牙鲆kiss2基因的分子鉴定及表达分析
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摘要
为克隆与牙鲆生殖相关的神经内分泌因子kiss2基因,并检测该基因在牙鲆成体不同组织及早期不同发育阶段的表达变化,本研究采用Trizol法提取牙鲆成鱼各组织及早期不同发育阶段样品总RNA;参照其他物种的kiss2 c DNA序列,设计并合成kiss2引物进行PCR扩增;运用实时荧光定量PCR检测kiss2基因在牙鲆成鱼各组织及早期不同发育阶段的表达情况。结果成功地获得长度为449 bp的牙鲆kiss2 c DNA序列,其中编码区序列为366 bp,编码121个氨基酸。在该序列中发现了高度保守的"FNYNPLTLRF"序列,此序列可以调节促性腺激素释放激素和黄体生成素的合成与释放。定量PCR结果显示,牙鲆kiss2基因在脑组织中的表达量最高,在卵巢和精巢中也均有表达;牙鲆kiss2基因在胚胎阶段表达量相对较低,而在孵化后第7 d其表达量显著增高,且在孵化后的第29 d达到高峰。说明牙鲆kiss2基因具有明显的时期和组织差异性表达,其在牙鲆脑中的丰富表达及在精巢和卵巢中的表达表明该基因可能在牙鲆神经系统与生殖中发挥重要作用。
A reproduction-related neuroendocrine gene,kiss2,was cloned and its expression changes in different adult tissues and developmental stages were examined by real-time quantitative PCR in Japanese flounder( Paralichthys olivaceus). A c DNA sequence of 449 bp was obtained,which included an open reading frame( ORF) of 366 bp encoding a polypeptide of 121 amino acids. A highly conserved Kisspeptin-10 region "FNYNPLTLRF ", a functional peptide stimulating the synthesis and release of Gn RH / LH,was found. The most abundant expression of kiss2 was detected in brain.The kiss2 was also expressed in gonad including ovary and testis. During embryonic and larval development,kiss2 showed low levels of expression. After 7-d hatching,the expression of kiss2 markedly increased and reached a peak at 29 d. These results demonstrated that the kiss2 gene exhibits stage- and tissuespecific expression. Its expressions in brain and the gonad suggest that the kiss2 gene might be involved in nervous system and reproductive development of Japanese flounder.
A reproduction-related neuroendocrine gene,kiss2,was cloned and its expression changes in different adult tissues and developmental stages were examined by real-time quantitative PCR in Japanese flounder( Paralichthys olivaceus). A c DNA sequence of 449 bp was obtained,which included an open reading frame( ORF) of 366 bp encoding a polypeptide of 121 amino acids. A highly conserved Kisspeptin-10 region "FNYNPLTLRF ", a functional peptide stimulating the synthesis and release of Gn RH / LH,was found. The most abundant expression of kiss2 was detected in brain.The kiss2 was also expressed in gonad including ovary and testis. During embryonic and larval development,kiss2 showed low levels of expression. After 7-d hatching,the expression of kiss2 markedly increased and reached a peak at 29 d. These results demonstrated that the kiss2 gene exhibits stage- and tissuespecific expression. Its expressions in brain and the gonad suggest that the kiss2 gene might be involved in nervous system and reproductive development of Japanese flounder.
引文
[1]SEMINARA S B.Metastin and its G protein-coupled receptor,GPR54:critical pathway modulating Gn RH secretion[J].Frontiers in Neuroendocrinology,2005,26:131-138.
[2]MATSUI H,TAKATSU Y,KUMANO S,et al.Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat[J].Biochemical and Biophysical Research Communications,2004,320:383-388.
[3]ROA J,AGUILAR E,DIEGUEZ C,et al.New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[J].Frontiers in Neuroendocrinology,2008,29(1):48-69.
[4]KOTANI M,DETHEUX M,COMMUNI D,et al.The metastasis suppressor gene Ki SS-1 encodes kisspeptins,the natural ligands of the orphan G-protein-coupled receptor GPR54[J].Journal of Biology and Chemistry,2001,276:34631-34636.
[5]OHTAKI T,SHINTANI Y,HONDA S,et al.Metastasis suppressor gene Kiss-1 encodes peptide ligand of a G-protein-coupled receptor[J].Nature,2001,411:613-617.
[6]FUNES S,HEDRICK J A,VASSILEVA G,et al.The Kiss-1 receptor GPR54 is essential for the development of the murine reproductive system[J].Biochemical and Biophysical Research Communications,2003,312:1357-1363.
[7]DE ROUX N,GENIN E,CAREL J C,et al.Hypogonadotropic hypogonadism due to loss of function of the Ki SS1-derived peptide receptor GPR54[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100:10972-10976.
[8]LAPATTO R,PALLAIS J C,ZHANG D,et al.Kiss1-/-mice exhibit more variable hypogonadism than Gpr54-/-mice[J].Endocrinology,2007,148:4927-4936.
[9]OAKLEY A E,CLIFTON D K,STEINER R A.Kisspeptin signaling in the brain[J].Endocrine Reviews,2009,30:713-743.
[10]NAVARRO V M,CASTELLANO J M,ROA J,et al.Advanced vaginal opening and precocious activation of the reproductive axis by Kiss-1 peptide,the endogenous ligand of GPR54[J].The Journal of Physiology,2004,561:379-386.
[11]SMITH J T.Kisspeptin signalling in the brain:steroid regulation in the rodent and ewe[J].Brain Research Reviews,2008,57:288-298.
[12]DHILLO W S,CHAUDHRI O B,PATTERSON M,et al.Kisspeptin-54 stimulates the hypothalamic-pituitary-gonadal axis in human males[J].The Journal of Clinical Endocrinology and Metabolism,2005,90:6609-6615.
[13]SEMINARA S B,MESSAGER S,CHATZIDAKI E E,et al.The GPR54 gene as a regulator of puberty[J].The New England Journal of Medicine,2003,349:1614-1627.
[14]FELIP A,ZANUY S,PINEDA R,et al.Evidence for two distinct Kiss genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[J].Molecular and Cellular Endocrinology,2009,312:61-71.
[15]BIRAN J,BEN-DOR S,LEVAVI-SIVAN B.Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[J].Biology of Reproduction,2008,79:776-786.
[16]KANDA S,AKAZOME Y,MATSUNAGA T,et al.Identification of Ki SS-1 product kisspeptin and steroidsensitive sexually dimorphic kisspeptin neurons in medaka(Oryzias latipes)[J].Endocrinology,2008,149:2467-2476.
[17]LI S,ZHANG Y,LIU Y,et al.Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish(Carassius auratus)[J].Journal of Endocrinology,2009,201:407-418.
[18]EIICHI Y.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus[J].Aquaculture,1999,173:235-246.
[19]佟雪红,雎燕,路瑛,等.牙鲆变态期间核酸、总蛋白的变化及其与生长的关系[J].江苏农业科学,2014,42(3):171-173.
[20]孙朝徽,刘海金,司飞,等.养殖牙鲆性腺的组织学观察[J].大连水产学院学报,2008,23(6):451-454.
[21]GOTTSCH M L,CUNNINGHAM M J,SMITH J T,et al.A role for kisspeptins in the regulation of gonadotropin secretion in the mouse[J].Endocrinology,2004,145:4073-4077.
[22]KITAHASHI T,OGAWA S,PARHAR I S.Cloning and expression of kiss2 in the Zebrafish and Medaka[J].Endocrinology,2009,150:821-831.
[23]SHIMIZU Y,TOMIKAWA J,HIRANO K,et al.Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major[J].General and Comparative Endocrinology,2012,175:432-442.
[24]SHAHJAHAN M,MOTOHASHI E,DO H,et al.Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season[J].General and Comparative Endocrinology,2010,169:48-57.
[25]MECHALY A S,VINAS J,PIFERRER F.Identification of two isoforms of the kisspeptin-1 receptor(kissir)generated by alternative splicing in a modern teleost,the Senegalese sole(Solea senegalensis)[J].Biology Reproduction,2009,80:60-69.
[26]MECHALY A S,VINAS J,MURPHY C,et al.Gene structure and regulation of the Kiss-1 receptor-2(Kiss1r-2)in the Atlantic halibut:insights into the evolution and regulation of Kiss1r genes[J].Molecular and Cellular Endocrinology,2010,317:78-89.
[27]SELVARAJ S,KITANO H,FUJINAGA Y,et al.Molecular characterization,tissue distribution,and m RNA expression profiles of two Kiss genes in the adult male and female chub mackerel(Scomber japonicus)during different gonadal stages[J].General and Comparative Endocrinology,2010,169:28-38.
[28]MOHAMED J S,BENNINGHOFF A D,HOLT G J,et al.Developmental expression of the G-protein-coupled receptor 54 and three Gn RH m RNAs inthe teleost fish cobia[J].Journal of Molecular Endocrinology,2007,38:235-244.
[2]MATSUI H,TAKATSU Y,KUMANO S,et al.Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat[J].Biochemical and Biophysical Research Communications,2004,320:383-388.
[3]ROA J,AGUILAR E,DIEGUEZ C,et al.New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[J].Frontiers in Neuroendocrinology,2008,29(1):48-69.
[4]KOTANI M,DETHEUX M,COMMUNI D,et al.The metastasis suppressor gene Ki SS-1 encodes kisspeptins,the natural ligands of the orphan G-protein-coupled receptor GPR54[J].Journal of Biology and Chemistry,2001,276:34631-34636.
[5]OHTAKI T,SHINTANI Y,HONDA S,et al.Metastasis suppressor gene Kiss-1 encodes peptide ligand of a G-protein-coupled receptor[J].Nature,2001,411:613-617.
[6]FUNES S,HEDRICK J A,VASSILEVA G,et al.The Kiss-1 receptor GPR54 is essential for the development of the murine reproductive system[J].Biochemical and Biophysical Research Communications,2003,312:1357-1363.
[7]DE ROUX N,GENIN E,CAREL J C,et al.Hypogonadotropic hypogonadism due to loss of function of the Ki SS1-derived peptide receptor GPR54[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100:10972-10976.
[8]LAPATTO R,PALLAIS J C,ZHANG D,et al.Kiss1-/-mice exhibit more variable hypogonadism than Gpr54-/-mice[J].Endocrinology,2007,148:4927-4936.
[9]OAKLEY A E,CLIFTON D K,STEINER R A.Kisspeptin signaling in the brain[J].Endocrine Reviews,2009,30:713-743.
[10]NAVARRO V M,CASTELLANO J M,ROA J,et al.Advanced vaginal opening and precocious activation of the reproductive axis by Kiss-1 peptide,the endogenous ligand of GPR54[J].The Journal of Physiology,2004,561:379-386.
[11]SMITH J T.Kisspeptin signalling in the brain:steroid regulation in the rodent and ewe[J].Brain Research Reviews,2008,57:288-298.
[12]DHILLO W S,CHAUDHRI O B,PATTERSON M,et al.Kisspeptin-54 stimulates the hypothalamic-pituitary-gonadal axis in human males[J].The Journal of Clinical Endocrinology and Metabolism,2005,90:6609-6615.
[13]SEMINARA S B,MESSAGER S,CHATZIDAKI E E,et al.The GPR54 gene as a regulator of puberty[J].The New England Journal of Medicine,2003,349:1614-1627.
[14]FELIP A,ZANUY S,PINEDA R,et al.Evidence for two distinct Kiss genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[J].Molecular and Cellular Endocrinology,2009,312:61-71.
[15]BIRAN J,BEN-DOR S,LEVAVI-SIVAN B.Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[J].Biology of Reproduction,2008,79:776-786.
[16]KANDA S,AKAZOME Y,MATSUNAGA T,et al.Identification of Ki SS-1 product kisspeptin and steroidsensitive sexually dimorphic kisspeptin neurons in medaka(Oryzias latipes)[J].Endocrinology,2008,149:2467-2476.
[17]LI S,ZHANG Y,LIU Y,et al.Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish(Carassius auratus)[J].Journal of Endocrinology,2009,201:407-418.
[18]EIICHI Y.Studies on sex-manipulation and production of cloned populations in hirame,Paralichthys olivaceus[J].Aquaculture,1999,173:235-246.
[19]佟雪红,雎燕,路瑛,等.牙鲆变态期间核酸、总蛋白的变化及其与生长的关系[J].江苏农业科学,2014,42(3):171-173.
[20]孙朝徽,刘海金,司飞,等.养殖牙鲆性腺的组织学观察[J].大连水产学院学报,2008,23(6):451-454.
[21]GOTTSCH M L,CUNNINGHAM M J,SMITH J T,et al.A role for kisspeptins in the regulation of gonadotropin secretion in the mouse[J].Endocrinology,2004,145:4073-4077.
[22]KITAHASHI T,OGAWA S,PARHAR I S.Cloning and expression of kiss2 in the Zebrafish and Medaka[J].Endocrinology,2009,150:821-831.
[23]SHIMIZU Y,TOMIKAWA J,HIRANO K,et al.Central distribution of kiss2 neurons and peri-pubertal changes in their expression in the brain of male and female red seabream Pagrus major[J].General and Comparative Endocrinology,2012,175:432-442.
[24]SHAHJAHAN M,MOTOHASHI E,DO H,et al.Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season[J].General and Comparative Endocrinology,2010,169:48-57.
[25]MECHALY A S,VINAS J,PIFERRER F.Identification of two isoforms of the kisspeptin-1 receptor(kissir)generated by alternative splicing in a modern teleost,the Senegalese sole(Solea senegalensis)[J].Biology Reproduction,2009,80:60-69.
[26]MECHALY A S,VINAS J,MURPHY C,et al.Gene structure and regulation of the Kiss-1 receptor-2(Kiss1r-2)in the Atlantic halibut:insights into the evolution and regulation of Kiss1r genes[J].Molecular and Cellular Endocrinology,2010,317:78-89.
[27]SELVARAJ S,KITANO H,FUJINAGA Y,et al.Molecular characterization,tissue distribution,and m RNA expression profiles of two Kiss genes in the adult male and female chub mackerel(Scomber japonicus)during different gonadal stages[J].General and Comparative Endocrinology,2010,169:28-38.
[28]MOHAMED J S,BENNINGHOFF A D,HOLT G J,et al.Developmental expression of the G-protein-coupled receptor 54 and three Gn RH m RNAs inthe teleost fish cobia[J].Journal of Molecular Endocrinology,2007,38:235-244.