Application Research Of A Novel Dominant Selectable Marker Gene In Magnaporthe Oryzae

Magnaporthe oryzae is one of the most devastating phytopathogens of rice plants worldwide,which caused 10%-30% yield loss annually.With the release of the fungus and the host genome,the identification of the pathogenitic gene and the investigation of interaction mechanism between the fungus and the rice plants were accelerated.In addition,analysis of pathogenesis is a prerequisite to provide novel strategies for disease management.Understanding the pathogenic mechanism of M.oryzae requires accurate manipulation of genomic integration,such as knockout,over-expressing and fluorescent fusion-protein constructs.However,such undefined random integration may results in alteration of gene expression or gene disruption caused by position effects.A carboxin-resistant strain with an amino acid substitution(H245L)in the Mo Sdi1 subunit was found under the ultraviolet(UV)radiation.In this study,we provided a recombinant vector p Mo C-e GFP,which was efficiently integrated into the Mosdi1 locus by using carboxin as the selectable marker.PCR and southern blot validation revealed that 96% of all transformants was integrated correctly into the Mosdi1 locus as single copies.Independent transformants derived from the ATMT showed undiversified phenotypes,including hyphae growth,conidiation and conidia morphology.Meanwhile,plant infection on rice and barely indicated that the integration of p Mo C-e GFP vector in M.oryzae does not affect the virulence of the transformants on host.Above results encouraged us to utilize the Mosdi1 locus for integrate the foreign DNA into M.oryzae.Furthermore,we provided a vector which carries a yeast recombination cassette and thus allows assembly of multiple overlapping DNA fragments by the yeast gap repair approach for high-throughput gene complementation and protein localization assay.Complemented and fluorescent fusion mutants of Morak1,Mopmt2 and Mokhd4 were obtained by using this integrated system.Southern hybridization and RT-PCR results showed that the vectors were integrated into the Mosdi1 locus as single copies.These results suggesting that the utility of Mosdi1 locus for targeted integration is a useful method for genetic complementation analyses in M.oryzae.