Establishment Of Liposome-mediated Genetic Transformation System Of Lentinula Edodes And Study On Regulation Mechanism Of FacB On Lignocellulose Degradation In L.edodes

Lentinula edodes is a very common edible mushroom with high nutritional and medicinal value.In a natural environment,it is able to degrade lignocellulose using its own secreted lignocellulose-degrading enzymes.The degradation of lignocellulose involves the participation of many genes.At present,many transcription factors that can positively or negatively regulating lignocellulose-degradation have been identified,but the molecular mechanism of lignocellulose degrading enzyme expression regulation is still unclear.The completion of whole-genome sequencing of L.edodes in recent years has led the research of L.edodes to the stage of molecular biology research.However,the lack of an efficient and stable genetic transformation system has limited the molecular research progress in L.edodes.In this paper,a genetic transformation system applicable to L.edodes was constructed for the first time using liposome-mediated transformation method,and the transformation efficiency of several transformation systems were compared.and it was found that using liposome-mediated transform protoplasts generated from L.edodes,the transformation efficiency was 53 transformants/10⁶ protoplasts,and there was obvious transformants germination in 14 days;while using electroporation to transform protoplasts,the transformation efficiency was 7 transformants/10⁶ protoplasts,and transformants started to germinate in 20 days;no transformants were seen in Agrobacterium-mediated transformation of protoplasts for 3 weeks;the transformation efficiency of Agrobacterium-mediated transformation of hyphae was 33%,showing that 22transformants containing the hygromycin resistance gene could be selected in 66 hyphae,and the time to obtain transformants was also 3 weeks.In comparison,the liposome-mediated transformation method is simple to perform,with high transformation efficiency and short incubation time,making it suitable for subsequent studies.The success of the genetic transformation system was initially verified by PCR verification of the hph fragment of transformants,which was able to amplify the hph gene.To further validate the genetic transformation system of L.edodes,this paper selected phosphoglucose isomerase（PGI）as a target gene.PGI is an important enzyme involved in glycolytic process and polysaccharide synthesis process,and it is responsible for the interconversion of glucose-6-phosphate（G-6-P）and fructose-6-phosphate（F-6-P）.In this study,the homologous gene identified in the whole-genome library of L.edodes,named pgi,was used to construct pgi-silenced strains and pgi-overexpressed strains using the genetic transformation system constructed in this study,23 and 47 transformants were screened,respectively.Among them,18 and 28 were positive transformants,respectively.PGI is a key enzyme in the glycolytic process and will be involved in the process of mycelial growth,so mycelial biomass was examined and found that the mycelial biomass of the pgi-silenced strains were significantly reduced by 30%-70%compared to the WT strain,while the mycelial biomass of pgi-overexpressed strains were 2.46-2.52 times higher than that of the WT strain.Then the effect of pgi on polysaccharides was explored.pgi-silenced strains had significantly higher production of extracellular polysaccharide（EPS）and intracellular polysaccharide（IPS）compared to WT strains,which were 1.5-2 and 1.3-1.6times higher,respectively.In contrast,the EPS and IPS contents of pgi-overexpressed strains were significantly reduced by 68%and 42%-63%,respectively.For cell wall polysaccharides,theβ-1,3-glucan contents of pgi-silenced strains were significantly reduced by 38%-43%,and the chitin contents were significantly higher,2.33-2.79 times higher than that of the WT strain,total cell wall polysaccharide content is elevated;while theβ-1,3-glucan contents of pgi-overexpressed strains were significantly higher,1.3-1.42times higher than that of the WT strain,and the chitin contents were significantly lower,37.5%-40%,total cell wall polysaccharide content is reduced.The effect on cell wall polysaccharide content implies a change in susceptibility to cell wall stress.pgi-silenced strains showed increased resistance to fluorescent brightener（CFW）and decreased resistance to Congo red（CR）,while pgi-overexpressed strains were exactly the opposite of pgi-silenced strains.The above results further validated the success of the genetic transformation system.In this study,we found that acetic acid significantly increased the lignocellulose degradation of L.edodes,and the transcriptional levels of degradation-related genes.On this basis,we further screened a transcriptional activator that can respond to both acetic acid and lignocellulose degradation,named FacB.The activities of cellulase and manganese peroxidase was significantly reduced in facB-silenced strains,which were 50-52%and72-74%of WT strain,respectively,and the activity of these enzymes was further reduced under acetic acid treatment,which were 45-47%and 67-73%of WT strain,respectively;In contrast,the activities of cellulase and manganese peroxidase were significantly increased in the facB-overexpressed strains,which were 1.31-1.67 times and 1.11-1.23 times that of WT strain,respectively,and the activities of the enzymes were further increased under acetic acid treatment,which was 1.48-1.94 times and 1.22-1.38 times that of WT strain,respectively.Among them,the change of facB gene expression has less regulation effect on manganese peroxidase than cellulase.facB did not have a significant regulatory effect on laccase.In conclusion,FacB can participate in the inducing effect of acetic acid on the degradation of lignocellulose.FacB acts as a transcription factor that functions by binding to the promoter of a gene to transcriptionally activate the gene expression.Therefore,this study predicted the promoter regions of cellulase and manganese peroxidase genes based on the reported FacB binding sequences(TCC/GN_8-10C/GGA)and found that some genes have the binding sequences of FacB.The cbh1 gene,which has the greatest effect on transcript levels,was selected and FacB was initially verified to bind to the promoter region of cbh1 by yeast single hybridization experiments.These results initially suggest that FacB can bind to the promoter regions of lignocellulose-degradation-related enzymes,transcriptionally activate the expression of these genes,and then increase cellulase and manganese peroxidase enzyme activities to promote lignocellulose degradation.In summary,in this paper,the genetic transformation system of L.edodes was established for the first time using liposome-mediated transformation method,and the success of the genetic transformation system was verified with pgi as a target gene.After that,a transcription factor,FacB,which responds to both acetic acid and lignocellulose degradation was screened on the basis that acetic acid can enhance lignocellulose degradation of L.edodes,and the role of FacB in the of lignocellulose degradation was initially explored,laying the foundation for further understanding of the process of lignocellulose degradation.