Plant Mol Biol Rep
DOI 10.1007/s11105-012-0441-7
ORIGINAL PAPER
Isolation and Activity Analysis of a Seed-Abundant soyAP1
Gene Promoter from Soybean
Yan Zhao & Shuli Shao & Xiaowei Li & Ying Zhai &
Qinglin Zhang & Dandan Qian & Qingyu Wang
# Springer-Verlag 2012
Abstract The soybean aspartic proteinase gene soyAP1 has
previously been shown to be expressed specifically in soybean seeds. To investigate the expression pattern and active
cis-elements of the soyAP1 promoter, the 1,650-bp 5′-upstream genomic DNA fragment named PS-552 was isolated
by PCR walking. Sequence analysis revealed that this fragment contains a series of motifs related to seed-specific
promoters and some pollen-expressed elements. Stable expression in transgenic Arabidopsis thaliana showed that the
PS-552 promoter can regulate beta-glucuronidase gene accumulation in mature seeds at much higher levels than other
tissues, especially vegetative tissues, and exhibits similar
activity to the 35S promoter in mature seeds. These results
show that the PS-552 promoter is a highly active promoter
controlling downstream gene expression, mainly in mature
seeds. The 5′-end deletion studies of PS-552 showed that the
cis-elements of CAAACAC, AACA, E-box, and CCAA
play a role in increasing the seed-specific activity. The
proportion of mature seed activity and flower activity was
increased as the deletion fragment lengthened, indicating
that seed cis-elements possibly lessen or suppress the effect
of pollen-expressed elements, increasing the activity of PS552 in mature seeds.
Electronic supplementary material The online version of this article
(doi:10.1007/s11105-012-0441-7) contains supplementary material,
which is available to authorized users.
Y. Zhao : X. Li : Y. Zhai : Q. Zhang : D. Qian : Q. Wang (*)
College of Plant Science, Agricultural Division, Jilin University,
Changchun 130062, China
e-mail: [email protected]
Y. Zhao : S. Shao
College of Life Science and Agroforestry, Qiqihaer University,
Qiqihaer 161006 Heilongjiang, China
Keywords Soybean . SoyAP1 . Promoter .
Transgenic Arabidopsis
Introduction
The seeds of alimentary and oil crops contain components that
are edible and useful for humans, and researchers have further
increased or decreased the contents of certain materials or
engineered seeds to produce new components for food crop,
industrial, and medical purposes. Seed-specific promoters can
regulate exogenous genes to express exclusively in seeds,
allowing foreign proteins to concentrate in the seeds and
avoiding unnecessary waste produced by constitutive promoters, such as the 35S promoter, which drives stable gene
expression in all tissues during all developmental periods
(Yang et al. 2008). Seed-specific promoters will help plant
seeds serve as bioreactors with special purposes.
Many seed-specific promoters have been obtained, with
most derived from the 5′-flanking sequences of proteins and
lipid genes stored in the seeds of alimentary or oil crops,
such as Brassica napin, rice glutelin and prolamin, Perilla
oleosin 19, and sunflower HaFAD2-1(Chandrasekharan et
al. 2003; Qu and Takaiwa 2004; Chung et al. 2008; Zavallo
and Bilbao 2010). Some of these seed-specific promoters
have displayed striking effects in transgenically engineered
plants. For instance, maize kernel structure and wet milling
properties were successfully modified by a maize γ-Zein
promoter, and phytic acid biosynthesis in transgenic rice
was effectively suppressed under the control of the rice
Ole18 promoter (Zhang et al. 2009; Kuwano et al. 2009).
Soybean is an important economic crop and a chief source
of nutrients such as proteins and lipids. In transgenic soybean
research, controlling the expression of different genes using
seed-specific promoters can effectively decrease homologous
Plant Mol Biol Rep
silence. The main soybean seed-specific promoters that
have been cloned and applied thus far are the promoters
of the oleosin gene, lectin gene, β-conglycinin α subunit gene,
and glycinin G1 gene (Sarmiento et al. 1997; Philip et al.
2001; Yoshino et al. 2006; Ding et al. 2006). Therefore,
more seed-specific promoters should be obtained from
soybean.
SoyAP1 encodes soybean aspartic proteinase (AP), which
occurs in a wide variety of plants (Mutlu and Gal 1999).
Plant APs have different functions, such as enhancing expression under unusual conditions, processing storage-protein
precursors, or involving in autolysis (Schaller and Ryan
1996; Asakura et al. 2000; Beers et al. 2000). According to
the study by Terauchi et al. (2004), soyAP1 is expressed
specifically in soybean seeds; it has especially been identified
in dry seeds through the Northern blot method. Thus, the
soyAP1 promoter is probably a seed-specific promoter, or it
may regulate gene expression mainly in seeds. To investigate
the activity of soyAP1 promoter and the expression pattern of
the corresponding gene, we isolated and identified the 5′flanking sequence of soyAP1 and performed stable expression
in Arabidopsis thaliana.
Materials and Methods
Plant Materials and cDNA Synthesis
Seeds of the soybean Glycine max (Jidou2) were obtained
locally and grown in outdoor natural conditions from early
May to late September in northeastern China. Total RNA
were extracted by the RNAiso Reagent (Takara, Dalian,
China) from roots, stems, leaves, flowers, and immature
seeds collected on the 30th day after flowering (DAF) and
mature seeds collected on the 90th DAF. Single-stranded
cDNA from all of the samples was synthesized from total
RNA with Reverse Transcriptase M-MLV and the oligo
(T)18 primer (Takara, Dalian, China).
Promoter Cloning and Sequence Analysis
Based on the cDNA sequence of soyAP1 gene (Terauchi et
al. 2004), the thermal asymmetric interlaced (TAIL)-PCR
method was used to isolate the upstream sequence of the
ATG start codon using the Genome Walker Universal Kit
according to the manufacturer's protocol (Takara, Dalian,
China) with six arbitrary primers (AD1: 5′-NTCGASTW
TSGWGTT-3′; AD2: 5′-NGTCGASWGANAWGAA-3′;
AD 3 : 5′-WGTGNAGWANCANAGA-3′; AD 4 : 5′A G W G N A G WA N C AWA G G - 3 ′ ; A D 5 : 5 ′ T G W G N A G WA N C A S A G A - 3 ′ ; A D 6 : 5 ′ STTGNTASTNCTNTGC-3′) and three gene-specific primers (S1: 5′-AAACAACTTTGGTGGCACTGAGG-3′; S2:
5′-GGTGGCACTGAGGAAACCGATAT-3′; S 3 : 5′ACTTCTCCTCAAAAGTTCGCTGT-3′). Then, the TAILPCR method was performed secondly according to the
isolated sequence firstly with the same six arbitrary primers
and three other gene-specific primers (P1: 5′-GAAACGCGG
TTAGGAAAACAGATGAG-3′; P2: 5′-GAGGAATAGG
TTTACCGATACGTGGAGA-3′; P3: 5′-CAGTAGTGTCA
CACCTTCCTTCCTCTTT-3′). The genetic DNA of
soybean as template, the primer set (Y1: 5′-GGGCTGCAG
A G T G G A G TA G C A A A G G A C G A - 3 ′ ; Y 5 : 5 ′ GGGCCATGGGTTCTCCTATTCATCCAAAT-3′), was
designed to amplify the full 5′ flanking sequence upstream
of ATG, which contains the promoter region of the soyAP1
gene and is named PS-552. The underlined letters represent
the restriction enzyme sites of PstI and NcoI. The PCR product was purified and cloned into pMD-18T for sequencing.
The knowledge of the promoter characteristics of eukaryotic
cells and the Neural Network Promoter Prediction software
were combined to analyze the putative transcription start site
(TSS), and PlantCARE websites (El-Shehawi et al. 2011)
were used to analyze cis-acting elements and related transcription factor bindin sites.
Construction of Expression Vectors for PS-552 and Deleted
Promoters
Quantitative Real-Time RT-PCR
The stable housekeeping gene soybean β-tubulin was selected
as a reference gene to detect the relative quantities of soyAP1
gene expression among different tissues using quantitative realtime RT-PCR (qRT-PCR). Using single-stranded cDNA as the
template, the primer pair (forward: 5′-GGAAGGCTTTCTTG
CATTGGTA-3′; reverse: 5′- AGTGGCATCCTGGTACTGC3′) was used to amplify the β-tubulin gene (accession number
GMU12286). The primer set (forward: 5′-GTGAGATGGCGG
TTGTTTGG-3′; reverse: 5′-CTCTGGGCTAAGGTC
GAAAG-3′) was used for qRT-PCR analysis of the soyAP1
gene.
In order to study the functional regions of PS-552 promoter,
5′-end deletion analysis (Saha et al. 2011) was carried out. The
deleted promoters PS-356, PS-157, and PS-34 (spanning
−356, −157, and −34 to +1,089 of the 5′-flanking region of
the PS-552 promoter) were obtained with the forward primers
(Y2: 5′-GGGCTGCAGTCACCCTGCAAAAAAG-3′; Y3:
5′-GGGCTGCAGTTAAATTTATTGATGAAAGG-3′; Y4:
5′- GGGCTGCAGAAATGAAGGTGTAACTG-3′) and the
reverse primer (Y5). The recognition sequence for the restriction enzyme PstI was added to the end of each of the forward
primers. A series of expression vectors was constructed containing PS-552 and the three deleted promoter fragments by
Plant Mol Biol Rep
replacing the 35S promoter of the pCAMBIA1301 vector
through digestion with PstI and NcoI followed by ligation.
To evaluate promoter activity, the expression of the gene
reporter beta-glucuronidase (GUS) was measured.
Stable Transformation of A. thaliana
A. thaliana, ecotype Columbia (Col-0), was transformed
with Agrobacterium tumefaciens strain EHA105 suspended
with four recombinant constructs and the pCAMBIA1301
vector using the floral dip method (Clough and Bent 1998).
Two generations of the regenerated plant were selected on MS
medium with 25 μg/ml of hygromycin, transferred to soil, and
grown in the greenhouse (16-h photo period at 20–24 °C). The
insertion of the PS-552, PS-356, PS-157, PS-34, and 35S
promoters in the pCAMBIA1301 vector was confirmed by
PCR amplification of genomic DNA using the primer sets
forward primers: Y 1, Y2 , Y 3, Y 4 and S: 5′-AGGACC
TAACAGAACTCGCCGTAA -3′, respectively, and the
GUS gene internal sequence as the reverse primer: 5′CCCACACTTTGCCGTAATGAG-3′, and RT-PCR amplification using the primer set forward: 5′-TTCTACACAG
CCATCGGTCCA-3′ and reverse: 5′-TGAAAAAGCC
TGAA CTCACCG-3′. Finally, three independent homozygous T2 transgenic lines for each promoter were used for
further analysis for GUS histochemical detection and fluorometric assay.
Histochemical Staining
Histochemical staining analysis for GUS activity was carried out as described by Jefferson et al. (1987). Various
tissues collected from transgenic A. thaliana were incubated
in 10 ml of GUS reaction buffer (2 mM of 5-bromo-4chloro-3-indolyl-D-glucuronide, 0.1 % of Triton X-100,
and 0.1 % of mercaptoethanol) for 10–12 h at 37 °C,
followed by fixation (0.3 % formaldehyde, 10 mM MES,
and 0.3 M mannitol) for 30 min at room temperature and
rinsing three times with 50 mM NaH2PO4. After GUS
staining, the materials were submerged in ethanol to remove
chlorophyll, and the images were then photographed using a
stereomicroscope (Nikon, Japan).
for the next step of total protein measurement by the Bradford
method (Bradford 1976). Another 50 μl of each of the above
supernatant solutions was added to an assay buffer containing
2 mM 4-methyl umbelliferyl glucuronide at 37 °C. The reaction was terminated by the addition of 0.2M Na2CO3.
Fluorescence was assessed on a fluorescence spectrophotometer (Shimadzu , Japan) with 4-methylumbelliferone (MU) as
the standard. GUS enzyme activity was expressed as nanomoles of 4-MU per minute per milligram protein. Three
replicates were performed for each sample.
Statistical Analysis
The statistical analysis of the relative amounts of soyAP1
gene expression in soybean tissues and the GUS activities
regulated by four promoter sequence (PS-552, PS-356, PS157 and PS-34) in transgenic A. thaliana lines were performed using the SPSS 12.0 (SPSS, Chicago, IL, USA)
software. The data shown represent the mean of three independent experiments. Statistically significant differences
were analyzed (P<0.05 and P<0.01).
Results
qRT-PCR Analysis and Promoter Cloning
To directly assess the pattern of soyAP1 gene expression in
soybean tissues, qRT-PCR was applied to determine the
levels of transcription in various tissues. In Fig. 1, the
relative soyAP1 gene expression levels in stems, leaves,
flowers, immature seeds, and mature seeds compared with
roots as the reference value are 4.4-, 1.2-, 9.3-, 7.7-, and
75.7-fold, respectively. The relative amounts of soyAP1
gene expression in mature seeds were extremely significant
(P<0.01) compared with other tissues, demonstrating abundant accumulation of the soyAP1 gene in mature seeds.
In order to obtain a more longer sequence upstream of the
ATG of soyAP1 gene, the TAIL-PCR experiment was performed two times. Two isolated sequences were spliced, and
the 1,650-bp PS-552 promoter sequence (GenBank accession
number HM208384; Fig. 2) was isolated with the primer set
(Y1 and Y5), which was the promoter of soyAP1 gene.
Fluorometric Assay
Sequence Analysis of the PS-552 Promoter
The fluorometric assay to detect the GUS activity was performed according to the method proposed by Jefferson et al.
(1987). Various tissues of transgenic A. thaliana lines were
extracted with GUS extraction buffer (50 mM phosphate buffer, pH 7.0; 10 mM EDTA; 0.1 % TritonX-100; 0.1 % sodium
lauryl sarcosine; and 10 mM β-mercaptoethanol). After centrifugation, the supernatant of various extraction liquids was used
Combining prediction software and our knowledge of the
characteristics of eukaryotic cells, the presumed TSS was located at 553 bp and designated as +1 (Fig. 2). Transcription
factors regulate gene expression by binding to cis-acting elements. Hence, determining the position and the number of
certain cis-elements from the promoter sequence would be
Plant Mol Biol Rep
Fig. 1 QRT-PCR detecting the
relative amounts of soyAP1
gene expression using the
reference gene β-tubulin as an
internal control. Comparing
with other tissues, the level of
significance of the relative
amounts in mature seeds was
P<0.01
helpful to predict the function of the promoter. The PS-552
promoter sequence contained seed-specific expression ciselements, such as an element required for high expression of
seed-specific genes (RY-repeated) (Risueno et al. 2008), an
element involved in triacylglycerol synthesis and present in
seed-specific promoters (E-box) (Kim et al. 2007), seedspecific elements (CCAA, ACGT, AACA, and Skn-1 motif)
(Chamberland et al. 1992; Niu et al. 1996; Washida et al.
1999; Fauteux and Stromvik 2009), and embryo-related elements (CAAACAC, SEF3, and SEF4) (Kim et al. 2006;
Chung et al. 2008). Some pollen-expressed elements (52box, GTGA and ntp303-box) (Eyal et al. 1995; Gupta et al.
2007; Zhang et al. 2008) were also present.
Functional Characterization of the PS-552 Promoter
in Transgenic A. thaliana
To determine the temporal and developmental activity of
PS-552 promoter, A. thaliana plants were transformed with
two constructs (PS-552-GUS, 35S-GUS), and the expression of the reporter gene GUS was tested. After selection for
resistance to Hyg and molecular detection, three independent transgenic lines of each construct were chosen for
fluorometric and histochemical GUS assay. According to
the fluorometric results (Fig. 3a), GUS activity was low in
roots, stems, leaves, and flowers of the transgenic PS-552
plant, while GUS activity in mature seeds was approximately 44-, 16-, and 34-fold higher than roots, stems, and leaves,
respectively, at the 20th DAF. P<0.01 was found between
mature seeds and other tissues (roots, stems, leaves, flowers,
immature seeds). These results showed that the PS-552
promoter controls gene expression mainly in mature seeds.
The same results were also obtained from GUS staining
assay (Fig. 3b). Moreover, both Fig. 3a and b show that
the expression level of GUS in mature seeds of PS-552 lines
was similar to that in 35S lines, and there was no significant
difference (P00.064), indicating that PS-552 promoter activity is as strong as 35S in mature seeds. Therefore, the PS-
552 promoter is a highly active promoter that produces
abundant downstream gene expression in mature seeds.
5′-End Deletion Analysis of the PS-552 Promoter
in Transgenic A. thaliana
The PS-552 promoter has strong activity in mature seeds, and
because there were many seed-specific cis-elements in the
promoter sequence, the series of expression vectors containing
PS-552 and three deleted promoters were designed and constructed to investigate the active cis-elements. Figure 4 shows
that the activities of all the four vectors in different tissues of
transgenic A. thaliana lines had similar tendency, and the
activity in the seed on the 20th DAF was significantly higher
than that in other tissues (P<0.01). Four vectors had similar
activity in roots, stems, and leaves (P>0.05), but there were
discrepancies in seeds and flowers. PS-552 and PS-157 had
similar activity in seeds in the 20th DAF (P>0.05) that was
higher than PS-356 (P<0.01) and about twofold higher than
PS-34 (P<0.01). These results indicated that the region −157
to −34 was crucial for promoting gene expression in seeds, and
it was presumed that the cis-elements of CAAACAC, AACA,
E-box, and CCAA play important roles in increasing the seedspecific activity. Comparing the four mutated lines, the flower
activity in PS-34 was higher than that in the other three mutated
lines (P<0.01), which may be related to the pollen-specific ciselements mainly located downstream of PS-34.
Discussion
In the researches of plant transgenic engineering, some foreign
genes needed to be expressed in various tissues, such as OxO
gene that resisted Septoria musiva and HAL1 gene related to
increased salt (Liang et al. 2001; Ellul et al. 2003), which could
be regulated by the constitutive promoter. However, some
target genes were expected to be expressed in specific tissues,
and a tissue-specific promoter would be considered firstly. In
Plant Mol Biol Rep
Fig. 2 Functional elements
analysis of the PS-552 promoter.
Putative TSS is indicated with
+1. All putative cis-elements
are underlined
this paper, a new promoter (PS-552 promoter) was isolated,
and the regulatory pattern was characterized. The PS-552
promoter can induce much higher GUS gene accumulation in
mature seeds than in other tissues (P<0.01), especially vegetative tissues, and it has similar activity to 35S, implying that
the PS-552 promoter is a highly active promoter that controls
downstream gene expression mainly in mature seeds.
The expression pattern of the soyAP1 gene basically agrees
with the regulatory pattern of the PS-552 promoter, as lower
activity was observed in roots, stems, leaves, and flowers, and
much higher activity was found in mature seeds. Compared
with Northern blotting (Terauchi et al. 2004), the qRT-PCR
method used here more accurately and directly detected the
discrepancies among soybean tissues of the soyAP1 gene.
The 5′-end deletion analysis showed that the activity in
mature seeds differs widely among PS-552 and the three
deleted fragments, especially the deletions of the regions
from −552 to −356 and −157 to −34. The CAAACAC motif
Plant Mol Biol Rep
Fig. 3 a GUS activity was
detected in transgenic A.
thaliana lines fused with PS552 and 35S promoters and
measured in nanomoles of 4MU produced per minute per
milligram of protein. The data
represent GUS activity±standard error measured (SEM)
with three replicates in three
independent transgenic lines.
R1, S1, and L1 represent roots,
stems, and leaves of the seedling; F represents fully developed flowers, and SI indicates
seeds on the 12th DAF. R2, S2,
and L2 represent roots, stems,
and leaves on the 20th DAF,
and PS-552 and 35S represent
seeds on the 20th DAF. b Spatial comparison of GUS expression between PS-552 and
35S in transgenic A. thaliana on
the 20th DAF
is located at −436 to −430 and served the dual function of
stimulating transcription in the embryo and repressing transcription in other tissues (Chatthai et al. 2004). Therefore,
the CAAACAC motif was presumed to play an important
role in increasing the activity in mature seeds.
Although the activity in flowers was low, the activity was
higher than other vegetative tissues regardless of the expression pattern of the soyAP1 gene or the regulatory pattern of the
PS-552 promoter, and the ratio of seed activity and flower
activity increased as the deletion fragment lengthened (Fig. 4),
Fig. 4 GUS activity was detected in transgenic A. thaliana lines fused
with PS-552, PS-356, PS-157, and PS-34 promoters and measured in
nanomoles of 4-MU produced per minute per milligram of protein. The
data represent GUS activity ± SEM with three replicates in three
independent lines of each promoter construction. R1, S1, and L1
represent roots, stems, and leaves of the seedling, and F represents
fully developed flowers. R2, S2, and L2 represent roots, stems, and
leaves on the 20th DAF, and SD indicates seeds on the 20th DAF
Plant Mol Biol Rep
indicating that seed cis-elements might lessen or suppress the
function of flower cis-elements, leading to an increase in the
activity in mature seeds of PS-552 lines. This point has not
been proposed before, so it is necessary to continue this study
through further cis-element deletion or point mutations.
As is widely known, ABA is an efficient inhibitor of
germination and occurs in high concentrations in dormant
seeds, and it sometimes affects some seed-specific promoters
(Ng et al. 2004; Luo et al. 2008; Kim et al. 2011). The core
sequence of the ABA-responsive element (ABRE) is ACGT
(Ng et al. 2004; Ross and Shen 2006). The PS-552 sequence
also contains ABRE-like cis-elements (C/G/TACGT) located
at +116, +224, +433, and +971. ABA treatment during GUS
transient expression in soybean seeds led to no change in the
soybean seed activities (data not shown), indicating that soyAP1
gene expression was not affected by ABA. Transcription factors
are able to bind to specific sets of short conserved sequences
contained in each promoter. WRKY-type transcription factors
have multiple roles in the plant defense response and developmental processes (Zhou et al. 2008), some of which can
bind the W-box (TGACY) that is also present in the PS-552
sequence. The PS-552 sequence also contains binding sites of
the transcription factors DOF (AAAG) and MYB (CNGTTR),
which play regulatory roles in the control of seed germination
and in multiple types of hormone and stress responses (Chen
et al. 2006; Gupta et al. 2011). These transcription factor
binding sites might be related to the characteristics and functions of the soyAP1 gene.
Acknowledgments This work was supported by a specific grant
from the Ministry of Agriculture (no. 2008ZX08004-003) and the
National Natural Sciences Foundation of China (grant no. 30971808).
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