黑鲷NKCC1分子特征及其对急性盐度胁迫的表达响应

Molecular Characteristics of NKCC1 in Black Sea Bream Acanthopagrus schlegelii and Its Expression in Response to Acute Salinity Stress

为了解黑鲷(Acanthopagrus schlegelii)在盐度胁迫过程中的适应机制,本研究利用基因扩增、聚类分析、荧光定量PCR等技术对NKCC1基因进行生物信息学分析以及在不同组织中的表达特征研究,并探讨其在急性盐度胁迫下的表达机制。结果表明NKCC1基因的开放阅读框(Open Reading Frame,ORF)为3504 bp,共编码1167个氨基酸,预测蛋白分子量为127.19 kDa,理论等电点为5.75。结构域分析表明黑鲷NKCC1分子为跨膜蛋白,并包含一个典型的Na^(+)-K^(+)-Cl^(-)共同转运蛋白SLC12A结构域,且在不同物种中具有很高的保守性;序列比对分析表明黑鲷NKCC1氨基酸序列与金头鲷NKCC1a的同源性最高,为97.8%;组织表达分析表明黑鲷NKCC1基因在所检测的12个组织,即肝脏、肾脏、心、脑、眼、鳃、鳍条、脾脏、皮肤、肠、性腺和白肌中均有表达,其中在肾脏、鳃和肠中表达量最高,并显著高于其他组织(P<0.05);在低盐度胁迫下,NKCC1基因在鳃组织中的表达量对盐度变化响应十分迅速,而在肾脏组织中,低盐度和高盐度胁迫都会在不同时间点抑制NKCC1基因的表达量。综上所述,本研究为解析NKCC1基因在黑鲷调节渗透压和离子平衡过程中的功能机制奠定了基础,并为促进黑鲷养殖产业的可持续发展提供一定的技术理论。

To understand the adaptation mechanism of black sea bream(Acanthopagrus schlegelii)under salinity stress,the bioinformatics analysis and the expression characteristics of NKCC1 gene in different tissues were carried out by using gene amplification,cluster analysis and fluorescence quantitative PCR techniques,and the expression mechanism of NKCC1 gene under acute salinity stress was also detected.The results showed that the open reading frame(ORF)of NKCC1 gene was 3504 bp,encoding 1167 amino acids.The predicted molecular weight of NKCC1 protein was 127.9 kDa,and the theoretical isoelectric point was 5.75.The domain analysis showed that NKCC1 was a transmembrane protein and contained a typical Na^(+)-K^(+)-Cl^(-)cotransporter SLC12A domain,which was highly conserved in different species.Sequence alignment analysis showed that the amino acid sequence of NKCC1 and NKCC1a had the highest homology,which was 97.8%.Tissue expression analysis showed that NKCC1 gene was expressed in the 12 tissues tested:liver,kidney,heart,brain,eye,gill,fin,spleen,skin,intestine,gonad and white muscle of black sea bream,and the highest expression was found in kidney,gill and intestine,and significantly higher than other tissues(P<0.05).When under low salinity stress,the expression of NKCC1 gene in gill tissue responded rapidly to salinity changes,while in kidney tissue,the expression of NKCC1 gene was inhibited at different time points by both low and high salinity stress.In conclusion,this study lays a foundation for exploring the mechanism of NKCC1 gene in regulating osmotic pressure and ion balance in black sea bream,and provides a certain technical theory for promoting the sustainable development of black sea bream farming industry.