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1 2013 1, 20(1): Journal of Fishery Sciences of China 研究论文 DOI: /SP. J 柳学周 1,2, 徐永江 1,2, 廖梅杰 1,2, 潘传燕 3, 王妍妍 1.,, ; 2., , ,2 摘要 : RACE, (Verasper variegatus) 3 GnRH cdna : cgnrh-Ⅱ sgnrh sbgnrh GnRH 1 1 Gly-Lys-Arg 1 GnRH, cgnrh-Ⅱ cdna 568 bp, 85, ORF 255 bp, 5 UTR 141 bp, 3 UTR 169 bp sgnrh cdna 457 bp, 90, ORF 270 bp, 5 UTR 41 bp, 3 UTR 143 bp sbgnrh cdna 381 bp, 98, ORF 294 bp, 5 UTR 48 bp, 3 UTR 36 bp 3 GnRH, GnRH, 3 GnRH, GnRH, PCR, 3 GnRH : GnRH mrna, cgnrh-Ⅱ mrna, sbgnrh, sgnrh sbgnrh mrna (P<0.05), GnRH mrna (P>0.05) 3 GnRH, sbgnrh GnRH, : ; GnRH; ; 中图分类号 : S917 文献标志码 : A 文章编号 : (2013) ,,, (BPG ), (GnRH) BPG, GnRH,, (GtH) (FSH) (LH),,, [1], 14 GnRH, GnRH GnRH 8 GnRH, GnRH 10,, GnRH [2], 收稿日期 : ; 修订日期 : 基金项目 : (CARS-50); 863 (2012AA10A413); ( );. 作者简介 : (1959 ),,,. Tel: ; liuxz@ysfri.ac.cn

2 1 : 13 2~3 GnRH [3] 3 GnRH, GnRH,, ; GnRH ( cgnrh-Ⅱ),, [4] ; 3 GnRH sgnrh, [2], (Verasper variegatus),,,,,, GnRH, GnRH, FSH LH, [1], GnRH,, m 3, 3 4, 1 500~ g, 2~3 /m 3 2, 2%~3% , 4 (2 : 2 ), MS222(400 mg/l),, GnRH, ,, ( ), 12 ( ) ( ) 3, 3 GnRH [5] 1.2 GnRH RNA cdna Trizol (Invitrogen, ) RNA RNA 1. 0%, (Nanodrop ND2000) DNase I(NEB, USA) RNA DNA SMART TM RACE cdna (Clontech, USA) 1 cdna, GnRH GnRH, PCR ( 1) GnRH cf1 cr1 cr2 cgnrh-Ⅱ, sbf sbr sbgnrh, sf sr sgnrh PCR : 94 5 min; 94 1 min, 55 1 min, min, 30 ; 72 5 min 1.3 GnRH 3 5 RACE 3 5 RACE cgnrh-Ⅱ cs1 ca1, sbgnrh sbs1 sba1, sgnrh ss1 ss2 sa1( 1) SMART TM RACE cdna (Clontech, USA), PCR 3 5 RACE 3 : 3 -Race ready-cdna, cf1 UPM cs1 NUP PCR cgnrh-Ⅱ 3 ; sbf1 UPM sbs1 NUP sbgnrh 3 ; ss1 UPM ss2 NUP sgnrh 3 5 :

3 14 20 Tab. 1 primer name UPM-long UPM-short NUP cf1 cr1 cr2 sbf sbr sf sr cs1 ca1 sbs1 sba1 ss1 ss2 SA1 表 1 圆斑星鲽 GnRH 基因 cdna 扩增表达所用引物 Primers used for PCR amplification of GnRH cdnas in Verasper variegatus (5 3 ) nucleotide acid(5 3 ) CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT CTAATACGACTCACTATAGGGC AAGCAGTGGTATCAACGCAGAGT CACCTGTGAACCTGTGAGAC GGTCACCAGGTAGAAAAGCA AGCTCTCTGGCCAAAGCATC AAGACCACCGAATCGGAGAC CTGTAAATTCCGGCAAAAGG TACCTTCCTCTGCAGCTCTG GCTCAGGAAGAGACACCACT GTTCTGCTGCTGGGGCTGCTTCT CTGAAATCTCTGACGTGCCAAAA CTTGTCAGTGTGGCTGCTCCTCG GAGCAGCCACACTGACAAGGTTT CTGATGGAAGCGAGCAGCAGACT GGATGATGGGCACAGGCGGAGTGGT AGACACCACTCCGCCTGTGCCCATC 3 GnRH 3 5 ( 1) 5 -RACE-Ready cdna, cr1 UPM ca1 NUP PCR, cgnrh-Ⅱ 5 sbr1 UPM sba1 NUP sbgnrh 5 sa1 UPM NUP sgnrh 5 3 GnRH PCR, PCR : s; s, 10, T m 1, 72 1 min; s, s, 72 60s, 20, 72 7 min PCR : 94 1 min; 58 1 min, 72 1 min, 30, min 1.5 % PCR, DNA (Montage, ), PCR peasy- T1 vector (Transgene Biotechnology, China), JM109, M GnRH PCR 3 GnRH RNA cdna PCR realplex PCR (Eppendorf, ) PCR 25 μl, μl2 SYBR Premix Ex Taq TM Ⅱ, 1 μl (1 μmol/l), 1 μl 1:100 cdna 9.5 μl (ddh 2 O) β-actin ( 2) GnRH cdna, Beacon designer 7.0 (Premier BioSoft) cf2 cr2 cgnrh Ⅱ, sf2 sr2 sgnrh, sbf2 sbr2 sbgnrh, β-actin F β-actin R β-actin PCR : s, 94 5 s, s, 40 PCR, β-actin 3 GnRH 1.5 GnRH mrna 3 GnRH, PCR, GnRH mrna

4 1 : 15 表 2 圆斑星鲽 GnRH 基因定量表达引物列表 Tab. 2 Primers used for quantitative expression of GnRH genes in Verasper variegatus primer name cf2 cr2 SbF2 sbr2 sf2 sr2 β-actin F β-actin R (5 3 ) nucleotide sequence (5 3 ) TGACTGTAGGACGAGCAGAG GCAGAACCAGCCGAGATG AAGGACTGAAGACCACCGAATC CACGAGGAGCAGCCACAC CAGGTGTTGGTGTTGATG AGGTCTCTCTTGGGTTTG TTCTGGTGATGGTGTGAC GTGGTGGTGAAGGAGTAG 1.1 PCR ( 2) PCR PCR DAMBE ( uottawa. ca/) GnRH Chromas( html), BLAST ( nlm.nih.gov/blast/) ; DNAStar ( GnRH, SignalP V3.0 ( GnRH Clustalx ( clustal.org/download/current/) MEGA 4.1( PCR Schmittgen [6], (SPSS 16.0), 0.05, P< GnRH cdna, cgnrh-Ⅱ sbgnrh sgnrh 3 GnRH 1 cdna 1 5 GnRH GnRH GnRH cdna cgnrh-Ⅱ cdna 568 bp, 85, bp Fig. 1 1 cgnrh-Ⅱ sgnrh sbgnrh cdna SP: ; GnRH: ; GAP: GnRH. Schematic structure of cgnrh-ii, sgnrh, and sbgnrh precursor cdnas of Verasper variegates and positions of primers used for RT-PCR SP: signal peptide; GnRH: gonadotropin-releasing hormone; GAP: GnRH-associated peptide.

5 16 20 (ORF), bp 5, bp 3, 1 (TGA), 23, 9.59 kd, 8.53 GenBank HM131601( 2-A) sgnrh cdna 457 bp, 90, bp (ORF), 1 41 bp 5, bp 3, 1 (TGA) 10 kd, GenBank HM131602( 2-B) sbgnrh cdna 381 bp, 98, bp (ORF), 1 48 bp 5 2 GnRH cdna A. cgnrh-ii; B. sgnrh; C. sbgnrh. (SP) GnRH (cgnrh-ii sgnrh sbgnrh) GnRH (GAP), TGA (*), AATAAA. Fig. 2 Nucleotide sequence of the cdna encoding the GnRH precursors of Verasper variegatus and the deduced amino acid sequence Nucleotides are numbered from 5 to 3 end, beginning with the initiation codon (ATG) in the coding region for the opening reading frame. Amino acid residues are numbered with the first residue (Met) in the open reading frame. Signal peptide (SP), GnRH core peptide (cgnrh-ii, sgnrh, sbfnrh) and GnRH-associated peptide (GAP) are underlined. The asterisk indicates the stop codon. The nucleotides corresponding to the polyadenylation signal in the 3 -untranslated region (AATAAA) are double underlined.

6 1 : 17, 1 36 bp 3, 1 (TGA) kd, 9.31 GenBank HM131603( 2-C) 2.2 cgnrh-Ⅱ cgnrh-Ⅱ GnRH Gly-Lys-Arg ( 3) cgnrh-Ⅱ 56.5%~100%, 97.6%, 25.9%~61. 2% sgnrh 42.2%~97.8%,, 97.8%, (78.9%~85.2%), 42.4%~44.4%, 61%~70% ( 3) sbgnrh, 90.8%~93.8%, Tab. 3 GnRH form cgnrh-Ⅱ sbgnrh sgnrh 表 3 圆斑星鲽 GnRH 氨基酸序列与其他硬骨鱼类的同源性比对分析 Amino acid identity between prepro-gnrhs of Verasper variegatus and those of other teleosts species Verasper variegatus cgnrh-Ⅱ sbgnrh sgnrh Verasper moseri Paralichthys olivaceus Sparus aurata Dicentrarchus labrax Carassius auratus 1/2 60/ / /20. 9 Cyprinus carpio Oncorhynchus mykiss Oncorhynchus keta Ictalurus punctatus Anguilla japonica Pseudotropheus crabro 鳉 Oryzias latipes Verasper moseri Paralichthys olivaceus Paralichthys orbignyanus Sparus aurata Pagrus major Dicentrarchus labrax Pseudotropheus crabro 鳉 Oryzias latipes Verasper moseri Paralichthys olivaceus Sparus aurata Dicentrarchus labrax Carassius auratus Oncorhynchus mykiss 1/ / / /61 Salmo salar 1/ / / /70 Pseudotropheus crabro 鳉 Oryzias latipes %

7 %~77.9% Clustalx Mega 4. 1 cgnrh-ii,, cgnrh-ii,, cgnrh-ii ( 3) sgnrh,, ( 4) sbgnrh Fig. 3 3 cgnrh-ii Phylogenetic tree of prepro-cgnrh-ii between Verasper variegatus and other vertebrates

8 1 : 19 GnRH( sbgnrh cfgnrh mgnrh pjgnrh ) sbgnrh,, sbgnrh ( 5) 2.3 GnRH mrna,, 3 GnRH mrna cgnrh-Ⅱmrna ( 6-A), sbgnrh mrna ( 6-B), sgnrh mrna, ( 6-C D) 3 GnRH, sbgnrh, mrna cgnrh-Ⅱmrna sgnrh mrna 30~ GnRH mrna, sbgnrh mrna, 8 ( ) (P<0.05), 12 ( ), 4, ( 7), sgnrh cgnrh-Ⅱ mrna (P>0.05) ( 8) 3 3 GnRH Fig. 4 4 sgnrh Phylogenetic tree of prepro-sgnrh between Verasper variegatus and other vertebrates

9 20 20 Fig. 5 5 sbgnrh Phylogenetic tree of prepro-sbgnrh between Verasper variegatus and other vertebrates 6 3 GnRH mrna A. cgnrh-Ⅱ; B. sbgnrh; C. sbgnrh ; D. sbgnrh. B: ; P: ; G: ; L: ; Sp: ; K: ; H: ; I: ; St:. Fig. 6 The mrna expression levels of three GnRH genes in various tissues determined by quantitative RT-PCR A. cgnrh-Ⅱ; B. sbgnrh; C. sbgnrh ; D. sbgnrh. B: brain; G: gonad; L: liver; Sp: spleen; K: kidney; H: heart; I: intestine; St: stomach.

10 1 : 21 7 cgnrh-Ⅱ sbgnrh mrna Fig. 7 8 Fig. 8 Fluctuation of brain cgnrh-Ⅱ mrna and sbgnrh mrna levels during ovary maturation cycle sgnrh mrna Fluctuation of brain sgnrh mrna level during ovary maturation cycle : sgnrh sbgnrh cgnrh-Ⅱ, 3 GnRH GnRH (SP) GnRH GnRH,, GnRH, 2 GnRH, 3 GnRH [7 9] GnRH ( ),, (Oreochromis niloticus), sbgnrh, [10], GnRH GnRH GnRH, GnRH, cgnrh-Ⅱ, [4,10], (Oncorhynchus masou) (Verasper moseri), cgnrh-Ⅱ, [7,11 12], [8] (Paralichthys olivaceus),, cgnrh-Ⅱ, cgnrh-Ⅱ, cgnrh-Ⅱ, cgnrh-Ⅱ, sgnrh,, sgnrh, sgnrh [13 15] sgnrh,,, sgnrh, sgnrh, [11,16], sgnrh mrna, sgnrh, [17], sgnrh,,,, sbgnrh, (Scophthalmus maximus) (Oncorhynchus mykiss) [7,17 21],, sbgnrh sbgnrh, [21 23] sbgnrh [7]

11 22 20, cgnrh-Ⅱ sgnrh sbgnrh [24 26],, [27 28] Collins [21] sbgnrh 鲉 (Sebastes rastrelliger), sbgnrh, (Pagrus major) sbgnrh 10 ( ), 4 ( ) [28] sbgnrh [18], sbgnrh,, cgnrh-Ⅱ sgnrh, sbgnrh GnRH,,, sbgnrh GnRH,, [1,4,29], GnRH,, 参考文献 : [1] Zohar Y, Muñoz-Cueto J A, Elizur A, et al. Neuroendocrinology of reproduction in teleost fish [J]. Gen Comp Endocrinol, 2010, 165: [2] Somoza G M, Lescheid D, Miranda L A, et al. Expression of pejerrey gonadotropinreleasing hormone (pjgnrh) in three orders of fish [J]. Biol Reprod, 2002b, 67: [3] Adams B A, Vickers E D, Warby C, et al. Three forms of gonadotropin-releasing hormone, including a novel form, in a basal salmonid, Coregonus clupeaformis [J]. Biol Reprod, 2002, 67: [4] Servili A, Lethimonier C, Lareyre J J, et al. The Highly conserved gonadotropin-releasing hormone-2 form acts as a melatonin-releasing factor in the pineal of a teleost fish, the European sea bass Dicentrarchus labrax [J]. Endocrinology, 2010, 151(5): [5],,,. [J]., 2011, 18(4): [6] Schmittgen T D, Livak K J. Analyzing real-time PCR data by the comparative CT method [J]. Nature Protocols, 2008, 3(6): [7] Amano M, Takahashi A, Yamanome T, et al. Molecular cloning of three cdnas encoding different GnRHs in the brain of barfin flounder [J]. Gen Comp Endocrinol, 2002, 126: [8],,,. cgnrh2ii sbgnrh [J]., 2006, 16(5): [9] Pham K X, Amano M, Amiya N, et al. Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder [J]. Fish Sci, 2007, 73: [10] Swapna I, Sudhakumari C C, Sakai F, et al. Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development [J]. J Exp Zool Part A, 2008, 309: [11] Holland M C, Gothilf Y, Meiri I, et al. Levels of the native forms of GnRH in the pituitary of the gilthead seabream, Sparus aurata, at several characteristic stages of the gonadal cycle [J]. Gen Comp Endocrinol, 1998, 112: [12] Okuzawa K, Kobayashi M. Gonadotropin-releasing hormone neuronal systems in the teleostean brain and functional significance[m]//prasada R, Peter R E. Neural regulation in the vertebrate endocrine system [M]. New York: Kluwer Academic Publisher/Plenum Press, 1999: [13] Yahalom D, Chen A, Ben-Aroya N, et al. The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform [J]. FEBS Lett, 1999, 463: [14] Montaner A D, Affanni J M, King J A, et al. Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia) [J]. Cell Mol Neurobiol, 1999, 19: [15] Okubo K, Amano M, Yoshiura Y, et al. A novel form of gonadotropin-releasing hormone in the medaka, Oryzias

12 1 : 23 latipes [J]. Biochem Bioph Res Co, 2000, 276: [16] Kobayashi M, Amano M, Kim M H, et al. Gonadotropin releasing hormone and gonadotropin in goldfish and masou salmon [J]. Fish Physiol Biochem, 1997, 17:1 8. [17] Uzbekova S, Lareyre J J, Madigou T, et al. Expression of prepro-gnrh and GnRH receptor messengers in rainbow trout ovary depends on the stage of ovarian follicular development [J]. Mol Reprod Devel, 2002, 62: [18] Anderson E, Fjelldal P G, Klenke U, et al. Three forms of GnRH in the brain and pituitary of the turbot, Scophthalmus maximus: Immunological characterization and seasonal variation [J]. Comp Biochem Physiol B, 2001, 129: [19] White R B, Fernald R D. Genomic structure and expression sites of three gonadotropin-releasing hormone genes in one species [J]. Gen Comp Endocrinol, 1998, 112: [20] Goos H, Bosma P, Zandbergen T, et al. Function and molecular aspects of the gonadotropin-releasing hormones in the African catfish, Clarias gariepinus [J]. Neth J Zool, 1995, 45: [21] Collins P M, Neill D F O, Barron B R, et al. Gonadotropin-releasing hormone content in the brain and pituitary of male and female grass rockfish (Sebastes rastrelliger) in Relation to seasonal changes in reproductive status [J]. Biol Reprod, 2001, 65: [22] Gothilf Y, Muñoz-Cueto J A, Sagrillo C A, et al. Three forms of gonadotropin-releasing hormone in a perciform fish (Sparus aurata): complementary deoxyribonucleic acid characterization and brain localization [J]. Biol Reprod, 1996, 55: [23] Lethimonier C, Madigou T, Muñoz-Cueto J A, et al. Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish [J]. Gen Comp Endocrinol, 2004, 135: [24] Kah O, Lethimonier C, Somoza G, et al. GnRH and GnRH receptors in metazoa: a historical, comparative, and evolutive perspective [J]. Gen Comp Endocrinol, 2007, 153: [25] Soga T, Ogawa S, Millar R P, et al. Localization of the three GnRH types and GnRH receptors in the brain of a cichlid fish: insights into their neuroendocrine and neuromodulator functions [J]. J Comp Neurol, 2005, 487: [26] Gonzalez-Martinez D, Zmora N, Mananos E, et al. Immunohistochemical localization of three different prepro- GnRHs in the brain and pituitary of the European sea bass (Dicentrarchus labrax) using antibodies to the corresponding GnRH-associated peptides [J]. J Comp Neurol, 2002, 446: [27] Okuzawa K, Granneman J, Bogerd J, et al. Distinct expression of GnRH genes in the red seabream brain [J]. Fish Physiol Biochem, 1997, 17: [28] Senthilkumaran B, Okuzawa K, Gen K, et al. Distribution and seasonal variations in levels of three native GnRHs in the brain and pituitary of perciform fish [J]. J Neuroendocrinol, 1999, 11: [29] Kim M H, Oka Y, Amano M, et al. Immunocytochemical localization of sgnrh and cgnrh-Ⅱ in the brain of goldfish, Carassius auratus [J]. J Comp Neurol, 1995, 356:

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吉林农业大学学报２０１６３８１８７９１ＪｏｕｒｎａｌｏｆＪｉｌｉｎＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｘｕｅｂａｏｊｌａｕｅｄｕｃｎＥｍａｉｌｊｌｎｄｘｂｖｉｐｓｉｎａｃｏｍ神经生长因子ｍｒｎａ在鹅等级前卵泡中的表达刘璐璐隋玉健朱海洋武惠岩杨辉孙永峰吉林农业大学动物科学技术学院长春１３０１１８