| With the improvement of living standard,consumers have become increasingly interested in the quality of Lentinula edodes.The studies on the flavor of L.edodes mainly involved in the effects of processing methods and cultivation substrates However,little is known of the genetic-mechanism of flavor formation.Therefore,studying the biological mechanisms has become important for quality improvement In this study,enzymes related to sulphur-containing compounds and transcriptional levels of related genes were investigated during hot-air drying processing of L.edodes fruiting bodies.Meanwhile,rice straw was substituted for sawdust at different ratios to cultivate L.edodes.The effects of adding cropped rice straw to substrate formulas on mushroom non-volatile taste compounds(soluble sugars/polyols,organic acids,5'-nucleotides,free amino acids)were investigated.Based on the genomic database and transcriptome data,the genes related to glutamic acid biosynthesis were explored and analyzedThe main results were as follows1.Transcriptome analysis of L.edodes during hot-air dry processing.The mushrooms were dried for 6 h at 50?.The moisture content,specific activities of?-glutamyltranspeptidase(GGT)as well as cysteine sulfoxide lyase(C-S lyase)and formaldehyde contents were measured.The results showed that the moisture content decreased approximately linearly from 86.83%(0 h)to 48.63%(6 h).Linear regression equation was Y=-0.0637 x+0.9009,with a high regression coefficient(R2=0.9561).The specific activity of GGT increased firstly and then decreased and peaked at 2 h.Nevertheless,the specific activity of C-S lyase and formaldehyde content displayed an "M" change trend,which reached their maximum value for C-S lyase at the third hour and the highest level of formaldehyde was at the fourth hour,respectivelyTo investigate the molecular mechanism of flavor compounds during the hot-air dry processing,four mushroom samples treated with hot-air at 0 h,2 h,4 h and 6 h were collected for transcriptome analysis and named as the control group,treat 1,treat 2 and treat 3,respectively.The average ratios of mapping into L.edodes genome W1-26 were 77.89%.A total of 20443 genes were detected.on the basis of the correlation analysis and PCA analysis,it was suggested that gene expression pattern are strikingly different between control group of three treatment groups.Compared with control group,different genes of 5934,6830 and 6527 were expressed for treat 1,treat 2 and treat 3,respectively.Moreover,DEGs were enriched in 22 metabolism pathways according to KEGG databaseFive C-S lyase family genes were identified in L.edodes genome,and named as Lecsl1,Lecsl2,Lecsl3,Lecsl4 and Lecsl5,respectively.According to the transcriptome analysis,these family members showed a total of 12 transcripts.Among them,the expression of Lecsl2 and Lecsl3 not only were up-regulated but also expressed at a relatively high expression levels during hot-air dry processing.The results suggested that Lecsl2 and Lecsl3 might be the crucial genes involved in the sulphur compound biosynthesis in L.edodes2.Clone and expression analysis of the gene Lecsl3.The ID of Lecsl3 was Le01Gene03297 in Wl-26 genome.The length of coding sequence was 966 bp by the result of T-A clone.Bioinformatics analysis showed that Lecsl3 contained 321 amino acids with a molecular weight of 36.28 kDa and a theoretical isoelectric point of 6.71 The protein encoded by Lecsl3 contained 29 potential phosphorylation sites with a stable hydrophilicity.As it had no signal peptides,it was assumed to be a non-secreted protein.Subcellular localization indicated that Lecsl3 protein played a role in peroxisomesAt 30 bp and 74 bp upstream of transcriptional start sites of Lecsl3 gene,TATA-box and CAAT-box which were predicted to be the basic elements of eukaryotic gene promoter.We found two elements that responded to heat shock,one biding site of transcription factor ATF,one biding site of transcriptional activator ABF1,2 biding sites of transcriptional activator GCN4 and 7 biding sites of TATA-B oxAn expression vector pGEX-6P-2-Lecsl3 was constructed and then the recombinant Lecsl3 protein was successfully expressed in Escherichia coli BL21(DE3).The purified Lecsl3 protein was a single band of 36 kDa,it was the same with the theory of molecular weight.The C-S lyase activity and endogenous formaldehyde contents were detected3.Analysis of taste components of L.edodes in different culture substrates.Rice straw was substituted for sawdust at five different ratios of 0,20%,40%,60%,and 80%to cultivate L.edodes and five kinds of mushrooms were successfully obtained.Mushroom yields varied between 155.08 and 202.03 g/kg substrate with the highest yield in the Control group.The higher the straw content in formulas,the slower the mycelium growth rate was found in plate.Protein,ash and fat contents in the five groups were between 13.24-16.05 g/100 g,5.79-7.49 g/100 g and 0.79-0.98 g/100 g,respectively.The decrease of protein levels and increase of ash levels were resulted from the supplementation of rice straw to substrateWith the addition of rice straw in culture substrates,the content of trehalose decreased,but both mannitol and arabitol increased.It showed the presence of 19 classes of amino acids and high levels of threonine(4.82-8.08 mg/g)and glutamic acid(2.00-3.90 mg/g)The contents of MSG-like amino acids in RS80?RS60?RS40?RS20 and control group were 5.12 mg/g,6.17 mg/g,3.62 mg/g,2.87 mg/g and 2.09 mg/g,respectively.The total 5'-nucleotide contents varied from 1.66 mg/g in RS20 group to 4.48 mg/g in RS80 group.Tartaric acid,malic acid,ascorbic acid,acetic acid,citric acid,fumaric acid and succinic acid were detected in each of sample.The results showed that the EUC values ranged from 10.42 to 84.49 g MSG/100 g,which increased with the addition of rice strawThe correlation analysis between rice straw ratios and taste components showed that rice straw contents in substrate had a significant positive correlation with the contents of glutamic acid,aspartic acid,MSG-like amino acids,5'-nucleotides and flavor 5'-nucleotides,arabitol,mannitol,fumaric acid,malic acid,GMP,UMP.4.Genome-wide identification of glutamate metabolism enzyme in L.edodes.We used W1-26 genome database to identify 4 candidate genes directly related to glutamate metabolism pathway.The candidate proteins included one glutamate synthase(Le01Gene05615),one NAD-specific glutamate dehydrogenase(Le01Gene 10227),one NADP-specific glutamate dehydrogenase(Le01Gene13517)and one ?1-pyrroline-5-carboxylate dehydrogenase(Le01Gene06992). |
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