太平洋真宽水蚤(Eurytemora pacifica)CRH-BP基因克隆及表达分析
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摘要
促肾上腺皮质激素释放激素结合蛋白(CRH-BP)是一种糖基化蛋白,与促肾上腺皮质激素释放激素(CRH)具有很高的亲和力,对CRH诱导的脑垂体促肾上腺皮质激素(ACTH)的释放起到抑制作用。本研究采用RT-PCR与RACE基因克隆方法首次获得太平洋真宽水蚤(Eurytemora pacifica)CRH-BP基因全长cDNA序列(GenBank登录号:MF289345)1950bp,其中ORF区1245bp编码415个氨基酸,5′非编码区841bp,3′非编码区294bp,理论分子量/等电点为45.816/5.87。生物学序列分析结果表明,太平洋真宽水蚤CRH-BP氨基酸序列与桡足类中近亲真宽水蚤同源性最高;具有6个蛋白修饰位点及蛋白家族标签序列,具有明显的跨膜结构域及信号肽。实时荧光定量PCR分析结果表明,太平洋真宽水蚤CRH-BP基因在盐度、温度及pH环境胁迫中的mRNA表达量均具有一定的时间梯度表达性与浓度梯度表达性特征。本文通过对太平洋真宽水蚤CRH-BP基因结构及不同环境因子刺激下的表达特点的研究为探究桡足类在环境应激、生殖生理及免疫调控等方面奠定了理论基础。
CRH-BP(Corticotropin releasing hormone binding protein) is a glycosylated protein in a high affinity with corticotropin releasing hormone(CRH).CRH induces the release of adrenocorticotropic hormone(ACTH),and CRH-BP can inhibit the release of ACTH by combining CRH.In this study,Eurytemora pacifica was selected as the research subject from which the full-length cDNA sequence of CRH-BP gene was obtained by RT-PCR and RACE methods.The full-sequence of CRH-BP(GenBank accession number:MF289345) was 1950 bp,having a complete open reading frame of 1245 bp encoding 415 amino acids,5′ and 3′ non-encoding ones for 841 bp and 296 bp,respectively,the molecular weight of 45.816 kDa,and isoelectric point of 5.89.Sequence comparison indicated high homology in amino acid sequence of CRH-BP cDNA to E.affinis,sharing 6 protein post-translational modification and protein family sequence tagged sites,an obvious signal peptide,and transmembrane domain.CRH-BP mRNA expression analysis of E.pacifica showed that Ep.CRH-BP gene expression could be up-regulated under salinity,temperature,or pH stress,depending on temporal and concentration conditions.Therefore,these results shall be helpful to further explore the structure and function of the copepod CRH-BP gene,and to provide a basis for studying the molecular mechanism of stress response from the copepod.
CRH-BP(Corticotropin releasing hormone binding protein) is a glycosylated protein in a high affinity with corticotropin releasing hormone(CRH).CRH induces the release of adrenocorticotropic hormone(ACTH),and CRH-BP can inhibit the release of ACTH by combining CRH.In this study,Eurytemora pacifica was selected as the research subject from which the full-length cDNA sequence of CRH-BP gene was obtained by RT-PCR and RACE methods.The full-sequence of CRH-BP(GenBank accession number:MF289345) was 1950 bp,having a complete open reading frame of 1245 bp encoding 415 amino acids,5′ and 3′ non-encoding ones for 841 bp and 296 bp,respectively,the molecular weight of 45.816 kDa,and isoelectric point of 5.89.Sequence comparison indicated high homology in amino acid sequence of CRH-BP cDNA to E.affinis,sharing 6 protein post-translational modification and protein family sequence tagged sites,an obvious signal peptide,and transmembrane domain.CRH-BP mRNA expression analysis of E.pacifica showed that Ep.CRH-BP gene expression could be up-regulated under salinity,temperature,or pH stress,depending on temporal and concentration conditions.Therefore,these results shall be helpful to further explore the structure and function of the copepod CRH-BP gene,and to provide a basis for studying the molecular mechanism of stress response from the copepod.
引文
王庆,刘丽君,杨宇峰,2012.南方某水库轮虫群落特征及其对环境因子的响应.生态学报,30(13):3385-3395
李光福,2015.四种环境因子对指状伪镖水蚤摄食、繁殖和发育的影响.青岛:中国海洋大学硕士学位论文,15-20
吴利,冯伟松,张堂林等,2011.春、秋季武湖浮游动物群落特征及其与环境因子的关系.水生态学杂志,32(2):31-37
陈光荣,雷泽湘,谭镇等,2010.环境因子对广东城市湖泊后生浮游动物的影响.水生态学杂志,3(4):28-32
夏品华,马健荣,李存雄等,2011.红枫湖水库冬春季浮游生物群落与环境因子的典范对应分析.环境科学研究,24(4):378-386
蒲龄,2014.草鱼CRH-BP基因cDNA全长克隆与表达分析.长沙:湖南农业大学硕士学位论文,38-40
Behan D P,Linton E A,Lowry P J,1989.Isolation of the human plasma corticotrophin-releasing factor-binding protein.Journal of Endocrinology,122(1):23-31
Chan R K W,Vale W W,Sawchenko P E,2000.Paradoxical activational effects of a corticotropin-releasing factorbinding protein“ligand inhibitor”in rat brain.Neuroscience,101(1):115-129
Chatzaki E,Margioris A N,Gravanis A,2002.Expression and regulation of corticotropin-releasing hormone binding protein(CRH-BP)in rat adrenals.Journal of Neurochemistry,80(1):81-90
Doyon C,Trudeau V L,Moon T W,2005.Stress elevates corticotropin-releasing factor(CRF)and CRF-binding protein mRNA levels in rainbow trout(Oncorhynchus mykiss).Journal of Endocrinology,186(1):123-130
Huising M O,Flik G,2005.The remarkable conservation of corticotropin-releasing hormone(CRH)-binding protein in the honeybee(Apis mellifera)dates the CRH system to a common ancestor of insects and vertebrates.Endocrinology,146(5):2165-2170
Lee K W,Rhee J S,Raisuddin S et al,2008.A corticotropinreleasing hormone binding protein(CRH-BP)gene from the intertidal copepod,Tigriopus japonicus.General and Comparative Endocrinology,158(1):54-60
Linton E A,Wolfe C D A,Behan D P et al,1988.A specific carrier substance for human corticotrophin releasing factor in late gestational maternal plasma which could mask the acth-releasing activity.Clinical Endocrinology,28(3):315-324
Liu L,Yu X L,Meng F et al,2011.Identification and characterization of a novel corticotropin-releasing hormonebinding protein(CRH-BP)gene from Chinese honeybee(Apis cerana cerana).Archives of Insect Biochemistry&Physiology,78(3):161-175
Majzoub J A,2006.Corticotropin-releasing hormone physiology.European Journal of Endocrinology,155(S1):S71-S76
Mazon A F,Verburgvan-Van Kemenade B M,Flik G et al,2006.Corticotropin-releasing hormone-receptor 1(CRH-R1)and CRH-binding protein(CRH-BP)are expressed in the gills and skin of common carp(Cyprinus carpio L.)and respond to acute stress and infection.Journal of Experimental Biology,209(3):510-517
Seasholtz A F,Burrows H I,Karolyi I J et al,2001.Mouse models of altered CRH-binding protein expression.Peptides,22(5):743-751
Seasholtz A F,Valverde R A,Denver R J,2002.Corticotropinreleasing hormone-binding protein:biochemistry and function from fishes to mammals.Journal of Endocrinology,175(1):89-97
Westphal N J,Seasholtz A F,2006.CRH-BP:the regulation and function of a phylogenetically conserved binding protein.Frontiers in Bioscience,11:1878-1891
李光福,2015.四种环境因子对指状伪镖水蚤摄食、繁殖和发育的影响.青岛:中国海洋大学硕士学位论文,15-20
吴利,冯伟松,张堂林等,2011.春、秋季武湖浮游动物群落特征及其与环境因子的关系.水生态学杂志,32(2):31-37
陈光荣,雷泽湘,谭镇等,2010.环境因子对广东城市湖泊后生浮游动物的影响.水生态学杂志,3(4):28-32
夏品华,马健荣,李存雄等,2011.红枫湖水库冬春季浮游生物群落与环境因子的典范对应分析.环境科学研究,24(4):378-386
蒲龄,2014.草鱼CRH-BP基因cDNA全长克隆与表达分析.长沙:湖南农业大学硕士学位论文,38-40
Behan D P,Linton E A,Lowry P J,1989.Isolation of the human plasma corticotrophin-releasing factor-binding protein.Journal of Endocrinology,122(1):23-31
Chan R K W,Vale W W,Sawchenko P E,2000.Paradoxical activational effects of a corticotropin-releasing factorbinding protein“ligand inhibitor”in rat brain.Neuroscience,101(1):115-129
Chatzaki E,Margioris A N,Gravanis A,2002.Expression and regulation of corticotropin-releasing hormone binding protein(CRH-BP)in rat adrenals.Journal of Neurochemistry,80(1):81-90
Doyon C,Trudeau V L,Moon T W,2005.Stress elevates corticotropin-releasing factor(CRF)and CRF-binding protein mRNA levels in rainbow trout(Oncorhynchus mykiss).Journal of Endocrinology,186(1):123-130
Huising M O,Flik G,2005.The remarkable conservation of corticotropin-releasing hormone(CRH)-binding protein in the honeybee(Apis mellifera)dates the CRH system to a common ancestor of insects and vertebrates.Endocrinology,146(5):2165-2170
Lee K W,Rhee J S,Raisuddin S et al,2008.A corticotropinreleasing hormone binding protein(CRH-BP)gene from the intertidal copepod,Tigriopus japonicus.General and Comparative Endocrinology,158(1):54-60
Linton E A,Wolfe C D A,Behan D P et al,1988.A specific carrier substance for human corticotrophin releasing factor in late gestational maternal plasma which could mask the acth-releasing activity.Clinical Endocrinology,28(3):315-324
Liu L,Yu X L,Meng F et al,2011.Identification and characterization of a novel corticotropin-releasing hormonebinding protein(CRH-BP)gene from Chinese honeybee(Apis cerana cerana).Archives of Insect Biochemistry&Physiology,78(3):161-175
Majzoub J A,2006.Corticotropin-releasing hormone physiology.European Journal of Endocrinology,155(S1):S71-S76
Mazon A F,Verburgvan-Van Kemenade B M,Flik G et al,2006.Corticotropin-releasing hormone-receptor 1(CRH-R1)and CRH-binding protein(CRH-BP)are expressed in the gills and skin of common carp(Cyprinus carpio L.)and respond to acute stress and infection.Journal of Experimental Biology,209(3):510-517
Seasholtz A F,Burrows H I,Karolyi I J et al,2001.Mouse models of altered CRH-binding protein expression.Peptides,22(5):743-751
Seasholtz A F,Valverde R A,Denver R J,2002.Corticotropinreleasing hormone-binding protein:biochemistry and function from fishes to mammals.Journal of Endocrinology,175(1):89-97
Westphal N J,Seasholtz A F,2006.CRH-BP:the regulation and function of a phylogenetically conserved binding protein.Frontiers in Bioscience,11:1878-1891