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《Acta Scientiarum Naturalium Universitatis Sunyatseni》 2009-06
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Cloning and Intergration Expression of Endo-β-glucanase Ⅲ Gene from Trichoderma viride in Saccharomyces cerevisiae

LIU Zehuan,TAI Yan,TANG Genyun,QUAN Yancai,WANG Junmei,XIAO Wenjuan,GONG Yingxue(Research Center for Molecular Biology,Institutes of Life andHealth Engineering,Jinan University,Guangzhou 510632,China)  
The endo-β-glucanase Ⅲ gene(eg3) was obtained from the total RNA of Trichoderma viride AS3.3711 by RT-PCR.Sequence analysis indicated that this eg3 gene was composed of 1 257 nucleotides,coding for 418 amino acid residues,and had its own signal peptide.The sequence homologies between this gene with Trichoderma Reesei eg3 were 99.6%.Then it was cloned into the high-copy integrative expression vector pScIKP,generating a recombinant plasmid named pScIKP-eg3,which was then transformed by electroporation into Saccharomyces cerevisiae AS2.489 after linearization.Transformants were detected by SDS-PAGE analysis,Congo Red method and enzyme activity assay.The results showed that transformants could secrete recombinant EG Ⅲ and produced hydrolysis halos on the Congo-Red-CMC plate,indicating that this eg3 gene was correctly expressed in S.cerevisiae.The highest enzyme activity of recombinant EG Ⅲ reached 120 U/mL after 60 h cultivation,and the optimum temperature and pH were 60 ℃ and 6.0 respectively.The genetic stability of transformants achieved 99.17% after 50 generations in nonselective medium.In conclusion,the eg3 gene of T.viride could be expressed in industrial yeast strain stably and efficiently.
【Fund】: 新世纪优秀人才支持计划资助项目(NCET-05-0745);; 广东省科技计划资助项目(2005B10401015);; 珠海市科技攻关资助项目(PC20071080)
【CateGory Index】: Q78
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