Development of a highly efficient gene targeting system in Penicillium oxalium HP7-1

【Author】

Ruijie Li;Qin Xiulin;Jia-Xun Feng;

【Abstract】

Background Gene targeting is necessary for analyzing of a gene,however gene targeting frequencies in filamentous fungi are very low.To develop a highly gene targeting system for Penicillium oxalium HP7-1,producing completely cellulolytic enzyme system,a AligD-Kan P.oxalicum strain was generated.Methods The P.oxalicum strain△ligD-Kan was generated by site-directed integrating the NHEJ(non-homologous end joining)pathway gene PoligD disruption cassette with kanr marker.The flanking regions(0.5/1.0/1.5 kb)of the agaA(arginase)and argB(acetylglutamate kinase)ORF were cloned and connected with the hph gene,respectively.The DNA fragments for disruption of the agaA and argB genes were introduced in to the PoligD disruptant,respectively.Results Deletion of PoligD in P.oxalicum significantly improved gene targeting frequencies(reached 90%)compared to that in the wild-type strain(73.3%for agaA and 12.5%for argB),when the 0.5 kb flanking regions were used.The obtained△ligD-Kan strain showed no apparent defect in vegetative growth including on solid or in liquild media and cellulose activity.Compared with the wild type strain,△ligD-Kan displayed similar sensitivity to ethyl methanesulphonate(EMS)and osmotic stress,while increased sensitivity to methyl methanesulfonate(MMS).Conclusion The results indicate that the△ligD-Kan strain is an efficient host for targeted gene disruption in P.oxalium HP7-1.

【Keywords】

Filamentous fungi;;Gene targeting efficiency;;ligD

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