【作者】 董志；

【导师】 裴真明； 何奕昆； 张飞云；

【作者基本信息】 首都师范大学 ， 植物学， 2008， 博士

【摘要】 一氧化氮(NO)是迄今发现的自然界中十个最小的化学分子之一,是一种易扩散的生物活性分子。NO在原生动物、细菌、酵母、动物和人体的作用机制已有大量研究。NO不仅在人体和哺乳动物的血管松弛、神经传导以及先天性免疫反应等动物生理代谢过程中是一种关键的信号分子,而且被认为是一种新的植物生长调节物质参与植物的生长发育。研究者发现NO在植物的抗病防御反应中也发挥着重要作用。目前NO药物学的作用在植物抗病反应中已有较多报道。然而,NO与SA、JA和ET植物激素相互作用如何调节植物抗病性的分子机制不清楚。本论文将利用我们已获得的多种突变体材料,采用反向遗传学,分子生物学和药物学等技术,重点解析NO与SA、JA和ET互作在植物抗病分子机制中作用。实验研究结果表明:(1)半定量RT-PCR分析发现NO含量升高的突变体cue1植株中组成型地表达抗病相关基因PR1和PDF1.2。在cue1中SA的含量大幅上升,细菌病原计数实验表明cue1对Pseudomonas syringae pv.macicola ES4326具有一定的抗性。用不同浓度NO供体SNP处理WT(Col),PR1和PDF1.2基因的表达与SNP的浓度有一定的依赖性。在cue1npr1和cue1nahG双突变体中,植株叶片NO含量与cue1基本一样,但是PR1基因的表达完全受到抑制,并且对P.s.m.ES4326表现出完全的感病。这些实验表明NO对抗病基因PR1的诱导是通过SA和NPR1来完成的。(2)外源NO供体SNP处理乙烯不敏感突变体ein2和ein3不能诱导PDF1.2的表达,而处理茉莉酸不敏感突变体jar1却能诱导PDF1.2的表达。半定量RT-PCR结果显示,双突变体cue1jar1不能阻断PDF1.2的表达,而双突变体cuelein2却阻断了PDF1.2的表达。荧光染料DAF-2DA对双突变体cue1ein2和cue1jar1染色发现NO含量和cue1的基本一样。细菌病原计数实验表明cue1ein2和cue1jar1对P.s.m.ES4326都具有抗性。双突变体cue1ein2表现出晚花现象,而cue1jar1依旧与jar1同样表现为早花。由此可见,NO对抗病相关基因PDF1.2表达的影响是通过EIN2来起作用,而不是JAR1,但是对ein2和jar1开花影响的机制可能完全不一样。(3) SNP处理突变体eds1和eds5,结果表明对抗病基因PDF1.2和PR1表达的影响和WT基本上一样,但是NO对eds1和eds5的叶片的发育有更重要的影响,当SNP的浓度高于20μM时,它们的叶片几乎全部都出现网状叶脉。因此,NO在EDS1和EDS5的上游起作用的可能性不大,却对它们的叶片发育过程有重要的作用。本论文初步通过反向遗传学,分子生物学和药物学的方法揭示了NO对抗病信号途径SA途径和JA/ET途径的调控作用。NO对SA抗病信号途径的标志基因PR1的表达调控主要是通过SA和NPR1来实现的,而对JA/ET抗病信号途径的标志基因PDF1.2的表达调控是通过EIN2和EIN3来完成的,而不是JAR1。这是首次通过遗传学的方法阐明NO在植物抗病信号途径中的机理研究,同时也提出了NO在JA/ET信号途径中的新观点和证据。更多还原

【Abstract】 Nitric oxide(NO) is one of the ten smallest chemical molecules found so far in nature and is a small highly diffusible bioactive molecule.NO has been most studied in protozoa,bacteria,yeast,animal and human.Further investigations led to the finding that NO is a multifunctional effector involved in numerous mammalian physiological processes,including the relaxation of smooth muscle,inhibition of platelet aggregation,neural communication and immune regulation.NO is also a novel regulation molecule in plant growth and development.Scientists considered that NO play an important role in plant defense.Recently,more studies have been done about NO pharmacological effect in plant defense.However,the underlying cellular and molecular mechanisms of NO and SA, JA/ET crosstalk in plant defense pathway remain largely unknown.Here we have use reverse genetics,molecular biology and pharmacology methods to elucidate NO signaling with SA and JA/ET in plant defense mechanisms.Three important findings were made.First,the cue1 plants were found to be constitutively resistant to virulent pathogen Pseudomonas syringae pv.maculicola ES4326;to have endogenous expression of the pathogensis-related gene 1(PR1) and plant defesin 1.2(PDF1.2) by Semi-Quantitative RT-PCR;and to have elevated level of nitric oxide(NO) and salicylic acid(SA).Homozygous lines for cue1 and either the SA-degarding bacterial gene nahG or the SA-insensitive mutation npr1 do not express PR1 or exhibit resistance to P.s.maculicola ES4326.Treatment WT with SNP induced PR1 and PDF1.2 expression in a dose-dependent manner.The double mutants of cue1npr1 and cue1nahG displayed an elevated level of endogenous NO.Therefore,we conclude that NO acts upstream of SA in inducing SAR.Second,treatment ein2 and ein3 plants with SNP could not induce PDF1.2 gene expression,however,treatment jar1 plants could induce PDF1.2 expression.The double mutant cue1jar1 blocked PDF1.2 expression,but cue1ein2 did not block PDF1.2 expression.Homozygous lines for cue1 and either the ethylene-insensitive mutant ein2 or the jasmonic acid-insensitive mutatant jar1 displayed an elevated level of endogenous NO or exhibit resistance to P.s.maculicola ES4326.It is surprise that the cue1ein2 plants delay flowering time,but cue1jar1 is early flowering as same as jar1.The result indicated that NO induced PDF1.2 expression by EIN2 not JAR1,but NO how to effect flowering time of ein2 and jar1,we do not know as far.Third,treatment salicylic acid-insensitive mutant eds1 and eds5 plants with SNP induced PR1 and PDF1.2 expression in a dose-dependent manner.Therefore,we conclude that NO does not act upstream of EDS1 and EDS5 in inducing SAR.NO play an important role in eds1 and eds5 leaf development.Treatment eds1 and eds5 with≥20μM concentrations of SNP completely induced rectilate pattern leaves compared with WT.We can conclude that NO is not upstream EDS1 and EDS5,but NO have more effection on leaves development in eds1 and eds5.In this thesis study,we primarily explained NO and SA,JA/ET defense pathway by genetics,molecular biology and pharmacology methods.We can conclude that NO acts upstream of SA in inducing SAR and NO induced PDF1.2 expression by EIN2 and EIN3 not by JAR1.This is first time using genetics to study NO in plant defense pathway.We also give new insights and evidence about NO and JA/ET crosstalk.更多还原