【作者】 刘方；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 Harpins是由革兰氏阴性植物病原细菌产生的一类蛋白类激发子,目前研究较多的有蔷薇科植物火疫病的病原细菌Erwinia amylovora产生的HrpN_Ea和水稻白叶枯病菌Xanthomonas oryzae pv.oryzae的HpaG_Xoo。当外源使用时,harpins可以诱导多种植物抗病、抗虫、耐旱、促进植物生长,乙烯信号传导因子EIN2和脱落酸（ABA）信号传导因子ABI2对harpin诱导的植物生长和对于耐旱性起关键调控作用。但是,harpins是否同时及如何同时行使这些功能,目前还不清楚。本文着重研究了乙烯和脱落酸对HrpN_Ea促进植物生长的调控作用;为深入研究乙烯和脱落酸信号传导对植物生长的交叉调控机制,产生了ein2 abi2双突变体;为深入研究harpin信号在植物体内传导的机制,产生了表达HpaG_Xoo。的转基因拟南芥,并测定了转基因植物的抗病防卫反应。1.脱落酸和乙烯信号互作对HrpN_Ea诱导拟南芥根生长的调控作用用HrpN_Ea处理植物可以激发乙烯和ABA的产生,从而诱导植物生长和对干旱的抗性。本文报道了乙烯和ABA两种激素的相互作用可以介导由HrpN_Ea引起的在拟南芥上促进根的生长作用。野生型拟南芥的种子Columbia（Col-0）和Landsberg erecta（Ler-0）用HrpN_Ea溶液浸泡处理后在促进乙烯和ABA水平升高的同时促进根的生长。当溶液中混合有合成乙烯或者感知乙烯和ABA信号的抑制剂时,这些反应会得到控制。HrpN_Ea影响植物根系生长的效应同样会在乙烯不敏感突变体etr1-1和ein5-1以及ABA不敏感突变体abi2-1中失效。我们的研究结果建立了乙烯和ABA信号与HrpN_Ea促进根长之间的相关性的一种机制。然而,当施用HrpN_Ea于植物叶片时,乙烯信号可以在缺失ABA信号的情况下起作用的,这就表明在HrpN_Ea促进植物生长时,在植物的不同组织叶子和根系中存在不同的信号机制。2.乙烯和ABA信号通路双突变体的产生乙烯是植物生长发育最重要的内源信号,乙烯介导的植物抗病防卫基本信号通路（Dangl and Jones,2001）对于植物的基本防卫十分重要。逆境激素ABA调控了植物营养生长和生殖生长过程中的许多重要事件,包括调节气孔关闭（Finkelstein and Rock,2002）,抵抗逆境,促进种子后熟和休眠等。在植物发育过程中,乙烯与ABA信号途径之间大多表现为协同作用,高浓度ABA抑制乙烯的合成。为了研究在植物抗病信号转导中的乙烯和ABA信号的上下游关系,以及解析乙烯信号的关键因子EIN2和脱落酸信号的关键因子ABI2之间的联系与作用,我们构建了RNAi的沉默载体,转化乙烯通路突变体ein2生态型植物,得到双突变体功效的植物,检测转基因植物中ABI基因的表达,为以后的表型分析,生理生化鉴定以及深入研究植物在胁迫信号转导过程中的级联关系提供了材料与依据。3.HpaG_Xoo在转基因拟南芥中表达诱导植物生长和抗病性我们将编码HpaG_Xoo的基因hpaG_Xoo转入拟南芥,发现不同的转基因株系都获得了对Pseudomonas syringae pv.tomato DC3000的抗性,并表达抗病防卫反应基因NPR1、PR-1和PR3b。但是,所有的转基因株系都没有发现HCD的发生。根据这些结果,我们认为HpaG_Xoo在拟南芥中转基因表达可以诱导抗病防卫基因的表达,诱导抗病性,但不发生HCD。另外,信号肽的有无似乎对HpaG_Xoo。在植物体内表达所启动的信号通路及多种表型没有影响。更多还原

【Abstract】 Harpins are glycine-rich,protease-sensitive,heat-stable,acidic proteins produced by Gram-negative plant pathogenic bacterial,and are required for induction of the hypersensitive response（HR） or hypersensitive cell death（HCD） in nonhost plants of bacteria.Application of harpins to many plants can enhance plant growth,induce resistance to pathogens,insects and drought.These effects have been observed in plants treated with HrpNEa from Eriwinia amylovora,HrpZ_Pss from Pseudomonas syringae pv.syringae, HrpZ_psph from P syringae pv.Phaseolicola,and HpaG_Xoo from Xanthomonas oryzae pv. oryzae.How harpins perform these diverse functions has not been well demonstrated.1.Root growth ofArabidopsis thaliana is regulated by ABA and ethylene signaling interaction in response to HrpN_EaTreating plants with HrpN_Ea stimulates ethylene and abscisic acid（ABA） to induce plant growth and drought tolerance,respectively.Herein,we report that both growth hormones cooperate to mediate the role of HrpNEa in promoting root growth of Arabidopsis thaliana seedlings.Root growth is promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking of germinating seeds of wild-type（WT） Arabidopsis in a solution of HrpN_Ea.However,these responses are arrested by inhibiting WT roots from synthesizing ethylene as well as sensing of ABA and ethylene.The effects of HrpN_Ea on roots are also nullified in ethylene-insensitive etr1-1 and ein5-1 mutants and in the ABA-insensitive mutant abi2-1 of Arabidopsis.These results provide evidence for presence of a relationship between root growth enhancement and signaling by ABA and ethylene in response to HrpN_Ea.Nevertheless,when HrpN_Ea is applied to leaves,ethylene signaling is active in the absence of ABA signaling to promote plant growth.This suggests the presence of a different signaling mechanism in leaves from that in roots.2.The construction and generation of a double mutant involving ABA and ethylene signal pathways.Ethylene is one of the most important endogenous signals in plant growth and development.It induces the primary disease-resistant response signaling pathway,which is essential for the fundamental self-defense by plants.Abscisic acid（ABA） as the stress hormone modulated many important events in plant growth and development which include modulating stoma closing,resistance to stresses,promoting seeds maturity and dormancy. The roles of ABA and ethylene in plant growth are mostly antagonistic.Specifically,ABA at high concentrations inhibits the synthesis of ethylene.We constructed ABI2-silencing vector,which was able to silence ABI2 in ein2-1 for further study on the roles of the interaction of ABA and Ethylene in plant development and signal transduction in resistance to disease.The silencing line we obtained was named ein2 abi2 Then distinctions of ein2-1 and ein2 abi2 were detected to study the function of ABI2 in Arabidopsis.This study will provide further materials and evidences for the research of relationship between ABA and Ethylene pathways in plant signal transduction in biostress.3.Transgenic expression of HpaG_Xoo of enhances plant growth and confers resistance.Harpins,a group of proteins produced by plant pathogenic bacteria,elicit hypersensitive cell death（HCD） in non-host plants of bacteria and induce resistance to pathogens and insects and enhance plant growth in many plants.HpaG_Xoo,encoded by the hpaG_Xoo gene of X.oryzae pv.oryzae,is a member of harpin group of proteins.Like others harpins,HpaG_Xoo induces HR and others various effects in the plant.Here we show that expression of the hpaG_Xoo gene in transgenic Arabidopsis enhances plant growth and confers pathogen defense without HCD.Resistance to Pseudomonas syringae pv.tomato DC3000 was enhanced at various levels in HATA1 lines.Genes NPR1,PR-1,PR3b,which are involved in pathogen defense,were expressed to various levels in HATA1 plants tested. However,cell death was not observed in HATA1 plants.Based on these data,we concluded that expression of HpaG_Xoo in transgenic Arabidopsis plants enhances plant growth and induces expression of defense-related genes and confers nonspecific resistance to pathogenic bacteria in the absence of HCD.In addition,plant growth and resistance to bacteria expression can be induced in transgenic plants expressing HpaG_Xoo constructed with or without a signal peptide in the transformation unit.更多还原