PMID- 23457794
OWN - NLM
STAT- MEDLINE
DCOM- 20130404
LR  - 20130305
IS  - 0001-6209 (Print)
IS  - 0001-6209 (Linking)
VI  - 52
IP  - 12
DP  - 2012 Dec 4
TI  - Agrobacterium tumefaciens-mediated transformation as a tool for insertional 
      mutagenesis in thermophilic fungus Thermomyces lanuginosus.
PG  - 1449-57
AB  - OBJECTIVE: To establish a stable transformation system of the thermophilic fungus 
      Thermomyces lanuginosus for its insertional mutagenesis. METHODS: Agrobacterium 
      tumefaciens-mediated transformation (ATMT) was applied to establish 
      transformation system of T. lanuginosus. Southern blotting of hph gene and 
      cloning of transforming DNA (T-DNA) flanking sequences were used to determine 
      insert number and site of T-DNA in the fungal genome, respectively. RESULTS: A 
      reliable transformation method is established for T. lanuginosus. Specifically, 
      pre-germinating spores of T. lanuginosus used at co-cultivated period was a 
      prerequisite. T. lanuginosus germinating spores co-cultivated with Agrobacterium 
      tumefaciens at 28 degrees C for 48 h achieved the highest transformation 
      efficiency. Addition of Acetosyringone (AS) during pre-culture of A. tumefaciens 
      and co-cultivation of T. lanuginosus germinating spores with A. tumefaciens was 
      essentially required, and the best results were obtained with AS at the 
      concentration of 500 microM. Southern blotting analysis showed that majority of 
      transformants (79.2%) contained a single insertion of T-DNA. Thermal asymmetric 
      interlaced PCR (TAIL-PCR) analysis showed random insertion of T-DNA in the fungal 
      genome. Using the transformation system, some stable phenotypic mutants of T. 
      lanuginosus were obtained. CONCLUSION: We report, for the first time, a simple 
      and efficient method for transforming T. lanuginosus by using ATMT. This approach 
      provides a tool for insertional mutagenesis gene tagging in this thermophilic 
      fungus.
FAU - Han, Hua
AU  - Han H
AD  - Department of Environmental Biology, Shandong Agricultural University, Tai'an 
      271018, China. jnhanhua@163.com
FAU - Xu, Xiaoxue
AU  - Xu X
FAU - Peng, Yanjie
AU  - Peng Y
FAU - Kong, Dehui
AU  - Kong D
FAU - Li, Duochuan
AU  - Li D
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - China
TA  - Wei Sheng Wu Xue Bao
JT  - Wei sheng wu xue bao = Acta microbiologica Sinica
JID - 21610860R
RN  - 0 (DNA, Bacterial)
RN  - 0 (T-DNA)
SB  - IM
MH  - Agrobacterium tumefaciens/*genetics/metabolism
MH  - Ascomycota/*genetics/growth & development/metabolism
MH  - DNA, Bacterial/genetics/metabolism
MH  - Genetic Vectors/genetics/metabolism
MH  - Mutagenesis, Insertional
MH  - *Transformation, Genetic
EDAT- 2013/03/06 06:00
MHDA- 2013/04/05 06:00
CRDT- 2013/03/06 06:00
PHST- 2013/03/06 06:00 [entrez]
PHST- 2013/03/06 06:00 [pubmed]
PHST- 2013/04/05 06:00 [medline]
PST - ppublish
SO  - Wei Sheng Wu Xue Bao. 2012 Dec 4;52(12):1449-57.