基于AMPK通路的沼泽型水牛与荷斯坦奶牛耐热差异的分子机理研究

Heat stress is an imbalance between heat production within the body and its dissipation in dairy cows, which is a major limiting factor in dairy industry production. AMP-activated protein kinase (AMPK) is involved in the regulation of HSP (Heat Shock Proteins) gene expression, and plays an important regulatory role in glucose metabolism and fat metabolism for ATP generation. Although swamp buffaloes (Bubalus bubalis) have strong heat tolerance compared to Holstein cows (Bos taurus), the molecular mechanisms of the difference in heat tolerance for swamp buffaloes and Holstein dairy cows are not yet fully understood. In this study, we plan to select Holstein cows (high milk yield and low heat stress tolerance) during the dry period as contrast control, and a combined transcriptomics and proteomcis approaches will be used to analyze the effects of environmental temperature (37℃ VS 20℃) on the gene transcription and protein expression for the swamp buffaloes and Holstein cows in the AMPK signalling pathway, analyze the roles of differential expression genes and proteins in AMPK signalling and validate expression level of important genes and proteins using Q-PCR and Western blot. Based on behaviour, physiological and biochemical parameters related to heat stress and the activity of rate-limiting enzymes in energy metabolism, these results will reveal the molecular mechanisms of the difference in heat tolerance for swamp buffaloes and Holstein dairy cows, and identify molecular markers related to thermotolerance and energy metabolism to settle the basis for the health development of dairy industry in tropical areas and explore and utilize characteristic gene of the swamp buffaloes.

奶牛热应激是体内产热与散热失衡造成的，严重制约奶业的健康发展。腺苷酸活化蛋白激酶（AMPK）涉及热休克蛋白基因表达的调控，对ATP生成的糖代谢和脂肪代谢关键限速酶的活性起重要调节作用。沼泽型水牛比荷斯坦奶牛耐热，但耐热差异的分子机理仍不明确。本研究以热敏感的荷斯坦干奶牛为对照，利用蛋白质组与转录组学关联分析技术在AMPK通路上研究环境温度（37℃ VS 20℃）对沼泽型水牛与荷斯坦干奶牛热应激和能量代谢相关基因的转录与蛋白质表达的影响；分析差异转录基因与差异表达蛋白的关联性及其在AMPK通路上的作用，对有关联的差异转录基因与表达的蛋白进行荧光定量PCR和Western blot表达量验证；结合耐热行为、热应激生理生化参数及能量代谢关键酶的活性，在AMPK通路上揭示耐热差异的分子机理，挖掘耐热分子标记。研究结果将为热带奶业健康的发展和沼泽型水牛种质特色基因的开发与利用奠定基础。

热应激危害奶畜健康，降低生产水平。荷斯坦奶牛与奶水牛是世界上最重要的两种奶畜，但是它们热应激环境适应的策略仍然未能明确。首先利用代谢组学技术测试比较荷斯坦奶牛与奶水牛在热中性与慢性热应激环境中的差异代谢物。荷斯坦奶牛在热应激状态下，升高体温与呼吸频率以实现散热。热应激影响血清氨基酸、脂肪酸与胆汁酸代谢。荷斯坦奶牛的适应策略是采取瓜氨酸与脯氨酸的代谢，而奶水牛采取支链氨基酸、生酮氨基酸与糖异生为主的代谢策略实现热应激。从生理与代谢策略来看，奶水牛较荷斯坦奶牛耐热。对慢性热应激与热舒适环境下的奶水牛血清（去除高丰度蛋白）开展非标蛋白质组学测试，获得17个差异表达蛋白，对其中的6个蛋白质进行PRM定量验证。结果发现，脂肪水解酶（LPL）、谷胱甘肽过氧化物酶（GPX3）、抗菌肽（CATHL2）、CP（血浆铜蓝蛋白）与血红蛋白（HBA1）在降低产热、增加血氧运输、提高机体病原菌的抵抗力等方面协同作用，增强奶水牛的耐热性能。