP analysis showed that the newly obtained ITS, cox1, cox2 and tRNA-nad2-tRNA sequences had 28, 7 and 12 SNP sites, respectively, compared to those of B. procyonis and B. columnaris previously deposited in GenBank. However, none of the SNP sites was suitable to distinguish B. procyonis from B. columnaris. Meanwhile, the newly obtained ITS sequences contained 7 to 9 GA tandem repeats, and the 7 and 8 repeats constituted the first records discovered in B. procyonis. Phylogenetic analysis inferred from ITS, cox1 or cox2 sequences indicated that B. procyonis and B. columnaris obtained from different geographical areas completely aggregated, and formed an individual clade, that clustered with B. devosi. Conclusion The markers sequences and SNP characteristics of B. raccoonus were highly similar to those of B. cylindrica, whereas phylogenetic analysis could not effectively differentiate B. procyonis from B. columnaris.

Key words: Raccoon, Baylisascaris procyonis, Nuclear and mitochondrial genes, Single nucleotide polymorphisms, Phylogeny

中图分类号:

S582.731

韦开文, 曾红霞, 何牧, 胡俊杰. 浣熊贝氏蛔虫核糖体基因和线粒体基因分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(5): 594-602.

WEI Kai-wen, ZENG Hong-xia, HE Mu, HU Jun-jie. Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2022, 40(5): 594-602.

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参考文献 21

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DNA 区域 DNA region	引物名字 Primer name	引物序列（5′→3′） Primer sequence (5′→3′)	参考文献 Reference
ITS	18sF	ACTGCTGTTTCGAGACCTTT-CGAG	[7]
	28sR	TAGCACCTTCTTTGGACTAT-AGCC
tRNA-nad2-tRNA	Amplicon 1F	CCGTTGGCCTTAACTGTTCG	[9]
	Amplicon 1R	GGCAACCCAACAACCATAG
cox1	Amplicon 2F	CGGGTTTTCTGTTCCTGTGG	[9]
	Amplicon 2R	ACCTGATTGGAAGTCAGGTG
cox2	Amplicon 3F	GTGGATAAGGGGCCTTGTG	[9]
	Amplicon 3R	ATAAACCCCCGCCAGTTCTC
cox3	BPCO3F	GGCTCTTTAGGTTTGACCA-GTT	a
	BPCO3R	GCAAACTCCAGACCAAGATGAT
cytb	BPCytbF	GGGTTCCAGATTTTAACA-GGT	a
	BPCytbR	ACCAAACACTGCCCAAGC
nad1	BPNAD1F	TTTTGAAGAAGGAGCAGATGAC	a
	BPNAD1R	CGAGGATAAGCCCTACGA-AC
nad5	BPNAD5F	TTGGGATAGTTGTAGAGGGG	a
	BPNAD5R	CAAAACGACCACAACAGA-AT

遗传标记 Genetic marker	本研究序列的登录号 Accession number of the sequence in this study	与GenBank序列的比对结果 Comparison result with sequences in GenBank
遗传标记 Genetic marker	本研究序列的登录号 Accession number of the sequence in this study	种名 Species	GenBank登录号 Accession number in GenBank	相似性/% （均值/%） Similarity/% (Average/%)
ITS	MZ092850~MZ092855	柱状贝氏蛔虫	KC543484、KC543485、MH030594、MH030595、LN600405、KX774493	98.6~100 (99.4)
		浣熊贝氏蛔虫	OU974763、MH030596、MH030597、MZ092854、JQ403615、KJ698593~KJ698602、KC543488	98.6~100 (99.2)
		德氏贝氏蛔虫	MN960312、KY465505、MH030598	96.1~97.3 (96.7)
cox1	MZ164969	柱状贝氏蛔虫	KY580736~KY580739	99.2~100 (99.6)
		浣熊贝氏蛔虫	MW385416~MW385530、MH795149、MH795150、KJ698533~KJ698567	98.5~99.5 (99.0)
		德氏贝氏蛔虫	MH795151	95.4
cox2	MZ172987	柱状贝氏蛔虫	FJ357429、KC543478、KC543480、KY580740~KY580742、MH469662、MH469663	99.4~100 (99.8)
		浣熊贝氏蛔虫	MH469664、MH469665、KP843602、KP843603、KC543482、AF179908、JF951366	99.4~99.7 (99.6)
		德氏贝氏蛔虫	MH469666	96.6
cox3	MZ172988	浣熊贝氏蛔虫	JF951366	98.3
		猪蛔虫	HQ912104	90.6
cytb	MZ172982~MZ172986	浣熊贝氏蛔虫	JF951366、JQ340492	98.8~99.0 (98.9)
		转移贝氏蛔虫	HQ671079、JQ340493、JQ340494	87.3~88.0 (87.5)
nad1	MZ172989	浣熊贝氏蛔虫	JF951366	98.2
		转移贝氏蛔虫	HQ671079	91.9
tRNA-nad2-tRNA	MZ172980	柱状贝氏蛔虫	KY580744~KY580747	99.6~99.9 (99.7)
		浣熊贝氏蛔虫	JF951366	99.2
		小熊猫贝氏蛔虫	HQ671080	89.7
nad5	MZ172981	浣熊贝氏蛔虫	JF951366	97.8
		转移贝氏蛔虫	HQ671079	88.1

种类（登录号） Species (Accession number)	ITS中的SNP位点 SNP site in ITS								ITS中的GA串联重复 GA tandem repeat in ITS
种类（登录号） Species (Accession number)	位置号 Position number								位置号（重复数） Position number (No. repeat)
	143^a	480^a (158)^b	523^a (201)^b	865^a	866^a	867^a	868^a	1 129^a	849~870
Bc (MH030594)	C	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (MH030595)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (KC543484)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bc (KC543485)	-	A	T	-	-	-	-	-	GAGAGAGAGAGAGAGAAA (7)
Bc (KX774493)	-	-	T	-	-	-	-	-	GAGAGAGAGAGAGAAA (6)
Bp (JQ403615)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (MH030596)	-	-	T	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (MH030597)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp (KC543488)	-	-	C	G	A	G	A	-	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#1 (MZ092850)	-	A	T	-	-	-	-	C	GAGAGAGAGAGAGAGAAA (7)
Bp#5 (MZ092851)	-	A	T	G	-	-	A	C	GAGAGAGAGAGAGAGAGAAA (8)
Bp#7 (MZ092852)	-	-	C	G	A	G	A	T	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#11 (MZ092853)	C	A	T	-	-	-	-	C	GAGAGAGAGAGAGAGAAA (7)
Bp#12 (MZ092854)	C	-	C	G	A	G	A	T	GAGAGAGAGAGAGAGAGAGAAA (9)
Bp#19 (MZ092855)	-	-	C	G	-	-	A	T	GAGAGAGAGAGAGAGAGAAA (8)

种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	37^a	168^a		201^a		231^a,c		378^a		429^a		484^a		549^a		696^a		786^a (43)^b		802^a (59)^b		804^a,d (61)^b		822^a (79)^b		832^a (89)^b
Bc (KY580736)	A	A		C		G		G		A		G		T		C		C		T		G		T		T
Bc (KY580738)	G	G		T		G		A		G		A		C		C		C		C		G		T		T
Bc (KY580739)	A	A		C		G		G		G		G		T		G		C		T		A		T		T
Bc (MH795147)	-	-		-		-		A		G		A		C		C		T		T		G		T		C
Bc (MH795148)	-	-		-		-		A		G		A		C		C		C		C		G		T		T
Bc (KC543475)	-	-		-		-		-		-		-		-		-		C		T		G		C		T
Bp (MH795149)	-	-		-		-		G		G		G		T		T		C		T		A		T		T
Bp (MH795150)	-	-		-		-		G		G		G		T		C		C		T		G		T		T
Bp (JF951366)	A	A		T		A		G		G		G		T		T		C		T		A		T		T
Bp#1 (MZ164969)	G	G		T		G		A		G		A		C		C		C		C		G		T		T
种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	834^a,d (91)^b		957^a (214)^b		972^a (229)^b		1 002^a,c,d (259)^b		1 046^a (303)^b		1 266^a,c		1 305^a		1 311^a		1 315^a,c		1 350^a		1 378^a		1 379^a		1 491^a,c		1 506^a,c
Bc (KY580736)	A		T		A		G		T		T		A		T		G		T		A		T		T		T
Bc (KY580738)	A		T		A		G		T		T		A		C		G		T		G		T		T		T
Bc (KY580739)	G		T		A		G		T		T		A		T		G		C		A		T		T		T
Bc (MH795147)	A		C		A		G		C		T		A		T		G		T		G		T		T		-
Bc (MH795148)	A		T		A		G		T		T		A		C		G		T		G		T		-		-
Bc (KC543475)	A		T		G		G		T		-		-		-		-		-		-		-		-		-
Bp (MH795149)	G		T		A		A		T		C		G		T		G		C		A		C		C		-
Bp (MH795150)	A		T		A		G		T		T		A		T		G		T		A		T		-		-
Bp (JF951366)	G		T		A		A		T		C		G		T		A		C		A		C		C		C
Bp#1 (MZ164969)	A		T		A		G		T		T		A		C		G		T		G		T		T		T

种类（登录号） Species (Accession number)	位置号 Position number
种类（登录号） Species (Accession number)	9^a	168^a (66)^b	219^a (117)^b	306^a (204)^b	348^a (246)^b	382^a (280)^b	582^a (480)^b
Bc (FJ357429)	-	G	A	A	T	C	G
Bc (MH469662)	-	G	A	A	C	C	G
Bc (MH469663)	-	G	A	A	T	C	G
Bc (KC543478)	-	G	A	A	T	C	G
Bc (KC543480)	-	G	G	A	T	T	T
Bc (KY580740)	T	G	A	A	C	T	G
Bc (KY580741)	T	A	A	A	C	T	G
Bc (KY580742)	G	G	A	A	T	C	G
Bp (MH469664)	-	A	A	A	T	T	G
Bp (MH469665)	-	G	A	A	C	T	G
Bp (KP843602)	-	G	A	A	C	T	G
Bp (KP843603)	-	A	A	G	T	T	G
Bp (KC543482)	-	A	A	A	T	T	G
Bp (AF179908)	-	A	A	A	T	T	G
Bp (JF951366)	T	A	A	A	T	T	G
Bp#1 (MZ172987)	G	G	A	A	T	C	G

浣熊贝氏蛔虫核糖体基因和线粒体基因分析

Analysis of ribosomal and mitochondrial genes of Baylisascaris procyonis

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种类（登录号） Species (Accession number)	位置号 Position number
	tRNA-Lys		tRNA-Leu	tRNA-Ser			nad2					tRNA-Arg
	4 117^a	4 171^a	4 222^a (47)^b	4 241^a (11)^b	4.243^a (13)^b	4 267^a (38)^b	4 345^a (63)^b	4 446^a (164)^b	4 632^a (350)^b	4 696^a (414)^b	5 031^a (749)^b	5239^a (54)^b
Bc (KY580744)	-	-	T	T	T	T	A	C	T	C	G	T
Bc (KY580745)	-	-	T	T	C	C	A	C	T	T	G	T
Bc (KY580746)	-	-	T	T	C	T	A	T	T	C	G	T
Bc (KY580747)	-	-	T	T	T	T	A	C	T	C	G	T
Bp (JF951366)	-	C	C	C	C	-	G	T	C	C	A	T
Bp#1 (MZ172980)	G	T	T	T	C	C	A	C	T	T	G	C

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