Gene Cloning and Purification of N-acetyltransferase from Trichoderma virens FT-333

• Main Authors: LIAN,GUAN-JHIH, 連冠智
• Other Authors: Chen, Ruey-Shyang
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/h82m24

碩士 === 國立嘉義大學 === 生化科技學系研究所 === 107 === 3,4-dichloroaniline (3,4-DCA) is derived from herbicides such as Diuron and Linuron, and has been known to be genetically toxic and cytotoxic to most organisms. Previous studies have found that Trichoderma virens FT-333 was highly resistant to 3,4-DCA, suggesting that FT-333 could detoxify 3,4-DCA due to its ability to acetylate 3,4-dichloroaniline by N-acetyltransferase (NAT). In this study, NAT1 and NAT2 of FT-333 were amplified by PCR using specific primer pairs. The sequence alignment analysis showed that the sequences between NAT1 and BN001413.1 in the GenBank were slightly different, but the identity of NAT2 and BN001414.1 were 100%. The full length NAT1 and NAT2 were further cloned into pQE30 vector and transformed into E. coli. However, the inserted NAT1 gene was mutated and the stop codon was generated. More, the recombinant protein from the inserted NAT2 gene could not be folded normally, resulting in an inclusion body. The NAT gene was then transferred to the pET32 vector with TRX-tag. Both recombinant proteins pET32-NAT1 and pET32-NAT2 produced inclusion bodies. By adding urea during the purification process, the NAT protein was obtained and used for enzyme activity analysis. In this study, N-acetyltransferase gene of T. virens FT-333 was successfully cloned, and the mechanism regarding how T. virens FT-333 detoxified 3,4-DCA will be further studied.