Analysis Of The Function Of C2H2-type Zinc Finger Transcription Factors Of Lenzites Gibbosa

Lenzites gibbosa is a white-rot fungus that has a strong ability to decompose wood and lignin.In order to dig deeper into its mechanism of degradation of lignocellulosic substrates at the level of transcriptional regulation,based on 15 transcriptomes under the treatment of non-sawdust and sawdust treatment at different times,C2H2-type zinc finger transcription factor(C2H2-ZFTF)genes,and cellulase genes were analyzed,and the structure and protein structure of C2H2-type transcription factor genes were analyzed,and the key enzyme genes and key pathways for degrading cellulose were excavated;the expression levels of C2H2-type zinc finger transcription factor genes that were differentially expressed(DEGs)and cellulase genes were detected by Real-time Quantitative PCR(RT-PCR),and it was found that the carbon catabolite repressor transcription factor gene(gene₅738)had a negative regulatory relationship with the expression of the cellulolytic enzyme genes.An RNAi silencing vector for this gene was constructed,and a mutant strain was obtained.Its function was verified and analyzed.The main research results are as follows:1.With the non-sawdust treatment as a control,the enzyme activity and hyphae content of L.gibbosa,cellulolytic enzymes and lignin degrading enzymes treated with the sawdust at different times were tested.The results showed that the wood matrix could make the activities of 3 kinds of cellulases[exoglucanase(EXGA),endoglucanase(ENGA),?-glucosidase(?-GA)],hemicellulase[xylanase(XYA)]and lignin degrading enzymes[manganese peroxidase(MnP)and laccase]increased.In the 13-day detection period,the activities of three cellulases and xylanase all showed an irregular increase trend,while the activities of MnP and LA showed a continuous increase trend.The amount of mycelium under the treatment of sawdust was significantly higher than that of the control group without the treatment of sawdust,indicating that L.gibbosa could secrete both cellulase and lignin degrading enzyme at the same time under the initial sawdust treatment condition to degrade the sawdust to obtain the nutrients needed for growth.The growth of mycelium on the lignocellulosic substrate and the regular pattern of the change of the enzyme activity in the initial degradation of wood laid the foundation for the subsequent screening of cellulose degrading enzyme genes and key pathways for cellulose degradation based on transcriptome data.2.The C2H2-ZFTF genes were screened,and the COG,GO and KEGG databases were used for function analysis.The results showed that L.gibbosa had 67 C2H2-ZFTF genes,of which there were 8 DEGs.42 C2H2-ZFTF genes were annotated in COG database,40 of which were related to the functions of replication,recombination and repair,and 2 were related to the functions of post-translational modification,protein turnover,and chaperone;40 C2H2-ZFTF genes were annotated in GO database,which were significant enriched in categories such as transcription-coupled nucleotide excision repair(GO:0006283),cellular processes(GO:0009987)and metal ion binding(GO:0046872);9 C2H2-ZFTF genes were annotated in KEGG database,which were significantly enriched in the pathway of protein processing in endoplasmic reticulum(ko04141).The analysis of the gene structure,protein structure and physicochemical properties of 67 C2H2-ZFTF genes showed that they all contained the Cys2-His2 zinc finger domain and belonged to the zf-C2H2 superfamily protein;one of DEGs(gene₅738)was a member of the C2H2-ZFTF genes,and its product is a carbon catabolite repressor,containing two "Cys2-His2" conserved domains.The functional analysis of C2H2-ZFTF genes lays the foundation for subsequent research on related gene functions.3.Combine transcriptome data and the enzyme activity detection data to construct and analyze a weighted gene co-expression network(WGCNA),and obtain the module with the highest correlation with the cellulose degrading enzyme activity,namely the turquoise module,which contains 304 module genes are mainly related to cellulose degradation.The COG,GO,and KEGG functional analysis of the turquoise modular genes show that genes are all related to the degradation of cellulose,and are significantly enriched in the starch and sucrose metabolism pathways(ko00500).Among the pathway,there are 4 routs that degrade cellulose to glucose,and a total of 20 genes for 3 cellulolytic enzymes(ENGA,EXGA and ?-GA)genes are enriched in the entire pathway.The candidate genes with a weight value greater than 0.2 in the turquoise module are screened,and 149 candidate genes are obtained.Among them,the module membership(MM)of gene₅738 is the largest,and the gene significance(GS)is also larger,indicating that it is the key hub gene.The GO and KEGG functional analysis of gene₅738 co-expression network genes(30)showed that gene₅738 co-expression network genes were significantly enriched in the GO database in the cellulose catabolic process(GO:0030245)and the polysaccharide catabolic process(GO:0000272),the cell wall macromolecular catabolic process involved in cytogamy(GO:0032219)and the cell wall chitin catabolic process(GO:0006039)have the largest number of genes,indicating that there are genes related to cellulose catabolism in the co-expression network;The gene₅738 co-expression network genes are significantly enriched in the KEGG database in the sucrose and starch metabolism pathway(ko00500)and the cell cycle-yeast pathway(ko04111)have the largest number of genes,indicating that gene₅738 is involved in the negative regulation of cellulose catabolism,and also has a certain relationship with growth and development.4.Using RT-PCR to detect the expression levels of 8 C2H2-XFTF DEGs and 20 cellulose degrading enzyme genes,joint analysis of expression trends and correlation analysis of expression levels,it is found that the expression patterns of both were consistent with the trend of expression in the transcriptome.The expression trends of C2H2-ZFTF DEGs(gene₅738 and gene₁1819)and some cellulolytic enzyme genes(gene₆13,gene₈814,and gene₁0281)were opposite.Correlation analysis showed that the two had a significant negative correlation.It was indicated that there was a negative regulatory relationship between the two,which laid the foundation for the subsequent in-depth study of the regulation of cellulase gene expression by the carbon catabolite repressor transcription factor gene.5.The carbon metabolism inhibitor Lg-cre1(gene₅738)was subjected to RNAi silent mutant construction,RT-PCR detection,restriction enzyme digestion identification and sequencing analysis showed that the silent expression vector pCAMBIA1301-TrpC-Hygro-gpdA-PZGJJ-TEF 1 was successfully constructed.The plasmid was transformed into Agrobacterium,and Agrobacterium mediated the mycelium of L.gibbosa,and finally the transformant strain of L.gibbosa was obtained.The growth phenotype of the mutant strain and the expression of growth and development-related genes(gene₂036,gene₉338,gene₁1991,and gene₉353)were verified,indicating that Lg-cre1 had a promoting effect on the growth of hyphae.In summary,L.gibbosa contains 67 C2H2-ZFTF genes,among which there are carbon metabolism inhibitor genes.WGCNA analysis indicated that the above-mentioned carbon metabolism inhibitory genes had a co-expression relationship with cellulase genes,and RT-PCR studies showed that there was a negative regulatory relationship between carbon metabolism inhibitors and cellulase genes.The construction of RNAi mutant strains indicated that the carbon metabolism inhibitor gene had a promoting effect on mycelial growth.