假单胞菌的氯儿茶酚1,2-双加氧酶基因的克隆和表达

 从土壤中分离得到1株降解2,4-二氯酚(2,4-DCP)能力较强的细菌菌株GT241-1,克隆了该菌株的3,5-二氯儿茶酚1,2-双加氧酶基因(dcpB),采用的克隆策略为:用Southern杂交对dcpB进行定位后,构建重组质粒,再用斑点杂交从重组质粒中筛选目的转化子.经序列测定得知dcpB亚克隆片段全长4303bp,其中dcpB基因编码区765bp.核苷酸和推测的氨基酸序列分析表明,dcpB与已在GenBank登记的相关基因有一定的差异.dcpB基因能够在大肠杆菌转化子中成功地表达有生物活性的酶.

Cloning and expression of chlorocatechol 1,2-dioxygenase gene of Pseudomonas GT241-1

A bacterium strain GT241-1 with fairly strong ability of degrading 2,4-dichlorophenol was isolated from soil, and the 3,5-dichlorocatechol 1,2-dioxygenase gene (dcpB) was cloned from this bacterium strain. The cloning strategy adopted was to construct recombinant plasmid after location of the dcpB using Southern blotting, and using again dot blotting to screen the aim transformant. Sequence determination showed that the whole length of dcpB subcloned fragment was 4303bp, in which the gene coding area was 765bp. The sequence analysis of nucleotide and the deduced amino acid showed that dcpB was different definitely from the relative genes registered in the GenBank. DcpB gene could express successfully the bio-active enzyme in colon bacillus transformant.