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研究生:賓秋雅
論文名稱:番椒與茄科疫病菌親合性與不親合性病程相關基因之表現差異
論文名稱(外文):The difference of pathogenesis-related gene expression in compatible and incompatible interactions of pepper and Phytophthora capsici leinian
指導教授:陳隆鐘
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:57
中文關鍵詞:茄科疫病菌Phytophthora capsici Leonian.病程相關基因pathogenesis-related genePR genePR-1PR-2β-13-葡聚糖酶β-13-glucanasePR-3幾丁質分解酶chitinase
外文關鍵詞:pathogenesis-related proteins ( PR proteins )Phytophthora capsici LeonianPR-1PR-2 ( β-13-glucanase )PR-3 ( chitinase )
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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
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