PEG模拟干旱胁迫下野生大豆转录组分析
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摘要
为研究耐旱型野生大豆在干旱胁迫下基因表达谱的变化,从备选野生大豆材料中筛选抗旱性强的野生大豆品种,同时探讨最适PEG胁迫浓度,而后以野生大豆永46为试验材料,利用RNA-seq技术对20%PEG6000处理不同时间的叶片进行基因表达谱差异分析。结果显示:获得39 183个序列信息,其中各时期共有序列27 875个。随干旱胁迫时间延长,差异表达基因数量发生变化,胁迫12 h达到最多。根据GO功能分析可将序列大致分为分子功能、细胞成分和生物学过程三大类,其差异表达基因广泛涉及糖、脂类、蛋白质和核酸等生物大分子代谢、能量代谢以及次生代谢过程。在KEGG数据库中,依据代谢途径可将其定位在127个分支,包括植物-病原体互作、植物激素信号转导、RNA降解、ABC转运蛋白等,其中植物激素信号转导途径在不同时间处理下的变化都显著。转录因子分析发现在干旱胁迫下变化明显的转录因子家族包括MYB、bHLH、AP2\EREBP、WRKY和NAC等。对4个不同功能基因干旱胁迫后不同时间的表达量进行荧光定量分析,其变化趋势与转录组数据相同。
In order to estimate the gene expression profiles affected by drought stress,a drought-resistant wild soybean accession Yong 46 was used to detect the differentially expressed genes under 20% PEG6000 by RNA-seq technology. A total of 39183 unigenes were screened out,of which 27 875 differentially expressed genes( DEGs) were coincident. All the assembled unigenes could be broadly divided into cellular component,molecular function and biological process by gene ontology,these DEGs were involved in metabolisms of carbohydrate,lipid,protein,nucleic acid and energy,and secondary metabolism. 127 classes according to their metabolic pathway were found by the analyze of Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway,including the plant-pathogen interaction,plant hormone signal transduction,RNA degradation and ABC transporters. The pathway of plant hormone signal transduction changed the most obviously under different times. The most evident change transcription factor families included MYB,bHLH,AP2-EREBP,WRKY and NAC under drought stress. The results of qRT-PCR were in conformity with the RNA-seq. These results provided a base for further study on drought stress functional genes screening in wild soybean.
In order to estimate the gene expression profiles affected by drought stress,a drought-resistant wild soybean accession Yong 46 was used to detect the differentially expressed genes under 20% PEG6000 by RNA-seq technology. A total of 39183 unigenes were screened out,of which 27 875 differentially expressed genes( DEGs) were coincident. All the assembled unigenes could be broadly divided into cellular component,molecular function and biological process by gene ontology,these DEGs were involved in metabolisms of carbohydrate,lipid,protein,nucleic acid and energy,and secondary metabolism. 127 classes according to their metabolic pathway were found by the analyze of Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway,including the plant-pathogen interaction,plant hormone signal transduction,RNA degradation and ABC transporters. The pathway of plant hormone signal transduction changed the most obviously under different times. The most evident change transcription factor families included MYB,bHLH,AP2-EREBP,WRKY and NAC under drought stress. The results of qRT-PCR were in conformity with the RNA-seq. These results provided a base for further study on drought stress functional genes screening in wild soybean.
引文
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[3] Lam H M,Xu X,Liu X,et al. Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection[J]. Nature Genetics,2010,42(12):1053-1059.
[4] Kim M Y,Lee S,van K,et al. Whole-genome sequencing and intensive analysis of the undomesticated soybean(Glycine soja Sieb.and Zucc.)genome[J]. Proceedings of the National Academy of Sciences of the United States of America,2010,107:22032-22037.
[5]王克晶,李向华.国家基因库野生大豆(Glycine soja)资源最近十年考察与研究[J].植物遗传资源学报,2012(4):507-514.(Wang K J,Li X H. Exploration and studies of wild soybean germplasm resources in the China genebank during recent decade[J]. Journal of Plant Genetic Resources,2012(4):507-514.)
[6] KosováK,Vítámvás P,Urban M O,et al. Plant abiotic stress proteomics:The major factors determining alterations in cellular proteome[J]. Frontiers in Plant Science,2018,9:122.
[7] Teng Z Q,Hui-Qing F U,Jia S H,et al. Review of current progress in the metabolomics for plant response to abiotic stress[J].Crop Research,2011,35(1):110-118.
[8] Fan X D,Wang J Q,Yang N,et al. Gene expression profiling of soybean leaves and roots under salt,saline-alkali and drought stress by high-throughput Illumina sequencing[J]. Gene,2013,512(2):392-402.
[9] Wang X,Oh M,Sakata K,et al. Gel-free/label-free proteomic analysis of root tip of soybean over time under flooding and drought stresses[J]. Journal of Proteomics,2016,130:42.
[10] Berretta J,Morillon A. Pervasive transcription constitutes a new level of eukaryotic genome regulation[J]. Embo Reports,2009,10(9):973-982.
[11] Liu X,Lyu S,Liu R,et al. Transcriptomic analysis reveals the roles of gibberellin-regulated genes and tran-scription factors in regulating bolting in lettuce(Lactuca sativa L.)[J]. Plos One,2018,13(2):e0191518.
[12] Gao C,Wang Y,Liu G,et al. Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamar&hispid[J]. Plant Molecular Biology,2008,66(3):245-258.
[13]姚敏磊,张璟曜,周汐,等.大豆响应低磷胁迫的数字基因表达谱分析[J].大豆科学,2016,35(2):213-221.(Yao M L,Zhang J Y,Zhou X,et al. The digital gene expression profiling analysis of genes in response to low phosphorus stress in soybean[J]. Soybean Science,2016,35(2):213-221.)
[14]任梦露,刘卫国,刘婷,等.荫蔽胁迫下大豆茎秆形态建成的转录组分析[J].作物学报,2016,42(9):1319-1331.(Ren M L,Liu W G,Liu T,et al. Transcriptome analysis of stem morphogenesis under shade stress in soybean[J]. Acta Automatica Sinica,2016,42(9):1319-1331.)
[15]张晓娜,朴春兰,董友魁,等.大豆根系应答重金属Cd胁迫的转录组分析[J].应用生态学报,2017,28(5):1633-1641.(Zhang X N,Piao C L,Dong Y K,et al. Transcriptome analysis of response to heavy metal Cd stress in soybean root[J]. Chinese Journal of Applied Ecology,2017,28(5):1633-1641.)
[16] Liu A,Xiao Z,Li M W,et al. Transcriptomic reprogramming in soybean seedlings under salt stress[J]. Plant Cell&Environment,2018,12(12):e0189159.
[17]吴倩,张磊,黄志平,等.转录组测序及其在野生大豆基因资源发掘中的应用[J].大豆科学,2013,32(6):845-851.(Wu Q,Zhang L,Huang Z P,et al. Transcription sequencing and its application on discovering the gene resources of wild soybean[J].Soybean Science,2013,32(6):845-851.)
[18] Wei C,Yao Q,Patil G B,et al. Identification and comparative analysis of differential gene expression in soybean leaf tissue under drought and flooding stress revealed by RNA-Seq[J]. Frontiers in Plant Science,2016,7:1044.
[19] Carter T E Jr,de Souza P I,Purcell L C. Recent advances in breeding for drought and aluminum resistance in soybean[C].Chicago:Proceedings of World Soybean Research Conference VI,1999:106-125.
[20]蒲伟凤.不同类型大豆种质资源抗旱性比较及野生大豆抗旱性筛选[D].秦皇岛:河北科技师范学院,2010.(Pu W F.Comparison of drought resistance in different soybean types and screening of drought resistance in Glycine soja[D]. Qinhuangdao:Hebei Normal University of Science and Technology,2010.)
[21]乔亚科,李桂兰,高书国,等.昌黎沿海野生大豆分布及其耐盐性[J].河北职业技术师范学院学报,2001(2):9-13.(Qiao Y K,Li G L,Gao S G,et al. Geographical distribution and salttolerance of wild soybean(Glycine soja)in inshore regions in Changli Hebei province[J]. Journal of Hebei Vocation-Technical Teachers College,2001(2):9-13.)
[22]王丹,乔亚科,韩粉霞,等.河北东部沿海地区野生大豆SSR多样性分析[J].大豆科学,2010,29(4):555-558.(Wang D,Qiao Y K,Han F X,et al. Diversity of Glycine soja in eastern coastal area of Hebei province[J]. Soybean Science,2010,29(4):555-558.)
[23] Zhang X,Jiang H,Wang H,et al. Transcriptome analysis of rice seedling roots in response to potassium deficiency[J]. Scientific Reports,2017,7(1):5523.
[24] Prince S J,Joshi T,Mutava R N,et al. Comparative analysis of the drought-responsive transcriptome in soybean lines contrasting for canopy wilting[J]. Plant Science,2015,240:65-78.
[25]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.)
[26]刘振山.小麦苗期干旱、高温和旱热共胁迫转录表达谱及ABD部分同源基因表达分化分析[D].北京:中国农业大学,2015.(Liu Z S. Transcriptome profiling and differential homeologous genes expression analysis of wheat(Triticum aestivum L.)seedlings during drought stress,heat stress and their[D]. Beijing:China Agricultural University,2015.)
[27] Fujita Y,Fujita M,Satoh R,et al. AREB1 is a transcription activator of novel ABRE-dependent ABA signal-ing that enhances drought stress tolerance in Arabidopsis[J]. Plant Cell,2005,17(12):3470-3488.
[28]周宏.桑树抗旱相关4个转录因子家族鉴定与表达分析[D].镇江:江苏科技大学,2017.(Zhou H. Identification and expression analysis of drought-resistant related 4 transcription factor families in mulberry(Morus L.)[D]. Zhenjiang:Jiangsu University of Science and Technology,2017.)
[29] Jiang J J,Ma S H,Ye N H,et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology,2017,59(2):86-101.
[30] Zhou Q Y,Tian A G,Zou H F,et al. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J]. Plant Biotechnology Journal,2008,6(5):486-503.
[31]刘鑫,邹郁陶,牟巍,等.玉米乙烯应答元件结合蛋白基因启动子克隆与功能验证[J].核农学报,2016,30(4):629-637.(Liu X,Zou Y T,Mu W,et al. Cloning and functional validation of promoter of ethylene-responsive element-binding protein gene in maize[J]. Journal of Nuclear Agricultural Sciences,2016,30(4):629-637.)
[32] Hu W,Hou X,Huang C,et al. Genome-wide identification and expression analyses of aquaporin gene family during development and abiotic stress in banana[J]. International Journal of Molecular Sciences,2015,16(8):19728-19751.
[33] Guerrero F D,Jones J T,Mullet J E. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes[J]. Plant Molecular Biology,1990,15(1):11.
[34]叶国良,宋娟娟,叶清.两种不同抗旱能力的豇豆根系水通道蛋白基因的克隆与分析[J].分子植物育种,2016,14(8):1977-1985.(Ye G L,Song J J,Ye Q. Cloning and analysis of aquaporins in roots of two cow pea(Vigna unguiculata L. Walp.)cultivars with contrasting drought tolerance[J]. Molecular Plant Breeding,2016,14(8):1977-1985.)
[2]杨如萍,包振贤,陈光荣,等.大豆抗旱性研究进展[J].作物杂志,2012(5):8-12.(Yang R P,Bao Z X,Chen G R,et al.The research progress in drought resistance of soybean[J]. Crops,2012(5):8-12.)
[3] Lam H M,Xu X,Liu X,et al. Resequencing of 31 wild and cultivated soybean genomes identifies patterns of genetic diversity and selection[J]. Nature Genetics,2010,42(12):1053-1059.
[4] Kim M Y,Lee S,van K,et al. Whole-genome sequencing and intensive analysis of the undomesticated soybean(Glycine soja Sieb.and Zucc.)genome[J]. Proceedings of the National Academy of Sciences of the United States of America,2010,107:22032-22037.
[5]王克晶,李向华.国家基因库野生大豆(Glycine soja)资源最近十年考察与研究[J].植物遗传资源学报,2012(4):507-514.(Wang K J,Li X H. Exploration and studies of wild soybean germplasm resources in the China genebank during recent decade[J]. Journal of Plant Genetic Resources,2012(4):507-514.)
[6] KosováK,Vítámvás P,Urban M O,et al. Plant abiotic stress proteomics:The major factors determining alterations in cellular proteome[J]. Frontiers in Plant Science,2018,9:122.
[7] Teng Z Q,Hui-Qing F U,Jia S H,et al. Review of current progress in the metabolomics for plant response to abiotic stress[J].Crop Research,2011,35(1):110-118.
[8] Fan X D,Wang J Q,Yang N,et al. Gene expression profiling of soybean leaves and roots under salt,saline-alkali and drought stress by high-throughput Illumina sequencing[J]. Gene,2013,512(2):392-402.
[9] Wang X,Oh M,Sakata K,et al. Gel-free/label-free proteomic analysis of root tip of soybean over time under flooding and drought stresses[J]. Journal of Proteomics,2016,130:42.
[10] Berretta J,Morillon A. Pervasive transcription constitutes a new level of eukaryotic genome regulation[J]. Embo Reports,2009,10(9):973-982.
[11] Liu X,Lyu S,Liu R,et al. Transcriptomic analysis reveals the roles of gibberellin-regulated genes and tran-scription factors in regulating bolting in lettuce(Lactuca sativa L.)[J]. Plos One,2018,13(2):e0191518.
[12] Gao C,Wang Y,Liu G,et al. Expression profiling of salinity-alkali stress responses by large-scale expressed sequence tag analysis in Tamar&hispid[J]. Plant Molecular Biology,2008,66(3):245-258.
[13]姚敏磊,张璟曜,周汐,等.大豆响应低磷胁迫的数字基因表达谱分析[J].大豆科学,2016,35(2):213-221.(Yao M L,Zhang J Y,Zhou X,et al. The digital gene expression profiling analysis of genes in response to low phosphorus stress in soybean[J]. Soybean Science,2016,35(2):213-221.)
[14]任梦露,刘卫国,刘婷,等.荫蔽胁迫下大豆茎秆形态建成的转录组分析[J].作物学报,2016,42(9):1319-1331.(Ren M L,Liu W G,Liu T,et al. Transcriptome analysis of stem morphogenesis under shade stress in soybean[J]. Acta Automatica Sinica,2016,42(9):1319-1331.)
[15]张晓娜,朴春兰,董友魁,等.大豆根系应答重金属Cd胁迫的转录组分析[J].应用生态学报,2017,28(5):1633-1641.(Zhang X N,Piao C L,Dong Y K,et al. Transcriptome analysis of response to heavy metal Cd stress in soybean root[J]. Chinese Journal of Applied Ecology,2017,28(5):1633-1641.)
[16] Liu A,Xiao Z,Li M W,et al. Transcriptomic reprogramming in soybean seedlings under salt stress[J]. Plant Cell&Environment,2018,12(12):e0189159.
[17]吴倩,张磊,黄志平,等.转录组测序及其在野生大豆基因资源发掘中的应用[J].大豆科学,2013,32(6):845-851.(Wu Q,Zhang L,Huang Z P,et al. Transcription sequencing and its application on discovering the gene resources of wild soybean[J].Soybean Science,2013,32(6):845-851.)
[18] Wei C,Yao Q,Patil G B,et al. Identification and comparative analysis of differential gene expression in soybean leaf tissue under drought and flooding stress revealed by RNA-Seq[J]. Frontiers in Plant Science,2016,7:1044.
[19] Carter T E Jr,de Souza P I,Purcell L C. Recent advances in breeding for drought and aluminum resistance in soybean[C].Chicago:Proceedings of World Soybean Research Conference VI,1999:106-125.
[20]蒲伟凤.不同类型大豆种质资源抗旱性比较及野生大豆抗旱性筛选[D].秦皇岛:河北科技师范学院,2010.(Pu W F.Comparison of drought resistance in different soybean types and screening of drought resistance in Glycine soja[D]. Qinhuangdao:Hebei Normal University of Science and Technology,2010.)
[21]乔亚科,李桂兰,高书国,等.昌黎沿海野生大豆分布及其耐盐性[J].河北职业技术师范学院学报,2001(2):9-13.(Qiao Y K,Li G L,Gao S G,et al. Geographical distribution and salttolerance of wild soybean(Glycine soja)in inshore regions in Changli Hebei province[J]. Journal of Hebei Vocation-Technical Teachers College,2001(2):9-13.)
[22]王丹,乔亚科,韩粉霞,等.河北东部沿海地区野生大豆SSR多样性分析[J].大豆科学,2010,29(4):555-558.(Wang D,Qiao Y K,Han F X,et al. Diversity of Glycine soja in eastern coastal area of Hebei province[J]. Soybean Science,2010,29(4):555-558.)
[23] Zhang X,Jiang H,Wang H,et al. Transcriptome analysis of rice seedling roots in response to potassium deficiency[J]. Scientific Reports,2017,7(1):5523.
[24] Prince S J,Joshi T,Mutava R N,et al. Comparative analysis of the drought-responsive transcriptome in soybean lines contrasting for canopy wilting[J]. Plant Science,2015,240:65-78.
[25]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.)
[26]刘振山.小麦苗期干旱、高温和旱热共胁迫转录表达谱及ABD部分同源基因表达分化分析[D].北京:中国农业大学,2015.(Liu Z S. Transcriptome profiling and differential homeologous genes expression analysis of wheat(Triticum aestivum L.)seedlings during drought stress,heat stress and their[D]. Beijing:China Agricultural University,2015.)
[27] Fujita Y,Fujita M,Satoh R,et al. AREB1 is a transcription activator of novel ABRE-dependent ABA signal-ing that enhances drought stress tolerance in Arabidopsis[J]. Plant Cell,2005,17(12):3470-3488.
[28]周宏.桑树抗旱相关4个转录因子家族鉴定与表达分析[D].镇江:江苏科技大学,2017.(Zhou H. Identification and expression analysis of drought-resistant related 4 transcription factor families in mulberry(Morus L.)[D]. Zhenjiang:Jiangsu University of Science and Technology,2017.)
[29] Jiang J J,Ma S H,Ye N H,et al. WRKY transcription factors in plant responses to stresses[J]. Journal of Integrative Plant Biology,2017,59(2):86-101.
[30] Zhou Q Y,Tian A G,Zou H F,et al. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54,confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants[J]. Plant Biotechnology Journal,2008,6(5):486-503.
[31]刘鑫,邹郁陶,牟巍,等.玉米乙烯应答元件结合蛋白基因启动子克隆与功能验证[J].核农学报,2016,30(4):629-637.(Liu X,Zou Y T,Mu W,et al. Cloning and functional validation of promoter of ethylene-responsive element-binding protein gene in maize[J]. Journal of Nuclear Agricultural Sciences,2016,30(4):629-637.)
[32] Hu W,Hou X,Huang C,et al. Genome-wide identification and expression analyses of aquaporin gene family during development and abiotic stress in banana[J]. International Journal of Molecular Sciences,2015,16(8):19728-19751.
[33] Guerrero F D,Jones J T,Mullet J E. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes[J]. Plant Molecular Biology,1990,15(1):11.
[34]叶国良,宋娟娟,叶清.两种不同抗旱能力的豇豆根系水通道蛋白基因的克隆与分析[J].分子植物育种,2016,14(8):1977-1985.(Ye G L,Song J J,Ye Q. Cloning and analysis of aquaporins in roots of two cow pea(Vigna unguiculata L. Walp.)cultivars with contrasting drought tolerance[J]. Molecular Plant Breeding,2016,14(8):1977-1985.)