Establishment of transformation and expression system for Rhizopus oryzae

• Main Authors: Yi-Ying Lin, 林宜穎
• Other Authors: 許文輝
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/66152384915588319838

碩士 === 中興大學 === 分子生物學研究所 === 94 === 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.