CAJ | 학술논문

为研究线粒体ATP酶F1-δ基因在鲢(Hypophthalmichthys molitrix)中的作用,采用RACE-PCR技术克隆出该基因全长,应用半定量 RT-PCR 法检测该基因在不同组织的表达,应用实时荧光定量 PCR 法检测急性低氧胁迫过程中不同溶氧浓度下该基因的组织表达变化。结果显示,鲢线粒体ATP酶F1-δ基因全长762 bp,开放阅读框480 bp,编码159个氨基酸残基,5′端非编码区114 bp,3′端非编码区168 bp。鲢与斑马鱼(Danio rerio)线粒体F1-δ编码氨基酸序列的相似性最高,达到89%；与大西洋鲑(Salmo salar)、罗非鱼(Oreochromis niloticus)、樱花钩吻鲑(Oncorhynchus masou formosanus)鲀、红鳍东方(Takifugu rubripes)的相似性分别为76%、75%、74%、69%；半定量RT-PCR结果显示,该基因在鲢心脏、脑、肝、脾和肌肉中均有表达,且心脏中最高,肌肉次之；实时荧光定量PCR结果表明,水中溶解氧(DO)分别为5.6(对照组)、4.38、3.37、2.11、1.12和0.54 mg/L时,随着溶解氧浓度的下降该基因在心脏中的表达逐渐下降且均显著低于对照组(P<0.05)；而在脑、肝、脾和肌肉中的表达则先升高后降低。寡霉素抑制法测得低氧胁迫过程中,鲢心脏等组织中 F1F0-ATP 酶活性均先升高后降低。这表明鲢线粒体 ATP 酶F1-δ基因在低氧胁迫中起到一定的作用,并对ATP酶的合成产生影响。
위연구선립체ATP매F1-δ기인재련(Hypophthalmichthys molitrix)중적작용,채용RACE-PCR기술극륭출해기인전장,응용반정량 RT-PCR 법검측해기인재불동조직적표체,응용실시형광정량 PCR 법검측급성저양협박과정중불동용양농도하해기인적조직표체변화。결과현시,련선립체ATP매F1-δ기인전장762 bp,개방열독광480 bp,편마159개안기산잔기,5′단비편마구114 bp,3′단비편마구168 bp。련여반마어(Danio rerio)선립체F1-δ편마안기산서렬적상사성최고,체도89%；여대서양해(Salmo salar)、라비어(Oreochromis niloticus)、앵화구문해(Oncorhynchus masou formosanus)돈、홍기동방(Takifugu rubripes)적상사성분별위76%、75%、74%、69%；반정량RT-PCR결과현시,해기인재련심장、뇌、간、비화기육중균유표체,차심장중최고,기육차지；실시형광정량PCR결과표명,수중용해양(DO)분별위5.6(대조조)、4.38、3.37、2.11、1.12화0.54 mg/L시,수착용해양농도적하강해기인재심장중적표체축점하강차균현저저우대조조(P<0.05)；이재뇌、간、비화기육중적표체칙선승고후강저。과매소억제법측득저양협박과정중,련심장등조직중 F1F0-ATP 매활성균선승고후강저。저표명련선립체 ATP 매F1-δ기인재저양협박중기도일정적작용,병대ATP매적합성산생영향。
We evaluated the effect of hypoxia on the mitochondrial ATPase F1-δ subunit gene in silver carp (Hy-pophthalmichthys molitrix). The full-length cDNA of this gene was cloned by rapid amplification of cDNA ends (RACE) PCR, and the expression of this gene was detected in different tissues by semi-quantitative RT-PCR. We then measured the relative expression of this gene in the different tissues following exposure to acute hypoxia stress of 5.6 mg/L (con-trol), 4.38 mg/L, 3.37 mg/L, 2.11 mg/L, 1.12 mg/L, or 0.54 mg/L dissolved oxygen [DO] using quantitative real-time PCR. The full-length cDNA of the mitochondrial ATPase F1-δ subunit gene was 762 bp, containing an open frame reading of 480 bp encoding 159 amino acids, and a 114 bp 5′-untranslated region and 168 bp 3′-untranslated region. The deduced amino acid sequence of the mitochondrial ATPase F1-δsubunit gene exhibited high similarity with Dario rerio (89%) and moderate similarity with Salmo salar(76%), Oreochromis niloticus(75%), Oncorhynchus masou formosa-nus(74%), and Takifugu rubripes(69%). Expression of the gene was highest in the heart followed by the muscle, with low levels of expression in the brain, liver, and spleen in silver carp. Expression of this gene decreased gradually as dissolved oxygen levels decreased. Expression in the heart was significantly lower (P<0.05) in the hypoxia-exposed fish than in the control group. Conversely, levels were initially significantly higher (P<0.05) in the brain, liver, spleen, and muscle of hypoxia-exposed fish, before decreasing significantly (P<0.05). F1F0-ATPase activity first increased then decreased in all the tissues during acute hypoxia stress. Changes in expression of the F1-δsubunit gene were consistent with changes in ATP enzyme activity in the brain, liver, spleen, and muscle, but not in the heart. The initial increase in expression ofδmay be caused by positive self-regulation of the gene in response to changes in desolved oxygen(DO). However, if damage to the inner mitochondrial membrane leads to loss of self-regulation when DO becomes too low,δexpression will rapidly decrease. This disrupts energy metabolism and blocks energy synthesis. Our results suggest that the mitochondrial ATPase F1-δsubunit gene may play a role in the response to hypoxia stress and influence the produc-tion and activity of ATPase in silver carp.