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为分离鹅(Anser cygnoides)微卫星序列片段,提取鹅基因组DNA,用Hae III和Rsa I内切酶消化并连接接头,再用接头特异引物进行PCR扩增。扩增产物与生物素标记的(AC)12探针杂交,杂交复合物用链霉亲和素包裹磁珠进行结合,得到单链DNA目标片段。再经PCR扩增,连接pMD19-T载体,转化入感受态大肠杆菌,得到微卫星富集小插入片段DNA文库。用Colony-PCR法筛选获得318个阳性克隆,并进行测序分析。结果表明,所测的318个序列有242个含微卫星序列,197个为有效微卫星序列,其中完全型(perfect)占60.9%,非完全型(imperfect)20.8%,混合型(compound)18.2%。(CA)n重复最为常见。文章为鹅种资源遗传多样性、分子进化、遗传图谱的构建及重要经济性状基因座定位等研究奠定了基础。
In order to isolate microsatellite sequences of goose (Anser cygnoides), goose genomic DNA was extracted, digested and ligated with Hae III and Rsa I endonucleases, and then PCR amplified using adapter-specific primers. The amplification product is hybridized with a biotin-labeled (AC) 12 probe, and the hybridization complex is bound with streptavidin-coated magnetic beads to obtain a single-stranded DNA target fragment. After PCR amplification, the pMD19-T vector was ligated and transformed into competent E. coli to obtain a microsatellite-enriched mini-insert DNA library. 318 positive clones were screened by Colony-PCR and sequenced. The results showed that there were 242 microsatellite sequences and 197 effective microsatellite sequences in 318 sequences, of which 60.9% of perfect, 20.8% of imperfect, 18.2 of compound %. (CA) n repetition is the most common. The article lays the foundation for genetic diversity, molecular evolution, construction of genetic map and locus of important economic traits in geese.