The Effect Of A Lys R-type Transcriptional Regulator Gene Of Pseudomonas Plecoglossicida On The Immune Responses Of Epinephelus Coioides

As an important pathogen in aquaculture,Pseudomonas plecoglossicida has caused heavy losses.The fulminant disease caused by P.plecoglossicida is temperature-dependent and mainly recorded during a seawater temperature range from 15-20°C.In order to explore the temperaturedependent pathogenicity of P.plecoglossicida,P.plecoglossicida in different temperature were determined with RNA-seq.The results revealed that the expression of a LysR-type transcriptional regulator gene（L321₂0267）of P.plecoglossicida at 18°C was significantly higher than that at 28°C,which was verified by quantitative real-time PCR（qRT-PCR）.Therefore,we predicted that L321₂0267 maybe related with the virulence of P.plecoglossicida.In order to uncover the effect of gene L321₂0267 in P.plecoglossicida on virulence,5 shRNAs were designed according to gene L321₂0267 and synthesized to construct recombinant pCM130/tac vectors.RNAi of 5shRNA could significantly reduce the content of L321₂0267 mRNA in P.plecoglossicida,with a maximal decrease of 90.63% by L321₂0267-RNAi（L321₂0267-shRNA882）.Compared with the wild-type strain,infection with the L321₂0267-RNAi strain resulted in a 50% reduction in mortality and an onset time delay of Epinephelus coioides,as well as alleviation of the symptoms in E.coioides spleens.The spleens infected by L321₂0267-RNAi strain or wild-type strain of P.plecoglossicida at 24 h were submitted into RNA-seq.Compared with the wild-type strain of P.plecoglossicida,the L321₂0267-RNAi strain resulted in a significant change in the spleen transcriptome of infected E.coioides.The results of GO and KEGG analysis showed that host genes of serine hydrolase activity,the antigen processing and presentation pathway,the B cell receptor signalling pathway and the chemokine signalling pathway were most affected by the L321₂0267 gene of P.plecoglossicida.Meanwhile,the immune genes were related to different numbers of miRNAs and lncRNAs,and some miRNAs were related to more than one gene.This paper indicated that 1.L321₂0267 is a virulence gene of P.plecoglossicida;2.the upregulation of the immune pathways facilitated E.coioides to remove the L321₂0267-RNAi strain compared with the wild-type strain of P.plecoglossicida.3.the immune genes were regulated by miRNA and lncRNA in a complex manner.