Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20872
標題: 環狀芽苞桿菌WL-12的幾丁質分解酵素A1基因在啤酒酵母菌內之表 現
Expression of Bacillus circulans WL-12 chitinase A1 gene in Saccharomyces cerevisiae
作者: 徐光明
Hsu, Kuang-Ming
關鍵字: chitinase A1
幾丁質分解酵素A1
Bacillus circulans WL-12
Saccharomyces cerevisiae
electroporation
環狀芽苞桿菌WL-12
啤酒酵母菌
電穿孔轉形
出版社: 植物學系
摘要: 環狀芽苞桿菌 (Bacillus circulans) WL-12 為革蘭氏陽性桿菌,最先是 從含有酵母菌細胞壁的土壤中分離出來,能分解真菌細胞壁。此菌株能產 生 A1、A2、B1、B2、C 及 D 六種不同的幾丁質分解酵素,其中以 A1 為 關鍵酵素,幾丁質分解酵素 A1 基因 (chiA1) 已被定序分析。由於幾丁 質分解酵素具有分解真菌細胞壁幾丁質的特性, 因此本實驗主要目的即 以此菌株的 chiA1 基因為材料,將這基因轉形至大腸桿菌 (Escherichia coli) 中,除了探討此基因在大腸桿菌中的表現情形外,並更進一步將此 段 chiA1 基因轉形至酵母菌中,希望能使其在酵母菌的系統中大量表現 ,以作為生物防治之基礎。 本實驗室前人首先依 Watanabe 等人的結果 (Watanabe et al., 1990 b ),根據 chiA1 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 ,將其連接於 pKK223-3 載體,成功地構築一個 pKK-chiA1 重組質體, 且轉形至大腸桿菌 (E. coli ) HB101 中。本實驗即利用此 pKK-chiA1 重組質體為材料,再將此chiA1 基因從 pKK-chiA1 重組質體中切割出來 ,連接於另一 YEp352 載體且轉形至大腸桿菌 (E. coli) JM109 (DE3), 將其培養在 LA-chitin 培養基,培養 3 日後形成澄清環。另外,為了更 進一步將 chiA1 基因轉形至酵母菌中表現,因此連接chiA1 基因於另一 pAAH5酵母菌表現載體,成功地構築另一個 pAAH5-chiA1 重組質體,並且 利用電穿孔 (electroporation) 方式將此重組質體送至啤酒酵母菌 (Saccharomyces cerevisiae ) AH22中,並且以 (YNB+histidine) 選擇 性培養基進行篩選。將酵母菌轉形株 pAAH5-chiA1 培養在 YPD-chitin 培養基中,於 30℃ 中培養 5 日後開始有澄清環形成。此外,在測定幾 丁質分解酵素活性的實驗中,發現酵母菌轉形株 pAAH5-chiA1有很高的幾 丁質分解酵素的活性,。由此實驗結果,可知道 Bacillus circulans WL-12 的 chiA1 基因,不僅能在原核生物 (如 E. coli) 中表現,也能 在真核生物 (如 Saccharomyces cerevisiae) 中表現。
Bacillus circulans WL-12, originally isolated from soil with yeast cell wall, is a gram positive bacterium which can degrade fungal cell wall. It can produce six different kinds of chitinase including A1, A2, B1, B2, C and D. Chitinase A1 is a key enzyme of chitinase system of B. circulans WL-12, and chitinase A1 gene (chiA1) have already been cloned and sequenced. Chitinase has a property of degrading fungal cell wall , so the main purpose of this study is to transform chiA1 gene into E. coli. Besides discussing the expression of chiA1 gene in E. coli, we go further to transform chiA1 gene into yeast, to make its expression numerously as the basis of bio- control. Accoding to the reports by Watanabe et al. (Watanabe et al., 1990 b), the predecessor of our laboratory designed two primers each on the opposite terminal of chiA1 gene ORF (open-reading frame), and had synthesized the chiA1 gene by PCR. The synthesized chiA1 gene was inserted into pKK223-3 vector. Then, the successfully constructed pKK-chiA1 plasmid was transformed into E. coli HB101.The chiA1 gene was isolated from pKK-chiA1 plasmid and religated with YEp352 vector, then transformed into E. coli JM109 (DE3).The clear zones appeared around bacterial colonies after 3 day incubation. Besides, we go further to transform chiA1 gene into yeast. The chiA1 gene was eluted from YEp352-chiA1 again, and ligated with pAAH5 expression vector. Then, transformed into Saccharomyces cerevisiae AH22 by electroporation. The transformants were screened on the YNB-histidin selective medium. Transformants containing pAAH5-chiA1 plasmid were selected out from YPD-chitin medium, the clear zones was observed after 5 day incubation at 30℃. Analysis the activity of chitinase indicated that pAAH5- chiA1 transformant have high activity of chitinase. So, we came to the conclusion that the chiA1 gene of Bacillus circulans WL-12 not only expresses in procaryotes (such as E. coli), but also expresses in eukaryotes (such as Saccharomyces cerevisiae).
URI: http://hdl.handle.net/11455/20872
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