CAJ | 학술논문

采用磁珠富集与放射性杂交相结合的方法开发黑斑狗鱼(Esox reieherti Dybowski)基因组微卫星资源.基因组DNA经Sau 3A Ⅰ限制性内切酶消化后,选取400-900bp的片段进行PCR全基因组扩增,并利用生物素标记的(CA)12、 (GA)12探针进行微卫星片段的富集.将得到的片段与pGEM-T载体连接后转入DH5α大肠杆菌中,然后利用γ-32P标记的放射性同位素探针进行第二次杂交.结果,共获得微卫星基因组文库1600个菌,杂交前菌落PCR检测阳性克隆率为90.91%;杂交后得到的阳性克隆为1300个,占87.25%.从中挑出196个进行测序,192(97.96%)个含有微卫星序列.在得到的微卫星序列中,重复单元除CA/GT、 GA/CT外,还观察到单碱基、四碱基、五碱基重复单元.根据侧翼序列应用引物设计软件Primer Premier 5.0设计引物70对,选择合成32对,通过优化PCR反应条件,结果有28对引物可扩增出清晰可重复的目的条带.本研究旨在对黑斑狗鱼基因组资源的开发利用起到一定的促进作用,并为黑斑狗鱼养殖品系的优化、遗传多样性的检测及遗传图谱的构建等奠定基础.
채용자주부집여방사성잡교상결합적방법개발흑반구어(Esox reieherti Dybowski)기인조미위성자원.기인조DNA경Sau 3A Ⅰ한제성내절매소화후,선취400-900bp적편단진행PCR전기인조확증,병이용생물소표기적(CA)12、 (GA)12탐침진행미위성편단적부집.장득도적편단여pGEM-T재체련접후전입DH5α대장간균중,연후이용γ-32P표기적방사성동위소탐침진행제이차잡교.결과,공획득미위성기인조문고1600개균,잡교전균락PCR검측양성극륭솔위90.91%;잡교후득도적양성극륭위1300개,점87.25%.종중도출196개진행측서,192(97.96%)개함유미위성서렬.재득도적미위성서렬중,중복단원제CA/GT、 GA/CT외,환관찰도단감기、사감기、오감기중복단원.근거측익서렬응용인물설계연건Primer Premier 5.0설계인물70대,선택합성32대,통과우화PCR반응조건,결과유28대인물가확증출청석가중복적목적조대.본연구지재대흑반구어기인조자원적개발이용기도일정적촉진작용,병위흑반구어양식품계적우화、유전다양성적검측급유전도보적구건등전정기출.
Esox reieherti Dybowsk genomic microsatellites were developed by using enrichment protocols combined with radioactive hybridization protocol. Four hundred to nine hundred base pair fragments were selected for the whole genome. DNA PCR amplification after digestion with restriction endonuclease Sau 3A Ⅰ, and (CA)12, (GA)12 probes marked with biotin were used for microsatellite DNA enrichment. The product fragments were connected with carrier pGEM-T and transferred into DHSα Escherichia coli competent cells, and radioactive isotope probes marked with γ-32 P were used for the second hybridization. As a result, a total of 1600 bacteria were obtained in the microsatellite genomic libraries, positive clones accounted for 90.91% before hybridization and 81.25% after hybridization, amounting to 1300. One hundred and ninety-six positive clones were selected for sequencing, and 192 clones included microsatellite sequences. The microsatellite sequences obtained, mono-nucleotide, quad-nucleotide and quint-nucleotide repeat motifs Were observed beside double-base-pairs CA/GT, GA/CT. Seventy primers were designed according to the flanking sequences by using software Primer Premier 5.0, and 32 primers were selected to be synthesized. After optimizing PCR reaction conditions, 28 primers were amplified and produced clear purpose bands. The aim of our research was to promote the development and utilization of E. reieherti genomic resource, and lay the foundation fur optimizing E. reieherti breeding strain in order to detect the genetic diversity and construct a genetic map.