琼氏不动杆菌GXP04的苯酚羟化酶基因的克隆及序列分析

Cloning and sequencing of phenol hydroxylase gene from Acinetobacter junii GXP04

提取琼氏不动杆菌GXP 04的总DNA,采用限制性内切酶E coRⅠ酶切处理后,构建以pLAFR 3为载体的基因组文库。通过TA IL-PCR扩增出苯酚羟化酶的上游基因,对菌落原位杂交获得的11个转化子进一步鉴定,最终确定其中的8个转化子含有完整的苯酚羟化酶基因,选取其中的pLAFR 3-7对GXP 04的苯酚羟化酶基因的核苷酸序列进行分析,并对其蛋白质基本性质进行预测。

Acinetobacter junii GXP04 is a phenol-degrading bacterium isolated before. DNA of GXP04 was digested with EcoR Ⅰ , and ligated with cosmid vector pLAFR3 to construct a genomic library. Colonies were screened by bolt hybridization, and 11 positive clones were selected. According to TAIL-PCR, the upstream gene of phenol hydroxylase was gained and was used to identify the credibility of the 11 positive clones. As a result, only 8 positive clones had the complete phenol hydroxylase. The pLAFR3-7 was selected for the analysis of nucleotide sequence and the basic character of protein.