- 學位論文
蝴蝶蘭抗細菌性軟腐病基因轉殖之研究
Study on Resistance of Bacterial Soft Rot in Transgenic Phalaenopsis
摘要
蝴蝶蘭 (Phalaenopsis spp.) 為台灣重要出口花卉，因栽培之高溫高濕環境易造成細菌性軟腐病 (bacterial soft rot)，細菌性軟腐病主要是由Erwinia spp. 所引起的病害，感染植物體的同時分泌細胞壁分解酵素，因此植物感病後葉片呈水浸狀。本研究之目的係將 Erwinia chrysanthemi 之果膠分解酶基因 pel (pectate lyase) E 及 pelZ，以農桿菌媒介轉殖法轉入蝴蝶蘭細胞，期望果膠分解酶將蝴蝶蘭細胞壁中的果膠分解成為寡糖 (oligosaccharides) 分子，誘導植株產生抗病反應，進而達到抗病之目的。經聚合酶連鎖反應 (polymerase chain reaction, PCR) 及南方氏雜交分析(Southern blot analysis) 檢測，得到35Spro:pelE 蝴蝶蘭轉殖株及35Spro:pelZ 蝴蝶蘭擬原球體，進一步以酵素聯結免疫吸附分析 (ELISA) 及西方轉漬分析 (western analysis)，確定 pel 基因在蝴蝶蘭細胞中確實進行果膠分解酶之轉譯。薄層色層分析法顯示35Spro:pelE 蝴蝶蘭轉殖株中具有果膠分解酶之活性，另外經Sulfamate / m-hydroxy diphenyl 法檢測，證實轉殖株葉片中亦具有果膠之分解產物。轉殖株與非轉殖株相比，葉片及氣生根有形態上之差異。利用掃描式電子顯微鏡分析葉面，轉殖株上下表皮之氣孔型態皆不正常。以流式細胞儀分析植物基因組，轉殖株具有較小片段之基因組，可能進行程序性細胞死亡 (programmed cell death, PCD)。35Spro:pelE 轉殖株馴化較未轉殖株困難，應與 CaMV 35S (cauliflower mosaic virus 35S) 啟動子大量持續果膠分解酶基因，大量的果膠造成轉殖株型態變異使馴化困難。針對 CaMV 35S 啟動子持續表現果膠分解酶基因，造成植株形態變異及馴化困難之現象，本研究已進一步使用誘導型的啟動子 PR1a (pathogenesis-related gene-1a) 來改善，理論上轉殖株只有在病原菌侵入，植物體本身產生水楊酸 (salicylic acid) 或是外施水楊酸誘導下，才會表現果膠分解酶基因。
並列摘要
The Phalaenopsis orchids are important export flowers in Taiwan. They are easily infected by bacterial soft rot under high temperature and high humidity during cultivation. The pathogen secrets plant cell wall degrading enzymes and causes plant tissue maceration and extensive cell death. The purpose of this study is to enhance disease resistance against soft rot disease. The pectate lyase genes pelE and pelZ isolated from Erwinia chrysanthemi were transformed into calli of Phalaenopsis using Agrobacterium-mediated transformation. In the 35Spro:pelE transgenic plants, the pectate lyase degrades the pectin in the cell wall into oligosaccharides. The oligosaccharide acts as signal molecules that lead to the disease-resistant response. Transgenic lines were confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Translation products of transgenes were dectected in transgenic plants by western bloting analysis and enzyme-linked immunosorbent assay (ELISA). The activity of pectate lyase enzyme in transgenic plant was confirmed by thin layer chromatography and the product in transgenic cell was confirmed by Sulfamate/m-hydroxy diphenyl assay. Compared with untransformed plant the leaf and aerial root of the 35Spro:pelE transgenic plants are peculiar. As revealed by the scanning electron microscope (SEM) analysis, the stomata on both upper and lower epidermis of leaves in transgenic Phalaenopsis are abnormal. Flow cytometric detection of DNA fragmentation indicate that programmed cell death (PCD) might happen in the cells of 35Spro:pelE transgenic plants. The continous expression of pelE driven by the CaMV 35S promoter may cause the transgenic plant abnormal. The inducible PR1a (pathogenesis-related gene-1a) promoter was hence used for plant transformation to solve this problem. The PR1a promoter can be induced under salicylic acid and pathogen infection.