Comparative Genomic Of Two Strains Of Vibrio Parahaemolyticus And Their Virulence Gene Expression And Transcriptome Studies In Different Salinity

Vibrio parahaemolyticus is one of a zoonoses bacteria which is widly living in seawater and brackish water.It will cause people diarrehea and vomiting and other gastrointestinal disease,it's also a pathogen of aquatic organisms such as fish,shrimp,crab and shellfish.With the development of farming techniques and research on organisms,the culture of marine shrimp in freshwater has become the mainstream of shrimp farming in China.Due to the culture environment changes,the original seawater pathogens entered the freshwater waters with the host and triggered a new disease in the culture process of Litopenaeus vannamei.Because of changes in environmental salinity,seawater pathogens have to change their living systems to adapt to low-salt environments,while bacterial value-adding capabilities and adhesion to the host will also increase.In this paper,selected two strains of Vibrio parahaemolyticus isolated from two different environments,then sequence the comparative genomic of two Vibrio parahaemolyticus.Detected the virulence factors and pathogenicity of two strains of Vibrio parahaemolyticus,and research the growth rate and virulence gene quantitatively expressed under different salinities.Combined with the transcriptomics information of two strains of Vibrio parahaemolyticus under different salinities,the effects of salinity on the transcription and growth of Vibrio parahaemolyticus were investigated.It provides a scientific basis for exploring the relationship between environmental factors and the pathogenicity of Vibrio parahaemolyticus.Whole genome sequencing of two Vibrio parahaemolyticus strains in this experiment using the Illumina Hiseq sequencing platform.The results showed that the Vibrio parahaemolyticus V9 genome of was 5489686 bp,the GC content was 45.23%,and 5202genes were predicted,the total length was 4645029 bp,with 108 tRNAs and 1 rRNA.The genome size of strain 383 is 5024488 bp,GC content is 45.16%,contains two circular chromosomes,and 4580 genes are predicted,the total length is 4324071 bp,and there are98 tRNAs and 16 rRNAs.The chromosome1 is 3172771 bp,the GC content is 45.11%,there are 84 tRNAs and 13 rRNAs;the chromosome2 is 1851717 bp,the GC content is45.26%,there are 14 tRNAs;3 rRNAs.The strain V9 has 14 gene islands.The strain 383has 13 gene islands on chromosome1 and 2 gene islands on chromosome2.Two incomplete phages were predicted with lengths of 7.8 kbp and 5.7 kbp.Combined the results of the growth rate of two strains of Vibrio parahaemolyticus in different salinity,found that the strain 383 bacterial concentration of OD600 was lower than 0.8 within 48 hours,that may be caused by the release of the carried prophage gene,phage can make bacterial death.A large number of virulence genes,secretory genes,and some drug resistance genes and genes used to invading hosts have been found in both strains of Vibrio parahaemolyticus.These genes may be play an important role in pathogenic and environmental tolerance of Vibrio parahaemolyticus V9 and Vibrio parahaemolyticus 383.Both strains contained virulence genes tlh,MAM7 and T3SS1.The common pathogenic genes tdh and trh and T3SS2 were not predicted,which was consistent with the results of PCR.Two strains have resistance genes for common aquatic drugs such as lactamase and tetracycline.More than50 secretory genes carried by the two strains contain most of the secretory systems currently known,which facilitate bacterial invasion of host cells to exert cytotoxicity and intestinal toxicity.This study found that freshwater strains have adapted low salinity environments,and their growth rate is faster than that of seawater strains in low salinity,while the tolerance to high salinity has decreased.Salinity has a greater effect on the expression of the pirA gene and the T3SS1 inner membrane protein gene vcrD1.The results showed that the expression of virulence genes not only had differences between strains,but also the effects of different virulence genes of the same strain are different.The expression levels of pirA gene and vcrD1 gene of the two strains differed by about 10 times.Combined with semilethal concentration(LD₅₀),seawater strain 383 48 h LD₅₀ is 1.73×10³ cfu/ml and,freshwater strain V9 48 h LD₅₀ is1.32×10⁵cfu/ml.The LD₅₀ of the two strains differed by nearly two orders of magnitude.Therefore,it is considered that the virulence of two strains of Vibrio parahaemolyticus is positively correlated with the expression of pirA gene and vcrD1 gene.At the same time,it is also suggested that freshwater culture of P.vannamei should be protected against the risk of Vibrio parahaemolyticus input.Using the Illumina Hiseq sequencing platform to sequencing transcriptome of strains V9 and strain 383 cultured at salinity 2 and strain V9 and strain 383 cultured at salinity 20,called 2-V9 and 2-383 and 20-V9 and 20-383.Compared the transcribed sequencing results with the transcriptome library,and the whole genome of the strain V9 and the strain 383cultured at a salinity of 10 was used as a comparison template.Screening,splicing,and assembly of the off-machine data,strain V9 yielded 4251 transcripts annotated as known functions,and strain 383 yielded 4694 transcripts annotated as known functions.Comparative sample 2-V9 and sample 20-V9 had 2758 genes are up-regulated and 1247 genes are down-regulated.The most up-regulated and down-regulated genes are cellular processes,metabolic processes,and single biological processes,all of which are biological processes.Comparative sample 2-383 and sample 20-383 had 3016 genes are up-regulated and 1425 genes are down-regulated.The most up-regulation of genes are cellular processes and metabolic processes in biological processes and the catalytic activities in molecular functions.The most down-regulated genes are cellular processes,metabolic processes,and single biological processes,all of which are biological processes.After GO annotation and KEGG pathway enrichment analysis,comparative sample 2-V9 and sample20-V9 differentially expressed genes are mainly enriched in starch and sucrose metabolism,phosphotransferase system?PTS?and valine,leucine and isoleucine degradation,comparative sample 2-383 and sample 20-383 differentially expressed genes are mainly enriched in starch and valine,leucine and isoleucine degradation,arginine and proline metabolism,salmonella infection.