| Postharvest fungal diseases cause substantial economic losses world-widely. In view of the fungicide toxicity on the environment and human health, and the development of fungicide resistance by pathogens, great efforts have been made to exploit alternatives to the synthetic fungicides. The biological control by using antagonistic yeasts is one of the most promising means. Strains of Cryptococcus laurentii are well known postharvest biocontrol yeasts, which have been shown high antagonistic activity in various fruit. Antagonistic yeasts have been selected mainly for their activity does not generally depend on the production of toxic metabolites, which could have a negative environment or animal toxicological impact, and for their ability to colonize and grow rapidly in surface wounds and for there are generally poorly sensitive to fungicides. Different mechanisms of action have been suggested for antagonist yeasts, which could be complementary to each other. Competition for space and nutrients, which is believed to be the major mode of action of antagonistic yeasts. In addition, several studies have shown that the capability of producing and secreted cell wall-degrading enzymes may involved in the action of antagonistic yeasts. Also the induce resistance in the host tissue play important role in the action of antagonistic yeasts. The objective of this paper were:(1) using SSH methods and Tomato Genechip to isolated different expression genes from tomato fruit after treat by Cryptococcus laurentii, (2) select four differently expressed genes from results of SSH and Tomato Genechip, clone Open Reading Frame (ORF) and then transform to Arabidopsis, for further function analysis. (3) detect the activity changes of defense-related enzymes include CAT, SOD, PPO, PAL, POD, LOX and concentration changes of MDA after treated by Cryptococcus laurentii or 1-MCP alone and combine.SSH was performed with cDNA from cherry tomato fruit inoculated by water as the "driver" and cDNA from tomato fruit inoculated by Cryptococcus laurentii as the "tester". A selected 50 clones in the SSH library were sequenced. BLASTX results revealed that 33 cDNAs had significant sequence homologies with known sequences in the NCBI database. The identified cDNAs encoded proteins involved in cellular processes such as:primary metabolism; Ripen-related; signal transduction; Defense and pathogen-related or stress stimuli or wounding. Six clones were selected for temporal expression analysis using RT-PCR based on the results of the reverse Northern blot screens. Results from RT-PCR confirmed the differential expressions of these transcripts. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated under some biotic and abiotic stress are also up-regulated after the application of biological control yeast to cherry tomato fruit. The expression of these proteins might increase the fruit resistance towards postharvest pathogen infection and damage.To obtain an overall picture of gene regulation during biocontrol yeast treated, two independent microarray analyses were performed. To reduce experimental variation, two sets of twenty fruits were harvested from biocontrol yeast-treated and untreated(wounding control) fruit, respectively, at 24h post treatment. 531 genes were selected from Tomato Genechip, the number of genes with increased signals was 219,123 of 219 have unknown function and that with decreased signals was 312,144 of 312 have unknown function. The majority of the up-regulated genes seem to be related to three major biological changes induced by biocontrol yeast in the tomato fruit, primer metabolism (10%), defense-related (7.7%) and transcription (4.5%). The three largest functional categories (except for unclassified protein) of the down-regulated genes are primary metabolism (13%), photosynthesis (7.7%) and energy metabolism(6.7%). Results from Tomato Genechip indicated that Cryptococcus laurentii can induced SA mediate SAR reaction and suppressed JA/ET mediate resistance reaction, at same time suppressed genes expression involved in energy metabolism and photosynthesis.For further analysis selected genes functions ORF of four selected genes were cloned and transformed in to Arabidopsis. The transgenic plants have been obtained.In order to got insight view to the relative between 1-MCP treatment and fruit resistance to postharvest pathogens. Effects of 1-MCP on resistance-related enzymes include catalase(CAT), Peroxidase (POD), Polyphenoloxidase (PPO), Phenylalanineammonialyase (PAL), Superoxide dismutase (SOD, linoleate:oxygen oxidoreductase (LOX) and malondialdehyde (MDA) concentration of cherry tomato fruit were determined. 1-MCP treated can induce PPO and POD activity, increased the concentration of MDA, and suppressed LOX and PAL activity. 1-MCP treated have on effect on SOD activity in cherry tomato fruit. The peak of CAT activity was bring forward 12h ahead after treated by 1-MCP compare with the control. |
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