【作者】 孙万春；

【导师】 梁永超；

【作者基本信息】 中国农业科学院 ， 植物营养学， 2008， 博士

【摘要】 尽管已有大量研究报道证明,硅对植物健康生长和发育是有益的,特别是在增强植物抗病性方面具有重要作用,但硅的抗病作用机制一直不清楚。传统观念认为,硅的抗病机制是其“机械或物理屏障”作用。但最近十多年的一些研究则显示,硅可能参与了植物的生理代谢过程,激活了植物的防卫机制,对硅的物理屏障作用这一假说提出了质疑。本文采用水培试验,研究了不同方式硅处理对稻瘟病的抗病效果以及对水稻生长的影响;硅对多种生理生化抗病机制的调控;利用荧光定量PCR技术,在水稻与稻瘟菌互作过程中硅对与有关防卫基因的调控;利用cDNA芯片技术分析在感病和未感病条件下硅调控的水稻基因表达图谱。从生理生化和基因水平对硅的抗病机制进行了系统而深入的研究,得到结果如下:(1)不加硅-加硅处理(Si-Si+)的植株与一直加硅处理(Si+Si+)具有同样高的抗性,防治效果分别为67.0%和69.3%;加硅-不加硅处理(Si+Si-)的植株失去部分抗性,只有47.7%的防治效果。硅作为“机械屏障”作用在提高水稻对稻瘟病的抗性中仍有一定作用,但非“机械屏障”作用具有更为重要的作用。硅在正常(非胁迫)条件下对水稻生长促进作用明显,也能缓解因稻瘟病菌胁迫所产生的对水稻生长的危害。无论接种与否,施硅处理的地上部和根部的硅含量显著高于不施硅处理;水稻地上部的硅含量明显高于根部。(2)施硅处理在水稻感染稻瘟病的初期(24 h)有个快速而短暂的H2O2积累,同时抑制过氧化氢酶(CAT)活性的反应。在水稻感染稻瘟病的后期(48 h以后),施硅能显著提高CAT活性,在72 h达到峰值,显著高于不施硅处理;而施硅处理水稻叶片中H2O2含量保持较低水平。在水稻感染稻瘟病的初期(48 h以前),施硅处理中的丙二醛(MDA)含量和质膜透性(相对电导率)快速升高,显著高于不施硅处理;但48 h以后,MDA含量和相对电导率快速下降,从96 h以后开始明显低于不施硅处理。施硅使脂氧合酶(LOX)活性的峰值出现得更早更快,其强度也显著高于不施硅处理。这些结果表明,在稻瘟病菌感染的初期,硅通过激发活性氧的爆发,提高膜脂过氧化水平和质膜透性,诱导过敏性反应的发生而增强水稻对稻瘟病的抗性;在稻瘟病菌感染的后期,硅能缓解因病菌侵染而产生的氧化伤害。(3)施硅处理的过氧化物酶(POD)活性显著低于不施硅处理,过氧化物酶活性与水稻对抗稻瘟病的抗性之间没有联系,甚至成负相关。水稻感染稻瘟病后,施硅能显著提高多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性以及总可溶性酚和木质素的含量。结果表明,硅能够调控与抗病有关的酚类物质代谢过程,参与植物防卫反应而增强水稻对稻瘟病的抗性。(4)施硅能使受稻瘟病菌侵染的水稻叶片中几丁质内切酶和外切酶活性更高,且保持更长时间。在稻瘟病菌感染的初期,β-1,3-葡聚糖酶活性增长缓慢,施硅和不施硅处理之间差异不显著;在感染的后期,施硅处理的β-1,3-葡聚糖酶活性显著低于不施硅处理。结果表明,硅能够通过提高几丁质酶活性来增强水稻对稻瘟病的抗性,但对β-1,3-葡聚糖酶没有调控作用。(5)感染稻瘟病菌后,无论施硅与否,PAL、CatA、Rcht2和Pr1a基因的相对表达量均上升;但在感病初期硅明显能更早更快提高这些基因的相对表达量,增长幅度也显著高于不施硅处理。施硅和接种稻瘟菌在感病初期对转录阻遏物OsCTBP-A基因有所抑制,但调控作用不明显。硅能更早更快激活转录因子OsBTF3基因的表达,相对表达量也显著高于不施硅处理。说明在稻瘟菌与水稻的互作过程中,硅积极参与对相关防卫基因的激活和调控,从而产生一系列生理生化抗病机制,这是硅抗稻瘟病的主要机制。(6)硅在正常(非胁迫)条件下能使36926个水稻基因中的1210个基因出现差异表达,其中126个基因表达上调,另外1084个基因表达下调。稻瘟菌接种12 h后,能诱导670个基因出现差异表达,其中346个基因表达上调,另外324个基因表达下调。在水稻感染稻瘟菌的情况下,硅使483个基因出现差异表达,其中27个基因表达上调,另外456个基因表达下调。这些差异表达的基因涉及到信号转导、抗病防御过程、转录调控、细胞生长与分裂、细胞内转运、细胞结构的组成、蛋白质合成、基础代谢和次级代谢以及能量利用等过程。cDNA芯片的结果表明,硅在正常(非胁迫)条件下对植物生长代谢具有明显作用,至少对于水稻这类单子叶植物是必需的;当植物受到病原物胁迫时,硅能调控植物本身产生更高效或更同步的防卫反应并减轻病原物的伤害。综上所说,硅对植物生长是非常重要和有益的,对水稻类单子叶植物也可能是必需的。硅能激活和调控植物的防卫基因,参与生理代谢活动,产生一系列的防御机制来阻止病原物的侵入和扩展。硅在抗病中的作用类似于植物诱导抗性的调节器,是主动过程,而不是仅仅限于“机械屏障”作用。更多还原

【Abstract】 Silicon (Si) has been proven to be beneficial for healthy growth and development of many plant species and plays an important role in enhancing plant resistance against fungal pathogens, however, the mechanisms involved are still unclear. Traditionally, it is thought that silicon acts as a mechanical or physical barrier in response to fungal attack. However, there have been increasing bodies of evidence showing that silicon is associated with host defense responses and plays an active and physiological role in enhancing resistance of the host plant. A series of hydroponics experiments were performed in a controlled rice-Magnaporthe grisea pathosystem to study the effects of silicon on rice growth, disease development and regulation of induced resistance. The effect of silicon on regulating defense-related genes in the rice-M. grisea interaction was conducted using real-time quantitative PCR approach. Using a cDNA chip containing 60727 rice cDNAs representing 36926 unique genes, we performed a comprehensive analysis of the influence of Si on gene expression of rice inoculated with or without M. grisea. The comprehensive and systematic study of mechanisms of silicon–enhanced resistance to rice blast was conducted. The results are presented as follows:(1) Rice plants that were switched from Si- (without Si added) to Si+ (with Si added) nutrient solution and simultaneously inoculated with M. grisea exhibited the same high resistance as the plants treated continuously with silicon, with control efficiencies of 67.0 % and 69.3%, respectively. However, rice plants switched from Si+ to Si- nutrient solution lost partial resistance to blast, with control efficiency of 47.7% only. It seems to suggest that there are still some roles that silicon plays as a physical barrier in controlling rice blast though these roles are very limited and not a major mechanism for Si-enhanced resistance to rice blast. Application of silicon was beneficial for rice growth and alleviated damage resulting from infection by M. grisea. Regardless of inoculation with M. grisea, Si concentration of shoots or roots in rice plants amended with 1.7 mM Si was significantly higher than that of the non-Si-amended plants. The Si concentrations in all treatments were significantly higher in rice shoots than in rice roots.(2) Silicon induced a rapid and transient burst of H2O2 at 24 h after inoculation. Catalase (CAT) activity in leaves of Si+ plants was lower at 24 h after inoculation compared with Si- plants, then rapidly increased and reached a peak at 72 h after inoculation, significantly higher than in Si- plants. Compared with Si- plants, malondialdehyde (MDA) concentration and membrane permeability in Si+ plants were significantly higher at 48 h after inoculation but lower from 96 h and onward. Two peaks of lipoxygenase (LOX) activity in Si+ plants were observed at 12 and 48 h after inoculation, respectively, and were earlier than in Si- plants which occurred at 24 and 72 h, respectively. The maximum LOX activity was significantly higher in Si+ plants than in Si- plants. In the early stages of infection by M. grisea, silicon induced a rapid and transient burst of H2O2, and consequently resulted in membrane lipid peroxidation and membrane damage, which may be closely correlated with HR. Silicon alleviated the oxidative damage during later periods of the rice-M. grisea interaction.(3) Silicon application significantly increased activities of polyphenoloxidase (PPO) and phenylalanine ammonia-lyase (PAL), as well as contents of total soluble phenolics and lignin. However, leaf peroxidase (POD) activity was significantly lower in Si+ plants than in Si- plants after inoculation and was not related directly to blast resistance. The results demonstrate that silicon actively participates in metabolism of phenolics to accelerate accumulation of antimicrobial compounds.(4) Exochitinase and endochitinase activities were significantly higher in Si+ plants than in Si- plants and maintained longer. However, silicon application decreasedβ-1, 3-glucanase activity in rice leaves infected by M. grisea. The results show that silicon improved chitinase activities to enhance resistance to rice blast. However, the influence of silicon onβ-1, 3-glucanase activity was not observed.(5) Regardless of silicon applied, the gene expression levels of PAL, CatA, Rcht2 and Pr1a all increased after inoculation with M. grisea. In the early stages of infection, increased expression of these genes in Si+ plants occurred earlier and faster with the extent of increment being also significantly higher than in Si- plants. Compared with Si- plants, expression of OsBTF3 in Si+ plants occurred earlier and faster, and its expression level was also significantly higher than in Si- plants. The results show that Si activated and regulated some defense-related genes in response to blast attack in rice.(6) We identified 1210 genes which were differentially expressed in the Si-amended plants without inoculation (Si/CK), with 126 genes being up-regulated and 1084 genes being down-regulated . Among 670 differentially-expressed genes in rice plants inoculated with M.grisea (M/ CK), 346 genes were up-regulated and 324 genes were down-regulated . After inoculation with M.grisea, 483 genes in Si+ plants were differentially expressed, compared with the Si- plants (Si+M/ M), with up-regulation of 27 genes and down-regulation of 456 genes. These genes included stress-related transcription factors, and genes involved in signal transduction, the biosynthesis of stress hormones (SA, JA, ethylene), the metabolism of reactive oxygen species, the biosynthesis of antimicrobial compound, primary and/or secondary metabolism, defense response, photosynthesis, and energy pathways, etc. On the basis of analyzing expression profile of rice genes, it can be concluded that silicon can exhibit obvious impacts on growth and development of plants, especially in rice plants, which were not infected by pathogens, but can regulate natural resistance mechanisms of plant to produce more efficient and timely defense response, and consequently alleviate damage caused by pathogens in plants subjected to pathogens stress.In conclusion, silicon is very important and beneficial for plant growth, and may be essential for gramineous plant species such as rice. Silicon can activate and regulate defense-related genes of plant, participate in physiological metabolism and induce a series of defense mechanisms to impede the invasion and cloning of pathogens. Silicon, acting as a regulator of induced resistance, is active in response to pathogens. The Si-enhanced plant resistance to pathogens is not solely limited to a mechanical barrier as previously proposed.更多还原