缢蛏生长因子受体结合蛋白2基因克隆、时空表达及SNP筛查
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摘要
为了探索生长因子受体结合蛋白2(GRB2)在缢蛏生长发育中的作用,本实验基于缢蛏转录组文库,利用SMART RACE技术克隆缢蛏GRB2(Sc-GRB2)基因的cDNA全长序列,分析其在不同组织和发育时期的表达差异,并在外显子中进行了SNP位点筛选。结果表明,Sc-GRB2基因cDNA全长1 223bp,开放阅读框711bp,编码236个氨基酸;氨基酸序列比对发现,缢蛏与文蛤、泥蚶等双壳贝类同源性较高(64%、59%),而与其他物种的同源性为45%~58%,表明GRB2基因比较保守;不同组织的荧光定量PCR(qRT-PCR)结果显示,Sc-GRB2基因在缢蛏7个组织中均有表达,其中足中的表达量极显著地高于其他6个组织(P<0.01);在8个发育时期的表达差异分析发现,该基因在稚贝期表达量极显著地高于其他7个时期(P<0.01)。SNP位点筛选结果表明,在Sc-GRB2基因的外显子区域共发现11个SNP位点。
To explore the molecular structure and biological function of GRB2 in Sinonovacula constricta(ScGRB2),its cDNA was cloned by SMART RACE techniques,then the bioinformatics,SNPs in exon and expression profiles in different tissues and developmental stages were analyzed.The results indicated that the full length cDNA of Sc-GRB2 gene was 1 223 bp,containing a complete 711 bp ORF encoding 236 amino acids.Comparisons of animal acid sequence,Sc-GRB2 has high homology with bivalves of Meretrix meretrixand Tegillarca granosa(64%and 59%).Sc-GRB2 has homologous with others share 45%-58%similarity,it indicate GRB2 is relatively conservative.The result of qRT-PCR in different tissues showed that Sc-GRB2 expressed in all seven tissues,the expression of foot was extremely significantly higher than others(P<0.01).The relative expression in different stages revealed that the expression of juvenile clams was extremely significantly higher than other stages(P<0.01).A total of 11 SNPs in the exon of Sc-GRB2 were identified.
To explore the molecular structure and biological function of GRB2 in Sinonovacula constricta(ScGRB2),its cDNA was cloned by SMART RACE techniques,then the bioinformatics,SNPs in exon and expression profiles in different tissues and developmental stages were analyzed.The results indicated that the full length cDNA of Sc-GRB2 gene was 1 223 bp,containing a complete 711 bp ORF encoding 236 amino acids.Comparisons of animal acid sequence,Sc-GRB2 has high homology with bivalves of Meretrix meretrixand Tegillarca granosa(64%and 59%).Sc-GRB2 has homologous with others share 45%-58%similarity,it indicate GRB2 is relatively conservative.The result of qRT-PCR in different tissues showed that Sc-GRB2 expressed in all seven tissues,the expression of foot was extremely significantly higher than others(P<0.01).The relative expression in different stages revealed that the expression of juvenile clams was extremely significantly higher than other stages(P<0.01).A total of 11 SNPs in the exon of Sc-GRB2 were identified.
引文
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[2]Zhang Yunda,Xu Guoxing,Liu Gang,et al.miR-411-5p inhibits proliferation and metastasis of breast cancer cell via targeting GRB2[J].Biochemical and Biophysical Research Communications,2016,476(4):607-613.
[3]Radtke D,Lacher S M,Szumilas N,et al.Grb2is important for T cell development,Th cell differentiation,and induction of experimental autoimmune encephalomyelitis[J].The Journal of Immunology,2016,196(7):2995-3005.
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[6]Avruch J,Zhang Xianfeng,Kyriakis J M.Raf meets Ras:completing the framework of a signal transduction pathway[J].Trends in Biochemical Sciences,1994,19(7):279-283.
[7]Li Liyan,Zhang Kai,Jiang Hong,et al.Quantitative proteomics reveals the downregulation of GRB2as a prominent node of F806-targeted cell proliferation network[J].Journal of Proteomics,2015,117:145-155.
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[13]谢淑媚,牛东红,阮海灯,等.缢蛏IGFBP基因结构及生长性状相关SNP筛选[J].水产学报,2015,39(6):799-809.Xie Shumei,Niu Donghong,Ruan Haideng,et al.Molecular characterization of IGFBPand association analysis with growth traits in the razor clamSinonovacula constricta[J].Journal of Fisheries of China,2015,39(6):799-809.
[14]Downward J.The GRB2/Sem-5adaptor protein[J].FEBS Letters,1994,338(2):113-117.
[15]Haines E,Minoo P,Feng Zhenqian,et al.Tyrosine phosphorylation of GRB2:role in prolactin/epidermal growth factor cross talk in mammary epithelial cell growth and differentiation[J].Molecular and Cellular Biology,2009,29(10):2505-2520.
[16]White M F.IRS2integrates insulin/IGF1signalling with metabolism,neurodegeneration and longevity[J].Diabetes,Obesity and Metabolism,2014,16Suppl 1:4-15.
[17]McDonald C B,Bhat V,Mikles D C,et al.Bivalent binding drives the formation of the Grb2-Gab1signaling complex in a noncooperative manner[J].FEBS Journal,2012,279(12):2156-2173.
[18]Liu S,Zhang H,Li M,et al.Recruitment of Grb2and SHIP1by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity of NK cells[J].Cell Death&Differentiation,2013,20(3):456-464.
[19]Birchmeier C,Birchmeier W,Gherardi E,et al.Met,metastasis,motility and more[J].Nature Reviews Molecular Cell Biology,2003,4(12):915-925.
[20]Belov A A,Mohammadi M.Grb2,a double-edged sword of receptor tyrosine kinase signaling[J].Science Signaling,2012,5(249):pe49.
[21]Senior M M,Frederick A F,Black S,et al.The three-dimensional solution structure of the Src homology domain-2of the growth factor receptorbound protein-2[J].Journal of Biomolecular NMR,1998,11(2):153-164.
[22]Jia C Y H,Nie Jing,Wu Chenggang,et al.Novel Src homology 3domain-binding motifs identified from proteomic screen of a Pro-rich region[J].Molecular&Cellular Proteomics,2005,4(8):1155-1166.
[23]潘智.Grb2与AMPK、TSC2的相互作用及其在细胞生长调控中的功能研究[D].保定:河北大学,2007.Pan Zhi.The function study of Grb2interactions with AMPK and TSC2[D].Baoding:Hebei University,2007.
[24]Simpson A,Uitto J,Rodeck U,et al.Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1and Mta3[J].Gene,2001,273(1):29-39.
[25]Hudson E R,Pan D A,James J,et al.A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias[J].Current Biology,2003,13(10):861-866.
[26]Cheng A M,Saxton T M,Sakai R,et al.Mammalian Grb2regulates multiple steps in embryonic development and malignant transformation[J].Cell,1998,95(6):793-803.
[27]Saxton T M,Cheng A M,Ong S H,et al.Gene dosage-dependent functions for phosphotyrosine-Grb2signaling during mammalian tissue morphogenesis[J].Current Biology,2001,11(9):662-670.
[28]Puto L A,Pestonjamasp K,King C C,et al.p21-activated kinase 1(PAK1)interacts with the Grb2adapter protein to couple to growth factor signaling[J].Journal of Biological Chemistry,2003,278(11):9388-9393.
[29]Morrison G M,Oikonomopoulou I,Migueles R P,et al.Anterior definitive endoderm from ESCs reveals a role for FGF signaling[J].Cell Stem Cell,2008,3(4):402-415.
[30]Chen Yali,Li Xiaofeng,Eswarakumar V P,et al.Fibroblast growth factor(FGF)signaling through PI 3-kinase and Akt/PKB is required for embryoid body differentiation[J].Oncogene,2000,19(33):3750-3756.
[31]刘阳.MicroRNA-200a调节Grb2抑制小鼠胚胎干细胞的中内胚层分化[D].上海:第二军医大学,2013.Liu Yang.MicroRNA-200aregulates Grb2and suppresses differentiation of mouse embryonic stem cells into endoderm and mesoderm[D].Shanghai:The Second Military Medical University,2013.
[32]Fulvio M D,Henkels K M,Gomez-Cambronero J.Short-hairpin RNA-mediated stable silencing of Grb2impairs cell growth and DNA synthesis[J].Biochemical and Biophysical Research Communications,2007,357(3):737-742.
[33]王如才,王昭萍.海水贝类养殖学[M].青岛:中国海洋大学出版社,2008.Wang Rucai,Wang Zhaoping.Science of Marine Shellfish Culture[M].Qingdao:China Ocean University of China Press,2008.
[34]Bao Yongbo,Li Peifen,Dong Yinghui,et al.Polymorphism of the multiple hemoglobins in blood clamTegillarca granosaand its association with disease resistance to Vibrio parahaemolyticus[J].Fish&Shellfish Immunology,2013,34(5):1320-1324.
[2]Zhang Yunda,Xu Guoxing,Liu Gang,et al.miR-411-5p inhibits proliferation and metastasis of breast cancer cell via targeting GRB2[J].Biochemical and Biophysical Research Communications,2016,476(4):607-613.
[3]Radtke D,Lacher S M,Szumilas N,et al.Grb2is important for T cell development,Th cell differentiation,and induction of experimental autoimmune encephalomyelitis[J].The Journal of Immunology,2016,196(7):2995-3005.
[4]Chardin P,Cussac D,Maignan S,et al.The GRB2adaptor[J].FEBS Letters,1995;369(1):47-51.
[5]Li N,Batzer A,Daly R,et al.Guanine-nucleotide-releasing factor hsos1binds to Grb2and links receptor tyrosine kinases to Ras signalling[J].Nature,1993,363(6424):85-88.
[6]Avruch J,Zhang Xianfeng,Kyriakis J M.Raf meets Ras:completing the framework of a signal transduction pathway[J].Trends in Biochemical Sciences,1994,19(7):279-283.
[7]Li Liyan,Zhang Kai,Jiang Hong,et al.Quantitative proteomics reveals the downregulation of GRB2as a prominent node of F806-targeted cell proliferation network[J].Journal of Proteomics,2015,117:145-155.
[8]Ahmed Z,Lin Chichuan,Suen K M,et al.Grb2controls phosphorylation of FGFR2by inhibiting receptor kinase and Shp2phosphatase activity[J].Journal of Cell Biology,2013,200(4):493-504.
[9]董迎辉,项翔,姚韩韩,等.泥蚶(Tegillarca granosa)生长因子受体结合蛋白2(GRB2)基因的克隆与表达分析[J].海洋与湖沼,2013,44(4):937-943.Dong Yinghui,Xiang Xiang,Yao Hanhan,et al.Cloning and expression of GRB2gene from the blood clamTegillarca granosa[J].Oceanologia et Limnologia Sinica,2013,44(4):937-943.
[10]高晓艳,董迎辉,施沈佳,等.文蛤生长因子受体结合蛋白2(GRB2)基因克隆、时空表达及SNP位点筛查[J].水产学报,2015,39(9):1324-1332.Gao Xiaoyan,Dong Yinghui,Shi Shenjia,et al.Cloning,spatiotemporal expression and SNPs identification of GRB2gene in hard clam Meretrix meretrix[J].Journal of Fisheries of China,2015,39(9):1324-1332.
[11]徐凤山,张素萍.中国海产双壳类图志[M].北京:科学出版社,2008:211-213.Xu Fengshan,Zhang Suping.An Illustrated Bivalvia Mollusca Fauna of China Seas[M].Beijing:Science Press,2008:211-213.
[12]牛东红,冯冰冰,李家乐.缢蛏铁蛋白基因的分子特性及其表达分析[J].上海海洋大学学报,2012,21(5):641-649.Niu Donghong,Feng Bingbing,Li Jiale.Molecular characteristics and expression analysis of ferritin gene fromSinonovacula constricta[J].Journal of Shanghai Ocean University,2012,21(5):641-649.
[13]谢淑媚,牛东红,阮海灯,等.缢蛏IGFBP基因结构及生长性状相关SNP筛选[J].水产学报,2015,39(6):799-809.Xie Shumei,Niu Donghong,Ruan Haideng,et al.Molecular characterization of IGFBPand association analysis with growth traits in the razor clamSinonovacula constricta[J].Journal of Fisheries of China,2015,39(6):799-809.
[14]Downward J.The GRB2/Sem-5adaptor protein[J].FEBS Letters,1994,338(2):113-117.
[15]Haines E,Minoo P,Feng Zhenqian,et al.Tyrosine phosphorylation of GRB2:role in prolactin/epidermal growth factor cross talk in mammary epithelial cell growth and differentiation[J].Molecular and Cellular Biology,2009,29(10):2505-2520.
[16]White M F.IRS2integrates insulin/IGF1signalling with metabolism,neurodegeneration and longevity[J].Diabetes,Obesity and Metabolism,2014,16Suppl 1:4-15.
[17]McDonald C B,Bhat V,Mikles D C,et al.Bivalent binding drives the formation of the Grb2-Gab1signaling complex in a noncooperative manner[J].FEBS Journal,2012,279(12):2156-2173.
[18]Liu S,Zhang H,Li M,et al.Recruitment of Grb2and SHIP1by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity of NK cells[J].Cell Death&Differentiation,2013,20(3):456-464.
[19]Birchmeier C,Birchmeier W,Gherardi E,et al.Met,metastasis,motility and more[J].Nature Reviews Molecular Cell Biology,2003,4(12):915-925.
[20]Belov A A,Mohammadi M.Grb2,a double-edged sword of receptor tyrosine kinase signaling[J].Science Signaling,2012,5(249):pe49.
[21]Senior M M,Frederick A F,Black S,et al.The three-dimensional solution structure of the Src homology domain-2of the growth factor receptorbound protein-2[J].Journal of Biomolecular NMR,1998,11(2):153-164.
[22]Jia C Y H,Nie Jing,Wu Chenggang,et al.Novel Src homology 3domain-binding motifs identified from proteomic screen of a Pro-rich region[J].Molecular&Cellular Proteomics,2005,4(8):1155-1166.
[23]潘智.Grb2与AMPK、TSC2的相互作用及其在细胞生长调控中的功能研究[D].保定:河北大学,2007.Pan Zhi.The function study of Grb2interactions with AMPK and TSC2[D].Baoding:Hebei University,2007.
[24]Simpson A,Uitto J,Rodeck U,et al.Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1and Mta3[J].Gene,2001,273(1):29-39.
[25]Hudson E R,Pan D A,James J,et al.A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias[J].Current Biology,2003,13(10):861-866.
[26]Cheng A M,Saxton T M,Sakai R,et al.Mammalian Grb2regulates multiple steps in embryonic development and malignant transformation[J].Cell,1998,95(6):793-803.
[27]Saxton T M,Cheng A M,Ong S H,et al.Gene dosage-dependent functions for phosphotyrosine-Grb2signaling during mammalian tissue morphogenesis[J].Current Biology,2001,11(9):662-670.
[28]Puto L A,Pestonjamasp K,King C C,et al.p21-activated kinase 1(PAK1)interacts with the Grb2adapter protein to couple to growth factor signaling[J].Journal of Biological Chemistry,2003,278(11):9388-9393.
[29]Morrison G M,Oikonomopoulou I,Migueles R P,et al.Anterior definitive endoderm from ESCs reveals a role for FGF signaling[J].Cell Stem Cell,2008,3(4):402-415.
[30]Chen Yali,Li Xiaofeng,Eswarakumar V P,et al.Fibroblast growth factor(FGF)signaling through PI 3-kinase and Akt/PKB is required for embryoid body differentiation[J].Oncogene,2000,19(33):3750-3756.
[31]刘阳.MicroRNA-200a调节Grb2抑制小鼠胚胎干细胞的中内胚层分化[D].上海:第二军医大学,2013.Liu Yang.MicroRNA-200aregulates Grb2and suppresses differentiation of mouse embryonic stem cells into endoderm and mesoderm[D].Shanghai:The Second Military Medical University,2013.
[32]Fulvio M D,Henkels K M,Gomez-Cambronero J.Short-hairpin RNA-mediated stable silencing of Grb2impairs cell growth and DNA synthesis[J].Biochemical and Biophysical Research Communications,2007,357(3):737-742.
[33]王如才,王昭萍.海水贝类养殖学[M].青岛:中国海洋大学出版社,2008.Wang Rucai,Wang Zhaoping.Science of Marine Shellfish Culture[M].Qingdao:China Ocean University of China Press,2008.
[34]Bao Yongbo,Li Peifen,Dong Yinghui,et al.Polymorphism of the multiple hemoglobins in blood clamTegillarca granosaand its association with disease resistance to Vibrio parahaemolyticus[J].Fish&Shellfish Immunology,2013,34(5):1320-1324.
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