Induction Of Apoptosis By Hydrogen Peroxide In Trichoderma Reesei And The Flow Cytometric Assessment Of Protoplast Viability

Trichoderma reesei (anamorph: Hypocrea jecorina) is an important filamentous fungus, which can produce a complete set of cellulases, hemicellulases, amylase and protease. In addition, it is an efficient host for heterologous protein expression. As a lower multicellular eukaryote, T. reesei has simple structure and fast growth rate, which make it significant in the researches of fungi heredity and metabolism.Apoptosis is a conserved mechanism that plays an essential role in eukaryotes. Malfunction of apoptosis contributes to many human diseases including cancer and AIDS. Research on yeast and filamentous fungi apoptosis is also expected to become new growth point in the biological field. During industrial fermentation, aerophilic fungus T. reesei is exposed to oxidative stress constantly, which can cause death of filament. It is speculated that apoptosis involves in this course.Here hydrogen peroxide was selected to mimic oxygen stress to study the mechanism of cell death. We have found that apoptosis can be induced in T. reesei by low external doses of H₂O₂. The accepted morphological markers of apoptosis in fungi were observed, including cytoplasmic vacuolization and extemalization of phosphatidylserine. DNA ladder, a commol/Lon phenomenon in metazoan apoptosis, is deficient in T. reesei. This research provides new insight into the death during fermentation.Flow cytometry is a powerful biomedical technology, which is also called Fluorescence-Activated Cell Sorting. Data are collected for each individual cell, which enables the investigator to measure the distribution of a property or properties within the population. However, because of the filamentous morphological limits, flow cytometry has little applicant in fungal research. This study assesses the potential of flow cytometric method to study the protoplast viability in filamentous fungi T. reesei. Cell viability after treatment with H₂O₂ was determined using the green fluorescent protein marker gene in combination with the viability indicator propidium iodide. This study demonstrates the successful application of flow cytometry to monitor filamentous fungal protoplasts.Our findings show that enough functional conservation exists between apoptotic machineries of mammol/Lals and fungi. We are trying to construct industrial strains by expression of anti-apoptotic proteins or deletion of pro-apoptotic genes in T. reesei. These strains are expected to have prolonged longevity and exhibited enhanced stress resistance. Besides, flow cytometric method to study fungal viability was successfully built up in this thesis. Since flow cytometric analysis does not cause any damage to cell viability, sorting these cells directly onto agar plates is advantageous to microbiologists.