Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (10): 1998-2007.doi: 10.3864/j.issn.0578-1752.2016.10.015

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Cloning, Sequence Analysis and Tissue Expression of IGFBP-2 Gene in Goat (Capra hirus)

ZHAN Si-yuan, DONG Yao, LI Li, ZHONG Tao, WANG Lin-jie, ZHANG Hong-ping   

  1. Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130
  • Received:2015-09-14 Online:2016-05-16 Published:2016-05-16

RichHTML

1

PDF

492

Abstract: 【Objective】The objectives of this study were to clone goat IGFBP-2 (Insulin-like growth factor binding protein -2) coding sequence (CDS), to analyze its sequence characteristics, mRNA and protein expression profiles. It could provide data for further investigation on the function and expression regulation of IGFBP-2 gene in the growth and development of postnatal goat. 【Method】The IGFBP-2 mRNA sequences of Ovis aries (NM_001009436) and Bos taurus (NM_174555) were downloaded from GenBank database, and the primers were designed according to the conserved regions by using Primer Premier 6.0 software after sequences alignment, then PCR amplification and the positive clones were obtained using TA clone technology. A complete coding sequence of IGFBP-2 gene was obtained from sequencing. The CDS and coding amino acid sequences were analyzed by using EditSeq, DNAMAN 6.0, MEGA 6.0 and ExPASy online software. The real-time quantitative PCR (Q-PCR) primers were designed using the acquired CDS sequence of the IGFBP-2 gene. The expression level of the IGFBP-2 gene and protein in postnatal Nanjiang brown goat’s different tissues (heart, liver, lung, longissimus dorsi muscle, semimembranosus muscle and triceps brachii muscle) and development stages (3 d, 30 d, 60 d, 90 d and 120 d) were detected using Q-PCR and western blotting. 【Result】A full-length CDS of 954bp was cloned for goat IGFBP-2, and encodes a protein of 217 amino acid. The GC and AT content is 69.39% and 30.61%. The molecular weight of goat IGFBP-2 protein is 33.8808 kD, and its theoretical isoelectric point is 7.82. The secondary structure of the protein contains random coil (68.14%), α-helix (18.30%) and extended strand (13.56%). Protein structure domain analysis showed that its amino acid sequence contains conserved IB (IGFBP homologues) and TY (Thyroglobulin type Ι repeats) domains, IB domain in the 37-125 amino acids and TY domain in the 250-302 amino acids. Phosphorylation sites prediction indicated that there are five Ser phosphorylation sites and seven Thr phosphorylation sites exists in IGFBP-2 protein sequence. Glycosylation sites prediction found that there are ten N-glycosylation sites and two O-glycosylation sitesexists in IGFBP-2 protein sequence. Sequence analysis indicated that the IGFBP-2 CDS sequence of the Nanjiang brown goat is similar with those of the sheep (98.99%), cattle (97.73%), pig (87.12%), human (78.33%) and mouse (76.26%). Sequence analysis revealed that the IGFBP-2 amino acid sequence of the Nanjiang brown goat is similar with those of the sheep (99.24%), cattle (98.10%), pig (87.07%), human (70.27%) and mouse (73.00%). Furthermore, goat IGFBP-2has the closest phylogenetic relationship to the sheep and cattle IGFBP-2 in amino acids sequences. The mRNA and protein levels of IGFBP-2 in the liver were significantly higher than that in other tissues (P＜0.01), and LD were the second (P＜0.01); in the liver, the mRNA and protein levels of IGFBP-2 were upregulated from 3 day to 120 day. In addition, the mRNA level of IGFBP-2 showed an “up-down-up” expression pattern during postnatal longissimus dorsi muscle development. 【Conclusion】IGFBP-2 was cloned and its tissue expression patterns were investigated.The sequence characteristics of IGFBP-2 is conserved in species, and liver is the main expression tissue of goat IGFBP-2 mRNA and protein. The expression of IGFBP-2 mRNA and protein occurred with some regularity and indicated that IGFBP-2 may play an important role in early growth and development of postnatal goat.

Key words: goat, IGFBP-2, gene cloning, real-time PCR, western blotting

[1]    FLORINI J R, EWTON D Z, COOLICAN S A. Growth hormone and the insulin-like growth factor system in myogenesis. Endocrine Reviews, 1996, 17(5): 481-517.
[2]    MOTTOLA C, MACDONALD R G, BRACKETT J L, MOLE J E, ANDERSON J K. Purification and aminoterminal sequence of an insulin-like growth factor-binding protein secreted by rat liver BRL-3A cells. The Journal of Biological Chemistry, 1986, 261(24): 11180-11188.
[3]    HWA V, OH Y, ROSENFELD R G. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocrine Reviews, 1999, 20(6): 761-787.
[4]    DUAN C, XU Q. Roles of insulin-like growth factor (IGF) binding proteins in regulating IGF actions. General and Comparative Endocrinology, 2005, 142(1-2): 44-52.
[5]    PRASONGSOOK S, CHOI I, BATES R O, RANEY N E, ERNST C W, TUMWASORN S. Association of Insulin-like growth factor binding protein 2 genotypes with growth, carcass and meat quality traits in pigs. Journal of Animal Sciences and Technology, 2015, 57: 31.
[6]    HOEFLICH A, WU M, MOHAN S, FOLL J, WANKE R, FROEHLICH T, ARNOLD G J, LAHM H, KOLB H J, WOLF E. Overexpression of insulin-like growth factor-binding protein-2 in transgenic mice reduces postnatal body weight gain. Endocrinology, 1999, 140(12): 5488-5496.
[7]    HOEFLICH A, NEDBAL S, BLUM W F, ERHARD M, LAHM H, BREM G, KOLB H J, WANKE R, WOLF E. Growth inhibition in giant growth hormone transgenic mice by overexpression of insulin-like growth factor-binding protein-2. Endocrinology, 2001, 142(5): 1889-1898.
[8]    WOOD T L, ROGLER L E, CZICK M E, SCHULLER A G, PINTAR J E. Selective alterations in organ sizes in mice with a targeted disruption of the insulin-like growth factor binding protein-2 gene. Molecular Endocrinology, 2000, 14(9): 1472-1482.
[9]    WHEATCROFT S B, KEARNEY M T. IGF-dependent and IGF-independent actions of IGF-binding protein-1 and -2: implications for metabolic homeostasis. Trends in Endocrinology and Metabolism, 2009, 20(4): 153-162.
[10]   FUKUSHIMA T, TEZUKA T, SHIMOMURA T, NAKANO S, KATAOKA H. Silencing of insulin-like growth factor-binding protein-2 in human glioblastoma cells reduces both invasiveness and expression of progression-associated gene CD24. Journal of Biological Chemistry, 2007, 282(25): 18634-18644.
[11]   ERNST C W, MCCUSKER R H, WHITE M E. Gene expression and secretion of insulin-like growth factor-binding proteins during myoblast differentiation. Endocrinology, 1992, 130(2): 607-615.
[12]   SHARPLES A P, AL-SHANTI N, HUGHES D C, LEWIS M P, STEWART C E. The role of insulin-like-growth factor binding protein 2 (IGFBP2) and phosphatase and tensin homologue (PTEN) in the regulation of myoblast differentiation and hypertrophy. Growth Hormone and IGF Research, 2013, 23(3): 53-61.
[13]   YAU S W, HENRY B A, RUSSO V C, MCCONELL G K, CLARKE I J, WERTHER G A, SABIN M A. Leptin enhances insulin sensitivity by direct and sympathetic nervous system regulation of muscle IGFBP-2 expression: evidence from nonrodent models. Endocrinology, 2014, 155(6): 2133-2143.
[14]   LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25(4): 402-408.
[15]   FIRTH S M, BAXTER R C. Cellular actions of the insulin-like growth factor binding proteins. Endocrine Reviews, 2002, 23(6): 824-854.
[16]   JOGIE-BRAHIM S, FELDMAN D, OH Y. Unraveling insulin-like growth factor binding protein-3 actions in human disease. Endocrine Reviews, 2009, 30(5): 417-437.
[17]   JONES J I, CLEMMONS D R. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews, 1995, 16(1): 3-34.
[18]   YAMADA P M, LEE K W. Perspectives in mammalian IGFBP-3 biology: local vs. systemic action. American Journal of Physiology Cell Physiology, 2009, 296(5): C954-976.
[19]   WILHELM F, KASSNER F, SCHMID G, KRATZSCH J, LANER A, WABITSCH M, KORNER A, KIESS W, GARTEN A. Phosphatidylinositol 3-kinase (PI3K) signalling regulates insulin-like- growth factor binding protein-2 (IGFBP-2) production in human adipocytes. Growth Hormone and IGF Research, 2015, 25(3): 115-120.
[20]   DELHANTY P J, HAN V K. The expression of insulin-like growth factor (IGF)-binding protein-2 and IGF-II genes in the tissues of the developing ovine fetus. Endocrinology, 1993, 132(1): 41-52.
[21]   KANG H S, KIM M Y, KIM S J, LEE J H, KIM Y D, SEO Y K, BAE J H, OH G T, SONG D K, AHN Y H, IM S S. Regulation of IGFBP-2 expression during fasting. Biochemical Journal, 2015, 467(3): 453-460.
[22]   HAZEL S J, NORDQVIST A C, HALL K, NILSSON M, SCHALLING M. Differential expression of IGF-I and IGF-binding protein-1 and -2 in periportal and perivenous zones of rat liver. Journal of Endocrinology, 1998, 157(2): 285-294.
[23]   PFAFFL M W, GEORGIEVA T M, GEORGIEV I P, ONTSOUKA E, HAGELEIT M, BLUM J W. Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species. Domestic Animal Endocrinology, 2002, 22(2): 91-102.
[24]   LUND J, SONDERGAARD M T, CONOVER C A, OVERGAARD M T. Heparin-binding mechanism of the IGF2/IGF-binding protein 2 complex. Journal of Molecular Endocrinology, 2014, 52(3): 345-355.
[25]   SHEN X, XI G, MAILE L A, WAI C, ROSEN C J, CLEMMONS D R. Insulin-like growth factor (IGF) binding protein 2 functions coordinately with receptor protein tyrosine phosphatase beta and the IGF-I receptor to regulate IGF-I-stimulated signaling. Molecular and Cellular Biology, 2012, 32(20): 4116-4130.
[26]   SILVERMAN L A, CHENG Z Q, HSIAO D, ROSENTHAL S M. Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells. Endocrinology, 1995, 136(2): 720-726.
[27]   OKSBJERG N, GONDRET F, VESTERGAARD M. Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system. Domestic Animal Endocrinology, 2004, 27(3): 219-240.
[28]   PENG M, PELLETIER G, PALIN M F, VERONNEAU S, LEBEL D, ABRIBAT T. Ontogeny of IGFs and IGFBPs mRNA levels and tissue concentrations in liver, kidney and skeletal muscle of pig. Growth Development and Aging, 1996, 60(3-4): 171-187.
[29]   KITA K, NAGAO K, TANEDA N, INAGAKI Y, HIRANO K, SHIBATA T, YAMAN M A, CONLON M A, OKUMURA J. Insulin-like growth factor binding protein-2 gene expression can be regulated by diet manipulation in several tissues of young chickens. Journal of Nutrition, 2002, 132(2): 145-151.
[30]   LEE H G, CHOI Y J, LEE S R, KUWAYAMA H, HIDARI H, YOU S K. Effects of dietary protein and growth hormone-releasing peptide (GHRP-2) on plasma IGF-1 and IGFBPs in Holstein steers. Domestic Animal Endocrinology, 2005, 28(2): 134-146.
[31]   CERRO J A, GREWAL A, WOOD T L, PINTAR J E. Tissue-specific expression of the insulin-like growth factor binding protein (IGFBP) mRNAs in mouse and rat development. Regulatory Peptides, 1993, 48(1-2): 189-198.
[32]   REHFELDT C, RENNE U, SAWITZKY M, BINDER G, HOEFLICH A. Increased fat mass, decreased myofiber size, and a shift to glycolytic muscle metabolism in adolescent male transgenic mice overexpressing IGFBP-2. American Journal of Physiology Endocrinology and Metabolism, 2010, 299(2): E287-298.
[1] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[2] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[3] LI Heng,ZI XiangDong,WANG Hui,XIONG Yan,LÜ MingJie,LIU Yu,JIANG XuDong. Screening of Key Regulatory Genes for Litter Size Trait Based on Whole Genome Re-Sequencing in Goats (Capra hircus) [J]. Scientia Agricultura Sinica, 2022, 55(23): 4753-4768.
[4] QU Cheng,WANG Ran,LI FengQi,LUO Chen. Cloning and Expression Profiling of Gustatory Receptor Genes BtabGR1 and BtabGR2 in Bemisia tabaci [J]. Scientia Agricultura Sinica, 2022, 55(13): 2552-2561.
[5] ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.
[6] DU Yu,WANG Yong,MENG QingYong,ZHU JiangJiang,LIN YaQiu. Knockdown Goat KLF12 to Promote Subcutaneous Adipocytes Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(1): 184-196.
[7] ZHAO Le,YANG HaiLi,LI JiaLu,YANG YongHeng,ZHANG Rong,CHENG WenQiang,CHENG Lei,ZHAO YongJu. Expression Patterns of TETs and Programmed Cell Death Related Genes in Oviduct and Uterus of Early Pregnancy Goats [J]. Scientia Agricultura Sinica, 2021, 54(4): 845-854.
[8] ZHANG Lu,ZONG YaQi,XU WeiHua,HAN Lei,SUN ZhenYu,CHEN ZhaoHui,CHEN SongLi,ZHANG Kai,CHENG JieShan,TANG MeiLing,ZHANG HongXia,SONG ZhiZhong. Identification, Cloning, and Expression Characteristics Analysis of Fe-S Cluster Assembly Genes in Grape [J]. Scientia Agricultura Sinica, 2021, 54(23): 5068-5082.
[9] FENG YunKui,WANG Jian,MA JinLiang,ZHANG LiuMing,LI YongJun. Effects of miR-31-5p on the Proliferation and Apoptosis of Hair Follicle Stem Cells in Goat [J]. Scientia Agricultura Sinica, 2021, 54(23): 5132-5143.
[10] TAN YongAn,JIANG YiPing,ZHAO Jing,XIAO LiuBin. Expression Profile of G Protein-Coupled Receptor Kinase 2 Gene (AlGRK2) and Its Function in the Development of Apolygus lucorum [J]. Scientia Agricultura Sinica, 2021, 54(22): 4813-4825.
[11] CHEN Yuan,CAI He,LI Li,WANG LinJie,ZHONG Tao,ZHANG HongPing. Alternative Splicing of TNNT3 and Its Effect on the Differentiation of MuSCs in Goat [J]. Scientia Agricultura Sinica, 2021, 54(20): 4466-4477.
[12] WANG Na,ZHAO ZiBo,GAO Qiong,HE ShouPu,MA ChenHui,PENG Zhen,DU XiongMing. Cloning and Functional Analysis of Salt Stress Response Gene GhPEAMT1 in Upland Cotton [J]. Scientia Agricultura Sinica, 2021, 54(2): 248-260.
[13] TAN YongAn,ZHAO XuDong,JIANG YiPing,ZHAO Jing,XIAO LiuBin,HAO DeJun. Cloning, Preparation of Antibody and Response Induced by 20-Hydroxyecdysone of Target of Rapamycin in Apolygus lucorum [J]. Scientia Agricultura Sinica, 2021, 54(10): 2118-2131.
[14] GU MingHui,LIU YongFeng,SHEN Qian,QIAO ChunYan. Improving Quality and Delaying Oxidation in Goat Meat Refrigeration by Polyphenols from Thinned Young Kiwifruit [J]. Scientia Agricultura Sinica, 2020, 53(8): 1643-1654.
[15] KunNeng ZHOU,JiaFa XIA,Peng YUN,YuanLei WANG,TingChen MA,CaiJuan ZHANG,ZeFu LI. Transcriptome Research of Erect and Short Panicle Mutant esp in Rice [J]. Scientia Agricultura Sinica, 2020, 53(6): 1081-1094.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd