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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Alcaligenes faecalis A15H1菌株总DNA的基因文库。用San3AⅠ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 BHB2688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nif H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-nif H DNA探针对所得重组噬菌体克隆之一(EMAFH1)进行Southern转移杂交,结果证实,其3.5kb的EcoRⅠ酶切片段为nif H阳性杂交带。将其克隆到质粒pUC19 DNA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(pAFH1)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 Alcaligenes faecalis A15H1 strain was constructed by using EMBL4, a bacteriophage replacement vector. Partially digested with San3A I restriction enzyme and cloned from a 13-20kb fragment. The vector DNA was completely double-digested by BamHⅠ and SalⅠ. The left and right arms were annealed to form the 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 packed extracts prepared with E. coli BHB2688 and E. coli BHB 2690 and the resulting library was assayed for potency 1.2 x 10 ~ 6 pfu, far exceeding the theoretically required pool capacity. The ~ (32) P-labeled nif H gene was used as a probe to screen clones containing the homologous sequences from the gene library by in situ 3-plaque in situ hybridization and verified by gradient point hybridization. Southern blotting of one of the resulting recombinant phage clones (EMAFH1) with the ~ (32) P-nif H DNA probe confirmed that the 3.5 kb EcoRI digestion fragment was a nif H positive hybridization band. After cloned into plasmid pUC19 DNA, it was transformed into recipient strain JM101 and then by Southern blotting to confirm that the resulting recombinant plasmid clone (pAFH1) contained a fragment homologous to nif H gene in Alcaligenes faecalis.