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1 39 2 Vol. 39, No ACTA HYDROBIOLOGICA SINICA Mar., doi: 0.754/205.4 cdna, 2 单喜双岳华梅陈细华叶欢杨晓鸽李创举 (.,, ; 2., 20306) : (Acipenser dabryanus) (Growth Hormone, GH), SMART cdna, GH cdna GH cdna 008 bp, 52 bp 5 (Untranslated region, UTR) bp (Open reading frame, ORF) 3 bp 3 UTR GH, PCR, GH mrna, GH 0 ; Western-blot qrt-pcr,, GH, GH, ; -, GH : ; ; qrt-pcr; Western-blot; 中图分类号 : Q 文献标识码 : A 文章编号 : (205) , 2, (Acipenser dabryanus), (Acipenser sinensis) (Psephurus gladius),, ( 5 7, 3 5 ), 20 70,,,, 988 [ 3] [4],,,,,,, - -, I(IGF-I), IGF-I [5 9],, [0, ] [2] [3],, 20 80, (Acipenser gueldenstaedtii) (Huso huso) 收稿日期 : ; 修订日期 : 基金项目 : (20AA0040); (203JBFZ0); (30004) 作者简介 : (989 ),, ; ; s9shanxishuang@26.com 通信作者 :,,, ; lcj@yfi.ac.cn

2 (Acipenser persicus) (Oncorhynchus keta) (Salmo gairdneri) (Ctenopharyngodon idellus) (Silurus meridionalis) (Monopterus albus) (Misgurnus anguillicaudatus) (Carassius auratus) [4 22] 984 [23],,,, [24], cdna, PCR (Quantitative real-time polymerase chain reaction, qrt-pcr) Western-blot mrna ;, HE. ( ),,, 80 GH GH,.2 RNA cdna SMART cdna RNA SV Total RNA Isolation System (Promega, ) ; PrimeScript RT reagent Kit With gdna Eraser (TaKaRa, ) cdna qrt-pcr, Super SMART PCR cdna Synthesis Kit SMART cdna.3 GH cdna GH cdna (GenBank : EU ) GH-F GH-R( ) PCR, GH cdna, spgh5 -R spgh5 - R2 spgh3 -F spgh3 -F2 GH cdna5 3, pmd9-t, (DH5α), DNA StarSeqMan, NCBI Blast ( gov/blast. cgi), SignalP4. ( cbs.dtu.dk/services/signalp/), ( services/ NetNGlyc/), ClustalX Primer GH-F GH-R spgh-5 -R spgh-5 -R2 spgh-3 -F spgh-3 -F2 RACE- RACE5 -AP RACE3 -AP RT-GHF RT-GHR 表本研究所用引物序列 Tab. Primers used in the present study Sequence (5-3 ) ATGGCATCAGGTCTGCTTCT CAGAGTACAGTTGCTCTCCACAA CTGGAACATAGCTGCGCTCAAAATCT GACACAGAAGCAGACCTGATGCCAT ATGTCAACCTAAGAAATGATGATGCTTTG CGTTTTGTGGAGAGCAACTGTACTCTG AAGCAGTGGTATCAACGCAGAGT ATCAACGCAGAGTACGCGGG GGTATCAACGCAGAGTACTT ATGGCATCAGGTCTGCTTCT CTGGAACATAGCTGCGCTCAAAATCT Production size (bp) Adβ-actin-F CCTTCTTGGGTATGGAATCTTGC 222 Adβ-actin-R CAGAGTATTTACGCTCAGGTGGG

3 2 : cdna 309, DNAMAN 6.0, Mega5 (Neighbor-Joining, NJ), (Bootstrap) ( 000).4 PCR GH cdna, RT-GHF RT-GHR ( ), ( )cdna qrt-pcr, SYBR Premix Ex Taq TM (Perfect Real Time), 3, Adβ-actin Bio-Rad CFX96TM Real Time System, 95 0min, 95 5s, 54 5s, 72 5s, 40.5 Western-blot (00 mmol/l β-, 20 mmol/l HEPES, 20 mmol/l EGTA, 5 mmol/l MgCl 2, ph7.3, mmol/l DTT, 2 mmol/l PMSF, 2.5 μg/ml leupetin, 2.5 μg/ml aprotinin),,, Loading Buffer 0min, SDS-PAGE, Mini Trans-Blot (Bio-Rad, ), PVDF (80 V, 0.5h) TBS (0. mol/l NaCl, 0. mol/l Tris, ph 7.5) 3, 5% (2h), GH 4 ; TBS , h, TBS NBT/BCIP, Adβ-actin, GH, , %,,,, 4 μm, HE HE,, PBS, 5min; 0min,, PBS, 5min; 3% 37 30min, PBS, 5min; 5% h, (.. 200), ; PBST 5, 0min, PBS, 0min; 2% FITC (.. 00), h; PBS 5, 0min, DAPI(.. 000) 30min; PBS 5, 0min,, 2 2. GH cdna 3 SMART cdna, GH cdna, Blast GenBank GH, GH cdna GH cdna 008 bp, 52 bp 5 UTR 3 bp 3 UTR, ORF 645 bp, 24, 24 kd SignalP4., 24, 90, N- (Asn-Cys-Ser) 4 Cys, 2 (GenBank : KJ650095) 2.2 GH GH,, GH, 95% GH (Xenopus laevis) (Boiga irregularis) (Gallus gallus) (Homo sapiens) (Mus musculus) 50%,, 40% 45% Mega 5 GH NJ ( ),, ( ), 2 : 7 ; GH mrna Adβ-actin, real-time qpcr GH mrna,,, GH mrna, GH 0, ( 2)

30 39 Fig. GH NJ Neighbor-Joining tree reconstructed based on putative GH amino acid residues between A. dabryanus and other vertebrates 2 PCR GH mrna Fig.

4 30 39 Fig. GH NJ Neighbor-Joining tree reconstructed based on putative GH amino acid residues between A. dabryanus and other vertebrates 2 PCR GH mrna Fig. 2 Transcription levels of GH mrna in various tissues of A. dabryanus by real-time qpcr 2.4 GH Western-blot GH GH mrna, GH, GH ( 3) 2.5 GH,, 4A, HE, ( 4B 4B) ( 4B 4B2) GH,, ( 4B2), Fig. 3 3 Western-blot GH Expression characterization of A. dabryanus GH in various tissues detected by Western-blot

5 2期单喜双等: 达氏鲟生长激素基因 cdna 克隆表达及免疫荧光定位研究图4 3 达氏鲟垂体和下丘脑中生长激素免疫荧光定位及生长激素分泌细胞的组织学观察 Fig. 4 Immunofluorescence localization of GH and histological observation of GH stimulating cells in the pituitary and hypothalamus of A. dabryanus A. 达氏鲟脑组切片位置示意图; B. 垂体及下丘脑荧光免疫组化图片, B 和 B2 分别为下丘和垂体部分区域放大, GH 抗体利用 FITC 绿色荧光标记, 蓝色荧光 DAPI 标记细胞核; C. 下丘脑及垂体组织 HE 染色, C 为局部区域放大图 C2 和 C3 分别为下丘脑和垂体部分区域放大, 图中强嗜酸性细胞(胞质红色)为生长激素分泌细胞; Pi. 垂体; Hy. 下丘脑; 图中标尺单位为 μm Schematic drawing of a sagittal section of the brain of A. dabryanus showing the location of the transverse section (A); Immunofluorescence signals stained with anti-a. sinensis Growth Hormone (GH) antiserum, showing GH immunoreactive cells in the pituitary and hypothalamus (B). The pituitary and hypothalamus stained with Harris s hematoxylin and eosin (C); Pi. pituitary; Hy. Hypothalamus; Bar μm (图 4B)信号细胞分布比较分散且数量较少 HE 染易被伊红着色, 在垂体(图 4C3)和下丘脑内(图 4C2) 色辅助定位生长激素分泌细胞(图 4C 4C 4C2 分布着强嗜酸性细胞, 区域位置与荧光免疫组化中 4C3), 生长激素分泌细胞含有致密的嗜酸性细胞质, 的特异性信号细胞一致, 可以确定为生长激素分泌

6 GH cdna, 24, GH GH,, Cys, 2, GH GH N-, NCT, N- Asn-X-Thr/Ser;, N- NCT, NDS,, [25, 26],,, GH,, 98%, 95%, GH GH, [4] [5] [6],,,,,, - -,,, (IGF-I),, GH [27 30],,,, real-time qpcr western-blot GH mrna, GH mrna, [26] GH mrna, GH, GH, GH-IGFI IGF, GH, GH, GH (GtH) (TSH) (ACTH) (PRL) (SL) (MSH),, [3 33], GH,, HE, HE,,,, ( 4), [3 33]

7 2 : cdna 33 [] Chen X H. Biology and Resources of Acipenseriformes Fishes [M]. Beijing: China Ocean Press. 2007, [.. :. 2007, 53 57] [2] Yangtze Aquatic Resources Survey Group. The Biology of the Sturgeons and Paddlefish in the Yangtze River and their Artificial Propagation [M]. Chengdu: Sichuan Scientific and Technical Publishing House. 988, [.. :. 988, 23 26] [3] Ding R H. The Fishes of Sichuan [M]. Chengdu: Sichuan Scientific and Technical Publishing House. 994, [.. :. 994, 29 30] [4] Zhang H, Wei Q W, Du H, et al. Present status and risk for extinction of the Dabry s sturgeon (Acipenser dabryanus) in the Yangtze River watershed: a concern for intensified rehabilitation needs [J]. Journal of Applied Ichthyology, 20, 27(2): 8 85 [5] Ahmed S F, Farquharson C. The effect of GH and IGF on linear growth and skeletal development and their modulation by SOCS proteins [J]. Journal of Endocrinology, 200, 206(3): [6] Reinecke M, Bjornsson B T, Dickhoff W W, et al. Growth hormone and insulin-like growth factors in fish: where we are and where to go [J]. General and Comparative Endocrinology, 2005, 42: [7] Canosa L F, Chang J P, Peter R E. Neuroendocrine control of growth hormone in fish [J]. General and Comparative Endocrinology, 2007, 5: 26 [8] Li W S, Lin H R. The endocrine regulation network of growth hormone synthesis and secretion in fish: emphasis on the signal integration in somatotropes [J]. Science China & Life Sciences, 200, 40(2): [,. :. :, 200, 40(2): 46 58] [9] Abu S I A, Yu L Q, Zhu Z Y, et al. Comparative expression analysis of GHR signaling related factors in zebrafish (danio rerio) and an in vivo model to study GHR signaling [J]. Acta Hydrobiologica Sinica, 20, 35(5): [Abu Shufian Ishtiaq Ahmed,,,. GHR., 20, 35(5): ] [0] Hull K L, Harvey S. GH as a co-gonadotropin: the relevance of correlative changes in GH secretion and reproductive state [J]. Journal of Endocrinology, 2002, 72: 9 [] Zhou L B, Liu X C, Ye W, et al. Sexual cycle changes of growth hormone in helmet catfish, Cranoglanis bouderius [J]. Acta Hydrobiologica Sinica, 2004, 28(6): [,,,.., 2004, 28(6): ] [2] Yada T. Growth hormone and fish immune system [J]. General and Comparative Endocrinology, 2007, 52(2-3): [3] Tatsuya S, Stephen D M. Prolactin and growth hormone in fish osmoregulation [J]. General and Comparative Endocrinology, 2006, 47(): [4] Cao H, Zhou R J, Wei Q W, et al. Molecular characterization of the growth hormone in Chinese sturgeon and its expression during embryogenesis and early larval stages [J]. Journal of Applied Ichthyology, 20, 27(2): [5] Pormehr L A, Pourkazemi M, Yarmohammadi M, et al. Phylogenetic and sequence analysis of the growth hormone gene of two sturgeons, Huso huso and Acipenser Gueldenstaedti [J]. Progress in Biological Sciences, 203, 3(2): [6] Salamroodi M, Pourkazemi M, Khoshkholgh M, et al. Molecular cloning and sequence analysis of growth hormone gene in Persian Sturgeon, Acipenser persicus Borodin, 897 (Pisces: Acipenseridae) [J]. Caspian Journal of Environmental Sciences, 203, (2): [7] Sekine S, Mizukami T, Nishi T, et al. Cloning and expression of cdna for salmon growth hormone in Escherichia coli [J]. PNAS, 985, 82: [8] Agellon L B, Chen T T. Rainbow trout growth hormone: molecular cloning of cdna and expression in Escherichia coli [J]. DNA, 986, 5(6): [9] Zhu Z Y, He L, Chen T T. Primary-structural and evolutionary analyses of the growth-hormone gene from grass carp (Ctenophavyngodon idellus) [J]. European Journal of Biochemistry, 992, 207: [20] Song P, Hu Y C, Xiang Z, et al. Cloning and sequencing of full Length cdna in Silurus meridionalis growth hormone [J]. Acta Genetica Sinica, 2002, 26(3): [,,,. cdna DNA., 2002, 26(3): ] [2] Zhang J N, Song P, Hu J R, et al. Molecular cloning and sequence analysis of full-length growth hormone cdnas from six important economic fishes [J]. Acta Genetica Sinica, 2005, 32(): 9 29 [,,,. 6 cdna., 2005, 32(): 9 29] [22] Lao H H, Bai J J, Ye X, et al. Molecular cloning and analysis of two distinct growth hormone cdnas from Carassius auratus auratus [J]. Acta Hydrobiologica Sinica, 2004, 28(): [,,,. cdna., 2004, 28(): 74 79] [23] Zhu Z Y, Li G, He L, et al. Novel gene transfer into the fertilized eggs of gold fish (Carassius auratus L. 758) [J].

8 34 39 Zeitschrift fur Angewandte Ichthyologie, 985, : 3 34 [24] Wang W, Wang Y P, Zhu Z Y. Growth hormone gene engineering in fish [J]. Progress in Natural Science, 2002, 2(5): [,,.., 2002, 2(5): ] [25] Guan J L, Machamer C E, Rose J K. Glycosylation allows cell-surface transport of an anchored secretory protein [J]. Cell, 985, 42: [26] Zhou L, Gu J X. N-glycosylation sites analysis and nonconsensus N-glycosylated sequences [J]. Chinese Bulletin of Life Sciences, 20, 23(6): [,. N- N-., 20, 23(6): 605 6] [27] Harvey S. Extrapituitary growth hormone [J]. Endocrine, 200, 38(3): [28] Yoshizato H, Fujikawa T, Soya H, et al. The growth hormone (GH) gene is expressed in the lateral hypothalamus: enhancement by gh-releasing hormone and repression by restraint stress [J]. Endocrinology, 998, 39(5): [29] Luna M, Martinez-Moreno C G, Ahumada-Solorzano M S, et al. Extrapituitary growth hormone in the chicken reproductive system [J]. General and Comparative Endocrinology, 204, 3: 9 [30] Cao Y C, Wen H B, Li W S, et al. The profile of growth hormone gene expression in extrapituitary tissues of Lepomis cyanellus [J]. Zoological Research, 2005, 26(2): [,,,. mrna., 2005, 26(2): 74 78] [3] Kasper R S, Shved N, Takahashi A, et al. A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β-tsh, β-fsh, β-lh, ACTH, and α-msh in the adenohypophysis of Oreochromis niloticus, the Nile tilapia [J]. Cell Tissue Research, 2006, 325: [32] Sánchez C F, Portillo A, Martıńdelrıó M P, et al. Immunocytochemical characterization of adenohypophyseal cells in the greater weever fish (Trachinus draco) [J]. Tissue and Cell, 2003, 35: [33] Mostafa A M, Noha A K, Amal M H. Immunocytochemical identification and distribution of the cell types in the pituitary gland of Bagrus bayad (Teleostei, Bagridae) [J]. World Journal of Neuroscience, 202, 2: 23 3 cdna CLONING, EXPRESSION AND IMMUNOLOCALIZATION OF GROWTH HORMONE GENE IN ACIPENSER DABRYANUS SHAN Xi-Shuang, 2, YUE Hua-Mei, CHEN Xi-Hua, YE Huan, YANG Xiao-Ge and LI Chuang-Ju (. Key Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture of China, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan , China; 2. College of Fisheries and Life, Shanghai Ocean University, Shanghai 20306, China) Abstract: The Dabry s sturgeon (Acipenser dabryanus Dumeril, 868) mainly distributes in the upper Yangtze River of China. It is currently endangered due to the destruction of their spawning grounds and other anthropogenic interferences. In this study, the full-length cdna sequence of GH was cloned from SMART cdnas of pituitary in A. dabryanus. The Dabry s sturgeon GH cdna was 008 bp in length, including a 52 bp 5 -untranslated region (UTR), a 3 bp 3 UTR and a 645 bp open reading frame (ORF) encoding a peptide of 43 amino acids. Amino acid sequence alignment revealed that A. dabryanus GH shared extremely high identities (over 95%) with its counterparts in other sturgeons. Moreover, sturgeons showed lower GH sequence identities with other bony fish than that of Amphibia, Reptilia, Aves, and Mammalia. By quantitative real-time PCR, GH mrnas were detected in the pituitary and hypothalamus but absent in any of other somatic organs examined. Moreover, the expression of GH mrnas in the pituitary was about 0 times to that of hypothalamus. Western-blot analysis suggested the GH expression in both the pituitary and hypothalamus, with higher expression level in the pituitary, which is consistent with the result of qrt-pcr. Immunofluoresence localization showed strong GH-positive signals in the pars intermedia of the pituitary, together with weak fluorescence signals detected in the hypothalamus. HE staining indicated that GH was mainly secreted by eosinophilic cells in the adenohypophysis. This study will benefit both the evolution study of vertebrate GH genes and the further biological function study of GH in cultured A. dabryanus. Key words: Acipenser dabryanus; Growth hormone; qrt-pcr; Western-blot; Immunolocalization

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)