Studies On The Regulation Of Pyoverdine In The Pathogenicity Of Pseudomonas Plecoglossicida

Large yellow croaker,one of the largest breeding marine fish species,was identified as one of six kinds of the most advantageous export aquatic products.With the development of modern intensive aquaculture,bacterial infections in large yellow croaker have become an increasingly important health problem.White-spots disease is one of serious epidemic of great yellow croaker cultured in net-cage and have contributed to severe economic losses.Recently,people think the pathogen of the white-spots disease in great yellow croaker is Pseudomonas plecoglossicida.The visceral white spot disease of large yellow croaker shows significant temperature dependence.Through high throughput transcriptome analysis technology and bioinformatics to analyze the difference of Pseudomonas deformation transcriptome cultured at 12°C,18°C and 28°C.Based on the results,five Pyoverdine（CVD）synthase encoding gene were found and the expression of the five genes in the pathogenic temperature（18°C）were higher than the non-pathogenic temperature（12°C and 28°C）.This means that the five kinds of PVD synthase encoding gene may be associated with the pathogenicity of Pseudomonas plecoglossicida.PVD and its synthase were variability and species-specific,and the information about the synthesis pathway and secretory pathway and pathogenesis of Pseudomonas PVD are still in the exploratory stage,and many of the specific molecular mechanism is not clear.Therefore,the in-depth research about PVD in Pseudomonas plecoglossicida is very meaningful.According to the previous findings we speculated that the synthesis of PVD regulated by temperature could be the reason of pathogenic in special tempreture of Pseudomonas plecoglossicida.The five PVD synthase encoding gene could be the key to the regulation mechanism.The transcription of PVD synthase genes,extracted from Pseudomonas plecoglossicida respectively cultured in the 12°C,18°C and 28°C,were detected using RT-PCR to corroborate the results in the transcriptome sequencing.Then the expression pattern of five genes in different temperatures were confirmed.OD₄₀₀ and OD₆₃₀ absorption spectrum and CAS plate assay were used to detect the synthesis of PVD in Pseudomonas plecoglossicida in different tempreture.The results showed that the expression of genes is much higher at 18°C than 12°C and 28°C.We constructed the five stable silencing PVD synthase encoding gene with shRNA in Pseudomonas plecoglossicida.Detecting the expression of genes by RT-PCR.OD₄₀₀ and OD₆₃₀absorption spectrum and CAS plate assay were used to detect the synthesis of PVD in Pseudomonas plecoglossicida with gene silencing.The functions of these genes were demonstrated with biofilm formation,growth and infection.Results showed that these PVD synthase genes play an important role in the process of growth,biofilm formation,iron uptake and toxicity in Pseudomonas plecoglossicida.