Fagopyrum 35: 5-17 (2018) 
  
5 
 
Analysing Structural diversity of Seed storage protein 
gene promoters: Buckwheat a case study 
 
Upasna Chettry, Lashaihun Dohtdong and N.K. Chrungoo 
Dept. of Botany, North Eastern Hill University 
Shillong-793002, Meghalaya, India. 
E-mail: nchrungoo@gmail.com 
DOI https://doi.org/10.3986/fag0004 
Received November 20, 2017; accepted August 12, 2018 
 
Keywords: buckwheat, cis-regulatory elements,phylogenetic profiling and nucleosome 
ABSTRACT
Multiple sequence alignment of 5’UTR of SSP 
genes from accessions of Fagopyrum esculentum 
revealed the invariant nature of sequences with the 
transcription start site at P761 and TATA box located 
-30bp upstream the TSS. Other cis-elements 
identified in the sequences included the legumin 
box (-581, -524, -184, -135, -91), the -131 prolamin 
box, DOF element (-718, -649, -540,-432, -272,-
225, -128) and CAAT box (-692, -530, -475, -411, -
282, -168, -54). Other elements identified included 
those involved in abscisic acid signalling viz., ABI3 
at P-470,-95,-68, RAV1 at P-694 and -543 and AGL15 at 
P-671. A comparative analysis of regulatory 
elements of SSP gene promoters of distantly 
related species the presence of five cis-regulatory 
elements viz. TATA BOX, E-BOX, RY- element, 
CAAT box and the Endosperm box, which interplay 
in seed specific SSP gene expression. Other 
modulators influencing seed specific gene 
expression detected in the sequences included the  
ABA-responsive elements ABI3, RAV1 and AGL15 
which play an integral role in seed maturation. 
Identification of potential nucleosome binding sites 
in SSP gene promoters of Cicer arietinum, 
Brassica napus, B. campestris, Vicia faba, and 
Pisum sativum at positions 78, 635, 195, 112 and 
152 respectively surmises the spatial fine tuning of 
SSP gene transcriptional regulation in these 
species. On the other hand, absence of 
nucleosome binding sites in the promoters of 
Fagopyrum esculentum, Zea mays, Avena sativa, 
Triticum aestivum and Oryza sativa may indicate 
relatively easier access of transcription factors to 
the proximal promoter, thereby providing higher 
level of gene expression. 
 
Chettry et al. 
  
6 
INTRODUCTION 
Seed storage proteins are a major class of proteins 
that not only serve as a source of nutrition to 
germinating seedlings but also as an important 
source of dietary proteins for human consumption. 
Genes encoding such proteins are under tight 
spatial and temporal transcriptional control. The 
basic building blocks for promoters of such genes 
are regions of cis-regulatory DNA, which in 
eukaryotes often comprises clusters of cis-
regulatory elements (CREs) that modulate gene 
expression through their interaction with trans-
acting factors. Plant cis-regulatory motifs are often 
reported as consensus sequences which are 
commonly delineated by reporter gene expression 
assays (Guilfoyle, 1997). Neverthless, PLACE 
database, a collection of experimentally 
characterized plant cis-regulatory elements 
sequences, remains an invaluable resource for 
annotating motifs discovered in sequences that 
have not been characterized experimentally (Higo 
et al.,1998). Majority of contemporary 
computational approaches for the discovery of cis-
regulatory elements use the position weight matrix 
(PWM) motif model, based on the frequencies of 
nucleotides at each position in a collection of 
regulatory elements (GuhaThakurta, 2006). The 
exponential growth in development of 
bioinformatics tools to discover specific motifs in 
DNA or protein sequences and creation of genomic 
resources representing specialized databases 
of plant cis-acting elements have greatly facilitated 
in silico analysis of promoters. However, discovery 
of such elements is hindered by the variability 
within their sequences, which typically tolerate 
nucleotide substitutions without loss of functionality. 
Further, in majority of the cases, and especially so 
in higher eukaryotes, TFs often regulate gene 
expression by binding to specific elements in the 
promoter regions of different genes independenty 
or in synergy with other regulatory proteins. 
Different cis-elements of a given promoter are also 
known to interact with different parts of cis-
regulatory module (CRM), where the relative 
positions of cis-elements and the distances 
between them are crucial.an overall regulatory 
complex (Arnone and Davidson, 1997).   
Detailed analysis of expression of genes coding for 
seed storage proteins has revealed that the 
expression of SSP genes and accumulation of the 
proteins is limited to the endosperm/ embryos or 
cotyledons of the seeds (Perez-Grau and Goldberg, 
1989; Fujino et al., 2001; Milisavljevic et al, 2004; 
Jain, 2004). Seed-specific expression has been 
shown to be conferred by the promoter regions of 
various storage protein genes (Devic et al, 1996; 
Lee et al, 2007; Moreno-Risueno et al, 2008). 
Signature cis-elements identified in the promoters 
of specific class of plant genes include the 
"legumin box" comprising of the core “RY motif " 
having the sequence 5’CATGCA3’ (Baumlein et al., 
1986, Dickinson et al, 1988, Forde et al, 1985) and 
the "vicilin box" having the core sequence 
5’GCCACCTCAT3’ in legumes (Vincente et 
al.,1997; Weschke et al., 1988) and the “prolamin 
box”( 5’TGTAAAG3’) or endosperm motif (E-motif) 
in cereals (Vicente et al., 1997; Shewry and 
Halford, 2003.). The promoter region of prolamin 
genes  comprises of three CREs including the 
Nucleosome binding potential of SSP gene promoters 
  
7 
GCN4-like (GLM) element (5’GRTGAGTCAT3’), 
the prolamin-box (5’TGTAAAGT3’) and the AACA 
(5’AACAAACTCTATC3’) element that respectively 
interact with bZIP, DOF and MYB family 
transcription factors (Fauteux and Strömvik, 2009). 
A comprehensive analysis of the napA gene 
promoter in rapeseed (Brassica napus L.) has 
revealed the presence of two regulatory complexes 
which include the B-box, that contains the distB 
element (5’GCCACTTGTC3’) together with the 
proxB element (5’TCAAACACC3’), and the RY/G 
complex which contains two RY repeats 
(5’CATGCA3’) and one G-box (5’CACGTG3’) 
(Ezcurra et al.,1999; Chandrasekharan et al., 
2003). G-box, CCAAAT box, E-box (5’CACCGT3’) 
and RY elements have been demonstrated to have 
a strong role in mediating gene expression in 
embryos (Lindstrom et al.,1990). Motifs conferring 
seed-specific expression are known to lie in the 
proximal region of the promoter, often within 500 
bp upstream of the transcriptional start (Wu et al., 
2000; Fujimori S et al., 2005).   
Although the availability of cis-acting regulatory 
element database and tools of bioinformatics help 
to predict the transcriptional properties of new 
entry sequences with considerable accuracy, 
understanding the structural features of DNA, such 
as GC skew, bendability, topography, free energy, 
curvature and nucleosome positioning gives a 
better understanding of the regulatory landscape 
of such genes (Florquin et al., 2005;  Kanhere and 
Bansal 2005b). The present study decribes the  
profiling of the 5’UTR of legumin-like seed storage 
protein gene in ten accessions of common 
buckwheat vis. a vis. seed storage protein gene 
promoters from distantly related species. This 
uncovered the presence of specific conserved 
motifs in SSP gene promoters across plant species 
and moderate nuclosome biding potential in 5’UTR 
of buckwheat legumin genes. 
MATERIALS AND METHODS 
Nucleotide sequences of the 5’UTR of legumin like 
seed storage proteins of ten accessions of 
common buckwheat viz. IC-107090, IC-107285, 
IC-107265, IC-108517, IC-79192, IC-16550, IC-
188669, IC-324313, IC-18864 and IC-363973 were 
generated by nucleotide sequencing of the 
relevant amplicons. Nucleotide sequences of the 
promoter regions of seed storage protein genes of 
other distantly related species were retrieved from 
Genbank database of NCBI for comparative 
analyses. The accession numbers of the 
sequences retrieved from Genbank data bases 
included EU595873 of Fagopyrum esculentum, 
AF420598 of Brassica napus, X67833.1 of B. 
junceae, Y13108 of B. campestris, Y13166 of Cicer 
arietinum, S60289.1 of Vicia faba, X02983.1 of 
Pisum sativum, X65064.1 of Hordeum vulgare, 
X65064.1 of Oryza sativa, EU189096.1 of Triticum 
aestivum and JQ241267 of Zea mays. 
 
 
Chettry et al. 
  
8 
Sequence analysis 
BLASTn analysis of the nucleotide sequences of 
the 5’UTR of legumin like seed storage proteins of 
ten accessions of common buckwheat was carried 
out using the BLAST tool of NCBI. The sequences 
were aligned using the multiple alignment too 
MULTALIN http://multalin.toulouse.inra.fr/multalin/). 
Distribution of cis-elements within the sequences 
was identified out by PLACE 
(http://www.dna.affrc.go.jp/PLACE/signalscan.htm
l) and AtPAN (http://atpan.itps.ncku.edu.tw). 
Neural Network Promoter Prediction tool 
(http://www.fruitfly.org/seq_tools/promoter.html) 
was used to identify the transcriptional start site in 
the target sequences. 
Nucleosome formation potential 
Comparative analysis on the nucleosome 
formation potential of the representative 
sequences from each species was performed  
with Strong Nucleosome tool (http://strn-
nuc.haifa.ac.il:8080/ mapping/home.jsf). 
Sequences with statistical value of scoring peaks 
between 50-65 were used to determine the 
potential position of nucleosome along the DNA 
sequence. 
Result and discussion  
Profiling of the 5’UTR of buckwheat legumin gene  
BLAST analysis of nucleotide sequences from all 
the accessions revealed more than 98% homology 
with 5’UTR of sequence bearing accession no. 
EU595873, the gene coding for legumin like seed 
storage protein gene of common buckwheat. 
Alignment of the sequences using MULTALIN 
clearly showed a highly conserved nature of the 
sequences (Fig. 1). Promoter prediction tool 
(Neural Network Promoter Prediction) identified 
three probable promoter regions between P’392-442, 
473- 523 and 721-771 in the sequences. Out of the three 
predicted transcription start sites, the TSS at P’761 
was located closest to the predicted ATG start 
codon at P’801. The TSS at P’761 also followed the 
YR rule (C-1A+1), having the pyrimidine 'C’ at -1 and 
the purine 'A' at +1 position (Yamamoto et al. 2007). 
Considering ‘A’ at position 761 (+1) as the 
predicted TSS and ATG at position 801 (+40) as 
the initiating codon, the TATA at position 731(-62) 
was identified as the TATA box of the promoter. 
Apart from TATA box, the sequences revealed 
several other cis-elements, that are involved in the 
regulation of eukaryotic gene expression in 
general and seed-specific expression in particular. 
The transcription start site predicted for the 
sequences of all the accessions followed the YR 
rule with the TATA box motif being localized at P’-30 
relative to the TSS. Alignment of the context 
sequences around TATA, TSS and ATG-start 
codon of buckwheat seed storage protein gene 
with the corresponding regions of seed storage 
protein genes from other accessions clearly 
Nucleosome binding potential of SSP gene promoters 
  
9 
established the high degree of conservation in 
spacing between these elements. Sequence 
analysis identified 3 legumin boxes comprised of 
the core sequence 5'CATGCA3' at P’-470, -95, and -
68, a single prolamin box, comprising of the 
sequence 5’TGTAAAG3’ at P-131 and 7 DOF motifs 
with the core sequence 5’AAAG3’ at P -718, -649, -540, 
-432, -272, -225, and -128 with respect to TSS. Legumin 
box is considered to be the key element in 
regulating seed specific expression of genes 
coding for legumin type proteins (Bäumlein et al., 
1992; Ellerström et al., 1996; Reidt et al., 2000). 
Destruction of the legumin box by a 6 bp deletion 
in an otherwise intact 2.4 kb 5'-noncoding 
upstream sequence of Vicia faba legumin gene 
LeB4 was shown to drastically reduce LeB4 
expression in seeds (Baumlein et al., 1992). 
Similar observations were made by Ezcurra et al. 
(1999) for RY elements in the promoter region of 
napin gene. Baumlein et al. (1992) has shown that 
the enhancer-like cis-elements in 5’UTR were fully 
functional only in conjunction with the core motif 
5’CATGCATG3’ of the legumin box, thereby 
indicating a possible role of legumin box in 
modulating enhancer activity in promoter of SSP 
genes. RY motif has been shown to interact with 
the conservative B3-domain of the transcriptional 
activators VP1 of maize (McCarty, 1995) and fus3 
and abi3 proteins of Arabidopsis (Ezcurra  et al., 
1999; Reidt et al., 2000). Analysis of several other 
seed specific promoters has confirmed the 
importance of RY elements for quantitative 
expression of seed specific genes as well as the 
potential of this motif in repression of gene 
expression in non-seed tissues (Mönke et al., 
2004; Singh, 1998) 
 While the “P-box” (5’TGTAAAG3’) is a -300 
enhancer element present in SSP genes of cereals 
and several other dicots (Vickers et al., 2006), we 
detected the “P- box” as a -131 element in the 
buckwheat legumin gene promoter. This element 
has also been reported to be involved in 
quantitative regulation of gene expression in seeds 
(Wu et al., 2000; Chandrasekharan et al., 2003). In 
many cases the “P-box” and “GCN4” motifs are 
coupled with each other with only a few nucleotides 
separating them. This module has been named as 
“bifactorial endosperm box”. The CAAT box, noted 
as an enhancer element involved in quantitative 
regulation of gene expression, was located at 
positions -692,-530,-475,-411,-282,-168, and -54. 
Sequence analysis also revealed the presence of 
SEF1 binding motif, having the core motif 
5’ATATTTATA3’ at P’-307. Lessard et al. (1990) have 
demonstrated strong interaction between SEF1 
and A-T rich sequences presnet far upstream in 
genes coding for α′ and β subunits of β-conglycinin. 
They suggested SEF1 recognizes its binding site 
with greater affinity than the other SEF factors and 
it may be involved in directing nucleosome phasing 
within the promoter region, analogous to 
mammalian high mobility group chromosomal 
proteins (HMG-1). Zhou et al. (2014) have reported 
that while deletion of SEF3 and SEF4 binding 
motifs from promoter of seed-specific allergen 
gene of Arachis hypogaea did not affect promoter 
activity, deletion of three E-boxes and one SEF1 
motif caused a marked decrease in promoter 
activity. Their results suggest the possibility of a 
role for E-box and SEF1 binding motifs in 
regulating seed-specific expression of genes. 
Chettry et al. 
  
10 
Seed storage protein gene promoters exhibit 
signature motifs 
Comparative analysis using AtPan sofware 
generated the co-occurrence of cis-motifs in the 
promoters of Cicer arietinum, Brassica.napus, 
B.campestris, Vicia faba, Pisum sativum, 
Fagopyrum esculentum, Zea mays, Avena sativa, 
Triticum aestivum and Oryza sativa (Table 1). The 
overall consensus generated from the SSP gene 
promoters investigated in the present study can be 
broadly divided into five conserved composite 
motifs (CREs). These included CRE1, which is 20 
to 30 bases upstream of the TSS. This element has 
been reported universally from all promoters (Joshi, 
1987). CRE2 included a G-box-like and a CAAT 
motif, nested into an E-box (5’CANNTG3’). CRE3 
comprised of the RY element (5’CATGCA3’) with 
core motif CATG. This motif is known to be highly 
conserved in seed specific promoters of both 
dicots and monocots (Dickinson et al., 1988). 
CRE4 comprised of the CAAT box which has been 
suggested to act as an an enhancer element 
involved in quantitative regulation of gene 
expression (Schirm et al, 1987; Wu et al., 2000). 
CRE5 included P-box or the endosperm box 
“5’TGTAAAG3’ that interacts with the transcription 
factor DOF which plays a key role in activating the 
expression of prolamin genes in cereals. Forde et 
al. (1985) has suggested the presence of atleast 
two types of controls operating on prolamin gene 
expression. While one was responsible for 
coordinating induction of genes during endosperm 
development, the other regulated subsequent 
rates of prolamin accumulation. It was suggested 
that these two controls have the ability to act 
differentially on subsets of prolamin genes. The 
two control systems were together named as the 
endosperm box which is a bipartite motif consisting 
of the prolamin box and the GCN4 like motif. The 
GCN4 motif has been reported to be a target of 
basic leucine zipper transcription factor that 
belongs to maize Opaque-2 (O2)-like protein family 
which is also known as RISBZ in rice. Yamamoto 
et al. (2006) have demonstrated that the prolamin 
binding factor transactivated several storage 
protein genes via an AAAG target sequence 
located within the promoters of such genes. They 
observed a synergism between RPBF and RISBZ1 
in recognizing the GCN4 motif (TGA(G/C)TCA) for 
inducing expression of SSP genes. It was 
suggested that RPBF gene, which predominatly 
expressed in maturing endosperm and 
coordinately expressed with seed storage protein 
genes, was involved in quantative regulation of 
genes expressed in the endosperm in cooperation 
with RISBZ1.  
 
 
 
 
 
 
 
Nucleosome binding potential of SSP gene promoters 
  
11 
Nucleosome Mapping determines potential 
nucleosome binding site for SSP promoters 
Variations in position of different cis-elements in 
the promoter sequences are expected to affect 
gene expression either through their interaction 
with transcription factors or through differences in 
nucleosome favouring and/or nucleosome 
excluding sequences (Tirosh et al.,2008). Besides 
the knowledge of cis-acting regulatory elements for 
prediction of transcriptional control of genes, 
information about structural features of DNA, such 
as GC skew, bendability, topography, free energy, 
curvature and nucleosome positioning would give 
a better understanding of the regulatory landscape. 
Therefore mapping of promoters for potential 
nucleosome binding sites would generate a deeper 
insight into long range interactions that may not be 
evident from sequence variations alone. 
Nucleosome positioning demarcates the promoter 
region and transcription start site. While promoters 
which confer ubiquitous gene expression are 
essentially free of nucleosomes, Levitsky et.al 
(2001) have suggested that promoters conferring 
tissue specific expression of genes display higher 
nucleosome formation potential. Nucleosome 
positioning map of each seed storage gene 
promoter, generated using Strong Nucleosome 
Mapping tool, revealed the highest scoring peak 
between 45-51. While a a scoring peak > 65 is 
considered to be statistally significant in 
determining potential position of the nucleosome 
along the DNA sequence, values between 50-60 
indicate a moderate affinity towards 
accommodating a nucleosome. This is due to 
involvement of determinants such as CpG islands 
or epigenetic regulation in modulating nucleosome 
positioning. With a scoring peak of >50, the 
promoters of seed storage proteins of C. arietinum, 
B. napus, B. campestris, V. faba, and P. sativum 
showed potential nucleosome binding sites at 
positions P’78, 635, 195, 112 and 152 respectively. The 
sites were located at positions -100 to -300 with 
respect to the TSS. On the other hand, analysis of 
nucleotide sequences of 5’UTR of genes coding for 
seed storage proteins in Fagopyrum esculentum, 
Zea mays, Avena sativa, Triticum aestivum and 
Oryza sativa by Strong Nucleosome tool revealed 
a score of <45, thereby indicating absence of 
nucleosome binding sites in the promoters of SSP 
genes of these crops. Jiang and Pugh (2009) has 
suggested that in comparison to the transcribed 
region of DNA, the UTR was essentially free of 
nucleosome biding potential. Our results indicate 
that compared to the promoters of genes coding 
for SSPs in C. arietinum, B. napus, B. campestris, 
V. faba, and P. sativum, those of Fagopyrum 
esculentum, Zea mays, Avena sativa, Triticum 
aestivum and Oryza sativa have lower accessibility 
to nucleosome, therby ensuring easy access to 
transcription factors and a consequent higher level 
of gene expression.  
 
 
 
Chettry et al. 
  
12 
ACKNOWLEDGMENTS: 
Financial support received from Department of 
Biotechnology, Govt. of India, New Delhi for 
undertaking the work under DBT Biotech Hub 
project vide grant no. BT/04/NE/2009 to NKC and 
INSPIRE fellowship from Department of Science & 
Technology, Govt. of India to UC is gratefully 
acknowledged. 
 
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Nucleosome binding potential of SSP gene promoters 
  
15 
 
Chettry et al. 
  
16 
 
 
 
Fig. 2: Nucleosome positioning map highlighting the scoring peak at the probable nucleosome binding site  in 
nucleotide sequences of 5’UTR of legumin genes of  (A) C.arietinum [acc. no. Y13166] ; (B) B.napus [acc. 
no. X67833.1], (C)  B.campestris [acc. no. Y13108], (D) V. faba [acc. no. X02983.1], and (E) P. sativum [acc. 
no. X02983.1]. A representative image of non existence of nucleosome binding site in Fagopyrum esculentum, 
Zea mays [acc. no. JQ241267],  Avena sativa [acc. no. EU595873], Triticum aestivum [acc. no. (EU189096.1] 
and Oryza sativa [acc. no. (X65064.1] is given in (F). The nuclosome binding site for the respective accessions 
is boxed.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Nucleosome binding potential of SSP gene promoters 
  
17 
Table1 :Tabular representation of the significant regulatory elements present in SSP promoter across 
different species. 
SITE MOTIF 
Oryza 
sativa Zea mays 
Hordeum
vulgare
 
Triticum 
aestivum 
Piscum sati
vum Vicia Faba 
Cicer arrient
um 
Brassica 
napus 
Brassica 
campestris
 
Brassica
juncaea 
Fagopyrum
esculentum 
SIGNIFICANCE
 
P
B
F
 
T
G
T
A
A
A
G
 -34,-
156,-
161,-
224,-
320,-
345,-
454,-
466,-
476 
-66,-136,-
158,-
173,-
315,-
540,-571 
-45,-
53,-
65,-
164,-
273,-
374 
-50,-58,-
69,-145,-
169,-
271,-
332,-336 
 
-124,-236,-
260,-294,-
308,-430,-
480 
--157,-
259,-
285,-
438,-
452,-
576,-
911,-
927,-
993,-
1135,-
1143,-
1159 
-155,-289,-
369,-429,-
786,-903,-
1052,-
1203,-
1224,-
1539,-
2050,-
2117,-2192 
-1064,-225 --161,-
259,-
327,-
611,-
659,-
726,-
827,-
1056,-
1139,-
1319,-
1386 
-225,-
1068 
-131 Core site 
required for 
binding of Dof 
proteins in 
maize 
E
-B
O
X
 
C
A
N
N
T
G
 --391,-
409 
-804,-
503,-
263,-517 
-355,-
379 
-182 -407,-824,-
1156,-
1124,-
1251,-1285 
-58,-
126,-
567,-
849,-901 
-517,-635,-
747,1060,-
1344,-2226 
-912,-887,-
594,-523 
-107,-
360,-
560,631,-
920,-
1143,-
1299,-
1336 
-58,-
80,-
138,-
598,-
651,-
841,-
917 
-581,-524,-
184,-135,-
91 
E-box of napA 
storage-protein 
gene of 
Brassica napus 
 
C
A
A
T
B
O
X
1
 
C
A
A
T
 -10,-
247,-
402,-
411 
-196,-
329,-
415,-
425,-
504,-
547,-
870,-
880,-940 
-72,-
109,-
147 
-77,-152 
 
-62,-83,-
493,-649,-
715,-798,-
904,-979,-
1109 
-370,-
549,-
676,-
757,-
938,-
1104,-
1130 
-198,-473,-
541,-951,-
1010,-
1100,-
1613,-
2023,-2122 
-926,-829,-
715,-345,-86 
-46,-
371,-
902,-
913,-
1095,-
1258,-
1409 
-86,-
347,-
405,-
720,-
827,-
897,-
924 
-692,-530,-
457,-411,-
282,-
168,54 
"CAAT 
promoter 
consensus 
sequence" 
found in legA 
gene of pea; 
D
O
F
 
A
A
A
G
  -34,-
156,-
161,-
224,-
320,-
345,-
454,-
466,-
476 
-66,-
135,-
158,-
174,-
258,-
315,-
540,-572 
-45,-
53,-
65,-
164,-
273,-
374 
169,-
271,-
332,-336 
-124,-236,-
260,-294,-
308,-430,-
480 
--157,-
259,-
285,-
438,-
452,-
576,-
911,-
927,-
993,-
1135,-
1143,-
1159 
-155,-289,-
369,-429,-
786,-903,-
1052,-
1203,-
1224,-
1539,-
2050,-
2117,-2192 
-225,-1064 --161,-
259,-
327,-
611,-
659,-
726,-
827,-
1056,-
1139,-
1319,-
1386 
-225,-
1068 
-131 Core site 
required for 
binding of Dof 
proteins in 
maize  
 
T
A
T
A
B
O X
 
T
T
A
T
T
T
  -23 -23 -20 -24 
 
 
 
23 -25 -23 -24 -21 -24 -30 TATA box 
elements are 
critical for 
accurate 
initiation 
O
P
A
Q
U
E
-2
 
T
G
A
G
T
C
A
 -210   -------- ------------ --------- --------------
---- 
---------- ---------- ------- ------ GNC4 motif is 
the recognition 
site forOpaque-
2 (O2)-like 
proteins 
R
IS
B
Z
I 
T
G
A
G
T
C
A
 -210   ---------    ----------- ---------- ------ ------ Required for 
the exp GNC4 
motif ression of  
S
E
F
1
     
 
 
 
 
  -1136 -803     -307 SEF1binding 
motif; sequence 
found-upstream 
region;ofsoybe
an βconglicinin 
gene