College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, 471023, People's Republic of China.
Laboratory of Aquatic Animal Nutrition and Physiology, Fisheries College, Jimei University, Xiamen, 361021, China.
Fish Physiol Biochem. 2020 Dec;46(6):2389-2402. doi: 10.1007/s10695-020-00883-9. Epub 2020 Oct 7.
The gene encoding HSP70 was isolated from Microptenus salmoides by homologous cloning and rapid amplification of cDNA ends (RACE). The HSP70 transcripts were 2116 bp long and contained 1953 open reading frames encoding proteins of 650 amino acids with a molecular mass of 71.2 kDa and theoretical isoelectric point of 5.22. The qRT-PCR analysis showed that the HSP70 gene was differentially expressed in various tissues under normal conditions, and the highest HSP70 level was observed in the spleen and the lowest levels in the muscle and heart. The clear time-dependent expression level of HSP70 was observed after bacterial challenge and heat stress. A significant increase in HSP70 expression level was detected and reached a maximum at 3 h and 6 h in liver, spleens and gill tissues after Aeromonas hydrophila infection and heat stress, respectively (P < 0.05). As time progressed, the expression of HSP70 transcript was downregulated and mostly dropped back to the original level at 48 h. The concentration of cortisol, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased as the time of stress progressed, with the highest level found on 3 h and later declined rapidly and reached to the control levels at the 48 h. Those results suggested that HSP70 was involved in the immune response to bacterial challenge and heat stress. The cloning and expression analysis of the HSP70 provide theoretical basis to further study the mechanism of anti-adverseness in Microptenus salmoides.
从麦穗鱼中通过同源克隆和快速扩增 cDNA 末端 (RACE) 分离出编码 HSP70 的基因。HSP70 转录本长 2116bp，包含编码 650 个氨基酸蛋白质的 1953 个开放阅读框，分子量为 71.2kDa，理论等电点为 5.22。qRT-PCR 分析表明，HSP70 基因在正常条件下在各种组织中差异表达，在脾脏中表达水平最高，在肌肉和心脏中表达水平最低。在细菌攻毒和热应激后，观察到 HSP70 的表达水平呈现明显的时间依赖性。在受到嗜水气单胞菌感染和热应激后，肝脏、脾脏和鳃组织中 HSP70 表达水平显著增加，并在 3 小时和 6 小时达到最大值（P<0.05）。随着时间的推移，HSP70 转录本的表达下调，大多数在 48 小时后恢复到原始水平。皮质醇、天冬氨酸转氨酶（AST）和丙氨酸转氨酶（ALT）的浓度随着应激时间的增加而增加，在 3 小时时达到最高水平，随后迅速下降，在 48 小时时恢复到对照水平。这些结果表明，HSP70 参与了对细菌攻毒和热应激的免疫反应。HSP70 的克隆和表达分析为进一步研究麦穗鱼抗逆性的机制提供了理论基础。
