日本蟳微卫星富集文库的建立与多态性标记的筛选  

Microsatellite-enriched library construction and polymorphic microsatellite marker isolation in Charybdis japonica

作　　者：宋春妮[1,2] 李健[1] 刘萍[1] 陈萍[1] 高保全[1] 

机构地区：[1]中国水产科学研究院黄海水产研究所,山东青岛266071 [2]大连海洋大学生命科学与技术学院,辽宁大连116023

出　　处：《水产学报》2011年第1期35-42,共8页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2006AA10A406);青岛市科技计划项目(07-2-3-5-jch)

摘　　要：采用磁珠富集法筛选日本蟳微卫星分子标记。日本蟳基因组DNA经Sau3 AⅠ酶切后,收集400～1 200 bp大小的片段并纯化,利用生物素标记的寡核苷酸探针(AC)15从中筛选出含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的基因组文库,经PCR检测筛选出阳性克隆进行测序。从随机挑选的970个菌落中筛选出369个阳性克隆进行测序,结果86.99%(321个)含有微卫星序列,其中完美型占80.54%,非完美型占15.95%,混合型占4.28%。除使用的探针AC重复外,还得到GA、CT等重复序列。共设计出102对微卫星引物,其中65对能扩增出清晰条带,27对具有多态性。同时筛选出的微卫星标记可为今后研究日本蟳的分子遗传育种提供有效的遗传标记。The method of magnetic bead hybridization enrichment was used to screen the microsatellite molecular markers of Charybdis japonica.Genomic DNA of Charybdis japonica was extracted and digested with restriction enzyme Sau3 A I.The fragments of 400-1 200 bp were recycled and purified.DNA fragments which containing microsatellite sequence were screened with（AC） 15 oligonucleotide probe and connected to pMD18-T vector.Genomic library contained the microsatellite sequence was constructed.Positive clones were isolated with PCR method and sequencing.After sequence analysis on 369 positive clones randomly picked from 970 colonies,321（86.99%） of the colonies were found to contain microsatellite sequences.Among the 321 microsatellites,80.54% were perfect,15.95% were imperfect and 4.28% turned out to be compound.From the primers designed for the 102 microsatellite loci,65 could amplify expected PCR products and 27 were found to be polymorphic.The results of this work may contribute to future studies of Charybdis japonica molecular genetic breeding.

关 键 词：日本蟳 微卫星 磁珠富集法 

分 类 号：Q785[生物学—分子生物学] S917[农业科学—水产科学]