摘要
为了克隆决定染色体形成的特异DNA片段YR-DNA．以λExCell为载体，克隆经限制性内切酶EcoRI消化后的水稻基因组DNA，λ噬菌体体外包装构建了滴度为1．2×10^6 pfu／ml，重组效率达86％的小型水稻基因组文库．随机选取了50个重组克隆。质粒释放后进行酶切鉴定，插入序列的大小为0．5～7kb，平均为3kb．以水稻基因组DNA为探针对基因组DNA文库进行筛选．从10000个重组子中选出200个杂交信号较强的阳性克隆，质粒释放后点渍杂交进行第2轮筛选．从中选取40个杂交信号较强的重组子．再次进行斑点杂交，选取10个杂交信号最强的重组子．酶切并回收基因组插入片段，标记特异的重组片段，与不同酶切后的水稻基因组DNA进行Southern杂交，其中有两个探针杂交后基因组酶切带上杂交信号呈弥散状，表明重组片段为散在重复序列．
Total genomic DNA of rice was digested with restriction endonuclease EcoRI. The DNA fragments were legated to the lambda ExCell. Then the legated DNA was packaged in vitro by a packagene Extract to construct the genomic library of rice which capacity was 1.2 × 106 pfu/ml and the recombination rate reached to 86 %. 50 clones were selected randomly for analysis. There insert size ranged from 500bp to 7kb with an average of 3kb. Labeled the total DNA of rice for probe with digoxingenin-11-dUTP （DIG） to screening the genomic library. 200 positive clones with strong hybridization signal were selected from about 10 000 plaque. The recombinants with the genomic DNA insert fragment were released from the 200 positive clones and were identified by dot blot hybridization with the probe of total DNA. 40 clones with strong hybridization signal were obtained. Then go on screening these clones by dot blot hybridization, 10 clones with the strongest hybridization signal were obtained at last. These 10 insert fragment were labeled and further applied for southern analysis with rice genomic DNA after digested with different restriction endonuclease. The hybridization signal of two fragment showed the selected DNA were interspersed repeat sequences.
出处
《河北北方学院学报（自然科学版）》
2006年第3期37-39,42,共4页
Journal of Hebei North University:Natural Science Edition
基金
"国家植物转基因技术研究开发与中试基地建设"专项课题(J99-B-001)
