广西南宁地区片形吸虫的分子鉴定及遗传多态性分析

Molecular identification and genetic polymorphism analysis of Fasciola flukes in Nanning,Guangxi

目的基于核糖体内转录间隔区(ITS+,包括ITS1、5.8S和ITS2及其侧翼序列)、烟酰胺腺嘌呤二核苷酸脱氢酶亚基Ⅰ(NADⅠ)、线粒体细胞色素c氧化酶亚基Ⅰ(COXⅠ)序列和磷酸烯醇式丙酮酸羧激酶基因(pepck)多重PCR初步鉴定广西南宁地区采集的片形吸虫的种类。方法于南宁市某屠宰场采集11头感染片形吸虫的牛肝脏,分离片形吸虫成虫,并孵育获得虫卵。提取片形吸虫成虫的基因组DNA,PCR扩增ITS+、NADⅠ和COXⅠ序列并测序,所得序列经BLAST在线工具进行比对,并结合pepck基因多重PCR鉴定虫种;提取部分片形吸虫的虫卵基因组DNA,PCR扩增ITS+序列并测序;采用BioEdit软件进行多序列比对,分析片形吸虫亲代及子一代的遗传变异情况。结果共获得151条片形吸虫成虫,ITS+序列扩增产物长964bp,仅NN20-2为Fh型,与GenBank中瑞士肝片形吸虫(登录号:MK321602)的同源性为100%;NN17-11、NN20-9和NN20-14携带巨片形吸虫和肝片形吸虫的双重序列特征,属于Fg/Fh杂合型,序列图谱上特定变异位点出现双峰,个体间双峰比值存在明显差异,NN17-11为Fg∶Fh=1∶1,NN20-9和NN20-14为Fg∶Fh=1∶5;其余147个样本是Fg型,可细分为19个亚型,其中NN4-1等100个虫体与GenBank中越南巨片形吸虫(登录号:MN970009)的同源性为100%,其余虫体中共出现14个变异位点,多为双峰。线粒体基因NADⅠ和COXⅠ序列扩增产物长度分别为535bp和438bp,分别有34、22个变异位点,细分为28、21种单倍型,所有虫体的NADⅠ和COXⅠ均属于巨片形吸虫类群。pepck PCR产物电泳显示NN17-11、NN20-2、NN20-9和NN20-14等4个虫体出现巨片形吸虫和肝片形吸虫的双重条带,其它样本只有巨片形吸虫的单条带。虫卵的ITS+序列结果显示,ITS-Fh型片形吸虫NN20-2成虫有1个虫卵(1/30)转变为Fg/Fh型;巨片形吸虫NN20-3成虫有5个虫卵(5/30)呈现Fg/Fh型,其余虫卵与母体基本一致。结论广西南宁地区片形吸虫种群以纯种巨片形吸虫为主,同时存在少量杂合型片形吸虫。

Objective To primarily identify the species of Fasciola flukes from Nanning,Guangxi Zhuang Autonomous Region based on sequences of ribosomal internal transcribed spacers(ITS,including ITS1,5.8S,and ITS2and their flanking sequences),NADH dehydrogenase subunitⅠ(NADⅠ),mitochondrial cytochrome c oxidase subunitⅠ(COXⅠ),and PCR results for phosphoenolpyruvate carboxykinase(pepck).Methods A total 11bovine livers were collected from a slaughterhouse in Nanning,adult flukes were isolated and incubated in PBS to obtain eggs.Genomic DNA of adult flukes was extracted,the ITS+,NADⅠ,and COXⅠgenes were amplified and sequenced,sequences were aligned with data from GenBank,and PCR results for the pepck gene were combined to determine the species of Fasciola flukes.Genomic DNA was extracted from the eggs of partial adult flukes,amplified,and sequenced along with the ITS+sequence.The software BioEdit was used to compare the sequencing results and further analyze genetic variation in 151adult flukes and eggs.Results In total,151Fasciola flukes were collected.The ITS+sequence was 964bp in length,NN20-2was the only member of F.hepatica,and it displayed 100%similarity to F.hepatica from Switzerland registered in GenBank(accession number:MK321602).NN17-11,NN20-9,and NN20-14displayed sequence characteristics from both F.gigantica and F.hepatica and were classified as a F.gigantica/F.hepatica heterozygote.There were 2peaks at specific points on the sequence map for ITS,and the proportion of points with 2peaks varied significantly among individuals.The proportion of F.gigantica/F.hepatica was 1:1in NN17-11,1:5in NN20-9,and 1:5 in NN20-14.One hundred and forty-seven flukes were F.gigantica and were subdivided into 19sub-genotypes.One hundred flukes(including NN4-1)had 100%similarity to F.gigantica from Vietnam registered in GenBank(accession number:MN970009);the remaining 47samples all had 14mutations that mostly produced 2peaks.After amplification with PCR,the NADⅠsequence was 535bp in length and the COXⅠsequence was 438bp in length.The NADⅠsequence had 34mutations while the COXI sequence had 21.The NADⅠsequence had 28haplotypes while the COXI sequence had 21.All samples belonged to the F.gigantica haplogroup.Electrophoresis of pepck indicated that 4samples had 2bands for F.gigantica and F.hepatica while the other samples had only 1band for F.gigantica.The ITS+sequence from eggs indicated that 1egg(1/30)of the ITS/F.hepaticafluke NN20-2was transformed into an F.gigantica/F.hepaticaform.Five eggs(5/25)of the F.giganticafluke NN20-3had an ITS+sequence that was F.gigantica/F.hepatica while the remaining samples were identical to the mother in type.Conclusion The population of Fasciola flukes in Nanning is dominated by pure F.gigantica,though there are a few heterozygous flukes.