低温胁迫下极地鱼抗冻基因ld4对细胞应激颗粒形成的影响
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摘要
为研究低温胁迫下抗冻基因ld4对细胞应激颗粒(Stress granules)形成的影响,从南极鱼Lycodichthys dearborn的多聚AFPⅢ基因ld12 c DNA中克隆得到AFPⅢ蛋白四聚体ld4,将其转染到He La细胞中,并采用反转录PCR(RT-PCR)、Western blot方法检测到目的基因ld4在细胞中成功表达,进而采用细胞免疫荧光方法检测细胞在低温(10℃)胁迫下细胞应激颗粒的形成情况。结果表明,与对照组相比较,在低温胁迫下,表达ld4基因的He La细胞中应激颗粒明显减少。研究表明,ld4基因的表达在低温胁迫下能显著降低He La细胞中应激颗粒的形成,试验结果可为ld4基因作为鱼类耐寒育种的候选基因提供理论依据。
An AFPⅢ tetramer gene ld4 from Antarctic eelpout Lycodichthys dearborni was transfected into He La cell( human cervical cancer cell) to investigate the relationship between antifreeze gene ld4 and cell stress granule formation in response to cold stress. Reverse Transcription PCR( RT-PCR) and Western blot were applied to verify the ld4 gene expression in mRNA and protein levels in ld4 transfected He La cell. Further,cellular stress granules formation was detected under cold stress( 10 ℃) and compared with that in the control cell by immunofluorescence. There was significant decrease in the stress granules formation in ld4 transfected He La cell under the cold stress than in the control,indicating that ld4 may enhance cellular cold resistance by reducing the formation of cellular stress granules. The findings provide a mechanism for ld4 gene as a candidate gene in breeding of cold resistant fishes.
An AFPⅢ tetramer gene ld4 from Antarctic eelpout Lycodichthys dearborni was transfected into He La cell( human cervical cancer cell) to investigate the relationship between antifreeze gene ld4 and cell stress granule formation in response to cold stress. Reverse Transcription PCR( RT-PCR) and Western blot were applied to verify the ld4 gene expression in mRNA and protein levels in ld4 transfected He La cell. Further,cellular stress granules formation was detected under cold stress( 10 ℃) and compared with that in the control cell by immunofluorescence. There was significant decrease in the stress granules formation in ld4 transfected He La cell under the cold stress than in the control,indicating that ld4 may enhance cellular cold resistance by reducing the formation of cellular stress granules. The findings provide a mechanism for ld4 gene as a candidate gene in breeding of cold resistant fishes.
引文
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[22]Wang Xin,De Vries A L,Chen C H.Genomic basis for antifreeze peptide heterogeneity and abundance in an Antarctic eel pout:gene structures and organization[J].Molecular Marine Biology and Biotechnology,1995,4(2):135-147.
[23]Wang Xin,De Vries A L,Chen C H.Antifreeze peptide heterogeneity in an Antarctic eel pout includes an unusually large major variant comprised of two 7 k Da typeⅢAFPs linked in tandem[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1995,1247(2):163-172.
[24]Baardsnes J,Kuiper M J,Davies P L.Antifreeze protein dimer:when two ice-binding faces are better than one[J].Journal of Biological Chemistry,2003,278(40):38942-38947.
[25]Yang Na,Peng Changlian,Cheng Deng,et al.The over-expression of calmodulin from Antarctic notothenioid fish increases cold tolerance in tobacco[J].Gene,2013,521(1):32-37.
[2]De Vries A L,Lin Yuan.Structure of a peptide antifreeze and mechanism of adsorption to ice[J].Biochimica et Biophysica Acta(BBA)-Protein Structure,1977,495(2):388-392.
[3]钟其旺,樊廷俊.鱼类抗冻蛋白的研究进展[J].生物化学与生物物理学报,2002,34(2):124-130.
[4]田云,卢向阳,张海文.抗冻蛋白研究进展[J].中国生物工程杂志,2002,22(6):48-53.
[5]Zhang Junfang,Deng Cheng,Wang Jianshe,et al.Identification of a two-domain antifreeze protein gene in Antarctic eelpout Lycodichthys dearborni[J].Polar Biology,2009,32(1):35-40.
[6]於静,Cheng C H,De Vries A L,等.南极eel pout(Lycodichthys dearborni)多聚体Ⅲ型抗冻蛋白基因的克隆及进化分析[J].遗传学报,2005,32(8):789-794.
[7]Montero H,Trujillo-Alonso V.Stress granules in the viral replication cycle[J].Viruses,2011,3(11):2328-2338.
[8]Emara M M,Brinton M A.Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly[J].Proceedings of the National Academy of Sciences of the United States of America,2007,104(21):9041-9046.
[9]Montero H,Rojas M,Arias C F,et al.Rotavirus infection induces the phosphorylation of e IF2αbut prevents the formation of stress granules[J].Journal of Virology,2008,82(3):1496-1504.
[10]Lindquist M E,Lifland A W,Utley T J,et al.Respiratory syncytial virus induces host RNA stress granules to facilitate viral replication[J].Journal of Virology,2010,84(23):12274-12284.
[11]Anderson P,Kedersha N.Stress granules:the Tao of RNA triage[J].Trends Biochemical Sciences,2008,33(3):141-150.
[12]Qin Qingsong,Hastings C,Miller C L.Mammalian orthoreovirus particles induce and are recruited into stress granules at early times postinfection[J].Journal of Virology,2009,83(21):11090-11101.
[13]Schütz S,Sarnow P.How viruses avoid stress[J].Cell Host&Microbe,2007,2(5):284-285.
[14]Solomon S,Xu Yaoxian,Wang Bin,et al.Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2α,entry to cytoplasmic stress granules,and selective interaction with a subset of mRNAs[J].Molecular and Cellular Biology,2007,27(6):2324-2342.
[15]Takahashi M,Higuchi M,Matsuki H,et al.Stress granules inhibit apoptosis by reducing reactive oxygen species production[J].Molecular and Cellular Biology,2013,33(4):815-829.
[16]Zhang Peifen,Li Yuye,Xia Jun,et al.IPS-1 plays an essential role in dsRNA-induced stress granule formation by interacting with PKR and promoting its activation[J].Journal of Cell Science,2014,127(11):2471-2482.
[17]Hofmann S,Cherkasova V,Bankhead P,et al.Translation suppression promotes stress granule formation and cell survival in response to cold shock[J].Molecular Biology of the Cell,2012,23(19):3786-3800.
[18]马战领,胡瑞芹,黄巧,等.齐口裂腹鱼Zona pellucida 3的克隆表达及其蛋白活性研究[J].大连海洋大学学报,2015,30(6):592-597.
[19]Shackleton N J,Opdyke N D.Oxygen isotope and palaeomagnetic evidence for early northern hemisphere glaciation[J].Nature,1977,270(5634):216-219.
[20]Snnichsen F D,De Luca C I,Davies P L,et al.Refined solution structure of typeⅢantifreeze protein:hydrophobic groups may be involved in the energetics of the protein-ice interaction[J].Structure,1996,4(11):1325-1337.
[21]Cheng C H,De Vries A L.Structures of antifreeze peptides from the Antarctic eel pout,Austrolycicthys brachycephalus[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1989,997(1-2):55-64.
[22]Wang Xin,De Vries A L,Chen C H.Genomic basis for antifreeze peptide heterogeneity and abundance in an Antarctic eel pout:gene structures and organization[J].Molecular Marine Biology and Biotechnology,1995,4(2):135-147.
[23]Wang Xin,De Vries A L,Chen C H.Antifreeze peptide heterogeneity in an Antarctic eel pout includes an unusually large major variant comprised of two 7 k Da typeⅢAFPs linked in tandem[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1995,1247(2):163-172.
[24]Baardsnes J,Kuiper M J,Davies P L.Antifreeze protein dimer:when two ice-binding faces are better than one[J].Journal of Biological Chemistry,2003,278(40):38942-38947.
[25]Yang Na,Peng Changlian,Cheng Deng,et al.The over-expression of calmodulin from Antarctic notothenioid fish increases cold tolerance in tobacco[J].Gene,2013,521(1):32-37.