鳜鱼T1R1启动子甲基化调控驯食分子机理研究

Mandarin fish 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. The intake and utilization of artificial diets as well as growth performance of mandarin fish were different in cultured stocks and wild populations, which were not only related to genetics, but also to learning, memory and adaption to accept artificial diets. Our previous studies have found that the methylation of T1R1 promoter and expression of learning and memory genes in brain were involved in feed habit domestication of mandarin fish. In the present study, the effects of learning and memory factors on T1R1 mRNA expression, T1R1 promoter activities and methylation status, methyltransferase expression and interactions between methyltransferase and T1R1 promoter will be examined by real-time quantitative PCR, Western blot, double luciferase reporter system, bisulfite sequencing PCR and chromatin immunoprecipitation. Correlation analysis between T1R1 promoter methylation pattern and feed habit demostration traits will be evaluated by ICV injection and early imprinting with recombinant protein of learning and memory factors. To investigate the relationship between feed habit domestication traits and epigenetic, the genetic pattern of T1R1 promoter methylation will be analyzed by comparing the methylation status of parents and offspring with different feed habit domestication traits. Our findings could lay the foundation for molecular marker-assisted selective breeding for good families or clones to greatly promote the intake and utilization of artificial diets in mandarin fishes.

鳜鱼食性奇特，自开食起终生以活鱼虾为食，通常情况下拒食配合饲料，鳜鱼人工饲料可控养殖问题成为水产界世界级的难题而亟待研究解决。本项目在发现鳜鱼驯食过程涉及脑T1R1启动子甲基化调控和学习记忆基因差异表达的基础上，从离体水平上，利用过表达载体、亚硫酸氢盐测序PCR、实时荧光定量PCR、Western blot、双荧光素酶报告系统及染色质免疫共沉淀等技术，深入研究鳜鱼驯食相关学习记忆因子对T1R1基因mRNA表达、T1R1基因启动子活性及其甲基化模式、甲基转移酶表达及其结合启动子情况的调控作用。从活体水平上，通过颅腔注射与早期印记处理，验证鳜鱼学习记忆因子调控脑T1R1启动子甲基化模式及其与驯食性状表型的关系。通过比较鳜鱼亲代父母本与后代脑T1R1启动子甲基化模式，探讨鳜鱼亲代基于个体学习记忆的驯食性状的后代表观遗传机制问题，为促进易驯食人工饲料鳜鱼新品系成功培育奠定必要的理论基础。

鳜鱼食性奇特，自开食起终生以活鱼虾为食，通常情况下拒食配合饲料，鳜鱼人工饲料可控养殖问题成为水产界世界级的难题而亟待研究解决。探讨鳜鱼对人工饲料的适应机理，特别是研究确定鳜鱼驯化适应中可遗传的表观遗传机制，对于培育易驯食饲料鳜鱼品系并最终解决鳜鱼人工饲料可控养殖问题具有重要意义。本项目研究发现，学习记忆在鳜鱼食性驯化中发挥重要作用，二次驯化成功率达100％，且二次驯化后鳜鱼对死饵料鱼的反应时间显著降低，二次驯化鳜鱼脑中与长时程记忆形成相关基因mRNA表达水平显著升高，鲜味受体t1r1基因启动子DNA甲基化水平和mRNA表达水平均显著降低，表明反复驯食可使鳜鱼形成特定记忆，提高驯化成功率。用学习记忆因子c-fos的抑制剂处理鳜鱼脑细胞后，c-fos和t1r1 mRNA表达水平均显著降低，用c-fos上游因子CaMKⅡ的抑制剂处理鳜鱼脑细胞后，c-fos和t1r1基因mRNA表达水平均显著升高，表明学习记忆因子c-fos可调控鳜鱼鲜味受体t1r1表达。鳜鱼T1R1对L-赖氨酸刺激反应最为强烈、其次为L-谷氨酸和L-精氨酸，饲料中添加赖氨酸和谷氨酸后，鳜鱼摄食量增加。同时，本项目研究发现亲代甲基化可遗传给子代，早期糖印记能够通过提高糖异生相关基因pepck1甲基化水平降低其mRNA表达水平，从而提高成年期鳜鱼对糖的利用，且亲代pepck1基因DNA甲基化可遗传到子代，提高子代糖利用能力。本项目研究结果可为促进易驯食人工饲料鳜鱼新品系成功培育奠定必要的理论基础。