牛ODF2和PDF2转录组测序筛选卵泡发育的相关基因
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摘要
为研究牛卵泡发育过程中影响卵泡发育的重要调控基因及其表达模式,通过B超声波监测并采集牛卵泡发育波中出现偏差期后的第二大卵泡(ODF2)和偏差期前的第二大卵泡(PDF2),建立卵泡颗粒细胞RNA文库并进行高通量测序;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通路分析,再经Gene Cards功能查询筛选牛卵泡发育的相关基因.结果表明:转录组测序共获得35 325个基因,其中,高表达基因15 536个,进一步筛选出504个差异表达基因;GO分析表明,504个差异表达基因中参与生物过程的基因占39.49%,细胞组分占46.96%,分子功能占13.55%;KEGG通路分析表明,共有97个差异表达基因通过10条信号通路参与牛卵泡发育调控(P<0.05),经Gene Cards进行功能查询后,共筛选出18个与牛卵泡发育密切相关的调控基因.在卵泡发育过程中,PYCARD、MYC、PRICKLE1、TGFBR3、PIK3R3、SOX4、TOPORS、KREMEN1、STK11和NTRK1可能直接参与细胞增殖或凋亡.
The aim of this study was to identify bovine follicular development important genes and their expression patterns. In bovine follicular development wave,the second largest follicle at onset of deviation( ODF2) and the second largest follicle at onset of predeviation( PDF2) follicles were collected after B ultrasound monitoring. RNA library of granulesa cells was established and carried out high-throughput sequencing. Sequencing results were searched by database and differentially expressed genes were screened,then analyzed by gene ontology( GO) and KEGG pathway. Bovine follicular development related genes were screened using GeneCards Function query. Results suggested that 35 325 genes were obtained from the RNA-seq,of which 15 536 significantly expressed genes were selected( fold change ≥2),and then 504 differentially expressed genes were selected. GO assignments were used to classify the functions of the 504 genes,and could be categorized into three main catrgories: biological process( 39.49%),cellular component( 49.96%),molecular function( 13.55%). KEGG pathway analysis indicated that 97 differentially expressed genes were involved in bovine follicular development by ten signal paths( P<0.05). Combining with Gene Cards,18 genes have been screened associated with follicle development. PYCARD,MYC,PRICKLE1,TGFBR3,PIK3 R3,SOX4,TOPORS,KREMEN1,STK11,NTRK1 may were directly participated in cell proliferation or apoptosis in the process of follicular development.
The aim of this study was to identify bovine follicular development important genes and their expression patterns. In bovine follicular development wave,the second largest follicle at onset of deviation( ODF2) and the second largest follicle at onset of predeviation( PDF2) follicles were collected after B ultrasound monitoring. RNA library of granulesa cells was established and carried out high-throughput sequencing. Sequencing results were searched by database and differentially expressed genes were screened,then analyzed by gene ontology( GO) and KEGG pathway. Bovine follicular development related genes were screened using GeneCards Function query. Results suggested that 35 325 genes were obtained from the RNA-seq,of which 15 536 significantly expressed genes were selected( fold change ≥2),and then 504 differentially expressed genes were selected. GO assignments were used to classify the functions of the 504 genes,and could be categorized into three main catrgories: biological process( 39.49%),cellular component( 49.96%),molecular function( 13.55%). KEGG pathway analysis indicated that 97 differentially expressed genes were involved in bovine follicular development by ten signal paths( P<0.05). Combining with Gene Cards,18 genes have been screened associated with follicle development. PYCARD,MYC,PRICKLE1,TGFBR3,PIK3 R3,SOX4,TOPORS,KREMEN1,STK11,NTRK1 may were directly participated in cell proliferation or apoptosis in the process of follicular development.
引文
[1]SPICER L J,ECHTERNKAMP S E.Ovarian follicular growth,function and turnover in cattle:A review[J].J Anim Sci,1986,62(2):428-451.
[2]ROMEREIM S M,SUMMERS A F,POHLMEIER W E,et al.Transcriptomes of bovine ovarian follicular and luteal cells[J].Data Brief,2016,10:335-339.
[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,MENG J,ZHU Z,et al.Detection of genes associated with follicle development through tran-scriptome analysis of bovine ovarian follicles GCs[J].Curr Bioinform,2018,13(2):127-140.
[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):1 961-1 966.
[6]LI P,YU X,XIE J,et al.Expression of cocaine-and amphetamine-regulated transcript(CART)in hen ovary[J].Biol Res,2017,50(1):18.
[7]李鹏飞,孟金柱,于雪静,等.外源性dsRNA对蛋鸡卵泡基因表达及发育的影响[J].畜牧兽医学报,2016,47(3):515-520.
[8]HUANG Y,YAO X L,MENG J Z,et al.Intrafollicular expression and potential regulatory role of cocaine-and amphetamineregulated transcript in the ovine ovary[J].Domest Anim Endocrinol,2016,54(1):30-36.
[9]AUDIC S,CLAVERIE J M.The significance of digital gene expression profiles[J].Genome Res,1997,7(10):986-995.
[10]SUN X,ZHOU S,MENG F,et al.De novo assembly and characterization of the garlic(Allium sativum)bud transcriptome by Illumina sequencing[J].Plant Cell Rep,2012,31(10):1 823-1 828.
[11]DIAS F C,KHAN M I,SIRARD M A,et al.Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle[J].Reproduction,2013,146(2):181-191.
[12]MIAO X,QIN Q L.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.
[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.
[14]FALCONI G,FABIANI E,FIANCHI L,et al.Impairment of PI3K/AKT and WNT/β-catenin pathways in bone marrow mesenchymal stem cells isolated from patients with myelodysplastic syndromes[J].Exp Hematol,2016,44(1):75-83..
[15]MA X,HAYES E,BISWAS A,et al.Androgens regulate ovarian gene expression through modulation of Ezh2 expression and activity[J].Endocrinology,2017,158(9):2 944-2 954.
[16]SHEN M,JIANG Y,GUAN Z,et al.Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy[J].Autophagy,2017,13(8):1 364-1 385
[17]HUANG Z,WANG W,MA J,et al.m TOR signaling pathway differently regulates central and peripheral axon regeneration[J].Acta Biochim Biophys Sin(Shanghai),2017,28:1-7.
[18]BEHRAM KANDEMIR Y,AYDIN C,GORGISEN G.The effects of melatonin on oxidative stress and prevention of primordial follicle loss via activation of m TOR pathway in the rat ovary[J].Cell Mol Biol(Noisy-le-grand),2017,63(2):100-106.
[19]张毅敏,于斌,陈佳,等.针刺治疗对卵巢早衰大鼠PI3K/Akt/m TOR信号通路的影响[J].中国针灸,2015,35(1):53-58.
[20]ZHANG H,ZHANG H,ZHANG Y,et al.Dishevelled-DEP domain interacting protein(DDIP)inhibits Wnt signaling by promoting TCF4 degradation and disrupting the TCF4/β-catenin complex[J].Cell Signal,2010,22(11):1 753-1 760.
[21]HOSSEIN G,KHANMOHAMMADI M,SAHRANAVARD FARD P,et al.Exogenous secreted frizzled-related protein-4 modulates steroidogenesis of rat granulosa cells through Wnt/β-catenin and PI3K/AKT signaling pathways[J].Avicenna J Med Biotechnol,2016,8(4):159-168.
[22]AYDOS A,GUREL A,OZTEMUR ISLAKOGLU Y,et al.Identification of polycystic ovary syndrome(PCOS)specific genes in cumulus and mural granulosa cells[J].PLo S One,2016,11(12):e0168875.
[2]ROMEREIM S M,SUMMERS A F,POHLMEIER W E,et al.Transcriptomes of bovine ovarian follicular and luteal cells[J].Data Brief,2016,10:335-339.
[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,MENG J,ZHU Z,et al.Detection of genes associated with follicle development through tran-scriptome analysis of bovine ovarian follicles GCs[J].Curr Bioinform,2018,13(2):127-140.
[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):1 961-1 966.
[6]LI P,YU X,XIE J,et al.Expression of cocaine-and amphetamine-regulated transcript(CART)in hen ovary[J].Biol Res,2017,50(1):18.
[7]李鹏飞,孟金柱,于雪静,等.外源性dsRNA对蛋鸡卵泡基因表达及发育的影响[J].畜牧兽医学报,2016,47(3):515-520.
[8]HUANG Y,YAO X L,MENG J Z,et al.Intrafollicular expression and potential regulatory role of cocaine-and amphetamineregulated transcript in the ovine ovary[J].Domest Anim Endocrinol,2016,54(1):30-36.
[9]AUDIC S,CLAVERIE J M.The significance of digital gene expression profiles[J].Genome Res,1997,7(10):986-995.
[10]SUN X,ZHOU S,MENG F,et al.De novo assembly and characterization of the garlic(Allium sativum)bud transcriptome by Illumina sequencing[J].Plant Cell Rep,2012,31(10):1 823-1 828.
[11]DIAS F C,KHAN M I,SIRARD M A,et al.Differential gene expression of granulosa cells after ovarian superstimulation in beef cattle[J].Reproduction,2013,146(2):181-191.
[12]MIAO X,QIN Q L.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.
[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.
[14]FALCONI G,FABIANI E,FIANCHI L,et al.Impairment of PI3K/AKT and WNT/β-catenin pathways in bone marrow mesenchymal stem cells isolated from patients with myelodysplastic syndromes[J].Exp Hematol,2016,44(1):75-83..
[15]MA X,HAYES E,BISWAS A,et al.Androgens regulate ovarian gene expression through modulation of Ezh2 expression and activity[J].Endocrinology,2017,158(9):2 944-2 954.
[16]SHEN M,JIANG Y,GUAN Z,et al.Protective mechanism of FSH against oxidative damage in mouse ovarian granulosa cells by repressing autophagy[J].Autophagy,2017,13(8):1 364-1 385
[17]HUANG Z,WANG W,MA J,et al.m TOR signaling pathway differently regulates central and peripheral axon regeneration[J].Acta Biochim Biophys Sin(Shanghai),2017,28:1-7.
[18]BEHRAM KANDEMIR Y,AYDIN C,GORGISEN G.The effects of melatonin on oxidative stress and prevention of primordial follicle loss via activation of m TOR pathway in the rat ovary[J].Cell Mol Biol(Noisy-le-grand),2017,63(2):100-106.
[19]张毅敏,于斌,陈佳,等.针刺治疗对卵巢早衰大鼠PI3K/Akt/m TOR信号通路的影响[J].中国针灸,2015,35(1):53-58.
[20]ZHANG H,ZHANG H,ZHANG Y,et al.Dishevelled-DEP domain interacting protein(DDIP)inhibits Wnt signaling by promoting TCF4 degradation and disrupting the TCF4/β-catenin complex[J].Cell Signal,2010,22(11):1 753-1 760.
[21]HOSSEIN G,KHANMOHAMMADI M,SAHRANAVARD FARD P,et al.Exogenous secreted frizzled-related protein-4 modulates steroidogenesis of rat granulosa cells through Wnt/β-catenin and PI3K/AKT signaling pathways[J].Avicenna J Med Biotechnol,2016,8(4):159-168.
[22]AYDOS A,GUREL A,OZTEMUR ISLAKOGLU Y,et al.Identification of polycystic ovary syndrome(PCOS)specific genes in cumulus and mural granulosa cells[J].PLo S One,2016,11(12):e0168875.