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標題: 聚合■連鎖反應在環狀芽孢桿菌 Bacillus circulans WL-12 的幾丁質分 解■基因選殖之應用
Chitinase gene cloning of Bacillus circulans WL-12 by PCR
作者: 陳韻如
Chen, Yun-ju
關鍵字: Bacillus circulans
Polymerase Chain Reaction
出版社: 植物學系
摘要: 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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