淡水养殖条件下急性缺氧对凡纳滨对虾表观特征、相关酶活性与基因表达的影响

Effects of acute hypoxia on apparent characteristics,relevant enzyme activity and gene expression of Litopenaeus vannamei under fresh water

【目的】深入挖掘凡纳滨对虾应答缺氧胁迫的生理调控机制,为凡纳滨对虾抗缺氧应激新品种的选育提供理论依据,也为营养素调控缺氧应激提供新的作用靶点。【方法】根据水体溶解氧(DO)含量,分别设对照组(5.00 mg/L DO)、低氧组(2.00 mg/LDO)和缺氧组(0.75 mg/LDO),观测急性缺氧胁迫下凡纳滨对虾的表观特征,于胁迫0、2、6、10和14 h采集凡纳滨对虾肝胰腺组织样品,通过ELISA试剂盒检测LDH、GCK、PK、Cyt c、SOD和CS活性,利用RT-PCR扩增HIF1-α、GLUT、BNIP3、p53、c-fos、Kv1.2、PTEN和VEGF等8个基因,并采用实时荧光定量PCR检测缺氧胁迫下的基因表达变化。【结果】急性缺氧胁迫下,凡纳滨对虾频繁游动,摄食量慢或不进食、蜕壳不遂,虾壳呈黄色或红色,生长速度缓慢并逐渐出现死亡。凡纳滨对虾肝胰腺中HIF1-α、GLUT、BNIP3和VEGF基因相对表达量随缺氧时间的延长呈显著上升趋势(P<0.05,下同),Kv1.2和PTEN基因相对表达量呈显著下降趋势,c-fos和p53基因相对表达量则表现为先升高后降低。缺氧组凡纳滨对虾肝胰腺中LDH、PK和Cyt c的活性随缺氧时间的延长呈显著下降趋势,且始终低于低氧组,而GCK、SOD和CS的活性呈先上升后下降趋势。【结论】凡纳滨对虾会通过产生各种生理生化调节策略来抵御急性缺氧胁迫,包括改变机体能量获取方式、增加能量积累、高表达糖代谢或有氧氧化过程的关键酶、抑制血红细胞凋亡、降低机体代谢率、刺激血管生成等,以降低机体对DO的消耗。

【Objective】To further explore the physiological regulatory mechanisms of Litopenaeus vannamei in response to hypoxic stress,and to provide a theoretical basis for breeding new L.vannamei varieties resistant to hypoxic stress,and a new target for nutrition regulation of hypoxic stress response.【Method】According to dissolved oxygen(DO)content,control group(5.00 mg/L DO),low oxygen group(2.00 mg/L DO)and hypoxia group(0.75 mg/L DO)were set to observe apparent characteristics of L.vannamei under acute hypoxic stress.The hepatopancreas tissue samples were collected at 0,2,6,10 and 14 h under hypoxic stress,then relevant enzyme activity of LDH,GCK,PK,Cyt c,SOD and CS was detected by ELISA reagent,and HIF1-α,GLUT,BNIP3,p53,c-fos,Kv1.2,PTEN and VEGF genes of L.vannamei were amplified by RT-PCR,and the real-time fluorescence quantitative PCR was used to detect the expression of these genes under hypoxia stress.【Result】Under hypoxic stress,the shrimp swam frequently,with slow or no food intake,started to grow slowly,shell unsuccessfully,and shrimp shell turned yellow or red then gradually died.The rela-tive expression of HIF1-α,GLUT,BNIP3 and VEGF genes in hepatopancreas of L.vannamei increased significantly as the hypoxia time went on(P<0.05,the same below);however,relative expression of Kv1.2 and PTEN genes showed a significant decreasing trend,while the expression of c-fos and p53 genes first increased and then decreased.The enzyme activity of LDH,PK and Cyt c all decreased significantly in the acute hypoxia group which were always lower than low oxygen group,the GCK,SOD and CS enzyme activity increased first and then decreased.【Conclusion】L.vannamei can resist acute hypoxic stress by producing various physiological and biochemical regulatory strategies,which includes changing the way of energy acquisition,increasing energy accumulation,high expression of key enzymes of glucose metabolism or aerobic oxidation process,inhibiting red blood cell apoptosis,reducing the body’s metabolic rate and stimulating angiogenesis,so as to reduce the body’s DO consumption.