綠色螢光蛋白與綠豆脂質運輸蛋白基因之結合表現於原生質體之分析

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研究生：	張楓瀅
論文名稱：	綠色螢光蛋白與綠豆脂質運輸蛋白基因之結合表現於原生質體之分析 Transient expression of the EGFP fused to mungbean VrLtp1 in protoplasts
指導教授：	林彩雲博士
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2005
畢業學年度：	93
語文別：	中文
論文頁數：	61
中文關鍵詞：	脂質運輸蛋白、綠色螢光蛋白、原生質體、PEG轉殖法
相關次數：	點閱：2 下載：0
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為了瞭解脂質運輸蛋白在細胞中的定位和傳送。我們使用本實驗之前從綠豆中分離的VrLtp1 cDNA複製VrspLtp1 DNA片段與綠色螢光蛋白結合，分別構築質體p35S□-VrspLTP1-EGFP-NOS，p35S□-EGFP-NOS，p35S□-Vrsp1-EGFP-NOS和p35S□-NOS。將以上四個質體DNA以PEG轉殖法分別送進菸草葉肉細胞原生質體與大豆懸浮細胞原生質體中，並分別以螢光顯微鏡觀察與螢光儀偵測。質體DNA p35S□-VrspLTP1-EGFP-NOS送進大豆懸浮細胞原生質體或菸草葉肉細胞原生質體，以藍光激發後，原生質體未呈現像p35S□-EGFP-NOS組的原生質體產生綠色螢光，而是呈現很亮的黃綠色，且細胞有變形的現象。接著利用螢光儀偵測對照組、與其他四個實驗組在大豆懸浮細胞原生質體其EGFP的表現量。p35S□-VrspLTP1-EGFP-NOS與p35S□-EGFP-NOS在大豆懸浮細胞原生質體的EGFP表現量相近，分別是對照組的3.2和3.4倍。實驗結果顯示p35S□-VrspLTP1-EGFP-NOS可以在原生質體表現，而將來可用以探討脂質運輸蛋白之定位和傳送。

The original objectives of this research were to examine the distribution of lipid transfer protein (LTP) and monitor the transport pathway of LTP in plants by means of fusion to enhanced green fluorescence protein (EGFP). This research was done using the mungbean VrLtp1 cDNA fused to EGFP. Plasmids p35S□-VrspLTP1-EGFP-NOS, p35S□-EGFP-NOS, p35S□-NOS and p35S□-Vrsp1-EGFP-NOS were constructed and transiently expressed in protoplasts prepared from tobacco mesophyll and soybean suspension cells. The transient expression was detected by fluorescence microscopy and measured using a multilabel counter fluorometer. The green light emission by the protoplasts transformed with p35S□-EGFP-NOS was detected under a fluorescent microscope. Interestingly, protoplasts transformed with p35S□-VrspLTP1-EGFP-NOS exhibited bright yellowish green fluorescence with deformed shape. Using the Multilabel Counter Victor3 to measure EGFP expression, the EGFP counts in protoplasts transformed with p35S□-VrspLTP1-EGFP-NOS and p35S□-EGFP-NOS were similar, about 3.2 and 3.4 fold of that of the control. Our results indicate that the p35S□-VrspLTP1-EGFP-NOS fusion protein was expressed in protoplasts and the fusion protein can be used for further study on localization of lipid transfer protein.

目    錄

摘要-------------------------------------------------------------------------------i
Abstract--------------------------------------------------------------------------ii
謝誌-----------------------------------------------------------------------------iii
縮寫-----------------------------------------------------------------------------iv
目錄-----------------------------------------------------------------------------v
圖目錄-------------------------------------------------------------------------vii

一、緒論-------------------------------------------------------------------------1
    1.1 脂質運輸蛋白-------------------------------------------------------1
    1.2綠色螢光蛋白基因與加強型綠色螢光蛋白--------------------4
二、材料與方法----------------------------------------------------------------7
2.1利用聚合酶連鎖反應製造VrLtp1 cDNA 片段----------------------7
   2.1.1模組DNA與設計之引子------------------------------------------7
   2.1.2 PCR擴增反應--------------------------------------------------7
2.2核酸接合反應------------------------------------------------------8
2.3 製備勝任細胞-------------------------------------------------------------9
2.4質體轉殖至細菌----------------------------------------------------------9
2.5小量製備質體DNA-----------------------------------------------------10
2.6雙股核酸自動序列定序------------------------------------------------11
2.7大量製備質體DNA-----------------------------------------------------12
2.8植物材料和生長條件---------------------------------------------------13
   2.8.1大豆懸浮細胞培養------------------------------------------------13
   2.8.2菸草培養------------------------------------------------------------14
2.9利用PEG轉殖法將質體DNA送進原生質體中------------------14
   2.9.1大豆懸浮細胞原生質體分離------------------------------------14
   2.9.2菸草葉肉細胞原生質體分離------------------------------------16
   2.9.3菸草葉肉細胞與大豆懸浮細胞原生質體之PEG轉殖法--17
2.10螢光顯微鏡和螢光儀分析--------------------------------------------18
三、結果------------------------------------------------------------------------20
3.1質體DNA的構築--------------------------------------------------------20
   3.1.1 p35S□-VrspLTP1-EGFP-NOS----------------------------------20
3.1.2 p35S□-EGFP-NOS-----------------------------------------------21
3.1.3 p35S□-Vrsp1-EGFP-NOS---------------------------------------22
3.1.4 p35S□-NOS--------------------------------------------------------24
3.2質體DNA分別表現於植物細胞--------------------------------------24
四、討論------------------------------------------------------------------------28
五、參考文獻------------------------------------------------------------------30
六、附錄------------------------------------------------------------------------61





圖  目  錄

圖一、引子序列設計相關資料---------------------------------------------36
圖二、質體 pVrspLTP1的構築--------------------------------------------37
圖三、質體pVrspLTP1-EGFP利用限制酵素分析正確性-------------38
圖四、質體pVrspLTP1-EGFP的構築與確定----------------------------39
圖五、質體p35S□-VrspLTP1-EGFP-NOS的構築與確定-------------41
圖六、質體p35S□-EGFP-NOS的構築與確定--------------------------44
圖七、質體pVrsp1的構築與確定------------------------------------------46
圖八、質體 pVrsp1-EGFP的構築與確定--------------------------------48
圖九、質體p35S□-Vrsp1-EGFP-NOS的構築與確定------------------50
圖十、質體p35S□- NOS的構築與確定----------------------------------52
圖十一、原生質體的照片 -------------------------------------------------54
圖十二、菸草葉肉細胞原生質體------------------------------------------55
圖十三、大豆懸浮細胞原生質體------------------------------------------56
圖十四、不同濃度的DNA對EGFP表現之影響------------------------59
圖十五、不同質體DNA表現於原生質體內之EGFP的表現量------60

                                

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