曾顯雄臺灣大學：植物病理與微生物學研究所吳秀芳Wu, Hsiu-FangHsiu-FangWu2007-11-272018-06-292007-11-272018-06-292004http://ntur.lib.ntu.edu.tw//handle/246246/58056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 -------------------------------------------------------------------------------------------63829431 bytesapplication/pdfen-USgene cloning3-glucanasebiocontrolSchizophyllum communemycoparasitismβ-1mycoparasitism biocontrolotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/58056/1/ntu-93-R89633007-1.pdf