NO和葡萄糖-6-磷酸脱氢酶在大豆铝胁迫响应中的调节作用研究

发布时间：2018-03-14 23:24

本文选题：铝胁迫　切入点：一氧化氮　出处：《河南师范大学》2017年硕士论文　论文类型：学位论文

【摘要】：铝胁迫是酸性土壤中限制植物正常生长和农作物产量的主要逆境因子,铝毒害对植物最初和最显著的一个特征就是抑制植物根的生长。一氧化氮(NO)作为信号分子参与了多种生物胁迫和非生物胁迫响应,然而其在铝胁迫中的调节作用仍不太清楚。作为戊糖磷酸途径的限速酶之一,葡萄糖-6-磷酸脱氢酶(G6PDH)可以催化产生还原力NADPH。有研究报道,G6PDH参与了多种环境胁迫的抗逆性反应,然而关于其对铝胁迫的响应及其调节作用还未见报道。本研究以大豆为实验材料,探讨了NO和G6PDH对铝胁迫的响应及其在铝毒害下的调控机制。主要结果如下:1.低浓度铝胁迫下NO对铝毒害的调节作用铝胁迫下,大豆幼苗根长和根尖伸长区细胞大小均受到抑制,膜脂过氧化程度增加,活性氧(H2O2和O2ˉ·)含量显著升高;抗氧化剂BHA处理则缓解了铝毒害引发的根生长抑制和膜脂过氧化程度。表明铝胁迫通过刺激根中活性氧(ROS)的累积,加深了膜脂过氧化的程度,从而导致氧化伤害,最终影响根尖伸长区细胞的形态大小,造成根生长抑制。铝胁迫下,NO供体(SNP)处理减轻了铝诱导的根伸长抑制、ROS积累以及膜脂过氧化程度,而NO清除剂(PTIO)处理则进一步加重了铝毒害。进一步研究结果显示,铝胁迫下SNP处理能显著增强抗氧化酶(POD、CAT)活性、抗氧化剂(GSH和ASA)含量以及GSH-ASA循环系统中的关键酶(APX、GR、DHAR和MDHAR)活性,而PTIO处理则抑制了铝诱导的抗氧化剂含量及抗氧化酶活性的增加。铝胁迫处理显著增加了大豆根中NO含量和硝酸还原酶(NR)活性,NR抑制剂处理则消除了铝诱导的NO含量的增加,而一氧化氮合酶(NOS)抑制剂处理后,NO含量没有明显改变,表明铝胁迫下NO的产生主要来自于NR途径。这些结果说明,铝胁迫下NR介导产生的NO通过调节抗氧化酶活性以及GSH-ASA循环中的GSH和ASA含量来清除过量的ROS,进而增强大豆对铝毒害的耐受性。2.高浓度铝胁迫下G6PDH对铝毒害的调节作用高浓度铝胁迫(≥200μM)处理能显著增加大豆根中总G6PDH活性,并随着铝浓度的增加酶活性逐渐升高;同时,胞质G6PDH(Cyt-G6PDH)活性与总G6PDH活性的变化趋势类似,而质体G6PDH活性则没有明显改变,表明铝胁迫引发的G6PDH活性的增加主要来源于Cyt-G6PDH。铝胁迫下,G6PDH抑制剂(GN6P)处理缓解了铝诱导的根生长抑制、膜脂过氧化以及ROS的积累,表明G6PDH参与了铝对大豆的毒害作用。进一步研究结果显示,NADPH氧化酶抑制剂(DPI)处理消除了铝胁迫引发的ROS积累,表明铝胁迫下ROS积累来自于NADPH氧化酶途径。铝胁迫下,GN6P处理降低了NADPH含量以及NADPH氧化酶活性,表明G6PDH调节了NADPH氧化酶的活性。随后的研究结果显示,铝胁迫提高了Cyt-G6PDH活性及编码基因(G6PDH1和G6PDH2)的表达,SNP处理进一步增强了其活性及基因表达,PTIO处理则抑制了其活性及基因表达,表明NO介导了铝胁迫诱导的Cyt-G6PDH活性的增强,该调节方式可能是通过增强G6PDH1和G6PDH2的基因表达实现的。此外,高浓度铝胁迫下,NO含量显著增强,其产生途径也是来源于NR途径。这些结果说明,铝胁迫下由NO介导的Cyt-G6PDH通过调节NADPH氧化酶依赖的ROS的积累,使得大豆遭受氧化伤害,最终导致大豆根生长抑制。综上所述,铝胁迫下,大豆幼苗能够通过NR途径产生NO,NO有两方面的作用,一方面是相对低浓度铝胁迫下,通过调节抗氧化酶活性和抗氧化剂含量来缓解铝胁迫造成的氧化损伤和根长抑制;另一方面,相对高浓度铝胁迫下,NO能够通过调节Cyt-G6PDH编码基因的表达来增强Cyt-G6PDH活性,由此引发NADPH的累积和NADPH氧化酶活性的增强,导致ROS的积累和氧化伤害程度的加深,最终引发根生长抑制。
[Abstract]:Aluminum stress is the main stress factors limiting plant growth and crop production in acid soils, aluminum toxicity to plants initially and one of the most significant feature is the inhibition of root growth. Nitric oxide (NO) as a signal molecule involved in various biotic and abiotic stress response, but its role in regulating aluminum stress is still not clear. As one of the rate limiting enzyme of the pentose phosphate pathway, glucose -6- phosphate dehydrogenase (G6PDH) catalyzes the generation of reducing power of NADPH. studies have reported that G6PDH is involved in the reaction resistance of various environmental stresses, but on the aluminum stress response and its regulation has not been reported. This study takes soybean as experimental materials. Effects of NO and G6PDH in response to Al stress and the regulation mechanism of aluminum toxicity. The main results are as follows: 1. the low concentration of aluminum stress under the regulation of NO on aluminum toxicity in the presence of Al Under the stress, soybean seedling root length and root elongation zone cell size was inhibited, the degree of membrane lipid peroxidation increased reactive oxygen species (H2O2 and O2 - -) content increased significantly; antioxidant BHA treatment alleviated the inhibition and lipid growth caused by Al peroxidation. Root showed that aluminum stress by stimulating root activity oxygen (ROS) accumulation, enhance the membrane lipid peroxidation, resulting in oxidative damage, and ultimately affect the shape and size of cells of root elongation zone, resulting in root growth inhibition. Under aluminum stress, NO donor (SNP) treatment reduced the Al induced inhibition of root elongation, ROS accumulation and membrane lipid peroxidation the degree of NO and scavenger (PTIO) treatment was further aggravated the aluminum toxicity. Further study showed that SNP treatment could enhance the antioxidant enzymes (POD, CAT) Al stress activity of antioxidants (GSH and ASA) and the GSH-ASA content of key enzymes in the circulatory system (APX, GR, DHAR and MDHAR) activity increased, while PTIO treatment inhibited the antioxidant content and antioxidant enzyme activity induced by aluminum. Aluminum stress significantly increased NO content and nitrate reductase activity in Soybean (NR), NR inhibitor treatment eliminates the increase of NO content induced by aluminum, and nitric oxide synthase (NOS) inhibitor, did not significantly change the content of NO, NO showed that under aluminum stress generated mainly from the NR pathway. These results suggest that the lead under aluminum stress NO NR mediated by regulating the activity of antioxidant enzymes and the GSH-ASA loop in GSH and ASA content to remove the excess ROS, thus enhancing the tolerance of.2. the high concentration of aluminum to aluminum toxicity in Soybean under G6PDH stress on aluminum toxicity in the regulating effect of high concentrations of aluminum stress (more than 200 M) treatment significantly increased the total G6PDH of soybean root activity, and with the increase of the concentration of aluminum enzyme activity gradually increased; at the same time, cell G6PDH (Cyt-G6PDH) activity and the change trend of total G6PDH activity and G6PDH activity were similar, plastids did not change significantly, that caused the G6PDH activity of aluminum stress increase comes mainly from Cyt-G6PDH. under aluminum stress, G6PDH inhibitor (GN6P) treatment alleviated the Al induced root growth inhibition, membrane lipid peroxidation and ROS the accumulation suggests that G6PDH is involved in the toxic effects of aluminum on soybean. Further study showed that NADPH oxidase inhibitor (DPI) treatment to eliminate aluminum stress caused by the accumulation of ROS, suggesting that the accumulation from the NADPH oxidase pathway ROS under aluminum stress. Under aluminum stress, GN6P treatment reduced the NADPH content and NADPH oxidase activity, suggesting that G6PDH regulates the activities of NADPH. The results show that aluminum stress increased Cyt-G6PDH activity and encoding gene (G6PDH1 and G6PDH2) expression of SNP further enhances its activity and base Because the expression of PTIO inhibited the activity and gene expression, suggesting that NO mediated enhancement of aluminum stress induced Cyt-G6PDH activity, the regulation may be enhanced by G6PDH1 and G6PDH2 gene expression. In addition, the high concentration of Al stress, the NO content increased significantly, which is derived from the NR pathway methods. These results indicated that under aluminum stress mediated by NO Cyt-G6PDH by regulating NADPH oxidase dependent ROS accumulation, the soybean suffered oxidative damage, eventually leading to root growth inhibition. In conclusion, under aluminum stress, soybean seedlings can produce NO through NR pathway, NO has two effects, one is relative low concentration of aluminum stress by regulating antioxidant enzyme activity and antioxidant content to alleviate oxidative damage and root growth inhibition caused by aluminum stress; on the other hand, relatively high concentrations of aluminum stress, NO could adjust Cy The expression of t-G6PDH coding gene enhances Cyt-G6PDH activity, which results in the accumulation of NADPH and the enhancement of NADPH oxidase activity, resulting in the increase of ROS accumulation and oxidative damage, and finally the growth inhibition of root growth.

【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.78;S565.1

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