Function Analysis Of Heat Shock Protein LeDnaJ-07 In Lentinula Edodes

Lentinula edodes,being famous for highly nutritional and pharmacological value,is overwhelmingly popular with consumers and manually cultivated by the large scale in the east-Asia regions such as China and Korea as well as Japan.Recently,Trichoderma species in L.edodes logs widely reported.Trichoderma sp.not only leads to the rotten logs for the part or all but also heavily affects the development of L.edodes fruiting bodies,reducing the production of L.edodes and resulting in a big loss in yield,which was induced by high temperature in our lab previous study.On the basis of L.edodes genome information,we analyzed the regulation mode of L.edodes on the response to heat stress and selected the genes and metabolites related to the thermotolerance by the proteomic and transcriptomic analysis,and verified their gene function.Meanwhile,the interaction among proteins involved in the response of L.edodes against heat stress was analyzed via the yeast two-hybrid system and bimolecular fluorescence complementation(Bi FC)assays.1.It was documented that 40? was lethal for 80% L.edodes strains using 36?-42?high temperature treatments on L.edodes mycelia for 24 h,indicating that 40? was used as the stress temperature in the later experiment.The thermotolerant strain S606 could regrow after 24 h of 40? heat stress,while the heat-sensitive strain YS3357 lost the recovery capacity.The mycelia of S606 and YS3357 were treated in a 40? incubator for24 h.Then,they were collected to extract protein and RNA.The proteome analysis exhibited that the contents of HSP40(Dna J),HSP70,HSP98 and the proteins(Letrp D,Le TAM-1 and Le YUCCA)related to tryptophan and IAA biosynthesis were significantly upregulated,and their contents in S606 were higher YS3357 after heat stress.Anthranilate synthase(Letrp E)had a about 500-fold increase in S606 after heat stress,but an obvious down-regulation with no detection.The transcriptomic analysis displayed that the transcript levels of many HSPs(s HSPs,HSP70,HSP98 and HSP104)were dramatical upregulated.0.01 mmol/L OABA and 0.01 mmol/L tryptophan could enhance the recovery capacity of S606 after 40? heat stress,but no effect on YS3357.0.01 mmol/L IAA conferred the recovery capacity to the heat-sensitive strain YS3357 after heat stress,yet inhibited the recovery capacity of S606.It was found that the intracellular IAA content with an about 10-fold increase after heat stress in S606 was higher than that with an about 2-fold increase in YS3357.2.On the basis of the function annotation information of L.edodes monokaryon strain W1-26,29 amino acid sequences containing J domain were identified.According to their position in L.edodes genome,29 genes encoding those J proteins were numbered from LeDnaJ01 to LeDnaJ29.The length of the LeDnaJ protein sequences varied from 102 to661 amino acids.The predicted molecular weights were between 11 k Da and 74 k Da,and the predicted p I values of LeDnaJ proteins ranged from 4.86 to 10.19.The analysis of gene structure showed that the most and least numbers of the introns were thirteen and one,respectively.Whether the amino acid sequences have the J domain and zinc finger domain as well as C-terminal domain or not,LeDnaJ proteins were classified into four groups: group A and group B had three genes,respectively;group C had nineteen genes;group E had four genes.Two major groups of cis-elements were observed in the promotor areas,including stress-responsive and hormone-responsive.During the development stage of L.edodes,29 LeDnaJ genes exhibited two different expression patterns: constitutive expression and tissue-specific expression patterns.The genes differently expressed by Cd and T.atroviride as well as heat stress accounted for 79.31%,55.17% and 37.93%,respectively,demonstrating that LeDnaJ participate in the regulation process of growth and development of L.edodes and the response to the stresses.3.The LeDnaJ07(LeDnaJ)was promoted by the promotor of the Legpd to construct the overexpression(OE)vector,and the antisence fragment(600bp)of the conservative function area in LeDnaJ07 was promoted by the promotor of the Legpd and Leactin to construct the RNAi vector.The vectors were transferred to L.edodes strain S606 mycelia via Agrobacterium tumefaciens-mediated transformation method.q RT-PCR result showed that the relative expression levels of two OE tranformants had approximate seven and thirteen-fold upregulation,the relative expression levels of two RNAi tranformants had more than 100-fold downregulation compared with those of S606.LeDnaJ RNAi inhibited the growth of L.edodes mycelia at 25?.After 41? heat stress for 24 h,mycelia of the two OE strains could regrow,but mycelia of the wild type(WT)strain S606 could not regrow.After 40? heat stress for 24 h,mycelia of the WT strain S606 could not regrow,but mycelia of the two RNAi strains could not regrow.After 30? and31? heat stress for 15 d,the colony diameters of two OE strains were larger than those of the WT strain S606 for 6 d and 15 d recovery time,yet no growth for the two RNAi strains.Intracellular IAA levels in the OE and RNAi strains were higher and lower than it in the WT strain S606,respectively.Antagonism experiments between L.edodes and T.atroviride mycelia revealed that the LeDnaJ07 overexpression in S606 enhanced the resistance of L.edodes to T.atroviride,but the LeDnaJ RNAi in S606 decreased the resistance of L.edodes to T.atroviride.The mycelia of two RNAi strains were fully overgrown by T.atroviride mycelia.0.01 mmol/L IAA could partly restore the resistance of the two RNAi strains to heat and T.atroviride stresses.The mcherry protein and LeDnaJ07 were promoted by the promotor of the Leactin to construct the fusion overexpression vector transferred to L.edodes strain YS55 mycelia via A.tumefaciens-mediated transformation method.9 OE positive transformants were gained by PCR method.q RT-PCR result showed that the relative expression levels of e two OE tranformants had approximate two and four-fold upregulation compared with those of YS55.Under UV view,the two OE strains showed the red fluorescence,yet no red fluorescence was found for the WT strain.LeDnaJ07 overexpression promoted the growth of L.edodes mycelia at 25?.After 30? and 31? heat stress for 15 d and 38?heat stress for 24 h,mycelia of the two OE strains could regrow,but mycelia of the WT strain YS55 could not regrow.Antagonism experiments between L.edodes and T.atroviride mycelia revealed that the LeDnaJ07 overexpression in YS55 enhanced the resistance of L.edodes to T.atroviride,and promoted the brow substance formation.The mycelia of the WT strain YS55 were fully overgrown by the mycelia of T.atroviride,and no obvious brown substance was found.The results indicated that LeDnaJ07 involved in the regulation of mycelial growth,the resistance of mycelia to heat and T.atroviride stresses and IAA biosynthesis.4.On the basis of the vector framework of p GADT7 and p GBKT7 plasmids,the ORFs of LeDnaJ07 and Letrp E were linked with the linearization vectors digested by Bam H-I and Eco R-I via homologous recombination to gain the bait vector plasmid(p GADT7-Letrp E)and library vector plasmid(p GBKT7-LeDnaJ07).Then,they were transferred to the yeast AH109 and Y187,respectively.After mating,the yeast cells containing p GBKT7-LeDnaJ07 and p GADT7-Letrp E mixture could grow on the 4-SD medium(Trp-,Leu-,His-,Ade-),and turn blue on the medium containing X-?-gal.Based on the vector framework of p GADT7 and p GBKT7 plasmids,the ORFs of LeDnaJ07 and Letrp E were linked with the linearization vectors digested by Bam H-I and Xho-I via homologous recombination to gain the C-terminal vector plasmid(p SCYCE-Letrp E)and N-terminal vector plasmid(p SCYNE-LeDnaJ07).Then,the two plasmids were transferred to the A.tumefaciens,respectively.Epidermal cell injection of tobacco leaves displayed that tobacco leaves infected by the test group(p SCYCE-Letrp E and p SCYNE-LeDnaJ07)showed the green fluorescence.The interaction between LeDnaJ07 and Letrp E was verified by the yeast two-hybrid and Bi FC assays.In this study,transcriptome and proteome were performed for the first time to analyze the gene and protein change of L.edodes mycelia after heat stress,select the genes related to thermotolerance and metabolites,identify Dna J protein family in L.edodes gemome,and perform gene colne and function analysis of LeDnaJ07.LeDnaJ07 not only directly regulates the termotolerance of L.edodes but also interactes with Letrp E,a rate-lating enzyme in IAA biosynthesis,to regulate the termotolerance of L.edodes via meditating IAA biosynthesis.This study preliminarily uncovered the molecular mechanism on the response to L.edodes mycelia to heat stress,laying the foundation for further studying the molecular mechanism on the resistance to stresses and resistance breeding.