关键词: 生物固氮, 电子传递体基因簇, NifA蛋白, 凝胶阻滞试验

Abstract: The electron transport complex gene cluster rnf1 was located within the nitrogen fixation island on the Pseudomonas stutzeri A1501 chromosome, and mutation of this cluster led to a sharply decrease of the nitrogenase activity. In this paper, a conserved binding site was identified in the promotor region of the rnf1 cluster. qRT-PCR results further indicated that the expression of rnf1 gene was dramatically down-regulated in the nifA mutant compared to the wild type, suggesting there was a regulatory role of NifA to the expression of rnf1 cluster. The interaction between NifA and the promoter region of rnf1 cluster was verified using bacteria one-hybrid system in vivo. Furthermore, the prokaryotic expression vector of nifA gene was constructed and the NifA protein was purified, and electrophoresis mobility shift assay showed that NifA and rnf1 promoter region could directly interact in vitro as well. Above results proved that NifA might recognize and bind to the promoter of rnf1 cluster specifically thereby regulating its transcription, and provided basis for further studying the regulation net of nitrogen-fixing genes.

Key words: biological nitrogen fixation, electron transport complex, NifA protein, electrophoresis mobility shift assay