Cloning And Function Of The Regulators For Nicotine Degradation In Mutant Strain N7-W13

The tobacco waste involving large quantities of nocitine can lead to seriously environmental pollution, biological methods employing microbes to degrade and recycle nicotine have gained wide attention.Study on microbial degradation mechanisms of nicotine at molecular level also provides its practical application with the theoretical basis and foundation.The Shinella sp. HZN7 in our lab has a high degradation rate of nicotine, and a variant of the pyridine and pyrrolidine pathway had been identified and characterized. In this paper, we explore the metabolic mechanism of nicotine degradation in Shinella sp. HZN7 further with molecular biology technique on the basis of the original research, and make the metabolic mechanism better in the genetic molecular level. We broke the gene of degradation of nicotine by inserting the transposon intoShinella sp. HZN7. Then we successfully screened goal deactivation mutant strains by using the method of photocopy tablet mutation. We created a library of nicotine degradation inactivation mutant, which involves 323 mutations.A typical mutant N7-W13 was chosen from the library for our study.The genes in Shinella sp. HZN7 we disrupted are the key to control the transformation of nicotine to 6HN. The mutant genes were successfully cloned from the strains Shinella sp. HZN7 through self-formed adaptor PCR which were named orfw13-1 and orfw13-2.Then genes orfw13-1 and orfw13-2 disruption and complementation suggested that the orfw13-1 and orfw13-2 gene are essential for the conversion of nicotine to 6HN in strain Shinella sp.HZN7. But the nucleotide and amino acid sequence showed small similarity to any gene or gene product with defined function in GenBank, the gene orfw13-1 and orfw13-2 were not the functional gene, and they worked like TCS(Two-Component Regulatory System).