The Transformation in Trichoderma viride Protoplasts by Restriction Enzyme Mediated Integration (REMI)

Abstract:

The transformation conditions of the protoplasts from Trichoderma viride mediated by restriction enzyme were studied in this paper. The optimum generation conditions of protoplasts were as followed: 8 mg/ml glucanex was added into the phosphate buffer (pH 6.98), the mycelial that cultured for 24 hr was hydrolyzed for 4 hr at 30°C under 40 r/min shaking speed. The protoplast yield was 4.7×107 cfu/mg. The regeneneration rate of protoplast was 14.5% on CM medium contained 0.3 mol/L KCl and 0.3 mol/L Inositol. Transformants were obtained by transfering hygromycin B resistance gene into T. viride by restriction enzyme mediated integration (REMI), The preliminary identification of the transformants indicated that the exogenous gene had been integrated into T. viride genome.

• Periodical: Advanced Materials Research (Volumes 518-523)
• Pages: 545-548
• DOI: https://doi.org/10.4028/www.scientific.net/AMR.518-523.545
• Online since: May 2012
• Authors: Yu Ru You, Shi Wang Liu, Yuan Feng Wu, Jun Huang, Gong Nian Xiao, Jian Wei Mao
• Keywords: Protoplasts, REMI, Transformation, Trichoderma Viride

References

[1] Shi Z.,Christian D. Leung H..Enhanced transformation in Magnaporthe grisea by restriction enzyme mediated integration of plasmid DNA. Phytopathology, 1995,85(3):329-333

DOI: 10.1094/phyto-85-329

[2] Giczey G, Kerenyi Z, Dallmann G, Hornok L. Homologous transformation of Trichoderma hamatum with an endochitinase encoding gene,resulting in increased leveeels of chitinase activity. FEMS Microbiology Letters , 1998,165:247-252

DOI: 10.1111/j.1574-6968.1998.tb13153.x

[3] Herrera-Estrella A.,Goldman G.H,Van Montagu M. High-efficiency transformation system for the biocontrol agents,Trichoderma spp. Mol. Microbiol, 1990,4:839-843

DOI: 10.1111/j.1365-2958.1990.tb00654.x

[4] Margolles-Clark E, Harman G E ,Penttila M. Enhanced expression of endochitinase in Trichoderma harzianum with the cbhI promoter of Trich oderma reesei. Appl.Environ. Microbil., 1996, 62:2152-2155

DOI: 10.1128/aem.62.6.2152-2155.1996

[5] Guerin NA & Larochelle DA， A user's guide to restriction enzyme-mediated integration in Dictyostelium. J. Muscle Res. Cell M， 2002， 23（7-8）:597-604.

[6] Moriguchi M, Kotegawa S.. Preparation and regeneration of protoplasts from mycelia of Morchella. Agric Biol Chem , 1985,49（9）:2791～2793.

DOI: 10.1271/bbb1961.49.2791

[7] Zhou XY， Xu SF， Liu LX & Chen J， Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration （REMI）. Bioresource Technology, 2007, 98 (15):2958-2962

DOI: 10.1016/j.biortech.2006.09.047