Research On The Molecular Mechanism Of Degradation Of Patulin By Pichia Caribbica

Patulin(PAT)is a highly toxic polyketide secondary metabolite which produced by PAT-producing fungi such as Penicillium expansum.P.expansum infects fruits and their derivatives and produces large amounts of PAT during the infection process.The blue mold disease generated by the global prevalence of P.expansum causes huge economic losses to the global fruit industry.Furthermore,PAT produced by P.expansum poses a serious threat to the health of consumers.With the developing of research on biological control of postharvest diseases of fruits,biocontrol or biodegradation of PAT in fruits and their derivatives by using antagonistic yeasts has become a hot topic in this field and shows a huge application prospects.A previous study of our group found that Pichia caribbica can significantly inhibits the accumulation of PAT in apples.In addition,P.caribbica can efficiently degrade PAT in vitro.However,degradation products of PAT by P.caribbica,the toxicity of these degradation products,and the molecular mechanism of PAT degradation by P.caribbica remaining unknown.Therefore,the main research points of this dissertation are as follows: identification of degradation products of PAT by P.caribbica;toxicity analysis of degradation products of PAT;studying the response of P.caribbica to PAT in genes and proteins expression levels using transcriptomics and proteomics techniques;roles of PcCRG1 in the degradation of PAT by P.caribbica.The results are shown as follows:1.P.caribbica degrades PAT and forms intermediates E-ascladiol and Z-ascladiol,E-ascladiol and Z-ascladiol are then eventually degraded to form unknown substances.Pretreated with PAT enhanced the capacity of intracellular enzymes of P.caribbica on degrading PAT,indicating that the PAT degradation of P.caribbica is an inducible process,and the induced enzymes of P.caribbica produced after PAT stimulation are involved in degradation of PAT.2.Toxicity analysis of the intermediate product ascladiol,and final degradation products of PAT degradation was performed.Ascladiol and the final product have no influence on the growth of Escherichia coli.However,the growth of E.coli was significantly inhibited by PAT.Seed germination and plant of Arabidopsis thaliana was significantly inbithed by PAT,but was not influenced by the ascladiol and the final product.Ascladiol and the final product had no effect on the growth of human esophageal epithelial cells(Het-1a),ROS accumulation,cell proliferation and apoptosis,whereas PAT significantly inhibited cell activity,stimulated ROS accumulation,and led to apoptosis.The result indictaed that ascladiol and the final product are both not toxic to E.coli,A.thaliana,and Het-1a.3.Studying was performed on analysising the response of P.caribbica to PAT in proteins expression levels based on proteomics technology.The results showed that 119 proteins expression levels was differentially down regulated and 51 proteins expression levels was differentially up regulated by PAT.Down-regulated of proteins are mainly involved in metabolism and biosynthesis.Among the 51 up-regulated proteins,22 genes were annotated to oxidoreductases.In addition,the expression level of superoxide dismutase(SOD),glutaredoxin(GRX)and thioredoxin(TRX)which were involved in defensing oxidative stress.The results show that P.caribbica responds to the oxidative stress caused by PAT by activating the expression of the above proteins to maintains the redox balance in P.caribbica.4.Studying was performed on analysising the response of P.caribbica to PAT in genes expression levels based on transcriptomics technology.The results showed that the expression of 151 genes were significantly down-regulated and 83 genes were significantly up-regulated after treatment with PAT.These down-regulated proteins are mainly involved in protein biosynthesis,amino acid metabolism and carbohydrate metabolism,which indicating PAT exhibits certain toxicity to P.caribbica.These up-regulated proteins are mainly involved in oxidreductase processes and biological reponse to chemical substances and toxin,which are consistent with proteomics results.In addition,the expression levels of PcCRG1,PcMDR1,and PcFMOL1 that are closely related to the removal or conversion of chemical substances were significantly up-regulated.5.The role of PcCRG1 gene in degrading PAT was studied.The results showed that the effect of PAT degradation by PcCRG1 knock-out mutants was significantly reduced,while the effect of PAT degradation by strians overexpressing of PcCRG1 was significantly enhanced.In vitro biochemical studies showed that PcCRG1 enzyme directly degrade PAT in the presence of the substrate S-adenosylmethionine(SAM).