鳜鱼驯食性状基因CREB信号通路及调控机理研究

The mandarin fish (Siniperca chuatsi) as an absolutely carnivorous fish, is the most famous high valued fish species in China. Under cultivation, mandarin fish only consume live prey fish throughout their life and refuse dead prey fish or artificial diets. This problem has been widely known as a hard nut to crack in the field of aquaculture in China. The live prey fish for the commercial production of mandarin fish not only consumes large amounts of fry and fingerlings, but also brings pathogens as well as toxic chemical drugs retained in the mandarin fish due to bioconcentration. Using transcriptome sequencing and digital gene expression profiling of mandarin fish, we identified 1,986 differentially expressed genes between feeders and nonfeeders of dead prey fish. The acquisition of novel food preference (dead prey fish) was found to be related to genes controlling learning and memory. We have identified the key gene (cAMP response element binding protein (CREB)) of learning and memory related to food habit domestication traits of mandarin fish. CREB phosphorylation is required for the transcription modulation in various neuronal processes in mammals，including neurogenesis, synaptic plasticity, learning and memory. Using inhibitor and activator of CREB signaling pathway, intracerebroventricular administration, eukaryotic expression vector of CREB, cell transfection, laser confocal microscopy, luminescence immunoassay, Real time-PCR, Western Blotting, immunohistochemical methods, we will continue to identify downstream target molecular of CREB, and upstream phosphorylation and dephosphorylation pathways of CREB about the food habit domestication traits of mandarin fish. Our findings could lay the foundation for molecular mechanism of food habit domestication and molecular marker-assisted selective breeding in mandarin fish.

鳜鱼是我国淡水鱼珍品，但自开食起终生以活鱼虾为食，鳜鱼驯食人工饲料问题是制约其集约化可持续健康养殖的关键难题之一，研究鳜鱼驯食人工饲料分子机理非常重要。申请人已通过对鳜鱼驯食相关转录组分析及功能研究，发现学习记忆关键基因CREB在鳜鱼驯食中发挥重要作用，但其相关信号通路及调控机理尚不清楚。本项目采用CREB信号通路抑制剂与激活剂进行鳜鱼脑室活体注射及离体培养细胞处理，通过发光免疫分析、共聚焦成像、Real time-PCR、Western Blotting、免疫组化等方法，检测CREB基因及其下游信号分子、上游活化与去活化CREB的关键因子mRNA、蛋白表达、蛋白磷酸化水平、关键酶活性及鳜鱼驯食行为与学习能力。本项目从活体和细胞水平研究确定鳜鱼驯食基因CREB发挥作用的下游信号通路及CREB上游活化与去活化途径，为进一步阐明鳜鱼驯食分子机理及基因辅助选育易驯食鳜鱼新品种奠定基础。

学习记忆在获得新的觅食技能和食物偏好方面扮演着重要的角色。然而，鱼类通过社会学习获得新食性及其分子机制尚不清楚。鳜鱼作为典型的凶猛性肉食鱼类，其食性特殊，自开食起终身以活鱼虾为食，拒食人工饲料。本研究针对鳜鱼驯食问题，研究了社会学习在鳜鱼从食活饵驯化至食死饵过程中的作用，发现学习组鳜鱼对死饵的驯化成功率和摄食量显著高于对照组，且食死饵的学会次数显著低于对照组。再次驯化组驯化率高于初次驯化组，反应时间低于初次驯化组。说明鳜鱼可以通过互相学习获得摄食死饵的新食性，并通过记忆增强驯食。通过Western blotting、转录组测序和信号通路分析，发现学习组及再次驯化组鳜鱼的学习记忆基因c-fos、fra2、zif268、c/ebpd和sytIV表达量显著增加，抑制食欲基因pomc、leptin a表达量显著减少；学习组鳜鱼的PKA和CaMKII磷酸化水平明显高于对照组。通过PKA和CaMKII信号通路抑制剂处理及染色质免疫沉淀技术分析，进一步发现鳜鱼驯食相关学习记忆通路是由CaMKII激活后，刺激c-fos基因表达，而c-Fos作为一个重要的转录因子抑制了抑食欲基因pomc的表达，从而提高鳜鱼食欲，促进驯食。