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Molecular Pathogens
MP2010, Vol.1, No.1
http://mp.sophiapublisher.com
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in lettuce (Di Gaspero et al., 2002).
Several features of the banana putative RGCs isolated in
this study suggest they are non-TIR-NBS-LRR disease
resistance genes. For example, the characteristic motifs
of the NBS domain of known resistance genes described
(Meyers et al., 1999; Pan et al., 2000; Peraza-Echeverria
et al., 2008) are present in each banana RGC at similar
positions. One of these motifs, the highly conserved
P-loop, has been shown to bind ATP in the NBS-LRR
resistance proteins I2 and Mi from tomato (Tameling et
al., 2002), suggesting the banana RGC proteins may also
bind ATP. The non-TIR (nT) motif (Bai et al., 2002),
which is associated only with the non-TIR subclass of
NBS sequences, is found in the N-terminal region of the
banana RGCs, while none of the motifs associated with
the TIR subclass are found in the corresponding region
of the banana RGCs. All of the amino acids deduced
from the RGAs shared homology (28%~54%) with those
from the known wilt resistance genes such as Fom-2,
I2C-1, I2C-2 and I2. Among them, the homology
between GF7 and I2 are relatively high (about 54%),
which indicated that it might be a possible function of
resistance to Fusarium wilt of banana.
3 Materials and methods
3.1 Plant material
‘Goldfinger’ (Musa spp., AAAB) plantlets resistant to
FOC subtropical race 4 (Smith et al., 1998) were
obtained from South Subtropical Crop Research
Institute, Chinese Academy of Tropical Agricultural
Science, and were maintained in a phytotron at 28
℃
.
Roots were harvested from 4-month-old plants, frozen
in liquid nitrogen and then stored at -80
℃
for use.
3.2 DNA extraction
DNA was extracted from plant tissue using a modified
CTAB method based on Doyle and Doyle (1987).
Fresh root tissues (2~3g) were ground to powder and
transferred to the extraction buffer [2% (w/v) CTAB
(cetyltrimethyl-ammonium bromide), 1.4 mol/L NaCl,
20 mmol/L EDTA, 100 mmol/L Tris-HCl (pH 8.0) and
2% (v/v) β-mercaptoethanol]. The suspension was
extracted
twice
with
equal
volume
of
chloroform:isoamyl alcohol (24:1). Dried pellet was
dissolved in an appropriate volume of double distilled
sterile water.
3.3 Degenerate Primers Design
According to the conservative regions of the
nucleotide-binding site and the leucine-rich repeat
(NBS-LRR) in cloned wilt resistance genes, five pairs
of primers were designed to isolate R gene analogues
(RGAs) from the genomic DNA of wilt resistance
germplasm ‘Goldfinger’ (AAAB) banana (Table 1).
Table1 Degenerate primers used in amplification
NBS name
Peptide
Primer sequences encoded (5’→3’)
References
F1(F)
P-loop
GGDGTDGGNAARACWAC
Deng et al., 2000
F2(R)
GLPL
AANGCHAGNGGYAANCC
Deng et al., 2000
F3(F)
P-loop
GGWATGGGWGGWRTHGGWAARACHAC
Lee et al., 2003
F4(R)
GLPL
ARNWYYTTVARDGCVARWGGVARWCC
Lee et al., 2003
F5(F)
P-loop
GGIGGIGTIGGIAAIACIAC
Peraza-Echeverria et al., 2008
F6(R)
GLPL
AAGIGCTAAGIGGIAAGICC
Peraza-Echeverria et al., 2008
F7(F)
Kinase
GTNYTNGAYGAYGTNTGG
self-designed
F8(R)
Kinase
TAGTTGTRAYDATDAYYYTRC
self-designed
F9(F)
P-loop
GGNGGNRTIGGIAARACIAC
self-designed
F10(R)
GLPL
GAGGGCNARNGGNAAICC
self-designed
Note: Codes for mixed bases: R=A/G, W=A/T, M=A/C, K=G/T, Y=C/T, S=C/G, H=A/T/C, D=A/T/G, V=A/C/G, B=C/G/T,
N=A/T/G/C, I=hypoxanthine
3.4 PCR and cloning
PCR mixtures (50 μL) contained 5 μL 10×buffer
(TaKaRa with MgCl2), 4 μL 2.5 mmol/L dNTP, 1 μL
of each degenerate primer, 5 U Taq polymerase