The Optimization Of Agrobacterium Tumefaciens Mediated Trichoderma Harzianum Transformation System And Analysis Of T-DNA Insertional Mutants

Trichoderma harzianum is an important biocontrol filamentous fungus which can antagonize more than 30 species of plant pathogens. Cloning functional genes can promote the study on biology, biocontrol mechanism and spore formation mechanism of T.harzianum. The knowledge can provide theoretical foundation for developing stable, high-efficiency Trichoderma agent. Producing large quantity of mutants by molecular tag method like ATMT is a high efficient method for cloning functional genes. In this study, ATMT method was studied and used to establish T.harzianum mutant library and then analyzed the related fungal phenotypes and functional genes.1. The optimization of Agrobacterium tumefaciens mediated transformation system and construction of T-DNA insertional mutant library of T. harziranum.The factors influencing the transformantion efficiency of A. tumefaciens mediated transformation of T.harzianum were studied. The results showed that the transformation efficiency was correlated with the 3'5'-Dimethoxy-4-Hydroxy Acetophenone (AS) concentration in induced medium and co-cultivated medium, the induced time of A. tumefaciens, pH of co-culture medium, co-cultivation time, co-cultivation temperature, and the initial concentration of A. tumefaciens and the initial concentration of T.harzianum conidia. According to these results, we established optimal conditions for transformation of T. harzianum as follows: A. tumefaciens cultured to OD600 0.30~0.45 in induced medium with 200μM AS for 6 hours and then concentrated by 4 to 5 times; mixing the A. tumefaciens ferment broth with same volume T.harzianum conidia suspension which concentration was 106 conidia per milliliter; spreading 400μL mixture on the co-cultivation medium (pH5.3, 200μM AS) with a filter paper on it; incubating 48h at 24℃. Under these conditions, the transformation efficiency of ATMT on T.harzianum is 60~150 mutants per 106 conidia.We constructed a T-DNA insertional mutant library of T. harzianum with 4500 transformants. The quality of the mutant library was evaluated. All the randomly selected resistant colonies were proved to be stable through mitotic cell division. PCR and southern blot analysis showed that the T-DNA was integrated into the genome via random recombination and about 80% tansfromants contained a single copy T-DNA.2. Screening and analysis of the spore formation mutants from T-DNA insertional library of T. harzianum920 transformants were screened for spore formation mutants. Eight mutants were selected from the transformants cultured on PDA medium. Compared with the wild type T.harzianum, these mutants, to some degree, showed difference in conidia number, colony phenotype and conidiophore phenotype.7 mutants with chlamydospore formation variation were selected from 2680 transformants cultured in CHM medium. Chlamydospore formation ability of four mutants was lower than the wild type T.harzianum. The other three mutants showed both lower chlamydospore formation ability and higher conidia formation ability.12 mutants with spore formation differences were selected from 1192 transformants cultured in PD liquid medium. The conidia formation ability of all these 12 mutants was significantly lower than that of the wild type T. harzianum. The characterization of these 12 mutants was analyzed. The results showed that as for the mutants 627, 840, 918, 1137, 1173, 1317 and 1373, chlamydospore developed in the mycelium and conidia formation decrease. However, as for the other 5 mutants 763, 772, 886, 917 and 1252, only the conidia number decreased.3. Screening and analysis growth rate mutants and antagonistic ability mutants from the T. harziamum T-DNA insertional mutant library.The growth rate of 600 randomly selected transformants cultured on PDA medium was measured. From this experiment, 3 mutants with lower growth rate and 4 mutants with higher growth rate were screened. T-DNA insertion can change the antagonistic ability of T.harzianum. We determined the antagonistic ability of 1260 transformants by co-culturing with Rhizoctonia solani on PDA plate. The results showed 6 mutants with higher antagonistic ability and 17 with lower antagonistic ability compared with the type T.harzianum.4. Characterization and analysis of T-DNA flanking sequence from T.harzianum transformantsWe successfully obtained 42 T-DNA right border flanking fragments which can be resolvable from 52 randomly selected transformants. In these DNA fragments, each of 33 sequences all corresponded to single sequence, 2 sequences corresponded to one same sequence and other 7 corresponded to plasmid sequence. The T-DNA right border sequences were shortened by some degree. 13 analyzed transformants (38%) were fully preserved. On the other hand, 8 border sequences have 1 to 2 nucleotides truncations, 4 border sequences have 8 nucleotides truncations, and 9 border sequences have more than 10 nucleotides truncations. The G+C content of sequences from all mutants was analyzed. The results showed the G+C content of sequenced fragments was 44%~56% for 92% sequence (mean=52.4). This was similar to the range of G+C content of the T.harzianum EST sequence available on the NCBI GenBank (92.5% of the EST sequences with a G+C content between 44 ~56%). From this we can deduce the T-DNA from A. tumefaciens was inserted into gene rich region.5. Cloning related gene from spore formation mutantsBy using TAIL-PCR to clone T-DNA right border sequences from spore formation mutants, we successfully obtained the fungal genomic DNA sequences flanking T-DNA right border from 9 transformants. The homologous sequences of 6 sequences were found by using Blastn and Blastx from NCBI GenBank. However, the homologous sequences of other 3 sequences were not found. They might be new genes related to spore formation which need to be further studied.