title: Screening and identification of insertion mutants from Bipolaris eleusines by mutagenesis based on restriction enzyme-mediated integration.
abstract: Ophiobolin A is sesterterpenoid-type phytotoxin and may be an important candidate for development of new crop protection and pharmaceutical products. The restriction enzyme-mediated integration (REMI) method was used to introduce the plasmid pSH75 into the ophiobolin A-producing filamentous fungus Bipolaris eleusines. A total of 323 stable transformants were obtained, all of which were capable of growing on potato-dextrose agar medium containing 200 μg mL(-1)  hygromycin B. The transformation frequency was about 4-5 transformants μg(-1)  plasmid DNA. An ophibolin A-deficient transformant (B014) was assessed and the presence of the hph gene in this transformant was confirmed by PCR. The cell-free cultural filtrates of this transformant showed significantly less inhibition on mycelial growth of the fungal pathogen Rhizoctoni solani but little effect on barnyard grass as opposed to that of the wild-type B. eleusines. There was no detectable amount of ophiobolin A in B014 samples measured with HPLC. This research suggests REMI as a potential approach for improving the production of ophiobolin A by B. eleusines via genetic engineering to upregulate certain genes responsible for desired biosynthetic pathways.
authors.affiliation: State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, China.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Jianping
authors.forename: Duan
authors.initials: D
authors.lastname: Guifang
authors.forename: Zhu
authors.initials: Z
authors.lastname: Kai
authors.forename: Zhou
authors.initials: Z
authors.lastname: Yongjun
authors.forename: Lu
authors.initials: L
authors.lastname: Yongliang
authors.forename: Yu
authors.initials: Y
authors.lastname: Liuqing
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D003470:Culture Media / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D031747:Echinochloa / Q000382:microbiology; D017353:Gene Deletion; D016254:Mutagenesis, Insertional / Q000379:methods*; D010957:Plasmids; D054830:Sesterterpenes / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D054830:Sesterterpenes; C040764:ophiobolin A; D004262:DNA Restriction Enzymes