水牛孤雌激活早期胚胎蛋白质表达谱的构建及生物信息学分析
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摘要
收集水牛孤雌激活后2细胞期早期胚胎500枚,利用0.1%链酶蛋白酶去除透明带后提取蛋白质,并使用胰蛋白酶进行酶解处理,再将酶解得到的多肽混合物经强阳离子交换色谱进行预分离,预分离后所收集的馏分通过液相色谱串联质谱(LC-MS/MS)技术进行质谱鉴定,原始数据通过数据库搜索获得该时期蛋白质表达谱。通过SEQUEST检索共得到778种蛋白,对其进行生物信息学初步分析,包括GO富集分析和蛋白互作网络分析等。结果发现,这些蛋白质主要参与氧化还原、蛋白质折叠、细胞内蛋白质运输等生物学过程,主要定位于线粒体和细胞膜上。本研究为后续以孤雌激活为研究模型探讨哺乳动物早期胚胎发育和母源性蛋白质调控机制提供了新的研究手段和方法。
The two-cell stage early embryo of buffalo is the research object.500 two-cell stage early embryo of buffalo after parthenogenetic activation were collected,and the zona pellucida was removed by using 0.1% pronase and then proteins were extracted then hydrolyzed by trypsin,the peptide mixture was separated by using the strong cation exchange chromatographic,the fraction after separation was indentified by using liquid chromatography-tandem mass spectrometry(LCMS/MS)technology for mass spectrometry,and the raw data was searched by the database then the protein expression profile of this period was done.A total of 778 protein by using SEQUEST search were obtained and then made preliminary bioinformatics analysis for this results.The results showed that these proteins were primarily involved in the oxidation and reduction processe,protein folding,intracellular protein transport and other biological processes,mainly in the mitochondria and the cell membrane.The research provided new research tools and methods for the study on parthenogenetic activation model of mammalian early embryonic and maternal protein regulation mechanism.
The two-cell stage early embryo of buffalo is the research object.500 two-cell stage early embryo of buffalo after parthenogenetic activation were collected,and the zona pellucida was removed by using 0.1% pronase and then proteins were extracted then hydrolyzed by trypsin,the peptide mixture was separated by using the strong cation exchange chromatographic,the fraction after separation was indentified by using liquid chromatography-tandem mass spectrometry(LCMS/MS)technology for mass spectrometry,and the raw data was searched by the database then the protein expression profile of this period was done.A total of 778 protein by using SEQUEST search were obtained and then made preliminary bioinformatics analysis for this results.The results showed that these proteins were primarily involved in the oxidation and reduction processe,protein folding,intracellular protein transport and other biological processes,mainly in the mitochondria and the cell membrane.The research provided new research tools and methods for the study on parthenogenetic activation model of mammalian early embryonic and maternal protein regulation mechanism.
引文
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[19]井洋洋,方南洙,张宝修,等.活性氧对鼠早期胚胎抗氧化酶基因表达的影响[J].中国兽医学报,2017,37(2):335-344.
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[2]黄右军,黄芬香,张秀芳,等.水牛卵母细胞体外受精与早期胚胎发育的研究[J].中国畜牧杂志,2002,38(4):10-12.
[3]SOUSA P A,WATSON A J,SCHULTZ G A,et al.Oogenetic and zygotic gene expression directing early bovine embryogenesis:a review[J].Mol Reprod Dev,1998,51(1):112-121.
[4]尹娜,沈朋雷,秦希玲,等.L-肉碱对水牛卵母细胞体外成熟的影响[J].中国兽医学报,2017,37(11):2215-2221.
[5]TELFORD N A,WATSON A J,SCHULTZ G A.Transition from maternal to embryonic control in early mammalian development:a comparison of several species[J].Mol Reprod Dev,1990,26(1):90-100.
[6]赵晓娥,刘玉太,惠琦辉,等.ROS诱导山羊卵母细胞核质同步成熟[J].中国兽医学报,2017,37(7):1401-1407.
[7]VIUFF D,AVERY B,GREVE T,et al.Transcriptional activity in in vitro produced bovine two-and four-cell embryos[J].Mol Reprod Dev,1996,43(2):171-179.
[8]DOMINKO T,MITALIPOVA M,HALEY B,et al.Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species[J].Biol Reprod,1999,60(6):1496-1502.
[9]FREI R E,SCHULTZ G A,CHURCH R B.Qualitative and quantitative changes in protein synthesis occur at the 8-16-cell stage of embryogenesis in the cow[J].J Reprod Fertil,1989,86(2):637-641.
[10]WILKINS M R,SANCHEZ J C,GOOLEY A A,et al.Progress with proteome projects:why all proteins expressed by agenome should be identified and how to do it[J].Biotechnol Genet Eng Rev,1996,13:19-50.
[11]COONROD S A,WRIGHT P W,HERR J C.Oolemmal proteomics[J].J Reprod Immunol,2002,53(1/2):55-65.
[12]YATES J R.Mass spectrometry and the age of the proteome[J].J Mass Spectrom,1998,33(1):1-19.
[13]LINK A J,ENG J,SCHIELTZ D M,et al.Direct analysis of protein complexes using mass spectrometry[J].Nat Biotechnol,1999,17(7):676-682.
[14]ZHANG P,NI X,GUO Y,et al.Proteomic-based identification of maternal proteins in mature mouse oocytes[J].BMC Genomics,2009,10(1):1-11.
[15]KIM J,KIM J S,JEON Y J,et al.Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation[J].Proteome Sci,2011,9(1):28.
[16]陈富美,付强,黄德伦,等.基于LC-MS/MS策略建立水牛卵母细胞蛋白质表达谱的方法研究[J].基因组学与应用生物学,2015(5):967-971.
[17]BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72:248-254.
[18]MASSICOTTE L,COENEN K,MOUROT M,et al.Maternal housekeeping proteins translated during bovine oocyte maturation and early embryo development[J].Proteomics,2006,6(13):3811-3820.
[19]井洋洋,方南洙,张宝修,等.活性氧对鼠早期胚胎抗氧化酶基因表达的影响[J].中国兽医学报,2017,37(2):335-344.
[20]CHRISTIANS E,CAMPION E,THOMPSON E M,et al.Expression of the HSP 70.1gene,a landmark of early zygotic activity in the mouse embryo,is restricted to the first burst of transcription[J].Development,1995,121(1):113-122.
[21]BENSAUDE O,BABINET C,MORANGE M,et al.Heat shock proteins,first major products of zygotic gene activity in mouse embryo[J].Nature,1983,305(5932):331-333.
[22]YOST H J,LINDQUIST S.RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis[J].Cell,1986,45(2):185-193.