中国水产科学2025，Vol.32Issue(9)：1224-1241,18.DOI:10.12264/JFSC2025-0115
日本沼虾线粒体分裂相关基因克隆及其碳酸盐碱度胁迫下表达分析
Molecular cloning and expression analysis of mitochondrial fission related genes in Macrobrachium nipponense in response to carbonate alkalinity stress
摘要
Abstract
Mitochondrial fission process 1(MTFP1)and mitochondrial fission protein 1(FIS1)are mitochondrial fission-related proteins that play crucial roles in mitochondrial homeostasis.Recent studies have reported that carbonate alkalinity stress damages mitochondrial structure and function in aquatic organisms.This study investigated the molecular response mechanisms of MTFP1 and FIS1 in the oriental river prawn,Macrobrachium nipponense,under carbonate alkalinity stress.To achieve this,the full-length cDNA sequences of MTFP1 and FIS1 genes were cloned using the cDNA terminal rapid amplification(RACE)technique.Semi-quantitative RT-PCR and real-time fluorescence quantitative PCR were used to analyze tissue-specific expression patterns and dynamic expression patterns during carbonate alkalinity stress.Immunofluorescence techniques were applied to examine protein expression and localization in the gill and hepatopancreas of prawn under 96 h carbonate alkalinity stress,while transmission electron microscopy(TEM)was utilized to observe mitochondrial structural damage in the gill and hepatopancreas of prawn under high carbonate alkalinity stress.The results show that the full-length cDNA of M.nipponense MTFP1(NCBI:OR209329)spans 1101 bp,comprising a 33 bp 5′-UTR,588 bp 3′-UTR,and 480 bp open reading frame(ORF)encoding 159 amino acids containing an MTP18 superfamily domain.The FIS1 gene(NCBI:PQ790281)spans 1933 bp with a 148 bp 5′-UTR,1324 bp 3′-UTR,and 480 bp ORF encoding 150 amino acids containing an FIS1 superfamily domain.Sequence alignment and phylogenetic analysis revealed high amino acid sequence similarity and close evolutionary relationships with MTFP1 and FIS1 from other crustaceans.RT-PCR analysis showed ubiquitous expression of both genes across eight tissues,with higher expression levels in gills,hepatopancreas,and gonads.During the 96 h acute carbonate alkalinity stress,two genes showed significantly higher(P<0.05)expression in the gill and hepatopancreas compared with the control group.Prokaryotic expression systems were constructed to generate polyclonal antibodies,with immunofluorescence confirming increased MTFP1 and FIS1 protein expression levels under stress.TEM observations revealed abnormal mitochondrial structures in the gill and hepatopancreas of prawn in response to 96 h acute carbonate alkalinity stress.This study demonstrates that higher concentration of carbonate alkalinity stress significantly affects the MTFP1 and FIS1 expression levels in the gill and hepatopancreas of M.nipponense,providing fundamental data for understanding the molecular characteristics of mitochondrial fission-related genes and their responses to high carbonate alkalinity stress in crustaceans.关键词
日本沼虾/碳酸盐碱度/MTFP1/FIS1/线粒体分裂Key words
Macrobrachium nipponense/carbonate alkalinity/mitochondrial fission protein 1/mitochondrial fission process 1/mitochondrial fission分类
农业科技引用本文复制引用
LYU Zhimin,LIU Jiaqi,WU Yijie,JIN Yiting,SUN Shengming..日本沼虾线粒体分裂相关基因克隆及其碳酸盐碱度胁迫下表达分析[J].中国水产科学,2025,32(9):1224-1241,18.基金项目
上海市农业科技创新项目(沪农科2024206) （沪农科2024206）
国家重点研发计划项目战略性科技创新合作专项(2023YFE0205100) （2023YFE0205100）
上海市教委东方学者特聘教授项目(TP2022078) （TP2022078）
上海市科技创新行动计划项目(23XD1421600) （23XD1421600）
宁夏回族自治区重点研发计划项目(202288F03034). （202288F03034）
