外源NO对PEG胁迫下苜蓿幼苗抗氧化酶及同工酶的影响
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摘要
为研究外源NO对聚乙二醇-6000(PEG)拟干旱胁迫下紫花苜蓿抗氧化酶活性及其同工酶的影响。本试验用一氧化氮供体硝普钠(SNP)或一氧化氮清除剂c-PTIO对干旱胁迫下紫花苜蓿幼苗进行处理,并采用分光光度法和聚丙烯酰胺凝胶电泳技术对抗氧化酶及其同工酶研究。结果表明:SNP+PEG处理能显著提高紫花苜蓿幼苗的地上部干重和根干重,分别增加了100.00%、61.54%;而不同处理的SOD、POD、CAT活性都随着处理时间的延长呈现先升高后降低的趋势,在处理的第4天,SOD、CAT活性达到最大值,在处理的第6天,SOD活性达到最高。在处理的第4天,SNP+NO处理POD活性降低了32.18%,而POD、CAT活性升高了9.81%、43.37%,说明SNP能够通过不同程度影响抗氧化酶活性而缓解PEG对紫花苜蓿的氧化损伤。PEG+c-PTIO则抑制了紫花苜蓿幼苗的抗氧化酶活性。不同时间各处理紫花苜蓿幼苗POD同工酶都呈现9条酶带,其中P1、P5、P6酶带不同处理之间酶活性明显不同;SOD同工酶在处理过程中产生相对迁移率分别为0.610、0.470和0.345的3条酶带。PEG+SNP处理酶带颜色较深。而CAT同工酶形成了两条相对迁移率分别为0.322、0.259的酶带,各处理之间无明显的变化。说明外源SNP能有效的缓解PEG对紫花苜蓿幼苗造成的氧化损伤,促进植物的生长。本研究结果为紫花苜蓿的耐旱生理机制研究提供了一定的科学依据。
In order to study the effects of exogenous NO on antioxidant enzyme activities and isozymes of Medicago sativa under the drought stress of polyethylene glycol-6000(PEG). This experiment used nitric oxide donor sodium nitroprusside(SNP) or nitric oxide scavenger c-PTIO to treat alfalfa seedlings under drought stress, and used spectrophotometry and polyacrylamide gel electrophoresis to study the antioxidant enzymes and their isozymes. The results showed that SNP +PEG treatment could significantly increase the aboveground dry weight and root dry weight of alfalfa seedlings, which increased by 100% and 61.54%, respectively, while the activity of SOD, POD and CAT in different treatments increased first and then decreased with the prolongation of treatment time. At 4 d of treatment, the activity of POD and CAT reached the maximum value. At 6 d of treatment, the activity of SOD reached the highest. On the 4 d of treatment, SNP+NO treatment decreased POD activity by 32.18%,while SOD and CAT activities increased by 9.81% and 43.37%, it indicated that SNP can alleviate the oxidative damage of PEG to alfalfa by affecting the activity of antioxidant enzymes to varying degrees. PEG+c-PTIO inhibited the antioxidant enzyme activities of alfalfa seedlings. The POD isozyme of alfalfa seedlings treated at different time showed 9 enzyme bands, and the enzyme activities of P1, P5 and P6 under different treatments were different.3 enzyme bands were produced in the process of treatment of SOD isozyme and the relative mobility was 0.610,0.470 and 0.345, respectively. The color of the enzyme band in PEG +SNP treatment was deeper. CAT isozymes formed two bands with relative mobility of 0.322 and 0.259, respectively. There was no significant change among the treatments. The results showed that exogenous SNP could effectively alleviate oxidative damage caused by PEG to alfalfa seedlings and promote plant growth. The results would provide a scientific basis for the physiological mechanism of drought resistance in alfalfa.
In order to study the effects of exogenous NO on antioxidant enzyme activities and isozymes of Medicago sativa under the drought stress of polyethylene glycol-6000(PEG). This experiment used nitric oxide donor sodium nitroprusside(SNP) or nitric oxide scavenger c-PTIO to treat alfalfa seedlings under drought stress, and used spectrophotometry and polyacrylamide gel electrophoresis to study the antioxidant enzymes and their isozymes. The results showed that SNP +PEG treatment could significantly increase the aboveground dry weight and root dry weight of alfalfa seedlings, which increased by 100% and 61.54%, respectively, while the activity of SOD, POD and CAT in different treatments increased first and then decreased with the prolongation of treatment time. At 4 d of treatment, the activity of POD and CAT reached the maximum value. At 6 d of treatment, the activity of SOD reached the highest. On the 4 d of treatment, SNP+NO treatment decreased POD activity by 32.18%,while SOD and CAT activities increased by 9.81% and 43.37%, it indicated that SNP can alleviate the oxidative damage of PEG to alfalfa by affecting the activity of antioxidant enzymes to varying degrees. PEG+c-PTIO inhibited the antioxidant enzyme activities of alfalfa seedlings. The POD isozyme of alfalfa seedlings treated at different time showed 9 enzyme bands, and the enzyme activities of P1, P5 and P6 under different treatments were different.3 enzyme bands were produced in the process of treatment of SOD isozyme and the relative mobility was 0.610,0.470 and 0.345, respectively. The color of the enzyme band in PEG +SNP treatment was deeper. CAT isozymes formed two bands with relative mobility of 0.322 and 0.259, respectively. There was no significant change among the treatments. The results showed that exogenous SNP could effectively alleviate oxidative damage caused by PEG to alfalfa seedlings and promote plant growth. The results would provide a scientific basis for the physiological mechanism of drought resistance in alfalfa.
引文
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Deng Z.,Bai D.,Zhai G.L.,Zhao H.S.,Feng J.J.,Han Q.B.,and Zhang W.Z.,2011,Study on the effect of plant growth regulators on dry matter accumulation,yield and quality of cotton in Xinjia ng,Ganhan Diqu Nongye Yanjiu(Agricultural Research in the Arid Areas),29(3):122-127(邓忠,白丹,翟国亮,赵红书,冯俊杰,韩启彪,张文正,2011,不同植物生长调节剂对新疆棉花干物质积累、产量和品质的影响,干旱地区农业研究,29(3):122-127)
Han R.H.,2006,Study on adaptive mechanisms of alfalfa(Medicago sativa)in drought stress at seedling stage,Dissertation for Ph.D.,Beijing Forestry University,Supervisor:Lu X.S.,pp.22-50(韩瑞宏,2006,苗期紫花苜蓿(Medicago sativa)对干旱胁迫的适应机制研究,博士学位论文,北京林业大学,导师:卢欣石,pp.22-50)
Kim K.Y.,Lee S.Y.,Cho Y.S.,Bang I.C.,Kim K.H.,Kim D.S.,and Nam Y.K.,2007,Molecular characterization and m RNAexpression during metal exposure and thermal stress of copper/zinc-and manganese-superoxide dismutases in disk abalone,Haliotis discus discus,Fish Shellfish Immunol.,23(5):1047-1059
Liang H.G.,Liu S.,Chen F.J.,and Xiong D.,2006,Development of peroxidase and isoesterase isozymes during seed germination of Tapiscia sinensis Oliv,Zhongzi(Seed),25(5):38-40(梁宏伟,刘姝,陈发菊,熊丹,2006,银鹊树种子萌发过程中的过氧化物酶和酯酶同工酶的变化,种子,25(5):38-40)
Liu G.L.,Li H.,LüL.T.,and Zhang M.,2018,Eaterase isozyme analysis of twenty strains of Auricularia auricular,Yingyong Shengtai Xuebao(Journal of Applied Ecology),(5):149-153(刘国丽,李红,吕立涛,张敏,2018,二十个黑木耳菌株的酯酶同工酶分析,应用生态学报,(5):149-153)
Liu W.Y.,Yang H.W.,Wei X.H.,Liu B.,Wang G.Q.,and Wu W.T.,2015,Effects of exogenous nitric oxide on seed germination physiological characteristics and active oxygen metabolish of Medicago truncatula under NaCl stress,Caoye Xuebao(Acta Prataculturae Sinica),24(2):85-95(刘文瑜,杨宏伟,魏小红,刘博,王高强,吴伟涛,2015,外源NO调控盐胁迫下蒺藜苜蓿种子萌发生理特性及抗氧化酶的研究,草业学报,24(2):85-95)
Liu X.M.,Liu C.,Liu Q.W.,Zhang Y.D.,and Li X.M.,2016,The effects of PEG pretreatment on the isozymes and expression of antioxidant enzymes in rice leaves under water stress,Zuowu Zazhi(Crops),(6):107-111(李雪妹,刘畅,刘倩雯,张煜笛,李雪梅,2016,PEG预处理对水分胁迫下水稻叶片抗氧化酶同工酶及其表达的影响,作物杂志,(6):107-111)
Sairam R.K.,Vasanthan B.,and Arora A.,2011,Calcium regulates Gladiolus flower senescence by influencing antioxidative enzymes activity,Acta Physiologiae Plantarum,33(5):1897-1904
Shao R.X.,Li L.L.,Zheng H.F.,Zhang J.Y.,Yang S.J.,Ma Y.,Xin L.F.,Su X.Y.,Ran W.L.,Mao J.,Zheng B.Y.,and Yang Q.H.,2016,Effects of exogenous nitric oxide on photosynthesis of maize seeding under drought stress,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(2):251-259(邵瑞鑫,李蕾蕾,郑会芳,张寄阳,杨慎娇,马野,信龙飞,苏小雨,冉午玲,毛俊,郑博元,杨青华,2016,外源一氧化氮对干旱胁迫下玉米幼苗光合作用的影响,中国农业科学,49(2):251-259)
Sun J.H.,Wang Y.R.,and Yu L.,2004,Growth characteristics and their correlation with the yield of Medicago sativa,Caoye Xuebao(Acta Prataculturae Sinica),13(4):80-86(孙建华,王彦荣,余玲,2004,紫花苜蓿生长特性及产量性状相关性研究,草业学报,13(4):80-86)
Tang S.H.,Zhou Q.G.,Sun M.,and Mao W.,2007,Effects of exogenous nitric oxide on seed germination,seedling growth and physiological characteristics of cucumber under osmotic pressure,Zhongguo Nongye Kexue(China Agricultural Sciences),40(2):419-425(汤绍虎,周启贵,孙敏,毛薇,2007,外源NO对渗透胁迫下黄瓜种子萌发、幼苗生长和生理特性的影响,中国农业科学,40(2):419-425)
Wang J.K.,Wang J.,Hu Y.P.,Li Y.,Chai W.X.,and Wang L.,2018,Responses of Medicago sativa seed to different salt stresses during germination,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):268-274(王建科,王钧,胡延萍,李毅,柴文秀,王莉,2018,紫花苜蓿种子萌发过程中对不同盐胁迫的响应,分子植物育种,16(1):268-274)
Xu Y.,Wei X.H.,Li B.B.,Cao L.,and Tang Z.M.,2013,Effects of exogenous nitric oxide on seed germination and seedling oxidative damage in Medicago sativa under NaCl stress,Caoye Xuebao(Acta Prataculturae Sinica),22(5):145-153(徐严,魏小红,李兵兵,曹丽,唐志敏,2013,外源NO对NaCl胁迫下紫花苜蓿种子萌发及幼苗氧化损伤的影响,草业学报,22(5):145-153)
Zhang X.Q.,Wang S.J.,and Sun M.,2015,Dynamic responses of active oxygen metabolism of Barbula fallax in rock desertification of Karst areas to drought stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):160-164(张显强,王世杰,孙敏,2015,喀斯特石生反叶扭口藓活性氧代谢对干旱胁迫的动态响应,基因组学与应用生物学,34(1):160-164)
Zhao M.,and Wei X.H.,2015 Effects of nitric oxide on Medicago sativa seed germination under imbibitional chilling,Caoye Xuebao(Acta Prataculturae Sinica),24(4):87-94(赵萌,魏小红,2015,吸胀冷害下外源NO对紫花苜蓿种子萌发及抗氧化性的影响,草业学报,24(4):87-94)
Deng Z.,Bai D.,Zhai G.L.,Zhao H.S.,Feng J.J.,Han Q.B.,and Zhang W.Z.,2011,Study on the effect of plant growth regulators on dry matter accumulation,yield and quality of cotton in Xinjia ng,Ganhan Diqu Nongye Yanjiu(Agricultural Research in the Arid Areas),29(3):122-127(邓忠,白丹,翟国亮,赵红书,冯俊杰,韩启彪,张文正,2011,不同植物生长调节剂对新疆棉花干物质积累、产量和品质的影响,干旱地区农业研究,29(3):122-127)
Han R.H.,2006,Study on adaptive mechanisms of alfalfa(Medicago sativa)in drought stress at seedling stage,Dissertation for Ph.D.,Beijing Forestry University,Supervisor:Lu X.S.,pp.22-50(韩瑞宏,2006,苗期紫花苜蓿(Medicago sativa)对干旱胁迫的适应机制研究,博士学位论文,北京林业大学,导师:卢欣石,pp.22-50)
Kim K.Y.,Lee S.Y.,Cho Y.S.,Bang I.C.,Kim K.H.,Kim D.S.,and Nam Y.K.,2007,Molecular characterization and m RNAexpression during metal exposure and thermal stress of copper/zinc-and manganese-superoxide dismutases in disk abalone,Haliotis discus discus,Fish Shellfish Immunol.,23(5):1047-1059
Liang H.G.,Liu S.,Chen F.J.,and Xiong D.,2006,Development of peroxidase and isoesterase isozymes during seed germination of Tapiscia sinensis Oliv,Zhongzi(Seed),25(5):38-40(梁宏伟,刘姝,陈发菊,熊丹,2006,银鹊树种子萌发过程中的过氧化物酶和酯酶同工酶的变化,种子,25(5):38-40)
Liu G.L.,Li H.,LüL.T.,and Zhang M.,2018,Eaterase isozyme analysis of twenty strains of Auricularia auricular,Yingyong Shengtai Xuebao(Journal of Applied Ecology),(5):149-153(刘国丽,李红,吕立涛,张敏,2018,二十个黑木耳菌株的酯酶同工酶分析,应用生态学报,(5):149-153)
Liu W.Y.,Yang H.W.,Wei X.H.,Liu B.,Wang G.Q.,and Wu W.T.,2015,Effects of exogenous nitric oxide on seed germination physiological characteristics and active oxygen metabolish of Medicago truncatula under NaCl stress,Caoye Xuebao(Acta Prataculturae Sinica),24(2):85-95(刘文瑜,杨宏伟,魏小红,刘博,王高强,吴伟涛,2015,外源NO调控盐胁迫下蒺藜苜蓿种子萌发生理特性及抗氧化酶的研究,草业学报,24(2):85-95)
Liu X.M.,Liu C.,Liu Q.W.,Zhang Y.D.,and Li X.M.,2016,The effects of PEG pretreatment on the isozymes and expression of antioxidant enzymes in rice leaves under water stress,Zuowu Zazhi(Crops),(6):107-111(李雪妹,刘畅,刘倩雯,张煜笛,李雪梅,2016,PEG预处理对水分胁迫下水稻叶片抗氧化酶同工酶及其表达的影响,作物杂志,(6):107-111)
Sairam R.K.,Vasanthan B.,and Arora A.,2011,Calcium regulates Gladiolus flower senescence by influencing antioxidative enzymes activity,Acta Physiologiae Plantarum,33(5):1897-1904
Shao R.X.,Li L.L.,Zheng H.F.,Zhang J.Y.,Yang S.J.,Ma Y.,Xin L.F.,Su X.Y.,Ran W.L.,Mao J.,Zheng B.Y.,and Yang Q.H.,2016,Effects of exogenous nitric oxide on photosynthesis of maize seeding under drought stress,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),49(2):251-259(邵瑞鑫,李蕾蕾,郑会芳,张寄阳,杨慎娇,马野,信龙飞,苏小雨,冉午玲,毛俊,郑博元,杨青华,2016,外源一氧化氮对干旱胁迫下玉米幼苗光合作用的影响,中国农业科学,49(2):251-259)
Sun J.H.,Wang Y.R.,and Yu L.,2004,Growth characteristics and their correlation with the yield of Medicago sativa,Caoye Xuebao(Acta Prataculturae Sinica),13(4):80-86(孙建华,王彦荣,余玲,2004,紫花苜蓿生长特性及产量性状相关性研究,草业学报,13(4):80-86)
Tang S.H.,Zhou Q.G.,Sun M.,and Mao W.,2007,Effects of exogenous nitric oxide on seed germination,seedling growth and physiological characteristics of cucumber under osmotic pressure,Zhongguo Nongye Kexue(China Agricultural Sciences),40(2):419-425(汤绍虎,周启贵,孙敏,毛薇,2007,外源NO对渗透胁迫下黄瓜种子萌发、幼苗生长和生理特性的影响,中国农业科学,40(2):419-425)
Wang J.K.,Wang J.,Hu Y.P.,Li Y.,Chai W.X.,and Wang L.,2018,Responses of Medicago sativa seed to different salt stresses during germination,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),16(1):268-274(王建科,王钧,胡延萍,李毅,柴文秀,王莉,2018,紫花苜蓿种子萌发过程中对不同盐胁迫的响应,分子植物育种,16(1):268-274)
Xu Y.,Wei X.H.,Li B.B.,Cao L.,and Tang Z.M.,2013,Effects of exogenous nitric oxide on seed germination and seedling oxidative damage in Medicago sativa under NaCl stress,Caoye Xuebao(Acta Prataculturae Sinica),22(5):145-153(徐严,魏小红,李兵兵,曹丽,唐志敏,2013,外源NO对NaCl胁迫下紫花苜蓿种子萌发及幼苗氧化损伤的影响,草业学报,22(5):145-153)
Zhang X.Q.,Wang S.J.,and Sun M.,2015,Dynamic responses of active oxygen metabolism of Barbula fallax in rock desertification of Karst areas to drought stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):160-164(张显强,王世杰,孙敏,2015,喀斯特石生反叶扭口藓活性氧代谢对干旱胁迫的动态响应,基因组学与应用生物学,34(1):160-164)
Zhao M.,and Wei X.H.,2015 Effects of nitric oxide on Medicago sativa seed germination under imbibitional chilling,Caoye Xuebao(Acta Prataculturae Sinica),24(4):87-94(赵萌,魏小红,2015,吸胀冷害下外源NO对紫花苜蓿种子萌发及抗氧化性的影响,草业学报,24(4):87-94)