Open AccessScreening and identification of insertion mutants from B ipolaris eleusines by mutagenesis based on restriction enzyme‐mediated integration
Author(s) -
Jianping Zhang,
Guifang Duan,
Kai Zhu,
Yongjun Zhou,
Yongliang Lu,
Liuqing Yu
Publication year - 2012
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2012.02537.x
Subject(s) - transformation (genetics) , biology , plasmid , mutant , phytotoxin , transposon mutagenesis , microbiology and biotechnology , mutagenesis , mycelium , gene , wild type , biochemistry , toxin , transposable element , botany
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 pSH 75 into the ophiobolin A ‐producing filamentous fungus B ipolaris 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 ( B 014) 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 R hizoctoni 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 B 014 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.