The size and ratio of homologous sequence to non-homologous sequence in gene disruption cassette influences the gene targeting efficiency in Beauveria bassiana.

Targeted gene replacement via homologous recombination (HR) is a conventional approach for the analysis of gene function. However, this event is rare in Beauveria bassiana, which hampers efficient functional analysis in this widely used entomopathogenic fungus. To improve homologous recombination frequency in B. bassiana, we investigated the effect of the ratio of homologous sequence to non-homologous sequence (HS/NHS) in gene disruption cassette upon the HR frequency by two gene loci BbNtl and BbThi, using the herpes simplex virus thymidine kinase as a negative selectable marker against ectopic transformants. Our data revealed that an increase of the ratio of HS/NHS achieved by either extending homologous sequence or decreasing non-homologous sequence could improve HR frequency in B. bassiana. We determined empirically that (1) at least 700 bp of homology to both sides of a target gene was needed to get a reasonable number of disruptants, e.g., 6.7 per thousand to 13.3 per thousand in B. bassiana. (2) When the ratio of HS/NHS was above 0.8, an acceptable HR frequency could be achieved for gene replacement in B. bassiana, while when the ratio was below 0.3, few gene disrupted mutants were obtained.

Characterization of a highly active promoter, PBbgpd, in Beauveria bassiana.

The promoter of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from Aspergillus nidulans (PgpdA) is widely used to direct expression of target genes constitutively in fungi. However, in some species, a heterogeneous promoter is found to be of low efficiency. To obtain a high-efficiency promoter for transformation of Beauveria bassiana, an entomopathogenic fungus widely used as an mycoinsecticide, a glyceraldehyde-3-phosphate dehydrogenase gene (Bbgpd) promoter, was cloned and characterized. Four deletion constructs (-2118, -1153, -726, and -354) of the 5'-upstream sequence of Bbgpd linked to a bar::gus fusion gene (phosphinothricin-resistance::beta-glucuronidase fused gene), which were used as selected marker gene and report gene, respectively, were generated. GUS activities of transgenic strains harboring -726, -1153, and -2118 deletion constructs were much stronger than that of the promoter of Aspergillus nidulans gpdA (PgpdA), with a twofold to threefold increase over that in the PgpdA construct. The -726 fragment was necessary to direct GUS expression in B. bassiana. No -354 transgenic progenies were obtained, possibly because it failed to initiate the transcription of bar::gus fusion gene. A remarkable increase of GUS activity was found between the -1153 and -726 constructs, indicating that some active transcriptional elements were located in this region. With a high expression level and relatively short sequence, PBbgpd can be used to drive target genes in B. bassiana transgenic research.