Microsoft Word 朱晓芳_new_.doc

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1 47 6 Vol.47, No OCEANOLOGIA ET LIMNOLOGIA SINICA Nov., 2016 (Pampus argenteus) -I * 喆 ( ) -I(Insulin-like growth factor-i, IGF-I) RT-PCR RACE, (Pampus argenteus) IGF-IcDNA, RT-PCR Real-time qpcr IGF-I IGF-I, IGF-I cdna 836bp, 5 128bp 3 92bp, (open reading frame, ORF)605bp, IGF-I 201 ; E, 59, 68, E 74 ; B C A D, E, IGF-I Ea-4, IGF-I, 83.52% 91.40%; RT-PCR Real-time qpcr, IGF-I mrna,,, ; RT-PCR Real-time qpcr, IGF-I mrna 30 50g (P<0.05); IGF-I mrna,,, ; -I ; ; Q953 doi: /hyhz (IGFs, insulin-like growth factors),, IGF-I IGF-II (Perks et al, 1995) IGF- (Wang et al, 2008; Li et al, 2011) IGF-I 70, 7.5kDa (Vong et al, 2003),, (Reinecke et al, 2005) Rinderknecht & Humber 1978 IGF-I, IGF-I, (Rinderknecht et al, 1978), Cao 1989 (Oncorhynchus tshauytscha) IGF-I cdna(cao et al, 1989),, (Cyprinus carpio) (, 2001) (Ctenapharyngodon idellus) (, 2001) (Myxocyprirms asiaticus) (, 2007), (Sparus aurata) (Tanaka et al, 1998) (Larimichthys crocea) (, 2012) (Lateolabrax japonicus) ( 焜, 2014) IGF-I (Pampus argenteus) (Perciformes), *, 2015GA ;, LY15C ;, 2015C110003, @qq.com :,, xushanliang@nbu.edu.cn;,, wangdanli@nbu.edu.cn : , :

2 (Stromateidae), (Pampus),, (, 2009, 2011;, 2010, 2013;, 2012, 2013;, 2014;, 2015;, 2015),, (IGF), RACE-PCR, IGF-I cdna, (Real-Time PCR) mrna, IGF-I 1 1.1, 1 5g 5 15g 15 30g 30 50g g g >150g , RNA, , ph 8.21±0.3, 20.3±0.8 傔 5# GenBank (Lateolabrax japonicus, JN ) (Siniperca chuatsi, HM ) (Epinephelus coioides, AY ) (Perca fluviatilis, AJ ) (Sparus aurata, AY ) IGF-I cdna, ClustalX1.83 IGF-I, Primer Premier 5 IGF-I-F IGF-I-R, PCR, 762bp, 5 RACE 3 RACE IGF-I-5 R1, IGF-I-5 R2, IGF-I-3 F1, IGF-I-3 F2, IGF-I cdna, PCR IGF-I-RT-F, IGF-I-RT-R 18S(AJ ) cdna, 18S F 18S R, 18S-F/R qigf-i-f/r IGF-I mrna, M13-R M13-47-F ( 1) 1.3 RNA cdna RNA (Axygen, RNA extraction kit) RNA TaKaRa RNA, cdna 37 15min, 85 5s cdna 20 PCR 1.4 IGF-I 基因 cdna 核心片段进行克隆 cdna, IGF-I-F/R, PCR PCR 50μL, : 94 5min, 94 30s; 55 30s, 72 1min, 30 ; 72 10min; 4 PCR 1.5%,, PCR,, Sunny 1.5 IGF-IcDNA NCBI ( nih.gov), BlastX, IGF-I IGF-I, 5 RACE 3 RACE IGF-I-5 R1, IGF-I-5 R2, IGF-I-3 F1 IGF-I-3 F2, RACE SMART TM RACE cdna (Clontech) 5 RACE 3 RACE, cdna 1.6 IGF-I Protparam ( protparam/) ; BlastX ; NCBI ORF Finder, ; ExPASy-PROSITE ( ; Signal P4.1 Server ; Smart ; ClustalW MEGA5.05 M-L(Maximum-Likelihood) (Bootstrap) 1000

3 6 : (Pampus argenteus) -I 1223 IGF-I-F1 IGF-I-R1 IGF-I-F2 IGF-I-R2 IGF-I-5'R1 IGF-I-5'R2 IGF-I-3'F1 IGF-I-3'F2 UPM IGF-I-S IGF-I-V M13-47-F M13-R qigf-i-f qigf-i-r 18S-F 18S-R IGF-I-S IGF-I-A : Y = C or T; R = A or G Tab.1 表 1 本实验所用引物及序列 Oligonucleotide primers used in the experiments (5 3 ) ATTGCCCGCATCTCATCYTC GCTGGGCATTTGTCCATTCG CAAGAGTGCGATGTGCTGTA CTATGTCCAGGTRAAGGTCT GTAGGAGTCAGGGTGAGGACGCAC CGCAACGCAGCTTGAAGACATCAC CCTGGACATAGTCATTCGACCTTC ACAGAATGTAGGGAAGGAGCGAATGG CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT ATTGCCCGCATCTCATCTTCTT ACAGTGAACCATTCCACAGGGGT CGCCAGGGTTTTCCCAGTCACGAC CAGGAAACAGCTATGAC TTGTGGATGAGTGCTGCTTC GCTGTACTAACCTTGGGTGCT GGTTTTCTCTCTGAACTGGGG CCTCCGACTTTCGTTCTTGAT ACCCAAGGTTAGTACAGCAGG TGAACCATTCCACAGGGGTA 1.7 PCR 100g g 5 15g 15 30g 30 50g g g PCR, 3 PCR,, 10 5,, : 95 30s; 95 5s, 59 30s, 40 ; 95 15s, 60 1min, 95 15s, Real-time PCR, ABI StepOnePlus TM Instrument ± (M±SD), SPSS17.0, One-Way ANOVA, Duncan Livak (2001), PCR, 2 Ct qrt-pcr, ΔCt : IGF-I Ct 18S Ct 1.8 IGF-I cdna IGF-I-S IGF-I-A, PCR 248bp, pgem-t,, Omega pgem-t Nco I Spe I,, PCR (AxyGen, ), 0.1% DEPC DNA, DIG RNA

4 Labeling Mix, Riboprobe System (Promega), T7 SP6 (Promega) DIG RNA RNA DNA DNA, 2.5μL 5mol/L ( DEPC ) 75μL, 20 DEPC 70%, RNA 30μL DEPC,, 5μL, 80 (2008) (2012) :, K 37 30min, 60 2h, 60 (.. = ) IGF-I cdan cdna, IGF-I-F IGF-I-R, 762bp, BlastX, (Larimichthys crocea, KKF ) (Epinephelus lanceolatus, ABZ ) (Siniperca chuatsi, ADO ) (Dicentrarchus labrax, AAV ) (Rachycentron canadum, ABG )IGF-I 98%, IGF-I IGF-I-3'F2 190bp cdna, 836bp IGF-I cdna (GenBank : KT210886), 1 IGF-I_CDS-F IGF-I_CDS-R IGF-I,,, cdna IGF-I cdna Pampus argenteus insulin-like growth factor-i 2.2 IGF-I IGF-I cdna 1 cdna 836bp, 128bp 5' (UTR) 605bp (ORF) 92bp 3'- (UTR), 201 IGF-I (pi) 9.72, 22.00kDa SignalP 4.1Server, 59, B 31, A 19, C 10, D 7, E 75 CysB6, CysB18, CysA6, CysA7, CysA11 CysA20 6 IGF-I ORF, 6 C (protein kinase C phosphorylation site); 1 N- (N-glycosylation site); 1 (casein kinase phosphorylation site); 1 (N-myristoylation site) ( 2) 2.3 IGF-I ClustalW IGF-I 20 IGF-I ( 3) 6 (Cys), 11 IGF-I 70.19%, (P. fluviatilis) 91.40%; 83.52% 91.40%; (Paralichthys lethostigma) (Cynoglossus semilaevis) 90.86% 83.96%; (Mugil cephalus) 86.02%; (Salmo salar) 81.82%; (Anguilla japonica) 73.75%; 70.19% 71.43%;, B A, C, D E ( 3) MEGA5.05, 21 IGF-I,, IGF-I, 4 4,,, (P. fluviatilis) (P. lethostigma) (S. aurata) (E. coioides) (S. chuatsi) (M. salmoides) (L. japonicus) (M. cephalus), (L. erythropterus) (C. semilaevis) (O. niloticus) (S. salar), (C. carpio) (Carassius auratus) (A. japonica) ;, (Homo sapiens) (Sus scrofa) (Mesocricetus auratus), (Gallus gallus) (Pelodiscus sinensis)

5 6 : (Pampus argenteus) -I IGF-I cdna Fig.1 The cdna sequence of P. argenteus IGF-I and deduced amino acids sequence : ; CysB6, CysB18, CysA4, CysA5, CysA9 CysA18 ; 2 IGF-I ORF Fig.2 The functional sites in ORF of IGF-I from P. argenteu : 1. C ( 29 31, , , , , ); 2. ( ); 3. N ( ); 4. ( ), (P. fluviatilis) (S. aurata) (E. coioides) ; ;, 2.4 IGF-I mrna RT-PCR Quantitative Real-time PCR IGF-I 10 ( 5) IGF-I

1226 47 10,, (P<0.05, n=3),, 2.5 IGF-I mrna RT-PCR Quantitative Real-time PCR IGF-I ( 6) 30g, IGF-I, 30 50g (P<0.05),, 1 30g 2.

6 ,, (P<0.05, n=3),, 2.5 IGF-I mrna RT-PCR Quantitative Real-time PCR IGF-I ( 6) 30g, IGF-I, 30 50g (P<0.05),, 1 30g ; g (P<0.05), 30 50g 1/2;, 3 IGF-I Fig.3 Multiple-sequence alignment of vertebrate IGF-I amino acids sequence : IGF-I ; ; IGFBP s ; IGF-I R ; 6

6 : (Pampus argenteus) -I 1227 4 IGF-I M-L Fig.4 The maximum-likelihood tree reconstructed based on putative IGF-I amino acids residues : MEGA 5.05 Maximum-Likelihood, 1000 ; 5 IGF-I mrna Fig.

7 6 : (Pampus argenteus) -I IGF-I M-L Fig.4 The maximum-likelihood tree reconstructed based on putative IGF-I amino acids residues : MEGA 5.05 Maximum-Likelihood, 1000 ; 5 IGF-I mrna Fig.5 Levels of IGF-I mrna transcription of different tissues with 18S acted housekeeping gene : M. Mark 2000, Li., Mu., Ki., In., Br., Ol., St., He., Gi., Ov. 2.6 IGF-I DIG RNA, DIG RNA, H.E 7,, ( ), ( 7, A1, A2, C1, C2), IGF-I RNA,,

8 1228 海图6 Fig.6 洋与湖沼 47 卷银鲳 IGF-I mrna 在不同生长阶段表达水平 Levels of IGF-I mrna transcription in different growth stages with 18S acted housekeeping gene 注: M: Mark 2000, 1: 1 5g, 2: 5 15g, 3: 15 30g, 4: 30 50g, 5: g, 6: g 图7 Fig.7 胰岛素样生长因子-I 原位杂交及 H.E 染色结果 In situ hybridization of P. argenteus, showing the location of insulin-like growth factor-i mrna in liver 注: A1 A4: IGF-I 反义探针杂交结果; B1 B2: IGF- I 正义探针杂交结果(对照组); C1 C3: H.E 染色结果(对照组); HC: 肝脏细胞, EC: 红细胞

9 6 : (Pampus argenteus) -I 1229 ( 7, A3, A4); ( 7, B1, B2) IGF-I mrna IGF-I mrna IGF-I cdna, (Pedroso et al, 2006) (, 2011) ( 焜, 2014) IGF-I, IGF-I E, B, C, A, D IGF-I, IGF-I 6 (Cys), 2 B, 4 A, (Hober et al, 1992; Magee et al, 1999), IGF-I 70.19%, 44.62%, IGF-I, ; B A 89.66% 100% 90.48% 100%,, IGF-IR IGFBPs (Zhang et al, 1994; Magee et al, 1999), IGF-I ; C (50.00% 100%) D (37.50% 100%) E (25.68% 97.3%) IGF-I 4 (Cao et al, 1989; Shamblott et al, 1993; Hashimoto et al, 1997), E 35 Ea-1, 47 Ea-2, 62 Ea-3, 74 Ea-4 IGF-I E 75, Ea-4 Duguay (1992) (Oncorhynchus keta) Ea-4 IGF-I, Chen (1998) (Acathopagrus schlegeli) Ea-4 IGF-I, Hashimoto (1997) (C. carpio) Ea-2 IGF-I, 3.2 IGF-I mrna RT-PCR quantitative real-time PCR IGF-I 10, (Carassius cuvieri)(, 2012),, IGF-I (Sjögren et al, 1999), IGF-I (2012) RT-PCR 鳡 (Elopichthys bambusa)β-actin, IGF-I mrna 鳡 6, IGF-I mrna,, ; 焜 (2014) (2012) (L. japonicus) (Scophthalmus maximus)igf-i mrna, IGF-I,, ; Otteson (Carassius auratus) IGF-I (Hitchcock et al, 2001), IGF-I mrna,, (Duan et al, 1998; Metón et al, 2000; Larsen et al, 2001; Gabillard et al, 2003) IGF-I, (Reinecke et al, 1998; Roith et al, 2001) 3.3 IGF-I mrna RT-PCR quantitative real-time PCR IGF-I, 30g, IGF-I, 30 50g (P<0.05),, 23 28, ( g), IGF-I, g,,, IGF-I (Wen et al, 2002) IGF-I, Shamblott (1993) (Oncorhynchus mykiss) IGF-I mrna, IGF-I mrna ; Duan (1998) IGF-I mrna 3.4 IGF-I IGF-I (GH),

10 , IGF-I (, 2008;, 2008; Patruno et al, 2008) DIG RNA IGF-I mrna, Patruno (2008) Patruno (2008) DIG RNA, IGF-I mrna, IGF-I mrna ; IGF-I, (2008) DIG RNA IGF-I mrna, IGF-I mrna,,, , 33(4): 83 89,,, I., 25(1): 1 4,, IGF-I mrna., 47(1): ,,, I cdna., 46(3): 28 36,,, , 27(12): , GHR IGF-1 IGF-1R GHR cdna. :, :,,, 鳡 1. ( ), 33(3): 32 39,,, ( ), 27(4): 1 6,,, I(IGF-I)cDNA., 42(2): 39 45, :,,, , 36(1): 18 21, 34,,, , 18(2): 焜,,, cdna., 44(2): 27 34,,, (Pampus argenteus), (P. cinereus) (P. sinensis)., 43(4): ,,, , 29(6): 53 58, GH/IGF : IGF GH. :,,, IGF-I cdna., 16(4): ,,, (Larimichthys crocea) IGF-., 43(1): 35 40,,, , 35(1): 72 76,,, , 29(7): , POMC TSHβ SL cdna. : Cao Q P, Duguay S T, Plisetskaya E et al, Nucleotide sequence and growth hormone-regulated expression of salmon insulin-like growth factor I mrna. Molecular Endocrinology, 3(12): Chen M H C, Lin G H, Gong H Y et al, Cloning and characterization of insulin-like growth factor I cdna from black seabream (Acanthopagrus schlegeli). Zoological Studies, 37(3): Duan C M, Nutritional and developmental regulation of insulin-like growth factors in fish. The Journal of Nutrition, 128(2): 306S 314S Duguay S J, Park L K, Samadpour M et al, Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon. Molecular Endocrinology, 6(8): Gabillard J C, Weil C, Rescan P Y et al, Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology, 133(2): Hashimoto H, Mikawa S, Takayama E et al, Molecular cloning and growth hormone-regulated gene expression of carp insulin-like growth factor-i. Biochemistry and Molecular Biology International, 41(5): Hitchcock P F, Otteson D C, Cirenza P F, Expression of the insulin receptor in the retina of the goldfish. Investigative Ophthalmology & Visual Science, 42(9): Hober S, Forsberg G, Palm G et al, Disulfide exchange folding of insulin-like growth factor I. Biochemistry, 31(6): Larsen D A, Beckman B R, Dickhoff W W, The effect of low temperature and fasting during the winter on metabolic stores and endocrine physiology (insulin, insulin-like growth factor-i, and thyroxine) of coho salmon, Oncorhynchus kisutch. General and Comparative Endocrinology, 123(3): Li J Z, Liu Z H, Wang D S et al, Insulin-like growth factor 3 is involved in oocyte maturation in zebrafish. Biology of Reproduction, 84(3):

11 6 : (Pampus argenteus) -I 1231 Livak K J, Schmittgen T D, Analysis of relative gene expression data using real-time quantitative PCR and the 2 ΔΔCt method. Methods, 25(4): Magee B A, Shooter G K, Wallace J C et al, Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues. Biochemistry, 38(48): Metón I, Caseras A, Cantó E et al, Liver insulin-like growth factor-i mrna is not affected by diet composition or ration size but shows diurnal variations in regularly-fed gilthead sea bream (Sparus aurata). The Journal of Nutrition, 130(4): Patruno M, Sivieri S, Poltronieri C et al, Real-time polymerase chain reaction, in situ hybridization and immuno histochemical localization of insulin-like growth factor-i and myostatin during development of Dicentrarchus labrax (Pisces: Osteichthyes). Cell and Tissue Research, 331(3): Pedroso F L, De Jesus-Ayson E G T, Cortado H H et al, Changes in mrna expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status. General and Comparative Endocrinology, 145(3): Perks C M, Denning-Kendall P A, Gilmour R S et al, Localization of messenger ribonucleic acids for insulin-like growth factor I (IGF-I), IGF-II, and the type 1 IGF receptor in the ovine ovary throughout the estrous cycle. Endocrinology, 136(12): Reinecke M, Björnsson B T, Dickhoff W W et al, Growth hormone and insulin-like growth factors in fish: where we are and where to go. General and Comparative Endocrinology, 142(1 2): Reinecke M, Collet C, The phylogeny of the insulin-like growth factors. International Review of Cytology, 183: 1 94 Rinderknecht E, Humbel R E, Primary structure of human insulin-like growth factor II. Febs Letters, 89(2): Roith D L, Scavo L, Butler A, What is the role of circulating IGF-I?. Trends in Endocrinology & Metabolism, 12(2): Shamblott M J, Chen T T, Age-related and tissue-specific levels of five forms of insulin-like growth factor mrna in a teleost. Molecular Marine Biology & Biotechnology, 2(6): Sjögren K, Liu J L, Blad K et al, Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice. Proceedings of the National Academy of Sciences of the United States of America, 96(12): Tanaka M, Taniguchi T, Yamamoto I et al, Gene and cdna structures of flounder insulin-like growth factor-i (IGF-I): multiple mrna species encode a single short mature IGF-I. DNA and Cell Biology, 17(10): Vong Q P, Chan K M, Leung K et al, Common carp insulin-like growth factor-i gene: complete nucleotide sequence and functional characterization of the 5'-flanking region. Gene, 322: Wang D S, Jiao B W, Hu C J et al, Discovery of a gonad-specific IGF subtype in teleost. Biochemical and Biophysical Research Communications, 367(2): Wen H S, Lin H R, Dong X et al, Seasonal changes and neuroendocrine regulation of growth hormone secretion in feral Catfish (Silurus asotus). Acta Zoologica Sinica, 48(2): Zhang W, Gustafson T A, Rutter W J et al, Positively charged side chains in the insulin-like growth factor-1 C- and D-regions determine receptor binding specificity. The Journal of Biological Chemistry, 269(14):

12 CLONG AND EXPRESSION ANAYSIS OF INSULIN-LIKE GROWTH FACTOR I GENE IN PAMPUS ARGENTEUS ZHU Xiao-Fang, GUO Xiao-Ge, ZHANG Ding-Yuan, YANG Jia-Zhe, XU Shan-Liang, WANG Dan-Li (School of Marine Science, Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo , China) Abstract Insulin-like growth factor-i (IGF-I) is an important regulator to growth, development, and metabolism of a vertebrate. To clone the IGF-I cdna sequences in liver of Pampus argenteus, we used RT-PCR (reverse transcription-polymerase chain reaction), RACE (rapid amplification of cdna ends). In addition, to detect the IGF-I expression in the tissues and IGF-I expression levels in the liver in six different growth stages and the positioning of the IGF-I gene in the liver, we used semi-quantitative RT-PCR, quantitative RT-PCR, and in-situ hybridization. Results show that the IGF-I cdna precursor consists of 836bp with a 605bp open reading frame, ORF, and 5' and 3' UTR (untranslated regions, 128bp and 92bp, respectively). Therefore, we deduce that the precursor protein of IGF-I consisted of 201 amino acids; the precursor peptide is composed of signal peptide, mature peptide and E peptide, signal peptide of 59 amino acids, mature peptide of 68 amino acids, and E peptide of 74 amino acids. The mature peptide was composed of four regions B, C, A, and D. Analysis on E peptide showed that IGF-I of pomfret was in Ea-4 type. The IGF-I coding sequence of pomfret had higher identity with the same mesh Perciformes fish, from 83.52% to 91.40%; and lower identity with mammals, birds and reptiles. Semi-quantitative RT-PCR and RT qpcr results show that IGF-I mrna was expressed in all 10 tissues, high in liver, and low in other tissues. Semi-quantitative RT-PCR and quantitative RT PCR in six different growth stages showed that IGF-I mrna expression level was the highest when fish weighed between 30 50g, and then decreased significantly between g (P<0.05). The in-situ hybridization to see IGF-I mrna expression in the liver indicated that GF-I positive cells were found throughout the liver; the signal intensity was significantly higher at cell edge, and positive signals were found mainly in cytoplasm. Key words Pampus argenteus; insulin-like growth factor-i gene; quantitative real-time PCR; in situ hybridization

Microsoft Word 曹伏君_new_.doc

Microsoft Word 曹伏君_new_.doc 45 2 Vol.45, No.2 2014 3 OCEANOLOGIA ET LIMNOLOGIA SINICA Mar., 2014 (Lethrinus erythropterus) * ( 524025) Wright (Lethrinus erythropterus),,,, ; ; ; Q954.56 doi: 10.11693/hyhz20140100011 (Lethrinus erythropterus)