| 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. |
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