Research On Cloning And Biological Control Function Of Mapks Gene From Trichoderma Asperellum

Trichoderma asperellum is important biocontrol fungus and control plant soil-borne pathogens through a variety of mechanisms. Though those mechanisms have been researched extensively, its biocontrol molecular mechanisms remain unclear. In order to master the biocontrol molecular mechanism of T. asperellum, the regulating genes related to biological control:taskl and taskkk were cloned based on cDNA library from T. asperellum mycelium and their biocontrol related functions were studied.Firstly, gene taskl, taskkk and their flanking sequences were cloned; the sequences of two genes were analysed. Recombinant binary vectors were constructed using hygromycin resistance gene as a selection marker. Transformation of T. asperellum utilized the method of Agrobacterium tumefaciens-mediated homologous recombination, the gene knock out mutants Ataskl and Ataskkk were obtainedThrough comparing mutant Δtask1and Δtaskkk with T. asperellum wild type, the influences of gene taskl and taskkk on T. asperellum growth were studied. The result showed that gene taskl had an effect on mycelia growth, mitosis, spore formation and colony growth state, did not affect colony growth speed and quality; gene taskkk was influential to spore formation, did not influence mycelia growth, mitosis, colony growth state, colony growth speed and quality.Then, the influences of gene taskl and taskkk on T. asperellum mycoparasitic effects were studied. The experiment results indicated that mutant Ataskl could recognize and adhere to R. solani, produce cell wall hydrolytic enzyme and hydrolyze R. solani, but could not mycoparasite it, that means gene taskl has anything to do with mycoparasitic effects of T. asperellum; mutant Ataskkk could not recognize and mycoparasite R. solani and growth separately, which means gene taskkk has anything to do with recognition and mycoparasitic effects of T. asperellum. The hydrolases gene expression and activity analysis of T. asperellum, mutant Ataskl and Ataskkk showed that gene taskl down regulated expression of cellulase, β-1,3glucanase, β-1,6glucanase, N-acetyl glucosaminidasel and β-1,4glucanase gene; taskkk gene up regulated expression of gene chitinase, N-acetyl glucosaminidase2, β-1,3glucanase and β-1,6glucanase gene. The hydrolases activity analysis related to biocontrol of T. asperellum, mutant Ataskl and Ataskkk indicated that gene taskl related to production of N-acetyl-glucosaminidase, β-1,3glucanase, cellulose and β-glucosidase, gene taskkk related to production of chitinase, N-acetyl-glucosaminidase and β-1,3glucanase. Finally, the influences of gene taskl and taskkk on T. asperellum antibiosis were studied. The experiment results indicated that gene taskl down regulated expression of polyketide synthase, but gene taskkk up regulated expression of it, which means that gene taskl and taskkk is not on the same signaling pathways. Antifungal ability of mutant Ataskl was higher than T. asperellum and mutant Ataskkk was lower than T. asperellum, which indicated the two genes related to the production of antifungal metabolites, only with the opposite effect. The influences of gene taskl and taskkk on6-amyl-a-pyrone from T. asperellum were studied. The yield of6-amyl-a-pyrone from T. asperellum wild type was1.32mg/g mycelial dry weight, mutant Ataskl was2.80mg6-amyl-α-pyrone/g mycelial dry weight,2.12times of T. asperellum wild type, and mutant Ataskkk was0.11mg6-amyl-a-pyrone/g mycelial dry weight,0.08times of T. asperellum wild type. Those showed that gene tasklwas negative regulation gene of6-amyl-a-pyrone, gene taskkk was up regulation gene of it, gene taskl and taskkk was not on the same signaling pathways.