膜结合血红蛋白在日本沼虾低氧应激中的作用及其调控机制

Oxygen is the most important critical factor on animal’s life activities. Hypoxia refers to the fact that the animal’s life activities can not get adequate oxygen, hypoxia stress mechanisms are involved in a series of physiological and biochemical processes and regulated by fine-tuning, and respiratory proteins play a vital role in this process of hypoxic stress. Compared with mammal, hypoxic stress molecular mechanism of crustaceans is unclear. The oriental river prawn Macrobrachium nipponense, is an economically important fresh-water species cultured in China in the last few decades. Our previous studies confirmed that high abundance of membrane-bound hemoglobin genes transcripts exist in the M. nipponense, and membrane-bound hemoglobin mRNA expression are individually induced by hypoxia. On that basis, membrane-bound hemoglobin gene sequence and structure are to analysis through molecular biology and biological information technology; Through environmental dissolved oxygen simulated experiment, we will determine hypoxic stress evaluation indicator system of M. nipponense; using quantitative RT-PCR and electrophoretic mobility shift assay (EMSA), we will verify membrane-bound hemoglobin is target genes regulated by hypoxia-inducible factor-1 of M. nipponense. The present project will analysis the function of membrane-bound hemoglobin in relation to molecular processes responsed to hypoxia by RNA interference, Western blot, Co-ImmunoPrecipitation (Co-IP), immunohistochemistry (IHC) and immunofluorescence (IF) and so on. The results of this project would reveal the molecular mechanism of hypoxic stress of M. nipponense, 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.

氧是动物生命活动最重要的关键因素。低氧指机体生命活动所需的氧不能得到充足的供给，低氧胁迫下的应激机制是涉及多基因参与的复杂的生理调控过程，其中动物体内呼吸蛋白在低氧应激过程中发挥至关重要的作用。鉴于甲壳动物低氧应激的分子机制尚不知晓，本研究在建立低氧胁迫下日本沼虾鳃组织转录组文库，发现低氧应激过程中大量高丰度表达膜结合血红蛋白基因的基础上，采用分子生物学技术分析日本沼虾膜结合血红蛋白的基因序列和结构；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；采用荧光定量RT-PCR和凝胶电泳迁移率等技术，阐明日本沼虾膜结合血红蛋白为缺氧诱导因子下游调控靶基因之一；采用RNA干扰、免疫共沉淀和免疫组化等技术，解析日本沼虾膜结合血红蛋白在低氧应激中的功能。研究结果不仅有助于深入揭示日本沼虾低氧应激分子机制，还有望为选育日本沼虾耐低氧新品种提供靶标基因，从而实现学术意义与应用价值的统一。

低氧指机体生命活动所需的氧不能得到充足的供给，低氧胁迫下的应激机制是涉及多基因参与的复杂的生理调控过程，其中动物体内呼吸蛋白在低氧应激过程中发挥至关重要的作用。鉴于甲壳动物低氧应激的分子机制尚不知晓，尤其我国淡水经济虾类日本沼虾不耐低氧易泛塘的特性一直是制约其集约化规模养殖的主要瓶颈。针对这一关键问题，本项目以日本沼虾为研究对象，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建立日本沼虾低氧应激评价指标体系，相继克隆得到日本沼虾低氧应激反应中最重要的缺氧诱导因子（HIF-1）、HIF-1抑制因子（FIH）、血蓝蛋白、膜结合血红蛋白和p53全长cDNA序列，并作组织表达和低氧时空表达差异分析。在发现低氧胁迫下日本沼虾鳃组织转录组中存在大量高丰度表达膜结合血红蛋白基因的基础上，通过阻断（RNA 干扰）和诱导（重组蛋白）膜结合血红蛋白基因，查明日本沼虾膜结合血红蛋白在低氧应激中的作用，通过生物信息学分析查明调控膜结合血红蛋白表达的microRNA，为深入研究日本沼虾低氧应激调控机制提供效应靶点。项目的研究结果不仅有助于深入揭示日本沼虾低氧应激分子机制，还有望为选育日本沼虾耐低氧新品种提供检测指标，从而实现该研究学术意义与应用价值的统一。