三疣梭子蟹微卫星文库的构建及序列分析

Construction of microsatellite library and sequences analysis of the swimming crab (Portunus trituberculatus)

采用磁珠富集法筛选三疣梭子蟹(Portunus trituberculatus)的微卫星序列。经Sau3AI酶切后的200～1000bp DNA纯化片段,与两端已知序列的人工接头连接,用含有生物素标记的(CA)12和(GA)12探针杂交,根据磁珠的链酶亲和素与生物素特异结合的特性,捕获含微卫星序列的单链DNA,以此为模板用人工接头序列为引物进行PCR扩增,随后将获得的片段连接到PMD18-T载体上,转化至DH5α感受态细胞中,成功构建了微卫星富集文库。测序其中的60个阳性克隆,得到42条微卫星序列(基因登录号为:HQ283153-HQ283194),除探针使用的CA/GT、GA/CT重复外,还得到GAGT重复序列。42条微卫星序列中,完美型31个(占73.8%),非完美型9个(占21.4%),混合型2个(占4.8%)。完美型的比例显著高于非完美型,这与其他真核生物微卫星序列的特征相一致。39条微卫星序列的重复次数大于10(占92.9%),其中,重复次数在10～19次之间的有27个,20次以上的有12个。筛选出的微卫星位点可为今后三疣梭子蟹遗传多样性评价、种群遗传结构鉴定及资源保护研究提供一套有用的分子标记。

Microsatellite sequences were isolated from swimming crab （Portunus trituberculatus） by using the magnetic bead hybridization enrichment method.Total genomic DNA was extracted and digested with restriction enzyme Sau3AI.Fragments in range of 200-1 000 bp were agarose-gel purified and ligated with special adaptors,and the combinations was hybridized with biotin-labelled microsatellite probe （CA） 12 and （GA） 12.By using the high binding affinity of biotin to streptavidin-coated magnetic beads,single-stranded DNA containing the selected microsatellite sequences were captured.The targeted DNA was used as the template to conduct PCR amplification using the adaptor as primers.The fragments were inserted into PMD18T vector,and thansformed into DH5α competent cell.Of the 60 positive clones for sequencing,42 microsatellite repeat were acquired （GenBank accession number： HQ283153-HQ283194） .Except the CA / GT and GA /CT,the microsatellite sequences also had repeat motif of GAGT.The 42 microsatellite sequences could be categorized into 31 perfect type （73.8% ） ,9 imperfect type （21.4% ） ,and 2 compound type （4.8%） .The percentage of perfect type was much higher than the imperfect type in swimming crab,which was same as other eukaryote.39 microsatellite sequences （92.9% ） had at least 10 times of repeats numbers.27 microsatellite containing sequences had 10-19 times of repeats numbers,and 12 microsatellite had more than 20 times of repeats numbers.The present study might provide a set of useful molecular markers for future studies on genetic diversity evaluation,population genetic structure identification and resources conservation of the swimming crab.