磁珠富集法制备大口鲶的微卫星分子标记

Microsatellite enrichment by magnetic beads in Silurus meriaionalis

通过磁珠富集的方法分离大口鲶（Silurus meriaionalis）的微卫星分子标记。将基因组DNA酶切，梯度离心收集400～900bp片段并纯化，连接Brown接头，用生物素标记的寡核苷酸（CA）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，然后将这些片段洗脱，PCR扩增，进行克隆构建“基因组文库”，再通过同位素标记的探针（CA）15进行2次杂交筛选，所得到的阳性克隆测序。从所获得593个阳性克隆中选取178个经测序，97．19％（173个）含有微卫星序列，90．60％重复数在10以上，75．98％为完美型。除探针中使用的CA重复单元外，还观察到Cr、GA、ATG的重复序列。设计获得120对微卫星引物，合成40对经PCR筛选，结果31对引物扩增出多态性条带。显示出，磁珠富集法是获得微卫星分子标记的一种有效的方法，可成为今后发展该标记的主要方法。同时，制备出的微卫星标记可为今后研究大口鲶的分子遗传育种提供有用的遗传标记。

Microsatellite enrichment by magnetic beads is a fast and efficient method for isolating this molecular genetic marker. Some microsatellite markers of Silurus meriaionalis were obtained by using the method. First, the genomic DNA was cut by the enzyme of Sau3AI, and targeted segments were collected with the size of 400 - 900 base pairs by centrifugation of sucrose density gradient. Then the segments were purified in a low melting agarose gel and ligated with a short linkers （20 bp）, from which, the ＂genomic PeR library＂ was created. These genomic DNA fragments were hybridized with a biotin-labeled SRS （simple repeat sequence） probe （CA）15. The hybrid mixture was incubated with magnetic beads coated with streptavidin. After washing to remove the non-SRS fragments, the eluted single-stranded DNA contained the selected microsatellite DNA. The selected DNAs were then amplified using primers designed complementary to the linkers, cloned into the pMD18-T vector and wansformed into competent E. coli DH5α. In this experiment, we performed the second screening with a radio labeled （CA）15 probe and obtained 178 positive clones. From these clones, 173 microsatellite sequences （about 97.19% ） were isolated, among which 90.60% microsatellites repeated more than 10 times and 75.98% microsatellites were perfect repeats. This allowed us to design 120 pairs of primers with the software Primer Premier 5.0. In addition, 40 pairs of primers were composed and screened. 38 pairs were used successfully to amplify special fragments,among which 31 pairs were polymorphism within species. These show microsatellite enrichment by magneticbeads is a suitable method to acquire microsatellite and it should become a main way to develop this molecular maker. At the same time, the microsatellite obtained can offer useful genetic markers for studying Silurus meriaionalis in the future.