HIF-1信号通路在低氧诱导日本沼虾线粒体自噬与凋亡中的作用及其机制

Oxygen is the most important critical factor on animal’s life activities. The oriental river prawn Macrobrachium nipponense, is an economically important fresh-water species cultured in China in the last few decades. However, dramatic die-offs are most common in M. nipponense cultured in anoxic pond that easily lead to huge economic losses. Compared with mammal, hypoxic stress molecular mechanism of crustaceans is unclear. Intriguingly, our preliminary results confirmed that hypoxia inducible factor-1 (HIF-1), autophagy and apoptosis related genes expression of M. nipponens were significantly induced by hypoxia, respectively. On that basis, the present project attempt to knowdown and induce oxygen receptor gene (proline hydroxylase) in order to clear outline HIF-1 alpha activate pathways of M.nipponense under hypoxic stress; through environmental dissolved oxygen simulated experiment, we will determine hypoxic stress evaluation indicator system, autophagy and apoptosis detection platform of M. nipponense; using dual luciferase activity assay and immuno co precipitation techniques, we will verify the BNIP3 and p53 is target genes regulated by HIF-1 of M. nipponense; the present project will analysis the function of BNIP3 and p53 in relation to molecular processes responsed to hypoxia by RNA interference, qRT-PCR, Western blot, pull down and so on. The results of this project would reveal the molecular mechanism of hypoxic stress of M. nipponense in detail, and would provide novel candidate target genes for hypoxia resistant varieties of M. nipponense, in order to achieve the unity of academic meaning and actual value.

氧是众多生物赖以生存的重要物质，日本沼虾养殖过程中经常因其不耐低氧的特性导致翻塘从而造成巨大的经济损失，迄今日本沼虾“特有”的低氧应激分子机制尚不知晓。本项目组最近发现HIF-1α、线粒体自噬与凋亡相关基因参与日本沼虾低氧应激分子过程，在此基础上，本项目拟通过阻断和诱导氧感受基因（脯氨酸羟化酶, PHD），清晰地勾勒出日本沼虾低氧应激下HIF-1α信号通路活化途径；通过环境溶解氧变化模拟实验建立日本沼虾低氧应激评价指标体系和线粒体自噬与凋亡检测平台；采用荧光素酶活性分析和免疫共沉淀等技术，阐明日本沼虾BNIP3和p53为HIF-1α潜在下游调控靶基因；采用RNA干扰、免疫印迹，pull down等技术，解析日本沼虾BNIP3和p53在低氧诱导线粒体自噬与凋亡中的功能及其分子机制。研究结果有助于解开日本沼虾不耐低氧分子机制这一谜团，同时可望为日本沼虾耐低氧新品种的选育提供靶标基因。

氧是众多生物赖以生存的重要物质，日本沼虾养殖过程中经常因其不耐低氧的特性导致翻塘从而造成巨大的经济损失，迄今日本沼虾“特有”的低氧应激分子机制尚不知晓。针对这一关键问题，本项目采用生物化学、生理学、分子生物学等技术手段，通过环境溶解氧变化模拟实验，探明HIF-1激活途径，并相继克隆得到日本沼虾低氧应激反应中线粒体自噬与凋亡相关基因（p53、caspase-3和ATG8）和糖酵解相关基因（AMPK, HK、LDH）全长cDNA序列，并作组织表达和低氧时空表达差异分析。在建立日本沼虾低氧应激评价指标体系和线粒体自噬与凋亡检测平台基础上，采用荧光素酶活性分析、免疫组化等技术，阐明日本沼虾LDH和p53为HIF-1α潜在下游调控靶基因；引进了线粒体自由基代谢的概念，并从自由基代谢失调的角度探讨了细胞凋亡蛋白在低氧应激过程中的作用；采用RNA干扰、免疫印迹，pull down等技术，解析日本沼虾细胞自噬在日本沼虾低氧应激中的保护作用。研究结果有助于解开日本沼虾不耐低氧分子机制这一谜团, 还有望为选育日本沼虾耐低氧新品种提供检测指标。