【作者】 周程爱；

【导师】 肖启明； 谢丙炎；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2006， 博士

【摘要】 诱导抗性或获得抗性是植物受病原物侵染或化学物质处理后能产生对随后病原物的侵染的抗性。不同的生物或非生物诱抗剂能诱导植物对病害获得局部或系统抗性,这些诱抗剂能通过不同的信号途径激活信号网络中的某一点从而诱导不同的植物产生特异性蛋白抵抗病虫害的攻击。系统获得抗性(SAR)是一种特殊形式的诱导抗性,具有系统、持久、抗病谱广等三大特点。研究开发诱导植物抗病性的物质具有重要的理论和实践意义。β-氨基丁酸(BABA)是一种具有广谱诱抗活性的植物化学诱抗剂,为了研制和开发该类氨基酸农药制剂,对BABA及其合成制剂温室诱导辣椒和番茄抗TMV、CMV、疫病、疮痂病和根结线虫病及田间诱导大白菜抗霜霉病的活性进行了研究,并且对BABA诱导辣椒抗病性的机理从酶(PR蛋白)活性及基因表达水平进行了研究。主要结果如下:1.β-氨基丁酸(BABA)喷雾处理辣椒叶片和茎秆后可诱导辣椒获得对TMV和CMV的抗性,并且BABA诱导茄门、931和21号等3个辣椒品种对TMV的抗性效果相同,其差异不明显;BABA的诱抗效果受诱抗剂的使用浓度所影响,诱导辣椒抗TMV的最佳处理浓度为1000μg/mL,并且对植株没有药害作用。2.茄门、931和21号等3个辣椒品种在BABA处理3d后即可产生对TMV的抗性,随着时间的推移,其抗性随之增高,BABA在第5～6d诱抗效果达到最高,之后开始下降,其持效期可达15d以上;随着生育期的增长,3种诱抗剂对甜辣椒的诱抗效果也随之增强,4叶期时,诱抗效果都相对较低:BABA诱导8叶期辣椒抗TMV的效果曲线呈现高-低-高的趋势;TMV接种浓度越高,BABA诱导辣椒抗TMV的效果越好。3.β-氨基丁酸(BABA)、茉莉酸(JA)及其甲酯(MeJA)喷雾处理辣椒叶片和茎秆后可诱导辣椒获得对TMV的抗性,三种诱抗剂对不同辣椒品种所表现的诱抗效果没有显著差异,最高的诱抗效果可达到50%以上;BABA、JA及等浓度混合溶液分别喷洒于6叶期茄门、931、21号辣椒植株的结果证明,1000μg/mL的BABA、JA及混合液在茄门上的诱抗效果分别为42.2%、35.2%和36.7%:对931的分别为34.9%、42.8%和44.5%;21号为46.2%、39.6%和43.6%,等浓度混合溶液的诱抗效果介于BABA和JA之间,说明BABA与JA既不可能存在拮抗作用,也不可能存在增效作用。4.10%BABA可溶性粉剂对多种病害的诱抗效果均在BABA纯剂之上,特别是对辣椒疫病的诱抗效果在接种后10d仍高达88%;对大白菜抗霜霉病的诱抗效果也较生产上常用药剂甲霜锰锌要好。BABA衍生物对TMV、CMV、疫病和根结线虫病亦表现出一定的诱抗潜能。5.对β-氨基丁酸诱导辣椒后叶片中β-1,3-葡聚糖酶、几丁质酶和过氧化物酶活性的动态变化进行测定,结果表明,BABA对不同抗、感辣(甜)椒β-1,3-葡聚糖酶、几丁质酶活性和过氧化物酶活性有不同程度的提高,但辣椒品种这3种PR蛋白酶活性升高的幅度高于甜椒品种;而且这三种PR蛋白活性变化与诱抗效果的表现相一致。6.BABA可以诱导几丁质酶ClassⅢ和脂氧合酶基因的表达,诱导处理辣椒后,几丁质酶ClassⅢ和脂氧合酶基因的表达都呈现低-高-低的抛物线型的发展趋势,6h时表达量较小或不表达,大多在12～24h间表达量达到最大,之后表达开始减弱,到48h基本停止表达或微弱表达。对所得差异片段进行了核苷酸序列同源性分析,发现与已经报道的辣椒的脂氧合酶的编码序列高度同源,同源性达到99%～100%。7.P450在植物体中担当着生物合成和代谢解毒两大类型功能,其中有一些P450在植物防御反应中具有重要的作用。用RT-PCR和RACE方法,从β-氨基丁酸(BABA)诱导辣椒叶片的基因表达体系中克隆出CYP92A的全长cDNA序列。生物信息学表明,该基因编码一条509个氨基酸残基的多肽,含有一段细胞色素P450保守的血红素结合区域。在二级结构上,该基因编码的蛋白质与已报道细胞色素P450的晶体结构相似。从而,CYP92A被确定是细胞色素P450。通过系统进化分析,CYP92A属于多基因家族P450的92A亚家族。结合己报道的92A亚家族成员的信息,推测CYP92A参与了植物的防御反应。β-氨基丁酸能够诱导辣椒对病毒病、细菌病害、真菌病害及线虫病害都具有较好的抗性,其抗性的产生与相关基因的表达及病程相关蛋白的产生有关。同时,BABA还能诱导辣椒叶片的细胞色素P450基因表达。本研究结果为BABA诱导机理的最终阐明提供了新的思路。更多还原

【Abstract】 The induced resistance or the acquired resistance is a kind of resistance produced by plant infected by pathogens or treated by chemistrical materials,which can protect plant from the subsquent infection of plant pathogens.Plants can acquire partial or systematic resistance when they were induced by different biological or abiotic elicitors, and these elicitors can activate a special dot of signal web by different signal route,then induce different plants to produce special protein for resisting the attack of pathogens and pests.Systematic acquired resistance(SAR) is a kind of special induced resistance, which has three charactoritics:systematic,persisting and broad spectrum of resist disease.It has significant theoretical and practical value to research and find materials that can induce plant resistance.DL-β-aminobutyric acid(BABA) is a kind of chemical elicitor for plant,which has widely induced resistance activities.In order to develop and exploit this kind of amino acid pesticides,a study on induced resistance produced by BABA and its preparations in pepper and tomato against TMV,CMV,Phytophthora capsici PC48,Xanthormonas vesicatoris(Boidge) Dowson and Meloidogyne incognita(Kofoid and white) Chitwood in greenhouse,and inducing resistance activation of cabbage against Peronospora parasitica in fields Were conducted.Furthermore,enzyme and moleculer research work with respect to mechanism of BABA inducing pepper to resist disease also had been done.The main results were as following:BABA could induce pepper to acquire resistance against TMV and CMV when pepper leaves and stems was sprayed with this kind of amino acid pesticides.The effects of BABA on resistance induction were not significantly different among the varieties of Qiemen,931 and 21.The effects of BABA on induction was influenced by concentrations of elicitors.The optimal treatment concentration was about 1000μg/mL, and the plants would not be damaged at this concentration.The resisitence was generated from three pepper strains 3 days later when they were treated by BABA.With time prolong,the resistance began to rise,and the induced resistance effect of BABA came to the highest after the plants were treated five or six days with BABA,then it began to decrease,the effect could remain more than 15days With the prolong of pepper reproductive period,the induced effect of BABA on sweet pepper strengthened.The resistance effect could be improved with the increasing of TMV concentration.BABA,JA and MeJA could induce pepper to generate resistance against TMV after the leaves and stems was treated.Inducedresistance effect &three elicitors didn’t show obvious diference,and the highest induced effect could reach above 50%.The effect of BABA,JA and blend solution on Qianmeng was 42.2%,35.2%and 36.7% repectively;the effect produced by above three elicitors on 931 was 349%,42.8%and 44.5%respectively;and the effect of them on 21 was 46.2%,39.6%and 43.6%.The induced resistene effect produced by blend solutiont with equal concentration was located in the middle of BABA and JA,which showed that resisting action and increasing effect action were not existeA between BABA and JA.The induced effect of 10%BABA powder against several plant diseases was better than that of the pure BABA,especially against pepper Phytophthora capsici.Its induced resistance effect against pepper Phytophthora capsici was still kept 88%after applied ten days.The induced effect of BABA on the cabbage against Peronospora parasitica was better than that of other usual pesticides used in production.BABA derivative also showed induced resistance ability to TMV,CMV,Phytophthora capsici and MeIoidogyne incognita.The results of enzymes assays showed that activities ofβ-1,3-glucanases, chitinases and POD all increased in different levels after BABA treated,but the increasing extent of PR protein activities in three peppers were higher than that of PR protein activities in sweat peppers,and the activity changes and induced effects of three PR protein concorded with each other.The expression of Chitinase ClassⅢgene and lipoxygenase gene could be expressed when plant was induced by BABA.Afterward treatment,the gene expression of Chitinase ClassⅢand lipoxygenase all showed a high-low-high developing trend,a few genes expressed six hours later afterward treatment,the expression quautities came to peak between 12 and 24 hours afterward treatment,then gene expression began to decrease,till 48 hours later,gene expression stopped or expressed a few Nucleotide sequence analysis of the differential cDNA fragment showed that the nucleotide sequence is highly homologous with the nucleotide sequence encoded lipoxygenase of Capsicum.P450 is responsible for biosynthesis and metabolism in plants.Some of P450 have significant actions in plant defence response.Through RT-PCR and RACE,The complete sequence of CYP92A was cloned from gene expression systems of pepper (Capsicum chmense) leaves induced by BABA,The analysis of bioinformatics showed that CYP92A gene encoded a 509-amino acid multi-peptide,which contained a conservertive Heme-binding region of cytochrome P450.This protein took on similarity of secondary structure with crystal structure of CYP450 reported,therefore,it was confirmed that CYP92A was cytochrome P450 in fact.By phylogenetic analysis,it was believed that CYP92A belong to 92A sub-family of P450.integrating with other reports with respect to 92A sub-family genes,it can assume that CYP92A may perticipate in plant defence response.Above results showed that BABA has a potential to induce pepper(Capsicum chinense) to produce good resistance against virus,bacteria and fungi deseases.The generation of resistance was related to the expression of corresponding gene and the production of relative proteins during the disease period.At the same time,BABA could induce gene expression of P450 in Capsicum chinense leaveso All these results provided a new concept for understanding the mechanism of the induced resistance of BABA.更多还原