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Streptomyces griseobrunneus S3 Family 19幾丁質分解酵素基因ChiF全長序列之選殖及重組蛋白質之分子特性檢測

Cloning of Full Length Sequence of a Family 19 Chitinase Gene ChiF from Streptomyces griseobrunneus S3 and the Molecular Characterization of the Recombinant Protein

摘要


本研究初步調查family 19幾丁質分解酵素,於台灣本土性具幾丁質分解能力鏈黴菌菌株之存在情形,以聚合酵素連鎖反應法(PCR),配合family 19幾丁質分解酵素專一性引子對的應用檢測,證實於21個供試菌株中,有11個可檢測到此酵素對應基因之存在。由其中憶證實於植物病害防治上,深具應用潛力之Streptomyces griseobrunneas S3菌株(SGS3),並進而選殖成功其family 19幾丁質分解酵素對應基因ChiF之全長核酸序列。所選殖之ChiF基因經解序,證實共計含891bp,所轉譯蛋白含296個胺基酸,分子大小約爲31.5kDa。此基因5’端之幾丁質結合區(ChtBD),具有與一般細菌幾丁質分解酵素type 3 ChtBD同樣之保留序列AKWWTQ。基因上游啓動子附近,並含有一已被證實與基因表現之幾丁質誘導與葡萄醣抑制調控攸關之核酸鹽基重複序列(T/A)GGTC(T/C)AGACC(T/A)。本研究已由SGS3 ChiF轉基因大腸桿菌中,成功萃取此ChiF重組蛋白,利用FPLC純化之蛋白製備,並進而製作成功其免疫檢測用多元抗血清。研究中繼而利用SDS-PAGE電泳分離,配合酵素活性染色以及西方漬染分析,且已證實其酵素活性及分子量確如預期,且此蛋白經SDS變性後仍可回復其活性。由已知同類酵素之胺基酸序列分析其親緣關係,所獲昨樹狀演化圖顯示,SGS3 ChiF與Nocardiopsis prasina chiB等均具高度同源性。本文中將討論此SGS3 chiF基因之分子特性及其與鏈黴菌屬成員抗真菌性表現之關係。

並列摘要


A preliminary survey of existence of family 19 chitinase gene among Taiwan native chitinolytic Streptomyces strains was conducted. Among 21 tested strains, 11 were detected positive by PCR (polymerase chain reaction) with the use of a family 19 chitinase gene specific primer pair. A full-length chitinase gene SGS3 ChiF was cloned from Streptomyces griseobrunneus S3-a chitinolytic strain known to have great potential in plant disease control application. Sequence analysis revealed that the gene contains 891 bp which encodes a 296 amino acid peptide with a molecular weight around 31.5 KDa. The gene contains a chitin binding domain (ChtBD) with ChtBD consensus sequence AKWWTQ. On its upstream was a promoter region with characteristic direct repeal sequence (T/A)GGTC(T/C)AGACC(T/A) known to he critical in chitin induction and glucose repression. From a SGS3 ChiF transformed Escherichia coli Rosetta(superscript TM) (DE3). the ChiF recombinant protein with a predicted size around 31.5 KDa was extracted and purified by Fast Performance Liquid Chromatography. The polyclonal antibody for immuno-detection of the recombinant protein was successfully prepared. The enzyme appeared to survive denaturing during SDS-PAGE analysis, the enzyme characteristics of recombinant protein was demonstrated by activity stain and western blotting. A phylogenetic analysis revealed that the cloned gene was highly homologous to the comparative gene known on S. griseus. S. coelicolor. and Nocardiopsis prasina. which might be adapted evolutionarily from the class IV chitinase of plant system. The molecular characteristics of the gene and its possible connection to antifungal characteristic of Streptomyces are herein discussed.

被引用紀錄


鄭雅丹(2010)。台北都會區進城費之最佳收費周界與費率水準之研究〔碩士論文,國立交通大學〕。華藝線上圖書館。https://doi.org/10.6842%2fNCTU.2010.00459

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