Construction Of ΔLg-cre1 Mutant Strain Of Lenzites Gibbosa And Function Study Of Lg-CRE1

Carbon catabolite repression（CCR）is a widespread regulatory mechanism in bacteria and fungi that inhibits the expression of enzymes related to carbon catabolism,such as cellulases.The key transcription factor is the carbon metabolism repressor Mig1/Cre A/CRE1.White rot fungi are rich in lignocellulose-degrading enzymes and play an important role in the carbon cycle.Investigating its CCR mechanism can help to clarify the carbon catabolism process and the relationship between the repression of holocellulose catabolism and lignin catabolism,and lay a theoretical foundation for the wood decomposition mechanism of white rot fungi.Lenzites gibbosa,a white rot fungus from the family Polyporacceae,has a high growth rate and a strong lignocellulolytic capacity.In this study,we used homologous recombination to construct a knockout mutant strain of CRE1 protein gene of L.gibbosa,and analyzed the growth and development of the mutant strain and the transcriptional expression of cellulase genes,so as to investigate the function of the carbon catabolite repressor CRE1 of L.gibbosa,and aimed to lay the foundation for revealing the carbon catabolite repression mechanism of L.gibbosa.The study results are as follows:（1）The coding sequence of Lg-cre1 was obtained by cloning and is 2148 bp in length.And phylogenetic and motif analysis of the Lg-CRE1 protein showed that its sequence length and conserved sequences differed significantly from those of Ascomycota,with some motif positions changed（e.g.motif 3,motif 5,motif 8,motif 12）,some motifs missing（e.g.motif 2,motif 14,motif 16）,and some motifs added（e.g.motif 7,motif 10,motif 11,motif 15,motif 17,motif 18,motif 19）,while its homology with proteins related to other species in Basidiomycetes is high;Prediction and analysis of the phosphorylation sites of Lg-CRE1 protein resulted in two phosphorylation sites,S151 and S493,which may play a key role.The results showed that Lg-CRE1 consists of 715 amino acids,has a molecular weight of 76.52 k Da,a theoretical isoelectric point of 9.24 and an instability index of 69.88,making it an unstable protein.Theα-helix accounted for 23.08%,the extended chain accounted for 7.97%,theβ-turned angle accounted for 4.90%and the random curl accounted for 64.06%.（2）An upstream sequence of 1222bp and a downstream sequence of 1261bp of the Lg-cre1gene were selected from the genome,and the knockout vector P-Δcre1 was constructed at both ends of the hygromycin resistance gene using the principle of homologous recombination.And PEG-mediated protoplast transformation was performed,and after screening and stable inheritance,a mutant of the Lg-cre1 gene of L.gibbosa was obtained.The morphology was observed and the mutant strain was found to have a lower growth rate and sparse mycelium than the wild type when cultured on solid complete medium,sodium carboxymethylcellulose medium and poplar wood powder glucose medium;when cultured on liquid complete medium,the mycelium of the mutant is looser than the wild type in the early growth stage.（3）RT-q PCR analysis and physiological assays were performed on the Lg-cre1 mutant and wild type strains under different treatment conditions.Under 2%sodium carboxymethylcellulose liquid culture condition,the expression of endoglucanase gene eg1,β-glucosidase gene bg1 and exoglucanase gene cbh1-2 in the mutant increased significantly,up to 171.57-fold,103.29-fold and 39.95-fold of 0d,respectively,while the expression of W-eg2,bg2 and cbh1-1 genes did not differ significantly.The highestβ-glucosidase activity in the mutant was 5.42-fold higher than that of the wild type,while the differences in filter paper enzyme activity,endoglucanase activity,exoglucanase activity and extracellular protein content between the two were not significant.In summary,the carbon catabolism repressor Lg-CRE1 is a C₂H₂-type zinc finger transcription factor with two C₂H₂ zinc finger structural domains.The present study demonstrated that the carbon catabolism repressor Lg-CRE1 of L.gibbosa has two functions:inhibition of cellulase gene expression and positive regulation of growth and development of L.gibbosa.