Regulationon Of Ethylene And Polyamines On Postharvest Senescence Of Pleurotus Eryngii And Preliminary Investigation On PeSAMDC

As one of the most promising medicinal and edible fungi in recent years,P.eryngii is increasingly favorable to consumers owing to its particular flavor and balanced nutrition.After harvest,the fruiting bodies are susceptible to dehydration softening,tissue browning and quality decaying,with extreme short shelf-life under ambient temperature mainly due to its high water content and lacking of protection on the surface,as well as high respiration rate.Therefore,further exploring the mechanism of postharvest senescence in P.eryngii is important for mataining storage quality and extending shelf-life of fruiting bodies.As a key regulator of ripening,ethylene plays a cruciall regulatory role in the maturation and senescence of both climacteric and non-climacteric fruits and vegetables.However,polyamines are ubiquitous polycationic nitrogenous bases of low molecular weight,which are regarded as multi-functional regulators of physiological processes and have been generally suggested to stabilize cell membranes and delay senescence.While S-adenosylmethionine（SAM）is a common precursor in the biosynthetic pathways of ethylene and polyamines,both of them have the opposite physiological function,which causing their regulation of postharvest senescence has received widespread attention in recent years.However,it is unclear what role of ethylene and polyamines play in the aging process of P.eryngii.In the present study,we further analyzed the regulation of ethylene and polyamines on postharvest senescence of P.eryngii respectively and preliminary investigated the S-adenosylmethionine decarboxylase gene cloned from P.eryngii（PeSAMDC）,the key enzyme of polyamines synthesis.The major results of this research were as follows:（1）The endogenous ethylene production as well as the effect of exogenous ethylene treatments on senescence process of postharvest P.eryngii was investigated.Ethylene production of postharvest P.eryngii fruiting bodies exposed to exogenous ethylene at concentrations of 0,1,10,100 and 1000μL/L respectively were detected by gas chromatography.Results showed that the biosynthesis of ethylene gradually increased with the continued maturation of P.eryngii throughout the postharvest storage and exhibited two peaks in 12 h and 36 h.Furthermore,the two peak values were as 12.53 and 7.57 times respectively,high as that of sporophores before storage（0 h）.Moreovre,exogenous ethylene speeded up the endogenous production of P.eryngii in varying degree,exacerbated the increase of PPO（polyphenol oxidase）and PAL（phenylalanineammonialyase）activities,moreover kept browning degree at a higher level,suppressed the decrease of relative electrical conductivity and MDA（malondialdehyde）,promoted the degradation of soluble sugar and protein,inhibited the rise of antioxidant enzymes activities（APX,CAT,POD,and SOD）,expedited the decline of DPPH（1,1-diphenyl-2-picryl-hydrazyl）scavenging ability,decreased total antioxidant capability（TAC）,total phenol and flavonoid contents,promoted the deterioration of the phenotype.The above mentioned indicated that ethylene probably participated in the postharvest senescence of P.eryngii.（2）The effect of preharvest exogenous polyamines sprayed on the contents of endogenous Spd and Spm,ethylene production,membrane system and deterioration phenotype of postharvest fruiting bodies were detected in P.eryngii.The results demonstrated that sprayed with 1 mmol/L exogenous Spd and Spm could siginificantly trigger the biosynthesis of endogenous Spd and Spm,suppress the increase of malondialdehyde（MDA）content and relative membrane permeability,maintain lower level of reactive oxygen species（ROS）,inhibit the decrease of S-adenosylmethionine decarboxylase（SAMDC）activity of P.eryngii,expedite the decline of the ratio of ethylene and polyamines（E/Spd and E/Spm）,moreover,promote more S-adenosylmethionine（SAM）turn to polyamine synthesis,which suppressed the release of ethylene and maintained a high storage quality of fruiting bodies.It can be seen that exogenous polyamines treatment could delay the postharvest aging of P.eryngii.（3）Protoplasts were obatained successfully using P.eryngii mycelium,which was digested for 2.0 h at 31℃ and 120 rpm in 2.0%lywallzyme with 0.6 mol/L of MgSO₄ as osmotic stabilizer.To determine the optimal concentration of hph（hygromycin B）in the screening medium,P.eryngii mycelium and protoplasts were inoculated on PDA plates with different concentrations（0,10,20,30,40,50,60 and 70μg/mL）of hph,indicating that the growth of mycelia and protoplasts was completely inhibited at a final concentration of 60μg/mL hph.A plasmid pYf11-hph,containing hph resistance gene（hygromycin phosphotransferase gene）,enhanced green fluorescent protein gene（egfp）and a strong promoter of RP27,was transferred into the protoplasts of P.eryngii by PEG/CaCl₂-mediated method.Results from hph resistance screening,PCR and real-time PCR indicated that 17 of these putative transformants had been definitely integrated of hph gene.Moreover,the mycelium of the transformants showed green fluorescence on fluorescence microscopy.These results showed that the exact integration of the selectable marker gene hph and reporter gene egfp into genomic DNA of P.eryngii.（4）The full length cDNA of PeSAMDC was 1318 bp,encoded for a protein of 423 amino acid residues,cloned from P.eryngii by sequence alignment with other basidiomycetes.The PeSAMDC gene was then transferred into the end site of strong promoter RP27 of pYf11-hph to construct the overexpression vector pYf11-hph-PeSAMDC though recombination technique.Then,the vetor was introduced into P.eryngii protoplasts though PEG/CaCl₂-mediated approach,and transformants were confirmed by hph resistance screening,PCR and real-time PCR verification.Concequently,we obtained the overexpression strains and fruiting bodies of PeSAMDC.