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研究生:邱偉中
研究生(外文):Wei-Chung Chiou
論文名稱:木黴菌Trichoderma harzianum ETS323植酸酶之基因選殖、表達、及基因特性分析
論文名稱(外文):Molecular Cloning, Expression and Characterization of Phytase Gene from Trichoderma harzianum ETS323
指導教授:周文敏
學位類別:碩士
校院名稱:國立虎尾科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:76
中文關鍵詞:Trichoderma harzianum ETS323 植酸酶RT-PCR5ˊRACE植酸酶篩選性培養基(PSM)誘導表達
外文關鍵詞:Trichoderma harzianumphytaseRT-PCR5ˊ-RACEQuantitive RT-PCR
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植酸酶(Phytase)是一類可以水解植酸(Phytic acid)生成肌醇與磷酸的酵素。 文獻報導指出:於自然界中有的微生物能分泌植酸酶分解難溶性植酸鹽並釋出磷酸,進而促使個體與植物生長。木黴菌Trichoderma harzianum為廣泛應用於生物防治的真菌之一,而其植酸酶的相關研究甚少。本論文研究成功地利用RT-PCR及5ˊRACE等技術由T. harzianum ETS323選殖出植酸酶的gene 和cDNA全長片段(Genomic-phytase-full 和phytase-full),其核苷酸長度分別為1918 及1680 bp。其中, 經分析後發現Genomic-phytase-full 含5段exons 與 4段introns 。 演譯後的Phytase經預測發現具有N端訊號胜肽,其成熟蛋白質的分子量為58.4 kDa,其pI值為5.3,並且具有10個可能的醣基化位以及 Histidine-acid phosphatases的高度保留區之胺基酸序列(RHGARYPT)。於大腸桿菌中以胞涵體形式表達出Phytase的部分蛋白 (即Phytase-m),亦完成了 Phytase-M抗體之製備。雖然於大腸桿菌中以水溶性蛋白形式表達出約全長的Phytase , 但是未偵測出此重組蛋白的大腸桿菌具有Phytase活性。此外,ETS323 Phytase無法在酵母菌Pichia分泌表達。 將T. harzianum ETS323 培養於液態PSM (phytase-screening medium)。西方墨點法分析發現Phytase-M抗體可以辨識到 ETS323菌絲體及培養液的蛋白質條帶。然而MALDI-TOF分析顯示這些蛋白質條帶不是Phytase 。而菌絲體及培養液皆未有明顯的Phytase 活性。RT-PCR顯示ETS323 Phytase基因在液固態培養條件中會受到Phytate誘導表現。綜合研究結果推測只有植酸為其磷來源時ETS323無法有效大量誘導分泌產生植酸酶,但仍然可以分泌少許的植酸酶。

The soil borne micro-organisms may secrete phytase to decompose insoluble phytate and release phosphate, subsequently promote plant growth. There is limited study with phytase from Trichoderma harzianum, a well know biological control against plant pathogens. In this study, the full-length of DNA fragment (phytase-full) encoding T. harzianum ETS323 phytase was obtained by RT-PCR and 5ˊ-RACE, as well as ETS323 phytase gene by PCR (Genomic-phytase-full ). The 1918 bp DNA fragment of Genomic-phytase-full is composed of five exons, separated by four introns. phytase-full comprises 1680 bp and its predicted protein has N-terminal signal peptide . The molecular mass of predicted mature ETS323 phytase is 58.4 kDa with 5.3 of pI. A high conserved regin (RHGARYPT) is found in ETS323 phytase, which is conserved among histidine acid phosphatases. ETS323 phytase is probably a glycoprotein as there are ten putative N-glycosylation sites. ETS323 phytase was expressed as soluble proteins in Escherichia coli; however, no phytase activity was detected. ETS323 phytase was not successfully expressed in yeast Pichia. The crude proteins were prepared from T. harzianum ETS323 liquid-cultured in phyate containing medium. Western blot analyses showed that the antibodies raised against EST323 phytase could recognize a protein bands from this culture medium. Moreover, Quantitive RT-PCR analyses indicated that the transcript level of ETS323 phytase could be induced by phytate.

目錄
摘要 ......................................................................................................................................i
Abstract .....................................................................................................................................ii
表目錄 .....................................................................................................................................iv
圖目錄 .....................................................................................................................................v
第一章 前言 .....................................................................................................................................1
第二章 文獻回顧 .....................................................................................................................................2
2.1 木黴菌 .....................................................................................................................................2
2.1.1 木黴菌之分類及生長型態 .....................................................................................................................................2
2.1.2 溶磷微生物之研究發展 .....................................................................................................................................2
2.2 植酸(phytate) .....................................................................................................................................2
2.3 植酸酶 (Phytase) .....................................................................................................................................4
2.3.1 最適pH 值 .....................................................................................................................................4
2.3.2 立體專一性 .....................................................................................................................................4
2.3.3 催化機制 .....................................................................................................................................5
2.3.3.1 HAP (Histidine -acid phosphatases) .....................................................................................................................................5
2.3.3.2 (BPP) β-propeller phytases .....................................................................................................................................6
2.3.3.4 Purple acid phosphatases (PAP) ........................................................................................................................ ............6
2.3.3.4 Cysteine phosphatase-like phytase (CP) .....................................................................................................................................6
2.4 Trichoderma spp.之 phytase 簡介 .....................................................................................................................................6
2.5 專利檢索 .....................................................................................................................................8
第三章、實驗材料與方法 ....................................................................................................................................11
3.1 菌種保存及活化 ....................................................................................................................................11
3.2 木黴菌T. harzianum ETS323 之植酸酶基因選殖 ....................................................................................................................................11
3.2.1 植酸培養基配製 ....................................................................................................................................11
3.2.2 ETS323 菌株的接種 ....................................................................................................................................11
3.2.3 ETS323 之Total RNA純化 ....................................................................................................................................11
3.2.4 反轉錄聚合鏈鎖酶反應(Reverse transcription-PCR) ....................................................................................................................................12
3.2.4.1 反轉錄反應 ....................................................................................................................................12
3.2.4.2 聚合鏈鎖酶反應 ....................................................................................................................................12
3.2.5 ETS323 植酸酶全長基因之選殖 ....................................................................................................................................13
3.2.5.1 反轉錄反應 ....................................................................................................................................13
3.2.5.2 5ˊ- RACE PCR 反應 ....................................................................................................................................14
3.2.6 PCR 產物之純化及回收 ....................................................................................................................................15
3.2.7 PCR 產物之選殖 ....................................................................................................................................16
3.2.7.1 DNA 連接反應 ....................................................................................................................................16
3.2.7.2 轉型作用-熱休克法 ....................................................................................................................................16
3.2.7.2.1 勝任細胞之製備 ....................................................................................................................................16
3.2.7.2.2 熱休克細胞轉型 ....................................................................................................................................16
3.2.7.3 重組質體之篩選確認 ....................................................................................................................................16
3.3 以大腸桿菌為宿主之誘導蛋白質表現 ....................................................................................................................................17
3.3.1 表現型載體之構築
....................................................................................................................................17
3.3.2 pET28a-Phytase-M 之誘導蛋白質表現 ....................................................................................................................................17
3.3.3 pET26bDsba-Phytase-A 之誘導蛋白質表現 ....................................................................................................................................17
3.4 多株抗體之製備 ....................................................................................................................................18
3.4.1 Electro-Eluter 回收蛋白質 ....................................................................................................................................18
3.4.2 蛋白質濃度測定 ....................................................................................................................................18
3.4.3 抗體血清的製備 ....................................................................................................................................18
3.4.3.1 注射 ....................................................................................................................................18
3.4.3.2 抽血及備製血清 ....................................................................................................................................19
3.5 西方墨點法 ....................................................................................................................................19
3.6 以酵母菌為宿主之誘導蛋白質表現 ....................................................................................................................................19
3.6.1 P. pastoris X-33、GS115 及KM71H 勝任細胞製備 ....................................................................................................................................19
3.6.3 P. pastoris 之轉型 ....................................................................................................................................20
3.6.4 重組菌株之篩選 ....................................................................................................................................20
3.7 木黴菌T. harzianum ETS323 之粗蛋白質收集及抗體辨識 ........................................................................21
3.7.1 PSM 液態培養基配製 ....................................................................................................................................21
3.7.2 T. harzianum ETS323 之液態接種 ....................................................................................................................................21
3.7.3 粗蛋白質之收集 ....................................................................................................................................21
3.7.3.1 培養液部分
....................................................................................................................................21
3.7.3.2 菌絲體部分分.
....................................................................................................................................21
3.7.3.3 抗體辨識部份 ....................................................................................................................................22
3.7.3.4 以MALDI-TOF-MS 分析蛋白質 ....................................................................................................................................22
3.8 植酸酶活性測定 ....................................................................................................................................22
3.8.1 活性測定方法 ....................................................................................................................................22
3.9 Quantitative RT-PCR ....................................................................................................................................22
3.9.1 以Quantitative RT-PCR 分析T. harzianum ETS323 Phytase 之基因表現 22
第四章、結果 ....................................................................................................................................24
4.1 Trichoderma harzianum ETS323 之植酸酶基因選殖 ....................................................................................................................................24
4.1.1 ETS323 之植酸酶基因選殖及序列分析 ....................................................................................................................................24
4.2 T. harzianum ETS323 之植酸酶基因重組 ....................................................................................................................................25
4.2.1 植酸酶基因之PCR 片段純化 ....................................................................................................................................25
4.3 表現型質體之構築 ....................................................................................................................................25
4.3.1 pET28a-Phytase-M 之表現型質體構築 ....................................................................................................................................25
4.3.2 pET26bDsba-Phytase-A 之表現載體構築 ....................................................................................................................................25
4.3.3 pGAPZαB、pGAPZB-Phytase -A 和pPICZαB、pPICZB-Phytase-A 之表現型載體構築 ....................................................................................................................................26
4.4 轉型後重組質體之確認 ....................................................................................................................................26
4.4.1 PCR 確認 ....................................................................................................................................26
4.4.2 限制酶確認 ....................................................................................................................................26
4.5 以大腸桿菌為宿主之蛋白大量表現 ....................................................................................................................................26
4.5.1 Phytase-M 重組蛋白質之誘導表達及純化 ....................................................................................................................................26
4.5.2 Phytase-A 重組蛋白質之誘導表達 ....................................................................................................................................27
4.6 以酵母菌P. pastoris 為宿主進行重組蛋白質之表達 ....................................................................................................................................27
4.6.1 Phytase 重組蛋白質之酵母菌分泌表達 ....................................................................................................................................27
4.6.2 Phytase-A 重組蛋白質之酵母菌胞內表達 ....................................................................................................................................28
4.7 以西方墨點法辨識Phytase ....................................................................................................................................28
4.7.1 以西方墨點法辨識重組Phytase-M ....................................................................................................................................28
4.7.2 以西方墨點法辨識重組Phytase-A ....................................................................................................................................28
4.7.3 抗體辨識T. harzianum ETS323 之Phytase ....................................................................................................................................28
4.7.4以Anti- L-amino acid oxidase(LAAO)抗體辨識T. harzianum ETS323 之胞外
蛋白質 ....................................................................................................................................29
4.8 植酸酶活性測定 ....................................................................................................................................29
4.8.1 重組Dsba-phytase 活性 ....................................................................................................................................29
4.8.2 T. harzianum ETS323 之粗蛋白質活性 ....................................................................................................................................29
4.9 Quantitative RT-PCR ....................................................................................................................................29
4.9.1 以Quantitative RT-PCR 分析T. harzianum ETS323 Phytase 之基因表現結
果 ....................................................................................................................................29
第五章 討論 ....................................................................................................................................30
第六章 結論 ....................................................................................................................................33
參考文獻 ....................................................................................................................................61
附錄 ....................................................................................................................................65
Extended Abstract ....................................................................................................................................68
簡歷(CV) ....................................................................................................................................76

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