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Universal plasmids to facilitate gene deletion and gene tagging in filamentous fungi
Longguang Qin1; Ang Li1; Kaeling Tan1,2; Shuhui Guo1; Yingying Chen1; Fang Wang1; Koon Ho Wong1,2
2019-04
Source PublicationFUNGAL GENETICS AND BIOLOGY
ISSN1087-1845
Volume125Pages:28-35
Abstract

Gene manipulation is an important routine technique and its efficiency is often a rate-limiting step in research. To facilitate gene manipulation in filamentous fungi, we adapted the S. cerevisiae Gene Deletion and Gene Tagging plasmid collections to include additional selectable markers that make the useful resources applicable to other fungi. Three markers for auxotrophic selection in Aspergillus and related species (the riboB, pyroA and pyrG genes of Aspergillus fumigatus) and a dominant selectable marker for glufosinate resistance (the Bar gene from Streptomyces hygroscopicus) were introduced to the collections. A total of fifty-six plasmids were constructed for all combinations between the four new markers and thirteen epitope tags (viz., 3xHA, 13xMYC, 3xFLAG, FLAG, MYC, T7, HIS, Strep, S, HSV, VSV-G, V5 and GFP). The selectable marker and epitope tag cassettes are positioned between two universal sequences in the plasmids, and therefore, can be amplified by PCR using the same pair of primers. With these plasmids, we have also established a simple and efficient procedure for making gene deletion and gene tagging transformation DNA constructs. The procedure, along with the universal flanking sequences, allows quick and easy interchange of selectable marker and epitope cassettes in transformation DNA constructs for different selection and/or tagging. To demonstrate utility and efficiency of the system, we simultaneously performed C-terminal tagging of HapB – a subunit of the highly conserved Aspergillus nidulans CCAAT binding complex that plays important transcriptional regulatory roles – using ten different epitopes in order to identify those neutral to HapB function in vivo. It is expected that the expanded plasmid collections coupled with the simple construction strategy would facilitate gene manipulation in many fungal species.

KeywordGene Deletion Epitope Tagging Selectable Markers Plasmid Collection Filamentous Fungi Aspergillus
DOI10.1016/j.fgb.2019.01.004
Indexed BySCI
Language英语
WOS Research AreaGenetics & Heredity ; Mycology
WOS SubjectGenetics & Heredity ; Mycology
WOS IDWOS:000463311000003
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Document TypeJournal article
CollectionFaculty of Health Sciences
Corresponding AuthorKoon Ho Wong
Affiliation1.Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau
2.Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau
First Author AffilicationFaculty of Health Sciences
Corresponding Author AffilicationFaculty of Health Sciences
Recommended Citation
GB/T 7714
Longguang Qin,Ang Li,Kaeling Tan,et al. Universal plasmids to facilitate gene deletion and gene tagging in filamentous fungi[J]. FUNGAL GENETICS AND BIOLOGY,2019,125:28-35.
APA Longguang Qin.,Ang Li.,Kaeling Tan.,Shuhui Guo.,Yingying Chen.,...&Koon Ho Wong.(2019).Universal plasmids to facilitate gene deletion and gene tagging in filamentous fungi.FUNGAL GENETICS AND BIOLOGY,125,28-35.
MLA Longguang Qin,et al."Universal plasmids to facilitate gene deletion and gene tagging in filamentous fungi".FUNGAL GENETICS AND BIOLOGY 125(2019):28-35.
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