Isolation And Identification Of Nitrogen-fixing Bacteria From Termites And Cloning And Analysis Of Nitrogenase Genes

Termites are typical organisms which can usually thrive on nitrogen-poor food.Such food is wood materials rich in cellulose?hemi-cellulose and lignin,and in which nitrogen is not very high.Previous research shows that the nitrogen content of termites'organs is similar to that of other animals,there is a lot of azotobacter in termite gut,and up to 60%of the nitrogen in termites' organs comes from the nitrogen fixation of symbiotic microorganisms which live in the hindgut.Odontotermes formosanus is a major pest of agriculture and forestry crops and they can destroy reservoir and dam.In this study,Odontotermes formosanus in Wu Han Luo Jia Mountain were used,nitrogen-free medium was adopted as the isolation medium,then by plate streaking,36 nitrogen-fixing strains of high nitrogenase activity were got from the Odontotermes formosanus gut.And then morphological observation,physiological and biochemical tests and molecular biologic detection were carrid out.The results show that strain HUB-IV-004 and strain HUB-IV-033 are Klebsiella pneumoniae,strain HUB-IV-03 3 is Klebsiella variicola,strain HUB-IV-006 is Klebsiella oxytoca.In addition,bacteriostatic experiment were carried out by oxford plate assay system.And in this experiment,endophytic bacteria HUB-I-004?HUB-I-032?HUB-I-034?HUB-I-047 which were preserved in our laboratory were adopted as functional bacterium,and the azotobacter which were isolated from the Odontotermes formosanus gut were adopted as the indication bacterium.The results showed that the fermentation product of endophytic bacteria have obvious inhibition effects on the azotobacter in the Odontotermes formosanus gut.Besides,in this study,the azotobacter isolated from termicides gut and the cellulose decomposing bacteria separated from termicides body were mixed-cultivated,the results of this experiment showed that the activity of cellulose in cellulose decomposing bacteria is significantly increased,thus the results provide a theoretical basis for the synergy between strains.Moreover,in this study,the full length of nifH gene was successfully cloned by PCR.Then it was linked with the expression vector pET28a and was transformed into E.coli BL21.The target protein was expressed by IPTG induction,and the purified protein was analyzed by mass spectrometry analysis.The results show that the target protein is the iron protein of nitrogenase and establish a basis for further study about the nitrogen fixing mechanism of termites.