The Gene Expression And Function Study Of Hydrophobin And Heat Shock Proteins From Lentinula Edodes

Lentinula edodes is an edible and medicinal fungi,which is fleshy and rich in aroma.It is deeply loved by consumers and consided as "the queen of mushroom ".L.edodes usually fruit under low and alternated temperature.In natural conditions,the optimal growth temperature for mycelia running is 25 ?.In summer the temperature in main producing areas of L.edodes is far higher than 25 ?.It causes severe consequences for L.edodes production due to the high temperature,which results in insufficient supply of fresh L.edodes in summer.So,if we want to fundamentally solve the problem of L.edodes under high-temperature cultivation,it is imminent that find out how to improve the ability of L.edodes in resistance to high temperature stress.Hydrophobins refers to a family of small proteins secreted by filamentous fungi,which show an extremely high surface activity.Hydrophobin has both hydrophobic structure and a hydrophilic structure,with strong amphipathic.Hydrophobin plays a role in protecting against fungi,covering the surface of the fungus,protecting the cell wall integrity and protecting fungal resistance to harsh environments.It has been reported that hydrophobin are associated with fungal resistance under high temperature stress,but at present these proteins are less studied related to high temperature stress.Current research of hydrophobins in edible fungi under high temperature stress is relatively are.Therefore,this paper mainly did an experiment on the gene expression differences of hydrophobins between hot-sensitive strain and hot-tolerant strain of L.edodes.Heat shock proteins(HSPs)are induced when temperature is higher than normal growth temperature above 5 ?,most of the normal proteins in transcription and translation systems are suppressed,but there is still some rapid large-scale synthesis of new proteins.Studies proved that HSPs can coordinate with other co-chaperone protein to repair denatured proteins and degrade misfolded proteins.The higher content of HSPs in plants,the stronger resistance in the face of adversity stress,therefore HSPs play a vital role in plant growth and adversity stress.In this study a research on gene the expression differences of HSPs between hot-sensitive strain and hot-tolerant strain of L.edodes was carried out.This study choosed hot-tolerant strain P18N44 and hot-sensitive strain P18 in L.edodes as the rexperiment materials.At first to filter high temperature condition by different treatment,ultimately high temperature condition(37 ?)and heat shock time(Oh,4h,6h,8h,12h,P18h)were determined.Main results were as follows:1.Hydl gene and hyd2 gene in L.edodes by GenBank were obtained.Heat shock protein genes in each closely genetic relationship species of L.edodes were also found out by NCBI.HSPs can be divided into Hsp 100?Hsp90?Hsp70?Hsp60?smHsp by molecular weight,then a local software blast-2.2.28 + was built by the genome of L.edodes.Finally the gene sequence of proteins were got by blasting in the genome of L.edodes.Primers f hyd1,hyd2,hsp100,hsp90,hsp70,hsp60,smhsp were designed for detection gene expression under high temperature stress by real-time fluorescent quantitative PCR.2.The bioinformatics analysis showed that the Hydrophobins and HSPs were acidic proteins,and Hyd1?Hyd2 and smHsp were unstable proteins;but Hsp100,Hsp90,Hsp70 and Hsp60 were stable proteins.Respective molecular weight and point isoelectric of each protein were given.Hyd1 and Hyd2 were bidirectional transmembrane proteins,but Hsp 100 and smHsp had no transmembrane structure,so they were not transmembrane proteins.Hsp90,Hsp70 and Hsp60 proteins were transmembrane proteins,and these were related to membrane localization and transmembrane transport.The signal peptide prediction showed that Hyd1 and Hyd2 contained a signal peptide,so they belonged to the secreted proteins.HSPs did not exist signal peptides,and it did not belong to a secreted protein.Finally,predicted phosphorylation sites and three-dimensional structural models of each protein were analyzed.3.RT-PCR results showed that the relative expression of hyd1 and hyd2 in the two strains under high temperature stress was significantly different,and the relative expression of the two genes in hot-tolerant strain P18N44 is far higher than that in hot-sensitive strain P18.Within 0 to 8h,the relative expression of hyd1 and hyd2 genes in the two strains was proportional to the treatment time;Within 8 toP18h,the relative expression of hyd1 and hyd2 genes in both strains was substantially inversely proportional to treatment time.During high temperature condition,the relative expression of the protein hsp100,hsp90,hsp60 in hot-tolerant strain P18N44 is far higher than that in hot-sensitive strain P18.At 8h,the relative expression of hsp100,hsp90 and hsp60 in both strains substantially reached the highest value.The relative expression of hsp70 and smhsp in both strains were shown unstable state;To sum up,hyd2 and hsp60 genes were picked out from in hydrophobin and HSPs,and then make the two genes heterologous expression were stydied to verify their functionality.4.Recombinant vect pET-32a/hyd2?pET-32a/hsp60 and empty vector pET-32a were introduced into E.coli BL21 respectively.E.coli BL21 was treated by heat shock(50?)at Omin,30min,60min,90min,120min.Results of survival rate showed that Hyd2 and HSP60 from L.edodes can improve the viability of E.coli.SDS-PAGE analysis proved that the protein of Hyd2 and Hsp60 indeed existed and these recombinants could improved the capacity of E.coli against higher temperature stress(50?)and could protected other proteins in cells.Hydrophobic protein and heat shock protein function studies showed that Hyd2 protein and Hsp60 had the potential to improve strains of L.edodes in adaptation to high temperature stress.