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目的研究广东省南澳岛广州管圆线虫(Angiostronglus cantonensis)遗传多样性,分析其与中国不同地区分离株间的系统发生关系。方法 2015-2016年采用分层随机抽样法在南澳岛抽取3个行政村(宫前村、金山村和六都村),选取55个采样点。用鼠笼捕鼠,解剖鼠类收集广州管圆线虫成虫。现场采集福寿螺(Pomacea canaliculata)和玛瑙螺(Achatina fulica),从螺类收集Ⅲ期幼虫,实验室感染SD大鼠后收集成虫。提取成虫基因组DNA,PCR扩增核糖体DNA内转录间隔区2(internal transcribed spacer 2,ITS2)和线粒体DNA细胞色素氧化酶Ⅰ亚基(cytochrome c oxidase subunitⅠ,COⅠ)并测序,运用Clustal X1.83比对序列,Dna SP5.10分析其序列组成及遗传多样性。从Gen Bank上获取浙江温州(登录号HQ540551.1)、广东深圳(登录号HQ540546.1)、台湾花莲(登录号KF591125.1)等中国不同地区分离株的ITS2序列,运用Mega6.06基于Kumara双参数模型计算遗传距离,用邻接法(NJ)和最大简约法(MP)构建系统进化树,分析各地区分离株的亲缘关系。结果从南澳岛共捕获鼠类110只,广州管圆线虫感染阳性率为29.1%(32/110);分别检测福寿螺和玛瑙螺1 190只和24只,广州管圆线虫感染阳性率分别为6.1%(72/1 190)和83.3%(20/24)。PCR结果显示,广州管圆线虫ITS2和COⅠ片段长度分别为693和1 174 bp,位点变异率分别为4.7%和1.0%。南澳岛广州管圆线虫ITS2基因单倍型多样性指数为0.927,核苷酸多样性指数为0.007,平均核苷酸差异指数为4.549。COⅠ基因单倍型多样性指数为0.440,核苷酸多样性指数为0.004,平均核苷酸差异指数为3.743。基于ITS2的分析结果显示,南澳岛与国内其他地区分离株遗传距离为0.181~0.775,NJ法MP法构建的系统进化树基本一致,南澳岛大部分虫体序列与广东深圳、福建福清、云南普洱、广西南宁的分离株同属一大分支,小部分则单独聚成一支。结论南澳岛广州管圆线虫种群内存在一定程度的遗传分化和遗传多样性,其虫体可能存在多种类型和来源,但尚需进一步研究。
Objective To study the genetic diversity of Angiostronglus cantonensis in Nan’ao Island, Guangdong Province and analyze its phylogenetic relationship with isolates in different regions of China. Methods From 2015 to 2016, stratified random sampling method was used to extract three administrative villages (Gongqian Village, Jinshan Village and Liudu Village) on Nan’ao Island and select 55 sampling points. Trapped with a squirrel cage, rats were collected to collect the adults of C. elegans. Pomacea canaliculata and Achatina fulica were collected on the spot. Stage III larvae were collected from the snails and adult worms were harvested after laboratory infection in SD rats. The adult genomic DNA was extracted and sequenced. The internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit Ⅰ (COⅠ) were amplified by PCR and sequenced. The Clustal X1.83 The alignment sequence, Dna SP5.10 analysis of its sequence composition and genetic diversity. The ITS2 sequences of isolates from different regions of China such as Wenzhou, Zhejiang (accession number HQ540551.1), Guangdong Shenzhen (accession number HQ540546.1) and Hualien, Taiwan (accession number KF591125.1) were obtained from Gen Bank, and were sequenced based on Mega6.06 based on Kumara Two-parameter model was used to calculate the genetic distance. The phylogenetic tree was constructed by using NJ method and MP method to analyze the genetic relationship among isolates from different regions. Results A total of 110 rodents were captured from Nan’ao Island. The positive rate of C. elegans infection in Guangzhou was 29.1% (32/110). The positive rates of infection were 1.19 and 24, respectively. The positive rates of C. elegans were 6.1 % (72/1 190) and 83.3% (20/24). The PCR results showed that the length of ITS2 and COI fragments of A. angiostrongis were 693 and 1 174 bp respectively, and the site variation rates were 4.7% and 1.0%, respectively. The haplotype diversity index of ITS2 gene of O. anglicis in Nan’ao Island was 0.927, the nucleotide diversity index was 0.007, and the average nucleotide difference index was 4.549. The haplotype diversity index of COⅠ gene was 0.440, the nucleotide diversity index was 0.004, and the average nucleotide difference index was 3.743. Based on the results of ITS2 analysis, the genetic distance between Nanao Island and the rest of China was 0.181 ~ 0.775. The phylogenetic tree constructed by NJ method was basically the same. Most of the sequences from Nanao Island were similar to those from Shenzhen, Guangdong, Fuqing, Yunnan, , Guangxi Nanning isolates belong to a major branch, a small part of a separate into a single. Conclusion There is a certain degree of genetic differentiation and genetic diversity in C. elegans population in Nan’ao Island. There may be many types and sources of its pathogens, but further study is needed.