Basic HTML Version
Plant Gene and Trait 2012, Vol.3, No.3, 13
-
17
http://pgt.sophiapublisher.com
16
the other hand, Ca
2+
accelerator alone could not
induce flower of
G.
‘Amaranth’ (data not shown), and
Ca
2+
agonist and Ca
2+
chelant could not inhibit the
flower induced with ethylene completely, just delay
flower. Consequently, Ca
2+
regulators had an important
regulated role in the flower induction with ethylene of
G.
‘Amaranth’, but not decisive effect. It meant that
ethylene could not induce the
G.
‘Amaranth’ flower
through Ca
2+
-CaM system. So, the regulating pathway
and molecular mechanism of flower induction of
bromeliads with ethylene need intensive study.
3 Materials and Methods
3.1 Materials
G.
‘Amaranth’ with 20 leaves, 11 month after
transplanting from culture bottle, and cultured in
the greenhouse of Tropical Crops Genetic Resources
Institute, Chinese Academy of Tropical Agricultural
Sciences.
Reagents ethylene (40%) was made in China, and Ca
2+
accelerator A23178, Ca
2+
agonist W7, TFP and Ca
2+
chelant EGTA were bought from Sigma Company.
Treatment methods: This experiment was arranged 5
treatments with 10 plants respectively (Table 1). Firstly,
we got rid of the water in the rosulated leaf 1 d before
treatment. Filling 5 mL ethylene solution into the
center of rosulated leaf at 9:00 pm and discarded the
solution after 24 h, and washed with water. The leaf
and shoot tip tissue were taken at 0 h, 1 h, 6 h, 24 h,
48 h, 72 h, 4 d and 5 d respectively and soon freeze in
nitrogen, and then stored at
-
80
℃
.
Table 1 Treatments of the experiment and the concentration of each reagent
Contents of the perfusion solution
Treatment No.
Ethrel (mL/L)
A
23187
(μmol/L)
EGTA (mmol/L)
TFP (mmol/L)
W7 (mmol/L)
CK
0
0
0
0
0
E (CK)
0.4
0
0
0
0
EA
0.4
2
0
0
0
EE
0.4
0
20
0
0
ET
0.4
0
0
10
0
EW
0.4
0
0
0
1
3.2 Cloning of
CaM
gene
Total RNA from young leaf and shoot tip was extracted
by using CTAB method, and cDNA was synthesized
using SuperScript™
Ⅲ
Reverse Transcriptase (Invitrogen).
The cDNAs were used for PCR with gene-specific
primer (cam1, 5'
-
CCAgAgCACCTACCCAgACT
-
3'
and cam2, 5'
-
ACCTCggAgCAgATgAcg
-
3') designed
according to the cDNA sequences of
CaM
genes in
GenBank by Primer Premier 5.0 software. The process
of amplification followed as: pre-denaturated 5 min at
94
℃
, and then consisted of 35 cycles of, 30 s at 94
℃
,
50 s at 60
℃
, 90 s at 72
℃
, and a 10 min extension at
72
℃
. The PCR products were stored at 4
℃
.
3.3 RT-PCR analysis of
CaM
gene
Using
β-actin
as reference gene, the primers were
5'
-
CAGTGGTCGTACAACTGGTAT
-
3' and 5'-ATCC
TCCAATCCAGACACTGT
-
3'. Primers for
CaM
were
cam1 and cam2, which were gave in previous
paragraph. The RT-PCR reaction, with a total volume
of 25 μL, consisted of 100 ng cDNA, 1×reaction
buffer, 2.5 mmol/L MgCl
2
, 0.25 mmol/L dNTP, 1 U of
Taq
DNA polymerase (Fermentas) and 0.5 μmol/L of
each primer. The amplification process consisted of 35
cycles of 30 s at 94
℃
, 50 s at 60
℃
, 90 s at 72
℃
, and
a 10 min extension at 72
℃
.
3.4 CaM expression analysis by Northern blot
Total RNA was extracted by CTAB method from
young leaf and shoot tip of plants treated with
ethylene for 0 h, 1 h, 6 h, 24 h, 48 h, 72 h, 4 d and 5 d,
respectively. The RNA was stored at
-
80
℃
.
For the Northern blot analysis, 10 μg of RNA were
separated in formal dehyde-agarose gels and transferred
into Nytran membranes (Schleicher & Schuell) and
fasten with Spectroline UV Crosslinker, Select TM
Series. We used the cDNA reversed from RNA as