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

作　　者：韦开文 曾红霞 何牧[2] 胡俊杰[3] WEI Kai-wen;ZENG Hong-xia;HE Mu;HU Jun-jie(School of Biological Sciences,Yunnan University,Kunming 650000,China;Kunming Zoological Garden,Kunming 650000,China;School of Ecology and Environmental Sciences and Yunnan Key Laboratory or Plateau Mountain Ecology and Restoration of Degraded Environments,Yunnan University,Kunming 650000,China)

机构地区：[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

出　　处：《中国寄生虫学与寄生虫病杂志》2022年第5期594-602,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：云南省教育厅科学研究基金(2021Y044)。

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)、 t RNA-nad2-t RNA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t RNA-nad2-t RNA (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t RNAnad2-t RNA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t RNA-nad2-t RNA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�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 t RNA-nad2-t RNA 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 cox2sequences 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 t RNA-nad2-t RNA, 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 t RNA-nad2-t RNA 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 s

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 

分 类 号：S582.731[农业科学—作物学]