The roles of insulin-like growth factor 5 (IGFBP5) on fish osmoregulation

胰岛素样生长因子 5 (IGFBP5) 对鱼类渗透压调节的作用

• 批准号: 22K06291
• 负责人: 黄 國成
• 金额: $ 2.66万
• 依托单位: Toho University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2022
• 资助国家: 日本
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22K06291/
• 关键词: osmoregulation; IGFBP5; medaka; chum salmon; ISH chain reaction; ionocyte; 成長ホルモン; 体液調節; IGF結合タンパク質; 塩類細胞

I have made IGFBP5a knockout (KO) medaka using CRISPR-Cas9 system. I have introduced a 5-nucleotide deletion in the IGFBP5a gene in drR medaka and established a heterologous KO line. With crossing between the heterologous line, it is in the process to breed homologous IGFBP5a KO medaka line.The salinity tolerance and transporter expression in the ionocytes in heterologous KO medaka was examined and no apparent difference was observed compared with the WT drR medaka.I have made two custom antibodies for the IGFBP5a in medaka and chum salmon respectively, but the antibodies for IGFBP5a were not desirable as the immunohistochemistry and Western blot results showed non-specific signals at the mucus.I have established a novel method to detect the transcript localization using in situ hybridization chain reaction (ISHCR). The method can distinguish closely resemble gene isoforms and I have demonstrated the distinct localization of Na-K-ATPase (NKA) alpha-1a and alpha-1b in chum salmon gill, which cannot be distinguished by conventional in situ hybridization method using cRNA probes. I have used the same method to study the localization of IGFBP5a in chum salmon ionocytes, and initial result suggested that the IGFBP5a is not colocalized with NKA, an observation different from that of zebrafish.I found a sequential increase in IGFBP5a expression during embryonic stages, in which the smoltification occurs in chum salmon. However, seawater treatment downregulated the IGFBP5a expression significantly in smolting chum salmon, an observation that is different from that of medaka.

我已经用CRISPR-CAS9系统制作了IGFBP5a淘汰赛(KO)青竹。我在青竹的IGFBP5a基因中引入了5个核苷酸的缺失，并建立了一个异源KO系。通过异源品系间的杂交，培育出同源的IGFBP5a KO青花鱼品系。检测了异源青花鱼的耐盐性和转运蛋白在离子细胞中的表达，并与野生型DRR青花鱼进行了比较，没有明显的差异。我已经分别制备了青花鱼和大马哈鱼的IGFBP5a抗体，但由于免疫组织化学和Western印迹结果显示粘液处有非特异性信号，所以IGFBP5a抗体并不理想。该方法可以区分非常相似的基因异构体，并且证明了Na-K-ATPase(NKA)α-1a和α-1b在鲑鱼鳃中的明显定位，这是传统的使用cRNA探针的原位杂交方法所不能区分的。我用同样的方法研究了IGFBP5a在鲑鱼离子细胞中的定位，初步结果表明IGFBP5a与NKA不是共定位的，这与斑马鱼的观察结果不同。我发现IGFBP5a在胚胎阶段表达依次增加，在鲑鱼中发生嗅化。然而，海水处理显著下调了熏鲑鱼中IGFBP5a的表达，这一观察结果与青鱼不同。