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Plant Gene and Trait 2012, Vol.3, No.5, 22
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27
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effects on transgenic plants. Therefore, the flower-
specific expression promoters have an important app-
lication potential to directionally improve the comer-
cial traits of landscape plants (Fan et al., 2010; Gao et
al., 2010). Currently,
AP3
promoter of
Arabidopsis
ecotype (Ler) has been cloned (Koch et al., 2001), and
site-specific mutation analysis revealed that
cis
-elements
such as CArG1, CArG2 and CArG3 are closely related
to flower-specific of
AP3
expression (Tilly et al., 1998).
Driven by
Ap3
promoter from Ler ecotype Arabidopsis,
the heterologous gene
ipt
from
Agrobacterium tume-
faciens
can specifically expressed in the floral organ
of transgenic petunia (
Petunia hybrida
), resulting in
the significant increase of flower diameter and fresh
weight, but almost same size of vegetative to that of
non-transgenic plants (Verdonk et al., 2008). This
study provides a theoretical and practical basis for
further application of flower-specific expression
promoter in improving the commercial floral traits of
landscape plants. The three most useful ecotypes in
Arabidopsis
are the Landsberg erecta (Ler), Columbia
(Col) and Wassilewskija (Ws), respectively, among
these different ecotypes, there are large differences in
morphological development and physiological response
(Zhang et al., 2006). So far,
AP3
promoter of Arabi-
dopsis ecotype (Col) has not been reported, so we
cloned the
AP3
promoter from
Arabidopsis
ecotype
(Col) (GenBank accession No. FJ619533), conducted
bioinformatics analysis and constructed
pAtAP3
::
GUS
plant expression vector so as to lay ground to
further explore the function and expression mechan-
ism of
AP3
promoter of
Arabidopsis
ecotype (Col)
and accumulate information on the directed impro-
vement of the commercial traits related to flower in
landscape plants.
23
1 Results and Analysis
1.1 PCR amplification of
pAtAP3
Taken total DNA extracted from young leaves of
Arabidopsis
ecotype (Col) as DNA template, we
successfully amplified a fragment of
AP3
gene promoter,
pAtAP3
, with high-fidelity KOD-plus DNA polymerase.
The product was about 1 800 bp, which was consistent
with the predicted length (Figure 1).
1.2 Cloning of
pAtAP3
After recovered with DNA purification Kit, the target
PCR products were added nucleotide A at 3' end. Then
Figure 1 Agarose gel separation of PCR product of
pAtAP3
Note: M: DNA marker
Ⅲ
; 1~2: PCR product of
pAtAP3
they were ligated to pMD18
-
T overnight and the
ligated products were transformed into competent
cells of
E. coli
DH5α by heat shock method and
incubated at 37
℃
overnight. The transformants were
firstly selected by colony PCR and plasmid PCR, and
then were identified by double enzyme digestion with
Hin
d
Ⅲ
and
Xba
Ⅰ
. The digest products of the
positive recombinants, 1 800 bp and 2 800 bp (Figure
2), were consistent with the predicted length, which
indicated that they were suitable to be sequenced.
Figure 2 Agarose gel electrophoresis of recombinants digested
by
Hin
d
Ⅲ
and
Xba
Ⅰ
Note: M: DNA marker
Ⅲ
; 1~2: Recombinants digested by
Hin
d
Ⅲ
and
Xba
Ⅰ
1.3 Sequencing and sequence analysis of
pAtAP3
The sequencing results of the two recombinants of
pAtAP3
were the same and the length was 1 767 bp
(Figure 3). Pairwise sequence alignment analysis based
on bl2seq program showed that the sequence similarity
between
pAtAP3
and U30729 was up to 98%, indicating
that
AP3
promoter sequences between different ecotypes