【作者】 任秀艳；

【导师】 董汉松；

【作者基本信息】 南京农业大学 ， 植物病理学， 2007， 博士

【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,它们的共同特征是:富含甘氨酸,热稳定,对蛋白酶敏感,在非寄主植物叶片上可以诱导Hypersensitive response（HR）或Hypersensitive cell death（HCD）。外源施用后,可以诱导多种植物抗病、抗虫、抗旱、促进植物生长。这些效应在不同harpins处理的植物上都有所发现,它们是来自于Eriwinia amylovora的HrpN_Ea、Pseudomonas syringae pv.syringae的HrpZ_Pss、P.syringae pv.phaseolicola的HrpZ_Psph和Xanthomonas oryzae的HpaG。但是,对于harpins如何行使自身的多重功能,目前还不清楚。本研究着重对以下几个方面进行了研究。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HrpN_Ea是由引起蔷薇科植物火疫病的病原细菌Erwinia amylovora产生的一种Harpin蛋白。HrpN_Ea处理植物可以激发乙烯和脱落酸分别诱导植物生长和抗旱。本文报道了脱落酸和乙烯两种激素同时应用时调节HrpN_Ea在拟南芥根生长中的促进作用。我们首先研究了野生型拟南芥Col-0和Ler-0从种子萌发结束到胚根伸出后,HrpN_Ea对根生长的作用,结果发现,与水和EVP处理相比,HrpN_Ea处理后明显促进了根的初期生长。在处理12h和24h后,HrpN_Ea的作用更明显。EVP同水处理一样不影响根的生长。用HrpN_Ea溶液浸泡处理野生型拟南芥种子在促进脱落酸和乙烯水平升高的同时促进根的生长。这些反应通过抑制剂AgNO₃或1-MCP抑制野生型种子对乙烯的感知及NDGA和AOA抑制野生型种子对ABA和乙烯合成而受到限制。HrpN_Ea处理拟南芥突变体etr1-1,ein5-1和ABA不敏感突变体abi2-1后,对根生长始终都没有影响。我们的结果建立了一个在HrpN_Ea的作用下,脱落酸和乙烯信号和促进根系生长之间的机械联系。然而,当在叶片上施用HrpN_Ea时,在促进植物生长过程中,乙烯信号是在没有脱落酸信号的情况下起作用的,这表明,HrpN_Ea在植物叶片和根中的信号机制是不同的。2.HpaG_Xoo转基因表达诱导植物生长和抗病性根据前人的报道,外源施用harpins时,可以启动SAR通路包括SA积累、EDSI和NDR1的调控、PR基因的表达及NPR1对PR基因表达的调控,其中HCD与抗病性同步发生、但没有必然的因果关系。HpaG_Xoo是水稻白叶枯病菌X.oryzae pv.oryzae的基因hpaG_Xoo编码的一种harpin蛋白,同其它harpins蛋白一样可以在非寄主植物上诱导产生过敏反应以及其它多种效应。我们将编码HpaG_Xoo的基因hpaG_Xoo与花椰菜花叶病毒35S启动子的融合基因单元通过真空花絮渗透法转入拟南芥,获得表达HpaG_Xoo的拟南芥(HpaG_Xoo-expressing Arabidopsis,HATA1)转基因系。对转基因系进行了以下分子验证:通过PCR方法,确定了hpaG_Xoo基因在拟南芥基因组中的稳定整合;用RT-PCR方法证明了hpaG_Xoo基因在拟南芥中可以转录;从转基因拟南芥中提取的HpaG_Xoo具有在烟草上诱导HR的能力;同时根据SDS-PAGE电泳,转基因表达的HpaG_Xoo大小没有改变（15.6 kD）。这些结果表明,hpaG_Xoo基因在拟南芥体内可以正常翻译为有活性的、完整的HpaG_Xoo分子。另外,我们选择了具有一定代表性的细菌菌株P.syringae pv.tomato DC3000,对转基因拟南芥的不同株系进行抗病性实验,结果发现不同的转基因株系都获得了对该细菌的抗性,但抗性水平有所差别。同时我们对抗病防卫反应基因NPR1、PR-1和PR3b的表达进行了测定,发现在转基因株系中,防卫反应相关基因都有一定的表达。但是,所有的转基因株系都没有HCD的发生,而外源喷施野生型拟南芥发生了明显的Micro-HR。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,诱导对细菌的抗病性,但不能诱发HCD。另外,是否带有信号肽对hpaG_Xoo基因在植物体内表达后所诱导的促生长和抗病没有影响。3.HpaG_Xooc及其功能片段对田间水稻产量的影响对水稻细菌性条斑病菌（X.oryzae pv.oryzicola）的HpaG_Xooc进行改造,产生了9个不同的功能片段,其中HpaG_10-42促进水稻生长、诱导水稻抗病性的效应最强。本研究在3个地区对9个水稻品种进行了田间小区实验,发现HpaG_10-42蛋白提高水稻产量的效应显著地优于HpaG_Xooc全长蛋白。根据水稻生长期确定处理时间,测定了两种蛋白18种浓度组合的田间效应。分别在苗期,返青后期,分蘖期和始穗期喷施不同组合的蛋白溶液,调查两种类型水稻产量。正交分析表明,功能片段HpaG_10-42的6μg/ml的浓度组合的效果最好,但是HpaG_10-42在最小剂量的施用效果仍显著高于HpaG_Xooc最高剂量的施用效果。HpaG_10-42和HpaG_Xooc在水稻海陆红2号的四个生长期单独施用,发现HpaG_10-42在苗期,返青后期和分蘖后期处理,都明显增加作物产量,而在始穗期施用效果不大,但都比全长蛋白的效果好。HpaG_10-42促进水稻产量增加效果好于常规农事操作和HpaG_Xooc处理。而HpaG_10-42对9个不同水稻品种产量的影响存在很大差别,而和品种类型无关。本研究结果为有效应用病原代谢物的有益功能片段来改良大田粮食作物,提供了范例。4.HpaG_Xoo蛋白表达及抗体制备HpaG_Xoo在植物上可以诱导产生多种效应,但是对于该蛋白在植物上是如何起作用,在什么部位起作用,目前还不清楚。已有人报道了harpins在植物细胞中的作用部位,但对不同harpins的研究结果不尽相同。为了下一步研究HpaG_Xoo在植物中的作用部位,构建了可用于HpaG_Xoo蛋白纯化的高效表达载体,并制备了抗血清。利用PCR方法从水稻白叶枯病菌中扩增得到hpaG_Xoo基因,连接pET30a（+）载体,获得了重组质粒pET30a（+）::hpaG_Xoo,该载体上保留了蛋白质纯化所需的His tag编码序列。转化宿主菌BL21（DE3）产生表达菌株BLHR4。表达菌株经IPTG诱导培养,进行SDS-PAGE电泳,产生分子量为21.6 kD大小的组氨酸标记的融合蛋白条带。利用HisTrap HP Kit试剂盒对HpaG_Xoo蛋白进行了纯化。SDS-PAGE结果表明,体外表达融合组氨酸的HpaG_Xoo经过Ni柱纯化同样得到21.6kD大小的单一条带。以牛血清蛋白（BSA）为标准,经凝胶成像系统BioImage软件测定,蛋白提取液中目的蛋白的浓度约为0.5-1.0 mg/ml,每升菌液可提纯蛋白约2-4 mg。该纯化蛋白在烟草上引起典型的过敏性反应。我们将浓度为1 mg/ml纯化的蛋白新西兰家兔进行免疫,获得了HpaG_Xoo蛋白的多克隆抗体,间接ELISA方法测定抗血清的效价,结果效价达到1:16,000以上,能够满足以后的实验需要。同时利用Western blot对蛋白与抗体之间的特异性结合进行了验证,结果表明,不论是粗蛋白还是纯化蛋白在相应的位置上都有很强的杂交信号,而空载体提取物在相应的位置没有任何信号。为了验证转基因植物中表达的HpaG_Xoo蛋白与抗血清之间是否特异性结合,我们提取了植物中表达的HpaG_Xoo蛋白,并对其进行了杂交分析,结果表明,转基因植物中表达的HpaG_Xoo蛋白和原核表达的一样可以和抗血清特异性识别并产生较强的杂交信号,由于该蛋白原核表达融合了His-tag,故比植物中表达的蛋白条带大,因此杂交信号不在一条直线上。5.HpaG_Xoo蛋白互作因子的筛选本研究使用酵母双杂交系统,以HpaG_Xoo为诱饵,对拟南芥cDNA文库进行筛选。根据营养缺陷型和β-半乳糖苷酶活性初步筛选到6个阳性克隆,测序并在Genbank中进行序列比对,结果表明它们分别编码拟南芥中的核黄素合酶（Riboflavin synthase,RS）、液泡膜嵌入蛋白（Tonoplast intrinsic protein 2,TIP2）、成束类阿拉伯半乳聚糖蛋白（Fasciculin-like arabinogalactan-protein,FLA8）、质膜嵌入蛋白（Plasma membraneintrinsic protein,PIP1）、核转运因子（Nuclear transport factor 2,NTF2）和碳酸酐酶（Carbonic anhydrase 2,CA2）蛋白,而且序列同源性达到100%。总结:本研究结果表明,harpins诱导植物生长和抗病的过程中,乙烯和脱落酸信号传导通路参与了这些反应的调控。植物与HpaG_Xoo互作的因子可能通过不同的机制在这些信号传导过程中起作用。对这些问题的研究,有助于深入了解harpins诱导植物多种反应的机制。本研究的创新点:（1）解析ABA和乙烯信号的互作对HrpN_Ea诱导拟南芥根生长的调控作用;（2）构建了带有His-tag的HpaG_Xoo表达载体并纯化出有活性的蛋白质;（3）筛选到了与HpaG_Xoo互作的植物因子,对其功能进行初步预测。不足之处:由于时间关系,没能对HpaG_Xoo在植物上的作用部位及筛选出的互作因子的功能做进一步研究。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 drough.These effects have been observed in plants treated with HrpN_Ea from Eriwinia amylovora,HrpZ_Pss from Pseudomonas syringae pv.syringae, HrpZ_Psph from P.syringae pv.phaseolicola,and HpaG from Xanthomonas oryzae.How harpins perform these diverse functions has not been clear completely.1.Root growth of Arabidopsis thaliana is regulated by ABA and ethylene signaling interaction in response to HrpN_EaApplying HrpN_Ea to plants can stimulate ethylene and abscisic acid to induce plant growth and drought tolerance,respectively.Here we report that both hormones cooperate to mediate the role of HrpN_Ea in promoting root growth of Arabidopsis thaliana（Arabidopsis）. Root growth was promoted coordinately with elevation in levels of ABA and ethylene subsequent to soaking seeds of wild-type Arabidopsis in a solution of HrpN_Ea.These responses were arrested by inhibiting wild-type seeds to sense ethylene or synthesize either of ABA and ethylene.Consistently,HrpN_Ea effects on roots were nullified in ethylene-insensitive etr1-1 and ein5-1 mutants,and ABA-insensitive mutant abi2-1 of Arabidopsis.Our results establish a mechanistic connection between enhancement of root growth and signaling by ABA and ethylene in response to HrpN_Ea.However,ethylene signaling was working in the absence of ABA signaling to promote plant growth when HrpN_Ea was applied to leaves,indicating different signaling mechanisms in leaves from roots.2.Transgenic expression of HpaG_Xoo enhances plant growth and confers plant resistance to bacteriaHpaG_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.The hpaG_Xoo gene was inserted into the transformation vector pBI121 between the 35S promoter and uidA（GUS） gene.Transformation was conducted by flower vacuum infiltration with recombinant Agrobacterium tumefaciens.Integration of hpaG_Xoo gene into Arabidopsis chromosomes were determined by polymerase chain reaction（PCR）.The expression of hpaG_Xoo gene in hpaG_Xoo-expressing transgenic Arabidopsis（HATA1） plants was detected by RT-PCR and assays for activity of HpaG_Xoo isolated from transgenic plants.T3 plants of HATA1 lines and transgenic lines containing the vector only were tested,together with the parent （ecotype Col-0） plants,for expression of defense-related genes,cell death and resistance to pathogen bacteria.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.3.HpaG affects grain yield of rice in extensive grower plantingsBased on works in our lab,HpaG_10-42 is most active among the nine fragments, generated from truncating the X.oryzae pv.oryzicola HpaG_Xooc protein,in enhancing growth and inducing disease resistance in rice.Here we show evidence that HpaG_10-42 significantly exceeds HpaG_Xooc to increase grain yield of rice under grower plantings based on 9 rice varieties growing at 3 locations.Application procedures were established by testing 18 combinations of two proteins doses with treating time according to rice growth stages.HpaG_10-42 was applied to nursery seedling 10 d before transplant,late turning-green stage,late tillering stage and early heading stage of indica and japonica rice varieties using different concentration arrays.Orthogonal experimental analysis on grain yield show that the optimized concentration array of HpaG_10-42 is 6μg/ml array.HpaG_10-42 used at the minimal dosage was significantly greater than HpaG_Xooc used at higher concentrations applied at the four stages of rice growth in the effects on yield of rice.HpaG_10-42 enhances gain yield higher than local agronomic measures,including use of chemicals and HpaG_Xooc. In addition,effect of HpaG_10-42 on 9 varieties of rice is different,and have no related to type of rice.Our results provide an example for effective use of beneficial fragments derived from pathogen metabolites to increase yield in the staple food crop.4.Expression and antibody preparation of HpaG_Xoo proteinToward cellular localization of HpaG_Xoo in plants,an hpaG_Xoo expression vector was made to involve a his-tag,which facilitates protein purification by affinity chromatography. In the study,hpaG_Xoo gene was amplified by PCR from JXOⅢgDNA,and PCR product was 420 bp.Purified hpaG_Xoo fragment was ligased to pET30a（+） vector,creating recombinant plasmid pET30a（+）::hpaG_Xoo,which contains a His-tag-encoding sequence. Recombinant HpaG_Xoo protein was produced and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis（SDS-PAGE）,which revealed the protein as 21.6 kD in size.After infiltrated into tobacco leaves,HpaG_Xoo caused typical HR.Recombinant HpaG_Xoo protein was purified through His Trap HP columns(pre-charged with Ni²⁺).The yield was 2-4 mg/L culture and the concentration of purified proteins was 0.5-1.0 mg/ml. The HpaG_Xoo-His fusion protein also caused typical HR on tobacco.Therefore,we made an effective construction and produced active HpaG_Xoo.The polyclonal antibody against HpaG_Xoo was produced with New Zealand White Rabbit.The titer of the polyclonal antibody against HpaG_Xoo was greater than 1:16,000.The anti-HpaG_Xoo polyclonal antibody reacted with HpaG_Xoo protein in the analysis of ELISA and Western blot hybridization analysis.Moreover,Western blot hybridization analysis also revealed specific presence of HpaG_Xoo in HATA1 lines.5.Screening of proteins that interact with HpaG_Xoo using yeast two-hybridTo explore plant proteins that could interact with HpaG_Xoo,a yeast two-hybrid system was used to screen Arabidopsis cDNA library.The bait and library plasmid were cotransformed into the yeast strain Y190.Six positive clones were obtained by the selection of autotrophic phenotype andβ-galactosidase assay.Sequencing and Genbank blasting comparison demonstrated that these six clones were distinct in sequences and encoded parts of Arabidopsis riboflavin synthase（RS）,tonoplast intrinsic protein 2（TIP2）,fasciculin-like arabinogalactan-protein（FLAS）,plasma membrane intrinsic protein（PIP1）,nuclear transport factor 2（NTF2）,or carbonic anhydrase 2（CA2） proteins.Moreover,similarity of the cloned sequences is 100%compared with the corresponding homologues reported previously.In conclusion,plant responses to harpins involve regulation by ABA and ethylene signaling pathways.Plant proteins interacting with HpaG may act in the pathways by different mechanisms.Characterization of how HpaG_Xoo and its interacting factors function will shed light on signaling regulation of diverse responses of plants to harpins.更多还原