Study The Function Of C2H2 Transcription Factor THA09974 From Trichoderma Harzianum TH-33

Trichoderma harzianum is an important kind of bio-control fungus.It has a broad spectrum of antagonism,strong adaptability and multiple action mechanisms.Knocking out G?-Thga1 of Trichoderma harzianum found that conidiophore and conidium amount were significantly reduced.We explored the transcriptome sequencing of TH-33 and TH-33-THGA1 knocked out strain respectively,founding the gene expression of C2H2 transcription factor THA 09974 has changed the most.In the past research of the lab,deletion mutant of THA 09974^-was built and the had the different clonony feacture.So in this study,we explored a set of researches on the THA 09974.For example,we constructed reversion mutant of THA09974 and compared the characteristics of the differences on TH-33,mutants THA09974^-and THA09974^+/-strains,several results of experiments were as the followings:1.Reversion mutant of THA09974^+/-of TH-33 was gotPgpda promoter fragment,tha09974,Ttrpc terminated fragments were connected by overlapping PCR to get tha09974 gene cassette.Then the gene cassette was connected into pKH-KO-G418 by infusion enzyme to get pKH-KO-G418-THA09974 vector.PCR and enzyme digestions were carried out for the identification.The THA09974^+/-was got by the transformation of pKH-KO-G418-THA09974 to THA09974^-via protoplast transformation.PCR and RT-PCR were explored to validate the existence of target genes in the mutant strain.The absolute fluorescence quantitative detected the one copy of tha09974 gene in THA09974^+/-mutant.These results showed that THA09974^+/-was built successfully with single copy.2.The effect of THA09974 on the biological characteristics of T.harzianumThe colony morphology of the knockout mutant THA09974^-had obvious changes with becoming deeper green on PDA medium,the growth rate and biomass of THA09974^-strain vs Th-33 strain fell 30%and 50%respectively,the amount of spores was increased 100%comparing with wild type,growth rate of THA09974^+/-and TH-33 were no significant difference.THA09974^-secreted 28.9%more pectin Enzyme than TH-33.THA09974^+/-and TH-33 has no significant differences in producing pectin enzyme.So the tha09974 gene had negatively effected on spore production and pectin Enzyme,and positively effected on strain growth.3.The effect of THA09974 on the stress resistance of T.harzianumUnder 2.4 mM Cu²⁺concentration vs 0mM Cu²⁺,Biomass of the TH-33,THA09974^-,THA09974^+/-were reduced were reduced by 50%,15.6%,50%respectively;The differences in growth inhibition rate were reached 78%,75%,80.9%respectively,All three strains don't produce sporulation.So under the stress of 2.4 mM Cu²⁺,tha09974 maybe have no significant effect on the growth of strain hyphae and spore production and play a positive role on strain mycelia yield.Under 5 mM H₂O₂ stress,the spore production of TH-33,THA09974^-,THA09974^+/-were reduced 65%,78.7%and 68%,comparing with 0 mM H₂O₂ concentrations,the biomass were decreased by 36.7%,69%and 43%respectively,while the growth inhibition rates were dropped to 67%,39%and 90%;In conclusion,the tha09974 maybe play a negative regulatory role for spore production under the stress of H₂O₂ in the strains and had positive effect on the mycelial growth and yield of strains.4.The effect of THA09974 on the antagonism of T.harzianumThe competition activity of knockout mutant THA09974^-has been enhanced 6%against Rhizoctonia solani than wide type TH-33,and reversion mutant THA09974^+/-.Moreover,the production of cellulose enzyme of THA09974^-was higher 40%and 39.7%than TH-33,THA09974^+/-,to sum it,loss of tha09974gene could increase the secretion of cellulase.