甲烷氧化细菌(Methylosinus methanica)81Z中与甲醇氧化有关基因的克隆

CLONING OF THE GENES ENCODING FOR METHANOL OXIDATION FROM METHANOTROPH METHYLOSINUS METHANICA 81Z

以大肠杆菌(E.coli)DH-1菌株和考斯质粒pLA 2917为宿主-载体系统,建立了甲烷氧化细菌(Methylosinus methanica)81Z染色体基因文库。甲醇利用菌(Methylobacterium organophilum)XX的甲醇脱氢酶(MDH)结构基因片段(2.5kb)经α-^(32)P标记后作为探针对该文库进行筛选,得到两个杂交阳性重组质粒,即pG5和pH11,其中pG5的插入片段为25.4kb,互补分析表明:pG5的插入片段能使包括MDH和细胞色素C(Cyt.c)基因突变株在内的5株甲醇利用菌(Methylobacterium sp.)AM1的甲醇氧化阻滞突变株恢复在甲醇上的生长能力,并且能使3株甲醇利用菌XX的甲醇氧化阻滞突变株恢复野生型生长。至少有4个与甲醇氧化有关的基因位于该片段上。Southern blot杂交进一步证实:该插入片段与探针同源的区域在其4.8kb Sal Ⅰ片段内。

E. coli DH-1 and cosmid pLA2917 was used as a host-vector system to construct genomic bank of Methylosinus methanica 81Z. Two recombinants, pG5 and pH11, were found containing inserts which give positive response when the bank was screened with a α-^(32)P labelled methanol dehydrogenase (MDH) structural gene probe from Methylobacterium organophilum XX. Complementation analysis shows that the insert of pG5, with size of 25.4 kb, can restore the growth on methanol of 5 MDH^- mutants which include MDH and Cyt.c structural gene mutants of Methylobacterium sp. strain AMI, as well as 3 MDH^- mutants of Methylobacterium organophilum XX. At least 4 genes encoding for methanol oxidation were proposed locating on this insert. Southern blot provided the further evidence that there was a homologous region of the MDH structural gene probe within the 4.8 kb Sal 1 fragment of the insert.