作　　者： ; ; ; ; ; ;

机构地区： 长沙大学生物工程与环境科学系

出　　处： 《中国水产科学》 2007年第4期608-614,共7页

摘　　要： 通过FIASCO（Fast Isolation by AFLP Sequences Containing repeats）法构建鳜鱼（Siniperca chuatsi）基因组微卫星富集文库，分离微卫星DNA序列并对其特征进行分析。鳜鱼基因组DNA经MseⅠ限制性内切酶消化后，选取200～800bp的片段与MseⅠ接头连接，用生物素标记的寡核苷酸探针（AC）8、（CT）8、（AT）7、（GATA）8、（GATT）7与其杂交，杂交复合物结合到包被有链霉亲和素的磁珠上，变性洗脱获得单链目的片段，经PCR扩增形成双链，然后克隆到pGEM-T载体上，转化至DH5α中，首次成功构建鳜鱼基因组微卫星富集文库。对其中100个阳性克隆进行测序，60个（60％）含有微卫星序列（GenBank Accession Number：DQ789247～DQ789306）。成功设计了47对鳜鱼微卫星引物，并合成21对引物进行PCR扩增，结果筛选出18个多态性微卫星标记。结果表明：FIASCO法能有效提高筛选微卫星标记的效率。本研究筛选的微卫星标记可以用于鳜鱼遗传背景分析和遗传连锁图谱构建，并将为鳜鱼基因组结构分析、标记辅助育种以及数量性状位点（QTL）基因的定位等研究提供候选微卫星标记。[中国水产科学，2007，14（4）：608—614] A microsatellite-enriched genomic library of Siniperca chuatsi was constructed by using the method of FIASCO （Fast Isolation by AFLP Sequences Containing Repeats）, and the microsaellite se- quences and their characteristics were analyzed in this study. Siniperca chuatsi＇ s genomic DNA was digested with restriction enzyme Mse Ⅰ, then the fragments of 200- 800 bp were ligated with Mse I adaptors, and hybridized with biotinylated oligoprobes（AC）8, （CT）8, （AT）7, （GATA）8 and （GATT）7. The hybrid mixture was attached to streptavidin-coated magnetic beads. After the non-SSRs fragments were removed by washing,single-stranded DNA containing the selected microsatellite DNA was collected. Then the single- stranded DNA was amplified using Mse Ⅰ - N primers to obtain double-stranded targeted fragments. The double-stranded fragments were cloned into pGEM-T vectors and transformed into DH5α strain, and then the microsatellite-enriched library of Siniperca chuatsi was triumphantly constructed. One hundred clones which were 120 - 500 bp in size were isolated and sequenced from Siniperca ckuatsi microsatellite libraries. Sixty clones （60%） of them contained microsatellites （GenBank Accession Number： DQ789247 - DQ789306）. We designed 47 pairs of SSR-primers and synthesized 21 pairs of them, 18 polymorphic microsatellite markers were screened out. The results indicate that FIASCO is a good method for enhancing the efficiency of development of microsatellite markers. These microsatellite markers are useful for genetic back-ground analyzing and genetic linkage mapping construction of Siniperca ckuatsi. Therefore, they provide various of candidates for comparative genome analysis, marker-assisted selection and analysis of quantitative trait loci location of Siniperca ckuatsi. [Journal of Fishery Sciences of China,2007,14（4）：608-614]

关 键 词： 鳜鱼 微卫星标记 富集文库

领　　域： [生物学]