三峡库区上、下游湖北钉螺基因组DNAITS2遗传多态性研究

Study on ITS2 gene diversity of Oncomelania hupensis from upstream and downstream of the Three Gorges Reservoir

目的分析三峡库区上、下游湖北钉螺核糖体DNA第2转录间隔区（rDNA-ITS2）基因的遗传变异。方法采集三峡库区上游四川、云南及下游安徽、湖北4省8地（市）钉螺,提取其基因组DNA,PCR特异性扩增rDNA ITS2基因并测序,ClustalX（1.81）软件进行多序列比对,MEGA（3.1）软件Kimura2-parameter法计算遗传距离,非加权组平均法（UPGMA）和最小进化法（ME）构建系统发生树。结果上游与下游不同地域株钉螺间ITS2基因差异约为11%,下游地区的肋壳与光壳钉螺ITS2基因差异约为3.8%,上游螺群间的遗传距离为0.001～0.013,下游螺群间的遗传距离为0.004～0.017,上游与下游螺群间的遗传距离在0.033～0.041之间。2种方法构建的系统发生树其拓扑结构一致,分为2大支,上游的云南和四川地理株形成一支系,下游的湖北、安徽地理株形成另一支系,既有肋壳钉螺又有光壳钉螺。结论上、下游地理株湖北钉螺间ITS2基因遗传差异较显著,但下游肋壳和光壳钉螺ITS2基因遗传变异小,表明地域距离和生态环境对湖北钉螺的遗传变异影响较大,不能以钉螺纵肋表型的有无作为钉螺种株的判断标准。

Objective To analyze the genetic variation of rDNA ITS2 of Oncomelania hupensis from upstream and downstream of the Three Gorges Reservoir. Methods Snails were collected from 8 different areas upstream and downstream of the Reservoir. Genomic DNA was extracted from the snails; ITS2 gene fragments were amplified by PCR and then purified and sequenced. Sequences were edited using ClustalX （vl. 81） software. The genetic distances of the various regions were calculated by the Kimura method and phylogenetic trees were constructed with the methods of UPGMA and ME using MEGA （v3. 1） software. Results The ITS2 gene difference between upstream and downstream population was found in 11% of nucleotides, and divergence of 3.8% was observed between smooth- and ribbed-shelled downstream populations. The genetic distance between upstream and downstream populations ranged from 0. 033 to 0. 041, while that of among upstream or downstream populations was 0. 001-0. 013 and 0. 004-0. 017. Phylogenetic trees constructed by UPGMA and MP took on a similar topo-structure： isolates of Yunnan and Sichuan clustered into one group, while the Hubei and Anhui isolates appeared as another group with both smooth- and ribbed-shelled snails. Conclusion There is a large genetic diversity between upstream and downstream snail populations, but there was little difference among smooth- and ribbed-shelled downstream populations. The geographical distance and ecological environment have a sub- stantial impact on genetic mutation, and the smooth- or rough-rib phenotype did not help to determine the Oncomelania hupensis snail species.