藏鸡与低地鸡胚胎NADH脱氢酶功能差异的分子机理研究

Tibet chicken has adapted itself well to hypoxia in the high altitude area. Our previous studies showed that embryos of Tibet chicken were higher in values of mitochondrial respiratory functions than those of the lowland chicken under hypoxic conditions, which suggested that keeping high mitochondrial respiratory functions be an effective approach to fit in the hypoxic environment. NADH dehydrogenase plays an important role in improving mitochondrial respiratory functions. The objective of the present project is to investigate how NADH dehydrogenase improves mitochondrial respiratory functions in Tibet chicken embryos under hypoxic conditions. Mitochondrial respiratory functions of chicken embryos including NADH dehydrogenase activity will be determined with Clark oxygen electrodes; differences of expression or/and phosphorylation of NADH dehydrogenase subunits will be detected with fluorescent dyes ( Pro-Q-Diamond / SYPRO ) combined with 2D-PAGE and biological mass spectrometry; key subunits will be found, and then confirmed by gene transfection, RNA interference and ELISA, etc. Using molecular biology and bioinformatics technology, DNA sequences of key subunits will be obtained, polymorphisms of these genes will be detected, and effects of gene mutations in key subunits will be predicted and then validated. The results will show an important molecular mechanism of Tibet chicken embryonic mitochondria for adaptation to hypoxia, which may be useful for chicken breeding at high altitude areas.

藏鸡对高原低氧环境具有可稳定遗传的适应能力。低氧条件下，较高的线粒体呼吸功能是机体低氧适应的有效途径。前期研究表明，NADH脱氢酶对提高低氧孵化的藏鸡胚胎线粒体呼吸功能有重要作用。本申请项目拟用Clark氧电极法测定鸡胚的线粒体呼吸功能及NADH脱氢酶活性；用荧光染料（Pro-Q-Diamond/SYPRO）染色定量法、双向凝胶电泳和生物质谱等技术方法分析藏鸡和低地鸡胚胎NADH脱氢酶的亚基表达和磷酸化差异；用基因转染技术、反义核酸技术和酶联免疫吸附测定等方法验证影响NADH脱氢酶功能的关键亚基；用分子生物学和生物信息学技术获得编码关键亚基基因的核苷酸序列，检测基因多态性，分析预测基因突变对基因表达水平和磷酸化位点的影响并加以验证，以期揭示NADH脱氢酶改善藏鸡胚胎线粒体呼吸功能的分子机理。所获结果可以揭示藏鸡胚胎线粒体低氧适应的重要分子遗传学基础，为培育高原优质高产鸡品种提供理论指导。

本项目的研究目标是利用转录组测序技术分析藏鸡胚胎和低地鸡胚胎在转录组上的差异，筛选与NADH脱氢酶功能有重要关系的差异基因（关键基因），并揭示藏鸡胚胎通过关键基因改善NADH脱氢酶功能的分子遗传学机理。总体来讲，本研究是按照研究计划进行的，然而，研究结果与预期并不一致。我们利用转录组测序技术分析了藏鸡胚胎和低地鸡胚胎在转录组上的差异，但未能筛选到与NADH脱氢酶功能有重要关系的差异基因（关键基因），甚至没有发现与线粒体电子传递相关的差异基因，所以我们未就NADH脱氢酶在低氧适应中的作用展开研究。尽管没有发现预期的差异基因，但是我们找到了一些与急性时相反应、脂肪代谢和机体抗氧化系统相关的一些差异基因，并进行了功能注释。以上的研究结果表明急性时相反应、脂肪代谢和抗氧化能力的差异可能是藏鸡胚胎低氧适应的主要原因，而以前发现的藏鸡胚胎较强的线粒体呼吸功能只是低氧适应的重要表现。由于本项资助时间较短强度低，我们未能对这些差异基因进行功能验证。