Overexpression of the Bivalent Antibacterial Peptide Genes in Pichia pastoris Delays Sour Rot in Citrus Fruit and Induces Geotrichum citri-aurantii Cell Apoptosis

摘要

Genetic manipulation of antagonists shows tremendous potential for improving biocontrol activity of antagonists. Here, bivalent antibacterial peptide Cecropin A/PsdI gene was transformed and expressed in methylotrophic yeast, Pichia pastoris GS115. The bivalent yeast GS115/CEC-PSD showed a great ability in delaying sour rot in citrus fruit and based on observations of restrained growth of geminated Geotrichum citri-aurantii spores relative to non-transformed strain. Although there was no remarkable discrepancy between growth rates of strain GS115 and GS115/CEC-PSD in vivo and vitro, plenty of vacuoles were observed in the cellular space of G. citri-aurantii treated with the CEC-PSD via transmission electron microscopy. Meanwhile, a gel-retardation assay exhibited that the DNA of G. citri-aurantii was degraded by CEC-PSD. Furthermore, our research revealed apoptosis genes in G. citri-aurantii were expressed at different levels with the CEC-PSD levels, among these genes, the expression level of RLM1 and RLM1 was decreased remarkably, and the expression level of others changed relatively a little. These findings suggest that bivalent antimicrobial peptides can inhibit G. citri-aurantii by harming cell membrane, disrupting the cell metabolism, inducing programmed cell death and can therefore heighten the inhibition effect of the biocontrol yeast against postharvest diseases and this provides a new approach for the biological control of postharvest diseases.