鳜鱼驯食性状相关基因功能分析与机理研究

Mandarin Fish, an absolutely carnivorous fish, usually refuse to eat dead food or man-made feed due to its specific feeding habit. 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. Our research group has obtained the transcriptome database of mandarin fish from the brain, liver and other tissues of two groups (200 individuals easy to domesticate and 200 individuals hard to domesticate). Based on the results of food habit domestication-related genes screened from the transcriptome database of mandarin fish, we have analyzed and identified the SNPs for the food habit domestication traits-related genes, and four SNPs have been found for the first time using methods including CRS-PCR, PCR-SSCP and SB-ASA. We will continue to explore the SNPs of the mandarin fish genes related to pathways of food appetite control, retinal photosensitivity, circadian rhythm, metabolism, learning and memory, in order to clarify the relation between gene expression and nucleotide polymorphism. In addition, in vitro cell system, isotope-tracing methods, confocal imaging, radioimmunoassay, real time-PCR, Western blotting, immunohistochemistry, enzyme assay and other test methods will be used to study the relationship between signal pathway and the traits of food habit domestication in mandarin fish. Our study will lay the foundation for the future work of molecular marker-assisted selective breeding in mandarin fish.

鳜鱼驯食人工饲料是水产界世界级难题，具重要理论与应用价值。项目组在易驯食与不易驯食鳜鱼脑、肝等组织转录组及数字表达谱测序基础上，已筛选得到鳜鱼驯食性状相关基因，并已克隆获得其中9个摄食相关基因的基因组全序列，采用易与不易驯食鳜鱼各200尾通过CRS-PCR、PCR-SSCP、SB-ASA等方法对其SNP位点进行了鉴定与相关性分析，得到了4个鳜鱼驯食相关SNP位点。本项目拟进一步对其它21个涉及摄食调控通路、视网膜感光通路、昼夜节律通路、消化代谢通路、学习记忆通路的鳜鱼驯食性状相关基因SNP位点进行鉴定与分析，并采用细胞转染、同位素标记法、放射性免疫分析法、共聚焦成像、Real time-PCR、Western Blotting、免疫组化、酶活性测定等方法，重点研究这些基因的信号通路及作用途径。本项目从基因水平上探讨鳜鱼驯食性状的决定机制，将为基因辅助选育易驯食优良鳜鱼新品种、品系奠定基础。

本项目以我国特有肉食性鳜鱼为试验材料，在易驯食与不易驯食鳜鱼脑、肝组织转录组及数字表达谱测序基础上，对其驯食性状相关基因进行功能分析及机理研究。通过转录组和数字基因表达谱测序，项目成员在易驯食与不易驯食鳜鱼中分别发现4,768和41个潜在SNP位点。对鳜鱼摄食调控通路(NPY、Leptin)、学习记忆通路(PP1)、消化代谢通路(PEP、TRY、AMY)、脂代谢通路(LPL、HL)、生长通路(GH)驯食性状相关基因SNP位点与鳜鱼驯食性状相关性研究表明：鳜鱼NPY有3个SNP位点，而LEP中未发现SNP位点。其中，位于NPY 5’调控区的-1258A/C SNP位点与鳜鱼驯食性状有强烈的相关性；克隆得到鳜鱼四个PP1亚型，在易驯食鳜鱼ppp1caa和ppp1cb中检测到SNP位点，且其在易驯食鳜鱼脑中mRNA表达显著升高，这可能是使易驯食鳜鱼对摄食活饵长期记忆衰退的一种潜在的机制；在鳜鱼PEP和TRP基因中分别发现2个和1个SNP位点，其中PEP基因D1和D5型与鳜鱼驯食性状有强烈相关性；在鳜鱼LPL基因中发现3个单核苷酸多态性位点，其中Diplotype 2与驯食性状显著相关，而HL基因中未发现单核苷酸多态性位点；在鳜鱼Gh基因中发现4个SNP位点，其中3个位点与其生长性状显著相关。此外，项目团队进一步对鳜鱼驯食性状相关基因UCP开展了基因功能研究，揭示了其在鳜鱼中的代谢调控作用。最后，项目团队开发了主要鳜亚科鱼类微卫星分子标记，并对鳜属5种鱼类进行微卫星遗传多样性分析，为鳜鱼分子标记辅助育种提供候选微卫星标记。本研究从分子水平上揭示了鳜鱼驯食性状的决定机制，并得到了一系列鳜鱼驯食性状遗传选育的候选分子标记，为基因辅助选育易驯食优良鳜鱼新品种、新品系奠定了基础。