蒙古扁桃菌根苗对干旱胁迫的分子响应机制
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摘要
【目的】探明菌根对蒙古扁桃(Prunus mongolica)抗旱能力影响的分子机制。【方法】对生长45 d的菌根化蒙古扁桃与非菌根化蒙古扁桃进行非干旱胁迫和干旱胁迫处理,非干旱胁迫蒙古扁桃在处理期间每天补充水分;干旱胁迫处理蒙古扁桃从培育45 d开始停止浇水,模拟自然干旱胁迫,持续时间15 d。试验结束后,每个处理选12株进行叶长、叶宽、叶片脱落数及生物量的测定和统计;另选12株采用高通量测序方法进行转录组测序,并对其差异表达转录本进行GO和KEGG富集分析。【结果】干旱胁迫条件下,菌根化苗木底部的一些叶片会脱落,而非菌根化苗木叶片几乎不脱落;同时,菌根化苗木的地下生物量显著高于非菌根化苗木。通过高通量测序发现,4个处理文库共获得43 641个转录本;在P<0.001时,菌根化蒙古扁桃干旱胁迫(MD)与非干旱胁迫(MCK)处理相比,存在820个差异表达转录本;干旱胁迫条件下,菌根化苗木(MD)与非菌根化苗木(ND)相比,存在3 751个差异表达转录本;非干旱胁迫条件下,菌根化苗木(MCK)与非菌根化苗木(NCK)相比,存在2 315个差异表达转录本。GO富集分析发现,MD与ND处理文库间的细胞组分、分子功能和生化过程3类主要功能分类的差异表达转录本,较MD与MCK处理文库间均增加;MCK与NCK处理文库间差异转录本的分类结果同MD与ND处理文库间基本相同,只是在分子功能分类中多出通道调节活性这一功能。经KEGG富集分析发现,天线蛋白、类胡萝卜素生物合成途径、激素信号传导途径、N代谢途径、过氧化物酶体、植物昼夜节律和MAPK信号途径等与干旱胁迫存在密切联系。实时荧光定量PCR表明转录组测序数据可靠。【结论】菌根化处理可以提高蒙古扁桃的抗旱能力。
【Objective】 This study aimed at exploring the molecular mechanism of mycorrhiza on drought resistance of Prunus mongolica.【Method】 The mycorrhizal P. mongolica and non-mycorrhizal P. mongolica almonds were treated with non-drought stress and drought stress for 45 days.The non-drought stress seedlings were replenished with water every day during the treatment to maintain the maximum water holding capacity(48.7%).The watering for seedlings in drought stress treatment were stopped 45 days after cultivation,and the natural drought stress was simulated for 15 days.After 60 days,twelve seedlings were selected from each treatment to measure and count leaf length,leaf width,leaf abscission number and biomass.Twelve seedlings were sequenced by high-throughput sequencing and their differentially expressed transcripts were analyzed by GO and KEGG enrichments.【Result】 Under drought stress,some leaves at the bottom of mycorrhizal seedlings fell off,while non-mycorrhizal seedlings barely fell off.The underground biomass of mycorrhizal seedlings was significantly higher than that of non-mycorrhizal seedlings.A total of 43 641 transcripts were obtained from the four processed libraries by high-throughput sequencing.At P<0.001,there were 820 differentially expressed transcripts in mycorrhizal P. mongolica under drought stress(MD) and non-drought stress(MCK) conditions.There were 3 751 differentially expressed transcripts compared to MD and non-mycorrhizal(ND) seedlings under drought stress.There were 2 315 differentially expressed transcripts in MCK and non-mycorrhizal seedlings(NCK) under non-drought stress conditions.GO enrichment analysis showed that the differential expression transcripts of the three major functional categories of cellular components,molecular functions and biological processes were increased between the MD and ND processing libraries compared to the MD and MCK processing libraries.The classification results of differential transcripts between MCK and NCK processing libraries were basically the same between MD and ND,but it only had a function of channel regulation activity in the molecular function classification.The KEGG enrichment analysis showed that antenna protein,carotenoid biosynthetic pathway,hormone signaling pathway,nitrogen metabolic pathway,peroxisomes,circadian rhythm-plant and MAPK signaling pathway were closely related to drought stress.Real time quantitative PCR showed that transcriptome sequencing data were reliable.【Conclusion】 Mycorrhiza can improve the drought resistance of P. mongolica.
【Objective】 This study aimed at exploring the molecular mechanism of mycorrhiza on drought resistance of Prunus mongolica.【Method】 The mycorrhizal P. mongolica and non-mycorrhizal P. mongolica almonds were treated with non-drought stress and drought stress for 45 days.The non-drought stress seedlings were replenished with water every day during the treatment to maintain the maximum water holding capacity(48.7%).The watering for seedlings in drought stress treatment were stopped 45 days after cultivation,and the natural drought stress was simulated for 15 days.After 60 days,twelve seedlings were selected from each treatment to measure and count leaf length,leaf width,leaf abscission number and biomass.Twelve seedlings were sequenced by high-throughput sequencing and their differentially expressed transcripts were analyzed by GO and KEGG enrichments.【Result】 Under drought stress,some leaves at the bottom of mycorrhizal seedlings fell off,while non-mycorrhizal seedlings barely fell off.The underground biomass of mycorrhizal seedlings was significantly higher than that of non-mycorrhizal seedlings.A total of 43 641 transcripts were obtained from the four processed libraries by high-throughput sequencing.At P<0.001,there were 820 differentially expressed transcripts in mycorrhizal P. mongolica under drought stress(MD) and non-drought stress(MCK) conditions.There were 3 751 differentially expressed transcripts compared to MD and non-mycorrhizal(ND) seedlings under drought stress.There were 2 315 differentially expressed transcripts in MCK and non-mycorrhizal seedlings(NCK) under non-drought stress conditions.GO enrichment analysis showed that the differential expression transcripts of the three major functional categories of cellular components,molecular functions and biological processes were increased between the MD and ND processing libraries compared to the MD and MCK processing libraries.The classification results of differential transcripts between MCK and NCK processing libraries were basically the same between MD and ND,but it only had a function of channel regulation activity in the molecular function classification.The KEGG enrichment analysis showed that antenna protein,carotenoid biosynthetic pathway,hormone signaling pathway,nitrogen metabolic pathway,peroxisomes,circadian rhythm-plant and MAPK signaling pathway were closely related to drought stress.Real time quantitative PCR showed that transcriptome sequencing data were reliable.【Conclusion】 Mycorrhiza can improve the drought resistance of P. mongolica.
引文
[1] 王进,张勇,赵刚,等.蒙古扁桃种子萌发和幼苗生长对渗透胁迫的响应 [J].干旱地区农业研究,2014,32(1):191-195.Wang J,Zhang Y,Zhao G,et al.Response of mongolian almond seed germination and seedling growth to osmotic stress [J].Agricultural Research in the Arid Areas,2014,32(1):191-195.
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[3] Smith S E,Read D J.Mycorrhizal symbiosis [M].New York:Academic Press,2008.
[4] Smith S E,Facellii E,Pope S,et al.Plant performance in stressful environments:interpreting new and established knowledge of the roles of arbuscular mycorrhizas [J].Plant and Soil,2010,326(1/2):3-20.
[5] Jayne B,Quigley M.Influence of arbuscular mycorrhiza on growth and reproductive response of plants under water deficit:a meta-analysis [J].Mycorrhiza,2014,24(2):109-119.
[6] Li T,Hu Y J,Hao Z P,et al.First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices [J].New Phytologist,2013,197(2):617-630.
[7] 王琚钢,高晓敏,白淑兰,等.丛枝菌根对蒙古扁桃抗旱性影响研究 [J].干旱区资源与环境,2014,28(12):138-142.Wang J G,Gao X M,Bai S L,et al.Effects of arbuscular mycorrhiza on the drought tolerance of Prunus mongolica [J].Journal of Arid Land Resources and Environment,2014,28(12):138-142.
[8] Alimohammadi A,Shiran B,Martínez-gómez P,et al.Identification of water-deficit resistance genes in wild almond (Prunus scoparia) using cDNA-AFLP [J].Scientia Horticulturae,2013,159(7):19-28.
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[10] Tisserant E,Kohler A,Dozolme-seddas P,et al.The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont [J].New Phytologist,2012,193(3):755-769.
[11] 王琚钢.基于高通量测序技术分析菌根对蒙古扁桃抗旱机制的影响 [D].呼和浩特:内蒙古农业大学,2015.Wang J G.Analysis the influence of mycorrhizae on the drought resistance mechanisms of Mongolian Almond (Prunus mongolica Maxim) by using the next generation sequencing technology [D].Hohhot:Inner Mongolia Agricultural University,2015.
[12] 刘润进,陈应龙.菌根学 [M].北京:科学出版社,2007.Liu R J,Chen Y L.Mycorrhizal [M].Beijing:Science Press,2007.
[13] 刘洪博,刘新龙,苏火生,等.干旱胁迫下割手密根系转录组差异表达分析 [J].中国农业科学,2017,50(6):1167-1178.Liu H B,Liu X L,Su H S,et al.Transcriptome difference analysis of Saccharum spontaneum roots in response to drought stress [J].Scientia Agricultura Sinica,2017,50(6):1167-1178.
[14] Langmead B,Salzberg S L.Fast gapped-read alignment with Bowtie 2 [J].Nature Methods,2012,9(4):357-359.
[15] Robinson M D,Mccarthy D J,Smyth G K.EdgeR:a bioconductor package for differential expression analysis of digital gene expression data [J].Bioinformatics,2010,26(1):139-140.
[16] Consortium U.The universal protein resource(UniProt) [J].Nucleic Acids Research,2008,36(Suppl1):D190-D195.
[17] Conesa A,G?tz S,García-gómez J M,et al.Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research [J].Bioinformatics,2005,21(18):3674-3676.
[18] Verda I,Abbott A G,Scalabrin S,et al.The high-quality draft genome of peach(Prunus persica) identifies unique patterns of genetic diversity,domestication and genome evolution [J].Nature Genetics,2013,45(5):487-494.
[19] Zhang Q,Chen W,Sun L,et al.The genome of Prunus mume [J].Nature Communications,2012,3(4):1318.
[20] Tang S,Liang H,Yan D,et al.Populus euphratica:the transcriptomic response to drought stress [J].Plant Molecular Biology,2013,83(6):539-557.
[21] Peng S,Jiang H,Zhang S,et al.Transcriptional profiling reveals sexual differences of the leaf transcriptomes in response to drought stress in Populus yunnanensis [J].Tree Physiology,2012,32(12):1541-1555.
[22] Rao D E,Chaitanya K V.Photosynthesis and antioxidative defense mechanisms in deciphering drought stress tolerance of crop plants [J].Biologia Plantarum,2016,60(2):1-18.
[23] Zhang J,Jia W,Yang J,et al.Role of ABA in integrating plant responses to drought and salt stresses [J].Field Crops Research,2006,97(1):111-119.
[24] Courtney,Lappas.The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation [J].Cellular & Molecular Immunology,2015,12(1):107-112.
[25] Miransari M.Arbuscular mycorrhizal fungi and nitrogen uptake [J].Archives of Microbiology,2011,193(2):77-81.
[26] 刘敏.灌木铁线莲根围AMF多样性及其菌根苗对干旱胁迫的响应机制 [D].呼和浩特:内蒙古农业大学,2017.Liu M.Research on the arbuscular mycorrhizal fungal diversity in the rhizosphere of Clematis fruticosa and the mechanisms of mycorrhizal seedings response to drought stress [D].Hohhot:Inner Mongolia Agricultural University,2017.
[27] Rivero R M,Mestre T C,Mittler R,et al.The combined effect of salinity and heat reveals a specific physiological,biochemical and molecular response in tomato plants [J].Plant Cell & Environment,2014,37(5):1059-1073.
[28] Filippou P,Bouchagier P,Skotti E,et al.Proline and reactive oxygen/nitrogen species metabolism is involved in the tolerant response of the invasive plant species Ailanthus altissima to drought and salinity [J].Environmental & Experimental Botany,2014,97(1):1-10.
[29] Kishor P B K,Sangam S,Amrutha R N,et al.Regulation of proline biosynthesis,degradation,uptake and transport in higher plants:its implications in plant growth and abiotic stress tolerance [J].Current Science,2005,54(4):271-275.
[30] Mo Y,Wang Y,Yang R,et al.Regulation of plant growth,photosynthesis,antioxidation and osmosis by an arbuscular mycorrhizal fungus in watermelon seedlings under well-watered and drought conditions [J].Frontiers in Plant Science,2016,7(454):1-15.
[31] Zou J J,Li X D,Ratnasekera D,et al.Arabidopsis calcium-dependent protein kinase 8 and catalase 3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress [J].Plant Cell,2015,27(5):1445-1460.
[32] Hammer E C,Nasr H,Pallon J,et al.Elemental composition of arbuscular mycorrhizal fungi at high salinity [J].Mycorrhiza,2011,21(2):117-129.
[2] 王进,马国泰,宋涛,等.干旱、半干旱地区蒙古扁桃种子萌发对土壤水分和播种深度的响应特征 [J].冰川冻土,2014,36(5):1313-1320.Wang J,Ma G T,Song T,et al.The response characteristics of germination of Prunus mongolica seed to soil moisture and sowing depth in the arid and semi-arid regions [J].Journal of Glaciology and Geocryology,2014,36(5):1313-1320.
[3] Smith S E,Read D J.Mycorrhizal symbiosis [M].New York:Academic Press,2008.
[4] Smith S E,Facellii E,Pope S,et al.Plant performance in stressful environments:interpreting new and established knowledge of the roles of arbuscular mycorrhizas [J].Plant and Soil,2010,326(1/2):3-20.
[5] Jayne B,Quigley M.Influence of arbuscular mycorrhiza on growth and reproductive response of plants under water deficit:a meta-analysis [J].Mycorrhiza,2014,24(2):109-119.
[6] Li T,Hu Y J,Hao Z P,et al.First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices [J].New Phytologist,2013,197(2):617-630.
[7] 王琚钢,高晓敏,白淑兰,等.丛枝菌根对蒙古扁桃抗旱性影响研究 [J].干旱区资源与环境,2014,28(12):138-142.Wang J G,Gao X M,Bai S L,et al.Effects of arbuscular mycorrhiza on the drought tolerance of Prunus mongolica [J].Journal of Arid Land Resources and Environment,2014,28(12):138-142.
[8] Alimohammadi A,Shiran B,Martínez-gómez P,et al.Identification of water-deficit resistance genes in wild almond (Prunus scoparia) using cDNA-AFLP [J].Scientia Horticulturae,2013,159(7):19-28.
[9] Mousavi S,Alisoltani A,Shiran B,et al.De novo transcriptome assembly and comparative analysis of differentially expressed genes in Prunus dulcis Mill.in response to freezing stress [J].PLoS One,2014,9(8):e104541.
[10] Tisserant E,Kohler A,Dozolme-seddas P,et al.The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont [J].New Phytologist,2012,193(3):755-769.
[11] 王琚钢.基于高通量测序技术分析菌根对蒙古扁桃抗旱机制的影响 [D].呼和浩特:内蒙古农业大学,2015.Wang J G.Analysis the influence of mycorrhizae on the drought resistance mechanisms of Mongolian Almond (Prunus mongolica Maxim) by using the next generation sequencing technology [D].Hohhot:Inner Mongolia Agricultural University,2015.
[12] 刘润进,陈应龙.菌根学 [M].北京:科学出版社,2007.Liu R J,Chen Y L.Mycorrhizal [M].Beijing:Science Press,2007.
[13] 刘洪博,刘新龙,苏火生,等.干旱胁迫下割手密根系转录组差异表达分析 [J].中国农业科学,2017,50(6):1167-1178.Liu H B,Liu X L,Su H S,et al.Transcriptome difference analysis of Saccharum spontaneum roots in response to drought stress [J].Scientia Agricultura Sinica,2017,50(6):1167-1178.
[14] Langmead B,Salzberg S L.Fast gapped-read alignment with Bowtie 2 [J].Nature Methods,2012,9(4):357-359.
[15] Robinson M D,Mccarthy D J,Smyth G K.EdgeR:a bioconductor package for differential expression analysis of digital gene expression data [J].Bioinformatics,2010,26(1):139-140.
[16] Consortium U.The universal protein resource(UniProt) [J].Nucleic Acids Research,2008,36(Suppl1):D190-D195.
[17] Conesa A,G?tz S,García-gómez J M,et al.Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research [J].Bioinformatics,2005,21(18):3674-3676.
[18] Verda I,Abbott A G,Scalabrin S,et al.The high-quality draft genome of peach(Prunus persica) identifies unique patterns of genetic diversity,domestication and genome evolution [J].Nature Genetics,2013,45(5):487-494.
[19] Zhang Q,Chen W,Sun L,et al.The genome of Prunus mume [J].Nature Communications,2012,3(4):1318.
[20] Tang S,Liang H,Yan D,et al.Populus euphratica:the transcriptomic response to drought stress [J].Plant Molecular Biology,2013,83(6):539-557.
[21] Peng S,Jiang H,Zhang S,et al.Transcriptional profiling reveals sexual differences of the leaf transcriptomes in response to drought stress in Populus yunnanensis [J].Tree Physiology,2012,32(12):1541-1555.
[22] Rao D E,Chaitanya K V.Photosynthesis and antioxidative defense mechanisms in deciphering drought stress tolerance of crop plants [J].Biologia Plantarum,2016,60(2):1-18.
[23] Zhang J,Jia W,Yang J,et al.Role of ABA in integrating plant responses to drought and salt stresses [J].Field Crops Research,2006,97(1):111-119.
[24] Courtney,Lappas.The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation [J].Cellular & Molecular Immunology,2015,12(1):107-112.
[25] Miransari M.Arbuscular mycorrhizal fungi and nitrogen uptake [J].Archives of Microbiology,2011,193(2):77-81.
[26] 刘敏.灌木铁线莲根围AMF多样性及其菌根苗对干旱胁迫的响应机制 [D].呼和浩特:内蒙古农业大学,2017.Liu M.Research on the arbuscular mycorrhizal fungal diversity in the rhizosphere of Clematis fruticosa and the mechanisms of mycorrhizal seedings response to drought stress [D].Hohhot:Inner Mongolia Agricultural University,2017.
[27] Rivero R M,Mestre T C,Mittler R,et al.The combined effect of salinity and heat reveals a specific physiological,biochemical and molecular response in tomato plants [J].Plant Cell & Environment,2014,37(5):1059-1073.
[28] Filippou P,Bouchagier P,Skotti E,et al.Proline and reactive oxygen/nitrogen species metabolism is involved in the tolerant response of the invasive plant species Ailanthus altissima to drought and salinity [J].Environmental & Experimental Botany,2014,97(1):1-10.
[29] Kishor P B K,Sangam S,Amrutha R N,et al.Regulation of proline biosynthesis,degradation,uptake and transport in higher plants:its implications in plant growth and abiotic stress tolerance [J].Current Science,2005,54(4):271-275.
[30] Mo Y,Wang Y,Yang R,et al.Regulation of plant growth,photosynthesis,antioxidation and osmosis by an arbuscular mycorrhizal fungus in watermelon seedlings under well-watered and drought conditions [J].Frontiers in Plant Science,2016,7(454):1-15.
[31] Zou J J,Li X D,Ratnasekera D,et al.Arabidopsis calcium-dependent protein kinase 8 and catalase 3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress [J].Plant Cell,2015,27(5):1445-1460.
[32] Hammer E C,Nasr H,Pallon J,et al.Elemental composition of arbuscular mycorrhizal fungi at high salinity [J].Mycorrhiza,2011,21(2):117-129.
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