生物絮团中聚β-羟基丁酸酯（PHB）对梭鱼免疫调控的研究

Biofloc technology is a zero-water exchange, environment-friendly farming system and it has been widely used in aquaculture since it could enhance the fish growth and immunity. Poly-β-hydroxybutyrate (PHB) in bioflocs is an importantly activated compound and it could stimulate the immune response of aquatic species, especially for fish. However, there is little known about its molecular mechanisms in fish upon PHB stimulation. In our laboratory, we have extracted the PHB from bioflocs and established PHB feeding methods. In this proposal, we will select the soiny mullet (Liza haematocheila), an important aquaculture species as model system to investigate the molecular mechanisms how PHB could enhance the immunity. The main research subjects include: (1) with biochemical analytic methods to analyze the immune-related enzymatic changes upon PHB treatment; (2) applying RNA-seq technology to comparatively analyze the transcriptomic profile in L. haematocheila upon PHB stimulation, and unigenes functional annotation and pathway will be predicted through bioinformatics methods; (3) the crucial genes related to immunity will be screened and their spatial differential expression status will be assayed using qRT-PCR technology, so as to reveal their potential function； Finally，(4) the promoters of target genes will be cloned and sequenced, and then plasmid without promoters will be constructed, with which how PHB could affect the activity of promoters will be assayed. Therefore, this study will elucidate the enzymatic and molecular mechanisms of L. haematocheila against PHB stimulation at both transcriptional level and post-transcriptional level. Thus, our study will provide a comprehensive understanding of the molecular mechanisms in fish with biofloc systems and it embraces great significances in both basic theory and practical application in aquaculture.

生物絮团技术是一种新兴的零排放生态养殖技术，在水产养殖中具有广阔的应用前景。聚β-羟基丁酸酯 (poly-β-hydroxybutyrate，PHB) 是生物絮团中的重要活性成分，可提高养殖动物免疫力，但与其作用相关的调控机制尚不清楚。项目组已从生物絮团中分离PHB并构建了鱼类最佳投喂模式。本项目拟以江苏重要养殖鱼类—梭鱼为养殖对象，研究PHB的免疫调控机制。主要内容：①采用生化技术分析PHB对梭鱼免疫相关酶酶活的影响；②采用RNA-seq技术构建PHB刺激后mRNA表达谱，筛选差异表达基因，分析其特征和调控网络；③采用qRT-PCR分析差异基因的时空表达，揭示PHB的空间作用靶点和影响度；④分子克隆靶基因启动子序列，分析启动子功能。本研究将从酶和基因水平揭示PHB对鱼类免疫的调控机制，有望为新型养殖技术的推广应用提供理论基础和技术支撑。因此，本项目具有重要的科学意义和较高的应用价值。

生物絮团技术是一种新兴的零排放生态养殖技术，在水产养殖中具有广阔的应用前景。聚β-羟基丁酸酯(poly-β-hydroxybutyrate，PHB)是生物絮团中的重要活性成分，可提高养殖动物免疫力，但与其作用相关的调控机制尚不清楚。本项目构建了生物絮团中PHB的最佳提取工艺；SEM-EDS观察显示，PHB中C、O的百分含量分别是63.3%和36.7%；红外光谱FTIR分析表明，PHB在1724.07 cm-1处有一强烈吸收峰值；GS-MS测定PHB保留时间为2.214。梭鱼PHB的最佳投喂方式为：2%添加量，30 d，或0.5%添加量，60 d。饲投PHB改变梭鱼肠道菌群，芽孢杆菌丰度显著上升；饲投PHB显著提高梭鱼的抗氧化能力；PHB的免疫调控作用与改善肠道菌群和提高抗氧化能力密切相关。RNA-seq测序表明，与免疫相关的差异表达基因主要富集于MAPK、PI3K-Akt、TNF、T cell receptor、Toll-like receptor、NF-κB等信号通路。以梭鱼原代培养巨噬细胞为研究模型，PHB促进巨噬细胞增殖、抑制LPS诱导的炎症反应，PHB通过调控促炎因子和抑炎因子的表达维持体内抗氧化能力的动态平衡，进而达到增强免疫力的功能。MAP3K8基因在PHB调控机体免疫功能中发挥重要作用，MAP3K8基因有3个核心启动子区和2个CpG岛；在第一、二和三个核心启动子区分别有11个、5个和4个转录因子结合位点，在第一和第二个CpG岛处分别有12个和23个转录因子结合位点。此外，PHB具有增强养殖动物抗病毒感染作用，抑制病毒增殖。本项目研究结果为深入揭示PHB对养殖动物的免疫调控机制具有重要的科学意义，对进一步饲用推广PHB及绿色水产养殖具有重要实际意义。. 本项目资助共发表学术论文9篇，其中SCI收录7篇；授权发明专利1件，申请发明专利2件；获中国商业联合会一等奖和市级自然科学优秀学术论文奖各1项。