云南省细粒棘球绦虫线粒体co1和nd1基因序列分析

doi:10.12140/j.issn.1000-7423.2023.03.007

摘要/Abstract

摘要：

目的分析云南省细粒棘球蚴线粒体细胞色素氧化酶1（co1）和NADPH脱氢酶第1亚基（nd1）的基因型和序列多态性。方法动物源棘球蚴采自云南省香格里拉市、大关县、洱源县、泸水市、维西县的屠宰场牛、羊和放养猪的肝脏或肺病灶组织分离的包囊，人源棘球蚴采自从剑川县、云龙县、隆阳区和玉龙县医院手术摘除的病灶组织。病原学鉴定后选取细粒棘球蚴，用DNA提取试剂盒提取基因组DNA，PCR扩增co1和nd1基因并测序，通过BLAST比对分析序列一致性和单核苷酸多态性，用MEGA-X软件采用邻接法构建基于co1和nd1的系统进化树。结果共采集62份棘球蚴样品，其中36份经病原学检测确定为细粒棘球蚴。co1基因测序成功32条，其中15条为G1型，17条为G5型，长度分别为824 bp和807 bp，提交至GenBank数据库获得的登录号分别为OP413393~OP413402、OP413498~OP413506和OP420520。nd1基因测序成功34条，其中11条为G1型，23条为G5型，长度分别为882 bp和888 bp，提交GenBank数据库获得的登录号为OP471626~OP471638。序列多态性分析结果显示，co1的G1、G5型序列的变异位点分别占1.94%（16/824）、3.10%（25/807）；nd1的G1、G5型序列的变异位点分别占0.45%（4/882）、2.93%（26/888）。基因型多序列比对结果显示，co1基因共有6条同源序列，其中G1型4条，同源序列的遗传距离分别为0.000 0~0.001 1、0.000 0、0.000 0~0.000 6和0.000 0~0.001 7；G5型2条，同源序列遗传距离分别为0.000 0~0.002 5和0.000 0~0.001 2。nd1基因共有3条同源序列，其中G1型1条，遗传距离为0.000 0~0.001 1；G5型2条，遗传距离为0.000 0和0.000 0~0.001 1。系统进化树分析结果显示，基于co1和nd1基因的系统进化树均形成G1和G5型2个分支，G1型与中国中西部（青海、宁夏、甘肃、四川），不丹、中东地区伊朗和约旦等的基因序列（GenBank登录号AF297617.1、KJ628328.1、EU072106.1、MW138946.1、AB688602.1）的亲缘关系较近；G5型与越南、中国广西、英国、波兰、赞比亚、法国、巴基斯坦、巴西、肯尼亚和纳米比亚等的基因序列（GenBank登录号MW558412.1、MN058591.1、KU378107.1、MZ322608.1、KU743915.1、KU743919.1、MN886291.1、KX010903.1、KU743918.1和KX138068.1）的亲缘关系较近。结论云南省细粒棘球绦虫流行株的基因型为G1和G5型，co1和nd1基因均存在单核苷酸多态性。

关键词: 细粒棘球绦虫, co1基因, nd1基因, 序列分析, 云南省

Abstract:

Objective To analyze the genotype and sequence polymorphisms of mitochondrial cytochrome oxidase 1（co1）and NADPH dehydrogenase subunit 1 (nd1) in Echinococcus granulosus from Yunnan Province. Methods Echinococcus of animal origin was collected from cysts isolated from liver or lung lesions of cattle, sheep and free-range pigs at slaughterhouses in Shangri-La, Daguan, Eryuan, Lushui and Weixi counties of Yunnan Province. Echinococcus of human origin was obtained from the focal tissues surgically removed from hospitals in Jianchuan, Yunlong, Longyang and Yulong Counties, then the E. granulosus was selected after pathogenic identification, and used to extract genomic DNA with a DNA extraction kit, followed by PCR amplification and sequencing of co1 and nd1 genes. The sequence homology and single nucleotide polymorphisms were analyzed using BLAST aligment. Phylogenetic trees based on co1 and nd1 were constructed by the neighbor-joining method using MEGA-X software. Results A total of 62 samples of Echinococcus were collected, 36 of which were pathogenically confirmed as E. granulosus. A total of 32 gene co1 sequences were successfully sequenced, of which 15 were type G1 and 17 were type G5, with a length of 824 bp and 807 bp, respectively. After submitting to GenBank database, the login numbers obtained were OP413393-OP413402, OP413498-OP413506 and OP420520. A total of 34 nd1 genes were sequenced successfully, of which 11 were type G1 and 23 were type G5, with lengths of 882 bp and 888 bp, respectively. The entry numbers obtained by submitting the GenBank database were OP471626-OP471638. The results of sequence polymorphism analysis showed that 1.94% (16/824) mutation sites in the co1 type G1 sequence and 3.10% (25/807) mutation sites in the G5 type sequence were found. In the nd1, 0.45% (4/882) and 2.93% (26/888) mutation sites were found in the type G1 sequence and the G5 type sequence, respectively. The results of multiple genotypic sequence comparison showed that there were 6 homologous sequences of the co1 gene, 4 of which were type G1, the genetic distances of homologous sequences were 0.000 0-0.001 1, 0.000 0-0.000 6, and 0.000 0-0.001 7, respectively. Also included two G5 types. The genetic distances of homologous sequences were 0.000 0-0.002 5 and 0.000 0-0.001 2, respectively. There were 3 homologous sequences in nd1 gene, of which 1 was type G1, and the genetic distance was 0.000 0-0.001 1, 2 of which were type G5, genetic distance was 0.000 0 and 0.000 0-0.001 1. Phylogenetic tree analysis showed that the phylogenetic tree based on co1 and nd1 genes formed two branches of type G1 and type G5, type G1 was closely related to the gene sequences of central and western China (Qinghai, Ningxia, Gansu, Sichuan), Bhutan, Iran and Jordan in the Middle East (GenBank entry number AF297617.1, KJ628328.1, EU072106.1, MW138946.1, AB688602.1). The G5 Type was closely related to Vietnam, China's Guangxi, the United Kingdom, Poland, Zambia, France, Pakistan, Brazil, Kenya and Namibia (GenBank login numbers MW558412.1, MN058591.1, KU378107.1, MZ322608.1, KU743915.1, KU743919.1, MN886291.1, KX010903.1, KU743918.1 and KX138068.1). Conclusion The prevalent isolate of E. granulosus in Yunnan Province are of genotype G1 and G5, and both co1 and nd1 gene reveals single nucleotide polymorphisms.

Key words: Echinococcus granulosus, co1 gene, nd1 gene, Sequence analysis, Yunnan Province

中图分类号:

R383.33

李奔福, 王正青, 徐倩, 字金荣, 严信留, 彭佳, 李建雄, 蔡璇, 吴方伟, 杨亚明. 云南省细粒棘球绦虫线粒体co1和nd1基因序列分析[J]. 中国寄生虫学与寄生虫病杂志, 2023, 41(3): 306-311.

LI Benfu, WANG Zhengqing, XU Qian, ZI Jinrong, YAN Xinliu, PENG Jia, LI Jianxiong, CAI Xuan, WU Fangwei, YANG Yaming. Sequence analysis of mitochondrial co1 and nd1 genes in Echinococcus granulosus in Yunnan Province[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2023, 41(3): 306-311.

图/表 2

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