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研究生:吳秀芳
研究生(外文):Hsiu-Fang Wu
論文名稱:裂褶菌Schizophyllumcommune之葡聚糖裂解酵素(β-1,3-glucanase)之基因選殖及其生化活性探討
論文名稱(外文):Cloning and characterization of β-1,3-glucanase gene from mycoparasitic Schizophyllum commune
指導教授:曾顯雄曾顯雄引用關係
指導教授(外文):Shean-Shong Tzean
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:80
中文關鍵詞:gene cloning3-glucanasebiocontrolSchizophyllum communemycoparasitismβ-1
外文關鍵詞:3-glucanaseβ-1Schizophyllum communemycoparasitism biocontrolgene cloning
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以Rhizoctonia solani為碳素源,培養Schizophyllum commune可促進產生β-1,3-glucanase。於25℃、培養八天,所得之粗酵素10,000ml以超音波震盪過濾,經減壓濃縮機濃縮10倍,進行硫酸銨分割,利用等電點分離、陰離子交換樹脂和疏水性層析管柱純化後,增加251.5倍純化度,增加比活性13,579 (unti/mg),其pI值為3.45。再由活性電泳檢測及10% SDS-PAGE電泳分析得四個主要蛋白質片段,其分子量分別為63kDa、58 kDa 、29 kDa 和27 kDa,63 kDa之蛋白質經N端定序後得到8個胺基酸序列LDNGVGAL;58 kDa之蛋白質經N端定序後得到8個胺基酸序列LDNGVGRL,根據此N端胺基酸序列設計引子;另以NCBI上已發表之植物與真菌β-1,3-glucanase胺基酸序列設計C端引子,以多種組合進行反轉錄聚合酶連鎖反應,可獲得約500 bp 產物,定序後與NCBI資料庫之BLAST軟體進行序列比對,但比對結果尚未在NCBI資料庫中收尋到相似性或已註冊之序列。此外在大腸桿菌中建構了約2,000 個Fosmid DNA library clones,將再以製成的探針進行雜合,藉此選殖出完整的β-1,3-glucanase基因。
Schizophyllum commune was enhanced to produce higher yield ofβ-1,3-glucanasewhile the basal medium was amended with the cell wall of Rhizoctonia solani.β-1,3-glucanasewas partially purified by anion-exchange, hydrophobic-interaction chromatography, which increase 251.5 fold purtification and 13,579 specific activity. The pI of the enzyme was pH 3.45. The partially purified endo-β-1,3-glucanase was assumed to be a tetraisomer, consisting of 63kDa、58 kDa、29 kDa and 27 kDa monomer, respectively, as shown by SDS-PAGE. The N-terminal amino acid residues of 63 kDa and 58 kDa protein were LDNGVGAL and LDNGVGRL, respectively by Edman degradation. By different combination of the degenerate primers from N-terminal amino acid residues plus 7 degenerate primers from C-terminal conservative region to process RT-PCR, a 500 bp cDNA product was obtained, which shows identical N-terminal sequence as the original primers, but however also contained unexpected stop codon . The cDNA sequence will be used to probe the newly constructed Fosmid library to access the possibly hidden β-1,3-glucanasegene.
中文摘要 ------------------------------------------------------------------------------ 1
英文摘要 -------------------------------------------------------------------------------2
壹、緒言 --------------------------------------------------------------------------------3
貳、前人研究 --------------------------------------------------------------------------5
一、真菌重寄生(Mycoparasitism)現象 -------------------------------------------5
(一) 殺生性重寄生(Necrotrophic myco-parasitism) -------------------------5
(二) 毀滅性重寄生(Biotrophic mycoparasites) -------------------------------5
二、真菌細胞壁之構造 ------------------------------------------------------------6
三、真菌細胞壁分解酵素之種類及特性 ---------------------------------------6
(一) 真菌胞壁分解酵素之種類 ------------------------------------------------6
(二) 真菌細胞壁分解酵素之特性 ---------------------------------------------8
四、重寄生菌產生葡聚糖分解酵素(β-1,3-glucanase)之研究 ------------10
(一) 葡聚糖分解酵素(β-1,3-glucanase)之種類 ---------------------------10
(二) 葡聚糖裂解酵素(β-1,3-glucanase)之特性 ---------------------------10
(三) 葡聚糖裂解酵素(β-1,3-glucanase) 基因之選殖 --------------------12
(四) 葡聚糖裂解酵素(β-1,3-glucanase)在植物病害防治之應用 ------13
五、Rhizoctonia solani 之簡介 -------------------------------------------------13
六、Schizophyllum commune 之簡介 -------------------------------------------14
參、材料方法 ------------------------------------------------------------------------16
肆、結果 ------------------------------------------------------------------------------34
伍、討論 -------------------------------------------------------------------------------41
陸、參考文獻 ------------------------------------------------------------------------44
柒、表 ---------------------------------------------------------------------------------50
捌、圖 ---------------------------------------------------------------------------------54
附錄 -------------------------------------------------------------------------------------------63
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