| Nowadays,due to the rapid development of industries,including cosmetics,medicine,plastics,electronics,and so on,the environmental pollution caused by the emission of organic solvents is gradually increasing,which leads to the decrease of available water resources.Organic solvents not only cause great damage to the water ecological environment,but also have irreversible negative effects on the human healthy and the normal growth of other organisms.Therefore,it is of great scientific value and practical significance to explore the environmental pollution control of organic solvents.C-di-GMP is a ubiquitous second messenger in bacteria,which can regulate biological functions such as bacterial biofilm formation,virulence,cell cycle,transcription,etc.In this paper,the organic solvent resistant bacteria R.ruber SD3 were screened as the research object,further c-di-GMP effector proteins were screened from the bacteria and identified.At the same time,the expression changes of the proteins under toluene and phenol stress were analyzed.The study deepens people’s understanding of the signaling pathway mediated by diguanosine cyclase of R.ruber SD3,provides new targets for the genetic modification of R.ruber SD3 and lays a foundation for the application of the strain in environmental management and other fields.This paper is consists of six chapters.Chapters 1: This chapter introduces the research progress on the tolerance of Rhodococcus genus,the hazardous situation of organic solvents,the status of c-di-GMP effector proteins,and the main research contents of this paper.Chapters 2: First,c-di-GMP labeled by streptavidin was used as the ligand,and total protein of Rhodococcus Ruber SD3 was used as the "prey" to screen candidate c-di-GMP effector proteins by affinity pull-down assay.After screening,59 effector protein candidates were obtained by LC-MS/MS analysis.Two effector protein candidates were selected as research objects and named CEPC1 and CEPC3,respectively.Chapters 3: First,cepc1 and cepc3 genes were cloned by TA cloning,and the CEPC1 and CEPC3 were analyzed by bioinformatics.The accession number of cepc1 in Gen Bank was MK721211,it was predicted that the molecule weight of CEPC1 protein was 11.95 k Da.The isoelectric point(p I)was 11.18,and CEPC1 protein belonged to 50 S ribosomal protein L22,which was related to the correct folding and stability of the 23 S r RNA conformation and the early assembly of the 50 S large subunit.Molecular docking showed that CEPC1 protein and c-di-GMP had interaction force.The accession number of cepc3 in Gen Bank was MT023096,it was predicted that the molecule weight of CEPC3 protein was 14.24 k Da.The p I was 10.83,and the CEPC3 protein belonged to 50 S ribosomal protein L20,which is related to the three-dimensional structure of r RNA and protein translation.Molecular docking showed that the CEPC3 protein and c-di-GMP had weak interaction force.Chapters 4: The cepc1 gene was codon-optimized,and the recombinant plasmid ocepc1-p ET-28 a was constructed,then the recombinant plasmid was transferred into E.coli BL21(DE3)for heterologous expression.When the final concentration of IPTG was 0.4 mmol/L,shaking culture conditions was 30℃,180 r/min,and the culture time was 10 h,SDS-PAGE electrophoresis showed that the CEPC1 was obtained successfully,and the CEPC1-His fusion protein was purified by Ni-chelating affinity chromatography and identified by mass spectrometry sequencing.SPR analysis showed that CEPC1 was bound to c-di-GMP,and the KD = 127±1.03 μM,indicating that CEPC1 was the effector protein of c-di-GMP in R.ruber SD3.The cepc3 gene was codon optimization,and the recombinant plasmid ocepc3-p ET-21 b was constructed,then the recombinant plasmid was transferred into E.coli BL21(DE3)for heterologous expression.When the final concentration of IPTG was 0.5mmol/L,shaking culture conditions was 25℃,180 r/min and culture time was 8 h,SDS-PAGE electrophoresis showed that the CEPC3 was expressed successfully,further obtained the purified CEPC3-His fusion protein by Ni-chelating affinity chromatography and identified by mass spectrometry sequencing.However,SPR analysis showed that CEPC3 had a weak binding force or no binding force with c-di-GMP,indicating that CEPC3 was not the effector protein of c-di-GMP in R.ruber SD3.Chapters 5: The m RNA transcription level of cepc1 and cepc3 under organic solvent stress(toluene,phenol)were compared by q PCR technology,respectively.Compared with the m RNA levels of the control.The m RNA levels of cepc1 and cepc3 were upregulated by 49.90 and 5.74 times under toluene stress,respectively.The m RNA levels of cepc1 and cepc3 were up-regulated 86.72 times and 2.95 times under phenol stress,respectively,indicating that the expression of CEPC1 and CEPC3 proteins is likely to increase under toluene and phenol stress.Chapters 6: The main results of this experiment was summarized.The innovation content and deficiency of this research was analyzed,and further research in the future was proposed.Conclusion: The cepc1 and cepc3 gene sequences have been submitted to Gen Bank with their accession Numbers MK721211 and MT023096,respectively.The CEPC1 belonged to c-di-GMP effector protein,CEPC3 had a weak binding force or no binding force with c-di-GMP,so it was regarded as the c-di-GMP effector protein.The protein expression levels of cepc1 and cepc3 increased under toluene and phenol stress,indicating that the cepc1 and cepc3 genes played a role in the mechanism of resistance to organic solvents in R.ruber SD3. |
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