剑尾鱼微卫星DNA的筛选

以剑尾鱼(Xiphophorus helleri)为材料,经MboⅠ限制性内切酶消化基因组DNA后,选取500～2 000 bp的片段连接到经BamHⅠ酶切的pUC18载体上,转化大肠杆菌DH5α构建部分基因组文库.采用设计合成的(AC)7、(GT)7重复序列为引物,PCR筛选部分基因组文库,对其中9个重组阳性克隆进行测序,结果共获得24个微卫星序列,其中Perfect(完美型)13个,占54.2%;Imperfect(非完美型)3个,占12.5%;Compound(混合型)8个,占33.3%.表明(AC/GT)n在剑尾鱼的基因组DNA中含量非常丰富.同时,根据其中3个克隆微卫星的侧翼序列设计引物,PCR扩增剑尾鱼基因组DNA,结果均扩增到目的片段.而且,这3对引物扩增出来的微卫星片段在非选育的剑尾鱼中显示出多态性,而在近交系19代则表现为单态,为剑尾鱼的实验动物化遗传研究提供了理论依据.

Isolation of microsatellite DNA in swordtail fish Xiphophorus helleri

Swordtail fish (Xiphophorus helleri Heckel 1848) is a kind of small-scaled tropic freshwater fish which belongs to Cyprindontiformes, Cyprinodontidae, Xiphophorus. Pearl River Fisheries Research Institute began to inbreed it as laboratory animal in 1987 and by now several inbred strains have been obtained. Researches on its biological character, diet and nutrition, disease and disease monitoring etc. indicate that swordtail fish is suitable for genetics, toxicology, bacteriology, malnutrition symptom study and so on. In this paper, we describe the first isolation of microsatellites from swordtail fish and their preliminary application on genetic structure analysis. A partial swordtail genomic library was constructed. Nine recombinant positive clones were isolated by screening 120 clones of the genomic library through PCR with simple tandem repeats primers(AC)_7 and(GT)_7. 24 microsatellites were obtained after these 9 positive clones were sequenced. Among the 24 microsatellites, there were 13 perfect ones(54.2%),3 imperfect ones(12.5%) and 8 compound ones(33.3%).The results indicate that microsatellite sequences characterized by (AC/GT)_n are abundant in genomic DNA of swordtails. Three pairs of primers were designed according to three of these microsatellite-flanking sequences to amplify the genomic DNA of swordtails by PCR and the target fragments were obtained. The amplified fragments are polymorphic in wild swordtails but monomorphic in inbred strain. Furthermore, mutations in flanking sequence were also found from the alignment of the three alleles for Clone 5, which indicates that the mechanism of polymorphism of microsatellites is more complicated than what we assumed. The difference of repeating numbers of core repeat sequence may cause the polymorphism. These microsatellite sequences isolated in this study are expected to be useful as marker for genetic study of swordtails.