Marking The Mycelium Of The Sclerotinia Sclerotiorum Using The Green Fluorescent Protein Gene

Sclerotinia sclerotiorum (Lib.) deBary is an important fungal pathogen that damages a wide variety of crops and vegetables throughout the world. Most monocotyledon and dicotyledon are infected by S.sclerotiorum (Lib.) deBary. At present, traditional agricultural measures are hardly reach good results. Chemical germicides mainly are used to resist the disease induced by Sclerotinia sclerotiorum happened seriously. However, chemic-control is not only costly but also polluted the environment and the effect cannot do execution effectively. At the same time, the food security is impacted. The public attaches importance to the study of bio-control along with concerning about environment and health. To provide the theory and testimony for bio-control, we need to investigate deeply the fundamental of infection and new technology.In this work, a new reporter plasmid had been engineered based on the pCSN43 vector. This plasmid has been designed to produce the highest levels of gene expression in fungus mycelia cells. It contains a modified promoter followed by PtrpC and a high-efficiency TrpC terminator. The plasmid includes GFP gene and hygromycin resistance gene as selected marker. The expression vector was under the control of the PtrpC promoter from Aspergillus nidulans. The expression vector is constructed in a plasmid backbone that has been extensively modified to achieve enhanced levels of transgene expression in mycelial fungus protoplast cells. This work suggests a simple strategy for the expression of GFP gene and marking the S. sclerotiorum mycelia.I have prepared the protoplasts of S. sclerotiorum by digesting the mycelia. Furthermore, the expression vector pPGT was transformed into the protoplasts with PEG transformation method. Positive transformant was harvested by regenerated method and selected method, and then we can observe green flurescence phenomena as positive transformant a response to blue light. Finally it suggested that GFP gene was cloned into genome DNA of S. sclerotiorum, expression and stable inheritance by isolating the genome DNA of positive transformant, Southern blotting and passage culturing.This study provides the premise for the better understanding of the...