Cloning And Functional Analysis Of NPS6Gene In Setosphaeira Turcica

Maize is the important cereal crop in the world after wheat and rice. Many countries in the worlduse maize for different purposes due to its economic important value domestically and industrially.Maize production is faced with many constraints in the field and post harvest. The major constrainthindering high production is Northern Corn Leaf Blight disease caused by the fungus Setosphaeriaturcica which is a member of ascomycetes filamentous fungi. In this group of fungi, the virulencecharacteristics is determined by NPS6gene which play roles in preventing Hydrogen peroxideproduced by the maize plants. However, NPS6gene in Setosphaeria turcica as not yet been cloneddespite the fact that NPS6gene is conserved throughout filamentous fungi.Therefore, in this study we aim at cloning and functionally characterize the NPS6gene inSetosphaeria turcica.Different experiments were conducted in such a way that this gene can be cloned and functionalcharacterized.Gene length9420bp was first cloned by applying a genome walking method and technique. Thegene information was available relying other filamentous fungi such as Magnaporthe oryzae,Cochliobolus heterostrophus and Neurospora crassa N PS6.Knockout vector pXEH-NPS6LRF was constructed by1350bp sequence for the left flank and1000bp for the right flank separated by Hygromycin phosphotranferase (hph) insert under trpcpromoter. Protoplast transformation method was used where by Fungal strain St28A was preparedthe protoplasts and used in the transformation with vector for regeneration of transformants.Screening for the mutants was performed by analyzing all collected transformants for the presenceof hph. The analysis revealed that, all transformants consisted the hph gene, not only that, they werealso analyzed for the presence of NPS6gene. We got some transformants right now. We are doingvalidation for the transformants looking for the real NPS6knockout mutants.