| 摘要: |
| 采用磁珠杂交选择和PCR筛选法，从长牡蛎DNA选择片段文库中，快速分离含有微卫星序列的阳性克隆。结果表明，在筛选的200个白色茵落中，56个克隆含有重复次数5以上的微卫星序列，其中41个(20.5%)有随机侧翼区，可以进行引物设计，12个缺乏足够的侧翼序列，3个为中断的微卫星序列。此外，还获得两个小卫星克隆。在获得的微卫星序列中，完全的占54.7% ，不完全的占20.8% ，复合的占24.5%。除探针中使用的CA重复单元外，还观察到CT、ACT、CGCA、CACT、GACT、GCAC、CC TTA和CCTCA的重复序列。微卫星重复次数主要集中在5—30次之间，占71.7% ，最高为60次。本研究中构建的长牡蛎(CA)n富集微卫星文库将为以后开发未知微卫星标记提供帮助。 |
| 关键词: 长牡蛎,微卫星,快速分离,特性分析
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| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金资助项目，30170735号；教育部留学回国人员科研启动基金资助 |
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| MICROSATELLITE CLONES IN PACIFIC OYSTER CRASSOSTREA GIGAS：RAPID ISOLATION AND CHARACTERISTIC ANALYSIS |
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LI Qi1, KIJIMA Akihiro2
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1.The Key Laboratory of Mariculture，Ministry of Education，Ocean University of China;2.Faculty of Agriculture，Tohoku University
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| Abstract: |
| Traditional approach to obtain microsatellites is to create a size-selected genomic library in a plasmid or phage vector and then screen clones by oligoprobes containing different repeat motifs. For microsatellite repeats which are less abundant in the genome, it is difficult to isolate using the method. As numerous microsatellite markers are needed for the studies such as linkage analysis and genomic mapping in the Pacific oyster Crassostrea gigas, it is necessary to establish an efficient method to clone oyster microsatellite sequences. In the present study, we isolated CA repeat DNA clones in the Pacific oyster using magnetic bead hybridization selection, and analyzed the characteristics of the clones. DNA was extracted from a live Pacific oyster, and digested with Hae III and Dra I. The restricted DNA was electrophoresed on a 2.5% agarose gel, and fragments of 300—1000 bp were excised, purified, and ligated with the Eco R I-Not I-Bam H I adapter. After magnetic isolation of target sequences and adapter PCR, the PCR-amplified DNA fragments were inserted into the pBluescriptⅡSK(+)vector, and the recombinant plasmid vector was transformed into XL1-Blue MRF′ supercompetent cells. The positive clones containing microsatellite sequences were screened by the PCR technique. In the 200 white colonies screened, 56 clones contained microsatellite repeat sequences with more than 5 repetitions. Of the 56 clones, 4 1 clones(20.5%)with unique regions flanking the microsatellite array appeared promising for primer design , 1 2 clones did not have sufficient quality sequences on both sides of the repeat, and 3 clones contained interrupted repeat. In addition, 2 minisatellite clones were obtained. Compared with the traditional colony hybridization method, the enrichment procedure is very efficient. According to Weber(1990), the microsatellite sequences could be categorized structurally as follows：perfect(54.7% ), imperfect(20.8%), and compound(4.9%). Besides the motif of CA contained in the oligoprobe, we also found CT, ACT, CGCA, CACT, GACT, GCAC, CCTTA, and CCTCA repeats. Among the microsatellite repeats, relatively short arrays(5—30 repeats)were most abundant(71.7% ), and the largest array contained 60 repeats. The (CA)n-enriched library created in this study will be useful resource for developing additional anonymous microsatellite markers for Pacific oyster in the future. |
| Key words: Pacific oyster Crassostrea gigas, Microsatellite, Rapid isolation, Characteristic analysis |
