Identification And Characterization Of A T-DNA Insertion Mutant, Mu301, From Beauveria Bassiana

Beauveria bassiana can invade the hosts by directly penetrating cuticles and cause insect epidemic. In addition, the hosts hardly produce resistance to the pathogen. Therefore, B. bassiana has great potential in the control for agricultural and clinical pests. However, the longer time to kill the insects and inconsistent results deterred the application of entomopathogenic fungi. One reason for the limitation of wide application of mycoinsecticides is that the mechanism in the development and pathogenicity of this kind pathogen is largely unknown.To understand the mechanisms of development and pathogenesis of B. bassian, in this paper, we constructed a T-DNA insertion mutant pool of B. bassiana by Agrobacterium tumefaciens-mediated transformation (ATMT). From the pool, a T-DNA mutant, named Mu301, showing retarded growth and reduced pathogenicity, was identified and its corresponding gene was cloned by T-DNA tagging approach.The main results are as follows:1. Identification of the T-DNA insertion mutant Mu301The T-DNA insertion mutant, Mu301, was obtained by Agrobacterium tumefaciens-mediated transformation in B. bassiana. This mutant displayed significant reduce in virulence and slow growth.2. Cloning and characterization of MCCB geneA fragment of 2760 bp containing an ORF was obtained by tagging the flanking sequence in the mutant using Sitefinding-PCR method. Blast result revealed that this ORF similarity (83%) to the biosynthetic gene of 3-methylcrotonyl-CoA carboxylaseβChain in A. nidulans. The inserted-site was located at the promoter region, (-485bp) upstream to initiation codon ATG of the gene). 3. Gene knock-out of MCCBTo investigate the function of MCCB, homologous recombination was used. The gene knock-out vector, pPZPtk8.10-MCCB51-bar-MCCB31, was constructed and delivered in to B. bassiana genome by Agrobacterium tumefaciens-mediated transformation. Three homologous recombination strains (△mccB 111,△mccB 205 and△mccB 277) were obtained.4. The phenotype of△mccB mutantsIt was found that when leucine was used as a sole carbon and nitrogen source, the growth of△mccB mutants was significantly retarded compared with the wild-type strain, suggesting that disruption of MCCB interfered the leucine metabolism in the fungi.