CAJ | 학술논문

以经过连续多代抗寒选育获得的尼罗罗非鱼(Oreochromis niloticus)耐寒品系为实验材料,克隆了尼罗罗非鱼水通道蛋白基因(AQP1)的cDNA序列,并采用实时荧光定量PCR分析方法探讨了低温胁迫对罗非鱼AQP1基因表达水平的影响。序列分析结果显示,尼罗罗非鱼AQP1 cDNA长度为1098 bp,包含36 bp的5′端非翻译区序列、783 bp开放阅读框(ORF)和279 bp的3′非翻译区序列,编码261个氨基酸。氨基酸序列比对表明,各物种间AQP1序列的同源性较高(96%~59%)。聚类分析表明,尼罗罗非鱼首先与同属鲈形目丽鱼科的斑马拟丽鱼(Maylandia zebra)聚在一起,再与其他硬骨鱼类聚为一类。实时荧光定量PCR 结果表明：在水温从30℃逐渐降到10℃过程中,肌肉和肝组织 AQP1基因的表达量从20℃开始均逐渐下调。其中肌肉组织中,与30℃时的表达量相比,耐寒品系在20℃时表达量大幅度下调,当水温为15℃时,其表达量下调幅度为22.16倍。当水温降到10℃时,其表达量下调了107.73倍。而对照组AQP1基因的表达量在15℃时,下调幅度为5.38倍。当水温达到10℃时,其表达量下调幅度为11.30倍。结果分析显示,耐寒品系与对照组AQP1基因在低温条件下的表达量存在显著性差异(P<0.05)。而在肝组织中,当水温降到15℃时,耐寒品系和对照组的表达量下调幅度分别为3.38倍、1.42倍。水温降到10℃时,其表达量上调幅度分别为18.85倍、9.01倍。结果分析表明,低温条件下耐寒品系与对照组AQP1基因在肝组织中的表达差异不显著(P>0.05)。结果表明, AQP1表达对温度敏感,耐寒品系较高的耐寒能力与其在低温条件下的肌肉组织大幅上调表达有关。由此可见, AQP1基因在罗非鱼低温适应过程中发挥重要作用,是潜在的研究罗非鱼耐寒机制的候选基因之一,本研究为进一步研究罗非鱼的耐寒分子机制和开展鱼类抗逆育种提供参考。以經過連續多代抗寒選育穫得的尼囉囉非魚(Oreochromis niloticus)耐寒品繫為實驗材料,剋隆瞭尼囉囉非魚水通道蛋白基因(AQP1)的cDNA序列,併採用實時熒光定量PCR分析方法探討瞭低溫脅迫對囉非魚AQP1基因錶達水平的影響。序列分析結果顯示,尼囉囉非魚AQP1 cDNA長度為1098 bp,包含36 bp的5′耑非翻譯區序列、783 bp開放閱讀框(ORF)和279 bp的3′非翻譯區序列,編碼261箇氨基痠。氨基痠序列比對錶明,各物種間AQP1序列的同源性較高(96%~59%)。聚類分析錶明,尼囉囉非魚首先與同屬鱸形目麗魚科的斑馬擬麗魚(Maylandia zebra)聚在一起,再與其他硬骨魚類聚為一類。實時熒光定量PCR 結果錶明：在水溫從30℃逐漸降到10℃過程中,肌肉和肝組織 AQP1基因的錶達量從20℃開始均逐漸下調。其中肌肉組織中,與30℃時的錶達量相比,耐寒品繫在20℃時錶達量大幅度下調,噹水溫為15℃時,其錶達量下調幅度為22.16倍。噹水溫降到10℃時,其錶達量下調瞭107.73倍。而對照組AQP1基因的錶達量在15℃時,下調幅度為5.38倍。噹水溫達到10℃時,其錶達量下調幅度為11.30倍。結果分析顯示,耐寒品繫與對照組AQP1基因在低溫條件下的錶達量存在顯著性差異(P<0.05)。而在肝組織中,噹水溫降到15℃時,耐寒品繫和對照組的錶達量下調幅度分彆為3.38倍、1.42倍。水溫降到10℃時,其錶達量上調幅度分彆為18.85倍、9.01倍。結果分析錶明,低溫條件下耐寒品繫與對照組AQP1基因在肝組織中的錶達差異不顯著(P>0.05)。結果錶明, AQP1錶達對溫度敏感,耐寒品繫較高的耐寒能力與其在低溫條件下的肌肉組織大幅上調錶達有關。由此可見, AQP1基因在囉非魚低溫適應過程中髮揮重要作用,是潛在的研究囉非魚耐寒機製的候選基因之一,本研究為進一步研究囉非魚的耐寒分子機製和開展魚類抗逆育種提供參攷。
이경과련속다대항한선육획득적니라라비어(Oreochromis niloticus)내한품계위실험재료,극륭료니라라비어수통도단백기인(AQP1)적cDNA서렬,병채용실시형광정량PCR분석방법탐토료저온협박대라비어AQP1기인표체수평적영향。서렬분석결과현시,니라라비어AQP1 cDNA장도위1098 bp,포함36 bp적5′단비번역구서렬、783 bp개방열독광(ORF)화279 bp적3′비번역구서렬,편마261개안기산。안기산서렬비대표명,각물충간AQP1서렬적동원성교고(96%~59%)。취류분석표명,니라라비어수선여동속로형목려어과적반마의려어(Maylandia zebra)취재일기,재여기타경골어류취위일류。실시형광정량PCR 결과표명：재수온종30℃축점강도10℃과정중,기육화간조직 AQP1기인적표체량종20℃개시균축점하조。기중기육조직중,여30℃시적표체량상비,내한품계재20℃시표체량대폭도하조,당수온위15℃시,기표체량하조폭도위22.16배。당수온강도10℃시,기표체량하조료107.73배。이대조조AQP1기인적표체량재15℃시,하조폭도위5.38배。당수온체도10℃시,기표체량하조폭도위11.30배。결과분석현시,내한품계여대조조AQP1기인재저온조건하적표체량존재현저성차이(P<0.05)。이재간조직중,당수온강도15℃시,내한품계화대조조적표체량하조폭도분별위3.38배、1.42배。수온강도10℃시,기표체량상조폭도분별위18.85배、9.01배。결과분석표명,저온조건하내한품계여대조조AQP1기인재간조직중적표체차이불현저(P>0.05)。결과표명, AQP1표체대온도민감,내한품계교고적내한능력여기재저온조건하적기육조직대폭상조표체유관。유차가견, AQP1기인재라비어저온괄응과정중발휘중요작용,시잠재적연구라비어내한궤제적후선기인지일,본연구위진일보연구라비어적내한분자궤제화개전어류항역육충제공삼고。
The full length cDNA of the Aquaporin gene (AQP1) was cloned and sequenced from cold tolerant Nile tila-pia (Oreochromis niloticus) strains developed by successive and directional selective breeding. In addition, the effects of cold stress on AQP1 gene expression levels under low temperature treatments were measured using real-time quan-titative PCR. Sequence analysis showed that Nile tilapia AQP 1 cDNA consists of 1 098 base pairs (bp) and encodes a protein of 261 amino acids that contain a 36 bp 5′untranslated region (UTR), a 783 bp open reading frame (ORF) and a 279 bp 3′UTR. Multiple alignment and homological analysis revealed AQP1 evolutionary conservation among verte-brates and showed that Nile tilapia AQP1 protein shared high levels of amino acid identity with AQP1 from Maylandia zebra (96%), Diplodus sargus (93%), Rhabdosargus sarba (92%), Dicentrarchus labrax (92%), Esox lucius (86%), Anguilla anguilla (83%), Danio rerio (74%), Gallus gallus (63%), Mus musculus (59%) and Homo sapiens (59%). A phylogenetic tree was constructed using the neighbor-joining method with the deduced amino acid sequences of AQP1 among vertebrates. As expected from the sequence alignment, tilapia AQP1 fell into the cluster with Maylandia zebra AQP 1 first, and then clustered with the other teleost species. To investigate the effects of low temperature on AQP1 expression, we examined the expression of AQP1 mRNA at different temperatures (30℃, 25℃, 20℃, 15℃, 10℃) for 48 h using real-time quantitative PCR. There was a gradual decrease in AQP1 mRNA levels in the muscle and liver from 20℃as the water temperature dropped gradually from 30℃ to 10℃. In the muscle of the cold-tolerant tilapia strain, the AQP1 mRNA levels decreased drastically at 20℃and were 22.16 times less at 15℃than at 30℃, and then at 10℃, 107.73 times less down-regulated compared with 30℃. The expression of the AQP1 gene in the control group was 5.38 times less at 15℃than at 30℃, followed by reduced expression at 10℃which induced an 11.30 fold decrease in transcript amounts. Analysis of variance showed that the expression levels of AQP1 in muscle under cold stress were significantly different between the cold-tolerant tilapia strain and the control group (P<0.05). In the liver of the cold-tolerant strain and the control, the AQP1 mRNA expressions were 3.38 and 1.42 fold down-regulated at 15℃, and then at 10℃, 18.85 and 9.01 fold down-regulated when compared with 30℃. This showed that cold stress had no sig-nificant effects on liver AQP1 mRNA expression between the cold-tolerant tilapia strain and the control group (P>0.05). The variation in expression levels suggest that the expression of AQP1 is sensitive to temperature, and the higher cold resistance of the cold-tolerant tilapia strain was closely associated with the drastically down-regulated expression of AQP1 at low temperatures. These results suggest that the AQP1 gene plays an important role in low temperature accli-mation and might be one of the cold tolerance-related genes in Nile tilapia. The present results provide a theoretical basis for studying the molecular mechanism of cold tolerance and fish stress resistance breeding.