Analysis Of The Genome Of Rhodococcus Ruber SD3 And Expression And Function Of DnaK Protein

The biodegradation method on which microbes primarily work is the main method for treating organic wastewater in China.As a gram-positive representative strain of Rhodococcus which degrades organic solvents,Rhodococcus ruber has great potential in organic solvent degradation owing to easy incubation and a high reproductive speed.One of the many organic solvent tolerance mechanisms in microorganisms is to eliminate the toxicity of organic solvents with the action of molecular chaperones,such as heat shock proteins,which effectively assist in the correct folding of certain related proteins.Heat shock protein 70s(Hsp70s)are central components of the cellular network of molecular chaperones and folding catalysts,and are also the most important class of heat shock proteins.DnaK is an important Hsp70 protein and is often used as a model for studying Hsp70 s in microorganisms.The current mechanism of action on the tolerance of organic solvents to Rhodococcus ruber is unclear,and there are few potential targets for further improving the tolerance of organic solvents in Rhodococcus ruber.The main research contents and results of this paper are as follows:1.Firstly,a single-molecule PacBio sequencing technology was used to sequence the organic solvent-resistant Rhodococcus ruber SD3 to obtain a full genome with a size of 5.37 Mb and a GC content of 70.625%.At the same time,bioinformatics analysis of the whole genome can provide a basis for the genetic modification of Rhodococcus ruber SD3 and revealing the organic solvent tolerance mechanism of the strain.2.Secondly,The dnak gene was obtained by T cloning,and the recombinant plasmid PET-28a-dnaK was constructed.The recombinant plasmid was heterologously expressed in E.coli BL21(DE3),and the recombinant protein with a size of about 65 kDa was obtained by nickel column affinity chromatography,which was consistent with the expected size.The recombinant protein was identified by LC-MS/MS and identified as the target protein DnaK.The obtained recombinant protein can lay foundation for further exploration of DnaK function.At the same time,the secondary structure of DnaK was analyzed by circular dichroism analysis.It was found that the secondary structure of DnaK protein was mainly ?-fold,which was consistent with the subsequent modeling of DnaK by bioinformatics.3.At the same time,the expression of DnaK in Rhodococcus ruber SD3 under organic solvent stress was studied by Quantitative Real-time PCR(QPCR)to study the role of DnaK and the mechanism of organic solvents tolerance in Rhodococcus ruber SD3.The results showed that the expression of DnaK increased to 29.87 under toluene stress compared with the control group.The expression of DnaK increased to 3.93 times under phenol stress,indicating that DnaK is closely related to the organic solvent tolerance of Rhodococcus ruber SD3.In addition,bioinformatics methods were used to search for DnaK interaction proteins in the STRING database and in differential proteins upon organic solvent stress from Rhodococcus ruber SD3.The results showed that DnaK interacts with many differential proteins,indicating that DnaK may play a role in the folding of protein related to organic solvents tolerance of Rhodococcus ruber SD3.The results can provide evidences for the further analysis of the relationship between DnaK and the organic solvent tolerance mechanism of Rhodococcus ruber SD3.4.Finally,the conclusion of the paper was made and the outlook for the further researches was given.