Insertion Mutagenesis of Trichoderma atroviride by Restriction Enzyme-mediated DNA integration

The integration of transforming DNA mediat-ed by the in vivo action of restriction enzymes(RE-MI) was first described in yeast. Insertion mutage-nesis by REMI is a method that is being used togenerate mutations whose molecular basis can beeasily identified. REMI mutagenesis has a majoradvantage over classical mutagenesis in that themutants are marked with a molecular tag by themutagenic event. REMI has been described inmany organisms including the filamentous fungi,and its technology ha…

Insertion Mutagenesis of Trichoderma atroviride by Restriction Enzyme-mediated DNA integration

Restriction enzyme-mediated integration(REMI) of DNA has been recently received attention as a new technique for the generation of mutants by transformation in fungi. Trichoderma atroviride strain T23 was transformed with linearized plasmid pV2, conferring resistance to hygromycin B, in the presence of restriction enzyme used to linearize the plasmid. A total of 172 regeneration transformants were detected by successive inoculation for seven times subcultivation on fresh PDA plate containing hygromycin B. The plasmid was integrated stably into the chromosome DNA, which was confirmed by PCR and southern analysis. The difference between 172 transformants and the parent strain was confirmed in colonial color, sporulation and growth rate. The results showed that the significant difference appeared in above mentioned characters between transformants and parent strain is sporulation capability. Transformants TC6, TD5, TE7, TF1 and TK1 produced higher amounts of conidia than the parent strain T23. In addition, transformants TK1and TC6 showed stronger inhibition to the growth rate of the cucumber wilt pathogen (Fusarium oxyporum) in vitro.