不结球白菜BcCNL基因的克隆及功能分析
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摘要
[目的]CC-NBS-LRR(卷曲螺旋-核苷酸结合位点-富含亮氨酸重复,CNL)是高度保守的植物抗病蛋白家族。本文旨在克隆和研究CNL基因在不结球白菜抗霜霉病中的作用。[方法]以‘苏州青’c DNA为模板克隆获得BcCNL基因全长,并进行生物信息学分析、实时荧光定量PCR(RT-qPCR)检测、亚细胞定位及病毒诱导基因沉默(VIGS)验证。[结果]BcCNL基因含有2 790 bp开放阅读框,编码929个氨基酸,蛋白质相对分子质量为226.7,等电点为2.7。系统进化树结果显示,不结球白菜BcCNL蛋白与野甘蓝的进化关系最近,含有RX-CC、NB-ARC和LRR-3结构域;亚细胞定位显示其定位于细胞膜上。霜霉菌侵染后,BcCNL基因在抗病品种‘苏州青’中的表达量高于感病品种‘矮脚黄’; BcCNL基因的沉默降低了‘苏州青’对霜霉病的抗性。[结论]BcCNL蛋白在进化过程中保守性较高,定位于细胞膜上,BcCNL基因的沉默降低了‘苏州青’对霜霉病的抗性。
[Objectives]The CC-NBS-LRR( coiled-coil-nucleotide binding-site-leucine-rich repeat,CNL) is a highly conserved plant disease resistance protein family. In this paper,we aimed to clone and study the role of CNL gene against downy mildew in non-heading Chinese cabbage( Brassica campestris ssp. chinensis). [Methods]The full length c DNA of BcCNL was cloned by variety‘Suzhouqing',analyzed by bioinformatics analysis,RT-qPCR detection,subcellular localization and virus-induced gene silencing( VIGS) validation. [Results]The BcCNL gene contained a 2 790 bp open reading frame,encoding 929 amino acids,with a molecular weight of 226.7 and an isoelectric point of 2.7. The evolutionary relationship between BcCNL and CNL protein of Brassica oleracea var. oleracea was the most recent,and BcCNL contained RX-CC,NB-ARC and LRR-3 domains. The subcellular localization of BcCNL protein was located in the cell membrane. The expression of BcCNL gene in resistance variety‘Suzhouqing'was higher than that of susceptible variety‘Aijiaohuang'after infected with downy mildew. Silencing of BcCNL gene reduced the‘Suzhouqing'against downy mildew. [Conclusions]The BcCNL protein was highly conserved during evolution and was located in the cell membrane. The silencing of the BcCNL gene reduced the‘Suzhouqing'against downy mildew.
[Objectives]The CC-NBS-LRR( coiled-coil-nucleotide binding-site-leucine-rich repeat,CNL) is a highly conserved plant disease resistance protein family. In this paper,we aimed to clone and study the role of CNL gene against downy mildew in non-heading Chinese cabbage( Brassica campestris ssp. chinensis). [Methods]The full length c DNA of BcCNL was cloned by variety‘Suzhouqing',analyzed by bioinformatics analysis,RT-qPCR detection,subcellular localization and virus-induced gene silencing( VIGS) validation. [Results]The BcCNL gene contained a 2 790 bp open reading frame,encoding 929 amino acids,with a molecular weight of 226.7 and an isoelectric point of 2.7. The evolutionary relationship between BcCNL and CNL protein of Brassica oleracea var. oleracea was the most recent,and BcCNL contained RX-CC,NB-ARC and LRR-3 domains. The subcellular localization of BcCNL protein was located in the cell membrane. The expression of BcCNL gene in resistance variety‘Suzhouqing'was higher than that of susceptible variety‘Aijiaohuang'after infected with downy mildew. Silencing of BcCNL gene reduced the‘Suzhouqing'against downy mildew. [Conclusions]The BcCNL protein was highly conserved during evolution and was located in the cell membrane. The silencing of the BcCNL gene reduced the‘Suzhouqing'against downy mildew.
引文
[1]侯喜林.不结球白菜育种研究新进展[J].南京农业大学学报,2003,26(4):111-115. DOI:10.7685/j.issn.1000-2030.2003.04.026.Hou X L. Advances in breeding of non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,2003,26(4):111-115(in Chinese with English abstract).
[2]刘克钧,朱月林,侯喜林,等.不结球白菜抗病育种的研究.Ⅳ:不结球白菜抗芜菁花叶病、霜霉病及黑斑病的多抗性鉴定及筛选[J].南京农业大学学报,1997,20(3):31-35. DOI:10.7685/j.issn.1000-2030.1997.03.006.Liu K J,Zhu Y L,Hou X L,et al. Studies on breeding for disease resistance in non-heading Chinese cabbage.Ⅳ:Identification and screening for multiple resistance to turnip mosaic virosis,downy mildew and black spot in non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,1997,20(3):31-35(in Chinese with English abstract).
[3]申姗娜,侯喜林.不结球白菜感染霜霉病菌后防御物质及酶的变化[J].南京农业大学学报,2009,32(1):23-26. DOI:10.7685/j.issn.1000-2030.2009.01.005.Shen S N,Hou X L. Changes of protective substances and enzymes in non-heading Chinese cabbage after infection by downy mildew[J]. Journal of Nanjing Agricultural University,2009,32(1):23-26(in Chinese with English abstract).
[4]刘世拓,肖栋,许玉超,等.不结球白菜过氧化还原蛋白基因Brc2-Cys Prx的克隆和表达分析[J].南京农业大学学报,2017,40(1):40-47. DOI:10.7685/jnau.201603040.Liu S T,Xiao D,Xu Y C,et al. Clone and expression analysis of Brc2-Cys Prx gene from non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,2017,40(1):40-47(in Chinese with English abstract).
[5] Xiao S,Ellwood S,Calis O,et al. Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8[J]. Science,2001,291(5501):118-120.
[6] Gururani M A,Venkatesh J,Upadhyaya C P,et al. Plant disease resistance genes:current status and future directions[J]. Physiological and Molecular Plant Pathology,2012,78:51-65.
[7] Meyers B C,Kozik A,Griego A,et al. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis[J]. Plant Cell,2003,15(4):809-834.
[8] Bent A F,Kunkel B N,Dahlbeck D,et al. RPS2 of Arabidopsis thaliana:a leucine-rich repeat class of plant disease resistance genes[J].Science,1994,265(5180):1856-1860.
[9] Hayashi N,Inoue H,Kato T,et al. Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication[J]. The Plant Journal,2010,64(3):498-510.
[10] Deyoung B J,Innes R W. Plant NBS-LRR proteins in pathogen sensing and host defense[J]. Nature Immunology,2006,7(12):1243-1249.
[11] Loutre C,Wicker T,Travella S,et al. Two different CC-NBS-LRR genes are required for Lr10-mediated leaf rust resistance in tetraploid and hexaploid wheat[J]. The Plant Journal,2009,60(6):1043-1054.
[12] Zhang S,Ding F,Peng H,et al. Molecular cloning of a CC-NBS-LRR gene from Vitis quinquangularis,and its expression pattern in response to downy mildew pathogen infection[J]. Molecular Genetics and Genomics,2018,293(1):61-68.
[13]程永安,柯桂兰.影响大白菜霜霉病抗性鉴定的因素[J].西北农业学报,1995,4(4):69-72.Cheng Y A,Ke G L. Factors affecting evaluation of Chinese cabbage resistance to downy midew[J]. Acta Agriculturae Boreali-Occidentalis Sinica,1995,4(4):69-72(in Chinese with English abstract).
[14]徐淑平,卫志明.基因枪的使用方法介绍[J].植物生理学通讯,1998,34(1):41-43.Xu S P,Wei Z M. Introduction to method of microprojectile bombardment and its application[J]. Plant Physiology Communications,1998,34(1):41-43(in Chinese).
[15]杨学东,戴薇,张昌伟,等.白菜病毒诱导基因沉默技术体系的建立[J].园艺学报,2012,39(11):2168-2174.Yang X D,Dai W,Zhang C W,et al. The technology system establishment of VIGS in non-heading Chinese cabbage[J]. Acta Horticulturae Sinica,2012,39(11):2168-2174(in Chinese with English abstract).
[16] Xiao D,Liu S T,Wei Y P,et al. c DNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica[J]. Horticulture Research,2016,3:16034.
[17] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[18] Jones J D G,Dangl J L. The plant immune system[J]. Nature,2006,444(7117):323-329.
[19]陈俏丽,王峰,李丹蕾,等.‘大白谷’CC-NBS-LRR蛋白编码基因抗干尖线虫侵染时空表达研究[J].中国农学通报,2015,31(21):279-283.Chen Q L,Wang F,Li D L,et al. Spatial-temporal expression of CC-NBS-LRR protein-coding gene resisting to infection of Aphelenchoides besseyi in Oryza sativa L. ssp. indica[J]. Chinese Agricultural Science Bulletin,2015,31(21):279-283(in Chinese with English abstract).
[20] Radwan O,Mouzeyar S,Nicolas P,et al. Induction of a sunflower CC-NBS-LRR resistance gene analogue during incompatible interaction with Plasmopara halstedii[J]. J Exp Bot,2005,56(412):567-575.
[21] Veena M,Melvin P,Prabhu S A,et al. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen[J]. Molecular Biology Reports,2016,43(3):117-128.
[22] Radwan O,Gandhi S,Heesacker A,et al. Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower[J]. Molecular Genetics and Genomics,2008,280(2):111-125.
[23]孙威,许奕,许桂莺,等.病毒诱导的基因沉默及其在植物研究中的应用[J].生物技术通报,2015,31(10):105-110.Sun W,Xu Y,Xu G Y,et al. Virus-induced gene silencing and its application in plant research[J]. Biotechnology Bulletin,2015,31(10):105-110(in Chinese with English abstract).
[24] Pflieger S,Blanchet S,Camborde L,et al. Efficient virus-induced gene silencing in Arabidopsis using a‘one-step’TYMV-derived vector[J].The Plant Journal,2008,56(4):678-690.
[25] Manmathan H,Shaner D L,Snelling J,et al. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance[J]. Journal of Experimental Botany,2013,64(5):1381-1392.
[26] Krishnan A,Mahadevan C,Mani T,et al. Virus-induced gene silencing(VIGS)for elucidation of pathogen defense role of serine/threonine protein kinase in the non-model plant Piper colubrinum Link.[J]. Plant Cell,Tissue and Organ Culture,2015,122(2):269-283.
[2]刘克钧,朱月林,侯喜林,等.不结球白菜抗病育种的研究.Ⅳ:不结球白菜抗芜菁花叶病、霜霉病及黑斑病的多抗性鉴定及筛选[J].南京农业大学学报,1997,20(3):31-35. DOI:10.7685/j.issn.1000-2030.1997.03.006.Liu K J,Zhu Y L,Hou X L,et al. Studies on breeding for disease resistance in non-heading Chinese cabbage.Ⅳ:Identification and screening for multiple resistance to turnip mosaic virosis,downy mildew and black spot in non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,1997,20(3):31-35(in Chinese with English abstract).
[3]申姗娜,侯喜林.不结球白菜感染霜霉病菌后防御物质及酶的变化[J].南京农业大学学报,2009,32(1):23-26. DOI:10.7685/j.issn.1000-2030.2009.01.005.Shen S N,Hou X L. Changes of protective substances and enzymes in non-heading Chinese cabbage after infection by downy mildew[J]. Journal of Nanjing Agricultural University,2009,32(1):23-26(in Chinese with English abstract).
[4]刘世拓,肖栋,许玉超,等.不结球白菜过氧化还原蛋白基因Brc2-Cys Prx的克隆和表达分析[J].南京农业大学学报,2017,40(1):40-47. DOI:10.7685/jnau.201603040.Liu S T,Xiao D,Xu Y C,et al. Clone and expression analysis of Brc2-Cys Prx gene from non-heading Chinese cabbage[J]. Journal of Nanjing Agricultural University,2017,40(1):40-47(in Chinese with English abstract).
[5] Xiao S,Ellwood S,Calis O,et al. Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8[J]. Science,2001,291(5501):118-120.
[6] Gururani M A,Venkatesh J,Upadhyaya C P,et al. Plant disease resistance genes:current status and future directions[J]. Physiological and Molecular Plant Pathology,2012,78:51-65.
[7] Meyers B C,Kozik A,Griego A,et al. Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis[J]. Plant Cell,2003,15(4):809-834.
[8] Bent A F,Kunkel B N,Dahlbeck D,et al. RPS2 of Arabidopsis thaliana:a leucine-rich repeat class of plant disease resistance genes[J].Science,1994,265(5180):1856-1860.
[9] Hayashi N,Inoue H,Kato T,et al. Durable panicle blast-resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication[J]. The Plant Journal,2010,64(3):498-510.
[10] Deyoung B J,Innes R W. Plant NBS-LRR proteins in pathogen sensing and host defense[J]. Nature Immunology,2006,7(12):1243-1249.
[11] Loutre C,Wicker T,Travella S,et al. Two different CC-NBS-LRR genes are required for Lr10-mediated leaf rust resistance in tetraploid and hexaploid wheat[J]. The Plant Journal,2009,60(6):1043-1054.
[12] Zhang S,Ding F,Peng H,et al. Molecular cloning of a CC-NBS-LRR gene from Vitis quinquangularis,and its expression pattern in response to downy mildew pathogen infection[J]. Molecular Genetics and Genomics,2018,293(1):61-68.
[13]程永安,柯桂兰.影响大白菜霜霉病抗性鉴定的因素[J].西北农业学报,1995,4(4):69-72.Cheng Y A,Ke G L. Factors affecting evaluation of Chinese cabbage resistance to downy midew[J]. Acta Agriculturae Boreali-Occidentalis Sinica,1995,4(4):69-72(in Chinese with English abstract).
[14]徐淑平,卫志明.基因枪的使用方法介绍[J].植物生理学通讯,1998,34(1):41-43.Xu S P,Wei Z M. Introduction to method of microprojectile bombardment and its application[J]. Plant Physiology Communications,1998,34(1):41-43(in Chinese).
[15]杨学东,戴薇,张昌伟,等.白菜病毒诱导基因沉默技术体系的建立[J].园艺学报,2012,39(11):2168-2174.Yang X D,Dai W,Zhang C W,et al. The technology system establishment of VIGS in non-heading Chinese cabbage[J]. Acta Horticulturae Sinica,2012,39(11):2168-2174(in Chinese with English abstract).
[16] Xiao D,Liu S T,Wei Y P,et al. c DNA-AFLP analysis reveals differential gene expression in incompatible interaction between infected non-heading Chinese cabbage and Hyaloperonospora parasitica[J]. Horticulture Research,2016,3:16034.
[17] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[18] Jones J D G,Dangl J L. The plant immune system[J]. Nature,2006,444(7117):323-329.
[19]陈俏丽,王峰,李丹蕾,等.‘大白谷’CC-NBS-LRR蛋白编码基因抗干尖线虫侵染时空表达研究[J].中国农学通报,2015,31(21):279-283.Chen Q L,Wang F,Li D L,et al. Spatial-temporal expression of CC-NBS-LRR protein-coding gene resisting to infection of Aphelenchoides besseyi in Oryza sativa L. ssp. indica[J]. Chinese Agricultural Science Bulletin,2015,31(21):279-283(in Chinese with English abstract).
[20] Radwan O,Mouzeyar S,Nicolas P,et al. Induction of a sunflower CC-NBS-LRR resistance gene analogue during incompatible interaction with Plasmopara halstedii[J]. J Exp Bot,2005,56(412):567-575.
[21] Veena M,Melvin P,Prabhu S A,et al. Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen[J]. Molecular Biology Reports,2016,43(3):117-128.
[22] Radwan O,Gandhi S,Heesacker A,et al. Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower[J]. Molecular Genetics and Genomics,2008,280(2):111-125.
[23]孙威,许奕,许桂莺,等.病毒诱导的基因沉默及其在植物研究中的应用[J].生物技术通报,2015,31(10):105-110.Sun W,Xu Y,Xu G Y,et al. Virus-induced gene silencing and its application in plant research[J]. Biotechnology Bulletin,2015,31(10):105-110(in Chinese with English abstract).
[24] Pflieger S,Blanchet S,Camborde L,et al. Efficient virus-induced gene silencing in Arabidopsis using a‘one-step’TYMV-derived vector[J].The Plant Journal,2008,56(4):678-690.
[25] Manmathan H,Shaner D L,Snelling J,et al. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance[J]. Journal of Experimental Botany,2013,64(5):1381-1392.
[26] Krishnan A,Mahadevan C,Mani T,et al. Virus-induced gene silencing(VIGS)for elucidation of pathogen defense role of serine/threonine protein kinase in the non-model plant Piper colubrinum Link.[J]. Plant Cell,Tissue and Organ Culture,2015,122(2):269-283.