【作者】 张莹丽；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 蔬菜学， 2013， 博士

【摘要】 辣椒是一种重要的蔬菜作物，具有非常高的经济价值，在我国及全世界范围内栽培广泛。辣椒疫病（Phytophthora blight）是由辣椒疫霉菌（Phytophthora capsici Leonian）引起的一种毁灭性的的土传病害，能侵染多种茄科及葫芦科植物，对我国及世界各国的辣椒生产造成巨大的经济损失，已经成为制约辣椒产业化发展的重要因素之一。目前，对于辣椒疫病的防治主要采用化学防治、轮作和选育抗病品种，但是农药残留超标对环境造成污染，P. capsici生理小种的变化造成抗病品种效果不稳定。因此，利用嫁接与分子生物技术等开展辣椒抗疫病育种具有重要的现实意义。本试验探讨了不同抗性辣椒品种与P. capsici的互作防御机制，抗性辣椒品种做砧木嫁接对P. capsici的抗性机制，利用VIGS结合转基因技术对辣椒疫病抗性相关基因CaRGA2的功能在辣椒和烟草上分别进行了验证，使用病原菌诱导型启动子prp1-1，使其调控CaRGA2基因的表达，旨在为今后的辣椒抗病育种提供理论依据。主要研究成果如下：1.通过对陕西病原物的分离培养和形态学观察，将其确定为辣椒疫霉菌。不同抗性品种CM334、PBC602、B27接种P. capsici后，抗性品种CM334的根系活力最强，感病品种B27的根系活力最弱；POD、PAL和β-1,3-葡聚糖酶活性与抗病性呈正相关关系，三个不同抗性品种的酶活差异明显；我们利用抗病品种CM334接种P. capsici后，提取PAL和β-1,3-葡聚糖酶的粗酶液来抑制P. capsici菌丝生长和孢子囊形成，结果显示两种粗酶液的混合液对P. capsici菌丝生长和孢子囊形成具有协同增效作用；防御相关基因CaPO1、CaBGLU、CaBPR1和CaRGA2在三个不同抗性品种叶片中的表达水平高于根系，在抗病品种中的表达水平高于感病品种，表明了不同抗性品种在辣椒疫病防御反应中有差异。2. P. capsici胁迫下，辣椒嫁接苗的抗性明显提高，表现在POD、PAL和β-1,3-葡聚糖酶活性明显高于辣椒接穗自根嫁接苗；光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)明显高于辣椒接穗自根嫁接苗；相对电导率和胞间CO2浓度(Ci)明显低于辣椒接穗自根嫁接苗。而嫁接苗之间也表现出抗性强的嫁接苗叶片中POD、PAL和β-1,3-葡聚糖酶活性高于抗性较弱的嫁接苗；光合速率(Pn)、蒸腾速率(Tr)和气孔导度(Gs)高于抗性较弱的嫁接苗；相对电导率和胞间CO2浓度(Ci)低于抗性较弱的嫁接苗。因此不能单一地以上述生理指标作为辣椒抗P. capsici胁迫的指标，应以综合评价指标来鉴定。3.对辣椒嫁接苗防御相关基因的表达分析显示，通过嫁接可以将砧木中的某些抗病物质传递到接穗中，从而提高接穗的抗病性，P. capsici胁迫下CaPO1、CaBPR1、CaBGLU和CaRGA2防御相关基因在叶片中的表达量明显高于根系和茎，不同抗性嫁接苗中的表达量明显高于接穗自根嫁接苗，嫁接过程中具体是砧木中的哪种抗性物质传递到了接穗中还有待于进一步的研究。4. CaRGA2基因从抗疫病材料CM334的叶片中克隆得到，cDNA全长3018bp，开放阅读框(ORF)2874bp，编码957个氨基酸，预测蛋白分子量为108.6kDa，等电点8.106。实时定量PCR结果显示，接种P. capsici后CaRGA2基因被迅速诱导，其在抗病和感病品种中的表达模式不同；接种P. capsici后24h，CaRGA2基因在抗病品种CM334中迅速表达并达到一个峰值，表达量是感病品种的5倍，表明CaRGA2基因在辣椒疫病抗性中起着重要的作用。5.利用VIGS技术对辣椒CaRGA2基因功能进行了分析，在辣椒中成功沉默VIGS报告基因-八氢番茄红素脱氢酶基因(PDS)，使辣椒叶片表现白化现象，为大规模辣椒基因功能分析提供了技术指导；将携带CaRGA2目的基因的重组病毒载体接种P. capsici抗病品种CM334，通过半定量RT-PCR和离体叶片接种结果显示，CM334抗病性降低表现为易感病，VIGS结果显示了CaRGA2基因在辣椒对疫病的抗性中具有重要作用，证明CaRGA2基因为辣椒疫病抗性相关基因。6.利用PCR方法从马铃薯块茎的总DNA中克隆了受病原菌诱导的prp1-1启动子片段，构建了辣椒抗疫病相关基因诱导型植物表达载体pVBG-prp1-1-CaRGA2，使prp1-1病原菌诱导型启动子片段调控CaRGA2基因的表达；同时构建了CaRGA2基因组成型植物表达载体pVBG-CaRGA2。将构建好的两个载体导入辣椒和烟草，分别得到了CaRGA2基因诱导性表达(pVBG-prp1-1-CaRGA2)和组成型表达(pVBG-CaRGA2)的辣椒和烟草转化植株。7.对于CaRGA2基因诱导性表达(pVBG-prp1-1-CaRGA2)和组成型表达(pVBG-CaRGA2)的辣椒和烟草转化植株，进行初步的PCR分子鉴定及抗病性分析。与阴性对照未转基因植株相比，CaRGA2转基因辣椒和烟草并没有发生表型上的变化；离体叶片接种辣椒P. capsici后，pVBG-prp1-1-CaRGA2转基因辣椒和烟草叶片上的坏死病斑小于pVBG-CaRGA2，未转基因植株上的坏死病斑最大，说明CaRGA2基因参与了辣椒与P. capsici的抗性反应过程，prp1-1病原菌诱导型启动子调控了CaRGA2基因的表达，CaRGA2基因在辣椒抵抗P. capsici侵染的过程中发挥正向调节作用。更多还原

【Abstract】 Pepper (Capsicum annuum L.) is an important vegetable crop with high economic value,widely cultivated in China and the worldwide. Phytophthora blight was caused byphytophthora capsici Leonian, is a destructive soli-borne disease. P. capsici is also pathogenicon several solanaceous and cucubit hosts except for pepper. Which impacted on agriculture ofChina and other countries badly, and caused enormous loss on economy, and is one of theimportant factors of restricting pepper development of industrial in China. At present, thephytophthora blight of peppers was controlled mainly by application of chemicals, croprotation and breeding resisitant host. However, phytotoxicity and chemical residues maypollution to the environment, strong variability of physiological strains of P.capsici caused theinstability of resistant host. Therefore, carry out pepper resistant to P. capsici by grafting andmolecular biological technique has the important practical significance. The objectives of thisstudy were: to explore the defence mechanism of different resistant cultivars interaction of P.capsici; the resistance mechanism of P. capsici by using resistant pepper varieties asrootstocks; to identify the function of resistance related gene CaRGA2in pepper and tobaccothrough VIGS combined with transgenetic technology; to make the regulation of CaRGA2gene expression by use of pathogen inducible promoter prp1-1, and to provide a theoreticalbasis for pepper disease-resistance breeding in the future. The main results of the study are asfollows:1. Based on culture isolation and morphological observation blight-infected pepperplants in province, P.R. China, we identified the pathogen causing pepper phytophthora blightas Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602and B27) wereinoculated with this pathogen. The root activity of resistant CM334variety was the highestwhile that of susceptible B27variety was the lowest. Also, significant differences in theactivity of POD, PAL and β-1,3-glucanase were found; there was a positive correlationbetween disease resistance and activity of these three enzymes. We inhibited mycelial growthand sporangia formation of P. capsici using crude β-1,3-glucanase and PAL enzymes isolatedfrom the resistant variety CM334after it had been inoculated with P. capsici. These twoenzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defense related genes CaPO1, CaBGLU, CaBPR1and CaRGA2is higher in the leaves of the three varieties than in the root. All three genes were up-regulatedin infected leaves and roots of the pepper plants, always expressing at higher levels in theresistant cultivar than in the susceptible cultivar, suggesting that the differences in resistanceamong the pepper genotypes involve differences in the timing and magnitude of the defenseresponse.2. Under P. capsici stress, the resistance of grafted pepper seedlings was higher thanself-grafted pepper seedling. Compared with self-grafted pepper seedling had higherphotosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Gs) in leaves andlower relative conductivity and intercellular CO2concentration (Ci). In addition, the graftedseedling with higher resistance had higher activities of POD, PAL and β-1,3-glucanase,higher photosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Gs) andlower relative conductivity and intercellular CO2concentration in leaves than those of graftedseedling with lower resisance. Therefore, should be identified by the comprehensiveevaluation parameter, but not a single to the physiological parameter as resistance to disease.3. On the expression of defense related genes in grafted pepper seedlings analysisshowed, some resistance materials in rootstock can be transmission to scion by grafting, so asto improve the disease resistance of scion. Under the stress of P. capsici, expression of thedefense related genes CaPO1, CaBGLU, CaBPR1and CaRGA2is higher in the leaves of thegrafted seedlings than that of root and stem, different resistance grafted seedlings in theexpression level was significantly higher than self-grafted pepper seedling, in whichresistance materials in rootstock transfer to scion in grafting process to be further study.4. We isolated and characterized a P. capsici resistance gene, CaRGA2, from a highresistant pepper (C. annuum CM334) and analyzed its function by the method of real-timePCR and virus-induced gene silencing (VIGS). The CaRGA2has a full-length cDNA of3018bp with2874bp open reading frame (ORF) and encodes a957-aa protein. The protein has apredicted molecular weight of108.6kDa, and the isoelectric point is8.106. Quantitativereal-time PCR indicated that CaRGA2expression was rapidly induced by P. capsici. The geneexpression pattern was different between the resistant and susceptible cultivars. CaRGA2wasquickly expressed in the resistant cultivar CM334, and reached to a peak at24h afterinoculation with P. capsici, five-fold higher than that of susceptible cultivar. Our resultssuggest that CaRGA2has a distinct pattern of expression and plays a critical role in P. capsicistress tolerance.5. The function of gene CaRGA2was identified by using VIGS technology. Using thisTRV derived vector, we successfully silenced the VIGS reporter gene-phytoene desaturase (PDS) gene, which result in photobleaching phenotype. The VIGS system will benefit thelarge scale gene function analysis in pepper. The CaRGA2gene recombinant virus vector wasinoculated with resistance material CM334, the resistance level was clearly suppressed, anobservation that was supported by semi-quantitative RT-PCR and detached leave inoculation.VIGS analysis revealed their importance in the surveillance to P. capsici in pepper. Ourresults support the idea that the CaRGA2gene may show their response in resistance against P.capsici.6. The promoter of prp1-1was cloned from the potato tuber DNA by the method of PCR,pVBG-prp1-1-CaRGA2, the inducible plant expression vector comprising prp1-1promoterand CaRGA2gene were constructed, as result CaRGA2gene was induced to express by plantpathogenic fungi under the control of the prp1-1promoter fragment. At the same time,pVBG-CaRGA2, the constructive plant expression vector comprising CaMV35S promoterand CaRGA2gene were constructed. The constructed vector pVBG-CaRGA2and inducedvector pVBG-prp1-1-CaRGA2were introducted into pepper and tobacco, respectively,transformed pepper and tobacco plants were obtained.7. The pepper and tobacco transgenic plants of constructed vector pVBG-CaRGA2andinduced vector pVBG-prp1-1-CaRGA2, was analyzed by PCR and disease resistance.Compared to the non-transgenic plants negative control, the CaRGA2transgenic line does nothave any phenotype change in the plant. In detached leaves inoculation of P. capsici, it wasfound that there were less necrotic lesions on the pVBG-prp1-1-CaRGA2transgenic pepperand tobacco leaves than that on the leaves of pVBG-CaRGA2transgenic plants, the necroticlesions of non-transgenic plants was larger than transgenic plants, these results suggested thatthe CaRGA2gene was involved in the pepper resistance response to the P. capsici infection.Pathogen inducible promoter prp1-1regulated the expression of CaRGA2gene, CaRGA2gene might play positive role in regulating the pepper plant resistance response to P. capsici.更多还原