粪产碱菌(Alcaligenes faecalix)基因文库的构建和nifH基因同源顺序的克隆  

The Construction of Genomic Library of Alcaligenes faecalis and Cloning of infH Gene Sequence Homologous to Klebsiella pneumoinae

作　　者：海伟力[1] 郑洪刚[2] 尤崇杓 王斌[2] 

机构地区：[1]中国农业科学院原子能应用研究所,北京100094 [2]中国科学院遗传研究所

出　　处：《Acta Genetica Sinica》1992年第4期369-377,共9页

摘　　要：采用λ噬菌体置换型载体EMBL4,构建了Alcaligenes faecalis A-15 H1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SaiⅠ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 BHB2690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1.2×10~6 pfu,远远超过理论上所需的库容量。以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。对所得重组噬菌体克隆之一进行Southern转移杂交,结果证实,其3.5kb的EcoRⅠ酶切片段为nif H阳性杂交条带。将其克隆到质粒pUC19 DNA上后,转入受体菌JM101中。再次经Southern转移杂交,证明所得重组质粒克隆(pAFH)含有粪产碱菌中的与nifH基因有同源顺序的片段。A genomic library of Alcaligenes faecalis A-15H1 which possesses raiher high nitrogenase activity has been constructed. The total DNA of A. faecalis A-15 HI was partially digested with Sou3AI. 13-20 kb of fragments recovered from agarose gel were cloned in bacteriophage EM-BL4 vector. A total number of 1.2×108 of recombinants was obtained. It is much beyond the desired capacity of a library. By using nifH gene of K. pneumoniae from plasmid pGBl as probe, we have successfully screened the clone containing its homologous sequence. The recombinant bacteriophage DNA was digested with EcoRI. A 3.5 kb of hybridizing band appeared after southern blotting and then was cloned in pUC19 DNA. The result of southern blotting indicated that the recombinant plasmid clone contained nifH gene sequence of A. faecalis. This clone was named as pAFH.

关 键 词：粪产碱菌 基因文库 nif H基因 

分 类 号：Q939.113[生物学—微生物学]