PDF2和ODF1转录组测序筛选牛卵泡发育相关基因
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摘要
旨在筛选牛卵泡发育相关基因及其表达模式。本研究选取9头青年母牛,同期发情处理后,采集卵泡波中出现偏差前的小卵泡PDF2(The small follicle at onset of predeviation)和出现偏差后的最大卵泡ODF1(The largest follicle at onset of deviation),分离颗粒细胞(Granulesa cells,GCs),提取总RNA,并进行转录组测序;经牛RefSeq数据库搜索和差异表达基因筛选,进行GO(Gene ontology)分析;随机选择5个基因(MAPK13、CYP19A1、GREB1、SERPINE2、GSTA5)进行qRT-PCR表达量验证分析。结果表明:PDF2和ODF1转录组中共获得RPKM(The reads per kilobase per million reads)值≥0.5的表达基因15 413个,其中表达差异倍数(Fold change)≥2的差异表达基因共651个;对651个差异表达基因进行GO分析表明,参与生物过程(Biological process,BP)的基因占41.66%,分子功能(Molecular function,MF)占17.24%,细胞组分(Cellular component,CC)占41.10%;GO分析结合Genecards基因功能查询,共筛选出13个下调基因和17个上调基因与牛卵泡发育直接相关;CYP19A1、GREB1、SERPINE2在优势卵泡(Donminant follicles,DF)的表达量极显著高于从属卵泡(Subordinate follicle,SF)(P<0.01),MAPK13在SF表达量极显著高于DF(P<0.01),GSTA5在SF表达量显著高于DF(P<0.05)。5个基因差异表达趋势与转录组测序结果完全相符,也进一步证明了转录组测序结果的可信度,为深入探讨基因调控牛卵泡发育奠定了基础。
This study aimed to screen genes associated with bovine follicular development and investigate their expression patterns.Nine young cows were collected.The small follicle at onset of predeviation(PDF2)and the largest follicle at onset of deviation(ODF1)follicles were collected after estrus synchronization.The total RNA was extracted from separated granulosa cells for the transcriptome sequencing.The Gene Ontology analysis was carried out after searching the bovine Refseq database and screening differentially expressed genes.MAPK13,CYP19 A1,GREB1,SERP I NE2,GSTA5 genes were selected randomly for verification using qRT-PCR technology.Results suggested that 15 413 genes were obtained from PDF2 and ODF1 transcriptomes(RPKM value≥0.5),of which 651 differentially expressed genes were selected(Fold change ≥2).GO assignments were used to classify the functions of the 651 genes,and which could be categorized into 3 main catrgories,biological process(41.66%),molecular function(17.24%),cellular component(41.10%).Combining with Genecards,13 down-regulated and 17 up-regulated genes screened were associated with follicle development.qRT-PCR results indicated that the expression of CYP19A1,GREB1 and SERPINE2 in dominant follicles were very significantly greater than that in subordinate follicles(P<0.01).MAPK13 mRNA amounts in subordinate follicles were very significantly greater than that in dominant follicles(P<0.01).GSTA5 mRNA amounts in subordinate follicles were significantly greater than that in dominant follicles(P<0.05).qRT-PCR results were entirely consisted with transcriptome sequencing results,and further proved the reliability of the transcriptome sequencing,which provided a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle.
This study aimed to screen genes associated with bovine follicular development and investigate their expression patterns.Nine young cows were collected.The small follicle at onset of predeviation(PDF2)and the largest follicle at onset of deviation(ODF1)follicles were collected after estrus synchronization.The total RNA was extracted from separated granulosa cells for the transcriptome sequencing.The Gene Ontology analysis was carried out after searching the bovine Refseq database and screening differentially expressed genes.MAPK13,CYP19 A1,GREB1,SERP I NE2,GSTA5 genes were selected randomly for verification using qRT-PCR technology.Results suggested that 15 413 genes were obtained from PDF2 and ODF1 transcriptomes(RPKM value≥0.5),of which 651 differentially expressed genes were selected(Fold change ≥2).GO assignments were used to classify the functions of the 651 genes,and which could be categorized into 3 main catrgories,biological process(41.66%),molecular function(17.24%),cellular component(41.10%).Combining with Genecards,13 down-regulated and 17 up-regulated genes screened were associated with follicle development.qRT-PCR results indicated that the expression of CYP19A1,GREB1 and SERPINE2 in dominant follicles were very significantly greater than that in subordinate follicles(P<0.01).MAPK13 mRNA amounts in subordinate follicles were very significantly greater than that in dominant follicles(P<0.01).GSTA5 mRNA amounts in subordinate follicles were significantly greater than that in dominant follicles(P<0.05).qRT-PCR results were entirely consisted with transcriptome sequencing results,and further proved the reliability of the transcriptome sequencing,which provided a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle.
引文
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[15]RISCO A,CUENDA A.New insights into the p38γand p38δMAPK pathways[J].J Signal Transduct,2012,2012:520289,doi:10.1155/2012/520289.
[16]SUMARA G,FORMENTINI I,COLLINS S,et al.Regulation of PKD by the MAPK p38δin insulin secretion and glucose homeostasis[J].Cell,2009,136(2):235-248.
[17]OESTERREICH S.Scaffold attachment factors SAFB1and SAFB2:innocent bystanders or critical players in breast tumorigenesis?[J].J Cell Biochem,2003,90(4):653-661.
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[20]ADAMSON E D,MERCOLA D.Egr1transcription factor:multiple roles in prostate tumor cell growth and survival[J].Tumour Biol,2002,23(2):93-102.
[21]SUKHATME V P,CAO X M,CHANG L C,et al.A zinc finger-encoding gene coregulated with c-fos during growth and differentiation,and after cellular depolarization[J].Cell,1988,53(1):37-43.
[22]RUSSELL D L,DOYLE K M H,GONZALESROBAYNA I,et al.Egr-1induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone:combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory element binding protein,serum response factor,sp1,and early growth response factor-1[J].Mol Endocrinol,2003,17(4):520-533.
[23]LIM B C,MATSUMOTO S,YAMAMOTO H,et al.Prickle1promotes focal adhesion disassembly in cooperation with the CLASP-LL5βcomplex in migrating cells[J].J Cell Sci,2016,129(16):3115-3129.
[24]OKUDA H,MIYATA S,MORI Y,et al.Mouse Prickle1and Prickle2are expressed in postmitotic neurons and promote neurite outgrowth[J].FEBS Lett,2007,581(24):4754-4760.
[25]TAO H,SUZUKI M,KIYONARI H,et al.Mouse prickle1,the homolog of a PCP gene,is essential for epiblast apical-basal polarity[J].Proc Natl Acad Sci U S A,2009,106(34):14426-14431.
[26]WILSKER D,PROBST L,WAIN H M,et al.Nomenclature of the ARID family of DNA-binding proteins[J].Genomics,2005,86(2):242-251.
[27]NISHIBUCHI G,NAKAYAMA J I.Biochemical and structural properties of heterochromatin protein1:understanding its role in chromatin assembly[J].J Biochem,2014,156(1):11-20.
[28]DOETSCHMAN T,BARNETT J V,RUNYAN R B,et al.Transforming growth factor betasignaling in adult cardiovascular diseases and repair[J].Cell Tissue Res,2012,347(1):203-223.
[29]LUO W X,WILTBANK M C.Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1in bovine granulosa cells[J].Biol Reprod,2006,75(2):217-225.
[30]CAO M J,NICOLA E,PORTELA V M,et al.Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro[J].Matrix Biol,2006,25(6):342-354.
[31]MOHAMMED H,D′SANTOS C,SERANDOUR A A,et al.Endogenous purification reveals GREB1as a key estrogen receptor regulatory factor[J].Cell Rep,2013,3(2):342-349.
[2]ROMEREIM S M,SUMMERS A F,POHLMEIER W E,et al.Transcriptomes of bovine ovarian follicular and luteal cells[J].Data Brief,2017,10:335-339.
[3]TERENINA E,FABRE S,BONNET A,et al.Differentially expressed genes and gene networks involved in pig ovarian follicular atresia[J].Physiol Genomics,2017,49(2):67-80.
[4]LI P F,MENG J Z,LIU W Z,et al.Transcriptome analysis of bovine ovarian follicles at predeviation and onset of deviation stages of a follicular wave[J].Int J Genomics,2016,2016:347248,doi:10.1155/2016/3472748.
[5]李鹏飞,孟金柱,谢建山,等.牛卵泡ODF1与ODF2转录组发育相关基因筛选及表达差异分析[J].畜牧兽医学报,2015,46(11):1961-1966.LI P F,MENG J Z,XIE J S,et al.Screening and analyse study of genes associated with follicular development in bovine ODF1and ODF2transcript[J].Acta Veterinaria et Zootechnica Sinica,2015,46(11):1961-1966.(in Chinese)
[6]AUDIC S,CLAVERIE J M.The significance of digital gene expression profiles[J].Genome Res,1997,7(10):986-995.
[7]XU Q,ZHAO W M,CHEN Y,et al.Transcriptome profiling of the goose(Anser cygnoides)ovaries identify laying and broodiness phenotypes[J].PLoS One,2013,8(2):e55496.
[8]HATZIRODOS N,IRVING-RODGERS H F,HUMMITZSCH K,et al.Transcriptome profiling of granulosa cells of bovine ovarian follicles during growth from small to large antral sizes[J].BMC Genomics,2014,15:24,doi:10.1186/1471-2164-15-24.
[9]GILBERT I,ROBERT C,DIELEMAN S,et al.Transcriptional effect of the LH surge in bovine granulosa cells during the peri-ovulation period[J].Reproduction,2011,141(2):193-205.
[10]MIAO X Y,LUO Q M,QIN X Y.Genome-wide transcriptome analysis of mRNAs and microRNAs in Dorset and Small Tail Han sheep to explore the regulation of fecundity[J].Mol Cell Endocrinol,2015,402:32-42.
[11]GILBERT I,ROBERT C,VIGNEAULT C,et al.Impact of the LH surge on granulosa cell transcript levels as markers of oocyte developmental competence in cattle[J].Reproduction,2012,143(6):735-747.
[12]LEI M M,CAI L P,LI H,et al.Transcriptome sequencing analysis of porcine granulosa cells treated with an anti-inhibin antibody[J].Reprod Biol,2017,17(1):79-88.
[13]CROUCHER N J,FOOKES M C,PERKINS T T,et al.A simple method for directional transcriptome sequencing using Illumina technology[J].Nucleic Acids Res,2009,37(22):e148,doi:10.1093/nar/gkp811.
[14]O′CALLAGHAN C,FANNING L J,BARRY O P.p38δMAPK:emerging roles of a neglected isoform[J].Int J Cell Biol,2014,2014:272689,doi:10.1155/2014/272689.
[15]RISCO A,CUENDA A.New insights into the p38γand p38δMAPK pathways[J].J Signal Transduct,2012,2012:520289,doi:10.1155/2012/520289.
[16]SUMARA G,FORMENTINI I,COLLINS S,et al.Regulation of PKD by the MAPK p38δin insulin secretion and glucose homeostasis[J].Cell,2009,136(2):235-248.
[17]OESTERREICH S.Scaffold attachment factors SAFB1and SAFB2:innocent bystanders or critical players in breast tumorigenesis?[J].J Cell Biochem,2003,90(4):653-661.
[18]IVANOVA M,DOBRZYCKA K M,JIANG S,et al.Scaffold attachment factor B1functions in development,growth,and reproduction[J].Mol Cell Biol,2005,25(8):2995-3006.
[19]TOWNSON S M,KANG K Y,LEE A V,et al.Structure-function analysis of the estrogen receptorαcorepressor scaffold attachment factor-B1:identification of a potent transcriptional repression domain[J].J Biol Chem,2004,279(25):26074-26081.
[20]ADAMSON E D,MERCOLA D.Egr1transcription factor:multiple roles in prostate tumor cell growth and survival[J].Tumour Biol,2002,23(2):93-102.
[21]SUKHATME V P,CAO X M,CHANG L C,et al.A zinc finger-encoding gene coregulated with c-fos during growth and differentiation,and after cellular depolarization[J].Cell,1988,53(1):37-43.
[22]RUSSELL D L,DOYLE K M H,GONZALESROBAYNA I,et al.Egr-1induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone:combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory element binding protein,serum response factor,sp1,and early growth response factor-1[J].Mol Endocrinol,2003,17(4):520-533.
[23]LIM B C,MATSUMOTO S,YAMAMOTO H,et al.Prickle1promotes focal adhesion disassembly in cooperation with the CLASP-LL5βcomplex in migrating cells[J].J Cell Sci,2016,129(16):3115-3129.
[24]OKUDA H,MIYATA S,MORI Y,et al.Mouse Prickle1and Prickle2are expressed in postmitotic neurons and promote neurite outgrowth[J].FEBS Lett,2007,581(24):4754-4760.
[25]TAO H,SUZUKI M,KIYONARI H,et al.Mouse prickle1,the homolog of a PCP gene,is essential for epiblast apical-basal polarity[J].Proc Natl Acad Sci U S A,2009,106(34):14426-14431.
[26]WILSKER D,PROBST L,WAIN H M,et al.Nomenclature of the ARID family of DNA-binding proteins[J].Genomics,2005,86(2):242-251.
[27]NISHIBUCHI G,NAKAYAMA J I.Biochemical and structural properties of heterochromatin protein1:understanding its role in chromatin assembly[J].J Biochem,2014,156(1):11-20.
[28]DOETSCHMAN T,BARNETT J V,RUNYAN R B,et al.Transforming growth factor betasignaling in adult cardiovascular diseases and repair[J].Cell Tissue Res,2012,347(1):203-223.
[29]LUO W X,WILTBANK M C.Distinct regulation by steroids of messenger RNAs for FSHR and CYP19A1in bovine granulosa cells[J].Biol Reprod,2006,75(2):217-225.
[30]CAO M J,NICOLA E,PORTELA V M,et al.Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro[J].Matrix Biol,2006,25(6):342-354.
[31]MOHAMMED H,D′SANTOS C,SERANDOUR A A,et al.Endogenous purification reveals GREB1as a key estrogen receptor regulatory factor[J].Cell Rep,2013,3(2):342-349.