Purpose:Salinity is an important environmental factor that affects the physiological activities of fish. Flavobacterium cloumnare is a major aquaculture pathogen infecting various saltwater and freshwater fish. The goals of this study are investigating the mechanism of the immune responses to Flavobacterium cloumnare in grass carp under saline-alkali stress. Methods: Grass carp individuals, averaging 12 cm in body length, were obtained from Duofu fish farm (Wuhan, China) and cultured at recirculating aquaculture system for 2 weeks before the experiment began. For the challenge, all grass carp were randomly divided into three groups, and then cultured at saline-alkali water with the concentration of 0, 3‰ and 6‰. After 30 days, Groups of grass carp were were infected with 2 × 105 CFU/mL Flavobacterium cloumnare G4 strains for 3 h .Gills from each group at 24 hpi, 3dpi and 6dpi were collected. Total RNA of all samples was isolated using TRIzol® Reagent (Invitrogen) according to the manufacturer's introduction. gill per group at 24 hpi and 48 hpi were rinsedRNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent, USA). Samples with RNA integrity numbers (RINs) = 7.5 were subjected to cDNA library construction using TruseqTM RNA sample prep Kit (Illumina). Results:A total of 27 were processed for transcriptome sequencing, generating 177.79Gb Clean Data. At least 5.73Gb clean data were generated for each sample with minimum 93.70% of clean data achieved quality score of Q30. Clean reads of each sample were mapped to specified reference genome. Mapping ratio ranged from 88.11% to 92.33%. The expression of genes was quantified and differentially expressed genes were identified based on their expression.Criteria for differentially expressed genes was set as Fold Change(FC)=1.5 and Pvalue<0.05. Fold change(FC) refers to the ratio of gene expression in two samples. These DEGs were further processed for functional annotation and enrichment analysis. Conclusions: Our study represents the immune response of zebrafish against Flavobacterium cloumnare infection in saline-alkali stress conditions, and reveal the discrepant expression pattern of NOD-like pattern recognition receptors in the gills. Overall design: Total RNA was solated from gills of grass carp at 24 hpi, 3dp and 7dpi after cutured at saline-alkali water with the concentration of 0, 3‰ and 6‰ for 30 days