Effects and molecular regulation mechanisms of saline-alkali stress on the healthy grass carp [exp2]. Effects and molecular regulation mechanisms of saline-alkali stress on the healthy grass carp [exp2]

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

研究背景与目的：盐度（Salinity）是影响鱼类生理活动的重要环境因子。柱状黄杆菌（Flavobacterium cloumnare）是一类可感染多种海水及淡水鱼类的主要水产养殖致病菌。本研究旨在探究盐碱胁迫（saline-alkali stress）下草鱼（grass carp）对柱状黄杆菌的免疫应答机制。
实验方法：选取平均体长12 cm的草鱼，采购自中国武汉多福渔场，于循环水产养殖系统（recirculating aquaculture system）中驯养2周后开展实验。攻毒实验前，将所有草鱼随机分为3组，分别置于盐度为0、3‰、6‰的盐碱水体中饲养。饲养30天后，用2×10^5 CFU/mL的柱状黄杆菌G4菌株侵染各组草鱼，侵染时长3小时。分别于感染后24小时（24 hpi）、3天（3 dpi）及6天（6 dpi）采集各组草鱼的鳃组织。所有样本的总RNA采用TRIzol®试剂（TRIzol® Reagent，Invitrogen公司）依照厂商说明书进行提取。于24 hpi及48 hpi采集的各组鳃组织经漂洗后，使用Agilent 2100生物分析仪（Agilent, 美国）评估RNA完整性。RNA完整性数（RINs）≥7.5的样本采用TruseqTM RNA样本制备试剂盒（TruseqTM RNA sample prep Kit，Illumina公司）进行cDNA文库构建。
实验结果：共计27个样本进行转录组测序，共产出177.79 Gb清洁数据（Clean Data）。每个样本的清洁数据量至少为5.73 Gb，且至少93.70%的清洁数据质量值达到Q30。将每个样本的清洁reads比对至指定参考基因组，比对率范围为88.11%~92.33%。对基因表达量进行定量，并基于表达量差异鉴定差异表达基因（DEGs）。差异表达基因的筛选标准设置为：倍数变化（Fold Change, FC）≥1.5且P值（Pvalue）<0.05，其中倍数变化指两组样本间的基因表达量比值。对上述差异表达基因进一步开展功能注释及富集分析。
研究结论：本研究揭示了斑马鱼（zebrafish）在盐碱胁迫条件下针对柱状黄杆菌感染的免疫应答，并阐明了鳃组织中NOD样模式识别受体（NOD-like pattern recognition receptors）的差异表达模式。
实验整体设计：将草鱼置于盐度为0、3‰、6‰的盐碱水体中饲养30天后，分别于感染后24 hpi、3天（3 dpi）及7天（7 dpi）采集其鳃组织，提取总RNA。