蝴蝶蘭抗細菌性軟腐病基因轉殖之研究

Abstract

為提升蝴蝶蘭植株抗細菌性軟腐病( soft rot )能力。本研究利用農桿菌媒介法，以蝴蝶蘭癒合組織為材料，分別進行Erwinia chrysanthemi的果膠分解酶( pectate lyase )基因pelE和pelZ之轉殖，並以菸草材料進行轉殖基因表達及抗病測試。pelE的轉殖質體具有nptII篩選基因，故以添加G418的培養基進行轉殖後之篩選。經檢測確定含pelE之植物材料，進一步轉殖含有hpt、gus和pelZ的質體，並以hygromycin進行篩選，即進行雙重轉殖。將篩選所得的擬轉殖蝴蝶蘭癒傷組織和菸草葉圓片，進行細胞分化培養成苗，轉殖株已經由GUS活性分析、聚合酶連鎖反應和南方氏雜交分析確認。並利用西方轉漬法和酵素聯結免疫吸附法偵測基因表現，可測到預期的43和49 kDa之表達蛋白。經過氧化氫染色和病原菌接種之抗病分析，顯示菸草轉殖株對病原菌產生過敏性反應。

The purpose of this study is to enhance the resistance of orchids to bacterial soft rot. Pectate lyase genes ( pelE and pelZ ) isolated from Erwinia chrysanthemi were transformed into calli of Phalaenopsis and leaves of tobacco by Agrobacterium-mediated transformation. Transformed calli were selected by medium containing G418 due to nptII selectable marker gene in pelE construct. PelE transgenic callus lines confirmed by molecular analysis were further transformed with pelZ construct containing hpt and gus genes. Therefore, survival transformed callus were double transformed after selection by hygromycin. After callus was selected with antibiotics and regenerated, transgenic lines were confirmed by β-glucuronidase ( GUS ) activity, polymerase chain reaction ( PCR ), and Southern blot analysis. Expression products of transgenes were detected 43 and 49 kD by Western blotting analysis and enzyme-linked immunosorbent assay ( ELISA ). Hydrogen peroxide staining and pathogen inoculation analysis assay revealed that transgenic lines exhibited hypersensitive response ( HR ) to phytopathogens.