Construction Of ΔLg-sfp1 Mutant Strain And Functional Verification Of Lg-sfp1 In Lenzites Gibbosa

Lenzites gibbosa is a fast-growing white rot fungi of the Polyporaceae family,which has a strong ability to decompose wood and lignin.In fungi,C2H2 type zinc finger transcription factors are mainly involved in regulating growth and development,sporulation,carbon metabolism,pathogenicity,and stress response.SFP1 protein is split finger protein 1 of C2H2type zinc finger transcription factor.Previous studies have shown that in Saccharomyces cerevisiae and Candida albicans,the SFP1 zinc finger transcription factor has functions such as regulating growth and development,biofilm formation,and oxidative stress responses,Currently,research on the structure and function of SFP1 protein in white rot fungi has not been carried out,and further research is needed.In this study,the Lg-sfp1 gene in Lenzites gibbosa was used as the research object.The gene was cloned,structurally identified,and bioinformatics analyzed.The△Lg-sfp1 gene knockout vector was constructed based on the principle of homologous recombination,and the Lg-sfp1 gene deletion mutant strain was successfully obtained by PEG-mediated protoplast transformation.Through comparative analysis with wild bacteria（WT）,the function of Lg-sfp1gene was preliminarily verified.The main test results are as follows:1、Lg-sfp1 gene was successfully cloned and identified.The c DNA of the gene is 1947bp in length.The physicochemical properties and structure of the protein indicate that the Lg-sfp1 gene encodes a hydrophilic intramembrane protein composed of 648 amino acids,with a protein size of 68.27 k D,an isoelectric point of 6.46,an instability index of 68.04,and a lipid index of 51.20.The average coefficient of hydrophilicity is-0.61,indicating a hydrophilic protein.The results of protein conservative domain prediction indicate that Lg-SFP1 protein contains two C2H2 type zinc finger domains at the C-terminal of the amino acid sequence,and there is a characteristic sequence of SFP1,which is a split finger protein 1 of C2H2 type zinc finger transcription factor.The phylogenetic tree construction of Lg-SFP1 protein showed that Lg-SFP1 has the closest genetic relationship with the SFP1 of Trametes pubescens.2、Preparation of Protoplasts of Lenzites gibbosa,and the constructed knockout vector p KOV21-sfp1 was transformed into protoplasts using PEG mediated genetic transformation.After resistance screening and PCR detection,6 strains of△Lg-sfp1 gene knockout mutants were obtained.The expression of Lg-sfp1 gene in 6 strains of△Lg-sfp1 gene knockout mutants was detected by real-time fluorescence quantitative assay（q RT-PCR）,and a mutant strain with the best knockout effect was finally selected for subsequent functional verification.3、The Lg-sfp1 gene is involved in regulating the morphology and growth of the colonies in Lenzites gibbosa.In PDA medium,compared with wild type strains,the growth rate of△Lg-sfp1 gene knockout mutant decreased,the biomass of the strain decreased,and the morphological changes of the colonies were significant.There were more longitudinally growing aerial hyphae,and the mycelium was thicker.The△Lg-sfp1 gene knockout and wild-type strain were cultured on LNAS basic medium,after 3 days of cultivation,the corresponding treated mycelium RNA was extracted at 0,2,4,6,8,and 10 days.The expression of 5 growth and development related genes during growth and development was detected using q RT-PCR.The expression of 5 growth and development related genes in the△Lg-sfp1 gene knockout mutant strain decreased compared to wild-type strain.Using qRT-PCR to detect the expression of 5 growth and development related genes,the expression of 5 growth and development related genes in the△Lg-sfp1 gene knockout mutant strains decreased compared to wild type strains.4、The Lg-sfp1 gene is involved in regulating the stress response in Lenzites gibbosa.Under oxidative stress,in PDA medium containing low concentration of H₂O₂,the tolerance of△Lg-sfp1 gene knockout mutants was lower than that of wild type,while with the increase of stress factor concentration,the tolerance of△Lg-sfp1 gene knockout mutants was significantly higher than that of wild type mutants.The△Lg-sfp1 gene knockout and wild-type strain were cultured on LNAS basic medium,after 3 days of cultivation,the corresponding treated mycelium RNA was extracted at 0,2,4,6,8,and 10 days.Using q RT-PCR to detect the expression of 6 antioxidant enzyme genes related to stress response,the expression of 6antioxidant enzyme genes related to stress response in the△Lg-sfp1 gene knockout mutant strains increased compared to the wild type strains,indicating that the Lg-sfp1 gene has a negative regulatory effect on the production of antioxidant enzymes.Under ion stress,in PDA media containing different concentrations of Na Cl and KCl,the growth speed of the△Lg-sfp1gene knockout mutant and the wild type were continuously decreased as the concentration of salt stress factors increased,but the growth speed of the△Lg-sfp1 gene knockout mutant and the wild type was significantly decreased compared to the wild type.To sum up,this study successfully cloned and identified the split finger protein 1 gene Lg-sfp1 of the C2H2 type zinc finger transcription factor from in Lenzites gibbosa,and preliminarily analyzed the function of the Lg-sfp1 gene,expanding the research on the growth,development,and stress response mechanism of Lenzites gibbosa at the molecular biological level,laying a strong theoretical basis for the functional research of the structure and function of SFP1 protein in white rot fungi.