| Trehalose is a nonreducing disaccharide that is widely distributed in plants,animals and microbiology,as regard to its physical and chemical stability,its role in enhancing tolerance to various stresses have been reported in many organisms.Heat stress is an environmental factor that couldn't be avoided in traditional bag cultivation methods,it is of great significance to agricultural production in studing trehalose metabolism in Pleurotus ostreatus.Four aspects are included in this research:(1)Trehalose-6-phosphate synthase from Pleurotus ostreatus(PoTPS1)was cloned and expressed in E.coli,enzyme kinetic analyses were conducted to the purified prokaryotic expressed PoTPS1 from E.coli,which were as followed:the Km value against G6P and UDPG were 0.14 and 0.17 mM,respectively,and the Vmax and Kcat values were 91.86 nkat·g-1 and 5.89 s-1,respectively.The optimum pH and temperature for the recombinant PoTPSl were 7.4 and 30?,respectively;furthermore,the trehalose content in the mycelia was measured after heat stress(HS)treatment at 40?,and the results showed that trehalose content was 158.88 mg·g-1 dry weight(DW)after HS treatment for 15 h,and the enzyme activity of PoTPS1 at that time point reached highest,enzyme activity for trehalose degradation maintained at a relatively low level,which further on demonstrated the existence of the TPS-TPP trehalose synthesis pathway in P.ostreatus.(2)An Agrobacterium tumefaciens-mediated transformation(ATMT)system has been established in P.ostreatus.GPD promoter in previous plasmid pGL-GPD was replaced by GPD promoter from P.ostreatus,and the recombinant plasmid was named as Po-gpdOE.Four parameters were optimized as followed:The strain LBA4404 was the most suitable strain for the transformation of P.ostreatus;a bacteria-to-protoplast ratio of 100:1;an acetosyringone(AS)concentration of 0.1 mM,and 18 h of co-culture showed the best transformation efficiency.Southern blot analysis showed that HPH gene was integrated in the genome of P.ostreatus;and EGFP fluorescence assay also showed positive results;the mitotic stability assay showed that more than 75%transformants were stable after five generations.These results showed that our transformation method is effective.(3)HS significantly affected the mycelia radical growth of P.ostreatus,a growth inhibition ring was appeared after HS treatment for a period.Exogenous adding of H2O2 or reactive oxygen species(ROS)scavenging agent N-Acetyl-L-cysteine(NAC)and vitamin C(VC)to the medium under HS,and the results showed that H2O2 severely affected the mycelia growth,while exogenous adding of NAC or VC and HS treatment at the same time showed partly recovered mycelia growth;the trehalose content of mycelia after above treatments were detected,and the results showed that adding of 2 mM and 8 mM H2O2 to the medium increased trehalose content to 117.01 and 130.15 mg·g-1 DW,while adding 2 mM NAC and 8 mM VC and HS treatment at the same time partly recovered trehalose content to 85.86 and 89.23 mg·g-1 DW;all these results indicated that in one aspect,the mycelia growth defect was partly due to the intracellular accumulation of ROS after HS;and in another aspect,ROS participated in inducing trehalose synthesis in P.ostreatus.(4)The role of trehalose in P.ostreatus was also elaborated in this study:exogenous adding of trehalose and HS treatment at the same time significantly relieved the growth defect of the mycelia after HS;further verification by constructing overexpression transformants of PoTPS1 and PoNTH,endogenous trehalose content increased in OE::TPS1-5 and OE::TPS1-9 by 13.60%and 13.42%,while decreased in OE::NTH-2 and OE::NTH-6 by 43.07%and 45.06%;in OE::TPS1-5 and OE::TPS1-9,the growth inhibition rate relived as compared with the control strains and WT strain,while even worse in OE::NTH-1 and OE::NTH-2.In all,the accumulated trehalose in the mycelia could relieve the mycelia growth defects after HS. |
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