Construction Of A T-DNA Insertion Mutant Library Of Fusarium Oxysporum F. Sp.Cubense Race4and Knockout Of Fopel Gene

A mutant library, which initiated from the wild type strain B2of the Fusarium oxysporum f.sp. cubense race4, was constructed by Agrobacterium Tumefaciens-Mediated Transformation （ATMT）. And after its quality evaluated, phenotype analyzed and pathogenicity tested, the mutant T1317was nonpathogenicity compared with the wild strain B2. And it’s flanking sequence was amplified by hiTAIL-PCR, and we named the sequence fopel after aglined with the whole genome of wild strain B2. Then we constructed the gene knockout vector and got the fopel gene knockout mutants.The main conclusions were showed as flowing:1. The Agrobacterium Tumefaciens-mediated transformation after optimizing was used to construct the wild type strain B2’s mutant library that contained2137mutants. The fresh wild type strain B2spore suspension at1×106cfu/mL, agrobacterium OD600reaching about0.6and the pH at about5.5,200uM Acetosyringone and48hours of co-cultivation period at25℃were optimal for the transformationsystem. Ratio could reach300transformants per106spores.2. Random selected50transformats to do hygromycin resistance expression stability experiments, only a failure to continue to grow, the stability was98%; seven days through observation, we gained the2137T-DNA insertion mutants reactivated, found45abnormal transformats;19showed obviously changes in the amount of spore production. Through growth rate test, we found that the wild type B2growth rate was1.398cm/d, the growth of most mutants was almost the same performance. Pathogenicity detection of the mutant library group by group,after vitro leaves experiment, living leaves experiment, banana leaf sheath experiment and hydroponic banana seedlings experiment. Finally, we got two strains witch loss of pathogenicity, respectively were T0258, T1317; three weakened mutant respectively were T0415, T1020.T2101.3.5strains with significant changes in pathogenicity tests were act further research. Using improved hiTAIL-PCR, sequencing and the mutant T1317, T2101’s T-DNA flanking sequence were obtained, compared with the B2sequenced whole genome, the two were positioned to FOC4₁0005136and FOC4₁0013034. After comparing homologous, we found they translated Pectin lyase and Acetyltransferase. In combination with other researchers in fungi pathogenic gene research, we decided to choose T1317flanking sequences as the following in-depth study of gene function, and named fopel, its gene length was825bp, consists of three two exons and introns.4. We use the pCX62plasmid to construct the gene knockout vector. The gene homologous fragments was obtained by PCR amplification, after the fungal protoplast transformation, we got the gene knockout mutants. The mutants DNA were extracted and used four pairs primers PCR for verification. At last, we got positive transformation for further research in the future.