Research On The Construction Of CRISPR/Cas9 And Overexpression Technology System In Trichoderma Citriodor

Trichoderma has a good antagonistic effect on the growth of plant pathogens and can inhibit the growth of pathogenic microorganisms by digesting pathogen filaments,releasing antibiotics and cell wall lytic enzymes,so it is often used as a production of biological control agents and industrial enzymes.Under natural conditions,the use of wood mildew biocontrol agents is greatly affected by environmental factors,and there is less anti-biomass secretion,which is difficult to meet the actual production needs.Therefore,it is important to study the growth and biocontrol related genes of Trichoderma and clarify the regulatory mechanism of Trichoderma to improve the control effect.This study intends to study the overexpression of Trichoderma citrinoviride GF-11 and the construction of CRISPR/Cas9 gene editing system:Firstly,the overexpressed plasmid was constructed by seamless cloning method,and the transformation conditions mediated by Agrobacterium were optimized.Under the optimized conditions,the constructed plasmid was successfully transferred into Trichoderma citrinoviride GF-11,and the expression quantity,phenotype,antibacterial effect and enzyme activity of the overexpressed strain were detected.Second,plasmid containing Cas9 was transferred into Trichoderma to obtain stable strain of Cas9.GRNA fragment with T7 promoter and dDNA with upstream and downstream homologous arms of target gene were constructed,and the obtained products were transferred into protoplasts together to complete gene editing.The contents and results of this study are as follows:1.Antibiotic screening and target gene analysisThe minimum concentration of 20μg/m L of Hygromycin B and 100μg/m L of G418 were screened;the minimum concentration of 150μg/m L of cephalosporin was screened.GZT00172 gene was selected from the genomic data of GF-11,and the bioinformatics analysis showed that the full length of the gene was 1478 bp,with three introns and 1275 bp of m RNA sequence.The protein exhibits hydrophobicity at the N-terminal end and hydrophilicity at the C-terminal end.Its conserved structural domain belongs to the Glyco＿18 superfamily and has a signal peptide without transmembrane structural domain,and the function of this gene is presumed to be related to the encoding of chitinase.2.Cloning and of GZT00172 gene and constructed of overexpressed plasmidPrimers were designed to amplify the CDS sequence of GZT00172 gene,the comparison results showed that the target sequence was successfully amplified.The sequence was constructed into pDHt/SK-PC plasmid by seamless cloning method,and the overexpression plasmid PC-GZT00172 was obtained.3.Acquisition of overexpressed mutant and optimization of Agrobacterium-mediated transformation systemThe overexpression plasmid was transferred into Trichoderma citrinoviride by Agrobacterium-mediated transformation to obtain GZT00172 overexpression strain.The overexpression strain and the wild type strain were detected,and it was found that the gene expression level of the overexpression strain GZT00172 was increased by 12.65 times compared with the wild type,and the chitinase activity was increased by 2.4 times.There is no big difference in the growth rate of mycelium between the overexpression strain and the wild type strain,but the overexpression strain has more conidia,and both strains have a certain inhibitory effect on the growth of pathogenic bacteria.up to 51.16%,and the bacteriostatic rate of GF-11 was 30.76%.Obtained Trichoderma citrinoviride with Cas9;successfully constructed gRNA fragment and dDNA plasmidIn this study,the Agrobacterium-mediated transformation system of Trichoderma citrinoviride GF-11 was successfully established and optimized,and the optimized transformation conditions were as follows:the transformation was mediated by EHA105,the co-culture medium was supplemented with AS to a final concentration of 200μmol/m L,the co-culture time was 6 h,the initial concentration of Agrobacterium tumefaciens in the co-culture was OD₆₆₀=0.8,the concentration of Trichoderma spores was 10⁶/m L,and the co-culture time was 48 h.The conversion rate was about 45 transformants/10⁶Trichoderma spores under these optimized conditions.The system was constructed to facilitate the subsequent study of the functional gene of Trichoderma citrinoviride GF-11.4.Obtained Trichoderma citrinoviride with Cas9The pDHt/sk-PC plasmid with Cas9 was transferred into Trichoderma citrinoviride GF-11using an optimised Agrobacterium-mediated transformation system,and the growth rate and colony morphology of the transformants were observed to be indistinguishable from those of the wild-type strain,providing transformation material for subsequent studies of the CRISPR/Cas9gene editing system for Agrobacterium tumefaciens.5.Constructed gRNA fragment and dDNA plasmidSuccessfully constructed a gRNA fragment targeting the GZT00172 gene gRNA fragment with T7 promoter and dDNA plasmid with G418 screening marker,which can be used for gene editing.6.Acquisition of the GZT00172 mutant strainIn this study,the protoplasts were prepared for the first time using Trichoderma citrinoviride GF-11,and the preparation conditions were optimized as follows:the optimal age of Trichoderma was 15 h,the ratio of cellulase to snailase was 2:1,the enzymatic digestion time was 2.5-3 h,and the enzymatic digestion temperature was 30℃.The overall quality of the protoplasts prepared according to the above optimized conditions was good,and the concentration reached 10⁷-10⁸/m L,which could be used for subsequent experiments.The Cas9 mutant strain,gRNA and dDNA obtained from the construct were transformed together using PEG-mediated protoplast transformation and no budding of mycelium and no knockout strain was obtained.This study will provide a reference for the construction of genetic transformation systems for Trichoderma or other filamentous fungi,and will also provides the foundation for further studies on the function of the Trichoderma citrinoviride GF-11 biocontrol gene.