在植物抵抗病原菌的攻擊中最常被研究的防禦機制是系統性後天抗性（systemic acquired resistance, SAR），而與SAR的表現又常以病程相關蛋白質（pathogensis-related proteins, PR proteins）的表現作為指標。為了解番椒抵抗茄科疫病菌（Phytophthora capsici Leonian）的抗性機制，先利用亞洲蔬菜研究發展中心提供的茄科疫病菌（Pc17E）和番椒之PI201234 與 C02227抗病品系，以及Early Calwonder感病品種進行初步觀察，再進行抗性機制的分析。經接種四種鑑別寄主（PI201234, PBC602, PBC137及Early Calwonder）後所暫時區分之茄科疫病菌三種生理小種菌株（Race 1, Race 2及Race 3），在培養基上之菌落形態與隨機增幅多型性核酸（random amplified polymorphic DNA, RAPD）反應結果，可明顯看出Pc33E與Pc17E菌株較相近，而Pc1E與兩者差異較大。而對茄科疫病菌分別具抗病或感病之三種番椒品系PI201234、C02227及Early Calwonder，由外觀可看出三種植株之性狀具有明顯差異，且經由接種測試結果顯示PI201234及C02227對茄科疫病菌Pc17E確實具有極高度的抗性，而Early Calwonder 則在接種後表現出明顯的感病性，顯示這三種番椒品系有許多差異存在；但在RAPD反應結果中，這三者的差異不大。以前人發表的病程相關基因（pathogenesis-related protein gene, PR gene）序列設計出專一引子對，再利用反轉錄聚合酶連鎖反應，增幅出專一性的核酸片段，再以TOPO TA Cloning®做為選殖系統，得到PR-1、PR-2（β-1,3-葡聚糖酶，β-1,3-glucanase）和PR-3（幾丁質分解酶，chitinase）基因的部分序列。其中，PR-1與Capsicum annuum的PR-1 precursor mRNA (accession no. AF053343）相似度（similarity）為100％、與Capsicum annuum的PR-1 precursor mRNA（accession no. AF348141）相似度為96％、與Solanum torvum的PR-1 mRNA（accession no. AB062566）相似度為88％。PR-2與Nicotiana plumbaginifolia的β-glucanase precursor exon 2（accession no. M23120）相似度為92％、與Nicotiana plumbaginifolia的β-glucanase（gn1 gene）（accession no. X07280）相似度為92％、與Capsicum annuum的β-1,3-glucanase mRNA（accession no. AF227953）相似度為96％。PR-3與Capsicum annuum的class II chitinase ( CAChi2 ) mRNA（accession no. AF091235）相似度為99％、與Lycopersicon chilense的endochitinase mRNA（accession no. L19342）相似度為87％、與Solanum tuberosum的class II chitinase ( ChtA4 ) mRNA（accession no. AF024538）相似度為87％。以這些序列做為探針，再經由親和性反應（compatible interaction）和不親和性反應（incompatible interaction）接種試驗後，以北方墨點分析法（Northern blot analysis）偵測各病程相關基因於接種後之植株體內的表現。實驗結果顯示，在不親合性反應植株的莖部中，PR-1、β-1,3-葡聚糖酶和幾丁質分解酶基因在接種6小時後就有表現；而在親合性反應植株的莖中，PR-1、β-1,3-葡聚糖酶和幾丁質分解酶的基因在接種後12小時才有表現。綜合以上結果，得知番椒確實會因疫病菌的侵害而誘導其病程相關基因的表現，且與番椒植株對茄科疫病菌的抗性有所關聯。

In the disease resistance, the most investigated plant defense mechanism is systemic acquired resistance ( SAR ). SAR is associated with the activation of a large number of genes encoding various types of stress proteins, including pathogenesis-related proteins ( PR proteins ). Two resistant line of peppers, PI201234 and C02227, and one susceptible pepper variety, Early Calwonder, provided by the Asian Vegetable Research and Development Center ( AVRDC ) were inoculated with a highly virulent isolate of Phytophthora capsici Leonian, Pc17E ( Race 3 ), to investigate the difference of the expression of PR genes in pepper. According to the disease reactions with four differential host ( PI201234, PBC602, PBC137 and Early Calwonder ), all P. capsici isolates collected in Taiwan were temporarily classified into three races ( Race 1, Race 2 and Race 3 ) by AVRDC. Colony morphology and the patterns of random amplified polymorphic DNA ( RAPD ) products from P. capsici races Pc33E and Pc17E were similar. There were significant difference of the phenotype among three distinct pepper genotypes, PI201234, C02227, and Early Calwonder. The inoculation test showed that PI201234 and C02227 were highly resistant to Pc17E, while Early Calwonder was susceptible. Although there were many differences among these pepper genotypes, no divergence was found on the RAPD analysis. The specific primers for reverse-transcript polymerase chain reaction ( RT-PCR ) to obtain partial PR gene sequences were designed according to the sequence published in the GenBank. The RT-PCR products amplified with the specific primers were cloned in TOPO TA Cloning® Kit. The obtained PR-1 fragment shares 100 % similarity with Capsicum annuum ( accession no. AF053343 ), 96 % similarity with Capsicum annuum ( accession no. AF348141 ), 88 % similarity with Solanum torvum ( accession no. AB062566 ). The obtained PR-2 fragment shares 92 % similarity with Nicotiana plumbaginifolia ( accession no. M23120 ), 92 % similarity with Nicotiana plumbaginifolia ( accession no. X07280 ), 96 % similarity with Capsicum annuum ( accession no. AF227953 ). The obtained PR-3 fragment shares 99 % similarity with Capsicum annuum ( accession no. AF091235 ), 87 % similarity with Lycopersicon chilense ( accession no. L19342 ), 87 % similarity with Solanum tuberosum ( accession no. AF024538 ). The mRNA accumulation of PR-1, β-1,3-glucanase and chitinase genes in the stems of PI201234 and C02227 during the incompatible interaction were detected by Northern blot assay at 6 hours after inoculation. In the compatible interaction, the accumulation of PR-1, β-1,3-glucanase and chitinase genes in the stems of Early Calwonder were detected at 12 hours after inoculation. The results indicated that the induction of PR genes may related to the resistance of peppers to P. capsici.

目錄
壹、中文摘要………………………………………………………………………………i
貳、英文摘要……………………………………………………………………………iii
參、前言………………………………………………………………………………1
肆、材料與方法………………………………………………………………………………6
一、病原菌之培養保存、供試植物之栽種與接種…………………………7
二、以隨機增幅多型性核酸（random amplified polymorphic DNA, RAPD）分析茄科疫病菌三種生理小種菌株之差異…………………………8
茄科疫病菌之總去氧核糖核酸（deoxyribonucleic acid, DNA）的萃取………………………………………………………………………………8
以隨機增幅多型性核酸分析茄科疫病菌三種生理小種菌株之差異……10
三、利用隨機增幅多型性核酸（RAPD）分析三種番椒品系或品種之差異……………………………………………………………………………10
植物之去氧核糖核酸萃取…………………………………………………10
以隨機增幅多型性核酸分析三種番椒品系或品種之差異……………………………………………………………………………11
四、植物總核醣核酸（total ribonucleic acid, total RNA）萃取……………………………………………………………………………12
五、利用反轉錄聚合酶連鎖反應（reverse-transcript polymerase chain reaction, RT-PCR）對番椒的病程相關基因進行核酸片段之篩選……………………………………………………………………………12
六、以北方墨點分析法（Northern blot analysis）偵測病程相關基因於接種之植株莖部的表現………………………………………………………………15
伍、結果……………………………………………………………………………17
一、茄科疫病菌（Phytophthora capsici）三種生理小種之培養型態及基本型態比較………………………………………………………………17
二、三種番椒品系或品種接種茄科疫病菌Pc17E後之抗、感病病徵反應差異……………………………………………………………………………18
三、茄科疫病菌三種生理小種菌株隨機增幅多型性核酸分析之結果……………………………………………………………………………18
四、三種番椒品系或品種之生育特性以及隨機增幅多型性核酸分析結果……………………………………………………………………………19
五、利用反轉錄聚合酶連鎖反應（reverse-transcript polymerase chain reaction, RT-PCR）對番椒的病程相關基因進行核酸片段之篩選……………………………………………………………………………19
六、以北方墨點分析法（Northern blot analysis）偵測病程相關基因於接種之植株莖部的表現……………………………………………………………………………21
陸、討論……………………………………………………………………………22
柒、參考文獻……………………………………………………………………………26
捌、圖表說明……………………………………………………………………………33
玖、附表一……………………………………………………………………………54
拾、附表二……………………………………………………………………………55
拾壹、附圖一……………………………………………………………………………56
拾貳、附錄……………………………………………………………………………57

王添成。1999。蔬菜抗病篩選技術。91-106頁。蔬菜育種技術研習專刊。台灣省農業試驗所編印。
高清文、呂理燊。1977。台灣未發表的三種疫病病菌。植保會刊19：302-303。
呂理燊、高清文。1981。Phytophthora capsici引起的甜椒及辣椒之疫病。植保會刊23：59-66。
蕭吉雄、楊偉正、王三太。1999。蔬菜抗病育種。33-69頁。蔬菜育種技術研習會專刊。台灣省農業試驗所編印。
黃靜芳。2002。番椒與茄科疫病菌親合性與不親合性反應之特性分析。碩士論文。國立中興大學植物病理學研究所。台中，台灣。54頁。
Alexander, D., Goodman, R. M., Gut-Rella, M., Glascock, C., Weymann, K., Friedrich, L., Maddox, D., Ahl-Goy, P., Luntz, T., Ward, E., and Ryals, J. 1993. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc. Natl. Acad. Sci. U.S.A. 90: 7327-7331.
Alexopoulos, C. J., Mims, C. W., and Blackwell, M. 1996. Phylum Oomycota. Pages 683-737. in: Introductory mycology. John Wiley & Sons, Inc. U.S.A.
Agrios, G. N. 1997. How plants defend themselves against pathogens Pages 93-114. In: Plant Pathology. 4th ed. Academic Press. San Diego, Calfornia. U.S.A.
Barksdale, T. H., Papavizas, G. C., and Johnston, S. A. 1984. Resistance to foliar blight and crown rot of pepper caused by Phytophthora capsici. Plant Dis. 68: 506-509.
Bartnicki-Garcia, S. 1968. Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu. Rev. Microbio. 22: 87-108.
Birnboim, H. C. and Doly, J. 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7: 1513-1523.
Black, L. L. 1998. Studies on Phytophthora blight in pepper. Pages 25-27 In: AVRDC Report 1998. Asian Vegetable Research and Development Center. Shanhua, Tainan, Taiwan, R. O. C.
Black, L. L. and Berke, T. 1998. Breeding for Phytophthora resistance in pepper. Pages 115-119. In: Synopsis of the Xth Meeting on Genetics and Breeding of Capsicum and Eggplant of EUCARPIA, Avignon, France. Sept. 7-11.
Buchter, R., Stromberg, A., Schmelzer, E., and Kombrink, E. 1997 Primary structure and expression of acidic (class II) chitinase in potato. Plant Mol. Biol. 35 (6): 749-761.
Chen, R. D., Yu, L. X., Greer, A. F., Cheriti, H., and Tabaeizadeh, Z. 1994. Isolation of an osmotic stress- and abscisic acid-induced gene. Mol. Gen. Genet. 245 (2): 195-202.
Chomczynski, P. and Sacchi, N. 1987. Single-step methods of RNA isolation by acid guanidinium thiocyanate-phenol chloroform extraction. Anal. Biochem. 162: 156—159.
De Wit, P. J. G. M. 1997. Pathogen avriulence and plant resistance: a key role for recognition. Trends Plant Sci. 2: 452-458.
de Loose, M., Alliotte, T., Ghenysen, G., Genetello, C., Gielen, J., Soetaert, P., van Montagu, M., and Inze, D. 1988. Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia. Gene 70 (1): 13-23.
Erwin, D. C. and Ribeiro, O. K. 1996. Phytophthora capsici. Pages 262-268. In: Phytophthora diseases worldwide. APS Press, Minnesota, U.S.A. 555 pp.
Eyal, Y., Sagee, O. and Fluhr, R. 1992. Dark-induced accumulation of a basic pathogenesis-related ( PR-1 ) transcript and a light requirement for its induction by ethylene. Plant Mol. Biol. 19 (4): 589-599.
Garber, R. C. and Yoder, O. C. 1983. Isolational of DNA from filamentous fungi and separation into nuclear, mitochondrial, ribosomal, and plasmid components. Anal. Biochem. 135: 416-422.
Ho, H. H., Ann, P. J., and Chang, H. S. 1995. Species descriptions. Pages 19-23. In: The genus Phytophthora in Taiwan. Institute of Botany, Academia Sinica, Taipei. 86 pp.
Hong, J. K., Jung, H. W., Kim, Y. J., and Hwang, B. K. 2000. Pepper gene encoding a basic class II chitinase is inducible by pathogen and ethephon. Plant Sci. 159: 39-49.
Ish-Horowicz, D. and Burke, J. F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9: 2989-2998.
Jean, B. R. and Stephen, A. J. 1999. Ecologically based approaches to management of Phytophthora blight on bell pepper. Plant Dis. 83: 1080-1089.
Jung, H. W. and Hwang, B. K. 2000. Isolation, partial sequencing, and expression of pathogensis-related cDNA genes from pepper leaves infected by Xanthomonas campestris pv. vesicatoria. Mol. Plant Microbe Interact. 13: 136-142.
Kim, Y. J. and Hwang, B. K. 1994. Differential accumulation of β-1, 3-glucanase and chitinase isoforms in pepper stems infected by compatible and incompatible isolates of Phytophthora capsici. Physiol. Mol. Plant Pathol. 45: 195-209.
Kim, Y. J. and Hwang, B. K. 2000. Pepper gene encodign a basic pathogenesis-related 1 protein is pathogen and ethylene inducible. Physiol. Plant. 108: 51-60.
Klessig, D. F. and Malamy, J. 1994. The salicylic acid signal in plants. Plant Mol. Biol. 26: 1439-1458.
Kombrink, E. 1997. Primary structure of potato acidic (class II) chitinase. Plant Mol. Biol. 35(6): 749-761.
Leonian, L. H. 1922. Stem and fruit blight of pepper caused by Phytophthora capsici species nov. Phytophthology 12: 401-408.
Lee, S. C., Hong, J. K., Kim,Y. J., and Hwang, B. K. 2000a. Pepper gene encoding thionin is differentially induced by pathogens, ethylene and methyl jasmonate. Physiol. Mol. Plant Pathol. 56: 207-216.
Lee, S. C., Hippe-Sanwald, S., Lee, Y. K., Hohenberg, H., and Hwang, B. K. 2000b. In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems with Phytophthora capsici. Protoplasma 211: 64-75.
Lee, S. C. and Hwang, B. K. 2003. Identification of the pepper SAR8.2 gene as a molecular marker for pathogen infection, abiotic elicitors and environmental stress in Capsicum annuum. Planta 216 (3): 387-396.
Liu, D., Raghothama, K. G., Hasegawa, P. M., and Bressan, R. A. 1994. Osmotin overexpression in potato delays development of disease symptoms. Proc. Natl. Acad. Sci. U.S.A . 91: 1888-1892.
Malamy, J. and Klessig, D. F. 1992. Salicylic acid and plant disease resistance. Plant J. 2: 643-654.
Mauch, F., Mauch-Mani, B., and Boller, T. 1988. Antifungal hydrolases in pea tissue II: inhibition of fungal growth by combinations of chitinase and β-1,3-glucanse. Plant Physiol. 88: 936-942.
Murray, M. G. and Thompson, W. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8: 4321-4325.
Orozco-Cardenas, M., Narvaez-Vasquez , J., and Ryan, C. 2001. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13: 179-91.
Parra, G. and Ristaino, J. 1998. Insensitivity to Ridomil Gold (mefenoxam) found among field isolated of Phytophthora capsici causing Phytophthora blight on bell pepper in North Carolina and New Jersey. Plant Dis. 82: 711.
Pieterse, C. M. J. and van Loon, L. C. 1999. Salicylic acid independent plant defence pathways. Trends Plant Sci. 4: 52-58.
Ryals, J. A., Neuenschwander, U. H., Willits, M. G., Molina, A., Steiner, H. Y., and Hunt, M. D. 1996. Systemic acquired resistance. Plant Cell 8: 1809-1819.
Sherf, A. F. and MacNab, A. A. 1986. Vegetable Diseases and Their Control. John Wiley & Sons. NY. U.S.A. 728 pp.
Schroder, M., Hahlbrock, K., and Kombrink, E. 1992. Temporal and spatial patterns of β-1,3-glucanase and chitinase induction in potato leaves infected by Phytophthora infestans. Plant J. 2: 161-172.
Sticher, L., Mauch-Mani, B., and Metraux, J. P. 1997. Systemic acquired resistance. Annu. Rev. Phytopathol. 35: 235-270.
van Loon, L. C. 1990. The nomenclature of pathogenesis-related proteins. Physiol. Mol. Plant Pathol. 37: 229-230.
van Loon, L. C. and van Kammen, A. 1970. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. “Samsun” and “Samsun NN”. II. Changes in protein constitution after infection with TMV. Virology 40: 199-211.
van Loon, L. C. and van Strien, E. A. 1999. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 55: 85-97.
van Loon, L. C., Pierpoint, W. S., Boller, Th., and Conejero, V. 1994. Recommendations for naming plant pathogenesis-related proteins. Plant Mol. Biol. Rep. 12: 245-264.
Vleeshouwers, V. G., van Dooijeweert, W., Govers, F., Kamoun, S., and Colon, L. T. 2000. The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans. Planta 210 (6): 853-64.
Ward, E. R., Uknes, S. J., Williams, S. C., Dincher, S. S., Wiederhold, D. L., Alexander, D. C., Ahl-Goy, P., Metraux, J. P., and Ryals, J. A. 1991. Coordinate gene activity in response to agents that induce systemic acquired resistance. Plant Cell 3: 1085-1094.
Woloshuk, C. P., Meulenhoff, J. S., Sela-Buurlage, M., van den Elzen, P. J. M., and Cornelissen, B. J. C. 1991. Pathogen-induced proteins with inhibitory activity toward Phytophthora capsici. Plant Cell 3: 619-628.
Yoon, C. S., Glawe, D. A., and Shaw, P. D. 1991. A method for rapid small scale preparation of fungal DNA. Mycologia 83: 835-838.