PCR Amplification and Bioinformatics of a Novel Gene Coding Hydroxylamine Oxidoreductase*

Abstract

A probable novel gene coding hydroxylamine oxidoreductase (HAO) was acquired by PCR and gene sequencing in Acinetobactor sp. YY-5. Four pairs of PCR primers were designed according to the conservative regions of reported HAO gene to amplify and sequence the partial sequence of the HAO gene; several primers for genome walking PCR were designed according to the partial sequence to acquire the flank region of the partial HAO sequence; and the acquired sequence was subjected to Blast analysis in NCBI and open read frame (ORF) prediction by the professional ORF -analyzing software, FGeneSB. A 462 bp DNA sequence was obtained by one of the 4 pairs of primers, and further 2 690 bp DNA sequence of the flank region of 462 bp sequence was acquired through two rounds of genome walking PCR. Four ORFs were found within the 3 152 bp DNA sequence, one of which was of 555 bp. The 555-bp ORF contained the original 462 bp sequence and corresponded to an amino acid sequence of Mr 20.2×103, and no obvious similar HAO gene was found in GenBank. Thus, a novel HAO gene was possibly acquired by PCR.