大仓鼠高变异微卫星座位的筛选及应用

【作者】 郭俣；

【导师】 徐来祥；

【作者基本信息】 曲阜师范大学 ， 动物学， 2005， 硕士

【摘要】 通过构建大仓鼠基因组部分文库和PCR扩增法,对大仓鼠基因组微卫星座位进行了筛选。PCR扩增中使用（CA）₈、（CT）₈、（AT）₈寡核苷酸引物和pUC19质粒多克隆位点两侧的M13 Primers M3和RV引物分别配对,对经双酶切验证确有插入片段的4000个阳性克隆进行PCR筛选,初步筛选的结果表明使用（CA）₈引物筛选效率最高,而（CT）₈、（AT）₈筛选出的阳性克隆很少。最终确定完全使用（CA）₈进行PCR大量筛选,共筛选得到74个阳性克隆。使用DNA序列自动分析仪对这74个可能含有微卫星的重组阳性克隆进行测序,除其中12个克隆的测序信号较弱,无法得到其插入片段的序列外,还有15个克隆不含有明显的微卫星序列。在其他47个克隆中共得到25个微卫星序列,并经过进一步反复测序确定序列长度。其中（CA）_n类型的微卫星有22个占88%,（CT）_n类型的微卫星只有3个占12%,没有单独（AT）_n类型的微卫星序列出现,（AT）_n大部分与（CA）_n连锁在一起。利用BLAST序列分析软件对GenBank数据库进行检索,没找到同源性一致的相应微卫星序列,说明本文得到的25个大仓鼠微卫星序列都是首次发现的。在GenBank注册序列号为AY780293～AY780309,AY864067～AY864074。这些微卫星可分成三类:完美型占40%,非完美型占4%,复合型占56%。另外可见重复次数在8～20的微卫星序列最多,占48%,而重复次数在25次以上的很少。 在这25个微卫星序列中除了9个微卫星因序列太短不能设计引物外,其余16个使用Oligo 6.0和Primer 5.0软件对微卫星序列设计引物,并事先设计好相应的参数,共设计出10对不含茎环和二级结构的引物。用这10对引物对顺义、固安、饶阳三地的大仓鼠种群进行初步PCR扩增,其中有6对能很好的应用于种群遗传多态性分析。遗传距离和聚类分析表明,饶阳大仓鼠种群的遗传多态性已经与顺义和固安两地大仓鼠种群出现了较大差异,引物PAY7802、PAY7807和P5022801可以用作鉴别这三个地区大仓鼠种群的特异性微卫星标记,扩增得到的等位基因可以作为种群特征用于群体间遗传结构的鉴别。更多还原

【Abstract】 In this study a partial common great long-tailed hamster（Criecetulus triton） genomic library including more than 4000 clones was constructed. About 74 recombinant positive clones containing microsatellite sequences of C. triton were obtained through rapid screening small size fractionated genomic libraries with PCR technique. The primer sets used in PCR were M13 Primers M3/RV located at both sides of multiple cloning site of pUC19 and （CA）₈, （CT）₈, （AT）₈. Sequencing of these clones was performed with automated sequencer, and 25 microsatellites were isolated. The sequence numbers submitted to the GenBank are AY780293-AY780309, AY864067-AY864074. Among them twenty-two microsatellites have （CA）_n motif, aacounted for 88%, only three microsatellites have （CT）_n motif, accounted for 12%. In the 25 microsatellites, perfect ones are 40%, imperfect 4%, compound perfect 56%. The motif number from 8 to 20 in most microsatelltes are 48%, few motif numbers exceed 25.Ten good microsatellite primer sets were designed based on unique sequences flanking each motif with the software Oligo 6.0 and Primer 5.0. PCR amplification of template DNA of C. triton populations in Shunyi, Gu’an, and Raoyang were carried out with these primers. The result indicated that six microsatellte markers were polymorphism. Cluster analysis showed that there were certain differences bewteen the poulation of Raorang and the populations of shunyi, gu’an. The same microsatellite markers showed different allele number in different population. The dominated alleles and the private alleles found in each population can be different. Primers of PAY7802, PAY7807 and P5022801 can be used to evaluate the genetic structure among different populations as a population character.更多还原