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Plant Gene and Trait 2012, Vol.3, No.3, 13
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Our earlier studies showed that Ca
2+
regulator could
regulate the contents of Ca
2+
and CaM during ethylene
induced flower in bromeliads (Yi et al., 2011). But
there were rarely reports about the effects of Ca
2+
on
the expression of CaM and differentiation of flower
buds. In order to further study the probable role of
Ca
2+
-CaM system in bromeliads flower induction with
ethylene, we researched the effects of Ca
2+
regulators
on the expression of CaM and the stage of flower buds
development by RT-PCR, Northern blot and anatomy
methods. The results might be helpful for controlling
flower time of bromeliads and clarifying the mechanism
of bromeliads flower.
1 Result and Analysis
1.1 Cloning and sequence analysis of
CaM
gene
fragment
The fragment of PCR was about 444 bp (Figure 1).
The size was the same as the fragment inserted in
T-vector (Figure 2). The supposed amino acid sequence
was highly homologious with that of wheat and maize
registered in GenBank after sequencing (Figure 3). So,
this fragment was thought to be from CaM clealy, and
could be used for gene expression analysis.
Figure 1 Agarose gel electrophoresis of PCR amplification
product of
CAM
Figure 2 Agarose gel electrophoresis of PCR product of CaM
from plasmid in
E. coli
Figure 3 The comparison of calmodulin amino acid sequence of
Guzmania
‘Amaranth’, corn and wheat and the position of the
homologous fragment of CaM in that of maize
Note: TA:
Triticum aestivum
; Zea:
Zea mays
1.2 Effects of Ca
2+
regulator on
CaM
gene expression
in
G
. ‘Amaranth’ treated with ethylene
1.2.1 Analysis of expression characteristics of
CaM
by RT-PCR
The RT-PCR results showed that the expression
abundance of CaM increased in the beginning, and then
became lower during the first 7 days flowering-inducing
treated with ethylene or ethylene and Ca
2+
regulator.
However, only treated with ethylene, the expression of
CaM increased at 24 h sharply; Treated with ethylene
and A23178 (Ca
2+
accelerator), the peak expression
increased obviously early at 6 h; When treated with
ethylene with W7 or TFP (Ca
2+
agonist), or ethylene with
EGTA (Ca
2+
chelant), the expression of CaM increasing
remarkable delayed to 72 h or later (Figure 4).
1.2.2 Northern blot analysis of the expression
characteristics of
CaM
The results of Northern blot analysis also indicated
that expression abundance of CaM increased in the
beginning, and then became lower during the first 5
days flowering-inducing treated by ethylene or
ethylene and Ca
2+
regulator. Only treated by ethylene,