| Hemicellulose is the second most abundant structural polysaccharide in nature after cellulose.It widely exists in plant cell wall,and together with cellulose and lignin forms lignocellulose,which is a rich renewable energy source on earth.Hemicellulose can be directly or indirectly used in food,medicine,chemical industry and other fields,so the degradation and utilization of hemicellulose has become a hot spot in the research field.White rot fungus is a kind of wood decay fungus,which is widely distributed and has a large number.It can degrade lignin,cellulose and hemicellulose,and has become the most concerned degradation microorganism.Lenzites gibbosa is a kind of white rot fungus of Polyporaceae with fast growth and strong wood decomposition ability.It is widely distributed in northeast China,especially in the fallen and dead wood of broadleaf forest.It can cause white rot of wood.In order to further explore the mechanism of hemicellulose degradation by L.gibbosa,this study used Hi Seq high-throughput sequencing technology and LC-MS metabolome sequencing technology to conduct transcriptome sequencing on L.gibbosa mycelia respectively treated with two important hemicellulose components,xylan and mannose at 3 time points.Non-target metabolomics detection was performed on mycelium culture medium.Through single omics data and multi-omics combined analysis,key pathways and key genes of L.gibbosa degradation of hemicellulose were found,and its molecular function and mechanism were further explored,laying an important molecular foundation for fully revealing the mechanism of L.gibbosa degradation of hemicellulose.The main research results of this paper are as follows:（1）Determination of enzyme activity under different woody substratesL.gibbosa was respectively treated with sawdust and two important hemicellulose components,xylan and mannan.The activity of two hemicellulose degradation enzymes produced by L.gibbosa was detected regularly for 25 days.The results showed that L.gibbosa could produce hemicellulose enzymes in both sawdust and single hemicellulose substrate.The activity of xylanase and mannanase was significantly higher than that of the control group,and the peak activity of xylanase and mannanase appeared on the 25th day of the last time point,which was also used for the selection of time points in subsequent omics studies.（2）Transcriptome analysis of L.gibbosa treated with xylanL.gibbosa treated with xylan was cultured,and mycelia at 0 d（CK）,13 d（MA）and 25 d（MB）were selected for transcriptome sequencing.A total of 12,826 genes were identified by transcriptome sequencing,among which 788 new genes were discovered,further enriching the genome of the original species.A total of 706 differentially expressed genes were screened in the CK vs MA group,1,047 differentially expressed genes were screened in the CK vs MB group,and 549 differentially expressed genes were screened in the MA vs MB group.According to GO functional annotation analysis,the differential genes were mainly attributed to single organism process,metabolic process and catalytic activity.KEGG enrichment analysis showed that carbon metabolism（ko01200）,glycosaminoglycan degradation（ko00531）,other polysaccharide degradation（ko00511）and other pathways were significantly enriched.A total of 366 differentially upregulated genes were screened out,and 12 key hemicellulose degradation pathways were identified by GO enrichment and KEGG enrichment analysis.8key hemicellulose degradation genes were screened out and their expression levels were analyzed by RT-q PCR.（3）Metabolomics analysis of L.gibbosa treated with xylanNon-target metabolomics analysis was performed on L.gibbosa culture medium treated with xylan at 0,13 and 25d by liquid chromatography-tandem mass spectrometry（LC-MS）.Using PCA and OPLS-DA analysis,1,223 metabolites were identified in positive ion mode and626 metabolites were identified in negative ion mode.According to KEGG enrichment analysis of differential metabolites,the three comparison groups were respectively enriched to 53,76and 64 enrichment pathways.On this basis,6 significant enrichment pathways were screened out in CK vs MA group and 13 significant enrichment pathways were screened out in CK vs MB group by setting P value≤0.05.The MA vs MB group contained seven significant enrichment pathways.Through multi-omics combined analysis,metabolites in 12 key pathways in the transcriptome under xylan treatment were found,and the co-expression network between key genes and metabolites was constructed.（4）Transcriptome analysis of L.gibbosa treated with mannanThe mycelium samples of L.gibbosa treated with mannan at 0 d（CK）,13 d（GA）and 25 d（GB）were sequenced.A total of 667 differential genes were screened in the CK vs GA group,1,378 differential genes were screened in the CK vs GB group,and 658 differential genes were screened in the GA vs GB group.According to GO functional annotation analysis,differential genes are mainly involved in metabolic process,cellular process and catalytic activity.KEGG enrichment analysis showed that carbon metabolism（ko01200）,glycosaminoglycan degradation（ko00531）,other polysaccharide degradation（ko00511）,pentose and glucuronic acid conversion（ko00040）were significantly enriched.Based on the differential gene trend analysis,498 continuously up-regulated genes were found for GO enrichment and KEGG enrichment analysis.A total of 8 key pathways of L.gibbosa degradation of hemicellulose were foundand,11 key genes related to hemicellulose degradation were screened out and their expression levels were verified by RT-q PCR.（5）Metabolomics analysis of L.gibbosa treated with mannanNon-target metabolomics analysis was performed on L.gibbosa culture medium treated with mannan at 0 d,13 d and 25 d.The differential metabolites in the metabolome were studied.Under the positive ion mode,the differential metabolites in three comparison groups were found,which were 58,36 and 37,respectively.The differential metabolites in the three comparison groups were found in negative ion mode,13,10 and 13 respectively.KEGG enrichment analysis of differential metabolites showed that the three comparison groups were enriched to 62,70 and 65 enrichment pathways,respectively.On this basis,16 significant enrichment pathways were screened out in CK vs GA group and 12 significant enrichment pathways were screened out in CK vs GB group by setting P value≤0.05.The GA VS GB group contained 7 significant enrichment pathways.The co-expression network of 11 key genes and metabolites of the pathway in the mannan treated transcriptome was analyzed by multi-omics combined analysis.（6）Identification and functional analysis of LG-M9878 geneThe product of LG-M9878 gene is endo-1,4-β-mannanase（EC 3.2.1.78）,which is an important hemicellulose degrading enzyme.LG-M9878 gene was cloned and bioinformatics analyzed.The length of LG-M9878 gene is 1,392 bp,encoding 463 amino acids,the molecular weight is 49.26 KDa,the molecular formula is C2,198H3,324N582O688S11,hydrophilic protein,predicted the second and third order structure,etc.The CDS sequence of LG-M9878 gene was cloned,the prokaryotic expression vector p ET-32a-M9878 was constructed,and transformed into EScherichia coli BL21（DE3）.The expression of p ET-32a-M9878 protein was successfully induced by IPTG.Using molecular docking,the binding position of mannan to Lg-M9878protein was confirmed.The overexpression vector PCAMBIA1301-gpd-M9878 was constructed by single enzyme digestion and ligating transformation,and transformed into Agrobacterium LBA4404.L.gibbosa was transfected by Agrobacterium tumefaciens mediated method,and two LG-M9878 overexpression transgenic strains Lg M1 and Lg M2 were obtained.The growth type of the transgenic strains was identified,and RT-q PCR was performed to verify the overexpressed transgenic strains treated with sawdust and mannan.The results showed that the relative expression level of LG-M9878 gene of the overexpressed strains was continuously up-regulated,and the relative expression level of the overexpressed strains under mannan treatment was higher than that under sawdust treatment.Enzyme activity analysis showed that the production capacity of mannanase in transgenic strains Lg M1 and Lg M2 was significantly higher than that of the wild-type strain,and the overexpression of LG-M9878 gene increased the hemicellulose degradation ability of L.gibbosa.In conclusion,in this study,hemicellulose degradation of L.gibbosa was confirmed by hemicellulose activity detection,and the metabolic pathways and key genes related to hemicellulose degradation were identified by transcriptomic and metabolomic analysis of L.gibbosa treated with xylan and mannan.The function of LG-M9878,an important hemicellulose degrading enzyme gene,was verified.It was shown that mannan can induce the expression of LG-M9878 gene,and the overexpressed LG-M9878 gene can make L.gibbosa produce more mannanase,thus enhancing its hemicellulose degrading ability.In this study,some molecular mechanisms of hemicellulose degradation by L.gibbosa were analyzed,which laid an important molecular foundation for further revealing the mechanism of hemicellulose degradation by white rot fungus. |
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