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采用入噬菌体置换型载体EMBL4,构建了水稻联合固氮菌Al-caligenes faecalis A15Hl菌株总DNA的基因文库。用Sau3AI限制酶完成部分酶切,取13～20kb大小的片段进行克隆。载体DNA经BamHI和SalI完全双酶切,左右臂“退火”形成左右臂载体分子后再与外源片段连接,左右臂载体分子与外源片段按照1:1的分子比进行体外连接。用E.coli BHB 2688和E.coli BHB 2690制备的包装抽提物进行体外包装,所得基因文库效价测定为1.2×10~6pfu,远远超过理论上所需的库容量。以~(32)P标记的nifH基因作为探针,经3轮噬菌斑原位杂交,从基因文库中筛选出含有其同源顺序的克隆,并得到了梯度点杂交的验证。以~(32)P-nifHDNA探针对所得重组噬菌体克隆之一(EMAFHl)进行Southern转移杂交,结果证实,其3.5kb的EcoRI酶切片段为nifH阳性杂交带。将其克隆到质粒pUC19DNA上后,转入受体菌JM101中,再经Southern转移杂交,证明所得重组质粒克隆(pAFHl)含有粪产碱菌中的与nif H基因有同源顺序的片段。
A gene library of total DNA of Al-caligenes faecalis A15H1 strain was constructed by using EMBL4, a bacteriophage replacement vector. Partially digested with Sau3AI restriction enzyme, cloned from 13 to 20 kb in size. The vector DNA was completely double-digested by BamHI and SalI. The left and right arms were annealed to form left and right arm carrier molecules, which were then ligated with the exogenous fragments. The left and right arm carrier molecules and exogenous fragments were ligated in vitro at a 1: 1 molecular ratio. In vitro packaging was performed with the package extracts prepared with E. coli BHB 2688 and E. coli BHB 2690 and the resulting library of genes was determined to be 1.2 x 10 ~ 6 pfu, far exceeding the theoretically required pool capacity. The ~ (32) P-labeled nifH gene was used as a probe to screen clones containing the homologous sequences from the gene library by in situ plaque in 3 rounds and verified by gradient dot blot hybridization. Southern blotting of one of the resulting recombinant phage clones (EMAFH1) with the ~ (32) P-nifHDNA probe confirmed that the 3.5 kb EcoRI fragment was nifH positive. After cloned into the plasmid pUC19DNA, it was transferred into the recipient strain JM101, and then Southern blotting hybridization showed that the resulting recombinant plasmid clone (pAFH1) contains a fragment of homology to the nif H gene in Alcaligenes faecalis.