Transformation Of Plant Related Adverse Environment Transcription Factor CBF1 To Trichoderma Viride And The Study Of The Transformant

Filamentous fungus Trichoderma can survive in various environments and grow rapidly,it also can inhibit many phytopathogens,promote plant growth and induce the resistance of plant against disease.In agriculture,Trichoderma has been used to be as biological control agent,and it is the most successful ffungal biocontrol agent in plant disease control.CBF1 is one of the cold related transcriptional factors in plant,till now,there is no report of CBF1 in filamentous fungus.In this paper,we cloned the CBF1 gene form cDNA library of Arabidopsis thaliana,and construct the entry clone pENTRTTM/D-TOPO(?)-CBFl,after LR reaction with ImpGWB502 and ImpGWB505,we constructed two expression vectors:ImpGWB502-CBF1 and ImpGWB505-CBF1-GFP with a GFP label,both of them were transformed into Agrobacterium tumefaciens EHA105.The transformants were used to transform CBF1 into the Trichoderma viride.By use of Agrobacterium-mediated transformation(ATMT),we obtain 11 CBF1 transformants and 7 CBF1-GFP transformants,all transformants are genetic stability and contain the CBF1 gene prove by PCR.By RT-PCR technology,we found that only 4 CBF1 transformants expressed the CBF1 gene,and all CBF1-GFP transformants issued fluorescence in fluorescence microscope.By the use of confocal laser scanning microscope,we found the fluorescence are everywhere in cells of T.viride transformants,that means the CBF1 protein is scatted everywhere in the fungi cell,the result is not consistent with the report that the CBF1 protein is concentrate in plant cell nucleus.All the transformants which express CBF1 gene were grow in adverse environment to investigate the adaptation ability.Both transformants were culture in PDA plate with 6%NaCl or 1 mol/L mannitol in 23℃,and recorded the diameters.We found the diameter of transformants were 7%-12%smaller than Wild Type(WT)after culture 10 days in NaCl and 4 days in mannitol,the result suggest that,the transformants unable to resistance the osmotic pressure.When the transformants were incubate in 4 ℃ for 10 days,and then grow in 23 ℃ for 24 hours,we measure the colony diameter,and found that the CBF1-GFP transformants’ diameter is 10%larger the WT,but the CBF1 transformants except the diameter of CBF1-4 is 12%larger than WT,the rest of transformants’ diameter is smaller than WT.The result suggests that,the CBF1 may influence T.viride to response cold stress.When CBF1 transformants fist incubate in 37 ℃ for 24 h,and then grow in 23℃ for 4 days,the CBF1 transformants’ diameter on PDA can 65%larger than WT,and the average diameter of transformants is 60%larger than WT.That means the expression of CBF1 gene confer the transformants higher resistant high temperature stress.Trichoderma is an important biological control agent,but its anti-pathogens ability will be influence by environment,especially the adverse environment will endanger its survival.In this paper,we transform a cold related transcriptional factor CBF1 from A.thaliana into T.viride by ATMT.The results suggest that CBF1 would influence T.viride to response adverse environment stress,especially enhance the ability of tolerance high temperature stress.Till now,there is no report about improve ability of T.viride to resistant adverse environment by transform a CBF1 into this fungi.Our study is meaningful not only for improve Trichoderma survival and anti-pathogens ability,but also extended application of these fungi in agriculture...