Construction And Characterization Of The Ammonium-Excreting And-Resistant Genetically Engineering Strain From Nitrogen-Fixing Pseudomonas Stutzeri

To deregulate the inhibition of ammonium and constructing ammonium excreting strains have been the focus of intense research in biological nitrogen fixation.The first?second generation of ammonium-excretion strains gained by chemical mutagenesis or genetic recombination were likely to grow poorly and had a range of defects,such as decreased nitrogenase activity and low ammonium secretion capacity for extensive field application.How to overcome these problems and construct a new generation of nitrogen-fixing process strain with high efficiency means important theoretical research and practical application value.In this study,on the basis of the completion of the whole genome sequencing of Pseudomonas stutzeri A1501 and the regulation of nitrogen fixation gene expression,nitrogen metabolism and ammonium transport mechanism.By artificially designing the nitrogen fixation regulation related gene functional modules,this research was intended to enhance the ammonium tolerance and secretion ability the strain.The specific contents and results are described as follows:1.According to A1501 whole genome sequence,the two components regulatory transcription activator protein Ntr C,sigma factor Rpo N,nitrogen fixation is regulatory proteins Nif A,and non-coding RNA Nfi S participated in the regulation of nitrogenase activity of function modules such as genes encoding corresponding to the clone to mobilizable vector p LAFR3 and then transferred into A1568(ammonium transport carrier protein Amt B deletion strain)and A1501 respectively,8 genetic engineering strain was obtained.2.q RT-PCR was used to detect the expression levels of the target genes in the recombinant strains.The results showed that the transcription levels of rpo N,ntr C and nfi S genes in the recombinant strains were more than 2 times higher than those in the control strains(A1501 or A1568)under the same culture conditions,indicating that the target genes were overexpressed in the bacteria.3.Three recombinant strains,A1501/nif A,A1568/nif A,and A1568/ntr C,maintained nitrogen fixation activity at the concentration of 2 m M NH₄^+,and the nitrogenase activity of A1501/nif A and A1568/nif A was 1.5 times as high as that in nitrogen-free medium.A1568/ntr C also maintained a certain nitrogen fixation capacity under the condition of 2 m M NH₄⁺concentration,while no nitrogenase activity was detected in other strains.These results indicated that the overexpression of nif A gene played a positive role in the ammonium tolerance of nitrogenase.The amount of ammonium secretion of each strain was determined after incubation in nitrogen-free medium.Wild type A1501 and its derivative strains had no obvious capacity of ammonium secretion,while the amount of ammonium secretion of A1568and A1568/ntr C reached 2.5?M.This indicated that the absence of ammonium transporter protein was beneficial to the secretion of ammonium.4.The recombinant strain was inoculated into the host plant rice,and the recombinant genetically engineered strain A1501/nif A had obvious advantages over other strains in plant height and dry weight.The nitrogenase activity of the 6 derived strains were reduced by10%-40%than that of the control strains.q RT-PCR results showed that in addition to A1568/ntr C,the transcription level of nif H in the nitrogenase structure gene of the recombinant strain was down-regulated,and the transcription level of nif H in A1568/ntr C was 3.2 times and 10.3 times of that in A1501 and A1568,respectively.However,in the phenotype of nitrogenase activity,it does not show high level of nitrogen fixation ability,which remains a question for how to improve the nitrogenase activity of the ammonium secreting strain or combine genes of different metabolic pathways in the future.