The Research On Manganese Biooxidation Mechanism Of Endophyte Salinicola Tamaricis F01

Salinicola tamaricis F01 is a bacterium isolated from the leaves of Tamarix chinensis growing in the Yellow River Delta of Dongying City,Shandong Province,and S.tamaricis F01 has high resistance ability to bivalent manganese metal ions.In this paper,the model of S.tamaricis F01 bivalent manganese metal ion resistance and biooxidation mechanism was established by qRT-PCR technique,RNA-seq technique,SEM technique,XRD technique,protein separation and extraction techniques,etc.And the theory of bivalent manganese metal ion biooxidation mechanism of endophytic bacteria S.tamaricis F01 is improved and completed.Firstly,the results of S.tamaricis F01 whole genome sequencing were used to predict partial bivalent manganese metal ion biooxidation-related genes,and partial genes such as catalase-peroxidase of S.tamaricis F01 was detected and calculated by qRT-PCR technique after 8 h,16 h,24 h,32 h,40 h and 48 h.Gene expression levels predicted and related bivalent manganese metal ion biooxidation revealed that genes selected such as manganese metal ion-binding lipoproteins,major outer membrane proteins,catalase,and extracellular metalloproteinases were involved in bivalent manganese metal ion biooxidation of S.tamaricis F01,and the test time of transcriptome sequencing was determined based on qRT-PCR technique test results.The early gene expression pattern with regard to bivalent manganese metal ion biooxidation of S.tamaricis F01 under divalent manganese metal ion stress was revealed by analyzing the transcriptome sequencing results under the stress of minimum concentration impact growth of bivalent manganese metal ions.Under the stress of divalent manganese metal ions,the expression levels of genes encoding manganese metal ion efflux pump,ternary tricarboxylic acid transporter substrate binding protein,ammonium transporter,type I secreted C-terminal targeting domain protein and bacterial ferritin were significant up-regulated,it indicated that S.tamaricis F01 may alter the internal and external environment of S.tamaricis F01 by efflux,isolation,biooxidation and release of ammonium ion to relieve the toxicity of divalent manganese metal ion to endophytic bacteria S.tamaricis F01 and Tamarix chinensis.In addition,numerous gene expression levels related ion transport,redox reaction,tricarboxylic acid cycle,glucose metabolism pathway,fatty acid metabolism pathway,especially amino acid metabolism pathway changes significantly,it indicated that S.tamaricis F01 played a role in bivalent manganese metal ion biooxidation through changing expression levels of a variety of crucial metabolic pathway genes to protect against non-specific bivalent manganese metal damage,or adjusting the metabolic levels of the entire bacteria to adapt to the external bivalent manganese metal ion stress.In this biological process,the bacteria changed the external environment of the bacteria by discharging the ammonium salt outward,excluding carboxylate outward,and providing a precursor substance for the biooxidation of the divalent manganese metal ion,which was beneficial to the biooxidation of the divalent manganese metal ions.Some transcriptional regulators,molecular chaperones and key enzymes of metabolic pathways were induced by divalent manganese metal ion stress,it indicated that the overall regulation of S.tamaricis F01 under bivalent manganese metal ion stress was complex and fine,which helped maintain the manganese metal ion homeostasis in S.tamaricis F01.To further confirm the manganese oxidation mechanism of S.tamaricis F01,I separated proteins from S.tamaricis F01 bacterial culture solution by ammonium sulfate precipitation technique,dialysis technique,ion exchange chromatography technique,gel filtration technique,ultrafiltration technique and polyacrylamide gel electrophoresis technique after long-term experimental treatment with divalent manganese metal ions.The method of the bivalent manganese ion solution incubating with the protein solution,the LBB assay was used to verify the bivalent manganese metal ion oxidation function of the protein of strip.The protein species in the polyacrylamide gel was detected by two-stage mass spectrometry technique,and it was determined that catalase-peroxidase and flagellin homologous proteins played a major role in the late stage under the stress of divalent manganese metal ions.SWISS-MODEL homology modeling results of two catalase-peroxidase revealed that the two catalase-peroxidases may be homodimers and the ligand molecule were most likely the oxygen atom.And a lot of effort has been made to produce the catalase-peroxidases by means of the cell-free protein synthesis system.XRD technique and SEM technique were used to detect the precipitation of bivalent manganese metal ion oxide from S.tamaricis F01 bacterial culture solution cultivated from PYCM medium,PYCM HEPES medium,modified PYCM medium,modified PYCM HEPES medium,K medium,K HEPES medium,NaCl（w/v）5%LB medium,and NaCl（w/v）5%LB HEPES medium.The divalent manganese metal ions was mainly oxidized to MnCO₃,while a very small amount of MnO₂,Mn₂O₃,Mn₃O₄ and MnO were produced.And the SEM technique observation result reveals manganese metal ion oxide formed a lot of small spherical-shaped,columnar-shaped and rod-shaped particles aggregated and joined to larger spherical-shaped,rod-shaped and column-shaped substance.The relationship between S.tamaricis F01 and salt-tolerant plants was studied.S.tamaricis F01 has a unique growth-promoting and anti-reverse nature,which can produce growth-promoting substances such as indoleacetic acid,ferricin and ACC deaminase,but S.tamaricis F01 was not beneficial to the plant height,root length and weight of Suaeda salsa,The growth-promoting characteristics had no significant effect,and it was unable to aggregate to form a bivalent manganese metal ion oxide coating at the roots of Suaeda salsa by watering the bacterial liquid.This may be related to Tamarix chinensis,the host of S.tamaricis F01,because S.tamaricis F01 is not separated from Suaeda salsa,and S.tamaricis F01 is not directly related to Suaeda salsa,so that it was impossible to construct S.tamaricis F01 and Suaeda salsa composite system by watering the bacterial liquid.The application of environmental management may require the S.tamaricis F01 to return inoculation treatment to woody willow.In summary,this topic revealed the molecular mechanism of manganese resistance and manganese oxidation of plant endophytic S.tamaricis F01,providing reference data for elucidating the biooxidation mechanism of microorganisms,especially endophytic bacteria.The application laid a good foundation for the environmental governance of endophytic.