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研究生:陳韻如
研究生(外文):Chen, Yun-Ru
論文名稱:聚合酉每連鎖反應在環狀芽孢桿菌BACILLUSCIRCULANSWL-12的機丁質分解酉每基因
論文名稱(外文):CHITINASE GENE CLONING OF BACILLUS CIRCULAN S WL-12 BY RCRZENG
指導教授:陳昇明陳昇明引用關係
指導教授(外文):Chen, Sheng-Ming
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1995
畢業學年度:83
語文別:英文
論文頁數:42
中文關鍵詞:環狀芽孢桿菌幾丁質分解酉每聚合每連銷反應選殖基因植物科學植物學幾丁質分解?聚合?連鎖反應基因幾丁質分解■聚合■連鎖反應
外文關鍵詞:BACILLUS CIRCULANSCHITINACHITINASEPOLYMERASECHAIN REACTION ACLONINGAGENEPLANT-SCIENCEBOTANYBacillus circulanschitinchitinasePolymerase Chain Reaction (PCR)cloninggenePolymerase Chain Reaction (PCR)Polymerase Chain Reaction (PCR)
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環狀芽孢桿菌(Bacillus circulans) WL-12 品系菌株為生長於土壤中
,能分解酵母菌細胞壁之革蘭氏陽性菌 o 此菌株能產生 A1,A2,B1,B2, C
及 D 六種不同的幾丁質分解■,其中以 A1 為關鍵■,基因已被定序分析
o 本論文主要目的即以此菌株為材料,以 PCR 選殖策略對此幾丁質分解
■ A1 基因進行選殖至大腸桿菌中 o 除了分析 PCR 選殖 A1 基因之可行
性與應用外,並探討其在大腸桿菌中基因的表現情形,作為往後將此基因轉
殖至真核系統使其大量表現以利用於生物防治之依據 o實驗中,首先依
Watanabe 等人 {Watanabe et al., 1990. J. Biol. Chem. pp.
15659-15665.} 的結果,根據 A1 基因的序列,在 5'端與 3'端各設計一
條26個鹼基的引子,以 PCR 合成出一段約 2.1 Kb 之 ORF o 將其連接於
質體 pKK223-3, 則成功地構築一個轉形質體 pKK223-chiA1 且轉形至大
腸桿菌中 o轉形株幾丁質分解■基因的表現以澄清環的形成來判定 o 培
養於 YNB- chitin 固體培養基的轉形株澄清環於培養 10 日後形成 o 相
較於 B. circulans WL-12 則轉形株幾丁質分解■基因的表現有延遲現
象 o 此延遲表現的原因可能為轉形株所產生的幾丁質分解■累積於
periplasmic space 所致 o

Bacillus circulans WL-12, one of the microorganisms which are
able to decompose and grow on yeast cell wall, was originally
isolated from soil. Because of the ability to degrade insoluble
chitin, which is the component of fungal cell wall, it was
interesting us that cloning the chitinase gene to an eukaryotic
system for overexpression and appling in bio-control system.
According to the reports by Watanabe et al. {Watanabe et al.,
1990. J. Biol. Chem. pp. 15659-15665.}, we designed two primers
each on the opposite terminal of chitinase A1 gene ORF (open-
reading frame), and synthesized the chitinase A1 gene by PCR.
For cloning in E. coli, the synthesized chitinase A1 gene was
firstly constructed with a protein expression vector, pKK223-3,
and named pKK223-chiA1. Then, the successfully constructed
plasmid was transformed in E. coli by competent cell trans-
formation. Finally, the correct transformants were cultured on
medium containing colloidal chitin to express the chitinase
activity. After 10 day culture, clear zones appeared around
bacterial colonies. It indicates that chitinase A1 gene could
work in E. coli. However, after comparing the clear zone
produced from B. circulans WL-12, it seemed that the expression
activity was too low. The reason may be deduced by the membrane
structure of E. coli.

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