基于Illumina平台转录组测序筛选牛卵泡发育调控基因
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摘要
运用超声波监测技术,采集母牛发情期出现偏差前的第一大卵泡PDF1和出现偏差后的第二大卵泡ODF2,分别分离卵泡颗粒细胞(GCs),构建RNA文库,用Illumina2000测序;测序结果经数据库搜索和差异表达筛选,采用DAVID软件GO和KEGG pathway分析,经Genecards功能查询,筛选牛卵泡发育相关基因。结果表明:数据库搜索共获得35 325个基因,其中有意义的表达基因15 645个,从中筛选获得差异表达基因608个;GO分析表明,608个差异表达基因中参与生物过程的基因占60.46%,细胞组分的基因占20.08%,分子功能的基因占11.46%;KEGG pathway分析表明,608个差异表达基因通过9条信号通路参与牛卵泡发育调控(P<0.05),经Genecards功能筛选,共获得11个与牛卵泡发育密切相关的调控基因——SESN3、COL3A1、CCNE1、CAV1、CACNA1H、ITPR1、PIK3R3、MYC、PTGFR、CDK6、GAPDH,它们直接参与细胞增殖分化及信号通路调节。
The largest follicle at onset of predeviation(PDF1) and the second largest follicle at onset of deviation(ODF2) during bovine estrous cycle were collected by ultrasonic monitoring technology. The isolated granulosa cells(GCs) were subject to RNA library construction and Illumina 2000 sequencing. The resulted sequences were analyzed by Internet database RNA sequence search, differential expression screening, gene ontology and KEGG pathway analysis method, and genes sorting using Genecards to explore follicular development associated gene. The results showed that a total of 35 325 genes were identified after database gene search, among which were 15 645 significant expression genes with differentially expressed genes. The main functions of the 608 genes could be categorized into three groups: biological process(60.46%), cellular component(20.08%), molecular function(11.46%). KEGG pathway analysis showed that the 608 genes were involved in the regulation of bovine follicle development by 9 signal pathways(P<0.05). A total of 11 regulatory genes were obtained through genecards——SESN3, COL3 A1, CCNE1, CAV1, CACNA1 H, ITPR1, PIK3 R3, MYC, PTGFR, CDK6 and GAPDH, which were closely related to bovine follicular development and directly involved in cell proliferation, differentiation and signal pathway regulation.
The largest follicle at onset of predeviation(PDF1) and the second largest follicle at onset of deviation(ODF2) during bovine estrous cycle were collected by ultrasonic monitoring technology. The isolated granulosa cells(GCs) were subject to RNA library construction and Illumina 2000 sequencing. The resulted sequences were analyzed by Internet database RNA sequence search, differential expression screening, gene ontology and KEGG pathway analysis method, and genes sorting using Genecards to explore follicular development associated gene. The results showed that a total of 35 325 genes were identified after database gene search, among which were 15 645 significant expression genes with differentially expressed genes. The main functions of the 608 genes could be categorized into three groups: biological process(60.46%), cellular component(20.08%), molecular function(11.46%). KEGG pathway analysis showed that the 608 genes were involved in the regulation of bovine follicle development by 9 signal pathways(P<0.05). A total of 11 regulatory genes were obtained through genecards——SESN3, COL3 A1, CCNE1, CAV1, CACNA1 H, ITPR1, PIK3 R3, MYC, PTGFR, CDK6 and GAPDH, which were closely related to bovine follicular development and directly involved in cell proliferation, differentiation and signal pathway regulation.
引文
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[23]BERTOLI C,SKOTHEIM J M,DE BRUIN R A.Control of cell cycle transcription during G1 and S phases[J].Nat Rev Mol Cell Biol,2013,14(8):518-528.
[24]SONG Y,LI Z X,LIU X,et al.The Wnt/β-catenin and PI3K/Akt signaling pathways promote EMT in gastric cancer by epigenetic regulation via H3 lysine 27acetylation[J].Tumour Biol,2017,39(7):1010428317712617.DOI:10.1177/1010428317712617.
[25]ADHIKARI D,LIU K.Molecular mechanisms underlying the activation of mammalian primordial follicles[J].Endocr Rev,2009,30(5):438-464.
[26]REDDY P,LIU L,ADHIKARI D,et al.Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool[J].Science,2008,319:611-613.
[27]MAKKER A,GOEL M M,MAHDI A A.PI3K/PTEN/Akt and TSC/mTOR signaling pathways,ovarian dysfunction,and infertility:an update[J].J Mol Endocrinol,2014,53(3):R103-R118.
[28]SHEN M,JIANG Y,GUAN Z Q,et al.Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy[J].Autophagy,2017,13(8):1364-1385.
[2]ROCHE J F,MIHM M,DISKIN M G,et al.A review of regulation of follicle growth in cattle[J].J Anim Sci,1998,76(suppl_3):16-29.
[3]PEDERSEN H G,WATSON E D,TELFER EE.Analysis of atresia in equine follicles using histology,fresh granulosa cell morphology and detection of DNAfragmentation[J].Reproduction,2003,125(3):417-423.
[4]WONGSRIKEAO P,KANESHIGE Y,OOKI R,et al.Effect of the removal of cumulus cells on the nuclear maturation,fertilization and development of porcine oocytes[J].Reprod Domest Anim,2005,40(2):166-170.
[5]卫晓红,祁丽花,迟晓春,等.Smad2/Smad3蛋白在大鼠卵巢颗粒细胞中的表达及FSH对其活化的影响[J].解剖学报,2007,38(2):205-208.
[6]CASEY O M,MORRIS D G,POWELL R,et al.Analysis of gene expression in non-regressed and regressed bovine corpus luteum tissue using a customized ovarian cDNA array[J].Theriogenology,2005,64(9):1963-1976.
[7]CHOUDARY J B,GIER H T,MARION G B.Cyclic changes in bovine vesicular follicles[J].J Anim Sci,1968,27(2):468-471.
[8]SPICER L J,ECHTERNKAMP S E.Ovarian follicular growth,function and turnover in cattle:a review[J].JAnim Sci,1986,62(2):428-451.
[9]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.
[10]ROMEREIM S M,SUMMERS A F,POHLMEIER WE,et al.Transcriptomes of bovine ovarian follicular and luteal cells[J].Data Brief,2017,10:335-339.
[11]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 JGenomics,2016,2016:3472748.DOI:10.1155/2016/3472748.
[12]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.
[13]AUDIC S,CLAVERIE J M.The significance of digital gene expression profiles[J].Genome Res,1997,7(10):986-995.
[14]CHEN C C,JEON S M,BHASKAR P T,et al.FoxOs inhibit m TORC1 and activate Akt by inducing the expression of Sestrin3 and Rictor[J].Dev Cell,2010,18(4):592-604.
[15]HU S F,RAO M,LEI H,et al.Expression patterns of p38αMAPK during follicular development in the ovaries of neonatal rats[J].Acta Histochem,2017,119(5):538-542.
[16]SUPERTI-FURGA A,GUGLER E,GITZELMANN R,et al.Ehlers-Danlos syndrome type IV:a multi-exon deletion in one of the two COL3A1 alleles affecting structure,stability,and processing of type III procollagen[J].J Biol Chem,1988,263(13):6226-6232.
[17]CHEN X P,ZHANG T H,SHI J J,et al.Notch1signaling regulates the proliferation and self-renewal of human dental follicle cells by modulating the G1/S phase transition and telomerase activity[J].PLo S One,2013,8(7):e69967.DOI:10.1371/journal.pone.0069967.
[18]LIU P S,RUDICK M,ANDERSON R G W.Multiple functions of caveolin-1[J].J Biol Chem,2002,277(44):41295-41298.
[19]KAUR S,ARCHER K J,DEVI M G,et al.Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance:identification of susceptibility gene sets through network analysis[J].J Clin Endocrinol Metab,2012,97(10):E2016-E2021.
[20]DIOUF M N,LEFEBVRE R,SILVERSIDES D W,et al.Induction of alpha-caveolin-1(αCAV1)expression in bovine granulosa cells in response to an ovulatory dose of human chorionic gonadotropin[J].Mol Reprod Dev,2006,73(11):1353-1360.
[21]CRIBBS L L,LEE J H,YANG J,et al.Cloning and characterization of alpha1H from human heart,a member of the T-type Ca2+channel gene family[J].Circ Res,1998,83(1):103-109.
[22]CONACCI-SORRELL M,MCFERRIN L,EISENMANR N.An overview of MYC and its interactome[J].Cold Spring Harb Perspect Med,2014,4(1):a014357.DOI:10.1101/cshperspect.a014357.
[23]BERTOLI C,SKOTHEIM J M,DE BRUIN R A.Control of cell cycle transcription during G1 and S phases[J].Nat Rev Mol Cell Biol,2013,14(8):518-528.
[24]SONG Y,LI Z X,LIU X,et al.The Wnt/β-catenin and PI3K/Akt signaling pathways promote EMT in gastric cancer by epigenetic regulation via H3 lysine 27acetylation[J].Tumour Biol,2017,39(7):1010428317712617.DOI:10.1177/1010428317712617.
[25]ADHIKARI D,LIU K.Molecular mechanisms underlying the activation of mammalian primordial follicles[J].Endocr Rev,2009,30(5):438-464.
[26]REDDY P,LIU L,ADHIKARI D,et al.Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool[J].Science,2008,319:611-613.
[27]MAKKER A,GOEL M M,MAHDI A A.PI3K/PTEN/Akt and TSC/mTOR signaling pathways,ovarian dysfunction,and infertility:an update[J].J Mol Endocrinol,2014,53(3):R103-R118.
[28]SHEN M,JIANG Y,GUAN Z Q,et al.Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy[J].Autophagy,2017,13(8):1364-1385.