【作者】 郭默然；

【导师】 陈靠山；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 植物在整个生长发育过程中,需要不断抵御各种病原微生物的侵染。在与病原微生物长期相互作用并且协同进化过程中,植物逐渐形成一套复杂精密的防御体系。当受到病原微生物侵染时,植物自身会产生多种抗性反应,例如,离子流动和活性氧爆发、抗病相关蛋白基因表达、木质素合成等。激发子(elicitor)是一类同样能够诱导这些抗性反应的分子和因素。按照其来源,激发子可分为非生物激发子和生物激发子。生物激发子主要来源于病原物、其他微生物以及植物提取物或代谢物等。其中,寡糖激发子是一种来源于植物或真菌细胞壁结构的多糖降解产物,具有化学性质稳定、环境友好等特点,在低浓度下就能够诱导植物产生显著的抗病性。牛蒡低聚果糖(Burdock fructooligosaccharide, BFO)是本课题组从牛蒡根组织中分离纯化的一种分子量均一的水溶性菊糖型低聚糖。BFO占牛蒡根干重的17%左右,是一种储存型低聚糖。前期研究发现,BFO能够显著增加黄瓜对白粉病、枯萎病和炭疽病,番茄对灰霉病,烟草对烟草花叶病毒病以及多种果实对采后病害的抗性。BFO作为植物源、储存型的功能性低聚糖能诱导植物抗性无疑是一个有趣而新颖的发现。研究证实BFO处理显著提高了黄瓜、番茄和烟草局部叶及系统叶内源水杨酸(Salicylic Acid, SA)的水平和SA信号转导途径下游部分病程相关蛋白(Pathogenesis Related Proteins, PRs)的表达,表明BFO可能是通过水杨酸信号转导途径诱导植物系统抗性。但是,BFO诱导植物系统抗性是通过哪些信号途径来实现的?在诱导植物系统抗性过程中,有哪些早期信号转导机制?这些问题尚未见报道。本文利用高通量测序技术研究了BFO诱导后烟草基因表达谱及其早期转导信号分子Ca2+、NO和ROS的变化。结果如下：1.BFO诱导烟草的基因表达谱分析。以蒸馏水处理为对照,利用Solexa技术研究了BFO处理烟草1h的基因表达谱变化,结果发现412个基因的表达出现显著变化。其中表达显著上调的169个基因,主要包括编码SA激活的蛋白激酶、丝氨酸蛋白激酶、苏氨酸蛋白激酶、茉莉酸信号转导途径的抑制因子、ACC氧化酶、MYB转录因子、病程相关蛋白等基因；243个基因表达显著下调,主要包括编码ABA羟化酶、WRKY转录因子、ERF转录因子、ERF类转录因子、锌指蛋白等基因。BFO诱导的差异基因的基因本体论分析显示,差异基因参与的生物过程,主要集中在植物应激反应、防御反应、次生物质及RNA合成、激素响应以及信号转导等方面。为了进一步验证表达谱测序的可靠性,我们利用半定量和实时定量PCR技术研究了8个差异表达基因的表达量变化,结果与表达谱数据基本一致。研究结果表明,BFO激活植物抗性机制主要依赖SA介导的系统获得性抗性,还通过诱导次生代谢防御物质和其它一些与抗病性相关的因子来提高植物的防御反应。2.以烟草为植物材料,研究了BFO处理对烟草抗性早期信号转导的影响。结果表明：BFO能够迅速诱导烟草悬浮细胞胞外碱化,pH值5min内由5.23升高为5.6,之后逐渐降低至初始水平。BFO能够诱导胞外Ca2+内流,致使细胞内Ca2+含量迅速升高,9min时约为对照组的1.3倍。荧光和试剂盒定量检测发现BFO处理提高了烟草叶片中NO和ROS的含量。Ca2+相关的抑制剂(EGTA和LaCl3)都能够明显抑制BFO诱导的NO的生成。通过使用NO抑制剂(L-NAME)、ROS抑制剂(catalase)与BFO共处理发现,L-NAME能够抑制NO以及ROS的产生。相反,catalase能够抑制BFO诱导ROS的升高,但不能阻止NO水平的升高,从而证实NO作用位置可能位于ROS的上游。BFO处理1h时,SA合成基因PAL和ICS的表达分别在在30mmin和20min时达到高峰,约为对照的5倍和3.5倍。SA含量在BFO处理180min时约为对照的1.5倍。说明BFO能够通过诱导PAL和ICS的表达从而促进SA的积累。3.研究了BFO处理烟草后,与抗病性相关的生理生化反应以及次生代谢物质的变化。结果显示：BFO处理可显著降低MDA的含量,3天时与对照相比,降低了40%,从而减少对细胞膜的伤害作用。BFO处理可显著地增加烟草叶片的叶绿素含量,尤其是叶绿素a含量提高幅度较大,48h时含量增加了约1.5倍,有利于植株光合作用。BFO处理可以增加植物体内可溶性酚、可溶性糖和可溶性蛋白的含量,4天时约为对照组的2.1、2.2和1.2倍,一定程度上提高植物抗病性。同时,BFO处理提高了烟草中抗病相关酶(SOD、POD、PAL)的酶活,其活力增强都与植物诱导抗性的提高密切相关。此外,BFO提高了总酚、类黄酮及木质素等次生代谢物质的含量。总酚和类黄酮分别在处理后2天和3天时达到最高值,为对照组的1.77和1.65倍。木质素含量在处理后3-5d则明显高于对照,并在5d内一直保持上升趋势。4.研究挥发性物质(E)-2-己烯醛对采后番茄控制病害的影响。(E)-2-己烯醛能够显著降低果实自然感病和接种灰霉病的发生率和病情指数。自然感病实验中,处理组番茄的发病率和病情指数分别是对照组的51.9%和59.8%。在接种灰霉病实验中,4天时处理组的发病率和病情指数分别是对照的43.5%和27.8%。空间浓度为0.1μM的(E)-2-己烯醛处理不仅能够提高番茄果实中抗病相关酶POD和PAL的活性,增加总酚和类黄酮在果实中的积累,也明显诱导LOX和ETR等JA/ET信号途径相关基因的表达,但是却不能诱导病程相关蛋白基因(PR1、PR5)等SAR的标记基因的增加。推测(E)-2-己烯醛可能通过激活茉莉酸/乙烯途径诱导果实产生系统抗性抵御病害。综上所述,BFO诱导增强植物抗性的机制是以SA介导的SAR途径为主,多种激素信号通路共同作用的结果。次生代谢产物的合成和抗性基因的表达也与其密切相关。BFO处理烟草后会产生一系列的信号转导,例如胞外介质碱性化,钙离子变化,一氧化氮和活性氧产生等。其中,NO的升高受到Ca2+的调控。同时NO可以调节ROS的生成,并与ROS都参与了诱导编码SA合成基因表达从而促进水杨酸的积累。此外,BFO处理可以降低细胞膜相对透性及MDA含量,减少对膜的伤害作用；提高叶绿素含量,增强植物的光合作用；诱导抗病相关酶(CAT、POD、PAL、PPO)活性的提高,提高烟草叶片中总酚、类黄酮和木质素的累积,增强对病害的抗性。更多还原

【Abstract】 In nature, plants are continuously infected by a wide range of harmful pathogenic microorganisms. The evolutionary between plants and their attackers provided plants with a highly sophisticated defense system. Plants have developed various defence responses against pathogen attack, including reactive oxygen species burst, the accumulation of salicylic acid (SA) and lignin synthesis. These defense responses could also be induced by a class of molecules named elicitor. Elicitor is a kind of compounds trigger a large array of defense responses in host plant, including biotic elicitor and abiotic elicitor. Among biotic elicitor, the oligosaccharide elicitors derived from fungal and plant cell wall polysaccharides can induce resistance to various pathogens at very low concentrations.Burdock fructooligosaccharide (BFO) is a plant reserve carbohydrate, first isolated from the roots of Arcitum lappa by our group. The amount of BFO in the air-dried root tissue is about17.0%. BFO is not only water-soluble, nontoxic and biocompatible, but also has versatile functional properties, which can agree with the demands of modern environmental protection. In our previous works, BFO could enhance plant resistance to several pathogens. In tobacco, BFO could increase the expression of PRs and SA to enhance resistance against tobacco mosaic virus. It is interesting that a reserve oligosaccharide could improve defense responses in plant. However, the transcriptional programming and early signaling pathway after BFO treatment remain unclear, and the underlying molecular mechanism of BFO in plants is still largely unknown. Therefore, we investigated the expressions of key genes involed in signaling pathways and defense reaction after BFO treatment in tobacco by High-throughput sequencing; we researched that the early signal transduction and the relation of NO and ROS in tobacco after BFO treatment by fluorescent probe and related inhibitors. In addition, we also examined the physiological and biochemical changes associated with resistance in plant after the treatment of BFO. The mainly results are as followed:1. In the present study, we compared differential expression profiling of tobacco after treatment with BFO or distilled water (DW). A number of candidate genes that had altered expression in response to BFO were identified. In all,169genes were identified as up-regulated genes. The expressions of243genes were decreased. We investigated the expressions of genes involved in defense responses and signaling pathways in BFO treated tobacco plants on a genome-wide scale. GO analysis showed that differentially expressed genes were involved in responses to stress, defense responses, biosynthetic processes, responses to hormone, RNA biosynthetic processes and signaling pathways. Eight differentially expressed genes were selected to test the reliability of Solexa sequencing. The fold changes of differential genes in transcription profile and results by qRT-PCR were similar. The results suggested that the mechanism of BFO enhanced plant resistance might be basically about the SA-mediated pathway, and other hormones also play a role in defense response. Moreover, secondary metabolites were related to constitutive activation of the host defense response in BFO treated tobacco.2. We used tobacco plant material, studied the effect of early signal transduction pathway after BFO treatment in tobacco. The results show that:BFO induced extracellular alkalization in tobacco, extracellular Ca2+influx in tobacco epidermal cells, Ca2+is involved in the nitric oxide (NO) generation after BFO treatment, NO and reactive oxygen species (ROS) generation in tobacco guard cells and leaf tissue; Secondly, we demonstrate NO may act as a messenger in regulating ROS. BFO also induced the expression of the genes encoding PAL and ICS to induce the accumulation of SA. Meanwhile, BFO also induced the expression of the genes encoding NPR1to activite SAR and enhance plant resistance to pathogen.3. The physiological and biochemical changes associated with disease resistance after BFO treatment in tobacco were investigated, and the accumulation of secondary metabolite was detected. The results indicated that BFO could significantly decrease MDA content, increase the content of chlorophyll, soluble protein, soluble sugar and soluble phenols. Meanwhile, BFO treatment could increase the content of total phenolic, flavonoids, lignin and other secondary metabolites. In addition, BFO treatment increased the activity of related enzymes (peroxidase, superoxide dismutase, phenol oxidase) and the expression of related enzyme gene in tobacco. But interestingly, BFO treatment was not able to induce the accumulation of callose.4. In this paper, the effects of trans-2-hexenal on the control of postharvest diseases in tomato fruit were investigated. We indicated that the incidence of natural infection and inoculation by Botrytis cinerea in tomato fruit was significantly decreased after treatment with trans-2-hexenal. Trans-2-hexenal increased the mRNA levels of genes encoding ethylene receptors, lipoxygenase, and the phenylalanine ammonia lyase (PAL), but did not induce mRNA accumulation of the pathogenesis-related proteins (PR-la or PR-5). The activities of peroxidases and PAL and the accumulation of phenolic compounds were significantly enhanced after trans-2-hexenal treatment. The findings suggest that the controlling effects of trans-2-hexenal in postharvest tomato fruit might be related to JA/ET-mediated induced systemic resistance.In summary, the mechanism of BFO enhanced plant resistance might be basically about the SA-mediated pathway, and secondary metabolites play an important role in the induced resistance. Treatment with BFO results in the activation of plant responses, such as medium alkalinization, elevation of cytoplasmic Ca2+concentrations, NO and ROS production. Ca2+is involved in the generation of NO after BFO treatment. NO may act as a messenger in regulating ROS, and induces the expression of PAL and ICS genes to promote the accumulation of salicylic acid. In addition, BFO treatment could reduce the content of MDA, and increase the content of chlorophyll and the activities of defense related enzymes (CAT, POD, PAL, PPO). BFO treatment can induce the accumulation of total phenolics, flavonoids and lignin in tobacco to enhance resistance.更多还原