Molecular Cloning And Functional Characterization Of PoLac2 In Pleurotus Ostreatus

Pleurotus ostreatus is a common edible fungus and typical white rot fungus,which can effectively degrade the lignin of crop straw.Non-specific oxidases,such as laccases(Lac),play an important role in the degradation of lignin.However,there are few reports on the function of specific laccase genes in lignin degradation.In this study,candidate PoLac2 genes,which may be related to the degradation of lignin,were screened combining with bioinformatics analysis and expression patterns analysis of PoLacs.The Ca MV35 S promoter in the binary expression vector of p Cambia1304 was replaced with the pro Posdi and pro Pogpd,respectively.Constructed PoLac2 overexpression vector was transformed into Pleurotus ostreatus through Agrobacterium-mediated genetic transformation,in order to clear the function of PoLac2 in Pleurotus ostreatus lignin metabolism.The main results are as follows:1.The degradation of lignin by Pleurotus ostreatus was mainly in the mycelium stage.On the 30 th day,the lignin content was decreased by about 10.01% compared with the 5th day,but the lignin content did not change significantly during the fruiting stage.In addition,during the fruiting stage,the activity of extracellular laccase was also significantly lower than that in mycelia stage.2.There are some differences in the gene structures,conserved sequences and loop sequences of twelve laccases.However,all of the twelve laccases have the signal peptide and no transmembrane region,which indicates that these laccases belong to the secretory protein.Twelve PoLacs were unevenly distributed among 5 scaffolds and clustered on scaffold 6 and scaffold 11.On the 10 th day of cotton straw culture,the relative expression of PoLac2 was the highest,and the expression level of PoLac2 was significantly up-regulated compared to PDA culture conditions,suggesting that PoLac2 may be closely related to degradation of lignin by Pleurotus ostreatus.3.Compared with the wild-type strain,the laccase activity and the PoLac2 expression level of transformants increased in varying degrees,and the lignin degradation rates of transformant OE L2-2 and OE L2-3 were increased by 6.36% and 2.36%,respectively.Overexpression of PoLac2 enhanced the lignin degradation of cotton-straw by Pleurotus ostreatus.4.The lignin G/S ratio of cotton-straw treated with OE L2-2 transformant(1.42)was significantly lower than that treated with wild-type(1.96).However,the ratio of protons on the syringyl aromatic ring(0.36)was almost unchanged from the wild type.Transformant OE L2-2 accelerated the degradation of G units in cotton stalk lignin.In addition,compared with the wild type,the proportion of protons in the β-O-4,β-1,and β-βstructures of the lignin in the experimental group were decreased.Overexpression of PoLac2 promoted the breakage of the main bond structure between the lignin units of cotton straw.5.Compared with the wild-type strain,the yield of OE L2-2 transformant was lower,the bud formation time and fruit body maturation time were longer,but the contents of nutrients and active ingredients were higher.