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应用实时荧光定量PCR技术,结合生物酶和代谢产物测定,研究了氨氮急性胁迫对红螯光壳螯虾幼虾代谢及免疫系统的毒性影响及其毒后恢复情况。实验首先进行3 d的氨氮胁迫,取样后剩余虾移入曝气自来水进行7 d的毒后恢复实验。结果表明,3 d氨氮胁迫后,肌肉ACP、AKP、SOD活性表达均受到显著影响,随着氨氮浓度的升高酶活性分别降低,最高浓度组(16 mg/L)降低为对照组的76%、68%和62%。线粒体MnSOD、胞外Cu/ZnSOD的mRNA表达量也随着氨氮浓度增加而下降,最高浓度组降低至对照组的69%和68%。CAT、GPX活性以及GPX和GST的mRNA表达量变化不显著。肝胰腺中可溶性蛋白和甘油三酯含量随着氨氮浓度升高而降低,最高浓度组分别降低至对照组的72%和59%,AST活性在12 mg/L浓度组显著升高至对照组的134%。7 d恢复期过后,ACP和AKP活性以及各代谢指标恢复到正常水平;而SOD和GPX活性高于对照组。各抗氧化基因的表达量都不同程度高于对照组。实验表明,高浓度氨氮胁迫能抑制部分免疫相关酶的活性及基因表达,对免疫系统造成损害。氨氮胁迫下,红螯光壳螯虾动员蛋白质和脂肪来供能应对胁迫。7 d的恢复时间不足以让红螯光壳螯虾从胁迫中完全恢复,其肌肉仍处于轻度氧化应激状态。
The effects of acute ammonia nitrogen stress on the metabolism and immune system of juvenile red-shelled crayfishes and their post-virulence recovery were studied by real-time fluorescence quantitative PCR and bioassay and metabolite determination. The experiments were first carried out for 3 days ammonia nitrogen stress, after sampling the remaining shrimp into aerated water 7 days after the poisoning recovery experiments. The results showed that the ACP, AKP and SOD activities were significantly affected by ammonia nitrogen stress for 3 days, and decreased with the increase of ammonia nitrogen concentration. The highest concentration (16 mg / L) was reduced to 76% , 68% and 62% respectively. The mRNA expression of mitochondrial MnSOD and extracellular Cu / ZnSOD also decreased with the increase of ammonia nitrogen concentration, and the highest concentration decreased to 69% and 68% of the control. CAT, GPX activity and GPX and GST mRNA expression did not change significantly. The content of soluble protein and triglyceride in hepatopancreas decreased with the increase of ammonia nitrogen concentration, and the highest concentration group decreased to 72% and 59% of the control group respectively. The AST activity increased significantly in 12 mg / L group to the control group 134%. After 7 days of recovery, ACP and AKP activity and the metabolic parameters returned to normal levels; while the activities of SOD and GPX were higher than those of the control group. The expression of each antioxidant gene was higher than the control group. Experiments show that high concentrations of ammonia nitrogen stress can inhibit part of immune-related enzyme activity and gene expression, causing damage to the immune system. Under ammonia nitrogen stress, red-chelonian shellfish mobilize protein and fat to cope with stress. The recovery time of 7 d was not enough for the red chelonian crayfish to completely recover from the stress, and the muscle was still in a mild state of oxidative stress.