紫花苜蓿MsOXO基因的克隆及表达分析
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摘要
草酸氧化酶(OXO)在包括核盘菌在内的多种病原体的防御和植物改良中有重大作用。以紫花苜蓿为材料,克隆得到紫花苜蓿草酸氧化酶基因MsOXO。利用烟草瞬时表达进行亚细胞定位,结果显示,MsOXO定位在细胞壁上。qRT-PCR结果显示,MsOXO在紫花苜蓿的根、茎、叶中均有表达,但在根茎中表达量较低,这可能是根颈和茎基部更易被病菌侵染的原因。外源草酸50μmol·L~(-1)处理根或100μmol·L~(-1)处理茎及外源水杨酸100μmol·L~(-1)处理根均可以诱导MsOXO基因的上调表达。
Oxalate oxidase plays an important role in defence of various pathogens such as Sclerotinia and can be used for plant improvement. To explore the role of oxalate oxidase in the defense of Sclerotinia sclerotiorum,alfalfa was used for material in this study,an oxalate oxidase gene,MsOXO,was successfully cloned. Subcellular localization of MsOXO was performed using the transient expression of tobacco,and the result showed that MsOXO was located in cell wall. The results of q RT-PCR showed that MsOXO was expressed in roots,stems and leaves of alfalfa,but was lower in roots and stems,which might be the reason why the root neck and stem base were more susceptible to infection by the pathogen. The up-regulated expression of MsOXO gene was induced by exogenous oxalic acid 50μmol·L~(-1)treated root or 100 μmol·L~(-1)treated stem and exogenous salicylic acid 100 μmol·L~(-1)treated root.
Oxalate oxidase plays an important role in defence of various pathogens such as Sclerotinia and can be used for plant improvement. To explore the role of oxalate oxidase in the defense of Sclerotinia sclerotiorum,alfalfa was used for material in this study,an oxalate oxidase gene,MsOXO,was successfully cloned. Subcellular localization of MsOXO was performed using the transient expression of tobacco,and the result showed that MsOXO was located in cell wall. The results of q RT-PCR showed that MsOXO was expressed in roots,stems and leaves of alfalfa,but was lower in roots and stems,which might be the reason why the root neck and stem base were more susceptible to infection by the pathogen. The up-regulated expression of MsOXO gene was induced by exogenous oxalic acid 50μmol·L~(-1)treated root or 100 μmol·L~(-1)treated stem and exogenous salicylic acid 100 μmol·L~(-1)treated root.
引文
[1]南志标.我国的苜蓿病害及其综合防治体系[J].动物科学与动物医学,2001,18(4):1-4.NAN Z B.Alfalfa diseases and integrated control system of China[J].Animal Science and Veterinary Medicine,2001,18(4):1-4.(in Chinese)
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[3]胡进玲,汪治刚,徐娜,等.防治紫花苜蓿真菌病害的生防菌筛选[J].草业学报,2017,26(6):145-152.HU J L,WANG Z G,XU N,et al.Screening of inhibitory bacteria for prevention and treatment of fungal diseases of Medicago sativa[J].Acta Prataculturae Sinica,2017,26(6):145-152.(in Chinese with English abstract)
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[5]张薇,魏海雷,张力群,等.紫花苜蓿菌核病病原鉴定及其主要生物学特性[J].草业学报,2005,14(2):69-75.ZHANG W,WEI H L,ZHANG L Q,et al.Identification and control of Medicago sativa sclerotinia stem rot occurred in south Jiangsu hilly area[J].Acta Prataculturae Sinica,2005,14(2):69-75.(in Chinese with English abstract)
[6]FOSTER J,LUO B,NAKATA P A.An oxalyl-CoA dependent pathway of oxalate catabolism plays a role in regulating calcium oxalate crystal accumulation and defending against oxalate-secreting phytopathogens in Medicago truncatula[J].PloS One,2016,11(2):15.
[7]MONAZZAH M,SOLEIMANI M J,ENFERADI S T,et al.Effects of oxalic acid and culture filtrate of Sclerotinia sclerotiorum on metabolic changes in sunflower evaluated using FT-IRspectroscopy[J].Journal of General Plant Pathology,2017,84(1):1-10.
[8]CESSNA S G,SEARS V E,DICKMAN M B,et al.Oxalic acid,a pathogenicity factor for Sclerotinia sclerotiorum,suppresses the oxidative burst of the host plant[J].Plant Cell,2000,12(11):2191-2199.
[9]HELLER A,WITTGEIGES T.Oxalic acid has an additional,detoxifying function in Sclerotinia sclerotiorum pathogenesis[J].Plos One,2013,8(8):e72292.
[10]GUIMARAES R L,STOTZ H U.Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection[J].Plant physiology,2004,136(3):3703-3711.
[11]KIM K S,MIN J Y,DICKMAN M B.Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development[J].Molecular Plant-Microbe Interactions,2008,21(5):605-612.
[12]DAVIDSON R M,REEVES P A,MANOSALVA P M,et al.Germins:a diverse protein family important for crop improvement[J].Plant Science,2009,177(6):499-510.
[13]THOMPSON E W,LANE B G.Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk m RNA from dry and imbibing embryos[J].Journal of Biological Chemistry,1980,255(12):5965-5970.
[14]LANE B G.Cellular desiccation and hydration:developmentally regulated proteins,and the maturation and germination of seed embryos[J].The FASEB Journal,1991,5(14):2893-2901.
[15]LANE B G,DUNWELL J M,RAY J A,et al.Germin,a protein marker of early plant development,is an oxlate oxidase[J].Journal of Biological Chemistry,1993,268(17):12239-12242.
[16]DUNWELL J M,GIBBINGS J G,MAHMOOD T,et al.Germin and germin-like proteins:evolution,structure,and function[J].Critical Reviews in Plant Sciences,2008,27(5):34.
[17]LIVINGSTONE D M,HAMPTON J L,PHIPPS P M,et al.Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene[J].Plant Physiology,2005,137(4):1354-1362.
[18]THOMPSON C,DUNWELL J M,JOHNSTONE C E,et al.Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase[J].Euphytica,1995,85(1/3):169-172.
[19]LIU F,WANG M,WEN J,et al,.Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape[J].Plant Pathology,2015,64(6):1407-1416.
[20]HU X,BIDNEY D L,YALPANI N,et al.Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower[J].Plant Physiology,2003,133(1):170-181.
[21]王冰山,窦道龙,张猛,等.Germin基因的克隆及其在烟草中的表达[J].农业生物技术学报,2003,11(3):233-235.WANG B S,DOU D L,ZHANG M,et al.Cloning of Germin gene and its expression in tobacco[J].Chinese Journal of Agricultural Biotechnology,2003,11(3):233-235.(in Chinese with English abstract)
[22]胡杰.草酸氧化酶基因(OXO)遗传转化拟南芥及其抗病机理的初步分析[D].郑州:郑州大学,2014.HU J.Oxalate oxidase gene(OXO)’s genetic transformation in Arabidopsis and its preliminary analysis of disease resistance mechanism[D].Zhengzhou:Zhengzhou University,2014.(in Chinese with English abstract)
[23]向妙莲,汪杰,阙海勇,等.水杨酸诱导车前草抗菌核病研究[J].江西植保,2009,32(3):111-113,116.XIANG M L,WANG J,QUE H Y,et al.Research of salicylic acid against sclerotiorum in Plantago[J].Jiangxi Plant Protection,2009,32(3):111-113,116.(in Chinese with English abstract)
[24]耿志卓,丁立人,逯亚玲,等.外源水杨酸对不同水分胁迫下分枝期紫花苜蓿生长和生理特性的影响[J].草地学报,2016,24(2):369-376.GENG Z Z,DING L R,LU Y L,et al.Effects of salicylic acid on growth and physiological properties of Medicago sativa in branching stage under different water stress[J].Acta Agrectir Sinica,2016,24(2):369-376.(in Chinese with English abstract)
[25]WOO E J,DUNWELL J M,GOODENOUGH P W,et al.Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities[J].Nature Structural&Molecular Biology,2000,7(11):1036-1040.
[26]REQUENA L,BORNEMANN S.Barley(Hordeum vulgare)oxalate oxidase is a manganese-containing enzyme[J].Biochemical Journal,1999,343(1):185-190.
[27]LI L,XU X,CHEN C,et al.Genome-wide characterization and expression analysis of the germin-like protein family in rice and Arabidopsis[J].International Journal of Molecular Sciences,2016,17(10):1622.
[28]李亚平.节瓜遗传转化体系建立及草酸氧化酶基因OsOx O1的转化[D].晋中:山西农业大学,2014.LI Y P.Establishment of transformation system in Chiehqua and transformtion of the Os Ox O1 gene[D].Jinzhong:Shanxi Agricultural University,2014.(in Chinese with English abstract)
[29]YAMCHI A,BEN C,ROSSIGNOL M,et al.Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways[J].Cellular Microbiology,2018,20(4):e12796.
[30]BRISSON L F,TENHAKEN R,LAMB C.Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance[J].Plant Cell,1994,6(12):1703-1712.
[31]PALMA F,L PEZ-G MEZ M,TEJERA N A,et al.Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition[J].Plant Science,2013,208:75-82.
[32]王英哲,任伟,徐安凯,等.低温胁迫下紫花苜蓿对外源SA和ABA的生理响应[J].华北农学报,2012,27(5):144-149.WANG Y Z,REN W,XU A K,et al.Physiological responses to exogenous SA and ABA in alfalfa varieties under chilling stress[J].Acta Agriculturae Boreali-Sinica,2012,27(5):144-149.(in Chinese with English abstract)
[33]WEI F,JIE H U,YANG Y,et al.Transgenic Arabidopsis thaliana,expressing a wheat oxalate oxidase exhibits hydrogen peroxide related defense response[J].Journal of Integrative Agriculture,2015,14(12):2565-2573.
[34]K RK NEN A,KUCHITSU K.Reactive oxygen species in cell wall metabolism and development in plants[J].Phytochemistry,2015,112(1):22-32.
[35]VIDHYASEKARAN P.Reactive oxygen species and cognate redox signaling system in plant innate immunity[M]//PAMPSignals in Plant Innate Immunity.Signaling and Communication in Plants.Dordrecht:Springer,2014:283-306.
[36]YARULLINA L,KASIMOVA R,BURKHANOVA G,et al.The effect of salicylic and jasmonic acids on the activity and range of protective proteins during the infection of wheat by the septoriosis pathogen[J].Biology Bulletin,2015,42(1):27-33.
[2]王传乐,玉永雄.紫花苜蓿菌核病研究进展[J].畜牧与饲料科学,2007,28(2):46-48.WANG C L,YU Y X.Research progress of alfalfa Sclerotini[J].Animal Husbandry and Feed Science,2007,28(2):46-48.(in Chinese)
[3]胡进玲,汪治刚,徐娜,等.防治紫花苜蓿真菌病害的生防菌筛选[J].草业学报,2017,26(6):145-152.HU J L,WANG Z G,XU N,et al.Screening of inhibitory bacteria for prevention and treatment of fungal diseases of Medicago sativa[J].Acta Prataculturae Sinica,2017,26(6):145-152.(in Chinese with English abstract)
[4]BOLTON M D,THOMMA B P H J,NELSON B D.Sclerotinia sclerotiorum(Lib.)de Bary:biology and molecular traits of a cosmopolitan pathogen[J].Molecular Plant Pathology,2006,7(1):1-16.
[5]张薇,魏海雷,张力群,等.紫花苜蓿菌核病病原鉴定及其主要生物学特性[J].草业学报,2005,14(2):69-75.ZHANG W,WEI H L,ZHANG L Q,et al.Identification and control of Medicago sativa sclerotinia stem rot occurred in south Jiangsu hilly area[J].Acta Prataculturae Sinica,2005,14(2):69-75.(in Chinese with English abstract)
[6]FOSTER J,LUO B,NAKATA P A.An oxalyl-CoA dependent pathway of oxalate catabolism plays a role in regulating calcium oxalate crystal accumulation and defending against oxalate-secreting phytopathogens in Medicago truncatula[J].PloS One,2016,11(2):15.
[7]MONAZZAH M,SOLEIMANI M J,ENFERADI S T,et al.Effects of oxalic acid and culture filtrate of Sclerotinia sclerotiorum on metabolic changes in sunflower evaluated using FT-IRspectroscopy[J].Journal of General Plant Pathology,2017,84(1):1-10.
[8]CESSNA S G,SEARS V E,DICKMAN M B,et al.Oxalic acid,a pathogenicity factor for Sclerotinia sclerotiorum,suppresses the oxidative burst of the host plant[J].Plant Cell,2000,12(11):2191-2199.
[9]HELLER A,WITTGEIGES T.Oxalic acid has an additional,detoxifying function in Sclerotinia sclerotiorum pathogenesis[J].Plos One,2013,8(8):e72292.
[10]GUIMARAES R L,STOTZ H U.Oxalate production by Sclerotinia sclerotiorum deregulates guard cells during infection[J].Plant physiology,2004,136(3):3703-3711.
[11]KIM K S,MIN J Y,DICKMAN M B.Oxalic acid is an elicitor of plant programmed cell death during Sclerotinia sclerotiorum disease development[J].Molecular Plant-Microbe Interactions,2008,21(5):605-612.
[12]DAVIDSON R M,REEVES P A,MANOSALVA P M,et al.Germins:a diverse protein family important for crop improvement[J].Plant Science,2009,177(6):499-510.
[13]THOMPSON E W,LANE B G.Relation of protein synthesis in imbibing wheat embryos to the cell-free translational capacities of bulk m RNA from dry and imbibing embryos[J].Journal of Biological Chemistry,1980,255(12):5965-5970.
[14]LANE B G.Cellular desiccation and hydration:developmentally regulated proteins,and the maturation and germination of seed embryos[J].The FASEB Journal,1991,5(14):2893-2901.
[15]LANE B G,DUNWELL J M,RAY J A,et al.Germin,a protein marker of early plant development,is an oxlate oxidase[J].Journal of Biological Chemistry,1993,268(17):12239-12242.
[16]DUNWELL J M,GIBBINGS J G,MAHMOOD T,et al.Germin and germin-like proteins:evolution,structure,and function[J].Critical Reviews in Plant Sciences,2008,27(5):34.
[17]LIVINGSTONE D M,HAMPTON J L,PHIPPS P M,et al.Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene[J].Plant Physiology,2005,137(4):1354-1362.
[18]THOMPSON C,DUNWELL J M,JOHNSTONE C E,et al.Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase[J].Euphytica,1995,85(1/3):169-172.
[19]LIU F,WANG M,WEN J,et al,.Overexpression of barley oxalate oxidase gene induces partial leaf resistance to Sclerotinia sclerotiorum in transgenic oilseed rape[J].Plant Pathology,2015,64(6):1407-1416.
[20]HU X,BIDNEY D L,YALPANI N,et al.Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower[J].Plant Physiology,2003,133(1):170-181.
[21]王冰山,窦道龙,张猛,等.Germin基因的克隆及其在烟草中的表达[J].农业生物技术学报,2003,11(3):233-235.WANG B S,DOU D L,ZHANG M,et al.Cloning of Germin gene and its expression in tobacco[J].Chinese Journal of Agricultural Biotechnology,2003,11(3):233-235.(in Chinese with English abstract)
[22]胡杰.草酸氧化酶基因(OXO)遗传转化拟南芥及其抗病机理的初步分析[D].郑州:郑州大学,2014.HU J.Oxalate oxidase gene(OXO)’s genetic transformation in Arabidopsis and its preliminary analysis of disease resistance mechanism[D].Zhengzhou:Zhengzhou University,2014.(in Chinese with English abstract)
[23]向妙莲,汪杰,阙海勇,等.水杨酸诱导车前草抗菌核病研究[J].江西植保,2009,32(3):111-113,116.XIANG M L,WANG J,QUE H Y,et al.Research of salicylic acid against sclerotiorum in Plantago[J].Jiangxi Plant Protection,2009,32(3):111-113,116.(in Chinese with English abstract)
[24]耿志卓,丁立人,逯亚玲,等.外源水杨酸对不同水分胁迫下分枝期紫花苜蓿生长和生理特性的影响[J].草地学报,2016,24(2):369-376.GENG Z Z,DING L R,LU Y L,et al.Effects of salicylic acid on growth and physiological properties of Medicago sativa in branching stage under different water stress[J].Acta Agrectir Sinica,2016,24(2):369-376.(in Chinese with English abstract)
[25]WOO E J,DUNWELL J M,GOODENOUGH P W,et al.Germin is a manganese containing homohexamer with oxalate oxidase and superoxide dismutase activities[J].Nature Structural&Molecular Biology,2000,7(11):1036-1040.
[26]REQUENA L,BORNEMANN S.Barley(Hordeum vulgare)oxalate oxidase is a manganese-containing enzyme[J].Biochemical Journal,1999,343(1):185-190.
[27]LI L,XU X,CHEN C,et al.Genome-wide characterization and expression analysis of the germin-like protein family in rice and Arabidopsis[J].International Journal of Molecular Sciences,2016,17(10):1622.
[28]李亚平.节瓜遗传转化体系建立及草酸氧化酶基因OsOx O1的转化[D].晋中:山西农业大学,2014.LI Y P.Establishment of transformation system in Chiehqua and transformtion of the Os Ox O1 gene[D].Jinzhong:Shanxi Agricultural University,2014.(in Chinese with English abstract)
[29]YAMCHI A,BEN C,ROSSIGNOL M,et al.Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways[J].Cellular Microbiology,2018,20(4):e12796.
[30]BRISSON L F,TENHAKEN R,LAMB C.Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance[J].Plant Cell,1994,6(12):1703-1712.
[31]PALMA F,L PEZ-G MEZ M,TEJERA N A,et al.Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition[J].Plant Science,2013,208:75-82.
[32]王英哲,任伟,徐安凯,等.低温胁迫下紫花苜蓿对外源SA和ABA的生理响应[J].华北农学报,2012,27(5):144-149.WANG Y Z,REN W,XU A K,et al.Physiological responses to exogenous SA and ABA in alfalfa varieties under chilling stress[J].Acta Agriculturae Boreali-Sinica,2012,27(5):144-149.(in Chinese with English abstract)
[33]WEI F,JIE H U,YANG Y,et al.Transgenic Arabidopsis thaliana,expressing a wheat oxalate oxidase exhibits hydrogen peroxide related defense response[J].Journal of Integrative Agriculture,2015,14(12):2565-2573.
[34]K RK NEN A,KUCHITSU K.Reactive oxygen species in cell wall metabolism and development in plants[J].Phytochemistry,2015,112(1):22-32.
[35]VIDHYASEKARAN P.Reactive oxygen species and cognate redox signaling system in plant innate immunity[M]//PAMPSignals in Plant Innate Immunity.Signaling and Communication in Plants.Dordrecht:Springer,2014:283-306.
[36]YARULLINA L,KASIMOVA R,BURKHANOVA G,et al.The effect of salicylic and jasmonic acids on the activity and range of protective proteins during the infection of wheat by the septoriosis pathogen[J].Biology Bulletin,2015,42(1):27-33.