转多基因1年生库安托杨对溃疡病的抗性分析
|
摘要
[目的]本研究对转多基因库安托杨株系的溃疡病抗病性检测及抗病相关基因的表达分析,为通过基因工程手段培育抗病林木新树种提供有价值的参考。[方法]以转JERF36等多个外源基因的1年生库安托杨株系D5-9、D5-20、D5-21及非转基因受体D5-0为材料,对主干人工接种溃疡病菌的各株系病情指数进行了测定,同时对接种5 d后树皮组织中5个抗病基因的表达情况进行了实时定量PCR(qPCR)分析。[结果]在溃疡病菌胁迫下,3个转基因株系对溃疡病菌的抗性显著优于非转基因受体株系,转基因株系间抗病性也具有一定差异,D5-21的病情指数显著低于其它2个转基因株系;肉桂醇脱氢酶基因在3个转基因株系树皮中表达量均高于对照,类甜蛋白基因仅在D5-19树皮中的表达量高于对照,其他3个基因在转基因株系中的表达量或与对照相当或低于对照。[结论]转多基因库安托杨株系的溃疡病抗性与非转基因对照相比均有显著提高,且转基因株系间差异显著;同时转基因株系中不同抗病基因的表达模式也有很大差异,说明溃疡病菌胁迫下转多基因库安托杨抗病调控的复杂性,其分子调控机制还有待深入研究。
[Objective] To evaluate the resistance to poplar canker and mRNA expression of disease resistance-related genes of three transgenic Populus× euramericana‘Guariento' lines so as to provide reference for breeding disease-resistant trees through transgenic technology.[Method] Using 1-year-old stem of multiple transgenic P. × euramericana ‘Guariento' clones(D5-9, D5-20, and D5-21) and the wild control(D5-0) as material, the disease index of canker was analyzed after inoculated with Botryosphaeria dothidea for 60 days, and the expression of five disease resistance related genes in bark of these clones after inoculated with B. dothidea for 5 days were detected by relative quantitative PCR(qPCR). [Result] The results showed that the resistance of the three transgenic clones to poplar canker was significantly higher than that of the non-transgenic clone and obvious difference in canker resistance was observed among the three transgenic poplar clones with D5-21 was higher than the other two clones. The expression of cinnamyl-alcohol dehydrogenase family protein gene in the bark of the 3 transgenic clones was higher than that of the control, and the expression of thaumatin-like protein gene was higher than control only in bark of D5-19, whereas the expressions of the other three genes were similar to or lower than the control. [Conclusion] The difference in the canker resistance of the three transgenic clones and various expression profiles of the five disease resistant genes indicates the regulation complexity in the disease resistance of the multiple transgenic poplars under stress of B. dothidea, suggesting further study is needed.
[Objective] To evaluate the resistance to poplar canker and mRNA expression of disease resistance-related genes of three transgenic Populus× euramericana‘Guariento' lines so as to provide reference for breeding disease-resistant trees through transgenic technology.[Method] Using 1-year-old stem of multiple transgenic P. × euramericana ‘Guariento' clones(D5-9, D5-20, and D5-21) and the wild control(D5-0) as material, the disease index of canker was analyzed after inoculated with Botryosphaeria dothidea for 60 days, and the expression of five disease resistance related genes in bark of these clones after inoculated with B. dothidea for 5 days were detected by relative quantitative PCR(qPCR). [Result] The results showed that the resistance of the three transgenic clones to poplar canker was significantly higher than that of the non-transgenic clone and obvious difference in canker resistance was observed among the three transgenic poplar clones with D5-21 was higher than the other two clones. The expression of cinnamyl-alcohol dehydrogenase family protein gene in the bark of the 3 transgenic clones was higher than that of the control, and the expression of thaumatin-like protein gene was higher than control only in bark of D5-19, whereas the expressions of the other three genes were similar to or lower than the control. [Conclusion] The difference in the canker resistance of the three transgenic clones and various expression profiles of the five disease resistant genes indicates the regulation complexity in the disease resistance of the multiple transgenic poplars under stress of B. dothidea, suggesting further study is needed.
引文
[1] 刘会香, 贾秀贞, 吕全, 等. 中国杨树溃疡病的发生与防治[J]. 世界林业研究, 2005, 18(4): 60-63.
[2] 黄逢龙,焦一杰,丁辉,等.不同林分密度下场树树冠结构与溃疡病的关系[J]. 南京林业大学学报, 2010,34(4): 79-82.
[3] 王孟昌, 梁军, 樊军锋, 等. 主要杨树生产品种对溃疡病田间抗性的调查[J]. 西北林学院学报, 2008, 23(5): 122-123.
[4] Yuan L, Wang L, Han Z, et al. Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants[J]. J Exp Bot, 2012, 63(7): 2513-2524.
[5] Ye S, Jiang Y, Duan Y, et al. Constitutive expression of the poplar WRKY transcription factor PtoWRKY60 enhances resistance to Dothiorella gregaria Sacc. in transgenic plants[J]. Tree Physiol, 2014, 34(10):1118-1129.
[6] Wang L, Ran L, Hou Y, et al. The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar[J]. New Phytol, 2017, 215(1):351-367.
[7] Jiang Y, Guo L, Ma X, et al. The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus[J]. Tree Physiol, 2017, 37(5):665-675.
[8] 王建革, 苏晓华, 纪丽丽, 等. 基因枪转多基因库安托杨的获得[J]. 科学通报, 2006, 51(23): 2755-2760.
[9] 张晓芬. 转基因抗虫杨的抗虫性测定及对节肢动物群落影响研究[D]. 北京: 北京林业大学, 2009.
[10] 李丹, 李环, 丁昌俊, 等. 涝渍胁迫对转多基因库安托杨生长及生理性状的影响[J]. 林业科学研究, 2010, 23(1): 44-52.
[11] 夏永刚, 李密, 李永进, 等. 转多基因库安托杨对昆虫群落的影响[J]. 中国农学通报, 2013, 29(19): 84-88.
[12] 李丹,黄绢, 张伟溪, 等. 盐胁迫条件下转多基因库安托杨根尖离子流变化[J]. 林业科学, 2015, 51(9): 35-41.
[13] 张秀琳. 番茄基因JERFs的功能分析[D]. 北京:中国农业大学,2003.
[14] Lorenzo O, Piqueras R, Sánchez-Serrano J J, et al. ETHYLENE RESPONSE FACTOR1 Integrates Signals from Ethylene and Jasmonate Pathways in Plant Defense [J]. The Plant Cell, 2003, 15: 165-178.
[15] 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.
[16] Dangl J L, Jones J D. Plant pathogens and integrated defence responses to infection [J]. Nature, 2001, 411(6839):826-833.
[17] Gurr S J, Rushton P J. Engineering plants with increased disease resistance: how are we going to express it?[J]. Trends in Biotechnology, 2005, 23(6):283-290.
[18] Fonseca J P, Menossi M, Thibaudnissen F, et al. Functional analysis of a TGA factor-binding site located in the promoter region controlling salicylic acid-induced NIMIN-1 expression in Arabidopsis[J]. Genetics & Molecular Research Gmr, 2010, 9(1):167-175.
[19] Gutterson N, Reuber T L. Regulation of disease resistance pathways by AP2/ERF transcription factors [J]. Current Opinion in Plant Biology, 2004, 7(4):465-471.
[20] Xu Z S S, Chen M C, Li L C C, et al. Functions of the ERF transcription factor family in plants [J]. Botany-botanique, 2008, 86(9):969-977.
[21] Nakashima K, Yamaguchi-Shinozaki K. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses [J]. Plant Physiology, 2009, 149(1):88-95.
[22] Berrocal-Lobo M, Molina A, Solano R. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi [J]. Plant Journal, 2002, 29(1):23-32.
[23] Berrocal-Lobo M, Molina A. Ethylene response factor 1 mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum[J]. Molecular Plant-Microbe Interactions, 2004, 17(7):763-770.
[24] Zuo K J, Qin J, Zhao J Y, et al. Over-expression GbERF2 transcription factor in tobacco enhances brown spots disease resistance by activating expression of downstream genes [J]. Gene, 2007, 391(1-2):80-90.
[25] Cao Y, Song F, Goodman R M, et al. Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress.[J]. Journal of Plant Physiology, 2006, 163(11):1167-1178.
[26] Cao Y, Wu Y, Zheng Z, et al. Overexpression of the rice EREBP-like gene OsBIERF3 enhances disease resistance and salt tolerance in transgenic tobacco[J]. Physiological and Molecular Plant Pathology, 2005, 67(3-5): 202-211.
[27] Chen L, Zhang Z Y, Liang H X, et al. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat[J]. Journal of Experimental Botany, 2008, 59(15):4195-4204.
[28] 杨国顺. 转 JERFs 基因提高辣椒抗病性的研究[D]. 长沙:湖南农业大学, 2003.
[29] 李文正, 张海文, 王俊英, 等. ERF 转录因子及其在烟草抗逆性改良中的应用[J]. 生物技术通报, 2006(4): 30-34.
[30] 李义良.转基因杨树的分子检测及抗逆性评价[D].北京:北京林业大学, 2008.
[31] Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, et al. Sensing the environment: key roles of membrane-localized kinases in plant perception and response to abiotic stress [J]. Journal of experimental botany, 2013, 64(2): 445-458.
[32] Anderson J P, Lichtenzveig J, Gleason C, et al. The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[J]. Plant Physiology, 2010, 154(2):861-873.
[2] 黄逢龙,焦一杰,丁辉,等.不同林分密度下场树树冠结构与溃疡病的关系[J]. 南京林业大学学报, 2010,34(4): 79-82.
[3] 王孟昌, 梁军, 樊军锋, 等. 主要杨树生产品种对溃疡病田间抗性的调查[J]. 西北林学院学报, 2008, 23(5): 122-123.
[4] Yuan L, Wang L, Han Z, et al. Molecular cloning and characterization of PtrLAR3, a gene encoding leucoanthocyanidin reductase from Populus trichocarpa, and its constitutive expression enhances fungal resistance in transgenic plants[J]. J Exp Bot, 2012, 63(7): 2513-2524.
[5] Ye S, Jiang Y, Duan Y, et al. Constitutive expression of the poplar WRKY transcription factor PtoWRKY60 enhances resistance to Dothiorella gregaria Sacc. in transgenic plants[J]. Tree Physiol, 2014, 34(10):1118-1129.
[6] Wang L, Ran L, Hou Y, et al. The transcription factor MYB115 contributes to the regulation of proanthocyanidin biosynthesis and enhances fungal resistance in poplar[J]. New Phytol, 2017, 215(1):351-367.
[7] Jiang Y, Guo L, Ma X, et al. The WRKY transcription factors PtrWRKY18 and PtrWRKY35 promote Melampsora resistance in Populus[J]. Tree Physiol, 2017, 37(5):665-675.
[8] 王建革, 苏晓华, 纪丽丽, 等. 基因枪转多基因库安托杨的获得[J]. 科学通报, 2006, 51(23): 2755-2760.
[9] 张晓芬. 转基因抗虫杨的抗虫性测定及对节肢动物群落影响研究[D]. 北京: 北京林业大学, 2009.
[10] 李丹, 李环, 丁昌俊, 等. 涝渍胁迫对转多基因库安托杨生长及生理性状的影响[J]. 林业科学研究, 2010, 23(1): 44-52.
[11] 夏永刚, 李密, 李永进, 等. 转多基因库安托杨对昆虫群落的影响[J]. 中国农学通报, 2013, 29(19): 84-88.
[12] 李丹,黄绢, 张伟溪, 等. 盐胁迫条件下转多基因库安托杨根尖离子流变化[J]. 林业科学, 2015, 51(9): 35-41.
[13] 张秀琳. 番茄基因JERFs的功能分析[D]. 北京:中国农业大学,2003.
[14] Lorenzo O, Piqueras R, Sánchez-Serrano J J, et al. ETHYLENE RESPONSE FACTOR1 Integrates Signals from Ethylene and Jasmonate Pathways in Plant Defense [J]. The Plant Cell, 2003, 15: 165-178.
[15] 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.
[16] Dangl J L, Jones J D. Plant pathogens and integrated defence responses to infection [J]. Nature, 2001, 411(6839):826-833.
[17] Gurr S J, Rushton P J. Engineering plants with increased disease resistance: how are we going to express it?[J]. Trends in Biotechnology, 2005, 23(6):283-290.
[18] Fonseca J P, Menossi M, Thibaudnissen F, et al. Functional analysis of a TGA factor-binding site located in the promoter region controlling salicylic acid-induced NIMIN-1 expression in Arabidopsis[J]. Genetics & Molecular Research Gmr, 2010, 9(1):167-175.
[19] Gutterson N, Reuber T L. Regulation of disease resistance pathways by AP2/ERF transcription factors [J]. Current Opinion in Plant Biology, 2004, 7(4):465-471.
[20] Xu Z S S, Chen M C, Li L C C, et al. Functions of the ERF transcription factor family in plants [J]. Botany-botanique, 2008, 86(9):969-977.
[21] Nakashima K, Yamaguchi-Shinozaki K. Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses [J]. Plant Physiology, 2009, 149(1):88-95.
[22] Berrocal-Lobo M, Molina A, Solano R. Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi [J]. Plant Journal, 2002, 29(1):23-32.
[23] Berrocal-Lobo M, Molina A. Ethylene response factor 1 mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum[J]. Molecular Plant-Microbe Interactions, 2004, 17(7):763-770.
[24] Zuo K J, Qin J, Zhao J Y, et al. Over-expression GbERF2 transcription factor in tobacco enhances brown spots disease resistance by activating expression of downstream genes [J]. Gene, 2007, 391(1-2):80-90.
[25] Cao Y, Song F, Goodman R M, et al. Molecular characterization of four rice genes encoding ethylene-responsive transcriptional factors and their expressions in response to biotic and abiotic stress.[J]. Journal of Plant Physiology, 2006, 163(11):1167-1178.
[26] Cao Y, Wu Y, Zheng Z, et al. Overexpression of the rice EREBP-like gene OsBIERF3 enhances disease resistance and salt tolerance in transgenic tobacco[J]. Physiological and Molecular Plant Pathology, 2005, 67(3-5): 202-211.
[27] Chen L, Zhang Z Y, Liang H X, et al. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat[J]. Journal of Experimental Botany, 2008, 59(15):4195-4204.
[28] 杨国顺. 转 JERFs 基因提高辣椒抗病性的研究[D]. 长沙:湖南农业大学, 2003.
[29] 李文正, 张海文, 王俊英, 等. ERF 转录因子及其在烟草抗逆性改良中的应用[J]. 生物技术通报, 2006(4): 30-34.
[30] 李义良.转基因杨树的分子检测及抗逆性评价[D].北京:北京林业大学, 2008.
[31] Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, et al. Sensing the environment: key roles of membrane-localized kinases in plant perception and response to abiotic stress [J]. Journal of experimental botany, 2013, 64(2): 445-458.
[32] Anderson J P, Lichtenzveig J, Gleason C, et al. The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[J]. Plant Physiology, 2010, 154(2):861-873.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |