Molecular Plant Breeding 2015, Vol.6, No.20, 1
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Table 1 Sequence of selective amplification primers
Name
EcoR I (EcoR I+00) sequence
Name
Mse I (Mse I+00) sequence
Esa1
5’-GACTGCGTACCAATTCAA
Msa1
5’-GATGAGTCCTGAGTAAAA
Esa2
5’-GACTGCGTACCAATTCAC
Msa2
5’-GATGAGTCCTGAGTAAAG
Esa3
5’-GACTGCGTACCAATTCAG
Msa3
5’-GATGAGTCCTGAGTAAAC
Esa4
5’-GACTGCGTACCAATTCAT
Msa4
5’-GATGAGTCCTGAGTAAAT
Esa5
5’-GACTGCGTACCAATTCCA
Msa5
5’-GATGAGTCCTGAGTAAGA
Esa6
5’-GACTGCGTACCAATTCCC
Msa6
5’-GATGAGTCCTGAGTAAGG
Esa7
5’-GACTGCGTACCAATTCCG
Msa7
5’-GATGAGTCCTGAGTAAGC
Esa8
5’-GACTGCGTACCAATTCCT
Msa8
5’-GATGAGTCCTGAGTAAGT
Esa9
5’-GACTGCGTACCAATTCGA
Msa9
5’-GATGAGTCCTGAGTAACA
Esa10
5’-GACTGCGTACCAATTCGC
Msa10
5’-GATGAGTCCTGAGTAACG
Esa11
5’-GACTGCGTACCAATTCGG
Msa11
5’-GATGAGTCCTGAGTAACC
Esa12
5’-GACTGCGTACCAATTCGT
Msa12
5’-GATGAGTCCTGAGTAACT
Esa13
5’-GACTGCGTACCAATTCTA
Msa13
5’-GATGAGTCCTGAGTAATA
Esa14
5’-GACTGCGTACCAATTCTC
Msa14
5’-GATGAGTCCTGAGTAATG
Esa15
5’-GACTGCGTACCAATTCTG
Msa15
5’-GATGAGTCCTGAGTAATC
Esa16
5’-GACTGCGTACCAATTCTT
Msa16
5’-GATGAGTCCTGAGTAATT
were mixed with 4 μL formamide dye solution (98%
formamide, 10 mM EDTA, 0.05% each of xylene
cyanol and bromophenol blue), heat-denatured for
5min at 100
℃
, and then placed on ice. The denatured
mix were loaded onto 6% denaturing polyacrylamide
gel and electrophoresed in 1×TBE electrohphoresis
buffer at 100 W for 2.5 h. The gels were silver-stained
following the procedures as described (Maheswaran et
al., 1997). All the reactions for digestion, ligation,
pre-amplification, and selective amplification were
performed following the procedures as described
(Subudhi et al., 1998).
Transcript-derived fragment isolation and re-am-
plification
The polymorphic transcript-derived fragments (ESTs)
gel bands, selected by presence, absence or
differential intensity, were cut from the gel with a
sharp blade carefully to avoid any contaminating
fragments, and eluted in 100μL sterile double distilled
water, then kept at 95
℃
for 20min and hydrated
overnight at 4
℃
. 10μL aliquot was used as template
for re-amplification in 25μL PCR reaction system,
using the same set of corresponding selective primers
and the same PCR conditions as selective amplification
(Jayaraman et al., 2008). The PCR products were
electrophoresed in 1.5% 1×TAE agarose gel, and each
single clear band was isolated and eluted using the
Bioteke gel extraction kit (DP1721, BioTeke Corporation,
Beijing, China).
Cloning and sequencing of ESTs
The eluted ESTs were cloned into the plasmid
pUCm-T vector (Sangon Biotech, Shanghai, China)
following the manufacturer’s protocol; the recombinant
plasmid was transformed into DH5α E
. coli
competent
cell. The positive clone was selected out for sequencing
in Sangon Biotech Co., Ltd (Shanghai, China).
Bioinformatic analysis of sequences
The sequences of the ESTs were analyzed for their
homology with the publicly available database,
including the Genbank (
and The Banana Genome Hub (
- genome.
cirad.fr/blast) using BLASTn and BLASTx algorithms,
to get the useful bioinformation about the ESTs.
Semi-quantitative RT-PCR
Reverse transcription-PCR was performed from
banana total RNA using RevertAid
TM
First Strand
cDNA Synthesis Kit (Fermentas, Beijing, China)
according to the manufacture’s manual. Primers were
designed from the sequences of the ESTs using
Primer5.0 software. The house-keeping gene 18S
rDNA was used as the internal standard for quantity
and quality checking of RNA template in RT-PCR.
The PCR condition was essentially same as described
earlier for re-amplification of ESTs, with corresponding
anneal temperature for different primer pairs as listed
in Table 2.
