溶解氧昼夜变化对鳜血液生理生化指标及血红蛋白基因表达的影响

Effects of diel variation of dissolved oxygen on blood physiologi⁃cal and biochemical indexes and hemoglobin genes response ex⁃pression in Siniperca chuatsi

【目的】探究养殖水体溶解氧昼夜变化下鳜(Siniperca chuatsi)血液生理生化指标及血红蛋白基因(Hb)应答的变化规律,为揭示鱼类的短期低氧适应机制提供理论依据。【方法】以健康的鳜幼鱼为研究对象,通过转录组测序和基因组测序数据匹配,鉴定出鳜Hb基因序列,并进行生物信息学分析。在7:00、11:00、17:00和23:00等4个时间点采集鳜血液样品,使用PROCANPE-6800血细胞分析仪测定红细胞数、血红蛋白含量、红细胞压积、平均红细胞血红蛋白浓度和平均红细胞体积,采用实时荧光定量PCR检测Hb基因的表达变化。【结果】共鉴定获得11个鳜Hb基因,包括6个α型Hb基因(hbae4、hbae、hbae1.1、hbae5、hbaa1和hbaa1.1)及5个β型Hb基因(hbbe2、hbba2.1、hbba1、hbba1.1和hbba2);除hbae4基因序列由6个外显子和5个内含子构成外,其余10个基因序列均由3个外显子和2个内含子构成。11个鳜血红蛋白均不存在跨膜结构和信号肽,亚细胞定位于细胞质,其二级结构由α-螺旋、β-折叠、延伸链和无规则卷曲构成。池塘水体溶解氧含量存在周期性的昼夜变化;溶解氧含量最低时(7:00),鳜血液红细胞数、红细胞压积、血红蛋白含量和平均红细胞血红蛋白浓度均最高,显著高于其他3个时间点(P<0.05,下同);溶解氧含量最高时(17:00),红细胞数、红细胞压积和血红蛋白含量则显著低于其他3个时间点;平均红细胞体积在4个时间点无显著变化(P>0.05)。hbae4、hbae、hbae1.1、hbae5、hbaa1.1、hbbe2、hbba2.1、hbba1和hbba2基因的相对表达量均随溶解氧昼夜变化呈先上升后下降趋势,于17:00时达最高值,且这9个Hb基因在4个时间点的表达模式基本一致,即低溶解氧时相对表达量最低,高溶解氧时相对表达量最高;而hbaa1和hbba1.1基因相对表达量与溶解氧含量成反比。【结论】鳜通过调节红细胞数、血红蛋白含量和平均红细胞血红蛋白浓度等机制及调控Hb基因表达,以适应溶解氧的昼夜变化,从而保持机体供氧稳定。

【Objective】This study aimed to investigate the response patterns of blood physiological and biochemical indexes and hemoglobin genes(Hb)expression of Siniperca chuatsi under the condition of diel variation of dissolved oxygen in aquaculture water,providing theoretical basis for further study on the short-term hypoxia adaptation mechanism in fish.【Method】Healthy juvenile S.chuatsi were used as research objects.The Hb genes sequences were identified by matching the transcriptome sequencing and genome sequencing data,and bioinformatics analysis was carried out.Blood samples were collected at 4 consecutive time points:7:00,11:00,17:00 and 23:00.Red blood cell(RBC)count,hemoglobin(HGB)concentration,hematocrit(HCT),mean corpuscular hemoglobin concentration(MCHC)and mean corpuscular volume(MCV)were determined by using a PROCANPE-6800 blood cell analyzer.The expression levels of Hb genes were detected by real-time fluorescence quantitatively PCR(qRT-PCR).【Result】A total of 11 Hb genes were identified,including sixα-Hb genes(hbae4,hbae,hbae1.1,hbae5,hbaa1 and hbaa1.1)and fiveβ-Hb genes(hbbe2,hbba2.1,hbba1,hbba1.1 and hbba2).Except the hbae4 gene sequence,which was composed of 6 exons and 5 introns,the other 10 gene sequences were all composed of 3 exons and 2 introns.The 11 S.chuatsi hemoglobin had no transmembrane structure or signal peptide and the subcell were located in the cytoplasm.Their secondary structures were composed ofα-helix,β-turn,extended chain and random coil.A periodic diel variation of dissolved oxygen content was observed in the pond water.RBC count,HCT,HGB concentration and MCHC were the highest when the dissolved oxygen concentration was the lowest(7:00),which were significantly higher than those at the other three time points(P<0.05,the same below).At the highest dissolved oxygen concentration(17:00),RBC count,HCT,and HGB concentration significantly lower than those at the other three time points.There was no significant change in MCV among the four time points(P>0.05).The relative expression levels of hbae4,hbae,hbae1.1,hbae5,hbaa1.1,hbbe2,hbba2.1,hbba1 and hbba2 genes showed an overall trend of first increasing and then decreasing with diel variation of dissolved oxygen,reaching the highest value at 17:00.The expression patterns of these nine Hb genes at the four time points were basically the same,that was,the relative expression level was the lowest at low dissolved oxygen concentration and the highest at high dissolved oxygen concentration.The relative expression of hbaa1 and hbba1.1 genes were inversely proportional to dissolved oxygen concentration.【Conclusion】S.chuatsi adapts to diel variation of dissolved oxygen by regulating RBC count,HGB concentration,MCHC and Hb gene expression to maintain stable oxygen supply to the body.