广西三地广州管圆线虫亲缘关系研究

Study on the phylogenetic relationship of three Angiostrongylus isolates from three counties of Guangxi

目的分析和判定广西钦州、靖西、藤县三地广州管圆线虫（Angiostrongylus cantonensis）分离株的基因差异和系统发生关系。方法从广西钦州、靖西、藤县采集野生蛞蝓。分离广州管圆线虫三期幼虫。提取广州管圆线虫三期幼虫的基因组DNA，特异性扩增内转录间隔区2（intemal transcribed spaeer2，ITS2）基因并测序，与GenBank中的广州管圆线虫序列进行比对．分析基因变异。应用Mega5．05软件构建系统发生树，分析系统发生关系。结果从三地共采集623条蛞蝓．其中阳性蛞蝓共283条．平均感染率为45．4％。其中靖西感染率最高，为52．4％；藤县和钦州感染率分别为49．1％和19．3％。特异性扩增的ITS2长度为700bp。GenBank检索获广州管圆线虫（A．cantonensis）、A．du9iardini以及脉居管圆线虫（A．vasomm）的相应序列为对照序列，枯病菌（Rhizoctonia solani）的相应序列为构建系统发生树的根（root）序列。广州管圆线虫钦州分离株、靖西分离株、藤县分离株与GenBank中的广州管圆线虫序列的同源性分别为99．5％、99．5％和99．7％；靖西分离株与藤县分离株、GenBank中广州管圆线虫序列的同源性分别为100％和99．5％；藤县分离株与及GenBank中广州管圆线虫序列的同源性为99．5％。三个分离株与A．dujardini、脉居管圆线虫（A．vasorum）的同源性低。以广州管圆线虫为标准株，靖西分离株有11处碱基变异，变异率为1．93％：钦州分离株有6处，变异率为1．05％；藤县分离株有15处，变异率为2．63％，TG短序列重复插入突变明显。在最大似然法（maximum likelihood method）和邻接法（neighbor-joining method）构建的系统发生树上，靖西、钦州及藤县的分离株与广州管圆线虫同属一个分枝，系统发生关系较近，A．duiardini、脉居管圆线虫居于另一分枝。结论广西三地的广州管圆线虫分离株存在较高的遗传多态性．与GenBank中的广州管圆线虫同源性高．亲缘关系近．初步判断三个分离株与GenBank中广州管圆线虫为同一虫种。

Objective To analyze the phylogenetic relationship of Angiostrongylus cantonensis isolated from Qinzhou, Jingxi and Tengxian counties of Guangxi. Methods Slugs were collected from Qinzhou, Jingxi and Tengxian counties of Guangxi and the third stage larvae ofA ngiostrongylus cantonensis were collected from positive samples. The genomic DNA was extracted from A ngiostrong＇ylus cantonensis. Internal transcribed spacer 2 （ITS2） was amplified and the amplicon was sequenced, then analysis was carried out and compared with those from Gen- Bank. Phylogenetic trees were constructed and phylogenetie relationship ofA ngiostrongylus cantonensis isolates was analyzed by Mega5.05 software. Results Totally 623 slugs from 3 counties were collected and 283 were infected with A ngiostrongylus cantonensis with average infection rate of 45.4%. Among three counties, the infection rate in Jingxi was the highest with 52.4%, followed by Tengxian and Qinzhou with 49.1% and 19.3%, respectively. Frag- ments of 700bp were specifically amplified. Corresponding sequence of A. cantonensis, A. dujardini and A. vasorum from GenBank were used as similarity compared sequence while Rhizoctonia solani as root sequence. QinzhouJingxi and Tengxian isolates was very like to A. cantonensis from GenBank with homology of 99.5%, 99.5% and 99.7%, respectively. The homology between Jingxi, Tengxian isolates and A. cantonensis was 100% and 99.5%, respectively, and 99.5% between Tengxian isolates and A. cantonensis. The homology between the three isolates and A. dujardini, A. vasorumwere was low. Compared with A. cantonensis from GenBank, 11 bp heteromorphosis was found in Jingxi isolate with the ratio of 1.93%, 6 bp in Qinzhou isolate with the ratio of 1.05% and 15 bp in Tengxian isolate with the ratio of 2.63%. Reduplicated TG acids were obvious. In the phylogenetic trees constructed by maximum likelihood method and neighbor-joining method, Jingxi, Qinzhou, Tengxian isolates and A. cantonensis from GenBank were separated into one branch with closer phenogenetic relationship, while A. dnjardini and A. vasorum were separated into another branch with far phenogenetic rela- tionship from the three isolates. Conclusion The three isolates from Guangxi were similar to A. cantonensis from GenBank with higher homology, closer phenogenetic relationship and genetic variation.