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Plant Gene and Trait 2012, Vol.3, No.5, 22
-
27
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26
menhoff and Koch, 1994), and the final concentration
of total DNA was adjusted to 50 ng/μL. Based on
AP3
promoter sequence of
Arabidopsis thaliana
ecotype
Ler reported in GenBank (U30729) and multiple cloning
sites on the upstream and downstream of Cauliflower
Mosaic Virus 35S promoter in pBI121 vector, a pair of
specific PCR primers was designed with Primer
Premier 5.0 software. Forward primer 5'
-
AAGCTTCT
TAAGAATTATAGTAGCACTTGTT
-
3', containing self
Hin
d
Ⅲ
restriction sites (underline), and the downstream
primer 5'
-
TGCTCTAGAATTCTTCTCTCTTTGTTTA
ATC
-
3', inserted with
Xba
Ⅰ
restriction sites (underline),
were synthesized by Beijing AuGCT Biotechnology
Co., Ltd.
3.4 PCR amplification of
pAtAP3
50 µL PCR reaction system was composed of 5 µL
10×PCR Buffer, 5 µL 2.0 mmol/L
each dNTP, 1 µL
total DNA, 3 µL 25 mmol/LMgSO
4
, 1.5 µL 10 pmol/µL
forward and reverse primer, respectively, 1 µL KOD-plus
DNA polymerase and 32 µL ddH
2
O. Thermocycling
was performed at 95
℃
for 4 min, then at 95
℃
for 30 s,
52
℃
for 30 s, 68
℃
for 2 min 30 s for 35 cycles, 68
℃
for 7 min and finally kept at 4
℃
. The PCR products
were separated by 1% Agarose gel electrophoresis.
3.5 Cloning and sequencing of
pAtAP3
The target PCR products were recovered with TaKaRa
TM
Agarose Gel DNA Purification Kit. Because nucleotide
A can't be added to the 3' end of PCR products amplified
by high-fidelity KOD-Plus DNA polymerase, it is
necessary to add nucleotide A at its 3' end before the
purified target fragment was cloned into pMD18
-
T
vector. The adding nucleotide A reaction system was
10 μL, containing purified PCR products 8 μL, 2.5
mmol/L each dNTP 0.8 μL, PCR Buffer 1 μL, and
Taq
DNA polymerase 0.2 μL. This reaction was carried
out at 72
℃
for 30 min. Then these products were
ligated to pMD18
-
T with T4 DNA ligase and the
ligated products were transformed into competent
cells of
E. coli
DH5α by using heat shock method.
The transformants were selected by colony PCR and
plasmid PCR, and then were identified by double
enzyme digestion of
Hind
Ⅲ
and
Xba
Ⅰ
. The positive
recombinants were named as pMD18
-
T-
pAtAP3
, and
were sequenced by Sangon Biotech (Shanghai) Co. Ltd.
3.6 Bioinformatic analysis of
pAtAP3
sequence
The sequence of
pAtAP3
was submitted to GenBank
for accession number (http://www.ncbi.nlm.nih.gov/).
Sequences of
pAtAP3
and U30729 were aligned by
online bl2seq program (http://blast.ncbi.nlm.nih.gov/
Blast.cgi).
cis
-elements of
pAtAP3
were analyzed with
online analysis tools PLACE (http://www.dna.affrc.go.
jp/PLACE/signalscan.html) (Higo et al., 1999).
3.7 Construction of plant expression vector
pAt-
AP3
::
GUS
In order to further explore the expression pattern of
pAtAP3
, it is necessary to construct plant expression
vector
pAtAP3
::
GUS
. Firstly, the positive recombinant
pMD18
-
T-
pAtAP3
and empty vector pBI121 were
digested by
Hin
d
Ⅲ
and
Xba
Ⅰ
. Then the small
digestion fragment of recombinant pMD18
-
T-
pAtAP3
and the large fragment digestion product of pBI121
were recovered, respectively. Later the purified
products were ligated overnight, the ligated products
were transformed into
E. coli
DH5α competent cells
and cultured on LB medium with 50 µg/mL
Kanamycin. The plant expression vector
pAtAP3
::
GUS
was identified by colony PCR and plasmid PCR.
Authors
'
Contributions
SPG, XGH and HWX conducted this experiment; GAS and
XSK participated in the experiment design; FBY was the
person who took charge of this project, including experiment
design, data analysis, writing and modifying of the manuscript.
All authors have read and approved the final manuscript.
Acknowledgements
This study was financially supported by the National Natural
Science Foundation of China (30740013) and Program for
young teachers in University and college of Henan Province
(2010GGJS
-
075). Authors appreciate two anonymous reviewers
for their useful critical comments and revising advice to this
paper.
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