Identification Of GlnA And GlnR Genes In Bacillus Subtilis And Their Responses To Ammonia Nitrogen

The glutamine synthetase of Bacillus subtilis was encoded by glnA gene.It has a good absorption effect on ammonium,which effectively degrades some harmful ammonia nitrogen substances in water.glnR is a global nitrogen regulator that can inhibit or activate the genes expression of nitrogen metabolism to absorb nitrogen sources.In this study,we used PCR and RT-PCR to amplify genes,and analyze glnA,glnR genes by bioinformatics methods.We detected expression patterns of glnA and glnR genes response to ammonia nitrogen??NH₄?₂SO₄?by qPCR.In order to explore the preliminary function of glnA and glnR protein,the prokaryotic expression vector was constructed and further induced by IPTG.The main results were as follows:The CDS of glnA gene of Bacillus subtilis R47 was 1335 bp,encoded 444 amino acids,which was a member of the Glutamine synthetase superfamily by prediction.The secondary structure of glnA protein was mainly composed of Alpha helix?42.57%?and Random coil?34.46%?.The CDS of the glnR gene was 408 bp,which encoded 135 amino acids.The glnR protein was a member of the MerR-SF superfamily by prediction.The secondary structure of glnR protein was mainly composed of Alpha helix?54.07%?and Random coil?36.30%?.The prediction result of STRING software showed that glnR protein mainly interacted with glnA,amtB and gltA proteins,and the glnA protein mainly interacted with nasB,nasD,nasE and glnR proteins.The expression levels of glnA gene in B.subtilis samples treated with 40mM,50 mM,100 mM and 125 mM?NH₄?₂SO₄ at 24 h was significantly higher than that in the control group?p<0.01?.The relative expression levels of samples treated with 125 mM?NH₄?₂SO₄ were significantly higher than that in 40 mM,and 100 mM?NH₄?₂SO₄,respectively?p<0.01?.For glnR gene,the expression patterns of samples treated with 100 mM and 125 mM?NH₄?₂SO₄ were significantly higher than that in the control group?p<0.01?.The relative expression at 125 mM was the highest,which was significantly higher than that at 20 mM,40 mM,50 mM,and 100 mM?p<0.01?.The results indicated that expression levels of glnR and glnA genes were positively correlated with ammonia nitrogen concentration.The SDS-PAGE results showed that glnA and glnR protein were about 50 kDa and 14 kDa,respectively.The results of mass spectrometry presented that,the highest reliability score of glnA peptide was glutamine synthetase,the reliability score,protein coverage and average molecular mass were 544.1,88%and 50.25 kDa,respectively.For the glnR peptide,the highest reliability score of glnR peptide was a global nitrogen regulator,the reliability score,protein coverage and average molecular mass were 412.12,97%and 14.87 kDa,respectively.The ammonia nitrogen were degraded from 0.8 mg/L to 0.01 mg,when treated by 19?g/mL glnA protein after 4 h.The ammonia nitrogen also were degraded from 0.8 mg/L to 0.01 mg,when treated by 2.72?g/mL glnR protein after 4 h.The induced proteins of glnA and glnR have obvious degradation effects on ammonia nitrogen in water.The cytotoxity results exhibited glnA and glnR protein had a positive inhibitory effect on Escherichia coli.The above results indicated that the glnA and glnR genes play a role in degradation of ammonia nitrogen,which provides important reference data for system further study of the functions of glnR and glnA protein.