【作者】 王军娥；

【导师】 巩振辉；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2013， 博士

【摘要】 辣椒(Capsicum annuum L.)是一种重要的蔬菜作物，具有极高的经济价值，在全世界范围内广泛栽培。然而，辣椒的病虫害却日益加重。尤其是以辣椒疫霉菌(Phytophthora capsici)引起的疫病(Phytophthora blight)，现已成为制约辣椒产业化发展的主要因素之一。人们采取各种方法来防治疫病的发生，但是效果不明显，甚至造成农药残留物超标的严重后果。因此，利用分子生物技术开展抗病育种具有很重要的现实意义。本研究探讨了辣椒与P. capsici亲和与非亲和的互作防御机制，建立了辣椒的病毒诱导基因沉默(VIGS)优化体系，利用VIGS结合转基因技术对辣椒基因CaRGA1和CaPOD的功能在辣椒上进行了鉴定，并在此基础上利用第二代高通量测序技术对辣椒与不同亲和组合P. capsic i互作的转录组进行测序分析，旨在为以后的辣椒抗病育种提供理论依据。主要研究成果如下：1.探讨了辣椒与不同亲和组合辣椒P. capsici互作过程中的防御机制。主要包括：防御相关酶(β-1,3-葡聚糖酶和过氧化物酶)活性的测定以及防御相关基因(CABPR1、CABGLU和CAPO1)的表达模式分析。结果表明，不同亲和组合辣椒P. capsici中防御酶活性高低及变化趋势不一致；实时定量RT-PCR分析认为，不同亲和组合中防御相关基因的表达模式不同，叶片中的表达量明显高于根系中，非亲和组合中的表达也明显高于亲和组合。2.建立了辣椒的VIGS优化体系。在前人研究的基础上，以辣椒的八氢番茄红素脱氢酶基因CaPDS为阳性报告基因，构建了重组病毒沉默载体pTRV2-CaPDS；利用农杆菌注射法，对影响沉默效率的主要因素：不同品种(A5、A3和EC)、植株苗龄(两叶、四叶、六叶和八叶期)、接种浓度OD600(0.5、0.8、1.0、2.0和3.0)和培养温度(18、20、22、25和28℃)进行了分析，最终得到了适合辣椒的VIGS优化体系，为今后辣椒基因的高通量功能分析提供了技术支持。3.对辣椒CaRGA1和CaPOD基因进行了生物与非生物因素逆境胁迫下的表达模式分析。结果表明：在辣椒P. capsici侵染下，CaRGA1和CaPOD基因均被不同程度的诱导表达；在非生物因素胁迫处理下，CaRGA1受高盐、干旱和茉莉酸甲酯(MeJA)的诱导而上调表达，但其表达却受到水杨酸(SA)的抑制，说明CaRGA1基因可能参与了MeJA的信号转导途径；CaPOD基因参与了辣椒的抗盐与抗旱反应，另外，该基因可能也与SA和MeJA介导的信号转导途径有关。4.利用VIGS技术对辣椒基因CaRGA1和CaPOD的功能进行了初步鉴定。对获得的CaRGA1和CaPOD基因沉默辣椒植株进行观察，发现沉默植株与阴性对照植株之间并没有明显的表型差异，实时定量RT-PCR结果显示基因在沉默植株中的表达有不同程度的降低；对沉默植株进行离体叶片抗病性鉴定，分别于接种后第3d和第5d在CaPOD和CaRGA1沉默植株的离体叶片上出现坏死病斑，从而初步推断出这两个目的基因的沉默降低了辣椒对P. capsici的抗性反应。5.构建了CaRGA1基因的过量植物表达载体，通过农杆菌介导的遗传转化方法，最终获得了4株转基因辣椒植株。与阴性对照相比，CaRGA1转基因株系并没有发生表型上的变化；离体叶片接种辣椒P. capsici后，我们发现转基因株系叶片上的坏死病斑少于阴性对照植株，但是防御酶活性却明显高于对照植株活性，说明CaRGA1基因参与了辣椒与P. capsici的抗性反应过程；采用离体叶圆片法对转基因植株进行了抗盐性分析，结果表明：与转基因株系相比，阴性对照植株的叶圆片有失绿现象，有些甚至呈水渍状。叶片总叶绿素含量测定数据显示，转基因植株的叶绿素含量高于阴性对照，说明CaRGA1基因可能与辣椒的抗盐反应有关。6.构建了CaPOD基因的过量表达载体，通过农杆菌介导的遗传转化法转到辣椒植株中，最终获得了6株CaPOD基因过量表达的转基因植株。通过表型观察，我们发现转基因与阴性对照植株之间没有差异；抗性鉴定结果表明：CaPOD转基因植株对辣椒P. capsici的抗性增强，而且提高了植株的耐盐力。以上结果，不仅说明了CaPOD基因在辣椒抵抗P. capsici的过程中发挥正向的调节作用，还参与了辣椒应答高盐胁迫的反应。7.利用第二代高通量Illumina/Solexa测序技术，构建了辣椒与P. capsici亲和与非亲和互作的转录组数据库。测序共获得了101,641个Unigene，其中HX-9转录组有50,795个，PC转录组有50,846个；对所获得的Unigene进行功能注释，其中33,365个Unigene被注释到NR数据库，34,658个被注释到NT数据库，20,747个比对到Swiss-Prot数据库中，18,654、11,698和27,087个Unigene分别用于KEGG代谢途径分析、COG和GO分类；在两个转录组中共找到8,144个差异表达基因，HX-9转录组中上调表达的有6,497个，PC转录组中上调表达的有1,647个；利用半定量PCR的方法对筛选出的6个候选差异表达基因进行表达分析验证，分析结果与测序结果基本一致，证明我们建立的辣椒与P. capsici互作的转录组数据库可靠，为深入挖掘辣椒疫病抗性基因及其相关基因奠定了基础。更多还原

【Abstract】 Pepper (Capsicum annuum L.), widely cultivated in the world, is an important vegetablecrop with high economic value, but it is prone to be damaged by diseases and insects,especially the phytophthora blight caused by Phytophthora capsici. Many chemicals wereused to control this disease but it might cause high pesticide reidues in the pepper productions.Therefore, the development of pepper resistant to P. capsici through molecular technique isan important and realistic approach. The objectives of this study were: to explore themechanism of compatible and incompatible interaction between the pepper plant and the P.capsici; to establish the optimized system of virus-induced gene silencing (VIGS) in pepper;to identify the function of CaRGA1and CaPOD genes in pepper through VIGS combinedwith transgenitic technology. Based on these results, pepper transcriptome was sequenced byusing the second generation of Illumina/Solexa high-throughput transcriptome sequencingtechnique, and to provide a theoretical basis for pepper disease-resistance breeding in thefuture. The main results of the study are as follows:1. The defense mechanism in the pepper plant and the different compatible interactionwith P. capsici was studied. The main conclusion: the defense-related enzymes (β-1,3-glucanase and peroxidase) activities were assayed and the expression pattern of thedefense-related genes (CABPR1, CABGLU and CAPO1) was analyzed. The results showedthat the defense-selated enzymes activities of pepper plants in the compatible and theincompatible interaction were different, and the change trend of defense-related enzymeactivities is not consistent. Real-time quantitative PCR results indicated that the expressionpattern of defense-related genes in the compatible interaction were different from the other.However, theexperession level of these genes in leaves was higher than that in roots, and alsothe expression level in the incompatible interaction was higher than that in compatiblecombination.2. The optimized system of virus induced gene silencing (VIGS) in pepper wasestablished. Based on the results of previous studies, the CaPDS gene was used as positivereport gene, construsting virus silencing vector pTRV2-CaPDS, to test the main factorsaffecting the efficiency of VIGS. The different varieties (A5, A3and EC), the different plant ages (two-, four-, six-and eight-true leaf stage), the OD600value of Agrobacteriumconcentration (0.5,0.8,1.0,2.0and3.0) and the culture temperature (18,20,22,25and28℃)were optimized. The establishment of an optimized system of VIGS for pepper might providetechnical support for high-throughput functional analysis of genes in pepper in the future.3. The expression patterns of genes CaRGA1and CaPOD induced by several biotic andabiotic stresses was analyzed. The results showed that the CaRGA1and CaPOD genes wereinduced at different expression levels by the P. capsici infection. Under the abiotic stresstreatment, the expression of gene CaRGA1was induced by high salt, drought and methyljasmonate (MeJA), but not significantly induced by salicylic acid (SA) stress, whichindicating that CaRGA1gene may be involved in the MeJA signal transduction pathway. TheCaPOD gene was involved in the resistance to salt and drought stress, and might be involvedin SA and MeJA mediated signal transduction pathway.4. The function of genes CaRGA1and CaPOD were identified by using VIGStechnology. The pepper plants with silenced CaRGA1and CaPOD genes were created. Therewere no clear phenotypic differences between the silenced plants and the negative controlplants. Real-time quantitative RT-PCR results showed that the gene expression leveldecreased up to some degree in silenced plants. The disease resistance of CaRGA1andCaPOD were detected through the detached-leaf method in the silenced plants. At the thirdday after inoculation, the necrotic lesion occurred in the leaves of CaPOD-silenced plants.But the necrotic lesion in the leaves of CaRGA1-silenced plants occurred at the fifth day, laterthan that in the leaves of CaPOD-silenced plants. So it was concluded that the CaRGA1andCaPOD gene silenced have decreased the pepper resistance to phytophthora blight.5. The plant over-expression vector of CaRGA1gene was constructed and transformedthrough the Agrobacterium-mediated genetic transformation method, of4transgenic pepperplants were obtained. Compared to the negative control, the CaRGA1transgenic 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 transgenic leaves than that on the leaves ofcontrol plants. However, the defense-related enzymes (β-1,3-glucanase and peroxidase)activities in transgenic plants were significantly higher than the control plants. These resultssuggested that the CaRGA1gene was involved in the pepper resistance response to the P.capsici infection. In addition, the salt tolerance was analyzed by using the leaf discs methodin the transgenic and the control pepper plants. It was showed that the leaf discs from controlplants turned yellow, some with chlorosis phenomenon, some even a water-soaked, while theleaf discs from transgenic plants were still green. The chlorophyll content of transgenic plants was higher than that of controls, which indicating that CaRGA1gene may be related with thepepper salt tolerance reaction.6. The over-expression vector of CaPOD gene was constructed, and transformed to thepepper B12by Agrobacterium-mediated genetic transformation method. We have obtained6CaPOD over-expressed transgenic plants. There was no phenotypic difference between thetransgenic plants and the negative controls. The results of detached leaves resistanceidentification showed that the CaPOD transgenic plants increased resistance to P. capsiciinfection and improved the salt tolerance. In conclusion, the above results indicated that theCaPOD gene might not only play positive role in regulating the pepper plant resistanceresponse to P. capsici, but also participated in response to high salt stress.7. The compatible and the incompatible transcriptome databases were sequenced by thesecond generation of Illumina/Solexa high-throughput sequencing technology. A total of101,641Unigenes was acquired after transcriptome sequencing, among of which were50,795and50,846Unigenes in HX-9and PC transcriptome database, respectively. Functionalannotation of the obtained Unigenes,33,365Unigenes were annotated to the NR database,34,658were annotated to the NT database,20,747compared to the Swiss-Prot database,18,654,11,698and27,087Unigenes were annotated to the KEGG metabolic pathways, COGand GO classification, respectively. There were8,144differentially expressed genes in thecompatible and incompatible transcriptome,6,497genes were up-regulated expression in theHX-9transcriptome, and1,647genes were up-regulation expressed in the PC transcriptome.Six differentially expressed genes from the compatible and incompatible transcriptomedatabases were selected and their expression patterns in response to P. capsici inoculationwere analyzed using the semi-quantitative PCR method. According to the results from PCRand sequencing results, it was indicted that the pepper transcriptome database about thepepper plant and P. capsici with different compatible interactions is confirmed, and whichmight support the further study of genes related to phytophthora blight resistance in pepperplant.更多还原