Mechanism Of Tolerance To Ultra High Pressure Of Vibrio Parahaemolyticus Based On Transcriptome

In this work, the pressure-resistant strain N11 is screened from the pressure-sensitive strain C4 which is aquatic pathogens by multiple high pressure treated. Then the effect and differences of cell wall membrane, cell damage, as well as mechanism of tolerance of the pressure-resistant strain to ultra high pressure treatment is studied based on C4 and N11 strains. Further Seq-RNA sequencing technology is used to analyze the difference expression genes between the two strain, then combine with the genomic data screened the mutation nucleotide sites in N11 strain, and examines the differences in expression of genes and gene regulation by mutations using real-time quantitative PCR technique. The specific conclusions are as follows:（1） When studied the tolerance of C4 and N11 strains to high pressure, all of C4 strain are killed at 200 MPa, however a small part of the pressure-resistant strain survive.（2） From the result of cell membrane permeability, Na+/K+-ATPase activity, surface zeta potential, cell ultrastructure, and intracellular CAT activity of two strains trested by ultra high pressure, we know the ultra high pressure could destroy the structure of cell wall membrane, increase the membrane permeability, decrease the activity of enzyme, leak of cell contents, and low electron region. However, the damage of cell wall membrance of C4 stain is higher than that of N11 stain under the same pressure, so it is think that the damage of cell wall may be the main reason of the high pressure sterilization. The catalase in N11 strains had higher resistance to high pressure, thus improving tolerance of the strain to ultra high pressure.（3） There were a series of differentially expressed genes found, using the RNA-Seq sequencing technique to analyzed the transcription of C4 and N11 strains which are before or after the ultra high pressure. From the result of COG-category and WEGO functional enrichment analysis of differentially expressed genes, it is found that the expression levels of the genes in C4 strains which related to cell membrance binding protein, metabolism and biological regulation were changed obviously in pressure stress. Meanwhile the expression of the gene which related to inorganic salt ion transport and metabolism, antioxidation activity, cell killing was down regulated, which may lead to the destruction of the cell membrane, and the death of the bacterial. While the genes which related to cell activity, transport protein, biological process are regulation, as well as containing CBS structural domain membrane proteins, ABC type phosphate transport system and the peripheral component,50S ribosome protein and peripheral protein, which contributed to the tolerance of N11 strain to the ultra high pressure.（4） Through the KEGG pathway analysis of the differentially expressed genes, it is found that the ability of N11 strain on carbon source and amino acids was decreased, and the enery production was reduced compared with C4 strain after high pressure treatment.（5） It is predicted that 16 nucleotide sites in 8 genes in encoding region from N11 strain, and 22 nucleotide sites mutation in non encoding region, from the results of comparative analysis of the whole genome between C4 and N11 strain. In turn, the primer was designed, and verification of mutation sites showed that 18 sites were mutated by PCR amplification, which occurred in the non encoding region, and it was speculated that these mutation sites may be enhanced the pressure-resistance ability of the cell by regulated the upstream and downstream genes.（6） The real-time PCR analysis of the upstream and downstream genes of the mutation sites can be inferred that the expression of C4₄362 gene significantly reduced in the C4 strain may improve pressure sensitivity. The expression of C4₄609 gene abundantly expressed in cells may result the death of the strain. Protein encoded by the gene C4₄522 current function is not yet clear, but it can be speculated that the expression level with down regulation can increase the tolerance of strain to pressure. On the contrary, C4₄795 gene may play a negative role in the regulation of pressure tolerance of strain. And the C4₄363 and C4₄794 genes were not related to the strain’s ability to withstand the pressure.