肺形侧耳低温胁迫时期的转录组分析
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摘要
本文以肺形侧耳栽培菌株X57为研究对象,利用高通量RNA测序技术对4℃低温处理0h、6h和12h后的肺形侧耳进行基因表达分析来探讨肺形侧耳低温应答机制。分析结果筛选出低温处理6h差异表达基因742个,其中上调表达基因374个,下调表达基因368个;低温处理12h差异表达基因1 489个,其中上调表达基因占53%,下调表达基因占47%。Gene Ontology(GO)功能聚类分析表明,差异表达基因主要富集在结合、催化分子功能组和代谢过程、细胞过程生物学过程中。KEGG功能富集分析结果显示,差异基因主要富集在氨基酸、核糖体和类固醇生物合成,及氮类物质代谢的通路上,富集到与低温胁迫相关通路MAPKsignalingpathway-yeast上的基因表达随低温处理时间的增加呈上调趋势。已报道HOG-MAPK通路是MAPK途径研究较为明确、信号传递单一的真菌低温胁迫中重要的通路,本文运用生物信息学软件构建肺形侧耳的HOG-MAPK通路,并利用荧光定量PCR对相关基因表达进行验证,结果显示以低温处理0h为参照,随着低温胁迫时间增加通路上大部分基因的表达量持续上升,且差异显著,与RNA-Seq分析结果一致。本文通过全面分析肺形侧耳低温胁迫时期基因表达情况,为进一步研究肺形侧耳低温胁迫相关重要基因的功能鉴定与信号通路调控机理提供基础。
Gene expression at low temperature(4°C) of Pleurotus pulmonarius strain X57 was investigated by RNA-seq technologyto explore the putative mechanism of low temperature response of P. pulmonarius. There were 742 differentially expressedgenes after treatment at low temperature for 6 h, of which 374 were up-regulated and 368 were down-regulated. After beingtreated for 12 h, a total of 1 489 genes were differentially expressed at low temperature, among which 53% were up-regulatedand 47% down-regulated. The Gene Ontology classification showed that the differentially expressed genes were mainly involvedin binding, catalytic activity molecular functions and metabolic process, cellular process and biological pathway. The KEGGpathway analysis has also revealed that the differential expressed genes are mainly related to biosynthesis of amino acids,ribosomes and steroids, and nitrogen metabolism. The results also showed that genes expression related to MAPK signalingpathway was increased with the increase of low temperature treatment duration. It has been reported that the HOG-MAPK is animportant pathway for MAPK pathway study in low temperature stress of fungi, which was more explicit and single in signaltransmission. Bioinformatics software was used to construct HOG-MAPK pathway in P. pulmonarius, and the expression ofrelated genes was verified by RT-qPCR. The results showed that with reference to cold stress of 0 h, the expression of most genesin the pathway increased continuously with the increase of cold shock duration, and the difference was significant and wasconsistent with RNA-Seq analysis. A comprehensive analysis of genes expression in the cold stress period of P. pulmonariusprovides a basis for further study on the functional identification of the important genes related to the cold stress and themechanism of cold signal pathway regulation in P. pulmonarius.
Gene expression at low temperature(4°C) of Pleurotus pulmonarius strain X57 was investigated by RNA-seq technologyto explore the putative mechanism of low temperature response of P. pulmonarius. There were 742 differentially expressedgenes after treatment at low temperature for 6 h, of which 374 were up-regulated and 368 were down-regulated. After beingtreated for 12 h, a total of 1 489 genes were differentially expressed at low temperature, among which 53% were up-regulatedand 47% down-regulated. The Gene Ontology classification showed that the differentially expressed genes were mainly involvedin binding, catalytic activity molecular functions and metabolic process, cellular process and biological pathway. The KEGGpathway analysis has also revealed that the differential expressed genes are mainly related to biosynthesis of amino acids,ribosomes and steroids, and nitrogen metabolism. The results also showed that genes expression related to MAPK signalingpathway was increased with the increase of low temperature treatment duration. It has been reported that the HOG-MAPK is animportant pathway for MAPK pathway study in low temperature stress of fungi, which was more explicit and single in signaltransmission. Bioinformatics software was used to construct HOG-MAPK pathway in P. pulmonarius, and the expression ofrelated genes was verified by RT-qPCR. The results showed that with reference to cold stress of 0 h, the expression of most genesin the pathway increased continuously with the increase of cold shock duration, and the difference was significant and wasconsistent with RNA-Seq analysis. A comprehensive analysis of genes expression in the cold stress period of P. pulmonariusprovides a basis for further study on the functional identification of the important genes related to the cold stress and themechanism of cold signal pathway regulation in P. pulmonarius.
引文
Aguilera J,Randez-Gil F,Prieto JA,2007.Cold response in Saccharomyces cerevisiae:new functions for old mechanisms.FEMS Microbiology Reviews,31(3):327-341
Bahn YS,Xue C,Idnurm A,Rutherford JC,Heitman J,Cardenas ME,2007.Sensing the environment:lessons from fungi.Nature Reviews Microbiology,5(1):57-69
Ballester M,Codon R,Folch JM,2013.DAG expression:high-throughput gene expression analysis of realtime PCR date using stand curves for relative quantification.PLoS One,8(11):e80385
Feng ZY,Mi SF,Chen MJ,Zhao MW,Li JH,Pan YJ,2006.Differences of gene expression in mycelia of Lentinula edodes under cold stress.Chinese Journal of Applied and Environmental Biology,12(5):614-617(in Chinese)
Feng ZY,Mi SF,Zhao MW,Chen MJ,Li JH,Tan Q,Pan YJ,2006.Cloning and expression pattern of a cold stress-responding gene from Lentinula edodes.Journal of Agricultural Biotechnology,14(3):614-617(in Chinese)
Fu JS,2007.Study on cross breeding and establishment of molecular genetic markers of straw mushroom.Master thesis,Fujian Agriculture and Forestry University,Fuzhou.1-55(in Chinese)
Hayashi M,Maeda T,2006.Activation of the HOG pathway upon cold stress in Saccharomyces cerevisiae.Journal of Biochemistry,139(4):797-803
Li QJ,2016.Study on related genes of cold-induced primordium formation of Flammulina velutipes.Master Thesis,South China Agricultural University,Guangzhou.1-94(in Chinese)
Pertea M,Kim D,Pertea GM,Leek JT,Salzberg SL,2016.Transcript-level expression analysis of RNA-seq experiments with HISAT,StringTie and Ballgown.Nature Protocols,11(9):1650-1667
Panadero J,Pallotti C,Rodriguez-Vargas S,Randez-Gil F,Prieto JA,2006.A downshift in temperature activates the high osmolarity glycerol(HOG)pathway,which determines freeze tolerance in Saccharomyces cerevisiae.Journal of Biological Chemistry,281(8):4638-4645
Shen YY,Gu M,Cai WM,Jin QL,Fan LJ,Feng WL,Song TT,Tian FF,2014.Construction and analysis of a suppressive subtractive hybridisation cDNA library related to fruiting body formation of Pleurotus pulmonarius after cold stimulation.Acta Agriculturae Zhejiangensis,26(2):330-334(in Chinese)
Sinclair BJ,Stinziano JR,Williams CM,MacMillan HA,Marshall KE,2013.Real-time measurement of metabolic rate during freezing and thawing of the wood frog,Rana sylvatica:implications for overwinter energy use.The Journal of Experimental Biology,216(Pt2):292-302
Soto T,Beltran FF,Paredes V,Madrid M,Millar JBA,Soler JV,Cansado J,Gacto M,2002.Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe.European Journal of Biochemistry,269(20):5056-5065
Teige M,Scheikl E,Eulgem T,Doczi R,Ichimura K,Shinozaki K,Dangl JL,Hirt H,2004.The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.Molecular Cell,15(1):141-152
Tereshina VM,Memorskaia AS,2005.Adaptation of Flammulina velutipes to hypothermia in natural environment:the role of lipids and carbohydrates.Mikrobiologiia,74(3):329-334
Wang H,Bao DP,Chen MJ,Chen H,Feng AP,2010.Cloning and sequence analysis of mitogen-activated protein kinase homolog from Volvariella volvacea(VV-MAPKKK).Microbiology China,37(11):1606-1609(in Chinese)
Wang P,Fan ZQ,Fan RQ,2015.Xianglian external lotion restored the sensitivity of drug-resistant Candida albicans strains to fluconazole:a tran-scriptomics study.Chinese Journal of Integrated Traditional and Western Medicine,35(12):1505-1509(in Chinese)
Wang Z,Gerstein M,Snyder M,2009.RNA-Seq:a revolutionary tool for transcriptomics.Nature Reviews Genetics,10(1):57-63
Wu XC,Hu SJ,Qian KX,2005.HOG-MAPK pathway in yeast.Chinese Journal of Cell Biology,27(3):247-252(in Chinese)
Xiang L,Li Y,Zhu Y,Luo HM,Li CF,Xu XL,Sun C,Song JY,Shi LC,He L,Sun W,Chen SL,2014.Transcriptome analysis of the Ophiocordyceps sinensis fruiting body reveals putative genes involved in fruiting body development and cordycepin biosynthesis.Genomics,103(1):154-159
Zerbino DR,Birney E,2008.Velvet:algorithms for de novo short read assembly using de Bruijn graphs.Genome Research,18(5):821-829
Zhang ZC,Yao YM,Mao ZL,Sun GS,Qin WB,Dai ZL,2018.Transcriptome analysis and gene function annotation of early developmental broccoli microspores based on high-throughput sequencing technology.Acta Agriculturae Nucleatae Sinica,32(5):848-855
冯志勇,米朔甫,陈明杰,赵明文,李军辉,潘迎捷,2006.低温胁迫下香菇基因表达差异研究.应用与环境生物学报,12(5):614-617
冯志勇,米朔甫,赵明文,陈明杰,李军辉,谭琦,潘迎捷,2006.香菇低温胁迫应答基因的克隆及其表达分析.农业生物技术学报,14(3):381-386
傅俊生,2007.草菇杂交育种研究及其分子遗传标记的建立.福建农林大学硕士论文,福州.1-55
李琼洁,2016.冷诱导金针菇原基形成的相关基因研究.华南农业大学硕士论文,广州.1-94
沈颖越,顾敏,蔡为明,金群力,范丽军,冯伟林,宋婷婷,田芳芳,2014.秀珍菇子实体发育相关消减杂交cDNA文库的构建与分析.浙江农业学报,26(2):330-334
王平,樊志奇,范瑞强,2015.香莲外洗液逆转白念珠菌耐药的转录组学研究.中国中西医结合杂志,35(12):1505-1509
汪虹,鲍大鹏,陈明杰,陈辉,冯爱萍,2010.草菇MAPKKK同源基因的克隆及序列分析.微生物学通报,37(11):1606-1609
吴雪昌,胡森杰,钱凯先,2005.酵母HOG-MAPK途径.细胞生物学杂志,7:247-252
Bahn YS,Xue C,Idnurm A,Rutherford JC,Heitman J,Cardenas ME,2007.Sensing the environment:lessons from fungi.Nature Reviews Microbiology,5(1):57-69
Ballester M,Codon R,Folch JM,2013.DAG expression:high-throughput gene expression analysis of realtime PCR date using stand curves for relative quantification.PLoS One,8(11):e80385
Feng ZY,Mi SF,Chen MJ,Zhao MW,Li JH,Pan YJ,2006.Differences of gene expression in mycelia of Lentinula edodes under cold stress.Chinese Journal of Applied and Environmental Biology,12(5):614-617(in Chinese)
Feng ZY,Mi SF,Zhao MW,Chen MJ,Li JH,Tan Q,Pan YJ,2006.Cloning and expression pattern of a cold stress-responding gene from Lentinula edodes.Journal of Agricultural Biotechnology,14(3):614-617(in Chinese)
Fu JS,2007.Study on cross breeding and establishment of molecular genetic markers of straw mushroom.Master thesis,Fujian Agriculture and Forestry University,Fuzhou.1-55(in Chinese)
Hayashi M,Maeda T,2006.Activation of the HOG pathway upon cold stress in Saccharomyces cerevisiae.Journal of Biochemistry,139(4):797-803
Li QJ,2016.Study on related genes of cold-induced primordium formation of Flammulina velutipes.Master Thesis,South China Agricultural University,Guangzhou.1-94(in Chinese)
Pertea M,Kim D,Pertea GM,Leek JT,Salzberg SL,2016.Transcript-level expression analysis of RNA-seq experiments with HISAT,StringTie and Ballgown.Nature Protocols,11(9):1650-1667
Panadero J,Pallotti C,Rodriguez-Vargas S,Randez-Gil F,Prieto JA,2006.A downshift in temperature activates the high osmolarity glycerol(HOG)pathway,which determines freeze tolerance in Saccharomyces cerevisiae.Journal of Biological Chemistry,281(8):4638-4645
Shen YY,Gu M,Cai WM,Jin QL,Fan LJ,Feng WL,Song TT,Tian FF,2014.Construction and analysis of a suppressive subtractive hybridisation cDNA library related to fruiting body formation of Pleurotus pulmonarius after cold stimulation.Acta Agriculturae Zhejiangensis,26(2):330-334(in Chinese)
Sinclair BJ,Stinziano JR,Williams CM,MacMillan HA,Marshall KE,2013.Real-time measurement of metabolic rate during freezing and thawing of the wood frog,Rana sylvatica:implications for overwinter energy use.The Journal of Experimental Biology,216(Pt2):292-302
Soto T,Beltran FF,Paredes V,Madrid M,Millar JBA,Soler JV,Cansado J,Gacto M,2002.Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe.European Journal of Biochemistry,269(20):5056-5065
Teige M,Scheikl E,Eulgem T,Doczi R,Ichimura K,Shinozaki K,Dangl JL,Hirt H,2004.The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis.Molecular Cell,15(1):141-152
Tereshina VM,Memorskaia AS,2005.Adaptation of Flammulina velutipes to hypothermia in natural environment:the role of lipids and carbohydrates.Mikrobiologiia,74(3):329-334
Wang H,Bao DP,Chen MJ,Chen H,Feng AP,2010.Cloning and sequence analysis of mitogen-activated protein kinase homolog from Volvariella volvacea(VV-MAPKKK).Microbiology China,37(11):1606-1609(in Chinese)
Wang P,Fan ZQ,Fan RQ,2015.Xianglian external lotion restored the sensitivity of drug-resistant Candida albicans strains to fluconazole:a tran-scriptomics study.Chinese Journal of Integrated Traditional and Western Medicine,35(12):1505-1509(in Chinese)
Wang Z,Gerstein M,Snyder M,2009.RNA-Seq:a revolutionary tool for transcriptomics.Nature Reviews Genetics,10(1):57-63
Wu XC,Hu SJ,Qian KX,2005.HOG-MAPK pathway in yeast.Chinese Journal of Cell Biology,27(3):247-252(in Chinese)
Xiang L,Li Y,Zhu Y,Luo HM,Li CF,Xu XL,Sun C,Song JY,Shi LC,He L,Sun W,Chen SL,2014.Transcriptome analysis of the Ophiocordyceps sinensis fruiting body reveals putative genes involved in fruiting body development and cordycepin biosynthesis.Genomics,103(1):154-159
Zerbino DR,Birney E,2008.Velvet:algorithms for de novo short read assembly using de Bruijn graphs.Genome Research,18(5):821-829
Zhang ZC,Yao YM,Mao ZL,Sun GS,Qin WB,Dai ZL,2018.Transcriptome analysis and gene function annotation of early developmental broccoli microspores based on high-throughput sequencing technology.Acta Agriculturae Nucleatae Sinica,32(5):848-855
冯志勇,米朔甫,陈明杰,赵明文,李军辉,潘迎捷,2006.低温胁迫下香菇基因表达差异研究.应用与环境生物学报,12(5):614-617
冯志勇,米朔甫,赵明文,陈明杰,李军辉,谭琦,潘迎捷,2006.香菇低温胁迫应答基因的克隆及其表达分析.农业生物技术学报,14(3):381-386
傅俊生,2007.草菇杂交育种研究及其分子遗传标记的建立.福建农林大学硕士论文,福州.1-55
李琼洁,2016.冷诱导金针菇原基形成的相关基因研究.华南农业大学硕士论文,广州.1-94
沈颖越,顾敏,蔡为明,金群力,范丽军,冯伟林,宋婷婷,田芳芳,2014.秀珍菇子实体发育相关消减杂交cDNA文库的构建与分析.浙江农业学报,26(2):330-334
王平,樊志奇,范瑞强,2015.香莲外洗液逆转白念珠菌耐药的转录组学研究.中国中西医结合杂志,35(12):1505-1509
汪虹,鲍大鹏,陈明杰,陈辉,冯爱萍,2010.草菇MAPKKK同源基因的克隆及序列分析.微生物学通报,37(11):1606-1609
吴雪昌,胡森杰,钱凯先,2005.酵母HOG-MAPK途径.细胞生物学杂志,7:247-252