MPB-2015v6n20 - page 7

Molecular Plant Breeding 2015, Vol.6, No.20, 1-10
http://mpb.biopublisher.ca
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expressed in banana leaves at different time points
after Foc-TR4 inoculation by cDNA-AFLP, verify
their expression patterns through semi-quantitative
RT-PCR, and provide new genetic information for
genetic improvement on banana disease-resistance.
Materials and Methods
Biological Materials
Micropropagated Cavendish banana plantlets of the
variety ‘Brazilian’ for our experiments was get from
banana seedling cultivation factory in Danzhou City,
Hainan Province. Fusarium oxysporum f. sp. Cubense
tropical race 4 (Foc-TR4) was separated and preserved
by our research group.
Pathogen inoculation treatment
Foc-TR4 strains were inoculated in PDA medium (200
g Potato, 20 g Glucose and 18g Agar per liter medium)
plate, and cultured in the 25℃ and dark conditions
for 4 days. The hyphae of Foc-TR4 picked up by
inoculating loop, was inoculated in the CMC liquid
medium (7.5 g C8H11O5Na, 0.5 g Yeast extract, 0.5 g
NH4NO 3, 0.5 g KH2PO4 and 0.25 g MgSO4 per liter
medium), and cultured 5 days in 28℃ shaking condition
(150 r/min) for conidia growth. The hyphae were filtered
by double layer of sterile gauze, the cultured medium
suspension was centrifuged for 5min at 12000rpm,
discarded the supernatant and the precipitate was conidia.
The conidia were suspended in sterilized H2O, detected
and counted under 400×ordinary light microscope, and
diluted to 1.0×105 spores/mL concentration (early
study data) in sterilized H2O.
When the banana plantlets grew to six leaves stage,
we cut the health and clean young banana leaves for
inoculation experiments. Banana leaves were placed
in the 20 cm×35 cm×50 cm plastic box, the petioles of
leaves were covered with absorbent paper sprayed
enough sterile water. Pinprick six wounded spots
evenly on each leaf and inoculate 20 µL Foc-TR4
conidia solution on wounded spots for disease
inducing. The experiment set up control and treatment
group for comparison, the control group was
inoculated with 20 µL sterile water, and the treatment
group with 20 µL Foc-TR4 conidia solution. We took
samples at 4 different time points after inoculation: 4h,
24h, 3d and 6d, to study the gene differential expression of
banana induced by Foc-TR4 infection.
RNA preparation and cDNA synthesis
Total RNA was isolated from 200 mg banana leaf
tissues with the CTAB extraction method. The CTAB
extraction solution contains 2.5% CTAB, 100mM
Tris-HCl pH8.0, 1.4M NaCl, 20mM EDTA, pH8.0
and 4% PVP. The steps were as follows. First, weight
200 mg banana leaf tissues and grind fully in liquid
nitrogen, add 1mL of 65℃ preheated CTAB extraction
and 20 µL β-mercaptoethanol, transfer the homogenate
to 2.0 mL eppendorf tubes in 65 ℃ water bath for
30min, mixing them every 5min. Extract them twice
with 0.6mL chloroform, then add 5/8 volume of 8M
LiCl to the supernatant, keep in -80℃ refrigerator for
1h, and centrifuge with 12000 rpm for 20min at 4℃.
Wash the precipitate with 75% ethanol, dissolve them
in 400 μL DEPC-treated water, add 250 μL 8 M LiCl,
place them at -20 ℃ for 4 h, centrifuge with
12000rpm for 20 min at 4℃. Finally, wash RNA with
75% ethanol and dissolve RNA in 50 μL DEPC-
treated water. Take 5 μL RNA samples for electrophoresis
analysis and concentration determination, adjust the
RNA concentration to the same level, and store the
RNA in -80 ℃ refrigerator for following experiments.
2 μg total RNA was used initially for first strand
cDNA synthesis for each sample, followed by second
strand cDNA synthesis, using RevertAid TM First
Strand cDNASynthesis Kit (Fermentas, Beijing, China).
cDNA-AFLP experiment
About 200ng double-stranded cDNA was subjected to
standard AFLP template production. The restriction
enzymes used for cDNA digestion were EcoR I and
Mse I (Fermentas, Beijing, China). The digested
products were ligated to adapters which sequences
were as follows: EcoR I adapter, 5’-CTCGTAGACT
GCGTACC-3’ and 5’-AATTGGTACGCAGTCTAC-3’;
Mse I adapter, 5’- GACGATGAGTCCTGA G-3’ and
5’-TACTCAGGACTCAT-3’.
The ligated products were pre-amplified with the
pre-amplification primers which sequences were as
follows: 5’-GTAGACTGCGTACCAATTC-3’, and
5’-GACGATG AGTCCTGAGTAA-3’. Equal amounts
of pre-amplified products were selective amplified
with the selective amplification primers. The sequences
of selective primers were listed in Table 1.
Totally 160 different primer pair combinations were
selectively amplified. 6 μL selectively amplified products