罗氏沼虾(Macrobrachium rosenbergii)卵巢发育不同时期转录组分析
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摘要
为发掘罗氏沼虾(Macrobrachiumrosenbergii)卵巢发育过程中的重要功能基因,采用IlluminaHiSeqTM4000高通量测序平台对罗氏沼虾卵巢发育四个时期卵巢组织进行转录组测序。所测序列经质控、组装后比对到NR、String、Swiss-prot、Pfam、GO、KEGG数据库中注释,并进行差异基因聚类分析。结果显示,四个样品共产生221580604个Cleanreads,拼接获得95379个Unigenes。分别对四个样品测序文库进行两两比较,共检测到6605个差异表达基因,其中显著上调基因2410个,显著下调基因4195个。GO功能分类显示,共有6422条unigenes可分为分子功能、细胞组分、生物学过程3大类59分支,差异基因主要涉及糖类转运、氨基酸合成、酶活性、细胞膜组成等。通过KEGG通路注释,共有8423条unigenes被注释,涉及184种代谢途径,有7个代谢通路与卵巢发育相关,差异基因的KEGG富集结果显示,药物代谢细胞色素P450通路、氨基丁酸神经元突触、鞘糖脂生物合成、氮素代谢等通路富集显著。此外,对SSR与SNP分子标记进行了鉴定。研究结果为进一步探索罗氏沼虾卵巢发育机制提供了基础信息。
To analyze important functional genes of Macrobrachium rosenbergii during ovarian development, we conducted the transcriptome sequencing by Illumina HiSeqTM 4000. After quality control and assembly, the sequences acquired were blasted against NR, String, Swiss-prot, Pfam, GO and KEGG databases, and then the cluster analysis was performed. In total, 221580604 clean reads and 89529 Unigenes were obtained, and 6605 genes were differentially expressed, among which 2410 were up-regulated and 4195 down-regulated. According to the results of gene ontology functional enrichment analysis, 6422 Unigenes in the transcriptome library could be divided into 3 categories(molecular function, cellular component and biological process) with 59 branches. Significant differently-expressed genes were mainly distributed in the pathways like carbohydrate transport, amino acid synthesis, enzyme activity and the composition of the cell membrane. KEGG pathway analysis showed that 8423 Unigenes were given the pathway annotations and related to 184 pathways, and 7 pathways were associated with ovarian development. Analysis of differentially expressed genes revealed that several pathways, such as Drug metabolism——cytochrome P450, GABAergic synapse, Glycosphingolipid biosynthesis——globo series and Nitrogen metabolism, were significantly enriched. In addition, SSR and SNP molecular markers were identified. The results provide basic information for further exploration of the ovary development mechanism of M. rosenbergii.
To analyze important functional genes of Macrobrachium rosenbergii during ovarian development, we conducted the transcriptome sequencing by Illumina HiSeqTM 4000. After quality control and assembly, the sequences acquired were blasted against NR, String, Swiss-prot, Pfam, GO and KEGG databases, and then the cluster analysis was performed. In total, 221580604 clean reads and 89529 Unigenes were obtained, and 6605 genes were differentially expressed, among which 2410 were up-regulated and 4195 down-regulated. According to the results of gene ontology functional enrichment analysis, 6422 Unigenes in the transcriptome library could be divided into 3 categories(molecular function, cellular component and biological process) with 59 branches. Significant differently-expressed genes were mainly distributed in the pathways like carbohydrate transport, amino acid synthesis, enzyme activity and the composition of the cell membrane. KEGG pathway analysis showed that 8423 Unigenes were given the pathway annotations and related to 184 pathways, and 7 pathways were associated with ovarian development. Analysis of differentially expressed genes revealed that several pathways, such as Drug metabolism——cytochrome P450, GABAergic synapse, Glycosphingolipid biosynthesis——globo series and Nitrogen metabolism, were significantly enriched. In addition, SSR and SNP molecular markers were identified. The results provide basic information for further exploration of the ovary development mechanism of M. rosenbergii.
引文
王跃,毛开云,王恒哲等,2017.转录组学测序技术应用与市场分析.生物产业技术,(5):11-17
邓泱泱,荔建琦,吴松锋等,2006.nr数据库分析及其本地化.计算机工程,32(5):71-73,76
刘金磊,邓思平,江东能等,2018.ERR-dsRNA对罗氏沼虾卵巢中ERR及生殖相关基因表达的影响.广东海洋大学学报,38(3):8-16
吴维福,陈娈娈,李郁娇等,2014.三丁基锡对罗氏沼虾血清中免疫酶活力的影响.广东海洋大学学报,34(3):17-21
张艳,1999.罗氏沼虾卵巢发育的内分泌调控.广州:中山大学硕士学位论文
陆洋,吴旭干,柳梅梅等,2018.雌激素拮抗剂对三疣梭子蟹卵巢发育及相关基因表达的影响.水产学报,42(5):653-662
陈娈娈,2014.壬基酚在罗氏沼虾体内代谢及其对性腺发育相关基因表达的影响.湛江:广东海洋大学硕士学位论文
陈雪峰,杨国梁,孔杰等,2012.人工养殖与选育对罗氏沼虾遗传多样性的影响.水生生物学报,36(5):866-873
周俊,周发林,杨丽诗等,2012.斑节对虾cdc25基因的表达分析.广东农业科学,39(12):148-150
房君江,2008.中华绒螯蟹、罗氏沼虾CyclinB基因cDNA克隆及其在卵子发生过程中表达分析.上海:上海海洋大学硕士学位论文
赵维信,魏华,汪志强等,1995.人工诱导罗氏沼虾同步产卵与卵巢组织学研究.水产学报,19(4):289-296
柳军涛,2017.基于高通量RNA-seq数据转录组拼接的算法研究.济南:山东大学博士学位论文
姚泊,1991.不同温泉水水温对罗氏沼虾卵巢发育的影响.广州师院学报(自然科学版),(1):68-72
贾复龙,孟宪亮,刘萍等,2016.三疣梭子蟹细胞周期蛋白H基因克隆及其在卵巢发育中的表达分析.中国海洋大学学报,46(7):62-69
郭松,傅明骏,赵超等,2014.斑节对虾CDK8基因的克隆表达和原核表达及蛋白纯化.见:2014年中国水产学会学术年会论文摘要集.长沙:中国水产学会
谢波波,2017.斑节对虾卵巢发育过程中RBL和下游Chk1基因的生物学功能分析.上海:上海海洋大学硕士学位论文
黎东,2013.Cu2+、Zn2+对罗氏沼虾生长以及卵巢发育的影响.湛江:广东海洋大学硕士学位论文
颜素芬,姜永华,2004.南美白对虾卵巢结构及发育的组织学研究.海洋湖沼通报,(2):52-58
Apweiler R,Bairoch A,Wu C H et al,2004.UniProt:the universal protein knowledgebase.Nucleic Acids Research,32(S1):D115-D119
Ashburner M,Ball C A,Blake J A et al,2000.Gene ontology:tool for the unification of biology.Nature Genetics,25(1):25-29
Chen J,Liu P,Li Z et al,2013.The cloning of the cdk2 transcript and the localization of its expression during gametogenesis in the freshwater giant prawn,Macrobrachium rosenbergii.Molecular Biology Reports,40(8):4781-4790
Eddy S R,1998.Profile hidden Markov models.Bioinformatics,14(9):755-763
Jung H,Yoon B H,Kim W J et al,2016.Optimizing hybrid de novo transcriptome assembly and extending genomic resources for giant freshwater prawns(Macrobrachium rosenbergii):the identification of genes and markers associated with reproduction.International Journal of Molecular Sciences,17(5):690
Kanehisa M,Goto S,Kawashima S et al,2004.The KEGGresource for deciphering the genome.Nucleic Acids Research,32(S1):D277-D280
Langmead B,Trapnell C,Pop M et al,2009.Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biology,10(3):R25
Liao X L,Cheng L,Xu P et al,2013.Transcriptome analysis of crucian carp(Carassius auratus),an important aquaculture and hypoxia-tolerant species.PLoS One,8(4):e62308
Rao R M,Zhu Y B,Alinejad T et al,2015.RNA-seq analysis of Macrobrachium rosenbergii hepatopancreas in response to Vibrio parahaemolyticus infection.Gut Pathogens,7(1):6
Thanh N M,Ponzoni R W,Nguyen N H et al,2009.Evaluation of growth performance in a diallel cross of three strains of giant freshwater prawn(Macrobrachium rosenbergii)in Vietnam.Aquaculture,287(1-2):75-83
邓泱泱,荔建琦,吴松锋等,2006.nr数据库分析及其本地化.计算机工程,32(5):71-73,76
刘金磊,邓思平,江东能等,2018.ERR-dsRNA对罗氏沼虾卵巢中ERR及生殖相关基因表达的影响.广东海洋大学学报,38(3):8-16
吴维福,陈娈娈,李郁娇等,2014.三丁基锡对罗氏沼虾血清中免疫酶活力的影响.广东海洋大学学报,34(3):17-21
张艳,1999.罗氏沼虾卵巢发育的内分泌调控.广州:中山大学硕士学位论文
陆洋,吴旭干,柳梅梅等,2018.雌激素拮抗剂对三疣梭子蟹卵巢发育及相关基因表达的影响.水产学报,42(5):653-662
陈娈娈,2014.壬基酚在罗氏沼虾体内代谢及其对性腺发育相关基因表达的影响.湛江:广东海洋大学硕士学位论文
陈雪峰,杨国梁,孔杰等,2012.人工养殖与选育对罗氏沼虾遗传多样性的影响.水生生物学报,36(5):866-873
周俊,周发林,杨丽诗等,2012.斑节对虾cdc25基因的表达分析.广东农业科学,39(12):148-150
房君江,2008.中华绒螯蟹、罗氏沼虾CyclinB基因cDNA克隆及其在卵子发生过程中表达分析.上海:上海海洋大学硕士学位论文
赵维信,魏华,汪志强等,1995.人工诱导罗氏沼虾同步产卵与卵巢组织学研究.水产学报,19(4):289-296
柳军涛,2017.基于高通量RNA-seq数据转录组拼接的算法研究.济南:山东大学博士学位论文
姚泊,1991.不同温泉水水温对罗氏沼虾卵巢发育的影响.广州师院学报(自然科学版),(1):68-72
贾复龙,孟宪亮,刘萍等,2016.三疣梭子蟹细胞周期蛋白H基因克隆及其在卵巢发育中的表达分析.中国海洋大学学报,46(7):62-69
郭松,傅明骏,赵超等,2014.斑节对虾CDK8基因的克隆表达和原核表达及蛋白纯化.见:2014年中国水产学会学术年会论文摘要集.长沙:中国水产学会
谢波波,2017.斑节对虾卵巢发育过程中RBL和下游Chk1基因的生物学功能分析.上海:上海海洋大学硕士学位论文
黎东,2013.Cu2+、Zn2+对罗氏沼虾生长以及卵巢发育的影响.湛江:广东海洋大学硕士学位论文
颜素芬,姜永华,2004.南美白对虾卵巢结构及发育的组织学研究.海洋湖沼通报,(2):52-58
Apweiler R,Bairoch A,Wu C H et al,2004.UniProt:the universal protein knowledgebase.Nucleic Acids Research,32(S1):D115-D119
Ashburner M,Ball C A,Blake J A et al,2000.Gene ontology:tool for the unification of biology.Nature Genetics,25(1):25-29
Chen J,Liu P,Li Z et al,2013.The cloning of the cdk2 transcript and the localization of its expression during gametogenesis in the freshwater giant prawn,Macrobrachium rosenbergii.Molecular Biology Reports,40(8):4781-4790
Eddy S R,1998.Profile hidden Markov models.Bioinformatics,14(9):755-763
Jung H,Yoon B H,Kim W J et al,2016.Optimizing hybrid de novo transcriptome assembly and extending genomic resources for giant freshwater prawns(Macrobrachium rosenbergii):the identification of genes and markers associated with reproduction.International Journal of Molecular Sciences,17(5):690
Kanehisa M,Goto S,Kawashima S et al,2004.The KEGGresource for deciphering the genome.Nucleic Acids Research,32(S1):D277-D280
Langmead B,Trapnell C,Pop M et al,2009.Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.Genome Biology,10(3):R25
Liao X L,Cheng L,Xu P et al,2013.Transcriptome analysis of crucian carp(Carassius auratus),an important aquaculture and hypoxia-tolerant species.PLoS One,8(4):e62308
Rao R M,Zhu Y B,Alinejad T et al,2015.RNA-seq analysis of Macrobrachium rosenbergii hepatopancreas in response to Vibrio parahaemolyticus infection.Gut Pathogens,7(1):6
Thanh N M,Ponzoni R W,Nguyen N H et al,2009.Evaluation of growth performance in a diallel cross of three strains of giant freshwater prawn(Macrobrachium rosenbergii)in Vietnam.Aquaculture,287(1-2):75-83
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