Cloning and Characterization of a salt-induced DhPEX11, a gene involved in peroxisomal proliferation, from the extremely halophilic yeast Debaryomyces hansenii

• Main Authors: Huang Yi-Cin, 黃乙芩
• Other Authors: Wen-Der Wang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/7q6699

碩士 === 國立嘉義大學 === 生物農業科技學系研究所 === 106 === The earliest Debaryomyces hansenii was found in sea, the tolerance mechanisms against salinity is different from Saccharomyces cerevisiae. D.hansenii is an extreme halophile species of yeast compare with S. cerevisiae and can survive 24% NaCl. It has become a model organism for the study of tolerance mechanisms against salinity, but the mechanisms of its salt tolerance are still unclear. The goal of this study was to identify salt-induced gene in D. hansenii, and to characterize one of the salt upregulated gene, DhPEX11. The full-lengthed DhPEX11 was cloned by RACE (rapid amplification of cDNA ends) from D. hansenii. The DhPEX11 gene was first constructed into the expression vector of pPICZα B which carries the inducible promoter to methanol, and the expression cassette PAOX1/DhPEX11/Myc/His was then excised from the vector and introduced into Pichia pastoris GS115 strain by EasyComp. To localize the DhPEX11 protein and peroxisomes in the cell, using specific anti-His and anti-pmp70 antibody detected with DhPEX11 and peroxisomes. The results showed that DhPEX11 location same with peroxisomes in P. pastoris and culture the over-expression DhPEX11 in media contain NaCl, confirm DhPEX11 can increase salt tolerance.