小麦近等基因系幼穗二棱期转录组分析
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摘要
抽穗期是与小麦适应性直接相关的一个很重要的性状,直接或间接影响小麦的产量和抗性。为明确控制抽穗期的关键表达基因,以抽穗期分别为196d和184d的小麦近等基因系Y57和96-2为材料,对其二棱期幼穗分别构建转录组测序文库,利用Illumina HiSeq 2500平台进行RNA-seq测序;将获得的clean read与中国春参考序列比对,得到unique read,采用FPKM法计算基因表达量,对差异表达基因进行功能注释、GO和COG功能分类以及KEGG通路分析。结果表明,供试材料经过测序获得质量不低于30的碱基比例(Q30)均高于85.5%;Y57和96-2分别有60 246 194和60 963 580条clean read,其中63.11%和69.51%能与参考基因组序列匹配。共筛选到395个差异表达基因,有382个基因得到功能注释;有298个基因获得GO功能注释,主要涉及细胞组分、结合、细胞反应和代谢过程;COG注释的基因主要涉及一般功能、信号转导机制和转录;KEGG功能注释分析发现,显著富集在光合作用-天线蛋白通路上的基因均上调表达。编码丝氨酸/苏氨酸蛋白磷酸酶2A的基因在Y57中几乎不表达,而在96-2中表达量较高。进一步分析发现,多个与生长发育、抽穗开花相关的转录因子在两个材料中的表达差异显著。此结果可为深入研究抽穗期相关基因和穗发育调控机制奠定基础。
Heading date is one of the most important traits directly relating to the wheat adaptability and affects the wheat yield and resistance traits.Therefore,it is of significance for breeding and improving the wheat varieties with high yield at molecular level by mining heading-related genes.As the double-ridge stage is the key period affecting the heading date,the RNA of immature spikes at the double-ridge stage from two near-isogenic lines Y57(196days)and 96-2(184days)with different heading date were used to construct the transcriptome libraries and sequenced by Illumina HiSeqTM2500 platform in order to identify the important differentially expressed genes(DEGs)for heading date.RNA-seq clean reads were aligned to the Chinese Spring genome sequences to obtain uniquereads.Gene expression level was counted by computing the FPKM value.The functions and pathways of these DEGs were annotated by searching against the GO,COG and KEGG public database.The results indicated that Q30 is over 85.5%.Clean reads from Y57 and 96-2were 60 246 194 and 60 963580,respectively.There were 63.11% and 69.51% clean reads mapped to the reference genome sequences,respectively.Three hundred and ninety five DEGs were identified after differential expression analysis.Among them,382 DEGs were annotated in Nr database,and 298 genes were annotated by GO functional enrichment analysis,which are primarily involved in cell part,binding,cellular process and metabolic process.COG analysis showed that DEGs are mainly involved in general function,signal transduction mechanisms and transcription.Through KEGG analysis,we found all DEGs enriched in photosynthesis-antenna protein pathway were up-regulated.Both genes encoding serine/threonine protein phosphatase 2A were highly expressed in 96-2but lowly expressed in Y57.Further analysis found that the expression level of many transcription factors associated with development and flowering were significantly different.These results would provide abundant resources for further studies on genes related to heading date and laid a foundation for molecular mechanisms.
Heading date is one of the most important traits directly relating to the wheat adaptability and affects the wheat yield and resistance traits.Therefore,it is of significance for breeding and improving the wheat varieties with high yield at molecular level by mining heading-related genes.As the double-ridge stage is the key period affecting the heading date,the RNA of immature spikes at the double-ridge stage from two near-isogenic lines Y57(196days)and 96-2(184days)with different heading date were used to construct the transcriptome libraries and sequenced by Illumina HiSeqTM2500 platform in order to identify the important differentially expressed genes(DEGs)for heading date.RNA-seq clean reads were aligned to the Chinese Spring genome sequences to obtain uniquereads.Gene expression level was counted by computing the FPKM value.The functions and pathways of these DEGs were annotated by searching against the GO,COG and KEGG public database.The results indicated that Q30 is over 85.5%.Clean reads from Y57 and 96-2were 60 246 194 and 60 963580,respectively.There were 63.11% and 69.51% clean reads mapped to the reference genome sequences,respectively.Three hundred and ninety five DEGs were identified after differential expression analysis.Among them,382 DEGs were annotated in Nr database,and 298 genes were annotated by GO functional enrichment analysis,which are primarily involved in cell part,binding,cellular process and metabolic process.COG analysis showed that DEGs are mainly involved in general function,signal transduction mechanisms and transcription.Through KEGG analysis,we found all DEGs enriched in photosynthesis-antenna protein pathway were up-regulated.Both genes encoding serine/threonine protein phosphatase 2A were highly expressed in 96-2but lowly expressed in Y57.Further analysis found that the expression level of many transcription factors associated with development and flowering were significantly different.These results would provide abundant resources for further studies on genes related to heading date and laid a foundation for molecular mechanisms.
引文
[1]FAO STAT.Food and agriculture organisation of the united nations[R].Rome,Italy,2014.
[2]ALLARD R W,JAIN S K.Population studies in predominantly self-pollinated spices.II.Analysis of quantitative genetic changes in bulk-hybrid population barely[J].Evolution,1962,16(1):90.
[3]KAMRAN A,IQBAL l M,SPANER D.Flowering time in wheat(Triticum aestivum L.):A key factor for global adaptability[J].Euphytica,2014,197(1):1.
[4]米国华.小麦春化研究进展[J].麦类作物学报,1993,13(5):32.MI G H.A research on vernalization of wheat[J].Journal of Triticeae Crops,1993,13(5):32.
[5]马建华,姚琴,张彭良等.温光条件对光不敏感春性小麦穗发育的影响[J].麦类作物学报,2011,31(5):941.MA J H,YAO Q,ZHANG P L,et al.Effect of temperature and photoperiod on ear differentiation of spring wheat[J].Journal of Triticeae Crops,2011,31(5):941.
[6]何立人,李来胜,李正玮.大、小麦生育阶段及穗原基发育特点的研究[J].北京农学院学报,1988,3(2):21.HE L R,LI L S,LI Z W.A study on the ear differentiation and development stage of wheat and barley[J].Journal of Beijing University of Agriculture,1988,3(2):21.
[7]YOSHID H,KAMIO M.Differences in characteristics of earliness partitioned by developmental stage between wheat and barley cultivars[J].Japanese Journal of Breeding,2008,35(3):323.
[8]YASUDA S.Differences in mechanism of early maturity between cultivated common wheat and barley[J].Japanese Journal of Breeding,2008,39(3):327.
[9]ZHANG H N,WEI Y Z,SHEN J Y,et al.Transcriptomic analysis of floral initiation in litchi(Litchi chinensis Sonn.)based on de novo RNA sequencing[J].Plant Cell Reports,2014,33(10):1723-1735.
[10]KUMAR R R,GOSWAMI S,SHARMA S K,et al.Harnessing next generation sequencing in climate change:RNA-seq analysis of heat stress-responsive genes in wheat(Triticum aestivum L.)[J].Omics A Journal of Integrative Biology,2015,19(10):632.
[11]LIU W,WU Z H,ZHANG Y F,et al.Transcriptome analysis of wheat grain using RNA-Seq[J].Frontiers of Agricultural Science and Engineering,2014,1(3):214.
[12]王建革,孙宝启,黄友志.小麦抽穗期的遗传控制[J].遗传,2002,24(2):193.WANG J G,SUN B Q,HUANG Y Z.The genetic control of heading time in wheat[J].Hereditas(Beijing),2002,24(2):193.
[13]JOHN R P,MEGAN G.Temperatures and the growth and development of wheat:A review[J].European Journal of Agronomy,1999,10(1):23.
[14]WORLAND A J.The influence of flowering time genes on environmental adaptability in European wheats vernalization sensitivity[J].Euphytica,1996,89(1):49.
[15]KATO K,YAMAGATA H.Method for evaluation of chilling requirement and narrow-sense earliness of wheat cultivars[J].Japanese Journal of Breeding,2008,38(2):172.
[16]FENG N,SONG G,GUAN J,et al.Transcriptome profiling of wheat inflorescence development from spikelet initiation to floral patterning identified stage-specific regulatory genes[J].Plant Physiology,2017,174:1779.
[17]WANG Y,YU H,TIAN C,et al.Transcriptome association identifies regulators of wheat spike architecture[J].Plant Physiology,2017,149(2):pp.00694.2017.
[18]KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:Accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biology,2013,14(4):R36.
[19]TRAPNELL C,WILLIAMS B A,PERTEA G,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,2010,28:511.
[20]LENG N,DAWSON J A,THOMSON J A,et al.EBSeq:An empirical Bayes hierarchical model for inference in RNA-seq experiments[J].Bioinformatics,2013,29(8):1035.
[21]CONESA A,GOTZ S,GARCIR-GOMEZ J M,et al.Blast2GO:A universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,2005,21(18):3674.
[22]YE J,FANG L,ZHENG H K,et al.WEGO:A web tool for plotting GO annotations[J].Nucleic Acids Research,2006,34:293.
[23]DEVOS K M,DOLEZEL J,FEULLET C.Genome organization and comparative genomics wheat,science and trade[J].Wiley-Blackwell,2009:327.
[24]LING H Q,ZHAO S C,LIU D C,et al.Draft genome of the wheat A-genome progenitor Triticum urartu[J].Science Foundation in China,2013,496(2):87.
[25]JIA J Z,ZHAO S C,KONG X Y,et al.Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation[J].Nature,2013,496(7443):91.
[26]MARTIN L B B,FEI Z J,GIOVANNONI J J,et al.Catalyzing plant science research with RNA-seq[J].Front Plant Science,2013,4:1.
[27]YU Y L,GUO G F,LV D W,et al.Transcriptome analysis during seed germination of elite Chinese bread wheat cultivar Jimai 20[J].BMC Plant Biology,2014,14(1):20.
[28]YU Y L,ZHEN S M,WANG S,et al.Comparative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2stresses during seed germination[J].BMC Genomics,2016,17(1):97.
[29]JANSSENS V,GORIS J.Protein phosphatase 2A:A highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling[J].Biochemical Journal,2001,353(3):417.
[30]YU R M K,ZHOU Y,XU Z F,et al.Two genes encoding protein phosphatase 2Acatalytic subunits are differentially expressed in rice[J].Plant Molecular Biology,2003,51(3):295.
[31]GARBERS C,DELONG A,DERUERE J,et al.A mutation in protein phosphatase 2Aregulatory subunit A affects auxin transport in Arabidopsis[J].EMBO Journal,1996,15(9):2115.
[32]KISELEVAL A A,SHCHERBAN A B,LEONOVAL I N,et al.Identification of new heading date determinants in wheat5Bchromosome[J].BMC Plant Biology,2016,16(S1):8.
[33]LUO X,SUN X,LIU B,et al.Ectopic expression of a WRKY homolog from glycine soja alters flowering time in Arabidopsis[J].Public Library of Science One,2013,8(8):e73295.
[34]YU Y,HU R,WANG H,et al.MIWRKY12,a novel miscanthus transcription factor,participates in pith secondary cell wall formation and promotes flowering[J].Plant Science,2013,212(212):1.
[35]ZHAO T,NI Z F,DAI Y,et al.Characterization and expression of 42 MADS-box genes in wheat(Triticum aestivum L.)[J].Molecular Genetics&Genomics,2006,276(4):334.
[36]HONMA T,GOTO K.Complexes of MADS-BOX proteins are sufficient to convert leaves into floral organs[J].Nature,2001,409(6819):525.
[37]FOLTER S D,COLOMBO L.Frontiers|Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis:New openings to the MADS world[J].The Astrophysical Journal,2015,800:63.
[38]NGUYEN H N,LEE H.MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis[J].Plant Signaling&Behavior,2016,11:e1139278.
[39]邵宏波.高等植物开花时程的调控与光受体Ⅰ.开花时程的基因与光受体调控[J].生命科学研究,2001,5(Z1):150.SHAO H B.The regulation and control of flowering time and photoreceptors in higher plants:I.The regulation and control of genes and photoreceptors in the flowering time[J].Life Science Research,2001,5(Z1):150.
[2]ALLARD R W,JAIN S K.Population studies in predominantly self-pollinated spices.II.Analysis of quantitative genetic changes in bulk-hybrid population barely[J].Evolution,1962,16(1):90.
[3]KAMRAN A,IQBAL l M,SPANER D.Flowering time in wheat(Triticum aestivum L.):A key factor for global adaptability[J].Euphytica,2014,197(1):1.
[4]米国华.小麦春化研究进展[J].麦类作物学报,1993,13(5):32.MI G H.A research on vernalization of wheat[J].Journal of Triticeae Crops,1993,13(5):32.
[5]马建华,姚琴,张彭良等.温光条件对光不敏感春性小麦穗发育的影响[J].麦类作物学报,2011,31(5):941.MA J H,YAO Q,ZHANG P L,et al.Effect of temperature and photoperiod on ear differentiation of spring wheat[J].Journal of Triticeae Crops,2011,31(5):941.
[6]何立人,李来胜,李正玮.大、小麦生育阶段及穗原基发育特点的研究[J].北京农学院学报,1988,3(2):21.HE L R,LI L S,LI Z W.A study on the ear differentiation and development stage of wheat and barley[J].Journal of Beijing University of Agriculture,1988,3(2):21.
[7]YOSHID H,KAMIO M.Differences in characteristics of earliness partitioned by developmental stage between wheat and barley cultivars[J].Japanese Journal of Breeding,2008,35(3):323.
[8]YASUDA S.Differences in mechanism of early maturity between cultivated common wheat and barley[J].Japanese Journal of Breeding,2008,39(3):327.
[9]ZHANG H N,WEI Y Z,SHEN J Y,et al.Transcriptomic analysis of floral initiation in litchi(Litchi chinensis Sonn.)based on de novo RNA sequencing[J].Plant Cell Reports,2014,33(10):1723-1735.
[10]KUMAR R R,GOSWAMI S,SHARMA S K,et al.Harnessing next generation sequencing in climate change:RNA-seq analysis of heat stress-responsive genes in wheat(Triticum aestivum L.)[J].Omics A Journal of Integrative Biology,2015,19(10):632.
[11]LIU W,WU Z H,ZHANG Y F,et al.Transcriptome analysis of wheat grain using RNA-Seq[J].Frontiers of Agricultural Science and Engineering,2014,1(3):214.
[12]王建革,孙宝启,黄友志.小麦抽穗期的遗传控制[J].遗传,2002,24(2):193.WANG J G,SUN B Q,HUANG Y Z.The genetic control of heading time in wheat[J].Hereditas(Beijing),2002,24(2):193.
[13]JOHN R P,MEGAN G.Temperatures and the growth and development of wheat:A review[J].European Journal of Agronomy,1999,10(1):23.
[14]WORLAND A J.The influence of flowering time genes on environmental adaptability in European wheats vernalization sensitivity[J].Euphytica,1996,89(1):49.
[15]KATO K,YAMAGATA H.Method for evaluation of chilling requirement and narrow-sense earliness of wheat cultivars[J].Japanese Journal of Breeding,2008,38(2):172.
[16]FENG N,SONG G,GUAN J,et al.Transcriptome profiling of wheat inflorescence development from spikelet initiation to floral patterning identified stage-specific regulatory genes[J].Plant Physiology,2017,174:1779.
[17]WANG Y,YU H,TIAN C,et al.Transcriptome association identifies regulators of wheat spike architecture[J].Plant Physiology,2017,149(2):pp.00694.2017.
[18]KIM D,PERTEA G,TRAPNELL C,et al.TopHat2:Accurate alignment of transcriptomes in the presence of insertions,deletions and gene fusions[J].Genome Biology,2013,14(4):R36.
[19]TRAPNELL C,WILLIAMS B A,PERTEA G,et al.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,2010,28:511.
[20]LENG N,DAWSON J A,THOMSON J A,et al.EBSeq:An empirical Bayes hierarchical model for inference in RNA-seq experiments[J].Bioinformatics,2013,29(8):1035.
[21]CONESA A,GOTZ S,GARCIR-GOMEZ J M,et al.Blast2GO:A universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,2005,21(18):3674.
[22]YE J,FANG L,ZHENG H K,et al.WEGO:A web tool for plotting GO annotations[J].Nucleic Acids Research,2006,34:293.
[23]DEVOS K M,DOLEZEL J,FEULLET C.Genome organization and comparative genomics wheat,science and trade[J].Wiley-Blackwell,2009:327.
[24]LING H Q,ZHAO S C,LIU D C,et al.Draft genome of the wheat A-genome progenitor Triticum urartu[J].Science Foundation in China,2013,496(2):87.
[25]JIA J Z,ZHAO S C,KONG X Y,et al.Aegilops tauschii draft genome sequence reveals a gene repertoire for wheat adaptation[J].Nature,2013,496(7443):91.
[26]MARTIN L B B,FEI Z J,GIOVANNONI J J,et al.Catalyzing plant science research with RNA-seq[J].Front Plant Science,2013,4:1.
[27]YU Y L,GUO G F,LV D W,et al.Transcriptome analysis during seed germination of elite Chinese bread wheat cultivar Jimai 20[J].BMC Plant Biology,2014,14(1):20.
[28]YU Y L,ZHEN S M,WANG S,et al.Comparative transcriptome analysis of wheat embryo and endosperm responses to ABA and H2O2stresses during seed germination[J].BMC Genomics,2016,17(1):97.
[29]JANSSENS V,GORIS J.Protein phosphatase 2A:A highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling[J].Biochemical Journal,2001,353(3):417.
[30]YU R M K,ZHOU Y,XU Z F,et al.Two genes encoding protein phosphatase 2Acatalytic subunits are differentially expressed in rice[J].Plant Molecular Biology,2003,51(3):295.
[31]GARBERS C,DELONG A,DERUERE J,et al.A mutation in protein phosphatase 2Aregulatory subunit A affects auxin transport in Arabidopsis[J].EMBO Journal,1996,15(9):2115.
[32]KISELEVAL A A,SHCHERBAN A B,LEONOVAL I N,et al.Identification of new heading date determinants in wheat5Bchromosome[J].BMC Plant Biology,2016,16(S1):8.
[33]LUO X,SUN X,LIU B,et al.Ectopic expression of a WRKY homolog from glycine soja alters flowering time in Arabidopsis[J].Public Library of Science One,2013,8(8):e73295.
[34]YU Y,HU R,WANG H,et al.MIWRKY12,a novel miscanthus transcription factor,participates in pith secondary cell wall formation and promotes flowering[J].Plant Science,2013,212(212):1.
[35]ZHAO T,NI Z F,DAI Y,et al.Characterization and expression of 42 MADS-box genes in wheat(Triticum aestivum L.)[J].Molecular Genetics&Genomics,2006,276(4):334.
[36]HONMA T,GOTO K.Complexes of MADS-BOX proteins are sufficient to convert leaves into floral organs[J].Nature,2001,409(6819):525.
[37]FOLTER S D,COLOMBO L.Frontiers|Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis:New openings to the MADS world[J].The Astrophysical Journal,2015,800:63.
[38]NGUYEN H N,LEE H.MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis[J].Plant Signaling&Behavior,2016,11:e1139278.
[39]邵宏波.高等植物开花时程的调控与光受体Ⅰ.开花时程的基因与光受体调控[J].生命科学研究,2001,5(Z1):150.SHAO H B.The regulation and control of flowering time and photoreceptors in higher plants:I.The regulation and control of genes and photoreceptors in the flowering time[J].Life Science Research,2001,5(Z1):150.