海底热液口蟹Gandalfus yunohana特异性基因的筛选和功能分析
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摘要
本研究采用双链特异性DNA酶介导的均一化消减杂交方法比较了海底热液口蟹Gandalfus yunohana和其在普通条件下生存的近缘种三疣梭子蟹Portunus trituberculatus肝胰腺的mRNA,以探求生物在热液口环境中的生存机制。通过构建全长的均一化cDNA消减文库,在GenBank中比对得到51个与已知基因具有相似性的不同转录本。从这些基因之中,我们选取了硫氧还蛋白基因进行了原核表达系统的构建,并对该基因的完整开放阅读框序列进行了确定、分析与表达产物活性的检测。同时,我们通过RACE的手段获得了G. yunohana的热休克蛋白90基因cDNA的完整序列并进行分析,构建了该基因的原核表达系统并在原核细胞中初步验证了该基因在高温胁迫下对细胞的保护作用。这是首次对海底热液口物种的硫氧还蛋白与热休克蛋白90基因信息的报道,我们得到的数据为今后海底热液口生物在特殊环境下的生存机制的研究奠定了基础。
MRNA transcriptional profiles between hepantopancreas of the hydrothermal vent crab, Gandalfus yunohana, and its close-related non-vent counterpart, Portunus trituberculatus, was compared by the method of DNSH [DSN (duplex-specific nuclease)-mediated normalization and subtractive hybridization] in our research to explore biological survival mechanism of vent animals. Through constructing the full-length normalized subtractive cDNA library, we identified 51 different transcripts which have similarity to known genes in GenBank. We selected thioredoxin from these genes to construct prokaryotic expression system. After identifying and analysising the complete open reading frame of thioredoxin, we detected the activity of its expression product. Meanwhile, we obtained the complete cDNA sequence of heat shock protein 90 in G. yunohana through the method of rapid amplification of cDNA ends. After analysis of its sequence, we construct the prokaryotic expression system and detected its cell protection function under heat stress in prokaryotic cells. Our research provides the first genetic information on thioredoxin and heat shock protein 90 in hydrothermal vent species and our data lay the foundation for future research of biological resilience and survival mechanism in the special environment of hydrothermal vents.
MRNA transcriptional profiles between hepantopancreas of the hydrothermal vent crab, Gandalfus yunohana, and its close-related non-vent counterpart, Portunus trituberculatus, was compared by the method of DNSH [DSN (duplex-specific nuclease)-mediated normalization and subtractive hybridization] in our research to explore biological survival mechanism of vent animals. Through constructing the full-length normalized subtractive cDNA library, we identified 51 different transcripts which have similarity to known genes in GenBank. We selected thioredoxin from these genes to construct prokaryotic expression system. After identifying and analysising the complete open reading frame of thioredoxin, we detected the activity of its expression product. Meanwhile, we obtained the complete cDNA sequence of heat shock protein 90 in G. yunohana through the method of rapid amplification of cDNA ends. After analysis of its sequence, we construct the prokaryotic expression system and detected its cell protection function under heat stress in prokaryotic cells. Our research provides the first genetic information on thioredoxin and heat shock protein 90 in hydrothermal vent species and our data lay the foundation for future research of biological resilience and survival mechanism in the special environment of hydrothermal vents.
引文
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