Construction Of Zfc1 Gene Knockout Strain Of Trichoderma Asperellum And Analysis Of Cadmium Tolerance Function Of Zfc1

Trichoderma strains are one of the most widely used biological control agents which also exhibit great potential in the field of bioremdiation since they can tolerate or degrade a variety of toxic pollutants.To fully exploit Trichoderma spp.,we need to explore the tolerance of Trichoderma spp.to harmful substances in the environment and reveal the underlying molecular mechanism.Therefore,this study takes T.asperellum as the research object,selects the typical environmental pollutant cadmium treatment as the stress condition,and explores the function of zfc1,a gene related to cadmium tolerance of T.asperellum.Results here laid the foundation for a better application of T.asperellum in biological control,heavy metal remediation under various complex environmental conditions,and multi-technology combined environmental remediation.The main researches are as follows:1.Full-length fragment of zfc1 was cloned,and the length of zfc1 was 3732 bp.Bioinformatics analysis software predicted that zfc1 encoded protein,which contained C₂H₂zinc finger and other DNA-binding domains,were located in the nucleus,thus it was speculated that zfc1 was a C₂H₂zinc finger protein gene.The results of real-time fluorescence quantitative PCR analysis showed that the expression of zfc1 was significantly increased under 150 mg/L Cd²⁺stress,suggesting that zfc1 might be a gene related to cadmium tolerance in T.asperellum.2.The zfc1 gene knockout strain（Δzfc1）was constructed by Agrobacterium tumefaciens-mediated transformation（ATMT）technique.The genetic transformation of T.asperellum was mediated by A.tumefaciens AGL-1 harboring the knockout vector p C1300qh-up-down constructed in this research,and 30 possible transformants were screened.Then the verification of the potential transformant was performed by PCR amplification with 3 pairs of primers（zfc1F/R,hph F/R,up F/down R）.Finally,the transformant T₁₇was confirmed to be a zfc1 gene knockout strain with single-copy integration of hph by Southern blot.3.The effects of zfc1 on growth phenotype and cadmium tolerance of T.asperellum were analyzed by comparing the differences betweenΔzfc1 and wild-type（WT）strain.The growth rate measurements showed thatΔzfc1 and WT had no significant difference in colony morphology when cultured on PDA medium,butΔzfc1 grew faster than WT.However,Δzfc1 grew slower than WT on PDA plates with different concentrations of cadmium,which phenomenon disappeared when these two strains were cultured on the PDA plates containing other environmental pollutants（eg.dichlorvos,BPA,and BPS）.These results indicated that zfc1 negatively regulated the growth of T.asperellum and played an important role in the response of T.asperellum to pollutant stress,especially the cadmium stress.4.The mechanism of cadmium tolerance in T.asperellum regulated by zfc1 was investigated by comparing the adsorption and enrichment capacity of WT andΔzfc1for cadmium,microscopic observation,and the metabolomics analysis of WT andΔzfc1 strains under cadmium stress.ICP-MS results suggested that the adsorption efficiency of T.asperellum for Cd²⁺decreased by 7.77%after zfc1 gene deletion,meanwhile,the enrichment capacity of theΔzfc1 strain dropped more than twice.Electron microscopy analysis showed that the subcellular structure of T.asperellum changed under cadmium stress,including cytoplasmic contraction,reduction of vacuoles number,increment of lipid droplet,and hyphae broken or even necrotic,which phenomena were more significant in the zfc1 deletion strain.Metabolomics research reflected that the deletion of zfc1 led to remarkable changes in metabolic components,of which 36 were significantly down-regulated and 23 were significantly up-regulated.These differentially expressed metabolites mainly participated in the ABC transporters pathway,in which proline is significantly downregulated.The results indicated that zfc1 gene expression of T.asperellum increased under cadmium stress,and its gene products may reduce cadmium toxicity by regulating proline content and other metabolic pathways,resulting in enhancing the cadmium tolerance of the strain.