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研究生:張瑋齊
研究生(外文):Wei-Chi Chang
論文名稱:在大腸菌系統建構重組綠豆澱粉磷解?cDNA
論文名稱(外文):Construction of a mungbean (Vigna radiata L.) starch phosphorylase cDNA in E. coli system
指導教授:柯源悌
指導教授(外文):Yuan-Tih Ko
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:66
中文關鍵詞:綠豆澱粉磷解?大腸桿菌重組質體
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澱粉磷解? (starch phosphorylase,EC 2.4.1.1,SP) 普遍存在於植物體中,為植物體內參與澱粉代謝的重要酵素之ㄧ,其具有催化澱粉降解與合成的雙向活性。先前利用MALDI-TOF與免疫分析方法,完成綠豆 (mungbean, Vigna radiata L.) 105-kDa SP的鑑定並選殖出其全長之SP cDNA (命名為Vrsp),本研究目的是將Vrsp選殖到以大腸桿菌為宿主的表現載體,以期在大腸桿菌系統表現出具活性的rVrSP重組蛋白。先以生物資訊軟體 SWISS-Model 預測VrSP 蛋白質3D結構及特異性胺基酸序列,當全長序列 VrSP 輸入時,分別預測出以兔子肌肉glycogen phosphorylase (GP) 與potent inhibitor之共同結晶 (1WW2A)為模版,由第92個殘基開始的411個胺基酸之包含部份starch-binding site區域,及預測出以兔子肌肉的GP結晶 (2GJ4A) 為模版,由第585個殘基開始的399個胺基酸包含711-727 catalytic site 之結構。接著在VrSP兩端設計帶有EcoRI 與 SalI 切位的引子,利用PCR方式使其兩端帶有限制?切位,載入兩端經限制?處理的pMal-C2X載體中,並轉型至 NovaBlue 宿主細胞,篩選含全長 ORF 之序列;結果篩選出一個株系之重組質體其接入大小與預期相近,經定序發現接入序列卻與宿主同品系之大腸菌的一段序列相似度達 97%,而其餘2群重組質體只接入約2.2 kb與1.4 kb片段,推測是受Vrsp 本身序列影響,使其在重組質體複製時產生與大腸菌宿主序列置換的重組現象。
Starch phosphorylase ( SP, EC 2.4.1.1 ) is commonly found in plants and is one of the vital enzymes in the starch metabolic pathway. It catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Previous studies have identified the fragments of mungbean 105-kDa SP by MALDI-TOF, conducted immunological analysis and amplified the full-length cDNA fragment of mungbean (Vigna radiata L.) SP (named VrSP). The objective of this study was to clone the sequence into an expression vector and further express into a biological-active recombinant protein in E. coli system. Its 3-D structure and functional features were predicted in silico by SWISS-Model. One matched template, rabbit muscle phosphorylase with potent inhibitor (IWW2A), predicted the N-terminal of VrSP from 92 residue to 411 residue, including partial starch-binding site. The other template, rabbit muscle phosphorylase (2GJ4A), predicted the C-terminal of VrSP from 585 residue to 984 residue, including catalytic site (711-727). VrSP insert was prepared by PCR using primers designed with EcoRI and SalI sites in the flanking region, and was ligated into the parallel sites on pMal-C2X vector, followed by transforming into Novablue cloning host and screening. Insert size of one recombinant plasmid was nearly closed to the expected 2.9 Kb, however after sequencing, 97% of its sequence is matching to the DNA sequences in E. coli cloning host. In addition, the other two major groups of recombinant plasmid had 2.2 kb and 1.4 kb insert fragments in pMal-C2X vector. It showed that the sequence of VrSP itself may cause recombination with the host E. coli chromosomal DNA during the growth of these recombinant clones.
總 目 錄
圖目錄------------------------------------------------------------------------------------ Ⅳ
表目錄------------------------------------------------------------------------------------Ⅴ
中文摘要---------------------------------------------------------------------------------Ⅵ
英文摘要---------------------------------------------------------------------------------Ⅶ
第一章 序論------------------------------------------------------------------------------1
第一節 綠豆 -1
1.1 綠豆介紹------------------------------------------------------------------------1
1.2 營養成分------------------------------------------------------------------------1
1.3 生理功效------------------------------------------------------- ------------ --- 2
1.4 物理與加工特性--------------------------------------------------------------- 3
第二節 澱粉介紹 3
2.1 澱粉的結構與特性------------------------------------------------------------ 3
2.2 澱粉生合成的相關酵素------------------------------------------------------ 4
第三節 澱粉磷解?研究歷史 6
3.1 澱粉磷解?背景--------------------------------------------------------------- 6
3.2 催化特性------------------------------------------------------------------------ 8
3.3 綠豆中澱粉磷解?------------------------------------------------------------9
第四節 宿主表現系統介紹 9
第五節 研究目的 11
第二章 材料與方法-------------------------------------------------------------------13
第一節 材料 13
1.1 樣品---------------------------------------------------------------------------- 13
1.2 藥品---------------------------------------------------------------------------- 13
1.3 儀器設備---------------------------------------------------------------------- 14
第二節 實驗流程設計 16
第三節 Total RNA 之抽取----------------------------------------------------------- 17
第四節 DNA 片段的擴增與純化 18
4.1 反轉錄?作用 (reverse transcriptase, RT)------------------------------- 18
4.2 聚合?鏈鎖反應 ( polymerase chain reaction, PCR)------------------- 19
4.3 DNA 片段之純化方法---------------------------------------------------- 20
4.4 DNA 洋菜膠電泳分析---------------------------------------------------- 21
第五節 重組質體DNA之建構 22
第六節 質體DNA抽取 28
第七節 重組質體DNA之限制?切割分析 29
第八節 DNA 序列定序---------------------------------------------------------------29
第三章 結果與討論------------------------------------------------------------------- 30
第一節 VrSP 序列特性分析與 VrSP 胺基酸序列結構預測------------30
1.1 各物種間 SP 之親緣性分析--------------------------------------------30
1.2 VrSP 內部胺基酸序列與其他物種之 SP 比較---------------------30
1.3 各物種間 SP 靠近 C 端的 17 個胺基酸殘基比較---------------31
1.4 Vrsp 蛋白質之結構預測-------------------------------------------------31
第二節 RNA 的萃取與反轉綠作用------------------------------------------32
第三節 Vrsp 之PCR 量化與片段純化--------------------------------------32
第四節 重組質體構築與分析--------------------------------------------------33
4.1重組質體構築-----------------------------------------------------------------33
4.2重組質體分析-----------------------------------------------------------------34
第四章 結論---------------------------------------------------------------------------- 35
第五章 結果圖與表-------------------------------------------------------------------36
參考文獻---------------------------------------------------------------------------------56






圖 目 錄
圖一、澱粉的生合成路徑圖----------------------------------------------------6
圖二、pMAL-c2X 載體結構圖----------------------------------------------27
圖三、根據各物種間 SP cDNA 演繹出之胺基酸序列所推測出之親緣 性分析--------------------------------------------------------------------36
圖四、各物種間 SP 靠近 C 端的 17 個胺基酸殘基比較------------37
圖五、 以兔子肌肉GP與potent inhibitor之共同結晶 (1WW2A) 為
模板預測出的 VrSP (92~503) N端之 3D 立體結構---------39
圖六、以兔子肌肉的Glycogen phosphorylase (2GJ4A) 為模板預測出
的 VrSP (585~984) C 端之 3D 立體結構及 catalytic site--40
圖七、綠豆 total RNA 為模板,經 RT-PCR 方法以放大綠豆�n��-actin
片段基因---------------------------------------------------------------- 41
圖八、Vrsp cDNA 基因選殖之 RT-PCR 片段排列對照---------------42
圖九、以 SP-EcoRI-F1 與 SP-SalI-R1 為引子進行溫度梯度 PCR 之
電泳圖 ------------------------------------------------------------------44
圖十、 使用first strand cDNA為模板,以SP-EcoRI-F1 and SP-SalI-R1
引子進行PCR擴增之產物電泳圖--------------------------------45
圖十一、 Gel extraction 純化由 VrSP 以 SP-EcoRI-F1 and
SP-SalI-R1 引子所 PCR 擴增產物之電泳圖---------------46
圖十二、 載體 pMal-c2x 經限制? EcoRI 及 SalI 處理後之電泳圖
---------------------------------------------------------------------- 47
圖十三、控制組利用熱休克方式轉型至選殖宿主 Novablue 所獲得之
positive clone------------------------------------------------------- -48
圖十四、以莫耳數比3:1進行接合反應並利用熱休克方式轉型至選殖
宿主 Novablue 所獲得之 positive clone---------------------- 48
圖十五、以莫耳數比3:1進行接合反應並利用電穿孔方式轉型至選殖
宿主 Novablue 所獲得之 positive clone---------------------- 49
圖十六、轉型進入 Novablue 宿主的重組質體 DNA 經限制? EcoRI
切割之洋菜膠體電泳圖Ι---------------------------------------- -50
圖十七、轉型進入 Novablue 宿主的重組質體 DNA 經限制? EcoRI
切割之洋菜膠體電泳圖Π---------------------------------------- -51
圖十八、轉型進入 Novablue 宿主的重組質體 DNA 經限制? EcoRI
與 SalI 進行 double digestion之洋菜膠體電泳圖Ι---------52
圖十九、轉型進入 Novablue 宿主的重組質體 DNA 經限制? EcoRI
與 SalI 進行 double digestion之洋菜膠體電泳圖Π---------53

表 目 錄
表一、根據分子量、對醣類親合性及來源的不同,可將 SP 分為兩
種型態---------------------------------------------------------------------8
表二、藉由生物技術革新使大腸桿菌製造具生物活性蛋白質---------11
表三、各物種間 SP 靠近 C 端的 17 個胺基酸殘基比較-------------38
表四、本探討之 Vrsp 所使用的基因特意引子列表---------------------43
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