印度华首线虫核糖体和线粒体基因序列多态性分析

doi:10.12140/j.issn.1000-7423.2019.01.015

中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (1): 81-86.doi: 10.12140/j.issn.1000-7423.2019.01.015

印度华首线虫核糖体和线粒体基因序列多态性分析

周成艳^1,²(), 马君², 刘伟^3,⁴, 徐前明¹, 朱兴全^1,^2,^*()

1 安徽农业大学动物科技学院,合肥 230036
2 中国农业科学院兰州兽医研究所,家畜疫病病原生物学国家重点实验室,甘肃省动物寄生虫病重点实验室,兰州 730046
3 湖南农业大学动物医学院,长沙 410128
4 兽用蛋白质工程疫苗湖南省重点实验室,长沙 410128

收稿日期:2018-09-19 出版日期:2019-02-28 发布日期:2019-03-18
通讯作者: 朱兴全
作者简介:
作者简介：周成艳（1993-）,女,硕士研究生,从事寄生虫学研究。E-mail: zcyzcy99@126.com
基金资助:
国家自然科学基金青年项目（No. 31702225）;中国农业科学院科技创新工程项目（No. CAAS-ASTIP-2016-LVRI-03）;中国农业科学院农科英才项目

Sequence polymorphisms within Kalicephalus indicus ribosomal and mitochondrial genes

Cheng-yan ZHOU^1,²(), Jun MA², Wei LIU^3,⁴, Qian-ming XU¹, Xing-quan ZHU^1,^2,^*()

1 College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
2 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
3 College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China
4 Key Laboratory of Animal Protein Engineering Vaccine, Hunan Province, Changsha 410128, China

Received:2018-09-19 Online:2019-02-28 Published:2019-03-18
Contact: Xing-quan ZHU
Supported by:
Supported by the National Natural Science Foundation of China (No. 31702225), the Agricultural Science and Technology Innovation Program (ASTIP) (No. CAAS-ASTIP-2016-LVRI-03) and the Elite Program of Chinese Academy of Agricultural Sciences

摘要/Abstract

摘要：

目的分析印度华首线虫的核糖体基因和线粒体基因序列,探讨其作为分子标记的应用潜力。方法 2016年6月,于湖南省长沙市长沙县榔梨镇捕捉到2条野生大王蛇,并从蛇体内分离获得7条印度华首线虫。提取7条印度华首线虫基因组DNA,PCR扩增核糖体DNA内转录间隔区1和2（ITS-1和ITS-2）、5.8S、线粒体DNA细胞色素c氧化酶亚基1（cox1）、核糖体RNA小亚基12S、ATP酶亚基6（atp6）、烟酰胺腺嘌呤二核苷酸脱氢酶亚基4（nad4）基因,测序。采用ClustalX 1.83软件拼接各基因测序结果,并进行多序列比对。基于cox1、12S、atp6、nad4线粒体基因序列,以旋盘尾丝虫为外群,采用贝叶斯（BI）法构建系统进化树,分析7条印度华首线虫PCR扩增序列的种内变异。结果 ITS-1、5.8S和ITS-2扩增产物长度约为900 bp,cox1、12S、atp6和atp4扩增产物长度依次约为400、600、500和400 bp。ITS-1、5.8S和ITS-2的A + T含量为52.6%~53.7%,高于G + C含量（46.3%~47.4%）,cox1、12S、atp6和atp4的A + T含量分别为67.1%~68.9%、76.9%~77.4%、73.0%~75.5%和70.3%~73.3%,均高于其G + C含量（31.1%~32.9%、22.6%~23.1%、24.5%~27.0%和26.7%~29.7%）。ITS-1、5.8S、ITS-2、cox1、12S、atp6和atp4的种内变异依次为0~2.5%、0、0~5.0%、0~2.6%、0~3.7%、0.7%~8.7%和0~11.0%。7条印度华首线虫线粒体基因序列聚于一支,与钩口科锡兰钩口线虫的亲缘关系最近。结论印度华首线虫核糖体基因序列中ITS-2变异程度最大,有望成为研究印度华首线虫种内变异的理想分子标记。线粒体基因序列变异程度从低到高依次为cox1 < 12S < atp6 < nad4,nad4是研究印度华首线虫遗传多样性的理想线粒体分子标记。

关键词: 印度华首线虫, 核糖体DNA, 线粒体DNA, 序列分析

Abstract:

Objective To analyze the ribosomal and mitochondrial genes sequences polymorphisms of Kalicephalus indicus, and discuss their application potential as molecular markers. Methods Two wild Elaphe carinata snakes were captured in June 2016, in Zhangli Town, Changsha County, Hunan Province, and seven K. indicus adult worms were isolated from the snakes. Total DNA from the worms was extracted. The sequences of the first and second internal transcribed spacer regions 1 and 2(ITS-1 and ITS-2), 5.8S, mitochondrial cytochrome c oxidase subunit 1 (cox1), small subunit 12S of ribosomal RNA, ATP synthase subunit 6 (atp6), nicotinamide adenine dinucleotide dehydrogenase subunit 4 (nad4) of K. indicus were amplified using PCR and subjected to DNA sequencing. The sequencing results of each gene were aligned using ClustalX 1.83. A phylogenetic tree based on the mitochondrial DNA sequences cox1, 12S, atp6 and nad4 was constructed using Bayesian method, using Onchocerca volvulus as an outgroup. Intraspecific variation in these sequences among the seven K. indicus samples were analyzed. Results The size of the amplified products of ITS-1, 5.8S and ITS-2 was approximately 900 bp. The size of the amplified products of cox1, 12S, atp6 and nad4 was approximately 400, 600, 500 and 400 bp, respectively. The content of A + T of ITS-1, 5.8S and ITS-2 is 52.7%-53.7%, higher than its content of G + C (46.3%-47.4%). The content of A + T of cox1, 12S, atp6 and nad4 is 67.1%-68.9%, 76.9%-77.4%, 73.0%-75.5%, and 70.3%-73.3%, respectively, higher than their content of G + C (31.1%-32.9%, 22.6%-23.1%, 24.5%-27.0% and 26.7%-29.7%). Intraspecific variation in ITS-1, 5.8S, ITS-2, cox1, 12S, atp6 and nad4 were 0-2.5%, 0, 0-5.0%, 0-2.6%, 0-3.8%, 0.7%-8.7% and 0-11.0%, respectively. The phylogenetic tree clustered the seven K. indicus individuals into the same branch, with Ancylostoma ceylanicum being the closest species. Conclusion The ITS-2 sequence with the largest variation is expected to be an ideal molecular marker for studying the intraspecific variation of K. indicus. The degree of its intraspecific variation in mitochondrial gene sequences is as follows: cox1 < 12S < atp6 < nad4, and nad4 is an ideal mitochondrial molecular marker for studying the genetic diversity of K. indicus.

Key words: Kalicephalus indicus, Ribosomal DNA, Mitochondrial DNA, Sequence analysis

中图分类号:

R383.19

周成艳, 马君, 刘伟, 徐前明, 朱兴全. 印度华首线虫核糖体和线粒体基因序列多态性分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(1): 81-86.

Cheng-yan ZHOU, Jun MA, Wei LIU, Qian-ming XU, Xing-quan ZHU. Sequence polymorphisms within Kalicephalus indicus ribosomal and mitochondrial genes[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(1): 81-86.

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引物Primer	序列(5′→3′) Sequence (5′→3′)
NC5	GTAGGTGAACCTGCGGAAGGATCATT
NC2	TTAGTTTCTTTTCCTCCGCT
JB3	TTTTTTGGGCATCCTGAGGTTTAT
JB4.5	TAAAGAAAGAACATAATGAAAATG
atp6F	AGAATTCAATTACCTTTAAGTTCAGTAA
atp6R	AAATAAATCAAACGAGAAAAAATATATC
12SF	ATAATACTTGTTCCAGAATAATCGGCTA
12SR	AAACCTACTTTACTACAACTTACTCCCC
nad4F	TTTTGGGCTTTGGTTCTCAAATTGAAAA
nad4R	ATGTGTGTGACCGAAGAATATGCCGCCA

种名 Species	分类地位 Taxonomic status	国家 Country	GenBank登录号 GenBank accession number
锡兰钩口线虫Ancylostoma ceylanicum	钩口科Ancylostomatidae	中国China	NC035142
松材线虫Bursaphelenchus xylophilus	滑刃科Aphelenchoididae	法国France	JQ429761
蛔虫属Ascaris sp.	蛔科Ascarididae	中国China	KC839987
似蚓蛔线虫Ascaris lumbricoides	蛔科Ascarididae	中国China	HQ704900
猪蛔虫Ascaris suum	蛔科Ascarididae	中国China	HQ704901
小熊猫贝蛔虫Baylisascaris ailuri	蛔科Ascarididae	中国China	NC015925
马副蛔虫Parascaris univalens	蛔科Ascarididae	澳大利亚Australia	NC024884
简单异尖线虫Anisakis simplex	异尖科Anisakidae	韩国Korea	NC007934
拟地新异尖线虫Pseudoterranova azarasi	异尖科Anisakidae	中国China	NC027163
猫弓首蛔虫Toxocara cati	弓首科Toxocaridae	中国China	NC010773
蠕形住肠线虫Enterobius vermicularis	尖尾科Oxyuridae	韩国Korea	NC011300
旋盘尾丝虫Onchocerca volvulus	盘尾科Onchocercidae	美国USA	NC001861

印度华首线虫核糖体和线粒体基因序列多态性分析

Sequence polymorphisms within Kalicephalus indicus ribosomal and mitochondrial genes

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