红鳍东方鲀GPI基因对急性低盐胁迫的响应机制
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摘要
为了获得红鳍东方鲀Takifugu rubripes葡萄糖-6-磷酸异构酶(Glucose-6-Phosphate Isomerase, GPI)的基因信息及其表达特性,研究采用RT-PCR和实时荧光定量PCR (qPCR)技术进行了GPI基因的克隆、组织表达分析及其在急性低盐胁迫下的基因响应研究。结果显示,所获得的红鳍东方鲀GPI基因序列长1736 bp,包含一个完整的开放阅读框(Open Reading Frame, ORF)。ORF由1662个核苷酸组成,编码553个氨基酸;预测的氨基酸序列中有2个糖异构域(Sugar Isomerase Domains),不存在信号肽和跨膜结构域。多序列比对结果表明物种间GPI具有较高的保守性。qPCR结果表明:GPI基因mRNA在红鳍东方鲀鳃、肌肉、脑、肠、肝及肾等组织中均有表达,其中肌肉中表达量最高。在不同盐度胁迫下,在鳃中,各低盐组GPI mRNA相对表达量均呈现先升高后降低又回升的趋势;在肾中,各低盐组GPI mRNA相对表达量变化趋势各有不同。由此推测, GPI基因在红鳍东方鲀对急性低盐胁迫的响应中发挥一定作用。
To investigate the effect of low salinity stress on Glucose-6-Phosphate Isomerase(GPI) in Takifugu rubripes,we cloned GPI and analyzed its expressions in tissues with and without acute low-salinity stress using RT-PCR and real-time quantitative PCR(qPCR) techniques. The results showed that the GPI cDNA of the T. rubripes was 1736 bp in length containing an Open Reading Frame of 1662 nucleotides that encodes 553 amino acids. The predicted amino acid sequence contains two Sugar Isomerase Domains without signal peptide and transmembrane domains. Results of multiple sequence alignment showed that GPI was highly conserved among species. qPCR results showed that GPI mRNA was expressed in all the tested tissues, with the highest expression level in muscle. Under low-salinity stress, the relative mRNA expression of GPI in gill increased firstly, then decreased and increased again in all low-salinity groups,and the relative mRNA expression of GPI in kidney differed among low-salinity groups. Therefore, it was speculated that GPI play a role in the response of T. rubripes to acute low-salinity stress.
To investigate the effect of low salinity stress on Glucose-6-Phosphate Isomerase(GPI) in Takifugu rubripes,we cloned GPI and analyzed its expressions in tissues with and without acute low-salinity stress using RT-PCR and real-time quantitative PCR(qPCR) techniques. The results showed that the GPI cDNA of the T. rubripes was 1736 bp in length containing an Open Reading Frame of 1662 nucleotides that encodes 553 amino acids. The predicted amino acid sequence contains two Sugar Isomerase Domains without signal peptide and transmembrane domains. Results of multiple sequence alignment showed that GPI was highly conserved among species. qPCR results showed that GPI mRNA was expressed in all the tested tissues, with the highest expression level in muscle. Under low-salinity stress, the relative mRNA expression of GPI in gill increased firstly, then decreased and increased again in all low-salinity groups,and the relative mRNA expression of GPI in kidney differed among low-salinity groups. Therefore, it was speculated that GPI play a role in the response of T. rubripes to acute low-salinity stress.
引文
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[23]Sangiao-Alvarellos S,Laiz-Carrión R,Guzmán J M,et al.Acclimation of S.aurata to various salinities alters energy metabolism of osmoregula-tory and nonosmoregulatory organs[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,2003,285(4):897-907
[24]Sangiao-Alvarellos S,Arjona F J,Martin del Rio M P,et al.Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus[J].Journal of Experimental Biology,2005,208(22):4291-4304
[2]Repiso A,Andrés R,Climent F,et al.Expression patterns in mouse embryos of neuroleukin/glucose-6-phosphate isomerase and autocrine motility factor receptor[J].Anatomia Histologia Embryologia,2008,37(5):380-382
[3]Xu W,Seiter K,Feldman E,et al.The differentiation and maturation mediator for human myeloid leukemia cells shares homology with neuroleukin or phosphoglucose isomerase[J].Blood,1996,87(11):4502
[4]Gurney M E,Heinrich S P,Lee M R,et al.Molecular cloning and expression of neuroleukin,a neurotrophic factor for spinal and sensory neurons[J].Science,1986,234(4776):566-574
[5]Liotta L A,Mandler R,Murano G,et al.Tumor cell autocrine motility factor[J].Proceedings of the National Academy of Sciences of the United States of America,1986,83(10):3302-3306
[6]Niinaka Y,Harada K,Fujimuro M,et al.Silencing of autocrine motility factor induces mesenchymal to epithelial transition and suppression of osteosarcoma pulmonary metastasis[J].Cancer Research,2010,70(22):9483-9493
[7]Kugler W,Breme K,Laspe P,et al.Molecular basis of neurological dysfunction coupled with haemolytic anaemia in human glucose-6-phosphate isomerase(GPI)deficiency[J].Human Genetics,1998,103(4):450-454
[8]Niinaka Y,Paku S,Haga A,et al.Expression and secretion of neuroleukin/phosphohexose isomerase/maturation factor as autocrine motility factor by tumor cells[J].Cancer Research,1998,58(12):2667-2674
[9]Chen F,Jiang X L,Xie X B,et al.Correlation between serum Glucose-6-phosphate Isomerase(GPI)and Autoimmune Disease(AID)[J].Practical Preventive Medicine,2009,16(1):235-237[陈锋,江训良,谢小兵,等.血清葡萄糖-6-磷酸异构酶(GPI)与自身免疫性疾病(AID)相关性研究.实用预防医学,2009,16(1):235-237]
[10]Sun L C,Zhou L G,Du C H,et al.Glucose-6-phosphate isomerase is an endogenous inhibitor to myofibril-bound serine proteinase of crucian carp(Carassius auratus)[J].Journal of Agricultural and Food Chemistry,2009,57:5549-5555
[11]Wang K Q,Chen M,Gao T X.Study on taxonomy and fauna of Takifugu[J].Journal of Ocean University of Qingdao,2001,31(6):855-860[王奎旗,陈梅,高天翔.东方鲀属鱼类的分类与区系分布研究.青岛海洋大学学报,2001,31(6):855-860]
[12]Sun M L,LüH Q,Bao N,et al.IgM,NKCC1,and Hsp70gene expression in juvenile Takifugu rubripes under acute low-salinity stress[J].Journal of Fishery Sciences of China,2017,24(1):65-72[孙梦蕾,吕绘倩,暴宁,等.急性低盐胁迫下红鳍东方鲀幼鱼IgM、NKCC1和Hsp70基因的表达.中国水产科学,2017,24(1):65-72]
[13]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408
[14]Cao M J,Osatomi K,Matsuda R,et al.Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker(Argyrosomus argentatus)[J].Biochemical&Biophysical Research Communications,2000,272(2):485
[15]Sun L C.Study of a endogenous inhibitor to myofibrilbound serine proteinase of crucian carp(Carassius auratus)[D].Xiamen:Jimei University.2009[孙乐常.鲫鱼肌原纤维结合型丝氨酸蛋白酶内源性抑制剂的研究.厦门:集美大学.2009]
[16]Su J X.Ichthyology and Marine Fish Culture[M].2nd edition.Beijing:China Agricultural Press.2003,88-90[苏锦祥.鱼类学与海水鱼类养殖.第2版.北京:中国农业出版社.2003,88-90]
[17]Luo M Z,Guan R Z,Jin H.Effects of the salinity on the growth performance and digestive enzyme activities of Anguilla marmorata elver and A.bicolor pacifica elver[J].Acta Hydrobiologica Sinica,2015,39(4):653-660[罗鸣钟,关瑞章,靳恒.盐度对花鳗鲡和太平洋双色鳗鲡幼鳗生长性能及消化酶活力的影响.水生生物学报,2015,39(4):653-660]
[18]Guo Q D,Wang Y J,LüW Q.Combined effects of temperature and salinity on the physiological osmotic induction and antioxidant responses in the juvenile japanese flounder(Paralichthys olivaceus)[J].Acta Hydrobiologica Sinica,2014,38(1):58-67[郭勤单,王有基,吕为群.温度和盐度对褐牙鲆幼鱼渗透生理及抗氧化水平的影响.水生生物学报,2014,38(1):58-67]
[19]Kirschner L B.The energetics of osmotic regulation in ureotelic and hypoosmotic fishes[J].Journal of Experimental Zoology Part A Ecological Genetics&Physiology,1993,267(1):19-26
[20]Kidder G W,Petersen C W,Preston R L.Energetics of osmoregulation:II.Water flux and osmoregulatory work in the euryhaline fish,Fundulus heteroclitus[J].Journal of Experimental Zoology Part A Ecological Genetics& Physiology,2006,305A(4):318-327
[21]Cui W X.Construction of transcriptome under salinity stress and research of related functional genes on Turbot(Scophthalmus maximus)[D].Shanghai:Shanghai Ocean University.2017[崔文晓.大菱鲆(Scophthalmus maximus)盐度胁迫转录组构建及相关功能基因研究.上海:上海海洋大学.2017]
[22]Fang Z H,Tian X L,Dong S L.The growth performance,osmotic physiology and carbohydrate metabolism activity of juvenile Cynoglossus semilaevis acclimated to low salinity water[J].Periodical of Ocean University of China,2016,46(8):19-27[房子恒,田相利,董双林.低盐驯化对半滑舌鳎幼鱼生长、渗透生理及糖代谢酶活力影响的研究.中国海洋大学学报(自然科学版),2016,46(8):19-27]
[23]Sangiao-Alvarellos S,Laiz-Carrión R,Guzmán J M,et al.Acclimation of S.aurata to various salinities alters energy metabolism of osmoregula-tory and nonosmoregulatory organs[J].American Journal of Physiology-Regulatory Integrative and Comparative Physiology,2003,285(4):897-907
[24]Sangiao-Alvarellos S,Arjona F J,Martin del Rio M P,et al.Time course of osmoregulatory and metabolic changes during osmotic acclimation in Sparus auratus[J].Journal of Experimental Biology,2005,208(22):4291-4304