| 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. |
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