甜瓜与白粉病菌非亲和互作的数字基因表达谱分析
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摘要
为探讨甜瓜与白粉病菌非亲和互作在转录水平的分子机制,本研究选用高抗甜瓜品种‘Yuntian930’,利用Solexa高通量测序技术分析甜瓜幼苗接种白粉病菌前0 h和接菌后24 h、48 h和72 h的基因表达谱。与未接种相比,接种后3个时间点分别鉴定出219个、1 784个和2 371个差异表达基因,且上调表达基因数多于下调表达基因数。GO功能分析表明,已注释的差异表达基因显著富集到代谢过程、生物学过程、磷代谢过程、磷酸化及蛋白磷酸化修饰、光合膜、类囊体、氧化还原酶活性和序列特异结合位点等生物学过程。Pathway分析显示,差异表达基因富集到多条物质代谢、次级代谢物合成、甘氨酸/丝氨酸/苏氨酸代谢、光和系统、植物激素信号转导等通路中。比较各接种时间的差异表达基因显示,接种后48 h鉴定出的差异基因和Pathway最多,并发现有7个涉及多胺代谢的差异表达基因,其中6个呈上调表达。这一结果进一步验证了多胺代谢途径基因能有效响应白粉病菌胁迫反应,从而提高对白粉病菌的抵抗能力。利用q RT-PCR验证4个多胺代谢相关基因在接种白粉病菌后的差异表达,其结果与DGE一致,证实了DGE测序结果的可靠性。
In order to investigate the molecular mechanisms of incompatible interaction between melon and powdery mildew pathogen at the transcriptional level, the high-resistant melon material‘Yuntian930'was used to analyze the gene expression profiling of melon seedlings before inoculation and 24 h, 48 h, 72 h after inoculation with Podosphaera xanthii by using Solexa high-throughput sequencing method. Compared with no inoculation,219, 1 784 and 2 371 differentially expressed genes were identified at three different time points after inoculation,respectively, and the numbers of up-regulated genes were more than that of down-regulated genes. GO function analysis showed that these annotated differentially expressed genes were significantly enriched in biological processes, including metabolic process, biological process, phosphorus metabolic process, phosphorylation and protein phosphorylation modification, photosynthetic membrane, thylakoid, oxidoreductase activity, sequence-specific binding sites, etc. The pathway analysis showed that the differentially expressed genes were significantly enriched in multiple processes of substance metabolism and secondary metabolite synthesis, glycine metabolism,serine metabolism and threonine metabolism, photosynthesis, signal transduction pathways of plant hormone, etc.The comparisons of differential expression genes at four time points indicated that the most differential genes and pathways were observed at 48 h after inoculation, and seven of these genes were involved in the polyamine metabolism, in which six genes were up-regulated. This result further indicated that genes related to polyamine metabolic pathways could effectively respond to powdery mildew stress reaction and improve the resistance of the melon plants to P. xanthii. Four genes related to polyamine metabolism were verified the differential expression after the inoculation of powdery mildew by Real-time quantitative PCR and the result was consistent with that of DGE which proved the dependability of the results of DGE sequencing.
In order to investigate the molecular mechanisms of incompatible interaction between melon and powdery mildew pathogen at the transcriptional level, the high-resistant melon material‘Yuntian930'was used to analyze the gene expression profiling of melon seedlings before inoculation and 24 h, 48 h, 72 h after inoculation with Podosphaera xanthii by using Solexa high-throughput sequencing method. Compared with no inoculation,219, 1 784 and 2 371 differentially expressed genes were identified at three different time points after inoculation,respectively, and the numbers of up-regulated genes were more than that of down-regulated genes. GO function analysis showed that these annotated differentially expressed genes were significantly enriched in biological processes, including metabolic process, biological process, phosphorus metabolic process, phosphorylation and protein phosphorylation modification, photosynthetic membrane, thylakoid, oxidoreductase activity, sequence-specific binding sites, etc. The pathway analysis showed that the differentially expressed genes were significantly enriched in multiple processes of substance metabolism and secondary metabolite synthesis, glycine metabolism,serine metabolism and threonine metabolism, photosynthesis, signal transduction pathways of plant hormone, etc.The comparisons of differential expression genes at four time points indicated that the most differential genes and pathways were observed at 48 h after inoculation, and seven of these genes were involved in the polyamine metabolism, in which six genes were up-regulated. This result further indicated that genes related to polyamine metabolic pathways could effectively respond to powdery mildew stress reaction and improve the resistance of the melon plants to P. xanthii. Four genes related to polyamine metabolism were verified the differential expression after the inoculation of powdery mildew by Real-time quantitative PCR and the result was consistent with that of DGE which proved the dependability of the results of DGE sequencing.
引文
Alcázar R.,Altabella T.,Marco F.,Bortolotti C.,Reymond M.,Knocz C.,Carrasco P.,and Tiburcio A.F.,2010,Polyamines:molecules with regulatory functions in plant abiotic stress tolerance,Planta,231(6):1237-1249
Bai T.T.,Xie W.B.,Zhou P.P.,Wu Z.L.,Xiao W.C.,Zhou L.,Sun J.,Ruan X.L.,and Li H.P.,2013,Transcriptome and expression profile analysis of highly resistant and susceptible banana roots challenged with Fusarium oxysporum f.sp.cubense tropical race 4,PLo S One,8(9):e73945
Cominelli E.,Galbiati M.,Vavasseur A.,Conti L.,Sala T.,Vuylsteke M.,Leonhardt N.,Dellaporta S.L.,and Tonelli C.,2005,A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance,Curr.Biol.,15(13):1196-1200
Consonni C.,Humphry M.E.,Hartmann H.A.,Livaja M.,Durner J.,Westphal L.,Vogel J.,Lipka V.,Kemmerling B.,Schulze-Lefert P.,Somerville S.C.,and Panstruga R.,2006,Conserved requirement for a plant host cell protein in powdery mildew pathogenesis,Nat.Genet.,38(6):716-720
Deinlein U.,Stephan A.B.,Horie T.,Luo W.,Xu G.,and Schroeder J.I.,2014,Plant salt-tolerance mechanisms,Trends Plant Sci.,19(6):371-379
Fernández-Ortu觡o D.,Pérez-García A.,López-Ruiz F.,Romero D.,De Vicente A.,and Torés J.A.,2006,Occurrence and distribution of resistance to Qo I fungicides in populations of Podosphaera fusca in south central Spain,Eur.J.Plan Pathol.,115(2):215-222
Franceschetti M.,Fornale S.,Tassoni A.,Zuccherelli K.,Mayer M.J.,and Bagni N.,2004,Effects of spermidine synthase overexpression on polyamine biosynthetic pathway in tobacco plants,J.Plant Physiol.,161(9):989-1001
Gill S.S.,and Tuteja N.,2010,Polyamines and abiotic stress tolerance in plants,Plant Signal.Behav.,5(1):26-33
Han Z.G.,Zhao Z.Q.,Li H.H.,Zhang X.,Li X.L.,and Zhang W.2015,Expression profiling analysis between resistant and susceptible cotton cultivars(Gossypium hirsutum L.)in response to fusarium wilt,Zuowu Xuebao(Acta Agronomica Sinica),41(8):1201-1211(韩泽刚,赵曾强,李会会,张析李潇玲,张薇,2015,枯萎病菌诱导感,抗陆地棉品种的基因表达谱分析,作物学报,41(8):1201-1211)
Hu J.B.,Ma S.W.,Jian Z.H.,Wang J.M.,Li Q.,and Su Y.,2013Analysis of genetic diversity of Chinese melon(Cucumis melo L.)germplasm resources based on morphological characters,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(4):612-619(胡建斌,马双武,简在海王吉明,李琼,苏艳,2013,中国甜瓜种质资源形态性状遗传多样性分析,植物遗传资源学报,14(4):612-619)
Ju L.F.,Duan S.Y.,Zhang Y.,Hasi Agula,and Niu Y.D.,2016Bioinformatics analysis of melon ethylene response factor gene Cm ERFⅠ-14,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2467-2474(鞠林芳,段尚尧,张妍,哈斯阿古拉,牛一丁,2016,甜瓜乙烯应答因子基因Cm ERFⅠ-14的生物信息学分析,基因组学与应用生物学,35(9):2467-2474)
Liu C.M.,Li X.L.,Yang R.P.,Mo Y.L.,Wang Y.Q.,Xian F.Zhang X.,and Wang F.,2014,The protective roles of S-adenosylmethionine decarboxylase(SAMDC)gene in melon resistance to powdery mildew infection,Hort.Environ.Biotechnol.,55(6):557-567
Liu C.M.,Xian F.,Tian Z.G.,Yang R.P.,Zhang J.X.,and Zhang X.,2014,Analysis of gene expression profiling in the incompatible interaction between Chinese wild melon‘Yuntian930’and Podosphaera xanthii,Zhiwu Bingli Xuebao(Acta Phytopathologica Sinica),44(1):65-73(刘长命,咸丰,田治国,杨瑞平,郑俊鶱,张显,2014,野生甜瓜‘云甜-930’与白粉病菌互作的基因表达特征,植物病理学报,44(1):65-73)
Liu C.M.,Yang R.P.,Mo Y.L.,Wang Y.Q.,Zhang J.X.,and Zhang X.,2016,Induction of endogenous polyamine contents and powdery mildew resistance by exogenous spermidine in melon seedlings,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),36(1):85-92(刘长命,杨瑞平,莫言玲,王永琦,郑俊鶱,张显,2016,外源Spd预处理对甜瓜白粉病抗性及其内源多胺的诱导分析,西北植物学报,36(1):85-92)
Perchepied L.,Bardin M.,Dogimont C.,and Pitrat M.,2005,Relationship between Loci conferring downy mildew and powdery mildew resistance in melon assessed by quantitative trait Loci mapping,Phytopathology,95(5):556-565
Rao X.Y.,Huang X.L.,Zhou Z.C.,and Lin X.,2013,An improvement of the 2魮(-delta delta CT)method for quantitative real-time polymerase chain reaction data analysis,Biostat Bioinforma Biomath.,3(3):71-85
Sautel C.F.,Ortet P.,Saksouk N.,Kieffer S.,Garin J.,Bastien O.,and Hakimi M.A.,2009,The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences,Mol.Microbiol.,71(1):212-226
Sharma S.S.,and Dietz K.J.,2006,The significance of amino acids and amino acid-derived molecules in plant response and adaptation to heavy stress,J.Exp.Bot.,57(4):711-726
Tao Y.,Xie Z.Y.,Chen W.Q.,Glazebrook J.,Chang H.S.,Han B.,Zhu T.,Zou G.Z.,and Katagiri F.,2003,Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen pathogen Pseudomonas syringae,Plant Cell,15(2):317-330
Wu J.,Zhang Y.L.,Zhang H.Q.,Huang H.,Folta K.M.,and Lu J.,2010,Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology,BMC Plant Biol.,10:234-250
Yu S.C.,Zhang F.L.,Yu Y.J.,Zhang D.S.,Zhao X.Y.,and Wang W.H.,2012,Transcriptome profiling of dehydration stress in the Chinese cabbage(Brassica rapa L.ssp.pekinensis)by tag sequencing,Plant Mol.Biol.Rep.,30(1):17-28
Bai T.T.,Xie W.B.,Zhou P.P.,Wu Z.L.,Xiao W.C.,Zhou L.,Sun J.,Ruan X.L.,and Li H.P.,2013,Transcriptome and expression profile analysis of highly resistant and susceptible banana roots challenged with Fusarium oxysporum f.sp.cubense tropical race 4,PLo S One,8(9):e73945
Cominelli E.,Galbiati M.,Vavasseur A.,Conti L.,Sala T.,Vuylsteke M.,Leonhardt N.,Dellaporta S.L.,and Tonelli C.,2005,A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance,Curr.Biol.,15(13):1196-1200
Consonni C.,Humphry M.E.,Hartmann H.A.,Livaja M.,Durner J.,Westphal L.,Vogel J.,Lipka V.,Kemmerling B.,Schulze-Lefert P.,Somerville S.C.,and Panstruga R.,2006,Conserved requirement for a plant host cell protein in powdery mildew pathogenesis,Nat.Genet.,38(6):716-720
Deinlein U.,Stephan A.B.,Horie T.,Luo W.,Xu G.,and Schroeder J.I.,2014,Plant salt-tolerance mechanisms,Trends Plant Sci.,19(6):371-379
Fernández-Ortu觡o D.,Pérez-García A.,López-Ruiz F.,Romero D.,De Vicente A.,and Torés J.A.,2006,Occurrence and distribution of resistance to Qo I fungicides in populations of Podosphaera fusca in south central Spain,Eur.J.Plan Pathol.,115(2):215-222
Franceschetti M.,Fornale S.,Tassoni A.,Zuccherelli K.,Mayer M.J.,and Bagni N.,2004,Effects of spermidine synthase overexpression on polyamine biosynthetic pathway in tobacco plants,J.Plant Physiol.,161(9):989-1001
Gill S.S.,and Tuteja N.,2010,Polyamines and abiotic stress tolerance in plants,Plant Signal.Behav.,5(1):26-33
Han Z.G.,Zhao Z.Q.,Li H.H.,Zhang X.,Li X.L.,and Zhang W.2015,Expression profiling analysis between resistant and susceptible cotton cultivars(Gossypium hirsutum L.)in response to fusarium wilt,Zuowu Xuebao(Acta Agronomica Sinica),41(8):1201-1211(韩泽刚,赵曾强,李会会,张析李潇玲,张薇,2015,枯萎病菌诱导感,抗陆地棉品种的基因表达谱分析,作物学报,41(8):1201-1211)
Hu J.B.,Ma S.W.,Jian Z.H.,Wang J.M.,Li Q.,and Su Y.,2013Analysis of genetic diversity of Chinese melon(Cucumis melo L.)germplasm resources based on morphological characters,Zhiwu Yichuan Ziyuan Xuebao(Journal of Plant Genetic Resources),14(4):612-619(胡建斌,马双武,简在海王吉明,李琼,苏艳,2013,中国甜瓜种质资源形态性状遗传多样性分析,植物遗传资源学报,14(4):612-619)
Ju L.F.,Duan S.Y.,Zhang Y.,Hasi Agula,and Niu Y.D.,2016Bioinformatics analysis of melon ethylene response factor gene Cm ERFⅠ-14,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(9):2467-2474(鞠林芳,段尚尧,张妍,哈斯阿古拉,牛一丁,2016,甜瓜乙烯应答因子基因Cm ERFⅠ-14的生物信息学分析,基因组学与应用生物学,35(9):2467-2474)
Liu C.M.,Li X.L.,Yang R.P.,Mo Y.L.,Wang Y.Q.,Xian F.Zhang X.,and Wang F.,2014,The protective roles of S-adenosylmethionine decarboxylase(SAMDC)gene in melon resistance to powdery mildew infection,Hort.Environ.Biotechnol.,55(6):557-567
Liu C.M.,Xian F.,Tian Z.G.,Yang R.P.,Zhang J.X.,and Zhang X.,2014,Analysis of gene expression profiling in the incompatible interaction between Chinese wild melon‘Yuntian930’and Podosphaera xanthii,Zhiwu Bingli Xuebao(Acta Phytopathologica Sinica),44(1):65-73(刘长命,咸丰,田治国,杨瑞平,郑俊鶱,张显,2014,野生甜瓜‘云甜-930’与白粉病菌互作的基因表达特征,植物病理学报,44(1):65-73)
Liu C.M.,Yang R.P.,Mo Y.L.,Wang Y.Q.,Zhang J.X.,and Zhang X.,2016,Induction of endogenous polyamine contents and powdery mildew resistance by exogenous spermidine in melon seedlings,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),36(1):85-92(刘长命,杨瑞平,莫言玲,王永琦,郑俊鶱,张显,2016,外源Spd预处理对甜瓜白粉病抗性及其内源多胺的诱导分析,西北植物学报,36(1):85-92)
Perchepied L.,Bardin M.,Dogimont C.,and Pitrat M.,2005,Relationship between Loci conferring downy mildew and powdery mildew resistance in melon assessed by quantitative trait Loci mapping,Phytopathology,95(5):556-565
Rao X.Y.,Huang X.L.,Zhou Z.C.,and Lin X.,2013,An improvement of the 2魮(-delta delta CT)method for quantitative real-time polymerase chain reaction data analysis,Biostat Bioinforma Biomath.,3(3):71-85
Sautel C.F.,Ortet P.,Saksouk N.,Kieffer S.,Garin J.,Bastien O.,and Hakimi M.A.,2009,The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences,Mol.Microbiol.,71(1):212-226
Sharma S.S.,and Dietz K.J.,2006,The significance of amino acids and amino acid-derived molecules in plant response and adaptation to heavy stress,J.Exp.Bot.,57(4):711-726
Tao Y.,Xie Z.Y.,Chen W.Q.,Glazebrook J.,Chang H.S.,Han B.,Zhu T.,Zou G.Z.,and Katagiri F.,2003,Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen pathogen Pseudomonas syringae,Plant Cell,15(2):317-330
Wu J.,Zhang Y.L.,Zhang H.Q.,Huang H.,Folta K.M.,and Lu J.,2010,Whole genome wide expression profiles of Vitis amurensis grape responding to downy mildew by using Solexa sequencing technology,BMC Plant Biol.,10:234-250
Yu S.C.,Zhang F.L.,Yu Y.J.,Zhang D.S.,Zhao X.Y.,and Wang W.H.,2012,Transcriptome profiling of dehydration stress in the Chinese cabbage(Brassica rapa L.ssp.pekinensis)by tag sequencing,Plant Mol.Biol.Rep.,30(1):17-28