斑马鱼中kdm4aa基因对低温耐受能力的影响

Effect of kdm4aa Gene on Low Temperature Tolerance of Zebrafish

为了研究赖氨酸特异性去甲基化酶4aa(lysine demethylase 4aa,kdm4aa)在斑马鱼(Danio rerio)低温耐受能力中的作用,本研究通过实时定量PCR检测kdm4aa^(-/-)斑马鱼中kdm4其他家族成员的表达水平和低温处理后野生型(wild type,WT)斑马鱼中kdm4aa基因的表达水平;通过斑马鱼行为轨迹跟踪系统检测低温下kdm4aa^(-/-)幼鱼的运动能力;采用逐级降温方法检测kdm4aa^(-/-)斑马鱼低温耐受能力;通过DCFH-DA荧光探针、ATP检测试剂盒、Western blot检测低温下kdm4aa^(-/-)斑马鱼中活性氧(reactive oxygen species,ROS)、腺苷三磷酸(adenosine triphosphate,ATP)、磷酸化组蛋白H2A.X(gamma histone H2A.X,γH2A.X)和微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)水平。实时定量PCR结果显示,kdm4aa^(-/-)斑马鱼中kdm4ab、kdm4b基因的mRNA水平补偿性增加,kdm4c基因的mRNA水平与野生型斑马鱼的相比无显著性差异。斑马鱼行为轨迹跟踪系统检测发现,在18℃低温下,kdm4aa基因失活后斑马鱼幼鱼的移动距离显著增加。8℃低温下,野生型斑马鱼中kdm4aa基因的mRNA水平显著升高。与野生型斑马鱼相比,低温处理后kdm4aa^(-/-)斑马鱼的半数致死时间LT50显著降低(P<0.0001);ROS水平显著升高(P<0.0001);ATP含量显著降低(P<0.01);γH2A.X蛋白水平上升;LC3蛋白水平下降。结果表明:在低温环境下,kdm4aa^(-/-)斑马鱼的ROS积累导致ATP合成能力降低和DNA损伤增加,同时kdm4aa^(-/-)斑马鱼自噬能力降低,从而使斑马鱼的低温耐受能力降低。

In order to study the function of kdm4aa on low temperature tolerance of zebrafish,the levels of kdm4 families in kdm4aa^(-/-)zebrafish and the level of kdm4aa in wild type(WT)zebrafish treated by low temperature were detected by real time quantitative PCR.The locomotor ability of kdm4aa^(-/-)juvenile fish at low temperature was detected by the behavior trajectory tracking system of zebrafish.The low temperature tolerance of kdm4aa^(-/-)zebrafish was measured by a stepwise cooling method.Reactive oxygen species(ROS),adenosine triphosphate(ATP),gamma histone H2A.X(γH2A.X),and microtubule-associated protein 1 light chain 3(LC3)levels in kdm4aa^(-/-)zebrafish were determined by DCFH-DA fluorescent probe,ATP detection kit and western blot,respectively.Real time quantitative PCR results showed that compared with WT zebrafish,the mRNA levels of kdm4ab and kdm4b in kdm4aa^(-/-)zebrafish were significantly increased,while no significantly different of kdm4c was observed.The detection of zebrafish behavior trajectory tracking system showed that the movement distance of juvenile zebrafish increased significantly after kdm4aa knockout at a low temperature of 18℃.At a low temperature of 8℃mRNA level of kdm4aa in wild type zebrafish was significantly increased.Compared to WT zebrafish,the median lethal time LT50 of kdm4aa^(-/-)zebrafish was significantly decreased after low temperature treatment(P<0.0001);ROS level was significantly increased(P<0.0001);ATP content was significantly decreased(P<0.01);The level ofγH2A.X protein increased;LC3 protein levels decreased.These results showed that ROS accumulation of kdm4aa^(-/-)led to decreased ATP synthesis and increased DNA damage,and the autophagy ability of kdm4aa^(-/-)zebrafish was decreased,thus reducing the low temperature tolerance of zebrafish.