臺灣博碩士論文加值系統

R. oryzae 為一種絲狀真菌，由於生長快速，且具有高效率蛋白質外分泌系統，因此常被使用來生產工業用酵素。本研究主要的目的是建立 R. oryzae 轉形及基因表現系統，以利於產生同源或異源性蛋白質。此系統利用 uracil營養要求變異株 R. oryzae pyrG- 及來自 R. oryzae BCRC31837 的 pyrG 基因作為篩選標誌，並成功利用Novezyme 234、Yatalase、chitinase RS及chitosanase建立R. oryzae 原生質體的製備方法。將 Aspergillus oryzae NCHU1002 leucine aminopeptidase (LAP)基因選殖入Escherichia coli－R. oryzae穿梭載體並以 α-amylase 及 3-phosphoglycerate kinase (pgk)、glucoamylase (gla)和 aspartic protease啟動子控制基因表現，這些穿梭載體以PEG方法轉形入 R. oryzae pyrG-，得到轉形株 R. oryzae pyrG- (pamyP-lap)-1、R. oryzae pyrG- (ppgkP-lap)-1、R. oryzae pyrG- (pglaP-lap)-6及R. oryzae pyrG- (pAP-lap)-1，其最高的 LAP 活性分別為34.2mU/ml、 82.4mU/ml、 152.4mU/ml 及15.2mU/ml。利用南方墨點雜交法分析，發現轉形株 R. oryzae pyrG- 所攜帶 pamyP-lap、ppgkP-lap、pglaP-lap及pAP-lap質體都是以游離的狀態存在。

Rhizopus oryzae is a filamentous fungus which grow rapidly and secret a large amount of various enzymes extracellularly. This fungus has been used in the production of industrial enzymes. To develop transformation and expression systems for the production of homologous and heterologous proteins by R. oryzae, a uracil-auxotrophic mutant, R. oryzae pyrG- , was used as host cell, and a pyrG gene cloned from R. oryzae BCRC31837 was used as selectable marker. An efficient method for the preparation of R. oryzae protoplast was also established by using four lytic enzymes in combination. Aspergillus oryzae NCHU1002 lap gene was cloned into Escherichia coli－R. oryzae shuttle vector under the control of α-amylase, 3-phosphoglycerate kinase(pgk), glucoamylase (gla) or aspartic protease promoter. The recombinant plasmids were introduced into R. oryzae pyrG- by CaCl2/PEG transformation method. The LAP activities in the transformants harboring pamyP-lap, ppgkP-lap, pglaP-lap or pAP-lap can reach 34.2mU/ml, 82.4mU/ml, 152.4mU/ml, and 15.2mU/ml, respectively. Southern blot analysis indicated that pamyP-lap, ppgkP-lap, pglaP-lap and pAP-lap existed as episomal form in R. oryzae pyrG- cells.

中文摘要------------------------------------------------1
英文摘要------------------------------------------------2
前言---------------------------------------------------10
材料方法
一. 藥品------------------------------------------15
二. 菌株、質體------------------------------------15
三. LAP 之活性測試--------------------------------15
四. R.oryzae 基因轉形之建立-----------------------16
結果
一.R.oryzae轉形系統之建立---------------------------22
（一）篩選標誌------------------------------------22
（二）原生質體的再生------------------------------23
（三）R.oryzae 的轉形-----------------------------23
二.表現 lap 基因載體之構築及 R.oryzae 之轉形--------24
（一）利用 Taka-amylase A 基因之啟動子------------24
（二）利用 phosphoglycerate kinase 基因之啟動子---26
（三）利用glucoamylase 基因之啟動子-----------------26
（四）利用aspartic protease基因之啟動子-------------27
（五）轉形效率--------------------------------------28
（六）轉形株在有絲分裂時之穩定度--------------------28
三. 起動子對於 A. oryzae lap 基因在R. oryzae 中表現之影-29
（一）Taa 基因之啟動子-------------------------------29
（二）pgk基因之啟動子--------------------------------30
（三）Gla 基因之啟動子-------------------------------31
（四）aspartic protease基因之啟動子---------------------32
討論----------------------------------------------------33
一. R.oryzae轉形系統之建立-------------------------33
二. A. oryzae lap基因在R.oryzae 中之表現-----------36
三. pH 值對啟動子表現 LAP 的影響-------------------38
圖表----------------------------------------------------40
參考文獻------------------------------------------------72

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