臺灣博碩士論文加值系統

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.

目錄目次
中文摘要----------------------------------------------------------------------------------1
英文摘要----------------------------------------------------------------------------------2
壹、前言----------------------------------------------------------------------------------3
貳、前人研究------------------------------------------------------------------------------5
一、軟腐病簡介----------------------------------------------------------------------------5
二、植物抗病反應--------------------------------------------------------------------------6
三、抗軟腐病的策略------------------------------------------------------------------------7
(一)、Ferredoxin-like protein基因轉殖---------------------------------------------------7
(二)、Sarcotoxin IA 基因轉殖------------------------------------------------------------8
(三)、Lysozyme基因轉殖------------------------------------------------------------------8
(四)、Cecropin MB39的基因轉殖-----------------------------------------------------------9
(五)、果膠分解酶 (Pectate lyases, pels) 基因轉殖----------------------------------------9
(六)、Quorum sensing (QS) signalling molecules基因轉殖----------------------------------10
四、果膠分解酶基因-pelE和pelZ基因---------------------------------------------------------10
五、蝴蝶蘭基因轉殖系統與抗生素篩選--------------------------------------------------------11
参、材料與方法----------------------------------------------------------------------------13
一、應用農桿菌轉殖法於菸草基因轉殖--------------------------------------------------------13
(一)、植物材料--------------------------------------------------------------------------13
(二)、農桿菌菌種------------------------------------------------------------------------13
(三)、轉殖流程--------------------------------------------------------------------------13
(四)、抗生素篩選與植株再生--------------------------------------------------------------15
二、應用農桿菌轉殖法於蝴蝶蘭基因轉殖------------------------------------------------------15
(一)、植物材料--------------------------------------------------------------------------15
(二)、農桿菌菌種------------------------------------------------------------------------16
(三)、轉殖流程--------------------------------------------------------------------------16
(四)、抗生素篩選與植株再生--------------------------------------------------------------16
三、擬轉殖植株分析------------------------------------------------------------------------17
(一)、GUS活性分析-----------------------------------------------------------------------17
(二)、植物基因組DNA之抽取---------------------------------------------------------------17
(三)、聚合酶連鎖反應--------------------------------------------------------------------18
(四)、探針製備--------------------------------------------------------------------------19
(五)、南方氏雜交分析--------------------------------------------------------------------19
(六)、植物蛋白質之抽取------------------------------------------------------------------20
(七)、蛋白質電泳分析--------------------------------------------------------------------20
(八)、西方轉漬法------------------------------------------------------------------------21
(九)、酵素聯結免疫吸附法----------------------------------------------------------------22
四、轉殖植株抗病分析----------------------------------------------------------------------22
(一)、菌種材料--------------------------------------------------------------------------22
(二)、抗病分析試驗----------------------------------------------------------------------23
(三)、過氧化氫染色定位分析--------------------------------------------------------------23
肆、結果----------------------------------------------------------------------------------24
ㄧ、應用農桿菌轉殖法於菸草基因轉殖--------------------------------------------------------24
(一)、轉殖pelE菸草之分析----------------------------------------------------------------24
(二)、轉殖pelE/pelZ菸草之分析-----------------------------------------------------------25
(三)、轉殖pelZ菸草之分析----------------------------------------------------------------26
二、應用農桿菌轉殖法於蝴蝶蘭基因轉殖------------------------------------------------------26
(一)、轉殖pelE蝴蝶蘭之分析--------------------------------------------------------------26
(二)、轉殖pelZ蝴蝶蘭之分析--------------------------------------------------------------27
(三)、轉殖pelE/pelZ蝴蝶蘭癒合組織之分析-------------------------------------------------27
三、轉殖植物抗軟腐病之分析----------------------------------------------------------------28
伍、討論----------------------------------------------------------------------------------56
ㄧ、基因轉殖效率--------------------------------------------------------------------------56
二、蛋白質表現----------------------------------------------------------------------------58
三、轉殖株的抗病分析----------------------------------------------------------------------59
四、未來研究方向--------------------------------------------------------------------------60
陸、参考文獻------------------------------------------------------------------------------61

圖表目次
表1、蘭花基因轉殖系統---------------------------------------------------------------------12
圖1、pBI121-PelE、pGpelZ 和 pGKZ3-8 質體構築。--------------------------------------------14
圖2、轉殖pelE ( pBI121-PelE ) 菸草T2植株之聚合酶連鎖反應分析。---------------------------29
圖3、轉殖pelE ( pBI121-PelE ) 菸草T2植株之南方氏雜交分析。--------------------------------30
圖4、轉殖pelE ( pBI121-PelE ) 菸草T2植株之西方轉漬分析和酵素聯結
免疫吸附分析。----------------------------------------------------------------------------31
圖5、轉殖pelZ ( pPGKZ3-8 ) 菸草T2植株之GUS活性分析。--------------------------------------32
圖6、轉殖pelZ ( pPGKZ3-8 ) 菸草T2植株之聚合酶連鎖反應分析。 --------------------------33
圖7、轉殖pelZ ( pPGKZ3-8 ) 菸草T2植株之南方氏雜交分析。 ----------------------------------34
圖8、轉殖pelZ ( pPGKZ3-8 ) 菸草T2植株之西方轉漬分析 和酵素聯結免疫吸附分析。------------------------------------------------------------------------35
圖9、轉殖pelZ ( pPGKZ3-8 ) 菸草 × 轉殖pelE ( pBI121-PelE ) 菸草之聚合酶連鎖反應分析。----------------------------------------------------------------36
圖10、轉殖pelZ ( pPGKZ3-8 ) 菸草 × 轉殖pelE ( pBI121-PelE ) 菸草之南方氏雜交分析。-------------------------------------------------------------37
圖11、轉殖pelZ ( pPGKZ3-8 ) 菸草 × 轉殖pelE ( pBI121-PelE ) 菸草之南方氏雜交分析。-------------------------------------------------------------38
圖12、轉殖pelE、pelZ 和 pelE/pelZ 菸草株之植株外觀。--------------------------------------39
圖13、轉殖pelE (pBI121-PelE ) 蝴蝶蘭之植株外觀。------------------------------------------40
圖14、轉殖pelE ( pBI121-PelE ) 蝴蝶蘭之南方氏雜交分析。-----------------------------------41
圖15、轉殖pelE ( pBI121-PelE ) 蝴蝶蘭之西方轉漬分析和酵素聯結免疫吸附分析。--------------------------------------------------------------------------42
圖16、蝴蝶蘭癒合組織對hygromycin濃度之抗性試驗。------------------------------------------43
圖17、轉殖pelE ( pBI121-PelE ) 蝴蝶蘭癒合組織再進行pelZ ( pPGKZ3-8 ) 轉殖於含hygromycin和 G418抗生素培養基上篩選情形。---------------------------44
圖18、轉殖pelZ ( pPGKZ3-8 ) 蝴蝶蘭癒合組織於含G418 抗生素培養基上篩選情形。--------------------------------------------------------------------------45
圖.19、轉殖pelZ (pGPelZ ) 蝴蝶蘭癒合組織於含G418 抗生素培養基上篩選情形。----------------------------------------------------------------------------46
圖20、轉殖pelZ ( pPGKZ3-8 ) 蝴蝶蘭癒合組織之GUS活性分析。---------------------------------47
圖21、轉殖pelZ ( pPGKZ3-8 ) 蝴蝶蘭癒合組織原球體和葉片之GUS活性分析。--------------------------------------------------------------------------------48
圖22、轉殖pelZ ( pPGKZ3-8 ) 蝴蝶蘭植株之南方氏雜交分析。----------------------------------49
圖23、轉殖pelZ ( pPGKZ3-8 ) 蝴蝶蘭植株之西方轉漬分析。------------------------------------50
圖24、轉殖pelE ( pPelE-1 ) 菸草葉片接種不同濃度Erwinia chrysanthemi PB1 ( Ech. PB1 ) 菌液之抗病分析。--------------------------------------------51
圖25、轉殖pelE ( pBI121-PelE ) 菸草葉片接種不同濃度Erwinia carotovora subsp. carotovora 1 ( Ecc. 1 ) 菌液之抗病分析。--------------------------------51
圖26、轉殖pelE ( pBI121-PelE ) 菸草葉片之過氧化氫染色定位分析-----------------------------52
圖27、轉殖pelZ ( pPGKZ3-8 ) 菸草葉片之過氧化氫染色定位分析。---------------------------53
圖28、轉殖pelZ ( pPGKZ3-8 ) 菸草 × 轉殖pelE ( pBI121-PelE ) 菸草葉片之過氧化氫染色定位分析。---------------------------------------------------54
圖29、轉殖pelE ( pBI121-PelE ) 蝴蝶蘭葉片接種Erwinia chrysanthemi PB1 (Ech. PB1)之抗病分析。---------------------------------------------------55

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