Abstract：
:Genetic engineering offers a practical route for enhancing the insect biological control potential of entomopathogenic fungi such as Beauveria bassiana. To date, however, such efforts have relied upon transformation protocols that utilize antibiotic or herbicidal resistance markers as selection agents for the introduction of genes into the fungus. In order to avoid the use of such markers for the development of field-usable fungal strains, a markerless transformation system based upon complementation of uridine auxotrophy was developed. A targeted gene deletion knockout of orotidine 5'-phosphate decarboxylase (ura3) was isolated using a positive screening protocol with 5'-fluoro-orotate. Although growth was restored when the mutant, ΔBbura3, was grown in the presence of exogenous uridine, conidiation remained impaired and conidial yield was reduced. Insect bioassays revealed that the ΔBbura3 strain was essentially avirulent using both topical and intrahemocoel injection assays, indicating that the deletion mutant was unable to scavenge uridine from the host during infection. A series of plasmid constructs were developed for complementation of the ura3 mutant, and complemented strains were restored to wild-type growth and virulence. These data indicate that the ura3 mutant and corresponding complementation vectors can be used to construct markerless strains for the bioengineering of desired traits in B. bassiana.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Ying SH,Feng MG,Keyhani NOdoi
10.1007/s00253-012-4426-0subject
Has Abstractpub_date
2013-04-01 00:00:00pages
3017-25issue
7eissn
0175-7598issn
1432-0614journal_volume
97pub_type
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