https://scholars.lib.ntu.edu.tw/handle/123456789/96195
標題: | 植物保護─BACILLUS幾丁質分解酵素基因在PSEUDOMONAS根圈細菌之表現分析(2/3).. | 作者: | 陳昭瑩 | 關鍵字: | 幾丁質分解酵素;幾丁質分解酵素基因;根圈細菌;遺傳工程;基因表現載體;生物防治;chitinase;chitinase gene;rhizobacteria;genetic engineering;gene expression vector;biocontrol | 公開日期: | 2000 | 出版社: | 臺北市:國立臺灣大學植物病理與微生物學系暨研究所 | 摘要: | 本研究目的希望將Bacillus chiA基 因導入根圈螢光假單胞細菌中,使之 表現並存在於細菌外膜上而仍具有分 解幾丁質的能力且能發揮抑制病原菌 的效果。目前已將ompA基因的分泌訊 息序列及轉膜功能區序列以PCR法擴 增出來並選殖於pGEM-T easy載體 上。接著以二對引子擴增不同長度的 Bacillus chiA基因與ompA分泌訊息序 列及轉膜功能區序列黏接,並次選殖 於pMMB6載體中。此質體構築已送入 E. coli TOP10F’中表現幾丁質分解 酵素,在glycol chitin-SDS-聚丙烯醯 胺膠體電泳分析、trypan blue-glycol chitin培養基的測試及螢光測定分 析均測得幾丁質分解酵素的活性, 顯示ompA基因的分泌訊息序列及轉 膜功能區序列並不影響幾丁質分解 酵素基因的表現。由E. coli TOP10F’ (pMMST2000)在幾丁質培養基上的 生長特性及造成菌落區透明化的特 性,初步判斷ompA基因的分泌訊息序 列及轉膜功能區序列應使細菌細胞中 所產生的幾丁質分解酵素移位於細胞 外膜上。進一步要將此質體構築送 入Pseudomonas根圈細菌中,以測試 其防禦植物病原真菌的效果。 We plan to transfer a Bacillus chiA gene into Pseudomonas rhizobacteria for the expression of chitinase and tranlocation of chitinase to the outer membrane of bacterial cells. We expect the transformed Pseudomonas rhizobacteria will express antifungal activity or be with enhanced antifungal activity. A DNA segment containing the 2 sequences for signal peptide and transmembrane domain (ST sequence) was amplified by PCR, cloned into pGEM-T easy vector and fused with chiA gene segment of different lengths. The construct was subcloned into pMMB6. The plasmid pMMST2000 containing full length of chiA gene with ST sequence expressed chitinase in E. coli Top10F’ as shown in glycol chitin- SDS-PAGE, on trypan blue-glycol chitin medium, and in fluorometric assay, indicating that ST sequence would not affect the expression of chitinase in bacterial cells. Since E. coli Top10F’(pMMST2000) had a good growth activity and made clearing of chitin medium just beneath the colonies, we assumed that the ST sequence could drive the chitinase to the membrane. In the next, plasmid pMMST2000 will be transferred into Pseudomonas rhizobacteria to examine the antifungl activity of transformants. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/17679 | 其他識別: | 892317B002002 | Rights: | 國立臺灣大學植物病理與微生物學系暨研究所 |
顯示於: | 植物病理與微生物學系 |
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892317B002002.pdf | 32.83 kB | Adobe PDF | 檢視/開啟 |
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