运用生物素与链霉亲和素的强亲和性原理,用生物素-磁珠吸附微卫星富集法,筛选企鹅珍珠贝(Pteria penguin)的微卫星分子标记序列,进而对南海区企鹅珍珠贝的遗传多样性进行分析。对136个菌落中的85个阳性克隆进行微卫星测序,获得64个序列,达到85.93%。所得到的55个重复6次以上的微卫星序列中,除生物素探针中使用的CA重复外,还得到TG、TC、GA的重复序列。重复序列中完美型36个,占65.5%;不完美型14个,占25.5%;混合型5个,占9.1%。利用Primer Premier5.0设计引物40对,合成其中的20对并进行PCR筛选,15对可扩增出特异性条带。研究表明,筛选出的企鹅珍珠贝微卫星分子标记可用于进一步的遗传多样性分析。
Using the principle of the strong affinity between biotin and streptomycin, an experiment was conducted to screen microsatellite primer sequences and then to analyze the genetic diversity of Pteria penguin in Nanhai District. Through assaying the microsatellite sequence of the 85 positive clones among the 136 bacterial colonies, there were 65 succeeded, the percentage was 85.93%.Fifty-five microsatellite sequences that the repetition were more 6 times,the repeat sequence included not only CA, but also TG, TC and GA. Among the microsatellites, 36 repeating motifs were perfect, occupying 65.5%;14 repeating motifs were imperfect, occupying 25.5%;5 repeating motifs were compound, occupying 9.1%. Forty pairs of primers were designed using the software Primer Premier 5.0,then 20 pairs of primers were successfully composed and screened by PCR,15 pairs of primers could be successfully amplified the special band. The conclusion was that the screened microsatellite molecular marker could be used to analyze the genetic diversity in Pteria penguin.