| Straw cellulose can be fully utilized with the help of white-rot fungi which degrade lignin,a "protective screen" in straw lignocellulose,with advantages such as mild conditions,high product yields,low energy consumption and environmentally friendli ness.However,the degradation might be slow with low efficiency due to the degradation efficiency,SF of lignin,other external factors and degradation pathway of white-rot fungi.It is still unclear about the molecular mechanism of lignin degradation metabolism of white-rot fungi,which needs to be further studied.Pleurotus ostreatus,a kind of widely planted white-rot fungus,completely degrades lignin into CO2 and H2O through oxidization.Currently,the metabolism pathway and molecular mechanism of decomposing lignin by Pleurotus ostreatus is still unknown.In this study,Pleurotus ostreatus strains with high SF for degrading lignin of cotton stalks were screened out and the technical parameters related to lignin degradation were optimized during the lignin degradtion by Pleurotus ostreatus strain with the highest SF.Based on this,characteristics variation of the degradation process of cotton straw lignin was focused on and phylogenetic analyses on its laccase gene family and manganese peroxidase gene family were carried out respectively.Combining the expression level of the two gene families on solid medium of cotton stalks,key enzyme genes,PoLAC1,PoVP3 for lignin degradation were selected to study the functions so as to discover their roles in lignin degradation and then clarify molecular mechanism for cotton stalk lignin degradation by white rot-fungi.The main findings and conclusions were as follows:1.Optimization of influencing factors on cotton stalk lignin degradation and laccase production by Pleurotus ostreatusBased on the measurement of the lignin degradation rate of 10 strains of Pleurotus ostreatus,3 strains with high lignin degradability and SF were screened out.They were "Suping No.1","Heiping No.1" and "Wanping No.1".Among them,"Suping No.1" was taken as the research object.The influence of stalk’s particle size,material-liquid ratio,temperature,surface active agent and other factors on its lignin degradation of cotton stalks and its laccase production was investigated by single factor experiment and orthogonal experiment.It was found that the suitable conditions for degrading lignin of cotton stalks were:stalk’s particle size:0.5 mm,material-liquid ratio:1:3,culture temperature:26℃,Tween content:0.25 g/L;and the suitable conditions for laccase were:stalk’s particle size:0.75 mm,material-liquid ratio:1:3,culture temperature:26℃ and Tween content:0.25 g/L.2.Characteristics analysis of the degradation process of cotton stalk lignin by Pleurotus ostreatus"Suping No.1" was inoculated and cultured in cotton stalks solid medium to measure the lignin content,degrading enzyme activity at different times and detect the structural changes of lignin as well as degradation products with low molecular weight.The results showed that Pleurotus ostreatus mainly degradedlignin in the stage of its mycelium growth,producing LAC and MnP with high activity,with no LiP detected.Compared with G-type lignin in cotton stalks,S-type lignin is easier to be degraded by Pleurotus ostreatus.G/S ratio of lignin of cotton stolk was 1.20 before"Suping No.1" culture;on the 30th day after culture,the ratio was 2.41;on the 60th day after culture,the ratio was 4.00,illustrating a gradual increasing trend in G/S ratio of lignin with the passage of culture.Some benzodiazepines compounds with low molecular weight,such as protocatechuic acid,2,6-di-tert-butylphenol oxygen derivatives,vanillic acid,syringic acid,benzoic acid,3,5-dimethoxy,benzoate,etc.were produced in the process of lignin cotton stalk degradation and these benzodiaze-pines compounds are structurally similar to lignin monomers,which clarified the depolymerization of guaiacyl,syringul monomers of lignin and the side chain oxidation of these monomers.3.Analysis of lignin degradability,agronomic trait and quality of overexpressed PoLAC1 strainThe relative expression levels of overexpressed PoLAC1 strains,OE L1-1 and OE L1-4 were 11 times and 13 times higher than that of the wild type respectively.Activities of their extracellular laccases were higher than that of the wild type,and their laccase activities were nearly 2 times higher than that of the wild typc on the 5th day.Compared with the wild type,lignin degradation rates of the two strains rose by 6.86%and 5.21%respectively.The role of OE L1-4 strain in lignin degradation was mainly in demethylation or demethoxy of lignin monomers which is beneficial to depolymerization of lignin units.Compared with the wild type,the proton fraction of aromatic nucleus of guaiacyl of lignin of cotton stalks with cultured OE L1-4 strain decreased to 0.64(the wild type:0.68)while that of aromatic nucleus of lilac rose to 0.45(the wild type:0.35).The results showed that OE L1-4 accelerated the degradation of G-type monomer in lignin while slowed down the degradation of S-type monomer.On the 30th day after OE L1-4 culture,G/S ratio of lignin of cotton stalks was 1.42,lower than 1.94 on the 30th day after WT culture but still higher than 1.20 before culture,illustrating overexpressed PoLAC1 did not change the characteristics of preferentially degraded S-type monomer in lignin of cotton stalks.Compared with the wild type,morphogenesis,growth cycle and nutrient content of OE L1-4 sporocarp changed significantly.4.Analysis of lignin degradability,agronomic trait and quality of overexpressed Po VP3 strainThe relative expression levels of overexpressed Po VP3 strains,OE VP3-1 and OE VP3-2were 11 times and 9 times larger than that of the wild type respectively.The degradation rates of lignin climbed by 7.46%averagely compared with that of the wild type.The role of OE VP3-1 strain in lignin degradation was mainly in accelerating the breakage of propyl side chains Cα-Cβ of lignin monomers,which directly resulted in depolymerization and facilitated oxidation of side chains of lignin monomers.Compared with the wild type,the proton fraction of aromatic nucleus of guaiacyl of lignin of cotton stalks with cultured OE VP3-1 strain declined to 1.57(the wild type:1.48)while that of aromatic nucleus of lilac dropped to 0.77(the wild type:1.14).This result illustrated OE VP3-1 not only promoted degradation of G-type lignin but also obviously enhanced degradation of S-type.G/S ratio of lignin of cotton stalks before culture was 1.20.On the 30th day after culture of the wild type,the ratio was 1.38 and on the 30th day after culture of OE VP3-1,the ratio was 1.92,illustrating overexpressed PoVP3 did not change the characteristics of preferentially degraded S-type monomer in lignin of cotton stalks.Compared with the wild type,except prolonged ripening time of OE VP3-1’s sporocarp,no other changes in growth and development,morphogenesis were observed.Yet nutrients content in sporocarp had significant changes except moisture and Vitamin C. |
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