基于转录组测序技术筛选花■卵巢发育相关基因
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摘要
为筛选影响花■卵巢发育相关的基因,采用Illumina Hiseq技术对花■脑、卵巢和肝脏组织进行高通量转录组测序。结果显示,3个组织分别产生了49 484 132、47 540 538和50 622 304个clean reads,组装后共获得了99 878个unigenes,平均长度为1 430 bp。DE seq分析发现,在脑vs.卵巢组中特异性表达的基因数为2 305个,脑vs.肝脏组中特异性表达的基因数为839个,卵巢vs.肝脏组中特异性表达的基因数为1 474个,3个比较组共有的差异表达基因数为860个。GO分析发现,上述差异表达基因主要集中在初级代谢过程(primary metabolic process)、单细胞过程(single-organism process)、有机物代谢过程(organic substance metabolic process)等生物学过程中。KEGG pathway分析显示,一些与卵巢发育和减数分裂相关的信号通路得到了显著富集,如GnRH信号通路、类固醇激素合成、TGF-β信号通路、卵母细胞减数分裂等代谢通路。本研究结果丰富了花■的基因资源,可为花■的繁殖生物学研究提供基础数据。
In order to identify candidate genes involved in ovary development, Illumina Hiseq technology was used to define the transcriptome of spotted steed brain, ovary and liver. A total of 49 484 132, 47 540 538 and 50 622 304 clean reads were generated from brain, ovary and liver, and then assembled into 99 878 Unigenes with an average length of 1 430 bp. DEseq analysis revealed that 2 305 were found only in the expression of brain vs. ovary group,839 were found only in the expression of brain vs. liver group, 1 474 were found only in the expression of ovary vs. liver group, and 860 genes were found to be co-expressed in the three comparison groups. Gene ontology analysis indicated that many of these genes encoded proteins involved in primary metabolic process, singleorganism process, organic substance metabolic process. Based on KEGG pathway analysis, putative genes are novel candidate regulators of oocyte development and meiosis, such as GnRH signaling pathway, Steroid hormone biosynthesis, TGF-beta signaling pathway and Oocyte meiosis. The spotted steed transcriptome data reported here will provide valuable new data to increase genomic resources of this species. The data will also provide a valuable basis for further reproductive molecular biology studies of this commercial species.
In order to identify candidate genes involved in ovary development, Illumina Hiseq technology was used to define the transcriptome of spotted steed brain, ovary and liver. A total of 49 484 132, 47 540 538 and 50 622 304 clean reads were generated from brain, ovary and liver, and then assembled into 99 878 Unigenes with an average length of 1 430 bp. DEseq analysis revealed that 2 305 were found only in the expression of brain vs. ovary group,839 were found only in the expression of brain vs. liver group, 1 474 were found only in the expression of ovary vs. liver group, and 860 genes were found to be co-expressed in the three comparison groups. Gene ontology analysis indicated that many of these genes encoded proteins involved in primary metabolic process, singleorganism process, organic substance metabolic process. Based on KEGG pathway analysis, putative genes are novel candidate regulators of oocyte development and meiosis, such as GnRH signaling pathway, Steroid hormone biosynthesis, TGF-beta signaling pathway and Oocyte meiosis. The spotted steed transcriptome data reported here will provide valuable new data to increase genomic resources of this species. The data will also provide a valuable basis for further reproductive molecular biology studies of this commercial species.
引文
[1]Colli-Dula R C,Martyniuk C J,Kroll K J,et al.Dietary exposure of 17-alpha ethinylestradiol modulates physiological endpoints and gene signaling pathways in female largemouth bass(Micropterus salmoides)[J].Aquatic Toxicology,2014,156:148-160.
[2]Guiguen Y,Fostier A,Piferrer F,et al.Ovarian aromatase and estrogens:a pivotal role for gonadal sex differentiation and sex change in fish[J].General and Comparative Endocrinology,2010,165(3):352-366.
[3]Hu Q M,Zhu Y,Liu Y,et al.Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2014,4:7167.
[4]Chen J H,Li Y L,Zhang J B,et al.Identification and expression analysis of two Wnt4 genes in the spotted scat(Scatophagus argus)[J].Electronic Journal of Biotechnology,2016,20:20-27.
[5]Yang Y J,Wang Y,Li Z,et al.Sequential,divergent,and cooperative requirements of Foxl2a and Foxl2b in ovary development and maintenance of zebrafish[J].Genetics,2017,205(4):1551-1572.
[6]Zhou L Y,Charkraborty T,Yu X G,et al.R-spondins are involved in the ovarian differentiation in a teleost,medaka(Oryzias latipes)[J].BMC Developmental Biology,2012,12:36.
[7]Wu L M,Yang P,Luo F,et al.R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts,nile tilapia(Oreochromis niloticus)[J].General and Comparative Endocrinology,2016,230-231:177-185.
[8]Ayers K L,Lambeth L S,Davidson N M,et al.Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-Seq[J].BMCGenomics,2015,16(1):1-19.
[9]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.
[10]Gardner L D,Jayasundara N,Castilho P C,et al.Microarray gene expression profiles from mature gonad tissues of Atlantic bluefin tuna,Thunnus thynnus in the Gulf of Mexico[J].BMC Genomics,2012,13:530.
[11]Tao W J,Yuan J,Zhou L Y,et al.Characterization of gonadal transcriptomes from nile tilapia(Oreochromis niloticus)reveals differentially expressed genes[J].PLoSOne,2013,8(5):e63604.
[12]Ma D Y,Ma A J,Huang Z H,et al.Transcriptome analysis for identification of genes related to gonad differentiation,growth,immune response and marker discovery in the turbot(Scophthalmus maximus)[J].PLoS One,2016,11(2):e0149414.
[13]Yu L Y,Xu D D,Ye H,et al.Gonadal transcriptome analysis of Pacific abalone Haliotis discus discus:identification of genes involved in germ cell development[J].Marine Biotechnology,2018,20(4):467-480.
[14]顾若波,闻海波,徐钢春.花■的肌肉营养成分与品质评价[J].大连水产学院学报,2006,21(4):378-382.Gu R B,Wen H B,Xu G C.Evaluation of nutritive quality and nutritional components in Hemibarbus maculates Bleeker muscle[J].Journal of Dalian Fisheries University,2006,21(4):378-382(in Chinese).
[15]陆红法,李智强,方美娟,等.花■的生物学特性和人工繁殖初步研究[J].浙江海洋学院学报(自然科学版),2004,23(3):211-213.Lu H F,Li Z Q,Fang M J,et al.A primary study on the biological characteristics and artificial breeding of Hemibarbus maculatus Bleeker in pond[J].Journal of Zhejiang Ocean University(Natural Science),2004,23(3):211-213(in Chinese).
[16]庹云,刘小高,郭冬生,等.袁河花■驯化及人工繁殖试验[J].宜春学院学报,2013,35(3):111-113.Tuo Y,Liu X G,Guo D S,et al.Study on domestication and artificial propagation of Hemibarbus maculates in Yuanhe River[J].Journal of Yichun College,2013,35(3):111-113(in Chinese).
[17]顾若波,闻海波,徐钢春,等.花■胚胎发育及卵黄囊仔鱼形态的初步观察[J].浙江海洋学院学报(自然科学版),2006,25(4):373-378.Gu R B,Wen H B,Xu G C,et al.Preliminary observation on the embryonic development and mmorphology of the yYolk-sac larva of Hemibarbus maculates Bleeker[J].Journal of Zhejiang Ocean University(Natural Science),2006,25(4):373-378(in Chinese).
[18]汪若彭.丰溪河花■繁殖生物学研究[D].武汉:华中农业大学,2008.Wang R P.Study on reproductive biology of Hemibarbus maculates Bleeker in the Fengxi river[D].Wuhan:Huazhong Agricultural University,2008(in Chinese).
[19]Mode A,Gustafsson J?.Sex and the liver-a journey through five decades[J].Drug Metabolism Reviews,2006,38(1-2):197-207.
[20]Grabherr M G,Haas B J,Yassour M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[21]林植华,雷焕宗,林植云,等.花■的两性异形和雌体繁殖输出[J].上海交通大学学报(农业科学版),2005,23(3):284-288.Lin Z H,Lei H Z,Lin Z Y,et al.Sexual dimorphism and female reproductive output of Hemibarbus maculates[J].Journal of Shanghai Jiaotong University(Agricultural Science),2005,23(3):284-288(in Chinese).
[22]毛媛媛,郑荣泉,张启鹏,等.转录组测序研究三角帆蚌珍珠颜色相关基因[J].水产学报,2018,42(3):322-333.Mao Y Y,Zheng R Q,Zhang Q P,et al.Transcriptome sequencing analysis on pearl color related genes in Hyriopsis cumingii[J].Journal of Fisheries of China,2018,42(3):322-333(in Chinese).
[23]Lovejoy D A,Michalec O M,Hogg D W,et al.Role of elasmobranchs and holocephalans in understanding peptide evolution in the vertebrates:lessons learned from gonadotropin releasing hormone(GnRH)and corticotropin releasing factor(CRF)phylogenies[J].General and Comparative Endocrinology,2018,264:78-83.
[24]Nagahama Y,Yamashita M.Regulation of oocyte maturation in fish[J].Development,Growth&Differentiation,2008,50(suppl 1):S195-S219.
[25]Tokarz J,M?ller G,de Angelis M H,et al.Steroids in teleost fishes:a functional point of view[J].Steroids,2015,103:123-144.
[26]Halm S,Iba?ez A J,Tyler C R,et al.Molecular characterisation of growth differentiation factor 9(gdf9)and bone morphogenetic protein 15(bmp15)and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass[J].Molecular and Cellular Endocrinology,2008,291(1-2):95-103.
[27]Patnaik S,Mohanty M,Bit A,et al.Molecular characterization of activin receptor type IIA and its expression during gonadal maturation and growth stages in rohu carp[J].Comparative Biochemistry and Physiology Part B-Biochemistry and Molecular Biology,2017,203:1-10.
[28]Wang Z P,Mu X Y,Guo M,et al.Transforming growth factor-βsignaling participates in the maintenance of the primordial follicle pool in the mouse ovary[J].Journal of Biological Chemistry,2014,289(12):8299-8311.
[2]Guiguen Y,Fostier A,Piferrer F,et al.Ovarian aromatase and estrogens:a pivotal role for gonadal sex differentiation and sex change in fish[J].General and Comparative Endocrinology,2010,165(3):352-366.
[3]Hu Q M,Zhu Y,Liu Y,et al.Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole(Cynoglossus semilaevis)[J].Scientific Reports,2014,4:7167.
[4]Chen J H,Li Y L,Zhang J B,et al.Identification and expression analysis of two Wnt4 genes in the spotted scat(Scatophagus argus)[J].Electronic Journal of Biotechnology,2016,20:20-27.
[5]Yang Y J,Wang Y,Li Z,et al.Sequential,divergent,and cooperative requirements of Foxl2a and Foxl2b in ovary development and maintenance of zebrafish[J].Genetics,2017,205(4):1551-1572.
[6]Zhou L Y,Charkraborty T,Yu X G,et al.R-spondins are involved in the ovarian differentiation in a teleost,medaka(Oryzias latipes)[J].BMC Developmental Biology,2012,12:36.
[7]Wu L M,Yang P,Luo F,et al.R-spondin1 signaling pathway is required for both the ovarian and testicular development in a teleosts,nile tilapia(Oreochromis niloticus)[J].General and Comparative Endocrinology,2016,230-231:177-185.
[8]Ayers K L,Lambeth L S,Davidson N M,et al.Identification of candidate gonadal sex differentiation genes in the chicken embryo using RNA-Seq[J].BMCGenomics,2015,16(1):1-19.
[9]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.
[10]Gardner L D,Jayasundara N,Castilho P C,et al.Microarray gene expression profiles from mature gonad tissues of Atlantic bluefin tuna,Thunnus thynnus in the Gulf of Mexico[J].BMC Genomics,2012,13:530.
[11]Tao W J,Yuan J,Zhou L Y,et al.Characterization of gonadal transcriptomes from nile tilapia(Oreochromis niloticus)reveals differentially expressed genes[J].PLoSOne,2013,8(5):e63604.
[12]Ma D Y,Ma A J,Huang Z H,et al.Transcriptome analysis for identification of genes related to gonad differentiation,growth,immune response and marker discovery in the turbot(Scophthalmus maximus)[J].PLoS One,2016,11(2):e0149414.
[13]Yu L Y,Xu D D,Ye H,et al.Gonadal transcriptome analysis of Pacific abalone Haliotis discus discus:identification of genes involved in germ cell development[J].Marine Biotechnology,2018,20(4):467-480.
[14]顾若波,闻海波,徐钢春.花■的肌肉营养成分与品质评价[J].大连水产学院学报,2006,21(4):378-382.Gu R B,Wen H B,Xu G C.Evaluation of nutritive quality and nutritional components in Hemibarbus maculates Bleeker muscle[J].Journal of Dalian Fisheries University,2006,21(4):378-382(in Chinese).
[15]陆红法,李智强,方美娟,等.花■的生物学特性和人工繁殖初步研究[J].浙江海洋学院学报(自然科学版),2004,23(3):211-213.Lu H F,Li Z Q,Fang M J,et al.A primary study on the biological characteristics and artificial breeding of Hemibarbus maculatus Bleeker in pond[J].Journal of Zhejiang Ocean University(Natural Science),2004,23(3):211-213(in Chinese).
[16]庹云,刘小高,郭冬生,等.袁河花■驯化及人工繁殖试验[J].宜春学院学报,2013,35(3):111-113.Tuo Y,Liu X G,Guo D S,et al.Study on domestication and artificial propagation of Hemibarbus maculates in Yuanhe River[J].Journal of Yichun College,2013,35(3):111-113(in Chinese).
[17]顾若波,闻海波,徐钢春,等.花■胚胎发育及卵黄囊仔鱼形态的初步观察[J].浙江海洋学院学报(自然科学版),2006,25(4):373-378.Gu R B,Wen H B,Xu G C,et al.Preliminary observation on the embryonic development and mmorphology of the yYolk-sac larva of Hemibarbus maculates Bleeker[J].Journal of Zhejiang Ocean University(Natural Science),2006,25(4):373-378(in Chinese).
[18]汪若彭.丰溪河花■繁殖生物学研究[D].武汉:华中农业大学,2008.Wang R P.Study on reproductive biology of Hemibarbus maculates Bleeker in the Fengxi river[D].Wuhan:Huazhong Agricultural University,2008(in Chinese).
[19]Mode A,Gustafsson J?.Sex and the liver-a journey through five decades[J].Drug Metabolism Reviews,2006,38(1-2):197-207.
[20]Grabherr M G,Haas B J,Yassour M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[21]林植华,雷焕宗,林植云,等.花■的两性异形和雌体繁殖输出[J].上海交通大学学报(农业科学版),2005,23(3):284-288.Lin Z H,Lei H Z,Lin Z Y,et al.Sexual dimorphism and female reproductive output of Hemibarbus maculates[J].Journal of Shanghai Jiaotong University(Agricultural Science),2005,23(3):284-288(in Chinese).
[22]毛媛媛,郑荣泉,张启鹏,等.转录组测序研究三角帆蚌珍珠颜色相关基因[J].水产学报,2018,42(3):322-333.Mao Y Y,Zheng R Q,Zhang Q P,et al.Transcriptome sequencing analysis on pearl color related genes in Hyriopsis cumingii[J].Journal of Fisheries of China,2018,42(3):322-333(in Chinese).
[23]Lovejoy D A,Michalec O M,Hogg D W,et al.Role of elasmobranchs and holocephalans in understanding peptide evolution in the vertebrates:lessons learned from gonadotropin releasing hormone(GnRH)and corticotropin releasing factor(CRF)phylogenies[J].General and Comparative Endocrinology,2018,264:78-83.
[24]Nagahama Y,Yamashita M.Regulation of oocyte maturation in fish[J].Development,Growth&Differentiation,2008,50(suppl 1):S195-S219.
[25]Tokarz J,M?ller G,de Angelis M H,et al.Steroids in teleost fishes:a functional point of view[J].Steroids,2015,103:123-144.
[26]Halm S,Iba?ez A J,Tyler C R,et al.Molecular characterisation of growth differentiation factor 9(gdf9)and bone morphogenetic protein 15(bmp15)and their patterns of gene expression during the ovarian reproductive cycle in the European sea bass[J].Molecular and Cellular Endocrinology,2008,291(1-2):95-103.
[27]Patnaik S,Mohanty M,Bit A,et al.Molecular characterization of activin receptor type IIA and its expression during gonadal maturation and growth stages in rohu carp[J].Comparative Biochemistry and Physiology Part B-Biochemistry and Molecular Biology,2017,203:1-10.
[28]Wang Z P,Mu X Y,Guo M,et al.Transforming growth factor-βsignaling participates in the maintenance of the primordial follicle pool in the mouse ovary[J].Journal of Biological Chemistry,2014,289(12):8299-8311.