Preliminary Study On Molecular Mechanism Of CYT B Gene1166bp Intron Infulencing The G143A Mutation In Monilinia Fructicola

Brown rot caused by Monilina spp. is one of the important diseases of peach. Monilinia fructicola is the dominant species in China which can be collected in many orchards in different areas. Currently, the most efficient method to control this disease is fungicide application such as benzimidazoles (BZIs), ergosterol demethylation inhibitors (DMIs) and the quinone outside inhibitors (QoIs). Resistant strains to BZI and DMI fungicides have been detected in the field, but the Qol resistant strains have not yet found. The cytochrome b (Cyt b) gene, the target gene of the QoI fungicides was cloned and sequenced. Interestingly, a1166bp intron was found at the position143of the Cyt b predicted amino acid sequence. QoI fungicide resistance is conferred by a single point mutation at position143from glycine to alanine (G143A) in the Cyt b gene of many pathogens. The presence of an intron at the position143has been shown to prevent the formation of the G143A mutation even the solid evidence is still limited. In the present study, preliminary study was carried out to elucidate if the intron at position143affect the occurrence of the G143A mutation.The PEG-mediated protoplast transformation system was constructed in M. fructicola. Protoplast preparation conditions were optimized:isolates were grown in CM liquid medium with glass beads shaking every8hours for three days to collect the mycelium. Mycelium was digested in1.5mg/ml Snailase and10mg/ml lysozyme for3h with vigorous shaking and high quality protoplasts could be obtained. Even the homologous replacement knockout vector pKOC was constructed, unfortunately the homologous replacement transformants were not obtained currently.It was confirmed that3-4nuclei were in individual M. fructicola conidial spores. Agrobacterium-mediated transformation system was preliminarily established. However, the transformants were unstable most likely as the M. fructicola is a typical microorganism with multi-nuclei cells.Using the yeast expression system, the Cyt b cDNA with the G143A mutation followed by the1166bp intron and wild type cDNA with the1166bp intron at position143were integrated into yeast DNA. The transformants were used to analyze if the G143A mutation influence the splicing of the1166bp intron by investigating the transcription of the Cyt b genes. Results showed that the1166bp intron could not cut either in mutated or wild type Cyt b genes, suggesting that the heterogenous intron could be spliced in yeast cells.