Study On Anti-filamentous Fungi Activity Of Tachyplesin And Constitutive Expression Of Tachyplesin In Trichoderma Reesei

Tachyplesin I is a 17-amino-acid residues cationic antibacterial peptide, exhibiting potent, broad-spectrum activities against bacteria, fungi, viruses, protozoa, and cancer cells, and induction of cell differentiation. Tachyplesin is a class of antimicrobial peptides with great potential value. This paper include:Study on Tachyplesin inhibition of several common genetically engineered bacteria, Antibacterial machanism of tachyplesin Trichoderma reesei and the gene expression of tachyplesin in Trichoderma reesei were investigated.This paper fristly studies the tachyplesin on several common genetic engineering bacteria(Escherichia coli BL21, Bacillus subtilis WB800, Bacillus subtilis BS168, Yeast GS115, Trichoderma reesei QM9414)killing and its sensitivity. Using the broth microdil-ution method and MTT method to determinate the minimum inhibitory concentration(MIC) and minimum bactericidal concentration(MBC) of the bacteria, MIC are 10,10,10,20,40μg/mL respectively, and MBC are 20,20,20,40,80μg/mL respectively. And on this basis,we study the relationship of antibacterial activity and effect, it show that Trichoderma reesei is less sensitivity to Tachyplesin I, and that the filamentous fungus Trichoderma reesei QM9414 as Tachyplesin I expression host has great potential.In order to study the effect mechanism of Tachyplesin I on Trichoderma reesei, this paper investigates the Tachyplesin I on Trichoderma reesei cell and wall membrance effec-ts,and further reveals the interaction effect between Tachyplesin I and Trichoderma reesei genome DNA. The results showed that cell membrane permeability were affected; Tachypl-esin I suppressed the protoplast recovery growth, reduce the rate of regeneration; phosph-orus and intracellular ultraviolet absorption substance leakage. These results indicate that Tachyplesin I on cell membrane function, because the film is destroyed the pore formation and achieve the purpose of sterilization. It was confirmed that Tachyplesin I can cross the membrance into the cell and damage the genome DNA, it can not only with genomic DNA binding, high concentration can also lead to genomic DNA frature. The function way of Tachyplesin I on DNA is that the interaction and combination between Tachyplesin I and the double spiral on phosphate groups of Trichoderma reesei genome DNA, and parter of the structure of embedded DNA molecular trench, on Trichoderma reesei genome DNA double helix structure had a destructive effect.To produce an antimicrobial peptide (tachyplesin I) via a gene engineering efficiently, this paper uses Trichoderma reesei as host, construct tachyplesin I constitutive expression vector pAN-PSGT-Tac, and the vector transformed Trichoderma reesei protoplast succ-essfully. Using SDS-PAGE electrophoresis analysis the expression product and RT-PCR de-tect the tachyplesin I RNA expression level. At the strip,the target protein size 2.3KDa were not found by using silver nitrate staining of SDS-PAGE electrophoresis. The gene Tac expression was nagetive or low volume at the protein level; The lever of RNA detection shows that, sequencing of the RT-PCR products and the gene Tac are different, the former is shorter, and the presence of multiple base substitution.