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研究生:連唯菁
研究生(外文):Wei-Ching Lien
論文名稱:鳳梨再生系統與ACC氧化酶反義基因轉殖系統之研究
論文名稱(外文):Regeneration and transformation of pineapple with an antisense ACC oxidase gene
指導教授:林金和林金和引用關係
指導教授(外文):Chin-Ho Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:37
中文關鍵詞:鳳梨台農17 號開英種器官發生再生轉殖農桿菌基因槍ACC 氧化酶
外文關鍵詞:Pineapple (Ananas comosus L. Merr.)Tainon 17Smooth CayenneOrganogenesisRegenerationTransformationAgrobacterium tumefaciensBombardmentACC oxidase
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本研究以〝台農17號〞鳳梨(Ananas comosus (L.) Merr. cv. Tainon 17)及〝開英種〞鳳梨(Ananas comosus (L.) Merr. cv. Smooth Cayenne)為材料,以其葉片基部之芽體作為培植體,建立其器官發生(organogenesis)之再生及基因轉殖系統。鳳梨葉片芽體所生成之葉片培養於MSB2D0.5培養基(MS培養基內含30 g/l蔗糖、2.0 mg/l之BAP及0.5 mg/l之2,4-D),經2個月後可獲得再生植株,〝開英種〞及〝台農17號〞鳳梨之葉片培養再生率分別為25.8及32.1 %。
本研究以RT-PCR方法,自受傷鳳梨葉片組織選殖出ACC氧化酶基因片段,並將其分別以正義或反義方向構築到ubi啟動子(maize ubiquitin promoter)及nos終結子(nopaline synthase terminator)之間,並插入到pCAMBIA1301及pCAMBIA3301載體上形成四個攜帶正義或反義ACC氧化酶基因之植物轉殖載體。
以農桿菌(Agrobacterium)或基因槍(gene gun)法將構築之轉殖載體轉移到〝台農17號〞鳳梨之葉片。培養於添加5.0 mg/l hygromycin或0.5 mg/l phosphinothricin(PPT)之MSB2D0.5培養基篩選成活之轉殖培植體,經4個月可獲得再生植株。以聚合酶鏈鎖反應(polymerase chain reaction, PCR)及GUS染色分析檢測農桿菌感染之擬轉殖再生植株,均無法檢測到轉殖之基因或GUS藍色反應;分析基因槍法轉殖後之擬轉殖再生植株,則可偵測到GUS藍色反應的表現,以抗生素hygromycin篩選之成活擬轉殖再生植株(pCAMBIA1301系列)基因轉殖率為0.3 %,以PPT篩選之成活擬轉殖植株(pCAMBIA3301系列)的基因轉殖率為1.1 %。本研究已成功建立鳳梨經由器官發生再生及基因轉殖系統。
We describe here, an efficient system of pineapple (Ananas comosus L. Merr. cv. Tainon 17 and Smooth Cayenne) regeneration and transformation through the process of direct organogenesis by leaf buds as explants. The cultivar〝Tainon 17〞and〝Smooth Cayenne〞used in this study show organogenesis response from leaf base derived explants. The cultivar 〝Smooth Cayenne〞showed 25.8 % and〝Tainon 17〞showed 32.1 % of shoots formation from leaf bases after 2 months of culturing on MSB2D0.5 medium supplemented with 30 g/l sucrose, 2.0 mg/l Benzlaminopurine (BAP) and 0.5 mg/l of 2,4-dichlorophenoxy acetic acid (2,4-D). In this research, because the higher regeneration rate in〝Tainon 17〞, we use leaf bases from〝Tainon 17〞as explants for transformation by Agrobacterium tumefaciens or bombardment with antisense ACC oxidase gene between ubi (maize ubiquitin) promoter and nos (nopaline synthase) terminator on pCAMBIA1301 or pCAMBIA3301 vectors. Leaf bases were developed one month after culturing on MSB2D0.5 medium containing 5.0 mg/l hygromycin or 0.5 mg/l phosphinothricin (PPT) to select transgenic plant. Transformation has been confirmed by GUS assay, and polymerase chain reaction (PCR). After culturing for 4 months, there is no transgenic plant through Agrobacterium-mediated transformation. Transformation rate by bombardment selected by hygromycin showed 0.3 % and which selected by PPT showed 1.1 %. The pineapple plants via organogenesis in this study were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration and transformation system of pineapple through organogenesis are discussed.
中文摘要-------------------------------------------------------------------------------i
英文摘要------------------------------------------------------------------------------ii
目錄-----------------------------------------------------------------------------------iii
圖表目錄------------------------------------------------------------------------------v

前言------------------------------------------------------------------------------------1
前人研究------------------------------------------------------------------------------3
ㄧ、鳳梨------------------------------------------------------------------------------3
二、促進果實後熟之基因---------------------------------------------------------4
三、植物組織培養再生系統之建立---------------------------------------------5
四、農桿菌轉殖技術---------------------------------------------------------------6
五、基因槍轉殖技術---------------------------------------------------------------8
六、結語----------------------------------------------------------------------------10
材料與方法--------------------------------------------------------------------------11
ㄧ、實驗材料----------------------------------------------------------------------11
(一) 轉殖材料------------------------------------------------------------------11
(二) 試驗藥品------------------------------------------------------------------11
(三) 載體及轉殖之媒介------------------------------------------------------11
(四) 培養基---------------------------------------------------------------------11
(五) 植物荷爾蒙之配製------------------------------------------------------12
(六) 抗生素母液之配製------------------------------------------------------13
(七) 其他緩衝液及試劑------------------------------------------------------13
二、實驗方法----------------------------------------------------------------------14
(一) 鳳梨經器官發生(organogenesis)再生系統之建立----------------14
(二) ACC氧化酶(acco)基因之選殖---------------------------------------15
(三) 轉殖基因與載體之組合------------------------------------------------19
(四) 轉殖媒介之製備---------------------------------------------------------19
(五) 鳳梨基因轉殖------------------------------------------------------------20
(六) 擬轉基因植株系之分子分析------------------------------------------22
結果與討論--------------------------------------------------------------------------24
ㄧ、鳳梨經器官發生(organogenesis)再生系統之建立--------------------24
(一) 田間鳳梨經無菌培養後植株之增生---------------------------------24
(二) 鳳梨植株之發根及馴化------------------------------------------------25
(三) 無菌培養鳳梨利用葉片基部再生植株之增生---------------------25
二、ACC氧化酶(acco)基因之選殖-------------------------------------------25
(一) ACC氧化酶基因之篩選-----------------------------------------------25
(二) 植物轉殖載體之構築---------------------------------------------------26
(三) 植物轉殖載體轉型至農桿菌------------------------------------------27
三、基因轉殖鳳梨之育成-------------------------------------------------------27
(一) 鳳梨對Hygromycin敏感度之測試-----------------------------------27
(二) 鳳梨對Phosphinothricin敏感度之測試------------------------------28
(三) 鳳梨轉殖ACC氧化酶基因--------------------------------------------28
(四) 擬轉基因鳳梨植株系之鑑定------------------------------------------29
結論-----------------------------------------------------------------------------------31
參考文獻-----------------------------------------------------------------------------32
表--------------------------------------------------------------------------------------38
圖--------------------------------------------------------------------------------------42
附錄-----------------------------------------------------------------------------------62


表目錄

表一、比較鳳梨〝台農17號〞及〝開英種〞再生系統中之再生率------------38
表二、鳳梨〝台農17號〞經農桿菌感染後,以hygromycin篩選之反義股ACC氧化酶基因轉殖鳳梨產生大量實生苗之轉殖率-----------39
表三、鳳梨〝台農17號〞經基因槍轉殖後,以hygromycin篩選之反義股ACC氧化酶基因轉殖鳳梨產生大量實生苗之轉殖率-----------40
表四、鳳梨〝台農17號〞經基因槍轉殖後,以PPT篩選之反義股ACC氧化酶基因轉殖鳳梨產生大量實生苗之轉殖率---------------------41

圖目錄

圖一、鳳梨〝台農17號〞莖頂分生組織作為培植體建立無菌再生系統---------------------------------------------------------------------------------42
圖二、鳳梨〝開英種〞葉片芽體作為培植體建立無菌再生系統------------43
圖三、鳳梨〝台農17號〞葉片基部為培植體建立器官發生再生系統------44
圖四、鳳梨〝台農17號〞葉片不同受傷時間處理後萃取出之總量RNA ---------------------------------------------------------------------------------45
圖五、鳳梨〝台農17號〞經RT-PCR方式生成之ACC氧化酶基因片段---------------------------------------------------------------------------------46
圖六、受傷處理鳳梨〝台農17號〞葉片之ACC氧化酶基因序列片段-----47
圖七、鳳梨轉殖載體pCAMBIA1301-s-acco及pCAMBIA1301-as-acco構築示意圖------------------------------------------------------------------48
圖八、鳳梨轉殖載體pCAMBIA3301-s-acco及pCAMBIA3301-as-acco構築示意圖------------------------------------------------------------------------50
圖九、構築於農桿菌Agrobacterium tumefaciens LBA4404之pCAMBIA 1301-s-acco及pCAMBIA1301-as-acco,ACC氧化酶基因經PCR分析結果------------------------------------------------------------------------51
圖十、構築於農桿菌Agrobacterium tumefaciens LBA4404之pCAMBIA 1301-s-acco及pCAMBIA1301-as-acco,標識基因hygromycin phosphor- transferase基因(hph)經PCR分析之結果------------------52
圖十一、構築於農桿菌Agrobacterium tumefaciens LBA4404之pCAMBIA 1301-s-acco及pCAMBIA1301-as-acco,標識基因gus經PCR分析之結果------------------------------------------------------------------------53
圖十二、鳳梨〝台農17號〞葉片對hygromycin敏感度測試結果-------------54
圖十三、鳳梨〝台農17號〞葉片對hygromycin敏感度測試之直條圖-----55
圖十四、鳳梨〝台農17號〞葉片對Phosphinothricin敏感度測試結果-------56
圖十五、鳳梨〝台農17號〞葉片對Phosphinothricin敏感度測試之直條圖---------------------------------------------------------------------------------57
圖十六、鳳梨〝台農17號〞農桿菌轉殖ACC氧化酶基因之轉殖再生系統---------------------------------------------------------------------------------58
圖十七、農桿菌感染反義股ACC氧化酶之鳳梨〝台農17號〞轉殖株,其gus基因PCR電泳圖--------------------------------------------------------59
圖十八、鳳梨〝台農17號〞基因槍轉殖ACC氧化酶基因之轉殖再生系統---------------------------------------------------------------------------------60
圖十九、鳳梨〝台農17號〞轉殖株葉片GUS染色結果------------------------61

附表目錄

附表一、本研究所使用之菌種及載體之來源---------------------------------62
附表二、農桿菌培養AB培養基之組成----------------------------------------63
附表三、植物組織培養MS培養基之組成-------------------------------------64
附表四、鳳梨組織培養再生系統所使用之培養基---------------------------65
附表五、鳳梨培植體經農桿菌轉殖所使用之培養基------------------------66
附表六、鳳梨培植體經基因槍轉殖所使用之培養基------------------------67
附表七、利用鳳梨葉片芽體經器官發生之再生系統流程表---------------68
附表八、利用鳳梨葉片基部經器官發生之再生系統流程表---------------69
附表九、聚合酶鏈鎖反應所使用之引子對及其核酸序列------------------70
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