The Construction Of A T-DNA Insertional Mutant Library Of Trichoderma Harziranum By ATMT And Characterization Of Phenotypic Mutants

Trichoderma harzianum is an important biocontrol fungus, cloning its biocontrol related functional genes can provide a theoretical basis for developing the high-efficiency, stable, Trichoderma agent. Producing large quantities of T-DNA insertional mutants by Agrobacterium tumefaciens-mediated transformation system (ATMT) is a high efficient method for cloning functional genes. A T.harzianum mutant library was created by ATMT in this study and the biocontrol related fungal phenotypes and functional gene was analysed as follows.1. For generation of T-DNA insertional mutant library of T. harziranum by ATMT.We optimized a protocol appropriate for T-DNA mediated mutation for T.harzianum . First, A. tumefaciens was cultured to reach an OD600 value at 0.20~0.30 and induced by 6 hours. After induction 220μL of A. Tumefaciens cell suspension with a OD600 value at 0.60~0.80 was mixed with 220μL of T.harzianum conidia suspension(105 conidia per milliliter) and spread the mixture on the co-cultivation medium .The AS concentration in both inducing medium and co-cultivation medium is 200 mmol?L-1, pH of co-culture medium and inducing medium is 5.3. Co-cultivation was conducted at 24℃for 48h, and then transformates were selected on a hygromycin containing medium on which transformates took 60 hours to develop visible colonies. Under the above conditions, A library of 2998 transformants was constructed with an transformation efficiency of 80~200 transformants per 106 spores. After three generations in PDA medium without hygromycin B, 2956 tranformants can still resist to hygromycin B, the false positive rate was low and more than 89 percent of the transformants was genetic stable.2. Screening the conidiation defective mutants in transformants of T. harzianum.1999 transformants of T. harzianum were screened for s conidiation defective mutants on PDA and 2 mutants #446 and #1413 showed dramatic defects in colony features, conidial yield,pycnidium morphology. For example, the conidial number of mutant 1413 reduced to less than 1/105 of that of wild type and almost didn't development pycnidium in the mycelium.3. Screening the antagonistic ability mutants in transformants of T. harzianum.1999 transformants of T. harzianum were screened for antagonistic defective mutants by testing their inhibitory effect against Rhizctonia solani. Among selected 31 mutants, 29 showed enhanced antagonistic ability against R. solani, while 2 showed weakened antagonistic ability. For mutants with enhanced antagonistic ability, when incubated with R.solani, the time for them to cover the whole plate and kill R.solani was 10~15h shorter than wide type T. harzianum's. For mutants with the weakened antagonistic ability, the time for them to cover the whole plate is 25h longer than wide type T. harzianum's, and theny couldn't make R. solani perish.4. Analysis of T-DNA insertion of screened mutants with molecular biology techniques.For all selected mutants, PCR and Southern-blotting analysis showed that the T-DNA was integrated into the Th-33 genome in all mutants and about 62.5% transformants contained a single copy T-DNA.5. Cloning and analysis of the spore formation related DNA sequences. We successfully obtained A 3725bp fungal flanking T-DNA sequences from the spore formation mutant #1413 by TAIL-PCR, which contains a part of gene that encodes a protein homologous to chromatin assembly protein.