不同地域株东方次睾吸虫线粒体nad1基因序列分析及种系发育研究

doi:10.12140/j.issn.1000-7423.2024.05.017

摘要/Abstract

摘要：

为研究不同地区东方次睾吸虫线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚单位Ⅰ基因（nad1）的部分序列（pnad1）遗传差异及种系发育情况，取福建、江西麦穗鱼中分离出的东方次睾吸虫囊蚴分别感染鹌鹑30 d后，收集成虫，各取3条成虫分别抽提线粒体基因组DNA，PCR扩增pnad1基因并测序。采用BioEdit软件将获得的序列与GenBank中来自黑龙江的东方次睾吸虫（GenBank：KY232061、KY232066、KY232071）和同属相关吸虫线粒体pnad1序列进行比对，分析其序列的种内差异和种间差异。采用MEGA 11.0软件计算不同地区序列遗传距离，用邻接法构建基于线粒体pnad1序列的后睾科吸虫系统进化树。结果显示，来自福建、江西的东方次睾吸虫样品均扩增出500 bp条带，测序结果为523 bp线粒体pnad1基因。序列比对分析结果显示，pnad1序列种内遗传变异率为0~0.8%，种间遗传变异率为 12.6%~13.9%。系统进化树结果显示，来自福建、江西与黑龙江株东方次睾吸虫聚于同一分支上，再与胆囊次睾吸虫（GenBank：KT740986）和黄体次睾吸虫（GenBank：KT40991）聚为一个分支。提示线粒体pnad1序列种内相对保守，种间差异较大，可作为种间遗传变异研究的标记。

关键词: 东方次睾吸虫, 线粒体DNA, 线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚单位Ⅰ基因, 序列分析, 种系发育

Abstract:

To examine the genetic variation and phylogenetic relationship of nicotinamide adenine dinucleotide dehydrogenase subunits 1 (pnad1) of Metorchis orientalis from different geographic regions. The quail were infected with M. orientalis metacercaria isolated from Pseudorasbora parvain in Fujian Province and Jiangxi Province, respectively. Adult worms were collected after 30 days, and the mitochondrial genomic DNA was extracted from 3 adults in each region. The pnad1 was amplified from M. orientalis by PCR and sequenced. The obtained sequences were compared with the mitochondrial pnad1 sequences from Heilongjiang (GenBank: KY232061, KY232066, KY232071) and published sequences of the same genus using BioEdit software and the species-specific and inter-species differences were analyzed. The phylogenetic distance of different regions was calculated by MEGA 11.0 software, and the phylogenetic tree of opisthorchidae based on mitochondrial pnad1 sequence was constructed by the adjacency method. The results showed that 500-bp bands were amplified in the mitochondria of the M. orientalis from Fujian and Jiangxi, and the mitochondrial pnad1 gene was 523 bp. The results of sequence comparison showed that the intraspecific genetic variation rate of pnad1 sequence was 0-0.8%, and the interspecific genetic variation rate was 12.6%-13.9%. The constracted phylogenetic tree based on the pnad1 gene sequences showed that M. orientalis isolates from Fujian, Jiangxi and Heilongjiang were clustered together, M. xanthosomus (GenBank: KT740991) and M. bilis (GenBank: NC079698) were clustered in a branch. It was concluded that the mitochondrial pnad1 sequence is relatively conserved within species and varies greatly between species, which can be used as a marker for studying interspecific genetic variation.

Key words: Meotrchis orientalis, Mitochondrial DNA, nad1 gene, Sequence analyses, Phylogeny

中图分类号:

R383.29

陈云虹, 谢贤良, 李燕榕, 陈宝建, 林耀莹, 余文武, 余海峰. 不同地域株东方次睾吸虫线粒体nad1基因序列分析及种系发育研究[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(5): 668-671.

CHEN Yunhong, XIE Xianliang, LI Yanrong, CHEN Baojian, LIN Yaoying, YU Wenwu, YU Haifeng. Phylogenetic and sequence analysis of mitochondrial nad1 of Metorchis orientalis strains from different geographic regions[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2024, 42(5): 668-671.

图/表 3

参考文献 17

[1]	Wang Y, Li X, Sun Q, et al. First case report of Metorchis orientalis from black swan[J]. Int J Parasitol Parasites Wildl, 2020, 13: 7-12.
[2]	Lin JX, Li YS, Cheng YZ, et al. Report on human body experimental infection with Metorchis orientalis[J]. Strail J Prev Med, 2001, 7(2): 9-11, 65. (in Chinese)
	(林金祥, 李友松, 程由注, 等. 东方次睾吸虫 (Metorchis orientalis) 人体实验感染报告[J]. 海峡预防医学杂志, 2001, 7(2): 9-11, 65.)
[3]	Li Y, Zhang Y, Gao ZY, et al. Its sequence analysis of Metorchis orientalis from Grus japonensis[J]. Chin J Vet Med, 2021, 57(12): 113-116. (in Chinese)
	(李烨, 张莹, 高忠燕, 等. 丹顶鹤源东方次睾吸虫ITS序列分析[J]. 中国兽医杂志, 2021, 57(12): 113-116.)
[4]	Lin JX, Cheng YZ, Li YS, et al. The discovery of natural infection of human with Metorchis orientalis and the investigation of its focus[J]. ChinJ Zoonoses, 2001, 17(4): 1921. (in Chinese)
	(林金祥, 程由注, 李友松, 等. 人体自然感染东方次睾吸虫的发现及其疫源地的调查研究[J]. 中国人兽共患病杂志, 2001, 17(4): 1921.)
[5]	Gao JF, Mao RF, Sun YY, et al. Establishment of an indirect ELISA method for diagnosis of Metorchis orientalis infection in duck[J]. Chin Vet Sci, 2021, 51(8): 947-954. (in Chinese)
	(高俊峰, 毛瑞锋, 孙云异, 等. 鸭东方次睾吸虫病间接ELISA诊断方法的建立[J]. 中国兽医科学, 2021, 51(8): 947-954.)
[6]	Chen BJ, Xie HG, Xie XL, et al. Investigation of Clonorchis sinensis infection in the national surveillance site of Pucheng County, Fujian Province during 2016-2020[J]. Chin J Parasitol Parasit Dis, 2021, 39(5): 716-719. (in Chinese)
	(陈宝建, 谢汉国, 谢贤良, 等. 2016—2020年福建省浦城县华支睾吸虫病国家监测点人群感染情况分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(0): 716-719.)
[7]	Zhang Y, Wang LK, Hou MR, et al. Seasonal analysis of fish-borne zoonoses infection with cysticercus[J]. Heilongjiang Anim Sci Vet Med, 2019(4): 166-168. (in Chinese)
	(张莹, 王丽坤, 侯美如, 等. 鱼源性人兽共患吸虫囊蚴感染的季节性分析[J]. 黑龙江畜牧兽医, 2019(4): 166-168.)
[8]	Chu JN, Xu HT, He ZJ, et al. A comparative study on mitochondrial genetic diversity between captive and wild amur tigers in China[J]. Chin J Wildl, 2024, 45(2): 231-241. (in Chinese)
	(褚佳宁, 徐海涛, 何志健, 等. 我国圈养和野生东北虎种群线粒体基因组遗传多样性的比较研究[J]. 野生动物学报, 2024, 45(2): 231-241.)
[9]	Liang X, Liu Y. Analysis of sequence variation in mitochondrial cox1 gene of Toxocara canis isolates in China[J]. China Anim Health Insp, 2015, 32(3): 66-69. (in Chinese)
	(梁轩, 刘毅. 犬弓首蛔虫线粒体cox1基因的克隆及序列分析[J]. 中国动物检疫, 2015, 32(3): 66-69.)
[10]	Zhu WJ, Han XM, Cai QG, et al. Study on the molecular phylogeny of Echinococcus multilocularis in Qinghai Province[J]. Chin J Schisto Control, 2019, 31(6): 628-634. (in Chinese)
	(朱文君, 韩秀敏, 蔡其刚, 等. 青海省多房棘球绦虫分子种系发生研究[J]. 中国血吸虫病防治杂志, 2019, 31(6): 628-634.)
[11]	Xie XL, Chen YH, Chen BJ, et al. Morphological characteristics and molecular identification of Metorchis orientalis infection in domestic cats[J]. Chin J Zoonoses, 2023, 39(3): 249-252. (in Chinese)
	(谢贤良, 陈云虹, 陈宝建, 等. 家猫感染东方次睾吸虫形态学特征及分子鉴定[J]. 中国人兽共患病学报, 2023, 39(3): 249-252.)
[12]	Chen BJ, Xie HG, Jiang DW, et al. Investigation on Metorchis orientalis and vector host in Pucheng county, Fujian province, 2016-2017[J]. Chin J Vector Biol Contr, 2018, 29(6): 92-94. (in Chinese)
	(陈宝建, 谢汉国, 江典伟, 等. 福建省浦城县2016—2017年东方次睾吸虫感染及其媒介宿主调查[J]. 中国媒介生物学及控制杂志, 2018, 29(6): 92-94.)
[13]	Gao JF, Zhang Y, Hou MR, et al. Phylogenetic analysis and sequence analysis of mitochondrial cox1 and nad1 for Metorchis orientalis isolates from different regions in Heilongjiang Province[J]. Chin J Prev Vet Med, 2017, 39(11): 943-945. (in Chinese)
	(高俊峰, 张莹, 侯美如, 等. 黑龙江省不同地区东方次睾吸虫线粒体cox1和nad1基因序列分析及种系发育研究[J]. 中国预防兽医学报, 2017, 39(11): 943-945.)
[14]	Tang CT, Tang ZZ. Trematology in China[M]. Fuzhou: Fujian Science & Technology Publishing House, 2005: 569. (in Chinese)
	(唐崇惕, 唐仲璋. 中国吸虫学[M]. 福州: 福建科学技术出版社, 2005: 569.)
[15]	Liu J, Li YL. Study on the genetic variance of different strains of Clonorchis sinensis[J]. J Pathog Biol, 2011, 6(6): 443-445, 482. (in Chinese)
	(刘娟, 李雍龙. 不同地域株华支睾吸虫基因差异的研究[J]. 中国病原生物学杂志, 2011, 6(6): 443-445, 482.)
[16]	Tan L, Wang AB, Kong XX, etal. Polymorphism of mitochondrial pnad1 gene of Spirometra erinaceieuropaei sparganum isolated from different species of snake in Hunan Province[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 448-452. (in Chinese)
	(谭磊, 王爱兵, 孔小鲜, 等. 湖南省不同种类蛇源裂头蚴线粒体pnad1基因序列多态性研究[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 448-452.) doi: 10.12140/j.issn.1000-7423.2019.04.014
[17]	Wu HL, Zhuang YH, HuangW. Cloning and phylogenetic analysis of mitochondrial cox1 and nad1 genes of Clonorchis sinensis isolates from Hunan Province[J]. Chin Vet Sci, 2012, 42(1): 8-12. (in Chinese)
	(伍慧兰, 庄远红, 黄文. 湖南省华支睾吸虫线粒体cox1和nad1基因的克隆及进化分析[J]. 中国兽医科学, 2012, 42(1): 8-12.)