花鲈ncc、nkcc基因在海、淡水适应中鳃组织的表达与定位分析  

Expression and localization analysis of ncc and nkcc genes in gill tissues of Lateolabrax maculatus during salinity adaptation

作　　者：王志远 李金库 李昀[1] 王灵钰 齐鑫 李吉方[1] 温海深[1] WANG Zhiyuan;LI Jinku;LI Yun;WANG Lingyu;QI Xin;LI Jifang;WEN Haishen(Key Laboratory of Mariculture,MOE,Fisheries College,Ocean University of China,Qingdao 266603,China)

机构地区：[1]中国海洋大学水产学院,海水养殖教育部重点实验室,山东青岛266603

出　　处：《水产学报》2023年第8期46-56,共11页Journal of Fisheries of China

基　　金：国家重点研发计划(2020YFD0900204);国家自然科学基金(32072947);现代农业产业技术体系-国家海水鱼产业技术体系(CARS-47)。

摘　　要：为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR(qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2691和3120bp,编码896和1039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na^(+)及Cl^(-)离子转运蛋白亚型,可作为区分淡水、海水养殖花鲈的分子标志物,在渗透调节及盐度适应中发挥重要作用。The ncc gene encodes Na+-Cl−synergistic transporter,while the nkcc gene encodes Na+-K+-2Cl−synergistic transporter.Both of them belong to the solute carrier family12(SLC12),and play key roles in the osmotic regulation mechanism of fish.In order to explore the roles of ncc and nkcc in the osmotic regulation of spotted sea bass in this study,whole genome identification,multiple sequence alignment,phylogenetic tree construction and protein structure prediction were performed to identify the ncc genes of spotted sea bass and analyse their sequence structure.The expression levels of ncc and nkcc genes in gill tissues were detected by qRT-PCR,and the sites of expression of ncc2 and nkcc1a in gills of spotted sea bass in freshwater and seawater were determined by in situ hybridization(ISH).The results showed that two ncc genes,ncc1 and ncc2,were identified in spotted sea bass.Their CDSs length were 2691 and 3120 bp,encoding 896 and 1039 amino acids,respectively.The expression of ncc2 gene in gill tissue of freshwater fish was significantly higher than that in seawater,while the expression of nkcc1a in gill tissue of seawater fish was significantly higher than that in freshwater.Moreover,the expression of ncc2 in gill was gradually up-regulated during freshwater adaptation,while the expression of nkcc1a was gradually down-regulated.Meanwhile,the opposite expression trend was showed in seawater adaptation process.In addition,the ISH results showed that ncc2 and nkcc1a genes were located in the epithelium between adjacent gill lamellae in freshwater and seawater,respectively.The above results indicate that ncc2 and nkcc1a genes encode important Na^(+)-Cl^(-)transporter subtypes in the gills of spotted sea bass in freshwater and seawater,respectively.They can be used as specific markers to distinguish the spotted sea bass in freshwater and seawater,and play an important role in osmotic regulation and salinity adaptation.

关 键 词：花鲈 NCC NKCC 鳃 渗透调节 

分 类 号：S917[农业科学—水产科学]