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Plant Gene and Trait 2012, Vol.3, No.5, 22
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27
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1.4 Construction of plant expression vector
pAt-
AP3
::
GUS
The empty vector pBI121 and recombinant pMD18
-
T-
pAtAP3
were all digested by
Hin
d
Ⅲ
and
Xba
Ⅰ
.
Then the large digestion product of pBI121 (Figure 4)
and the small digestion fragment of pMD18
-
T-
pAtAP3
(Figure 2) were recovered, respectively. The recovered
products were ligated and transformed into
E. coli
DH5α. And then the colony PCR and plasmid PCR
were carried out to characterize the correct construct
of
pAtAP3
::
GUS
. The electrophoresis result showed
that the 1 800 bp target fragment was amplified
(Figure 5), which revealed that the plant expression
vector
pAtAP3
::
GUS
was successfully constructed.
25
Figure 4 Agarose gel separation of pBI121 digested by
Hin
d
Ⅲ
and
Xba
Ⅰ
Note: M: DNA marker
Ⅲ
; 1~2: pBI121 digested by
Hin
d
Ⅲ
and
Xba
Ⅰ
Figure 5Agarose gel separation of PCR products by plasmid PCR
Note: M: DNA marker
Ⅲ
; 1~4: PCR products
2 Discussion
The genetic engineering of landscape plant was
involved in improvement of lots of traits such as flower
color, flower type and flower diameter, so cloning and
identification of flower-specific expression promoter
has some important application potential on oriented
improvement of the commercial traits related to floral
organs (Fan et al., 2010; Gao et al., 2010). Cucumber
(
Cucumis sativus
L.) has served as a model plant to
understand hormone regulation in unisexual flower
development. It has been confirmed the role of
ethylene in the promotion of female cucumber flowers.
Under the control of
AP3
promoter of
Arabidopsis
ecotype (Ler), cucumber gene
CsACO2
was overe-
xpressed in transgenic Arabidopsis. It showed that
transgenic
Arabidopsis
displayed normal vegetative
morphology, but all flowers were sterile, which was
exactly caused by abnormal stamen. However, carpels
retained normal function with no obvious morpho-
logical change. The abnormal stamen morphological
change was resulted from increasing ACO2 enzyme
activity about 20% in floral bud (Duan et al., 2008),
which confirmed that the potential application of
AP3
promoter in fine regulation of floral traits.
In this study, we cloned
pAtAP3
from
Arabidopsis
ecotype (Col), which was assigned GenBank accession
as FJ619533. Bioinformatic analysis showed that it
contained several
cis
-elements related to flower-specific
expression. And the plant expression vector
pAtAP3
::
GUS
was successfully constructed. Therefore, it
would provide a material to further explore the
expression pattern and the mechanism of flower-
specific expression of
pAtAP3
. It also would lay the
foundation to apply
pAtAP3
for directly improvement
the commercial traits of landscape plant in the future.
3 Materials and Methods
3.1 Plant materials
Young rosette leaves of
Arabidopsis thaliana
ecotype
Col were used to extract total DNA.
3.2 Strain, plasmid and reagent
KOD-plus DNA polymerase was purchased from
TOYOBO Co. LTD. Plant expression vector pBI121
and Escherichia coli strain DH5α were kept in our
own laboratory. Cloning vector pMD18
-
T, Agarose
Gel DNA Purification Kit, MiniBEST Plasmid Purifi-
cation Kit, restriction endonucleases
Hin
d
Ⅲ
and
Xba
Ⅰ
were from TaKaRa Biotechnology (Dalian) Co.
LTD. DNA Marker
Ⅲ
and
Taq
DNA polymerase were
bought from Tiangen Biotech (Beijing) Co., LTD.
3.3 Total DNA extraction of
Arabidopsis
leaves and
primer design
Total DNA was isolated from young
Arabidopsis
leaves according to modified CTAB method (Mum-