外源NO及反向调控PEG胁迫下苜蓿萌发种子抗氧化酶及其同工酶动态的研究

doi:10.11686/cyxb2017478

摘要/Abstract

摘要： 以紫花苜蓿为材料,用一氧化氮供体硝普钠(SNP)及一氧化氮清除剂c-PTIO对苜蓿种子进行浸种处理,采用分光光度法和同工酶电泳技术来研究外源NO及反向调控对PEG胁迫下紫花苜蓿抗氧化酶活性及其同工酶的影响,并探讨NO调控苜蓿种子耐旱性的生理机制。结果表明:PEG胁迫下外施0.1 mmol·L^-1 SNP,第6天时较PEG处理POD活性降低了32.72%;第4天时SOD、CAT活性较PEG处理升高了10.48%、23.60%,有效缓解了PEG对紫花苜蓿萌发中种子的氧化损伤,提高其抗氧化能力。PEG胁迫下添加 c-PTIO抑制了苜蓿萌发期的抗氧化系统活性。从同工酶的谱带数量和强弱来看,POD同工酶各区带活力均随PEG胁迫时间的延长而增加,在第2天酶带只有1条,而第4天酶带呈现9条;SOD和CAT同工酶表达量变化不显著,但酶带强弱有一定变化,S3酶带随处理时间的延长表达量逐渐减弱;CAT同工酶谱带则一直保持2条带,无明显强弱变化。因此,外源NO在PEG胁迫下紫花苜蓿萌发中抗氧化酶快速响应并在维护氧自由基代谢平衡中起重要保护作用。

关键词: 一氧化氮, PEG胁迫, 紫花苜蓿, 抗氧化酶, 同工酶电泳

Abstract: To study the effects of exogenous NO and reverse regulation antioxidant enzyme activities and isoenzymes of alfalfa under PEG stress, alfalfa seeds were used as experimental materials, they were pretreated with nitric oxide donor sodium nitroprusside (SNP) or nitric oxide scavenger c-PTIO, the methods of spectrophotometry and isoenzyme electrophoresis were used to analyze the activities of antioxidant enzymes. The results showed that: under the PEG stress, the external application of 0.1 mmol·L^-1 SNP, the activities of POD were 32.72% lower than the PEG treatment on the 6th day; the activities of SOD and CAT were 10.48% and 23.60% higher than the PEG treatment on the 4th day respectively, so they could alleviate the oxidation damage on alfalfa seeds which caused by PEG stress. In addition, the exogenous of c-PTIO under drought stress inhibited the activity of antioxidant system during alfalfa germination stage. According to the number and intensity of isozymes, with prolonging the PEG stress time, the bands of POD isoenzymes activity increased, and only one strip on the second day, while 9 strips on the 4th day; the expression of isoenzymes of SOD and CAT were not significantly, but the strength of the enzyme band changed to some extent, the expression of S3 enzyme band was gradually weakened with prolonged the treatment time; CAT isoenzyme band had been kept at 2 bands all through, showed no significantly changes in strength. Therefore, exogenous NO rapidly responded to antioxidant enzymes during the germination of alfalfa under PEG stress and played an important protective role in the maintenance of oxygen free radical metabolism.

Key words: nitric oxide, PEG stress, Medicago sativa, antioxidant enzymes, isozyme electrophoresis

辛夏青, 魏小红, 韩厅, 岳凯, 赵颖. 外源NO及反向调控PEG胁迫下苜蓿萌发种子抗氧化酶及其同工酶动态的研究[J]. 草业学报, 2018, 27(10): 105-112.

XIN Xia-qing, WEI Xiao-hong, HAN Ting, YUE Kai, ZHAO Ying. Research on exogenous NO and reverse regulation to the dynamic changes of antioxidant enzymes and isoenzymes in alfalfa seed germination under PEG stress[J]. Acta Prataculturae Sinica, 2018, 27(10): 105-112.

参考文献

[1] Zheng G H, Liang Z, Lin J.Study on chilling injury and osmotic control of seed. Journal of the Chinese Academy of Sciences, 2001, 3: 182-187.
郑光华, 梁峥, 林坚. 种子吸胀冷害和渗透调控的研究. 中国科学院院刊, 2001, 3: 182-187.
[2] Yi Q, Wei X H, Qiang X, et al. Investigation into the mechanism of Mo-mediated Ca²+ signaling during seed germination and antioxidation in Medicago sativa past dust stress. Acta Prataculturae Sinica, 2016, 25(11): 57-65.
弋钦, 魏小红, 强旭. NO介导的Ca²+信号在干旱胁迫下紫花苜蓿种子萌发及抗氧化酶中的传导作用研究. 草业学报, 2016, 25(11): 57-65.
[3] Dein U, Stephan A B, Horie T, et al. Plant salt-tolerance mechanism. Trends in Plant Science, 2014, 19: 371-379.
[4] He Q Y, Xiang S H, Wu P, et al. Effects of hydrogen sulfide on antioxidant enzyme activities in soybean under salt stress. Soybean Science, 2015, 34: 427-431.
何庆元, 向仕华, 吴萍, 等. 硫化氢对盐胁迫条件下大豆抗氧化酶活性的影响. 大豆科学, 2015, 34: 427-431.
[5] Sairam R K, Vasanthan B, Ajay A.Calcium regulates Gladiolus flower senescence by influencing antioxidative enzymes activity. Acta Physiologiae Plantarum, 2011, 33: 1897-1904.
[6] Beligni M V, Lamattina L.Is nitric oxide toxic or protective. Trends Plant Science, 1999, 4(8): 229-300.
[7] Beligni M V, Lamattina L.Nitric oxide protects against cellular damage produced by methylviologen herbicides in potato plants. Nitric Oxide, 1999, 3(3): 199-208.
[8] Xu Y, Wei X H, Li B B, et al. Effects of exogenous nitric oxide on seed germination and seedling oxidative damage of alfalfa under NaCl stress. Acta Prataculturae Sinica, 2013, 22(5): 145-153.
徐严, 魏小红, 李兵兵, 等. 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, 2013, 22(5): 145-153.
[9] Li H, Zhao W C, Zhao H J, et al. Effects of exogenous nitric oxide donor sodium nitroprusside on ATPase activity and membrane lipid peroxidation in leaves of wheat seedlings under drought stress. Plant Physiology Newsletter, 2009, 45(5): 455-458.
李慧, 赵文才, 赵会杰, 等. 外源一氧化氮供体硝普钠对干旱胁迫下小麦幼苗叶中ATP酶活性和膜脂过氧化的影响. 植物生理学通讯, 2009, 45(5): 455-458.
[10] Toyomasu T, Zennyoxi A.On the application of isoenzyme electrophoresis to identification of strains in Leurinus edodes. Mushroom Science, 1981, 11: 675-684.
[11] Jie S, Fu X Z, Peng T, et al. Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modylation of amtioxidative capacity and stomatal response. Tree Phusuology, 2010, 30(7): 914-922.
[12] Chen J X, Wang X F.Guide of plant physiological experiments. Guangzhou: South China University of Technology Press, 2006.
陈建勋, 王晓峰. 植物生理学实验指导. 广州: 华南理工大学出版社, 2006.
[13] Hu N S, Wan X G.Isoenzyme technology and its application. Changsha: Hunan Science and Technology Press, 1985: 3-105.
胡能书, 万贤国. 同工酶技术及其应用. 长沙: 湖南科学技术出版社, 1985: 3-105.
[14] Liang H W, Liu S, Chen F J, et al. Changes of peroxidase and esterase isoenzymes in the process of germination of silver magpie trees. Seed, 2006, 25(5): 38-40.
梁宏伟, 刘姝, 陈发菊, 等. 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, 2006, 25(5): 38-40.
[15] He Z X, Zhang S Z.Electrophoresis. The Second Press. Beijing: Science Press, 1999.
何忠效, 张树政. 电泳. 第二版. 北京:科学出版社, 1999.
[16] Cai X Y, Chen X D, Li C Z, et al. Effects of exogenous calcium on seed germination of phyllostachys heterocycla in limestone region under drought stress. Chinese Journal of Applied Ecology, 2013, 24(5): 1341-1346.
蔡喜悦, 陈晓德, 李朝政, 等. 干旱胁迫下外源钙对石灰岩地区富羽叶亲树种子萌发期的影响. 应用生态学报, 2013, 24(5): 1341-1346.
[17] Tang S H, Zhou Q G, Sun M, et al. Effects of exogenous nitric oxide on seed germination, seedling growth and physiological characteristics of cucumber under osmotic stress. China Agricultural Sciences, 2007, 40(2): 419-425.
汤绍虎, 周启贵, 孙敏, 等. 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, 2007, 40(2): 419-425.
[18] Zhang H S, Zhao G Q, Li M F, et al. Physiological response of medium pennisetum seedlings to PEG, low temperature and salt stress. Acta Prataculturae Sinica, 2014, 23(2): 180-188.
张怀山, 赵桂琴, 栗孟飞, 等. 中型狼尾草幼苗对PEG、低温和盐胁迫的生理应答. 草业学报, 2014, 23(2): 180-188.
[19] Jiang Y B, Yang Y R, Zheng Q H.Effects of exogenous nitric oxide on antioxidase and chlorophyll fluorescence of seedling of alfalfa under drought stress. Agricultural Research in Arid Areas, 2008, 26(2): 65-67.
姜义宝, 杨玉荣, 郑秋红. 外源一氧化氮对干旱胁迫下苜蓿幼苗抗氧化酶活性和叶绿素荧光特性的影响. 干旱地区农业研究, 2008, 26(2): 65-67.
[20] Moreau M, Lindermayr C, Durner J, et al. NO synthesis and signaling in plants-where do we stand. Physiologia Plantarum, 2010, 138(4): 372-383.
[21] Wang S H, Zhang H, He Q Y.Effects of copper stress on antioxidant system of alfalfa seedling leaves. Chinese Journal of Applied Ecology, 2011, 22(9): 2285-2290.
王松华, 张华, 何庆元. 铜胁迫对紫花苜蓿幼苗叶片抗氧化系统的影响. 应用生态学报, 2011, 22(9): 2285-2290.
[22] Liu S C, Xiao L T, Liao B H, et al. Effects of lead stress on antioxidant enzyme activities and isoenzymes in cucumber seedlings. Chinese Journal of Applied Ecology, 2006, 17(2): 300-304.
刘素纯, 萧浪涛, 廖柏寒, 等. 铅胁迫对黄瓜幼苗抗氧化酶活性及同工酶的影响. 应用生态学报, 2006, 17(2): 300-304.
[23] Chen S P, Gao Y B, Liang Y, et al. Effects of endophytic fungal infection on protective enzyme activities in leaves of ryegrass under water stress. Journal of Applied and Environmental Biology, 2001, 7(4): 348-354.
陈世苹, 高玉葆, 梁宇, 等. 水分胁迫下内生真菌感染对黑麦草叶内保护酶系统活力的影响. 应用与环境生物学报, 2001, 7(4): 348-354.
[24] Mo R, Zheng C M.Analysis of esterase isozymes in rice seedlings under water stress. Journal of Tropical Crops, 1999, 20(4): 32-36.
莫饶, 郑成木. 水分胁迫下水稻幼苗酯酶等同工酶的分析. 热带作物学报, 1999, 20(4): 32-36.
[25] Li S Y, Qiao C, Hu R P, et al. Study on POD, CAT and SOD isozymes of spirulina platensis. Microbiology Bulletin, 2004, 31(1): 82-85.
栗淑媛, 乔辰, 扈瑞平, 等. 螺旋藻POD, CAT和SOD同工酶的研究. 微生物学通报, 2004, 31(1): 82-85.
[26] Li Y W.Comparative analysis of hydrogen peroxidease gene (CAT) isozymes in rice under adversity stress. Harbin: Northeast Forestry University, 2009.
李耀文. 水稻过氧化氢酶基因(CAT)同工酶在逆境胁迫下的比较分析. 哈尔滨: 东北林业大学, 2009.
[27] Lu Z Q.Study on the functional characteristics of APX and CAT isoenzymes in rice and their relationship with salt stress. Harbin: Northeast Forestry University, 2005.
鲁振强. 水稻APX与CAT同工酶的功能特性及其与盐胁迫关系的研究. 哈尔滨: 东北林业大学, 2005.