In recent years, acid rot is one of the main diseases found in fruit ripening and storage and transportation. It not only causes rotten fruit in late harvest, but also seriously affects the quality of wine grape and its products. Grape acid rot is caused by acid rot fungus (Geotrichum citri auranti). At present, chemical fungicides are mainly used to control acid rot, but this chemical method will not only lead to the production of resistant strains, but also the fungicides left on the surface of fruits will seriously harm human health. In recent years, more attention has been paid to phytoalexin. pterostilbene is an important phytoalexin produced by grapes under the pressure of resisting external biological or non biological stress. In the early stage, it was found that pterostilbene can effectively inhibit the activity of acid rot fungi through in vitro antibacterial experiment. However, the defense mechanism of pterostilbene against acid rot fungi in grape is not clear, so this experiment focused on the mechanism of pterostilbene against acid rot fungi, isolated and identified the secondary metabolites of grape defense acid rot fungi, cloned and analyzed the key genes of pterostilbene defense acid rot fungi, and The research results include the following aspects: 1.
1. The response of objective gene wgreom in the process of pterostilbene defense against acid rot was detected by fluorescence quantitative PCR. The results showed that the expression of wgreom gene changed with the infection time, showing a trend of first increasing and then decreasing. When infected for 24 hours, the expression of the target gene wgreom reached the highest level. After 48 hours, the expression decreased rapidly. After 72 hours, it was almost not expressed.
2. Using eukaryotic expression system and HPLC technology to verify the function of the target gene in vitro. Firstly, the eukaryotic expression vector ppic9k-reom was constructed, and the vector containing the target gene wgreom was transferred into Pichia pastoris expression system to induce its expression. The corresponding protein was detected by SDS-PAGE and Westerm blot technology, with a size of about 34.4kda. Then, the substrate was fed to the bmgy medium containing recombinant yeast, and pterostilbene was detected by HPLC technology. In vitro, it was proved that wgreom has the function of oxygen methylation to catalyze the production of pterostilbene.
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