Gao Weifeng, Chen Lu, Ding Qianqian, Gao Xiaojian, Zhang Yao, Zhang Xiaojun, Jiang Qun
College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu 225009, China.
Comp Biochem Physiol B Biochem Mol Biol. 2025 Aug-Sep;279:111123. doi: 10.1016/j.cbpb.2025.111123. Epub 2025 Jul 3.
The full-length cDNA sequences of two Na-K-2Cl cotransporter (NKCC) isoforms, MrNKCC1 and MrNKCC2, were identified in Macrobrachium rosenbergii. MrNKCC1 contains a 3183 bp open reading frame (ORF) encoding 1060 amino acids, while MrNKCC2 has a 2679 bp ORF encoding 892 amino acids. Both isoforms share a similar predicted secondary structure, featuring 12 transmembrane domains and a C-terminal, and their amino acid sequences and domain arrangement are highly conserved. Phylogenetic analysis groups fish and crustacean NKCC isoforms into distinct branches, each containing two isoforms. Expression analysis using real-time quantitative PCR (qPCR) revealed distinct tissue distribution patterns. MrNKCC1 was broadly expressed, with the highest levels in the intestine, whereas MrNKCC2 was predominantly found in the gill, hepatopancreas, and epidermis, with peak expression in the gill. To examine their roles in osmoregulation, prawns were exposed to 24 ‰ seawater for 96 h. Hemolymph concentrations of Cl, K, and Na increased, peaking at 24, 24, and 12 h, respectively. During this period, MrNKCC1 expression was suppressed, while MrNKCC2 expression significantly increased at 2 h after seawater exposure and then returned to initial levels. Gill filaments showed significant histological changes under seawater exposure, including the expansion and proliferation of chloride-secreting cells. Following a 96-h seawater exposure, prawns were transferred to freshwater. This transition led to a decline in plasma Cl, K, and Na concentrations, accompanied by a marked increase in MrNKCC1 and MrNKCC2 expression. The rapid transcriptional response of both MrNKCC isoforms to salinity fluctuations highlights their critical role in the osmoregulatory mechanisms of M. rosenbergii.
在罗氏沼虾中鉴定出了两种钠-钾-2氯协同转运蛋白（NKCC）亚型MrNKCC1和MrNKCC2的全长cDNA序列。MrNKCC1包含一个3183 bp的开放阅读框（ORF），编码1060个氨基酸，而MrNKCC2有一个2679 bp的ORF，编码892个氨基酸。两种亚型都具有相似的预测二级结构，有12个跨膜结构域和一个C末端，它们的氨基酸序列和结构域排列高度保守。系统发育分析将鱼类和甲壳类NKCC亚型分为不同的分支，每个分支包含两种亚型。使用实时定量PCR（qPCR）进行的表达分析揭示了不同的组织分布模式。MrNKCC1广泛表达，在肠道中水平最高，而MrNKCC2主要存在于鳃、肝胰腺和表皮中，在鳃中表达量最高。为了研究它们在渗透压调节中的作用，将对虾暴露于24‰的海水中96小时。血淋巴中Cl、K和Na的浓度升高，分别在24、24和12小时达到峰值。在此期间，MrNKCC1的表达受到抑制，而MrNKCC2的表达在海水暴露后2小时显著增加，然后恢复到初始水平。在海水暴露下，鳃丝出现明显的组织学变化，包括分泌氯离子细胞的扩张和增殖。在96小时海水暴露后，将对虾转移到淡水中。这种转变导致血浆Cl、K和Na浓度下降，同时MrNKCC1和MrNKCC2的表达显著增加。两种MrNKCC亚型对盐度波动的快速转录反应突出了它们在罗氏沼虾渗透压调节机制中的关键作用。
