Downloads: 51

Files in This Item:
File Description SizeFormat 
s13568-019-0818-1.pdf1.15 MBAdobe PDFView/Open
Title: Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora
Authors: Honda, Yoichi  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0002-7218-7367 (unconfirmed)
Tanigawa, Eiji
Tsukihara, Takahisa
Nguyen, Dong Xuan
Kawabe, Harunori
Sakatoku, Naofumi
Watari, Junko
Sato, Hideaki
Yano, Shigekazu
Tachiki, Takashi
Irie, Toshikazu
Watanabe, Takahito  kyouindb  KAKEN_id  orcid https://orcid.org/0000-0002-2770-6818 (unconfirmed)
Watanabe, Takashi  kyouindb  KAKEN_id
Author's alias: 本田, 与一
渡邊, 崇人
渡邊, 隆司
Keywords: Basidiomycete
Genetic transformation
Transient gene expression
Promoter assay
TATAA sequence
Issue Date: 24-Jun-2019
Publisher: Springer Science and Business Media LLC
Journal title: AMB Express
Volume: 9
Thesis number: 92
Abstract: A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl₂ treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.
Rights: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
URI: http://hdl.handle.net/2433/245407
DOI(Published Version): 10.1186/s13568-019-0818-1
PubMed ID: 31236750
Appears in Collections:Journal Articles

Show full item record

Export to RefWorks


Export Format: 


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.