【摘要】 以具有高吸Ｈ２活性的花生根瘤菌Ｌ８－３为出发菌株提取总ＤＮＡ，经ＢａｍＨＩ不完全酶切后，获得２０ｋｂ左右的ＤＮＡ片段，按Ｉｓｈ－Ｈｏｒｏｗｉｃｚ和Ｂｕｒｋｅ方法，与具有多克隆位点的ＣｏｓｍｉｄｐＬＡＦＲ３克隆载体连接，经体外包装、转导Ｅ．ｃｏｌｉＨＢ１０１，在选择培养基平板上获得１万多个重组克隆子．随机挑选２２个克隆子进行分析，其中１９个克隆子携带外源ＤＮＡ片段，平均长度为２１．４ｋｂ，文库有效克隆子数和插入ＤＮＡ片段均达到建库要求．ＰＣＲ筛选和生物素探针杂交初步结果显示，重组质粒中所携带的外源ＤＮＡ可能含有我们所需的吸Ｈ２基因．更多还原

【Abstract】 A genomic library of the hydrogen uptake positive (hup +) strain L 8 3 of Rhizobium arachis has been constructed by using the broad host range mobilizable cosmid pLAFR 3. The cosmid pLAFR 3 containing the multiple restriction enzyme coloning sites was digested with restriction enzyme Hind Ⅲ and EcoRI, respectively, and the linear DNA of pLAFR 3 was treated with alkaline phosphorylase. The dephosphorylated pLAFR 3 was digested with restriction enzyme BamHI and the large fragment of pLAFR 3 BamHI digested was isloated from Low point melting gel (0.3% agrose) by electrophoresis. Total DNA of Rhizobium arachis strain L 8-3 was partly digested with restriction enzyme BamHI and the 10 to 30 kb fragments were isolated and collected. After being dephosphorylated with alkaline phosphorylase, the fragment of DNA ligated with vectors. The ligated high molecular weight of DNA ligated with vectors. The colonies were selected on LB agar plates containing tetracycline. More than ten thousand clones were obtained and eighty six percent of the clones contained cosmid with insert DNA averaging 21.4 kb in length. By using the PCR screening and biotin probe hybridization, six recombinant cosmids were isolated from the genomic library. These six recombinants possibly contained complete hup genes. It is necessary for examining the expressions of hydrogen uptake activity after being transduced into the hup - Rhizobium .更多还原