15(2)(Agrobacterium tumefaciens)

polyethylene glycol (PEG) (restriction
enzyme-mediated integration, REMI)(Agrobacterium
1,2 ( E-mail: yuchu@mail.kdais.gov.tw)

1,000 (gene disruption cassette) 0.5-30% transposon (restriction
enzyme-mediated integration, REMI) transporson transposon (non-coding region) REMI REMI DNA genome 1995 Bundock (Agrobacterium
tumefaciens) DNA (T-DNA) 1998 de Groot (Ascomycetes)
(Basidiomycetes) (Zygomycetes) (Oomycetes) Agaricus bisporus, Calonectria morganii, Fusarium
circinatum Helminthosporium turcicum (protoplast) (ref.)
(Agrobacterium tumefaciens) (crown gall disease) (Agrobacterium
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242 Research Bulletin of KDARES Vol.24(2)
tumefaciens) (tumer-inducing plasmid) Ti-plasmid 200kbpTi-plasmid
T-DNA ( 1)T-DNA 24bp
left border right border border encoding auxin cytokinin
Ti-plasmid
virulence region vir genes T-DNA virulence region T-DNA
( encodign auxin
) T-DNA
1. (Agrobacterium tumefaciens) Vir (Michielse et al. 2005b)
T-DNA T-DNA virulence region plasmid T-DNA virulence region plasmid helper plasmid disarmed plasmid A. tumefaciens T-DNA plasmid E. coli T-DNA electroporation A. tumefaciens binary vector system
T-DNA
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242 Research Bulletin of KDARES Vol.24(2)
autophosphorylation VirA phosphoryl group VirG VirG protein DNA binding VirG vir genes activator ( 1)
T-DNA Vir C Vir D Vir D2 Vir
D1 right border 3 4 nucleotide right border 3’OH left border T-strand VirD2-T-strand complex type IV T-pilus
virulence protein VirB1-11 VirD4 T-strand VirB protein transport pore “T-pilusVirB2 protein T-pilin T-pilin T-pilus VirD4 protein T-strand VirB complex VirD2/T-strand complex VirE2VirE3 VirF T-pilus VirE2 DNA VirE2 T-strand T-strand nucleases
T-strand transport poreVirD2 C-terminal nuclear localization signal T-strand 5’ VirD2 T-strand T-DNA
de Groot et al. 1998
protoplast acetosiringone virulence systemMichielse
(2004) vir gene Aspergillus awamorni vir gene ( 2 1)
2. Ti-plasmid virulence region (Toro et al. 1988)
VirCEFH T-DNA VirFH VirE3
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VirC2
1. vir(Michielse et al. 2004)
Aspergillus Toro (1988) Vir C1 Vir C2
T-DNA nickingVirCT-DNA
nicking Michielse VirC2
T-DNA T-DNA VirABD G

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Blastomyces dermatitidis acetosyringone(AS)
vir
genes
( 2) pre-culture AS Beauveria
bassianaFusarium
oxysporum Magnaporthe grisea
pre-culture AS
2. (Agrobacterium tumefaciens) Aspergillus awamorini (Toro et al. 1988)

(Trihcoderma atroviride) 1993 Suominen T. reesei calcium/polyethylene glycol 32-52% T. atroviride Zeilinger (2004) T. atroviride (gene replacement cassette) tmk1 ( encodign MAP
kinase) ( 3A) tga3 ( encoding heterotrimeric G protein apha-subunit)( 3B) tmk1 disruption cassette EcoRI disruption cassette
genome EcoRI
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242 Research Bulletin of KDARES Vol.24(2)
3. (A)pTSZtmk1 (B)pTSZ tga3 DNAs EcoRI (A, C)ApaI/NheI (B) SmaI/ClaI (D) (Zeilinger et al. 2004)
tmk1 ( ) ApaI NheI ApaI NheI tga3 Zeillinger (2004)
50 T. atroviride 12wild type southern blot genomic DNA EcoRIApaI/NheI SmaI/ClaI genome
EcoRI tmk tga 4.2 kb/3.2
kb 5.7 kb/4.1 kb southern blot wild type 4.2 kb 5.7 kb tmk tga tmk1 6 4.2kb/3.2kb 6 tmk
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242 Research Bulletin of KDARES Vol.24(2)
tga wild type 5.7kb tga 5 5.7 4.1 kb tmk
tga tmk
tga 5’ 3’ non-coding region primer hygromycin primer PCR
Aspergillus awamori
( Saccharomyces cerevisiae
S. pombe) 50-100 bp 50-100%(1,16) (>1000 bp) 1030% G/C contant chromatin
Michielse (2005 a,b) Aspergillus awamori
pyrG 1000500250100 50 bp 5’pyrG 3’pyrG 5’pyrG 3’pyrG
500 bp ( 4)
4. Aspergillus awamori pyrG (Michielse et al. 2005a)
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CaCl2/PEG 1000500 250 bp
pgd promoter hygromycin 5’pyrG 3’pyrG 5’pyrG 5’ amdS Amds gene encode acetamidase
(E.C3.3.1.4) Aspergillus acetamide Acetamidase (acetamide) (acetate)
(ammonium) amds gene acetamide (8) fluoroacetamide fluoroacetamide acetamidase fluoroacetate Amds gene amds gene ( 5)
5. amds Michielse et al ( . 2005 b)
amds gene fluoroacetami-de Aspergillus awamori CaCl2/PEG 1000 bp (29%)(10%) 500 250bp 1000 bp 29% 500 bp 5% 500 bp
3’ 500 bp (outer) 34% 1000 bp ( 3)
250 bp ( ) CaCl2/PEG
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(Michielse et al. 2005 b)
southern
analysis NcoI 2659 bp 3091 bp 3091 bp 3091 bp
( 6) pyrG 1000 bp 500 bp 29% 5% 500 outer ( 500 bp 6) 34% amds amds pyrG
6% pyrG terminator
8% amds 1000 bp pyrG amds
3. CaCl2/PEG (Michielse et al. 2005 a)

AMT CaCl2/PEG pyrG
1000bp pyrG flank
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CaCl2/PEG 3 gfaA gfaA CaCL2/PEG 6 Kluyveromyces lactis 10 71 (4)
CaCL2/PEG CaCL2/PEG DNA-protein complex VirD2 VirE2 Streptomyces spp.
S. cerevisiae DNA DNA (6, 13) DNA VirD2 VirE2 CaCL2/PEG nucleases
Michielse (2005) 1000 bp pyrG 500 bp inner flagement 500bp outer flagement 5% 34% 500bp inner 500bp outer motif G/C content G/C content 46% 53% chromatin

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