鹅食欲素及其受体2基因克隆、序列和组织表达分析
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摘要
旨在根据GenBank公布的人、原鸡、绿头鸭等物种HCRT和HCRTR2基因序列设计特异性引物克隆扩增鹅HCRT和HCRTR2基因编码区序列,并进行生物信息学分析;同时应用RT-qPCR技术检测HCRT和HCRTR2 mRNA在四川白鹅19个组织中的表达情况。成功获得鹅HCRT和HCRTR2基因完整编码区序列,分别为456,1 506 bp,各自编码151,501个氨基酸。其中,HCRT基因编码的前体食欲素蛋白水解后可产生OXA和OXB 2种多肽;序列分析表明,鹅OXA和OXB分别为含34,28个氨基酸的残留肽。序列比对结果显示,鹅OXA、OXB和HCRTR2氨基酸序列在近源物种间呈高度保守,提示OXA、OXB和HCRTR2可能在物种的进化中扮演着重要的角色;但各物种间食欲素蛋白的信号肽保守性较低,暗示其信号肽序列可能对于各物种的功能特性具有重要的意义。进化树分析表明,HCRT和HCRTR2两者进化较为相似,其中,鹅与鸡的亲缘关系最近,而与斑马鱼的亲缘关系最远。RT-qPCR结果显示,鹅HCRT和HCRTR2基因在下丘脑中的表达量均达到最高,且在其他组织中也有不同程度的表达,推测食欲素系统可能通过下丘脑-垂体-性腺轴(HPG)参与动物生殖机能的调节。为进一步研究HCRT和HCRTR2基因在鹅繁殖活动中的作用奠定了基础。
The aim of this study was to clone the complete coding sequences of HCRT and HCRTR2 genes in goose by designing the specific primers according to their respective reference sequences of humans,chicken and ducks on NCBI website,and then they were analyzed by corresponding bioinformatic method. The expression levels of HCRT and HCRTR2 mRNA in nineteen tissues of Sichuan White Goose were detected. The complete coding regions of HCRT and HCRTR2 genes in goose were 456,1 506 bp,respectively,encoding 151 and 501 amino acids. Among them,the precursor orexin protein encoded by HCRT gene was hydrolyzed to produce OXA and OXB polypeptides. Sequence analysis showed that goose OXA and OXB contained 34 and 28 amino acid residues,respectively. The sequence alignment showed that the amino acid sequences of OXA,OXB and HCRTR2 were highly conserved among the nearest species,suggesting that OXA,OXB and HCRTR2 might play an important role in the evolution of species. However,the signal peptide conservatism of orexin protein among species was low,suggesting that the signal peptide sequence of orexin protein might be important to the functional characteristics of species. The evolutionary tree analysis showed that the evolution of HCRT and HCRTR2 were similar. Goose had the closest relationship with chicken and the farthest with zebra fish. RT-qPCR results showed that the expression levels of HCRT and HCRTR2 genes in the hypothalamus were the highest,though they were also expressed in other tissues to a certain degree. It is speculated that orexin system might participate in the regulation of animal reproductive function through hypothalamus-pituitary-gonad axis(HPG). The results of this study laid the foundation for further studying the role of HCRT and HCRTR2 genes in goose reproductive activities.
The aim of this study was to clone the complete coding sequences of HCRT and HCRTR2 genes in goose by designing the specific primers according to their respective reference sequences of humans,chicken and ducks on NCBI website,and then they were analyzed by corresponding bioinformatic method. The expression levels of HCRT and HCRTR2 mRNA in nineteen tissues of Sichuan White Goose were detected. The complete coding regions of HCRT and HCRTR2 genes in goose were 456,1 506 bp,respectively,encoding 151 and 501 amino acids. Among them,the precursor orexin protein encoded by HCRT gene was hydrolyzed to produce OXA and OXB polypeptides. Sequence analysis showed that goose OXA and OXB contained 34 and 28 amino acid residues,respectively. The sequence alignment showed that the amino acid sequences of OXA,OXB and HCRTR2 were highly conserved among the nearest species,suggesting that OXA,OXB and HCRTR2 might play an important role in the evolution of species. However,the signal peptide conservatism of orexin protein among species was low,suggesting that the signal peptide sequence of orexin protein might be important to the functional characteristics of species. The evolutionary tree analysis showed that the evolution of HCRT and HCRTR2 were similar. Goose had the closest relationship with chicken and the farthest with zebra fish. RT-qPCR results showed that the expression levels of HCRT and HCRTR2 genes in the hypothalamus were the highest,though they were also expressed in other tissues to a certain degree. It is speculated that orexin system might participate in the regulation of animal reproductive function through hypothalamus-pituitary-gonad axis(HPG). The results of this study laid the foundation for further studying the role of HCRT and HCRTR2 genes in goose reproductive activities.
引文
[1] 路海,姜云璐,陈京,白波.食欲素研究现状及展望[J].济宁医学院学报,2015,38(5):339-343.doi:10.3969/j.issn.1000-9760.2015.05.011.Lu H,Jiang Y L,Chen J,Bai B.Current situation and prospect of orexin research [J].Journal of Jining Medical College,2015,38(5):339-343.
[2] 贺婵婵,张蓉,张华,罗明,呼圣娟.食欲素与肿瘤关系的研究进展[J].中国全科医学,2016,19(8):986-988.doi:10.3969/j.issn.1007-9572.He C C,Zhang R,Zhang H,Luo M,Hu S J.Research progress of the relationship between orexins and tumors[J].Chinese General Practice,2016,19(8):986-988.
[3] Sharko A C,Fadel J R,Kaigler K F,Wilson M A.Activation of orexin/hypocretin neurons is associated with individual differences in cued fear extinction[J].Physiology & Behavior,2017,178:93-102.doi:10.1016/j.physbeh.2016.10.008.
[4] Yi C X ,Serlie M J ,Ackermans M T ,Foppen E,Buijs R M,Sauerwein H P,Fliers E,Kalsbeek A.A major role for perifornical orexin neurons in the control of glucose metabolism in rats[J].Diabetes,2009,58(9):1998.doi:10.2337/db09-0385.
[5] Kapica M,Puzio I,Kato I,Foppen E,Buijs R M,Sauerwein H P,Fliers E,Kalsbeek A.Role of feed-regulating peptides on pancreatic exocrine secretion[J].Journal of Physiology & Pharmacology,2008,59(S2):145-159.doi:10.2170/physiolsci.RP006108.
[6] 郝贺玲.LHA-DVC Orexin-A通路构成及对胃牵张敏感神经元放电活动和胃运动调控研究[D].青岛:青岛大学,2016.Hao H L.Study on the structure of the lha-dvc orexin-a pathway and the regulation of gastric motor and the discharge activity of gastric stretch sensitive neurons[D].Qingdao:Qingdao University,2016.
[7] Tamura T,Irahara M,Tezuka M,Kiyokawa M,Aono T.Orexins,orexigenic hypothalamic neuropeptides,suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats[J].Biochemical & Biophysical Research Communications,1999,264(3):759-762.doi:10.1006/bbrc.1999.1573.
[8] Hoskins L J,Xu M,Volkoff H.Interactions between gonadotropin-releasing hormone (GnRH) and orexin in the regulation of feeding and reproduction in goldfish (Carassius auratus)[J].Hormones & Behavior,2008,54(3):379-385.doi:10.1016/j.yhbeh.2008.04.011.
[9] 陈丽.增食欲素A通过增食欲素受体1和AKT信号通路影响大鼠胰岛INS-1细胞凋亡的研究[D].辽宁:中国医科大学,2014.Chen L.Effects of orexin A on apoptosis of insulin-1 cells in rats through orexin receptor 1 and AKT signaling pathway[D].Liaoning:China Medical University,2014.
[10] 罗鸿.人肾上腺H295R细胞中OrexinA调节3β-HSD的分子机制研究[D].辽宁:中国医科大学,2014.Luo H.Molecular mechanism of OrexinA regulating 3 -hsd in human adrenal H295R cells[D].Liaoning:China Medical University,2014.
[11] De Lecea L,Kilduff T S,Peyron C,Gao X,Foye P E,Danielson P E,Fukuhara C,Battenberg E L,Gautvik V T,Bartlett F S,Frankel W N,van den Pol A N,Bloom F E,Gautvik K M,Sutcliffe J G.The hypocretins:hypothalamus-specific peptides with neuroexcitatory activity[J].Proc Natl Acad Sci USA,1998,95(1):322-327.doi:10.1073/pnas.95.1.322.
[12] Willie J T,Chemelli R M,Sinton C M,Yanagisawa M.To eat or to sleep?Orexin in the regulation of feeding and wakefulness[J].Annu Rev Neurosci,2001,24:429-458.doi:10.1146/annurev.neuro.24.1.429.
[13] Miranda B,Esposito V,de Girolamo P,Peter J Sharp,Peter W Wilson,Ian C Dunn.Orexin in the chicken hypothalamus:immunocytochemical localisation and comparison of mRNA concentrations during the day and night,and after chronic food restriction[J].Brain Res,2013,1513:34-40.doi:10.1016/j.brainres.2013.03.036.
[14] Ohkubo T,Tsukada A,Shamoto K.cDNA cloning of chicken orexin receptor and tissue distribution:sexually dimorphic expression in chicken gonads[J].J Mol Endocrinol,2003,31(3):499-508.doi:10.1677/jme.0.0310499.
[15] Fern? J,Seňarís R,Diéguez C,Manuel T,Miguel L.Orexins (hypocretins) and energy balance:More than feeding[J].Mol Cell Endocrinol,2015,418:17-26.doi:10.1016/j.mce.2015.07.022.
[16] Nakabayashi M,Suzuki T,Takahashi K,Totsune K,Muramatsu Y,Kaneko C,Date F,Takeyama J,Darnel A D,Moriya T,Sasano H.Orexin-A expression in human peripheral tissues[J].Molecular & Cellular Endocrinology,2003,205 (1-2) :43-50.doi:10.1016/s0303-7207(03)00206-5.
[17] 王伟平.食欲素A对绵羊卵巢颗粒细胞增殖、成熟及功能影响的研究[D].呼和浩特:内蒙古农业大学,2015.Wang W P.Effect of orexin A on proliferation,maturation and function of ovary granulosa cells in sheep[D].Hohhot:Inner Mongolia Agricultural University,2015.
[18] 王露,赵玉岩.增食欲素调节下丘脑-垂体-肾上腺轴的研究进展[J].国际内分泌代谢杂志,2010,30 (S1):22-25.doi:10.3760/cma.j.issn.1673-4157.2010.s1.008.Wang L,Zhao Y Y.Research progress of orexin regulating hypothalamic-pituitary-adrenal axis[J].International Journal of Endocrinology and Metabolism,2010,30 (S1):22-25.
[19] 何珲,姜冬梅,康波.褪黑素调控动物繁殖功能的途径[J].中国生物化学与分子生物学报,2014,30 (2):144-149.He H,Jiang D M,Kang B.The pathway by which melatonin regulates animal reproductive function[J].Chinese Journal of Biochemistry and Molecular Biology,2014,30 (2):144-149.
[2] 贺婵婵,张蓉,张华,罗明,呼圣娟.食欲素与肿瘤关系的研究进展[J].中国全科医学,2016,19(8):986-988.doi:10.3969/j.issn.1007-9572.He C C,Zhang R,Zhang H,Luo M,Hu S J.Research progress of the relationship between orexins and tumors[J].Chinese General Practice,2016,19(8):986-988.
[3] Sharko A C,Fadel J R,Kaigler K F,Wilson M A.Activation of orexin/hypocretin neurons is associated with individual differences in cued fear extinction[J].Physiology & Behavior,2017,178:93-102.doi:10.1016/j.physbeh.2016.10.008.
[4] Yi C X ,Serlie M J ,Ackermans M T ,Foppen E,Buijs R M,Sauerwein H P,Fliers E,Kalsbeek A.A major role for perifornical orexin neurons in the control of glucose metabolism in rats[J].Diabetes,2009,58(9):1998.doi:10.2337/db09-0385.
[5] Kapica M,Puzio I,Kato I,Foppen E,Buijs R M,Sauerwein H P,Fliers E,Kalsbeek A.Role of feed-regulating peptides on pancreatic exocrine secretion[J].Journal of Physiology & Pharmacology,2008,59(S2):145-159.doi:10.2170/physiolsci.RP006108.
[6] 郝贺玲.LHA-DVC Orexin-A通路构成及对胃牵张敏感神经元放电活动和胃运动调控研究[D].青岛:青岛大学,2016.Hao H L.Study on the structure of the lha-dvc orexin-a pathway and the regulation of gastric motor and the discharge activity of gastric stretch sensitive neurons[D].Qingdao:Qingdao University,2016.
[7] Tamura T,Irahara M,Tezuka M,Kiyokawa M,Aono T.Orexins,orexigenic hypothalamic neuropeptides,suppress the pulsatile secretion of luteinizing hormone in ovariectomized female rats[J].Biochemical & Biophysical Research Communications,1999,264(3):759-762.doi:10.1006/bbrc.1999.1573.
[8] Hoskins L J,Xu M,Volkoff H.Interactions between gonadotropin-releasing hormone (GnRH) and orexin in the regulation of feeding and reproduction in goldfish (Carassius auratus)[J].Hormones & Behavior,2008,54(3):379-385.doi:10.1016/j.yhbeh.2008.04.011.
[9] 陈丽.增食欲素A通过增食欲素受体1和AKT信号通路影响大鼠胰岛INS-1细胞凋亡的研究[D].辽宁:中国医科大学,2014.Chen L.Effects of orexin A on apoptosis of insulin-1 cells in rats through orexin receptor 1 and AKT signaling pathway[D].Liaoning:China Medical University,2014.
[10] 罗鸿.人肾上腺H295R细胞中OrexinA调节3β-HSD的分子机制研究[D].辽宁:中国医科大学,2014.Luo H.Molecular mechanism of OrexinA regulating 3 -hsd in human adrenal H295R cells[D].Liaoning:China Medical University,2014.
[11] De Lecea L,Kilduff T S,Peyron C,Gao X,Foye P E,Danielson P E,Fukuhara C,Battenberg E L,Gautvik V T,Bartlett F S,Frankel W N,van den Pol A N,Bloom F E,Gautvik K M,Sutcliffe J G.The hypocretins:hypothalamus-specific peptides with neuroexcitatory activity[J].Proc Natl Acad Sci USA,1998,95(1):322-327.doi:10.1073/pnas.95.1.322.
[12] Willie J T,Chemelli R M,Sinton C M,Yanagisawa M.To eat or to sleep?Orexin in the regulation of feeding and wakefulness[J].Annu Rev Neurosci,2001,24:429-458.doi:10.1146/annurev.neuro.24.1.429.
[13] Miranda B,Esposito V,de Girolamo P,Peter J Sharp,Peter W Wilson,Ian C Dunn.Orexin in the chicken hypothalamus:immunocytochemical localisation and comparison of mRNA concentrations during the day and night,and after chronic food restriction[J].Brain Res,2013,1513:34-40.doi:10.1016/j.brainres.2013.03.036.
[14] Ohkubo T,Tsukada A,Shamoto K.cDNA cloning of chicken orexin receptor and tissue distribution:sexually dimorphic expression in chicken gonads[J].J Mol Endocrinol,2003,31(3):499-508.doi:10.1677/jme.0.0310499.
[15] Fern? J,Seňarís R,Diéguez C,Manuel T,Miguel L.Orexins (hypocretins) and energy balance:More than feeding[J].Mol Cell Endocrinol,2015,418:17-26.doi:10.1016/j.mce.2015.07.022.
[16] Nakabayashi M,Suzuki T,Takahashi K,Totsune K,Muramatsu Y,Kaneko C,Date F,Takeyama J,Darnel A D,Moriya T,Sasano H.Orexin-A expression in human peripheral tissues[J].Molecular & Cellular Endocrinology,2003,205 (1-2) :43-50.doi:10.1016/s0303-7207(03)00206-5.
[17] 王伟平.食欲素A对绵羊卵巢颗粒细胞增殖、成熟及功能影响的研究[D].呼和浩特:内蒙古农业大学,2015.Wang W P.Effect of orexin A on proliferation,maturation and function of ovary granulosa cells in sheep[D].Hohhot:Inner Mongolia Agricultural University,2015.
[18] 王露,赵玉岩.增食欲素调节下丘脑-垂体-肾上腺轴的研究进展[J].国际内分泌代谢杂志,2010,30 (S1):22-25.doi:10.3760/cma.j.issn.1673-4157.2010.s1.008.Wang L,Zhao Y Y.Research progress of orexin regulating hypothalamic-pituitary-adrenal axis[J].International Journal of Endocrinology and Metabolism,2010,30 (S1):22-25.
[19] 何珲,姜冬梅,康波.褪黑素调控动物繁殖功能的途径[J].中国生物化学与分子生物学报,2014,30 (2):144-149.He H,Jiang D M,Kang B.The pathway by which melatonin regulates animal reproductive function[J].Chinese Journal of Biochemistry and Molecular Biology,2014,30 (2):144-149.
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