苯胺双加氧酶基因的克隆与序列分析

Cloning and Sequence Analysis of Aniline Dioxygenase Gene

通过设计苯胺双加氧酶基因特异引物,以苯胺降解菌株ANA5基因组DNA为模板,PCR扩增出目的基因片断。然后利用粘粒pLAFR3作为载体,以E.coliEPI100作为受体,构建了菌株ANA5的基因组粘粒文库。以PCR扩增产物作为探针,通过菌落原位杂交筛选得到两个阳性克隆,经Southern杂交及亚克隆测序分析,初步确认克隆到苯胺双加氧酶基因。同时完成了苯胺双加氧酶基因atdA3A4A5序列的测定,并对其核苷酸及其推导的氨基酸序列进行分析,结果表明克隆到的苯胺双加氧酶基因与GenBank报道的基因有一定的差异,同时体现了该基因在进化上的保守性。

With specific primers designed, the aimed DNA fragment was amplified by PCR from aniline-degrading strain ANAS. A genomic library of strain ANA5 was constructed in the cosmid vector pLAFR3 using E. coli EPI100 as the host strain. Two recombinants were identified from the genomic library using in situ hybridization and Southern blotting probed with the PCR product. By the analysis of subclone sequencing, it was sure that the aniline dioxygenase gene of strain ANA5 was cloned. The sequence analysis and the deduced amino acid showed that they were different from the relative sequences registered in the C, enBank and revealed that aniline dioxygenase gene was conserved through evolution.