Molecular Plant Breeding 2015, Vol.6, No.20, 1
-
10
4
Table 2 Primers for semi-quantitative RT-PCR of ESTs
NO.
Primer name
Primer sequence(5'-3')
T(
℃
)
Length
E87
E87-R
ATACAGTGCGTTGTTGCC
51.1
262 bp
E87-F
CTTGCGAGCATTTCTTCT
E96
E96-R
CCGCTTCAATTCTAACAG
48.3
296 bp
E96-F
AGTAGGGCAAGAAGGAGA
E108
E108-R
GATGAGTCCTGAGTAAATACGGGCT
53.1
278 bp
E108-F
GGGAAGCAAAATCACAAACAAGAAG
E142
E142-R
TCAACTGTCCATGTGTCCTCCTAGA
50
169 bp
E142-F
GCACTTCAAGTTCAGGAATATCTCA
E205
E205-R
AAGACCCGCCCTACAAAC
50.7
211 bp
E205-F
GCGGCAGTTCTATTCTTTCT
E220
E220-R
GACTGCGTACCAATTCGCGGATACG
59.9
137 bp
E220-F
CTGAGTAACGACACCACAGAGCCCT
18S rRNA
18S rRNA-R
CACCAGACTTGCCCTCCA
62
171 bp
18S rRNA-F
CCTGAGAAACGGCTACCACAT
Results
Disease infection to banana leaves
Banana leaves from uniform seedlings were cut for in
vitro pathogen inoculation experiment. Foc-TR4
conidia was inoculated on leaves as infection treatment,
the sterile H2O was inoculated as control treatment.
The leaves of infection treatment showed typical
disease symptoms of Fusarium wilt disease at 3 dpi
(days post inoculation); the lesion expanded on leaves
with yellow color changes. At the same time, the
control treatment showed no disease symptom (Figure 1).
Screening of differential expressed ESTs by
cDNA-AFLP
High quality RNA of 4 different time points (4 h, 24 h,
3d and 6 d after inoculation) were extracted for
cDNA-AFLP experiment. A total of 256 primer
combinations were used for cDNA-AFLP analysis
from both control and infection treatment. On average,
Figure 1 The pathogenic symptom of banana leaves at 3 dpi.
The left leaf was inoculated with Foc-TR4 as infection
treatment, the right leaf inoculated with sterile H2O as control.
60-80 clear and unambiguous bands (ESTs) were
generated by each primer combination, which yielded
more than 10,000 bands totally. There were 223
differentially expressed ESTs isolated from silver-stained
cDNA-AFLP polyacrylamide gels (Figure 2), according
to their presence/absence, or quantitative difference
between the infection treatment and control at
different time points. All isolated EST fragments were
eluted from the polyacrylamide gel, and these fragments
were reamplified by selective amplification condition.
Sequence analysis of ESTs
223 re-amplified ESTs were linked to pUCm-T vector
and transformed into E. coli competent cell. The
positive clones were sequenced and compared with
bioinformatic database including Genbank of NCBI
and The Banana Genome Hub (
- genome.
cirad.fr/) by BLAST program, as results, 137 unique
ESTs were found. Most of ESTs had homology to
genes with known functions (Figure 3), whereas, 18%
of them were unclear classification, and 17% were
new EST with no similarity to any known function
genes. According to the functional catalogues, among
the known function ESTs, 7% belonged to disease
defense, 10% belonged to signal transduction, 13%
belonged to transcription, and so on. So presumably,
the molecular regulation mechanism of banana
responded to Foc- TR4 disease was very complicated.
The special genes related to disease and defense
response
When plant suffered from pathogenic stress or
infection, its first response is to stimulate the cell
