喜马拉雅旱獭（Marmota Himalayana）微卫星位点的筛选与应用
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摘要
以青藏铁路沿线喜马拉雅旱獭为材料，从肌肉组织中提取基因组DNA。由喜马拉雅旱獭近缘种文献及Genebank中筛选出多态性信息含量高的近缘种微卫星序列，以其相应的18对引物对基因组DNA进行扩增。这18个近缘种微卫星序列为AF259372、AF259373、AF259374、AF259375、AF259376、AY197780，AY197781、AY197782，AY197783、AY197784、AY197785、AY702707、AY702708、AY702709、AY702710、AY702711、AY702712及SS-Bible。将扩增出来的序列回收，经pBS-T载体连接，转化入大肠杆菌DH5α菌株，以(CA)。寡聚核苷酸引物和PUC19质粒多克隆位点两侧的M13primersM3和RV引物分别配对，PCR法筛选含微卫星序列的阳性克隆并进行测序。通过对几百个已鉴定为重组阳性克隆的筛选，获得21个含有微卫星的重组阳性克隆。将首次得到的21个喜马拉雅旱獭微卫星序列在GenBank注册，序列号为DQ888321～DQ888327，EF125660～EF125666，EF520117～EF125122。在21个微卫星序列中完美型14个，占67％；非完美型3个，占14％；复合型4个，占19％。利用BLAST序列分析软件对GenBank数据库进行检索，没有发现同源性一致的相应微卫星序列，表明本文得到的喜马拉雅旱獭微卫星序列都是首次发现的。此外，喜马拉雅旱獭微卫星的重复次数主要集中在20次以下，而20次以上的少见。
从这21个微卫星序列中选取重复区域长、侧翼序列好的座位使用Oligo6.0和Primer 5.0软件设计微卫星序列引物，共设计出5对不含茎环和二级结构的引物，长度在20bp左右。将5对引物进一步经过3％琼脂糖凝胶电泳检验筛选，其中4对引物具有多态性，4对引物为JA5F／JA5R，JA9F／JA9R，JD3F／JD3R，JD9F／JD9R。用这4对引物对沱沱河、海西洲、安多和乌兰四地的喜马拉雅旱獭种群进行遗传多样性分析，海西洲喜马拉雅旱獭种群遗传多样性最高，其次为乌兰、沱沱河和安多地理种群。聚类分析发现，沱沱河与安多两地种群的遗传距离相对较小，但是总体而言四个种群之间遗传差异较小。本实验首次建立了喜马拉雅旱獭的微卫星分子标记，为进一步开展喜马拉雅旱獭及其近缘种群的分子生物学研究奠定了基础。
Relative species' microsatellite primers were used to find out microsatellite loci in Himalayan Marmot. The 18 microsatellite sequences of relative species areAF259372、AF259373、AF259374、AF259375、AF259376、AY197780、AY197781、AY197782、AY197783、AY197784、AY197785、AY702707、AY702708、AY702709、AY702710、AY702711、AY702712 and SS-Bible. After gene application, connection and translation, hundreds of clones were screened with PCR technique for recombinant positive clones containing microsatellite sequences. The primers used in PCR were M13 Primers M3/M13 Primers RV which located at both sides of multiple clone site of pBS-T and microsatellite primers (CA)8 designed according to core repeats motifs of microsatellite. 21 microsatellite loci were found. GenBank accession numbers of these microsatellite sequences are DQ888321-DQ888327, EF125660-EF125666, EF520117-EF520122. Among these microsatellite loci there are 14 perfect ones in 67%; 3 imperfect ones in 14%; 4 compound ones in 19%.Repeat number of most microsatellites varies from 3 to 20, few microsatellites exceed 20.
Four microsatellite sequences were selected for designing oligonucleotide primers,using Oligo 6.0 and Primer 5.0 software.After extensive optimization, 4 pairs of primers gave satisfactory results and were polymorphism. Genetic polymorphism of Himalayan Marmot in Tuotuo River , Haixizhou, Wulan and Anduo were studied with these primers .The result indicate that genetic variation of Haixizhou population were highest, then were Wulan population, Tuotuo River and Anduo population. Cluster analysis showed that there were differences between four population, but differences between four populations were small. This experiment has made the foundation for further reserch of Himalayan Marmot in molecular ecology.
从这21个微卫星序列中选取重复区域长、侧翼序列好的座位使用Oligo6.0和Primer 5.0软件设计微卫星序列引物，共设计出5对不含茎环和二级结构的引物，长度在20bp左右。将5对引物进一步经过3％琼脂糖凝胶电泳检验筛选，其中4对引物具有多态性，4对引物为JA5F／JA5R，JA9F／JA9R，JD3F／JD3R，JD9F／JD9R。用这4对引物对沱沱河、海西洲、安多和乌兰四地的喜马拉雅旱獭种群进行遗传多样性分析，海西洲喜马拉雅旱獭种群遗传多样性最高，其次为乌兰、沱沱河和安多地理种群。聚类分析发现，沱沱河与安多两地种群的遗传距离相对较小，但是总体而言四个种群之间遗传差异较小。本实验首次建立了喜马拉雅旱獭的微卫星分子标记，为进一步开展喜马拉雅旱獭及其近缘种群的分子生物学研究奠定了基础。
Relative species' microsatellite primers were used to find out microsatellite loci in Himalayan Marmot. The 18 microsatellite sequences of relative species areAF259372、AF259373、AF259374、AF259375、AF259376、AY197780、AY197781、AY197782、AY197783、AY197784、AY197785、AY702707、AY702708、AY702709、AY702710、AY702711、AY702712 and SS-Bible. After gene application, connection and translation, hundreds of clones were screened with PCR technique for recombinant positive clones containing microsatellite sequences. The primers used in PCR were M13 Primers M3/M13 Primers RV which located at both sides of multiple clone site of pBS-T and microsatellite primers (CA)8 designed according to core repeats motifs of microsatellite. 21 microsatellite loci were found. GenBank accession numbers of these microsatellite sequences are DQ888321-DQ888327, EF125660-EF125666, EF520117-EF520122. Among these microsatellite loci there are 14 perfect ones in 67%; 3 imperfect ones in 14%; 4 compound ones in 19%.Repeat number of most microsatellites varies from 3 to 20, few microsatellites exceed 20.
Four microsatellite sequences were selected for designing oligonucleotide primers,using Oligo 6.0 and Primer 5.0 software.After extensive optimization, 4 pairs of primers gave satisfactory results and were polymorphism. Genetic polymorphism of Himalayan Marmot in Tuotuo River , Haixizhou, Wulan and Anduo were studied with these primers .The result indicate that genetic variation of Haixizhou population were highest, then were Wulan population, Tuotuo River and Anduo population. Cluster analysis showed that there were differences between four population, but differences between four populations were small. This experiment has made the foundation for further reserch of Himalayan Marmot in molecular ecology.
引文
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