Functional Characterization And Transcriptomic Analysis Of Site-2 Protease Like Protein From Rhodococcus Ruber

Site-2 protease is widely distributed in bacteria and participates in regulating various physiological functions of bacteria.These physiological functions include maintenance of membrane integrity,spore formation,lipid synthesis,pheromone production and virulence.Therefore,in-depth exploration of the zinc-containing S2P functions of various bacteria has important scientific value for further understanding of the various physiological functions of bacteria.There are few reports on the study of S2P-like proteins in Rhodococcus ruber.Expression change of S2plp was found to be highest in the proteomic study of Rhodococcus ruber SD3.The function of the protein was studied in the paper.The main research contents and results of this paper are as follows:1.Using bioinformatics method to analysis the s2plp gene,it was found that the protein was composed of 252 amino acids.Molecular formula was C1206 H1924N334O323S2,molecular weight was 26.335 k Da,and the theoretical isoelectric point was10.96.This protein was highly hydrophobic and stable.Structural analysis showed that S2plp had no signal peptide and disulfide bond with 6 transmembrane regions.Homologous alignment and phylogenetic tree analysis inferred that S2plp belonged to the M50 superfamily.Robetta comprehensive method was used to predict the tertiary structure of s2plp.Protein interaction predicted that S2plp might be related to signal transduction and material transport on the cell membrane.2.The expression variation of s2plp gene under the stress of various organic solvents was assayed by real-time fluorescent quantitative PCR technology(q PCR).The results showed that compared with the strains without organic solvent stress,s2plp was up-regulated under different organic solvent stress.In addition,the experiment also studied the correlation between the s2plp gene expression level and the concentration of organic solvents by setting a phenol concentration gradient.The results showed that within a certain range,when the phenol concentration increases,the expression level of s2plp gene was also be up-regulated.3.The p NV18-s2plp-s2plp recombinant plasmid was constructed and electro-transformed into the R.ruber SD3 to obtain the s2plp enhanced strain.Controled with the wild-type R.ruber SD3,the s2plp expression level in the enhanced strain was up-regulated to 248.52 times as q PCR detected.It was found that the growth advantage of the s2plp enhanced strain was more obvious than wild-type when cultured under the stress of various organic solvents such as isooctane and n-hexane.It is speculated that the s2plp has a certain promoting effect on the organic solvent tolerance of R.ruber SD3.4.The transcriptome analysis of the s2plp enhanced strain and the wild-type was performed using high-throughput sequencing technology.4846 unigenes was obtained,taking the wild-type R.ruber SD3 strain as a reference,under phenol stress,using p-adjust<0.05&|log₂FC|?1 as the screening criteria,a total of 13 genes with significant differential expression were screened,but the function of 8 genes is unknown.Function genes mainly focus on DNA repair,mismatches and DNA recombination.Combined with the previous research on the organic solvent tolerance of R.ruber SD3,it is considered that the s2plp gene may promote the repair of cell DNA damage by R.ruber SD3 in organic solvents.These results of transcriptomics will lay the foundation for further research on the organic solvent tolerance mechanism of R.ruber SD3 in the future.In this paper,the s2plp was found to improve the organic solvent tolerance of R.ruber SD3 using fluorescence quantitative PCR,gene enhancement and transcriptome analysis.The project laid a foundation for revealing s2plp-mediated signal pathway by analyzing the function of s2plp.It also provided potential molecular target for genetic engineering of R.ruber SD3.