杜仲内生拮抗细菌DZSY21诱导玉米抗病基因表达变化的转录组学研究
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摘要
杜仲内生拮抗细菌DZSY21可在玉米中稳定定殖并增强玉米植株的抗病能力。试验从基因水平上对内生拮抗细菌DZSY21诱导玉米产生抗病性的分子机制进行预测,利用转录组测序技术对内生拮抗细菌DZSY21处理玉米后不同生长时间段叶片的总mRNA进行差异表达分析,以Fold change≥2且FDR<0.01为标准,挑选差异表达基因(DEGs)。结果显示:以处理0 h作为对照,处理12 h时有2 413个差异表达基因,其中上调基因1 278个,下调基因1 135个;处理24 h时有737个差异表达基因,其中上调基因538个,下调基因199个。在此基础上,根据DEGs功能注释分类,在差异表达基因中筛选与抗病相关的基因,最终获得267个抗病基因。处理12 h筛选得到218个基因,主要涉及脂质转移蛋白、MATE转运蛋白及LysM受体蛋白激酶等26个抗病途径;处理24 h获得71个基因,主要涉及异黄酮还原酶、纤维素合成酶、苯丙氨酸解氨酶、L-抗坏血酸过氧化物酶、GLK转录因子及ACC氧化酶等30个抗病途径,其中不同处理时间段内重复基因23个。上述结果表明,将杜仲内生拮抗细菌DZSY21引入玉米,可调节玉米叶片中抗病相关基因的表达,为进一步寻找抗病基因及其功能鉴定奠定了基础。
Endophytic bacteria DZSY21 can stably colonize in maize and enhance the maize resistance to disease. Therefore, this study predicted the molecular mechanism of maize disease resistance induced by endophytic bacteria DZSY21 at the genetic level. The transcriptome sequencing technology was used to analyze the differential expression pattern of total genes in different periods of maize leaves inoculated with DZSY21. Cufflinks software was used to process the data, and the fold change≥2 and FDR<0.01 were set as standards to select DEGs. The results showed that there were 2 413 DEGs after inoculated with DZSY21 at 12 h compared with the control group inoculated with DZSY21 at 0 h, including 1 278 up-regulated DEGs and 1 135 down-regulated DEGs. There were 737 DEGs after inoculation at 24 h, including 538 up-regulated DEGs and 199 down-regulated DEGs. According to the results of functional annotation, 218 DEGs were obtained involved in 26 resistance pathways after inoculation at 12 h, including bifunctional inhibitor/lipid-transfer protein, MATE efflux family protein and LysM domain receptor-like kinase. There were 71 DEGs involved in 30 resistance pathways after inoculation at 24 h, including isoflavone reductase, probable cellulose synthase, phenylalanine ammonia-lyase, L-ascorbate peroxidase, probable transcription factor GLK and ACC oxidase. There were 23 DEGs duplicated in different treatment periods. The above results showed that the introduction of endophytic bacteria DZSY21 into maize could regulate the expression of resistance-related genes in maize leaves. This work provided a theoretical basis for further searching for resistance genes and their function identification.
Endophytic bacteria DZSY21 can stably colonize in maize and enhance the maize resistance to disease. Therefore, this study predicted the molecular mechanism of maize disease resistance induced by endophytic bacteria DZSY21 at the genetic level. The transcriptome sequencing technology was used to analyze the differential expression pattern of total genes in different periods of maize leaves inoculated with DZSY21. Cufflinks software was used to process the data, and the fold change≥2 and FDR<0.01 were set as standards to select DEGs. The results showed that there were 2 413 DEGs after inoculated with DZSY21 at 12 h compared with the control group inoculated with DZSY21 at 0 h, including 1 278 up-regulated DEGs and 1 135 down-regulated DEGs. There were 737 DEGs after inoculation at 24 h, including 538 up-regulated DEGs and 199 down-regulated DEGs. According to the results of functional annotation, 218 DEGs were obtained involved in 26 resistance pathways after inoculation at 12 h, including bifunctional inhibitor/lipid-transfer protein, MATE efflux family protein and LysM domain receptor-like kinase. There were 71 DEGs involved in 30 resistance pathways after inoculation at 24 h, including isoflavone reductase, probable cellulose synthase, phenylalanine ammonia-lyase, L-ascorbate peroxidase, probable transcription factor GLK and ACC oxidase. There were 23 DEGs duplicated in different treatment periods. The above results showed that the introduction of endophytic bacteria DZSY21 into maize could regulate the expression of resistance-related genes in maize leaves. This work provided a theoretical basis for further searching for resistance genes and their function identification.
引文
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[21] 朱巍巍,马天意,张梅娟,等.类受体蛋白激酶在植物中的研究进展[J].基因组学与应用生物学,2018,37(1):451-458. ZHU W W, MA T Y, ZHANG M J, et al. Research progress of receptor-like protein kinases in plants[J]. Genomics and Applied Biology, 2018,37(1):451-458. (in Chinese with English abstract)
[22] 刘俊芳,张佳,李贺,等.植物GOLDEN2-Like转录因子研究进展[J].分子植物育种,2017,15(10):3949-3956.LIU J F, ZHANG J, LI H, et al. Research progress of plant GOLDEN2-Like transcription factor[J]. Molecular Plant Breeding, 2017,15(10):3949-3956. (in Chinese with English abstract)
[23] SAVITCH L V, SUBRAMANIAM R, ALLARD G C, et al. The GLK1‘regulon’ encodes disease defense related proteins and confers resistance to Fusarium graminearum in Arabidopsis[J]. Biochemical & Biophysical Research Communications, 2007, 359(2):234-238.
[24] SCHREIBER K J, NASMITH C G, ALLARD G, et al. Found in translation: high-throughput chemical screening in Arabidopsis thaliana identifies small molecules that reduce Fusarium head blight disease in wheat[J]. Molecular Plant-Microbe Interactions, 2011,24(6):640-648.
[25] 刘蓉蓉,胡鸢雷,林忠平.合成大豆异黄酮的植物基因工程研究进展[J].农业生物技术学报,2007,15(5):888-895. LIU R R, HU Y L, LIN Z P. Progress in genetic engineering of soybean isoflavone biosynthesis in plants[J]. Journal of Agricultural Biotechnology,2007,15(5):888-895. (in Chinese with English abstract)
[26] 赵利锋,柴团耀. AP2/EREBP转录因子在植物发育和胁迫应答中的作用[J].植物学通报,2008,25(1):89-101.ZHAO L F, CHAI T Y. Roles of AP2/EREBP family of transcription factors in development and stress response of plants[J]. Chinese Bulletin of Botany, 2008,25(1):89-101. (in Chinese with English abstract)
[27] 刘茜,王爱云,荣玮. ERF转录因子在作物抗病基因工程中的研究进展[J].种子,2014,33(1):48-53.LIU X, WANG A Y, RONG W. Progress of ERF transcription factors in genetic engineering for disease resistance in crops[J].Seed, 2014,33(1):48-53. (in Chinese with English abstract)
[28] ZHANG X, LIU C J. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoida[J].Molecular Plant, 2015, 8(1):17-27.
[29] ZHANG X B, GOU M, LIU C J. Arabidopsis Kelch repeat F-Box proteins regulate phenylpropanoid bio-synthesis via controlling the turnover of phenylalanineammonia-lyase[J].The Plant Cell,2013,25: 4994-5010.
[30] ZHANG G X,GOU M,GUO C, et al. Down-regulation of Kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly) phenols and tolerance to ultraviolet radiation[J].Plant Physiology,2015,167(2):337-350.
[31] 陈嘉贝,张芙蓉,黄丹枫,等.盐胁迫下两个甜瓜品种转录因子的转录组分析[J].植物生理学报,2014,50(2):150-158. CHEN J B, ZHANG F R, HUANG D F, et al. Transcriptome analysis of transcription factors in two melon (Cucumis melo L.) cultivars under salt stress[J]. Plant Physiology Journal, 2014, 50(2):150-158. (in Chinese with English abstract)
[2] STURZ A V, CHRISTIE B R, NOWAK J. Bacterial endophytes: potential role in developing sustainable systems of crop production[J].Critical Reviews in Plant Sciences,2000,19(1):1-30.
[3] DING T, SU B, CHEN X J, et al. An endophytic bacterial strain isolated from Eucommia ulmoides inhibits southern corn leaf blight[J]. Frontiers in Microbiology, 2017, 8: 903.
[4] MARELLA S. Bacterial endophytes in sustainable crop production: applications, recent developments and challenges ahead[J]. International Journal of Life Sciences Research, 2014,2(2):46-56.
[5] 徐鹏,李浩然,曹志艳,等.玉米抵御鞘腐病菌侵染的生理机制[J].植物保护学报,2013,40(3):261-265.XU P, LI H R, CAO Z Y, et al. The physiological mechanism of corn against Fusarium proliferatum infection[J]. Acta Phytophylacica Sinica, 2013, 40(3):261-265. (in Chinese with English abstract)
[6] 苏博,江胡彪,乔建礼,等.玉米纹枯病生防菌DZSY21的抗病机制[J].浙江农业学报,2017,29(3):450-459.SU B, JIANG H B, QIAO J L, et al. Resistance mechanism of bio-control strain DZSY21 against maize sheath blight[J]. Acta Agriculturae Zhejiangensis, 2017, 29(3):450-459. (in Chinese with English abstract)
[7] SEKI M, NARUSAKA M, ISHIDA J, et al. Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray[J].The Plant Journal,2002,31(3):279-292.
[8] KREPS J A, WU Y, CHANG H S, et al. Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress[J]. Plant Physiology, 2002, 130(4):2129-2141.
[9] KUMARI S, SABHARWAL V P, KUSHWAHA H R, et al. Transcriptome map for seedling stage specific salinity stress response indicates a specific set of genes as candidate for saline tolerance in Oryza sativa L.[J]. Functional & Integrative Genomics, 2009, 9(1):109-123.
[10] WANG H, MIYAZAKI S, KAWAI K, et al. Temporal progression of gene expression responses to salt shock in maize roots[J]. Plant Molecular Biology, 2003, 52(4):873-891.
[11] LIANG C, FENG R, HUI Z, et al. Identification and expression analysis of genes in response to high-salinity and drought stresses in Brassica napus[J]. Acta Biochimica et Biophysica Sinica, 2010, 42(2):154-164.
[12] 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: R36.
[13] FLOREA L, SONG L, SALZBERG S L. Thousands of exon skipping events differentiate among splicing patterns in sixteen human tissues[J]. F1000 Research,2013,2: 188.
[14] ANDERS S, HUBER W. Differential expression analysis for sequence count data[J]. Genome Biology, 2010,11: R106.
[15] ALTSCHUL S F, MADDEN T L, ZHANG J, et al. Gapped BLAST and PSI BLAST: a new generation of protein database search programs[J]. Nucleic Acids Research, 1997, 25(17):3389-3402.
[16] 邓泱泱,荔建琦,吴松锋,等.nr数据库分析及其本地化[J].计算机工程,2006, 32(5):71-74.DENG Y Y, LI J Q, WU S F, et al. Integrated nr database in protein annotation system and its localization[J]. Computer Engineering, 2006, 32(5):71-74. (in Chinese with English abstract)
[17] APWEILER R, BAIROCH A, WU C H, et al. UniProt: the universal protein knowledgebase[J]. Nucleic Acids Research, 2004, 32: D115-D119.
[18] ASHBURNER M, BALL C A, BLAKE J A, et al. Gene ontology: tool for the unification of biology[J]. Nature Genetics, 2000, 25(1):25-29.
[19] KANEHISA M, GOTO S, KAWASHIMA S, et al. The KEGG resource for deciphering the genome[J]. Nucleic Acids Research, 2004, 32: D277-D280.
[20] 刘永杰,马传禹,马雪娜,等.玉米抗禾谷镰刀菌的转录组分析[J].作物学报,2016,42(8):1122-1133.LIU Y J, MA C Y, MA X N, et al. Transcriptional analysis of maize resistance against Fusarium graminearum[J]. Acta Agronomica Sinica, 2016, 42(8):1122-1133. (in Chinese with English abstract)
[21] 朱巍巍,马天意,张梅娟,等.类受体蛋白激酶在植物中的研究进展[J].基因组学与应用生物学,2018,37(1):451-458. ZHU W W, MA T Y, ZHANG M J, et al. Research progress of receptor-like protein kinases in plants[J]. Genomics and Applied Biology, 2018,37(1):451-458. (in Chinese with English abstract)
[22] 刘俊芳,张佳,李贺,等.植物GOLDEN2-Like转录因子研究进展[J].分子植物育种,2017,15(10):3949-3956.LIU J F, ZHANG J, LI H, et al. Research progress of plant GOLDEN2-Like transcription factor[J]. Molecular Plant Breeding, 2017,15(10):3949-3956. (in Chinese with English abstract)
[23] SAVITCH L V, SUBRAMANIAM R, ALLARD G C, et al. The GLK1‘regulon’ encodes disease defense related proteins and confers resistance to Fusarium graminearum in Arabidopsis[J]. Biochemical & Biophysical Research Communications, 2007, 359(2):234-238.
[24] SCHREIBER K J, NASMITH C G, ALLARD G, et al. Found in translation: high-throughput chemical screening in Arabidopsis thaliana identifies small molecules that reduce Fusarium head blight disease in wheat[J]. Molecular Plant-Microbe Interactions, 2011,24(6):640-648.
[25] 刘蓉蓉,胡鸢雷,林忠平.合成大豆异黄酮的植物基因工程研究进展[J].农业生物技术学报,2007,15(5):888-895. LIU R R, HU Y L, LIN Z P. Progress in genetic engineering of soybean isoflavone biosynthesis in plants[J]. Journal of Agricultural Biotechnology,2007,15(5):888-895. (in Chinese with English abstract)
[26] 赵利锋,柴团耀. AP2/EREBP转录因子在植物发育和胁迫应答中的作用[J].植物学通报,2008,25(1):89-101.ZHAO L F, CHAI T Y. Roles of AP2/EREBP family of transcription factors in development and stress response of plants[J]. Chinese Bulletin of Botany, 2008,25(1):89-101. (in Chinese with English abstract)
[27] 刘茜,王爱云,荣玮. ERF转录因子在作物抗病基因工程中的研究进展[J].种子,2014,33(1):48-53.LIU X, WANG A Y, RONG W. Progress of ERF transcription factors in genetic engineering for disease resistance in crops[J].Seed, 2014,33(1):48-53. (in Chinese with English abstract)
[28] ZHANG X, LIU C J. Multifaceted regulations of gateway enzyme phenylalanine ammonia-lyase in the biosynthesis of phenylpropanoida[J].Molecular Plant, 2015, 8(1):17-27.
[29] ZHANG X B, GOU M, LIU C J. Arabidopsis Kelch repeat F-Box proteins regulate phenylpropanoid bio-synthesis via controlling the turnover of phenylalanineammonia-lyase[J].The Plant Cell,2013,25: 4994-5010.
[30] ZHANG G X,GOU M,GUO C, et al. Down-regulation of Kelch domain-containing F-box protein in Arabidopsis enhances the production of (poly) phenols and tolerance to ultraviolet radiation[J].Plant Physiology,2015,167(2):337-350.
[31] 陈嘉贝,张芙蓉,黄丹枫,等.盐胁迫下两个甜瓜品种转录因子的转录组分析[J].植物生理学报,2014,50(2):150-158. CHEN J B, ZHANG F R, HUANG D F, et al. Transcriptome analysis of transcription factors in two melon (Cucumis melo L.) cultivars under salt stress[J]. Plant Physiology Journal, 2014, 50(2):150-158. (in Chinese with English abstract)