Functional Analysis Of Copper Stress Response Related Transcription Factor Gene Thmea1 In Trichoderma Harzianum

Trichoderma spp.,as active components in biological fungicides and fertilizers,has great potential in biological control of plant diseases and improvement of soil environment.In recent years,copper and copper compounds have been widely used in fungicides,chemical insecticides and animal husbandry food additives,which made the increase of soil copper content and producing negative effects on soil microorganism,imbalance of ecological system and declining of the soil quality.Study on copper tolerance and metabolism related mechanism of Trichoderma is of great significance to improve the effectiveness of disease prevention and soil ecological environment.Recently,a C2H2-type transcription factor gene thmea1 was cloned from T.harzianum th33 in our laboratory.The deduced amino acids of the coding region of thmea1 contained three C2H2-type zinc finger domains which are conserved with the yeast metallothionein expression activator ACE2,and was speculated to be involved in copper stress response and metabolic process in T.harzianum.In this study,the function of thmea1gene was investigated by gene knockout and overexpression techniques and combined with RNA-seq analysis.The results were as follows:1.According to the genomic information of T.harzianum Th33,specific primers were designed and the coding region of thmea1 gene was amplified by PCR with the cDNA of Th33 as template,and the sequence analysis of the PCR product showed that the coding region of thmea1 was 1446 bp in length with one exon and no intron.The deduced amino acids sequence contained three C2H2-type zinc finger domains which are conserved with those in the yeast metallothionein expression activator ACE2 and its homologous protein SWI5,and thmea1 gene was deduced to be a C2H2-type transcription factor gene.The sequence was submitted to NCBI and got the GenBank accession number MF802279.2.The gene knock-out vector pKH-KO-?thmea1 was constructed by the principle of homologous recombination,then the knock-out vector was transformed into the protoplast of T.harzianum Th33 by PEG-mediated protoplast transformation and getting the thmea1 gene knockout mutant?thmea1.The thmea1 gene expression box was constructed with gpdA as promoter and trpC as terminator which were cloned from Aspergillus nesteurus and inserted into the downstream of G418 resistance gene,generating the overexpression vector pKH-KO-Othmea1.The overexpression vector was transformed into Th33protoplasts and getting the thmea1 gene overexpression mutant Othmea1.3.Copper ion tolerance test of the wild Th33 and the thmea1 gene knockout mutant?thmea1 and overexpression mutant Othmea1 showed that the growth rate of?thmea1 was significantly faster than Th33 and Othmea1 and produced more airborne mycelium on PDA medium containing 0².4mM/L copper ions.The median concentration of copper ion tolerance(MIC₅₀)of?thmea1 was 1.92 mM/L,while the MIC₅₀ of Th33 and Othmea1 were 1.70 mM/L and 1.74 mM/L respectively,the copper resistance of?thmea1 was increased by about 12.9%.Copper ion adsorption results showed that there was no significant difference in the adsorption rate of copper ions between the above wild Th33 and mutant strains,which suggested that the absence of thmea1 gene might improve the tolerance of T.harzianum to copper ions.4.qRT-PCR analysis of copper metabolism-related genes showed that under the stress of copper,the expression level of metallothionein gene?Tha₀7074?in?thmea1 was significantly higher than that in Th33 and Othmea1,and the expression level was about 5 times higher than that of Th33.It is speculated that thmea1 gene may negatively regulate the expression of Tha₀7074.The expression level of p-ATP enzyme gene?Tha₀8191?in Th33 and Othmea1 was significantly higher than that in?thmea1,which can facilitate the entry of copper ions into the Golgi secretion and release system,suggesting that thmea1 may regulate the extracellular release of copper ions in T.harzianum.5.The RNA-seq was performed for wild Th33 and knockout mutant?thmea1 under treatment of 0.8mM/L copper ion.The results showed that those genes related ribosomal protein synthesis and stress repair functional heat shock protein and ubiquitin protein were up-regulated in?thmea1 under copper stress.It was speculated that thmea1 may regulate the regenerative cycle of functional proteins,and its regulatory mechanism needs further study.