Construction Of Recombinant Yeast GS115/PSD,GS115/CEC And Study On Their Inhibition Effect On Fruit Postharvest Decay

Considerable amount of fruits and vegetables is lost due to decay after harvest. Biological control of postharvest decays (BCPD) with antagonistic yeasts have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables. However, the effectiveness of general yeast on postharvest decay control is not comparable to fungicides. The future expansion of it will largely depend on discovering new antagonists or improving its effectiveness. Using genetic manipulation to improving antagonists in BCPD is a new way in biological control of postharvest fruit. The current study was undertaken to investigate the possibility of enhance biocontrol activity of antagonists against fungal pathogens by expressing a DNA sequence in yeast to allow for the production of an antifungal peptide to produce an improved biocontrol. According to the experiment design, Cecropin A and PsdI gene was successfully expressed in methylotrophic yeast Pichia pastoris GS115. Recombination yeast GS115/CEC and GS115/PSD showed great biocontrol efficiency in inhibiting postharvest diseases of fruits and vegetables. The capability of growth of recombination yeast, the safety of yeasts, the efficacy of recombination yeasts alone or in combination with other control means for controlling postharvest disease of cherry tomatoes, oranges, pears and apples were tested. Meanwhile, the antibacterial mechanism of yeast and fermentation condition optimization of recombination yeasts were studied.The results were shown as below:1. We synthesized the gene fragment of peptides cecropin A and psdI encoding the mature peptide gene according to the genebank sequence of its, and then the fragments was connect to PGEM-T vector. Connect cecropin A and psdI gene to the expression vector of Pichia Pastoris after constructing on pPIC9K. After the linearization of it, punch with electricity law channel P. Pastoris GS115 into. Adopt G418 to gradient screen high transformation son of resisting etc. Through Triciene-SDS-PAGE, we confirmed the expression success of recombination yeasts after 72h induced by methanol. The experiment of fermentation and PCR method showed that recombination yeasts had a good stability.2. Improved expression level of recombinant yeasts was achieved by optimization both the growth phase and induction phase culture conditions. The results indicated that the optimum fermentation condition of the growth phase is as follows:2% glycerol,2% (NH4)2SO4, pH=6.5-7.5,30℃,72h. The test results of induction phase showed that the optimum desired fermentation condition is as follows:1% methanol,2%(NH4)2SO4, pH=7.0,30℃,72h. The fermented condition of recombinant yeasts after adopting the orthogonal experiment to optimize, The main factor that its influence ferments is as follows, temperature(30℃)> concentration of methanol (1%)> ferment time (4 d)> pH of the solution (5.5). Five times repeat fermentation experiments of strain GS115/CEC and GS115/PSD under the optimum conditions show that the average protein content is 15.1 and 11.3mg/L correspond with results of the orthogonal test.3. Recombination strain GS115/CEC and GS115/PSD showed great inhibition activity on four kinds of fungus(Alternaria alternata, Botrytis cinerea, Botrytis cinerea and Geotrichum citri-aurantii) in vitro tests. Recombination strains also showed a significant inhibitory effect against all four pathogens in fruits. In reducing the development of postharvest diseases of fruits and vegetables, including:black rot of cherry tomato, caused by A. alternata; gray mold decay of cherry tomato, pear and apple, caused by B. cinerea; green mold of apple and pear caused by P. expansum. The concentrations of yeasts had significant effects on biocontrol effectiveness to postharvest diseases of fruits:the higher the concentrations of recombination strains, the better the control efficacy.4. The results about the inhibition mechanism of the recombination yeasts showed that: besides of tranditional competing for nutrients and space, the results obtained by scanning electron microscopy and transmission electron microscopy showed that the morphology and inner structure of the fungi treated with recombination strain GS115/CEC changed obviously. Some of the cells were made abnormality, rupture or lysed.5. In an attempt to clarify the molecular mechanism of action, we examined the binding properties exerted by the peptide expressing by recombination yeast on DNA and RNA of fungal cells. Results showed the expression product can inhibit the migration of RNA. With the increasing of the quantity of peptides, the moving speed of nucleic acids in agarose became slower. Meantime, DNA and RNA of the fungal cells were degraded by the peptide. And then the metabolic process of fungi was destroyed and the fungi were killed. Moreover, the expression level of PR proteins that play an important role in the plant pathogen attack response was induced by recombination yeast. So, enhancing the resistance activity of fruits to pathogens may be another mechanism of control postharvest disease of fruits and vegetables of recombination strain.6. The efficacy of recombination yeasts on the control of postharvest decay of fruits was enhanced significantly by combining with metal salts (CaCl2), carbonate and ethanol in vivo test. The efficacy of GS 115/CEC for control of fruits postharvest disease was significantly improved when combined with these allogenic materials.7. On the safety test, the results of acute toxicity to mice (food standard) showed the dried powder of recombination yeasts were safe. The quality parameters of fruits treated with recombination yeasts were similar to those of control fruit, which suggested that the transformed yeast did not impair quality parameters of fruits.