【作者】 梁元存；

【导师】 张天宇； 董汉松；

【作者基本信息】 山东农业大学 ， 植物病理学， 2002， 博士

【摘要】 Phytophthora和Pythium的许多种能向细胞外分泌一类小分子量的蛋白质，统称为elicitins。Elicitins在烟草上能引起过敏反应（hypersensitive response，HR）和系统获得抗性（systemic acquired resistance，SAR）。Elicitins-烟草互作体系是研究信号识别、信号传导等诸多问题的良好体系。本论文报告作者对烟草寄生疫霉（Phytophthora parasitica var. nicotianae）中国菌株的elicitin基因进行克隆、表达及其诱导植物抗病性方面的研究结果。根据已报道的寄生疫霉parA1基因的序列设计引物，从4株寄生疫霉不同寄主分离物（3株来自烟草，1株来自刺槐）中克隆到此基因，序列分析表明4株寄生疫霉parA1基因序列高度保守。然后研究了parA1在大肠杆菌（Escherichia coli）中的表达。对表达载体pET30a(+)双酶切(NdeⅠ-BamHⅠ)，构建表达elicitin蛋白的表达载体pET-eli，用CaCl2法转化大肠杆菌BL21菌株，通过IPTG诱导在大肠杆菌中产生的蛋白在烟草上引起过敏性坏死反应。对重组蛋白性质测定表明，重组蛋白有一定的耐热性，并对蛋白酶K敏感。至此，我们已成功地从寄生疫霉中国菌株中克隆到了parA1基因，并建立了大肠杆菌重组表达体系。在获得重组蛋白的基础上，测定了其诱导烟草和番茄对真菌、细菌和病毒病的抗病诱导作用。对不同浓度的elicitin进行诱导作用比较，此重组蛋白在30nM时，喷洒诱导烟草后产生最高的抗TMV的诱导作用；诱导作用可维持至少20天，其中在第4-5天诱导效果最好。因此，在elicitin诱导植物时，最好使用30nM浓度，并在诱导后第4-5天挑战接种可产生最大的诱导效果。用30nM的elicitin处理烟草和番茄，第4天接种测试的病原物，烟草经诱导后，烟草能抗TMV(tobacco mosaic virus)、烟草黑胫病菌(P. parasitica)、烟草赤星病菌(Alternaria longipes)和烟草青枯病菌(Pseudomonas solanacearum)，诱导作用分别是74.1%、63.8%、50.6%和45.0%；番茄经诱导处理后抗青枯病（P. solanacearum），诱导作用为43.1%。因此elicitin的抗病诱抗作用是非特异的。植物诱导抗性通常与过敏反应（HR）及病程相关（PR）等基因的表达相关联，而这些反应受信号分子水杨酸的调控，在不同体系中，还有H2O2等信号分子及Ca2+通路因子的参与。为了明确elicitin诱导HR的上游信号传导因子，对elicitin处理后<WP=8>植株体内SA、H2O2的积累及Ca2+通道相关反应作了测定。烟草经elicitin诱导处理后，体内水杨酸含量比对照提高了1.65-2.88倍，其中在诱导后的第1天和第4天各有一个峰值。用Ca2+通道阻断剂La3+与elicitin同时注射烟草叶片发现过敏基因HIN1表达被抑制，表明HIN1的表达依赖于胞外Ca2+。在elicitin处理烟草后，过氧化氢酶（CAT）的活性下降1.15-1.62倍；相应地，elicitin处理的烟草植株体内H2O2迅速积累，但在测定的时间内没有出现“氧化迸发”，H2O2含量只是在elicitin注射叶片后的第5和9小时各出现了两个小峰，并且H2O2的增加是系统性的，即不但处理叶片有量的积累，而且非处理叶片也有量的增加。根据这些现象，elicitin诱导HR的过程有SA、Ca2+通道因子和活性氧的参与。为了明确上述信号分子的变化是否与HR相关，我们研究了elicitin诱导的细胞快速死亡及HR的分子表征。用elicitin注射烟草后0.5小时，HR的表征基因HIN1表达，在7小时内表达量逐渐变弱，而最终在12-16小时可观察到HR。HR的另外的表征分子苯丙氨酸裂解酶（PAL）和PR基因的表达。在烟草受elicitin处理后的6天内，PAL活性出现了2个峰，活性水平高出对照1.13-2.44倍。Elicitin喷洒诱导后，检测了防卫基因PR-1b的表达情况。PR-1b在诱导1天后就有表达而并在测定的5天内都有一定量的表达。用150nM的elicitin喷洒烟草叶片，经trypon-blue染色，在12小时可观察到有细胞坏死，说明elicitin引起了micro-HR，而水和低浓度的elicitin不能引起micro-HR；同时发现注射引起HR的elicitin浓度远比引起micro-HR的浓度低。证实能产生HR的激发子的诱导作用不见得要伴随micro-HR更多还原

【Abstract】 Elicitins, proteinaceous elicitors produced by species of two oomycetes genera, Phytophthora and Pythium, induce hypersensitive reaction (HR) and systemic acquired resistance (SAR) in tobacco. They can be classified into basic elicitins produced by species such as Phytophthora crytogea and acidic elicitin produced by species such as P. parasitica. P. parasitica var. nicotianae, the causal agent of tobacco black shank disease, produces an α-elicitin, parasiticein. This study was aimed to determine mechanisms in some respects, by which elicitins activate plant defense.We have firstly cloned and characterized parA1 homologies from 4 isolates of P. parasitica isolated from tobacco and locust in China. Amplification products were got by polymerase chain reaction (PCR) of DNA from the 4 isolates, using primers specific to a parA1 reported previously. Their nucleotide sequences are 354 bp in size and identical one to another and to the parA1 gene previously cloned. Therefore, we have successfully cloned the parA1 gene from the Chinese isolates of P. parasitica. These homologies were cloned into the vector pET30a (+) and transformed them into Escherichia coli BL21. Elicitin proteins produced by the recombinant bacteria contained 118 amino acids. The proteins induced HR in tobacco, were thermostable and sensitive to protease K. Therefore, we have established a recombinant E. coli that is capable of producing the elicitin. This is the first report of cloning of elicitin genes from locust strain of the pathogen.Then, experiments to determine whether the elicitin produced by recombinant E. coli can induce resistance to pathogen were performed. Application of elicitin at 30 nM resulted in increased resistance to tobacco mosaic virus (TMV), as compared with the treatment sprayed with water; Induced resistance was strongest at 4-5 days and lasted over 20 days after inoculation. Moreover, tobacco and tomato displayed enhanced resistance to<WP=81>fungal, bacterial, and viral pathogens after treatment with elicitin, pathogens tested include Alternaria longipes, Phytophthora parasitica, Pseudomonas solanacearum and TMV.To investigate molecular signals and whether they have effects on the occurrence of the HR in tobacco leaves treated with elicitin, investigations of SA accumulation、Ca2+ channel response and H2O2 accumulation were carried out. The in vivo SA in tobacco treated and nontreated with the elicitin was tested based on determination by high-performance liquid chromatography (HPLC). Concentration of SA in leaf tissues treated with the elicitin increased by 1.65-2.88 fold compared with the control in 6 days, SA concentration showed two peaks, the first was at 1st day and the second one at 4th day after treatment. In an attempt to determine whether Ca2+ plays a role in elicitin-induced HR, a Ca2+channel inhibitor, La3+was used in injecting the tobacco leaves. The results that La3+ inhibited expression of HIN1, an HR-associated gene, indicated that the HR induction is dependent on Ca2+ channel. Meanwhile, Catalyase (CAT) activity in tobacco leaves tested conspicuously decreased. Consistently, H2O2 concentration increased in elicitin-treated leaves to a limited level, which could not be thought as an "oxygen burst" characterized by many-fold increase in active oxygen species. Based on these data, SA, Ca2+ channel, and H2O2 seems to affect the formation of the HR induced by the elicitin.To determine whether the alterations of the molecular markers mentioned above affect the HR, we assayed elicitin and water-treated tobacco for expressions of HIN1 and PR-1b genes and changes of phenylalanine ammonia lyase (PAL) activity. HIN1 was expressed in tobacco leaves infiltrated with elicitin; the optimal level of HIN1 gene expression was found by 30 minutes after treatment. PR-1b gene started to express in plants at 24h after treatment with elicitin. Meanwhile, PAL activity increased after treatment with elicitin. To illustrate whether the elicitin induces microscopic hypersensitive response (micro-HR), tobacco leaves sprayed更多还原