Agrobacterium tumefaciens-mediated transformation of the entomopathogenic fungus Nomuraea rileyi.
An Agrobacterium-mediated genetic transformation system for the entomopathogenic fungus Nomuraea rileyi was established. Three binary T-DNA vectors, pPZP-Hph, pPZP-Hph-RNAi and pPZP-Hph-DsRed2, were constructed. The trpc promoter from Aspergillus nidulans was used as the cis-regulatory element to drive the expression of hygromycin phosphotransferase (hph) gene and DsRed2, which conferred the hygromycin B (Hyg B) resistance and red fluorescence visualization, respectively. The blastospores and conidia were used as the recipients. The blastospores' transformation efficiency reached ∼20-40 transformants per 10(6) blastospores, whereas the conidia were not transformed. Based on an analysis of five generations of subcultures, PCR and Southern blotting assays, the Ptrpc-hph cassette had integrated into the genomes of all transformants, which contained single copy of the hph gene and showed mitotic stability. Abundant altered morphologic phenotypes in colonies, blastospores and hyphae formations were observed in the arbitrary insertional mutants of N. rileyi, which made it possible to study the relationships between the functions and the interrupted genes over the whole genome. The transformation protocol will promote the functional characterization of genes, and the construction of genetically engineered strains of this important entomopathogenic fungus, and potentially of other similar fungal pathogens.
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China; College of Life Science, Linyi University, Shandong 276000, China. Electronic address: changwen2000@sina.com.
Changwen
C
Shao
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: ypy128@vip.sina.com.
Youping
Y
Yin
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: qzr20101901003@163.com.
Zhaoran
Z
Qi
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: 19881021.ren@163.com.
Ren
R
Li
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: szy83529@163.com.
Zhangyong
Z
Song
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: sungq520@yeah.net.
Yan
Y
Li
Chongqing Engineering Research Center for Fungal Insecticides, School of Life Science, Chongqing University, Chongqing 400030, China. Electronic address: w-zk@163.com.
Zhongkang
Z
Wang
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000818:Animals; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics; D006999:Hypocreales / Q000235:genetics*; D007313:Insecta / Q000382:microbiology; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D014170:Transformation, Genetic
D004269:DNA, Bacterial; C036483:T-DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase
Agrobacterium tumefaciens-mediated transformation (ATMT); Hygromycin phosphotransferase gene (hph); Nomuraea rileyi; Red fluorescent gene (DsRed2); Vector construction