摘要
采用磁珠富集法构建哲罗鱼（Hucho taimen Pallas）荩因组微卫星文库。哲罗鱼基因组DNA经MboⅠ限制性内切酶消化后，选取400-900bp的片段，用生物素标记的简单重复序列（ACA）15作探针与其杂交，杂交复合物结合到包被有链霉亲和素的磁珠上，获得目的片段，连接T载体克隆，构建基因组微卫旱富集文库。再用同位素标记的（ACA）15探针进行二次筛选，筛选出686个阳性克隆，阳性克隆率为35．94％。对其中140个阳性克隆进行测序，共获得149个微卫星序列，4个小卫星序列，其GenBank Accession Number为DQ110955～DQ121108。其中perfect（完美型）62个，占41．61％；imperfect（非完美型）92个，占61．74％；compound（混合型）5个，占3．36％，重复次数主要分布于6～45（81．21％），平均重复次数为32．5，这表明（ACA／TGT）。在哲罗鱼基因组DNA中含最非常丰富。本研究旨为从DNA水平上研究哲岁鱼种群结构、遗传多样性及目前群体现状等方面提供有效工具。
Enrichment of microsatellite DNA with magnetic beads is a simple and efficient DNA isolating method, which was used to construct microsatellite DNAs library from Hucho tairnen Pallas genome in this study. This project was designed to isolate microsatellite markers from Hucho taimen Pallas genome using the biotin capture method, which had been developed as a convenient and efficient method in recent years. Firstly, genomic DNA was extracted and digested with Mbo Ⅰ. And 400-900 bp fragments were recovered and ligated to a short linker （23 bp）. Secondly, the ligated DNAs were hybridized with biotin-labeled simple sequence repeats（SSR）probes （ACA）15 and the mixture was incubated with streptavidin coated magnetic beads. The selected single-strand microsatellite DNA was obtained by removing the SSR uncombined fragments. After the amplification of the .selected microsatellite DNA, the PCR products were ligated to Tvector and transformed to E. coli competent cells. Finally, a library containing 2 100 colonies was constructed. Six hundred and eighty six positive colonies were obtained after the screening of the library by radioactive labeling hybridization. Sequencing of 140 positive colonies confirmed 149 microsatellite loci. Of these sequences, only 4 contained compound repeat motifs （2.61 % ）, and the others included 60 perfect （39.22%） and 89 imperfect （58.17%） repeat motifs. In addition, relatively short arrays （6-45 repeats） were most abundant （71.7 % ） and the largest array contained 131 repeats in the microsatellite repeats. According to unique sequences flanking each motif, 117 primers were designed using the software Primer Premier 5. The results indicated that microsatellite sequences were characterized by （ACA）, abundant in ge nomic DNA of Hucho taimen pallas. The （ACA）15-enriched library constructed in this study will be a useful resource for screening additional microsatdlite markers from Hucho taimen Pallas for further study.
出处
《中国水产科学》
CAS
CSCD
北大核心
2006年第2期181-186,共6页
Journal of Fishery Sciences of China
基金
国家"973"重点基础研究发展计划项目资助(2004CB117405)
关键词
哲罗鱼
微卫星
磁珠富集
Hucho taimen Pallas
microsatellite
enrichment with magnetic beads
