| Lentinula edodes(Berk.Pegler)is a kind of important cultivated edible fungi in the world.The main cultivated material of Lentinula edodes is oak sawdust,and the substrate conversion rate is low,causing waste of forest resources.But the straw and other resources are still unable to replace the oak sawdust for cultivation.In previous studies,we founded that six lignin-degradation enzymes(LCC9,LCC12,LCC13,LCC14,MNP2 and GLOX)in strain W1 of Lentinula edodes were associated with straw degradation.In this study,we analyzed the expression patterns of the lignin-degrading enzyme genes of Lentinula edodes in oak sawdust and straw substrate,and the functions of lcc12 and glox.In this study,we detected the differences of the expression of six enzyme genes among 38 core collection strains(22 wild strains and 16 cultivated strains)in oak sawdust and straw substrates by real-time quantitative PCR.The results showed thant the expression of 6 lignin-degrading enzyme genes of core collection of Lentinula edodes in the straw substrate was generally higher than that in the oak sawdust.Three wild strains(Y1,Y78,Y94)each had 4 lignin degrading-enzyme genes expressed in the straw substrate being more than 5 times higher than that in oak sawdust substrate(Y1(glox,lcc12,lcc13,lcc14);Y78(lcc9,lcc12,lcc13,lcc14);Y94(lcc9,lcc13,lcc14,mnp)).Among the cultivated strains,only glox,lcc13,and lcc14 of strain Z10 expressed in the straw substrate were more than 5 times higher than that in oak sawdust substrate.Correlation analysis showed thant the expression of mnp2 gene was significantly correlated with the laccase genes,and there was also a significant correlation between the four laccases(lcc9,lcc12,lcc13,lcc14).We constructed overexpression vector of p1300-g-lcc12 and p1300-g-glox and transformed the vectors into W1 by ATMT.Five lcc12 transformants and six glox transformants were selected and tested by Real Time-PCR,and named the transformants lcc12-A1,lcc12-A4,glox-B3,glox-B4 etc.The results showed that the gene lcc12 expression levels in the transformants lcc12-A1 and lcc12-A4 were 3.75 times and 1.97 times than that of the wild-type strain,and the remaining three strains had no changes.The expression levels of the glox gene in the transformants glox-B3 and glox-B4 were down-regulated compared to the wild-type strain,down regulated 13.69 times and 4.14 times,respectively.The remaining 4 strains were down-regulated below 2 times.The laccase activity of the transformants lcc12-A1,lcc12-A4,glox-B3,glox-B4 and the manganese peroxidase activity of the transformants glox-B3,glox-B4 were measured in CYM,oak sawdust and straw substrate respectively.The laccase activity of four transformants in CYM and oak sawdust were not significantly different from the wild-type strain.The laccase activity of lcc12-A4 in the straw was significantly higher than that of the wild type strain,and increased by 60%.Manganese peroxidase activity of glox-B3 and glox-B4 measured was not significantly different from that of the wild-type strain in CYM and straw substrate,and glox-B3 was significantly higher than the wild-type strain in sawdust substrate.The agronomic traits of fruit bodies of four transformants in sawdust substrate showed that lcc12-A4 was significantly higher than the wild-type strain W1 in the number of fruiting bodies of transformants per bag,total weight of fruiting bodies per bag and the weight of single fruiting body.This study is of great significance to breeding new varieties of Lentinula edodes suitable for straw substrate cultivation,improving the transformation and utilization efficiency of oak sawdust substrates,and revealing the expression and regulation of lignin-degrading enzyme genes in Lentinula edodes. |
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