| There are many nutrient-feeding insect that feeds on less nitrogen food in nature.Nitrogen is an essential element for the growth and metabolism of synthetic proteins and nucleotides in organisms.In order to maintain normal growth metabolism,these insects are biologically fixed by nitrogen by converting N2 in the air into NH3-N available by the action of microorganisms in the body.It is a very common phenomenon that insects bite plastics.Tenebrio molitor is a widely distributed storage pest.Tenebrio molitor could maintain normal survival with polystyrene plastic as the sole food.Polystyrene is an N-free polymer composed of C and H,it was speculated that Tenebrio molitor also had the biological nitrogen-fixing ability similar to that of the azo-trophic insect when biting polystyrene plastic.This paper used 3 to 5 weeks Tenebrio molitor larvae from the eastern part of China?Shanghai,Ningbo?,the west?Sichuan?,the South?Guangzhou?,the North?Beijing?and the Central?Luoyang?as the test object.The biological nitrogen fixation activity of Tenebrio molitor biting polystyrene plastic was first verified by acetylene reduction method,and confirmed that intestinal microbes play a decisive role in biological nitrogen fixation.The diversity and expression of nitrogenase in intestinal microorganisms of Tenebrio molitor were analyzed by PCR and RT-PCR and nifH gene amplicon high-throughput sequencing technique.The nitrogen-fixing microorganisms in the intestinal tract of Tenebrio molitor were isolated by the pure culture method of the characteristic medium,and the nitrogen-fixing activity of the dominant bacteria was verified in vitro.The main results achieved are as follows:?1?The nitrogen fixation activity of Tenebrio molitor,which was fed with polystyrene plastic and antibiotic-containing feed,was determined by acetylene reduction method.It was found that the six areas of the polystyrene-resistant Tenebrio molitor had nitrogen fixation activity,and the Beijing group had the highest nitrogen-fixing activity,which was 20.5123 nmol ethylene·h-1.The nitrogen fixation activity of antibiotic-treated worms in all groups decreased significantly,and the nitrogen fixation rate was basically maintained at 8.8 nmol ethylene·h-1.The difference in nitrogen fixation activity between the two groups of insects was analyzed,and it was confirmed that the bite-degrading polystyrene plastic Tenebrio molitor can perform biological nitrogen fixation under the action of intestinal microorganisms.?2?Using the primer set nifH1-nifH2 to amplify the nitrogenase nifH gene in the intestinal tract of Tenebrio molitor,and it was confirmed the presence of nitrogen-fixing microorganisms in the intestinal tract of insects.Detection of the product of RT-PCR revealed the band of the target gene,which fully indicated that the expression of nitrogenase was observed in the intestinal tract of Tenebrio molitor.Amplicon high-throughput sequencing technology was used to obtain 13 unique nifH gene sequences from the intestinal DNA samples of the six regions of Tenebrio molitor.Seven sequences were closely related to the?-Proteobacteria strains,including the nifH gene sequences in the intestine were highly similar to Klebsiella,Citrobacter,and Kluyvera.Klebsiella and uncultured cloned strains were cloned from the RT-PCR product.?3?101 strains of bacteria were isolated and purified from the intestinal tract of Tenebrio molitor under aerobic conditions using a nitrogen-free medium.After 16S rRNA amplification and sequencing analysis,it was divided into 22 different bacteria which were closely related to the strains of Proteobacteria,Actinobacteria and Bacillus.Proteobacteria occupied the largest abundance?72.7%?,mainly?-proteobacteria.The nitrogen-fixing activity of the potential nitrogen-fixing bacteria Klebsiella,Citrobacter,Pantoea,and Kluyveria and the positive control Azospirillum brasiliensis were determined.The nitrogen fixation activity of Klebsiella was found to be second only to Azospirillum,and the nitrogen fixation rate was 8.72 nmol ethylene·h-1. |
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