Download Isolation of pigeon pea (Cajanus cajan L.) legumin gene promoter

Indian Journal of Biotechnology
Vol 6, October 2007, pp 495-503
Isolation of pigeon pea (Cajanus cajan L.) legumin gene promoter and
identification of conserved regulatory elements using tools of bioinformatics
Rajani Jaiswal1, Vikrant Nain1, M Z Abdin2 and P A Kumar1*
1
NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012, India
Centre for Transgenic Plants Development, Department of Biotechnology, Jamia Hamdard, New Delhi 110 062, India
2
Received 5 January 2006; revised 28 October 2006; accepted 25 January 2007
A seed specific legumin gene promoter from pigeon pea was isolated by PCR amplification. Database assisted sequence
analysis of this promoter revealed several putative cis-acting regulatory elements. Comparative analysis of 15 seed-specific
legumin gene promoters from six species, viz. Cajanus cajan, Cicer arietinum, Pisum sativum, Glycine max, Vicia
faba and Arachis hypogaea, revealed several conserved motifs in promoter sequences; maximum conservation was
observed upstream to transcription start site. Most of the conserved motifs have known transcription factor binding
sites. One unknown conserved motif of seven base pair (AG/TGTGTA) was found 19 bp upstream to legumin box,
putatively named as L-19. Study of nucleosome formation potential showed that putative linker DNA is more prone to
mutations as compared to DNA involved in nucleosome formation. A chimeric construct was made with pigeonpea
legumin promoter and β-glucuronidase (GUS) gene. Analysis of GUS expression at different developmental stages of
transgenic tobacco plant’s parts revealed that the reporter gene was expressed at a high level only in mature seeds,
specifically in embryo, endosperm and in cotyledonary leaves of developing seedling. These data showed that GUS gene
transcription was regulated in a tissue specific and temporally regulated manner.
Keywords: β-glucuronidase (GUS), legumin gene, nucleosome positioning, phylogenetic footprinting
IPC Code: Int. Cl.8 C12N15/09, 15/29
Introduction
The genes encoding legumin seed storage
proteins are under seed specific developmental
regulation. Synthesis of these proteins is regulated
at transcription level and continues until they
comprise 60-80 percent of the protein in mature
seed1,2. Thus, legumin seed specific promoters
provide an excellent system to study the control of
expression of plant genes and use them in
development of transgenics for seed quality
improvement. Promoters governing developmental
regulation have been identified for other storage
protein genes from Cicer arietinum3, Pisum
sativum4,5, Glycine max6,7 and Vicia faba8,9, which
are expressed specifically in seeds.
Transcription regulatory regions in higher
eukaryotes, represented by cis-regulatory modules,
___________
*Author for correspondence:
Tel: 91-11-25841787; Fax: 91-11-25766420
E-mail: [email protected]
The sequence data reported has been submitted in GenBank nucleotide
sequence database under accession no. AY623813
are responsible for the formation of specific spatial
and temporal gene expression patterns. With the
availability of large number of sequences,
phylogenetic footprinting has become an important
tool to identify cis-regulatory elements in the
promoter
sequences 10,11. Potential cis-acting
elements have been identified as highly conserved
sequences in the promoters of specific class of plant
genes, such as ‘vicilin box’ and ‘legumin box’ in
storage protein of legumes2,8, and ‘prolamin box’ in
cereal storage proteins12, ‘G box’ in promoters of
genes responsive to stress and physiological
cues13,14,15, ‘W box’ in pathogen responsive genes16
and ‘I box’ in light inducible gene promoters 13 .
However, authenticity and certainty of a newly
identified conserved motif increase as the number of
species providing nucleotide sequence increases in the
analysis. In addition, nucleosome positioning has
been proposed to be a potential mechanism for
regulating gene expression17,18.
Although not complete, the availability of cisacting regulatory databases and tools of
bioinformatics help to predict the transcriptional
496
INDIAN J BIOTECHNOL, OCTOBER 2007
properties
of
new-entry
sequences
with
considerable accuracy19. The present study describes
the PCR based isolation of a pigeonpea legumin seed
specific (PLeg) promoter, sequence analysis for
potential regulatory elements and phylogenetic
relationship with other legume seed storage gene
promoters. The expression analysis of GUS reporter
gene under its control was carried out in transgenic
tobacco.
Materials and Methods
Isolation of PLeg Promoter
The PLeg promoter from pigeonpea was isolated
on the basis of conserved DNA sequences in the
homologous promoters of closely related species.
Primers were designed based on the sequence of
chickpea leg3 seed specific promoter20. Genomic
DNA was extracted from in vitro grown seedlings
of pigeonpea (cv. Pusa 855) by CTAB method 21 .
Polymerase chain reaction (PCR) was performed with forward primer sequence (5'GGCTGCAGGCAGAGTCCTTTATTCATTG-3')
containing PstI and reverse primer sequence (5'GGGGATCCGATGACAGATTTTGAAAAAG-3')
containing BamHI restriction sites to facilitate
directional cloning. The promoter sequence was
amplified from pigeonpea DNA using 2.5 units Pfu
DNA polymerase in a 50 µL reaction. Amplification was carried with 10 ng of genomic DNA and
0.2 M dNTP mix at 58°C annealing temperature in a
Thermocycler (Biometra) for 25 cycles. The purified
PCR product was cloned in pBluescript SK+ vector
with PstI and BamHI restrictions sites. DNA
sequencing of PLeg promoter was carried out by an
automated
DNA
Sequencer
(DNASeqC,
MegaBACE 1000). Nucleotide sequence was
determined for both strands. The PLeg promoter
was also cloned upstream to GUS gene by
replacing 35S CaMV promoter of pBI121 binary
vector, using HindIII and BamHI restriction sites. The
construct was designated as pPLeg::GUS.
Sequence Analysis
Various promoter sequences homologous to PLeg
promoter were extracted from GenBank. Chickpea
legumin (Y13166) and legumin3 (Y15527); pea
legA (X02982), legB (X02983), legC (X02984)
and legA2 (X57666); and soybean glycinin
(E07850), glycinin GY (E07852) glycinin GY1
(X15121), glycinin GY2 (X15122) and glycinin
A(2)B(1)A (X53404) sequences were obtained
by using WU-BLAST (http://www.arabidopsis.org
/wublast/index2.jsp). As promoter sequences are expected to share small conserved sequence motifs,
which may not figure in a BLAST search, MEME
(Multiple Expectation Maximization of Motif
Elicitation)22 was used to identify 50 conserved
motifs of 20 bp length in sequences selected in WUBLAST search. These 50 motifs selected in MEME
were used in motif alignment search tool (MAST) against
Eukaryotic Promoter Database (EPD) (http://www.
epd.isb-sib.ch/)23. The EPD MAST selected pea legJ
(X07014) in addition to WU-BLAST selected pea
leg (A, B & C) sequences. To get additional sequences, chickpea leg3 protein nucleotide sequence
was used in NCBI BLAST search. Faba bean
glycinin LeB4 (X03677) and peanut ArAh3/
ArAh4 (AF510854) were also found to have promoter
sequence in addition to protein sequence. All these
15 sequences selected were aligned by ClustalX24
and used to shade conserved regions by Bioedit
or convert to sequence logo by 'Weblogo' (http://
weblogo.berkeley.edu/logo.cgi)25. The PLeg promoter
sequence was analyzed using various database
search programs such as PLACE (http://www.
dna.affrc.go.jp)26, plant CARE (http://intra.psb.ugent.
be:8080/PlantCARE/)27, TRRD http://wwwmgs.
bionet.nsc.ru/mgs/papers/goryachkovsky/plant-trrd/)28
and matinspector (www.genoma tix.de)29.
Tobacco Transformation
The modified binary vector pBI121 containing
PLeg::GUS construct was mobilized into Agrobacterium tumefaciens strain LBA4404 by freeze thaw
method 30 . The transformed A. tumefaciens strain
was used to infect tobacco (Nicotiana tabacum cv.
Petit Havana SR-1) leaf discs and transgenic plants
were regenerated according to Horsch et al31. All
cultures were incubated under a 16 h photoperiod
(50 µE m-2 s-1, provided by cool-white and day light
Sylvania fluorescent lamps) at 27°C. All the
transgenic plants were grown and allowed to selfpollinate. Transgenic plants were analyzed by
PCR and Southern hybridization for the integration of
the gene construct.
GUS Assay
Twenty five independent transformants carrying
PLeg::GUS construct were analyzed. Seeds,
seedlings, petals, androecium and gynoecium from
the transgenic tobacco plants were analyzed for in
JAISWAL et al.: PIGEONPEA LEGUMIN GENE PROMOTER
situ GUS expression according to Jefferson et al32.
Plant materials were stained overnight with 2 mM
X-Gluc, 100 mM Tris-HCL (pH 7.0), 50 mM NaCl,
2 mM potassium ferricyanide, 2 mM potassium
ferrocyanide and 0.1% (v/v) Triton X-100 at 37°C.
After staining, tissues were incubated in 70%
ethanol to clear chlorophyll and were subsequently
fixed in 70% ethanol.
Results and Discussion
Isolation of PLeg Promoter Sequence
The complete sequence of PLeg promoter (AY
623813) was found to be 808 bp long as compared to
812 bp long chickpea leg3 gene promoter20. It has
98.5% similarity to chickpea leg3 gene promoter.
Comparison of the two sequences revealed
presence of different nucleotides at twelve
positions. The promoter sequence was highly AT
rich (65% AT and 34% GC) as observed in other
regulatory sequences.
Database Assisted Sequence Analysis
The results of identified general transcription and
potential regulatory elements have been
summarized in Table 1. The nucleotide sequence of
PLeg promoter revealed several characteristic
497
features. Transcription start site is located within
octanuclotide CTCCGCAT. The sequence analysis
showed that consensus sequence for ‘TATA was
TATAAA, preceded by dinucleotide CC at position
-33 bp to cap site. Typical animal gene promoter
sequence ‘CAT’ box was found at -49 bp of cap
site. In some plants this consensus sequence has
been found but the homology is often poor or no
‘CAT’ box is apparent 33 .
Several cis-elements were identified in PLeg
promoter sequence that are similar to those previously
described in storage protein and defense related gene
promoters. The presence of ‘Legumin box’, a 28 bp
conserved motif at -118 bp, which is present in
promoters of various legumin seed storage protein
genes8 and ‘prolamin box’, a conserved motif
present in cereal storage protein gene promoters 12 ,
suggests the specific regulatory function important
for expression of seed storage protein. ‘Opaque-2’
binding site and ‘AAGAA motif’ are other
regulatory sequences present in this promoter that
are found in promoters of genes expressed in seeds34,35.
‘G Box’, a cis-regulatory element involved in
various stress responsive gene promoters, including
UV light and abscisic acid (ABA)13,14,15, is present at
two positions -66 bp and -159 bp. Presumably, ‘The
Table 1—Cis-acting regulatory elements found in PLeg promoter sequence
Site
Sequence*
Position
Function
Transcription start site
Important for recognition by RNA polymerase II
Common cis-acting element in promoter and enhancer regions
Conserverd element in promoters of genes inducible by various
stress and physiological cues
Part of an auxin-responsive element
cis-acting regulatory element involved in the MeJA-responsiveness
WRKY plant specific zinc-finger-type factor associated with
pathogen defense
cis-acting element involved in the abscisic acid responsiveness
Highly conserved sequence element about 100bp upstream of TSS
in legumin gene promoters
Ethylene-responsive element
ABA insensitive protein 4
Cis-element involved in seed specific expression
CAP site
TATA box
CAT box
G-box
ctccgcAt
tcccTATAaataa
gCCAAc
tgACGTgt
TGA-box
TGACGW Box
TGACgtgt
TGACg
cttctTTGAcgtgtcca
+1
-33
-49
-66
-158
-66
-66
-72
ABRE
Legumin box
acaccttctttgACGTGtccatccttc
tccatacCCATgcaagctgaagaatgtc
-76
-118
ERE
ABI4
AAGAAmotif
I-box
WUN-motif
Opaque-2
A box
Prolamin box
TCA-element
ATTTcaac
CACCg
agaAAGAa
-240
-245
-294
gATATga
tAATTacac
TAATtacacatatttta
TATCaagcact
TTaaaTGTAAAAAgtAa
gAGAAgagaa
-302
-348
-348
-362
-385
-646
Covered in light inducible gene promoters
Wound-responsive element
Cis-element involved in seed specific expression
Sequence conserved in alpha-amylase promoters
Conserved in cereal seed storage protein gene promoters
Cis-acting element involved in the wound and pathogen responsive
genes.
*Base pair in capital letter denote the core sequence used in the search programs.
498
INDIAN J BIOTECHNOL, OCTOBER 2007
G box’ functions in combination with other regulatory
elements and gets activated under specific stress36.
Another cis-element ‘ABA insensitive’ (ABI4),
which works in combination with ‘G Box’ is
present at –245 bp position.
Phylogenetic Footprinting
Isolation of PLeg promoter allowed the
comparison of legumin seed specific promoters
sharing same specificity from distantly related
species. Seventeen legumin gene promoter
sequences from six species, viz Cajanus cajan, C.
arietinum, P. sativum, G. max, V. faba and Arachis
hypogaea were used. It was observed from
multiple sequence alignment of these sequences
that the frequency of conservation within the
promoter sequence decreases as distance from
transcription-start site to 5' increases. The highest
conserved regions were found upto -160 bp from
transcription-start site. Upstream vicinity of cap
site showed considerable conservation in the all
legume seed storage gene promoters analyzed,
followed by TATA box (TATAAA) and the ‘G
Box’ motif. Another conserved region represented
the G Box motif, suggesting legumin promoters
have retained their functional sites during the
course of evolution. The longest conserved region
between -118 to -91 bp position represents the
legumin box. Out of 28 contiguous nucleotides
present, 19 are perfectly invariant. In the present
phylogenetic footprinting analysis, an unknown motif
of seven bp (AG/TGTGTA) is present 19 bp upstream
of legumin box except in A. hypogaea where it is 20
bp upstream, of all legumin promoter sequences
analyzed. The evolutionary conservation of motif
putatively named as 'legumin minus nineteen' (L-19)
motif indicates its regulatory role in promoters of
legumin seed specific proteins.
Nucleosome-Formation Potential of PLeg Promoter
The promoter sequences may exhibit high or low
nucleosome-forming tendencies compared to
random DNA 17,18 . This could mean that
nucleosomes, whose positions are influenced by the
underlying DNA sequence, can in turn govern the
accessibility of regulatory DNA sequences. This
sequence-directed nucleosome positioning can help
to either selectively expose functionally important
DNA sequences by constraining their locations to
the linker region or impede accessibility to
functionally important sequences by constraining
their location to within the core particle18,37. This
forms the basis of search for evidence of
nucleosome positioning and consequently building
models to predict and investigate such locations.
Nucleosome-formation potential profile of
pigeonpea promoter was generated using
RECON
(http://www.mgs.bionet.nsc.ru/mgs/programs/recon/)38. The mean value of nucleosome
formation potential is +1 for set of nucleosome site
and -1 for the set of random sequence. A higher
probability of nucleosome positioning correlates
with the nucleosome-formation potential value close
to +1. Nucleosome formation potential profile of
PLeg promoter sequence showed three tentative
nucleosome binding regions, with the value ranging
between +0.5 to +1 (Fig. 1B). The first
nucleosome-binding region spanned approximately
from 80 bp to 225 bp (≈145 bp). The other two regions
showing potential sites for nucleosome formation
were found at 335 bp to 485 bp (≈150 bp) and 590
bp to 730 bp (≈145 bp). The nucleosome
formation potential value decreased from +0.5 to
-0.5 in the putative linker DNA region, 225 bp to
335 bp (≈110 bp). The second linker DNA ranged
from 485 bp to 590 bp (≈105bp). A random
sequence similar to PLeg promoter sequence in
length (808 bp) and AT/GC content was generated
(http://www.llamastar.com/phptest/dna.php)
and
used to generate nucleosome formation potential
profile for comparison. In the random sequence there
are some regions showing the value above 0.5 but
there is no characteristic pattern of nucleosome
formation as it is observed in promoter sequence
(Fig. 1A).
Comparison of nucleosome-formation potential
with conserved regions in multiple sequence
alignment of legumin promoters shows that both
linker DNA regions are more prone to mutations as
compared to DNA region involved in nucleosome
formation (Fig. 1C). A region of 15 nucleotides at
-220 bp position in PLeg promoter is absent in
other leg promoter sequences. It appears that
during evolution there might be a deletion of this
fragment in other sequences or addition in
pigeonpea and chickpea sequences. The second
linker DNA also shows more mutations as
compared to DNA involved in nucleosomeformation. However, both the linker DNA regions
have at least one conserved motif (Fig. 1C), which
indicates that linker DNA may not be entirely
JAISWAL et al.: PIGEONPEA LEGUMIN GENE PROMOTER
Fig. 1-Prediction of nucleosome formation (X-axis nucleosome formation potential, Y-axis nucleotide sequence: A. Random nucleotide
sequence; B. PLeg promoter sequence; C. Multiple sequence alignment of closely related promoter sequence (Pigeonpea PLeg; Chickpea
legurnin, legumin3; Pea legA, legB, legC, legA2; Soybean glycinin, glycinin GY, glycinin GYl, glycinin GY2 and glycinin A(2)B(l)A;
cl is conserved linker DNA region).
dispensable and possibly have some proteinbinding site.
It has been observed that a typical promoter has a
specific nucleosome positioning around transcription
start site39. It has been 'demonstrated that tissue
specific promoters display higher nucleosomeformation potential as compared with the potentials
of genes expressed in many tissues and house
keeping genes. A trend to increase the nucleosome
density might have occurred in promoters of
genes requiring fine-tuning, i.e. tissue specificity.
GUS Expression Analysis
The integration -of PL.eg::GUS gene in transgenic
tobacco plants were confirmed by Southern
hybridization and subsequently analyzed for GUS
expression (Fig. 2). Expression of GUS gene in
different parts, viz. seeds, seedlings, petals, androecium and gynoecium, was examined. There was no
GUS expression in the developing seeds. However,
mature seeds collected from the fully ripened (35-45 d
after flowering) pods showed strong staining
(Fig. 3). After dissecting the X-Gluc treated seeds, it
was observed that expression was localid in the
INDIAN J BIOTECHNOL, OCTOBER 2007
whole embryo and endosperm tissue (Figs 4A & B).
GUS analysis of the transgenic seedlings at 1 wk
interval was also carried out. At 0 d, whole seed
exhibited dark blue colour and after 7 d, the expression was localized to plumule of the germinating seed
(Fig. 4C). There was no GUS expression in root system
(Fig. 4C). After 14 d, it was observed that the
expression was localized in cotyledonary leaves of the
seedling (Fig. 4D). The seedling did not show any
GUS activity in any part after 21 d of germination. No
GUS activity was observed in non-transgenic plants.
I
rn
I
Fig. 2--South~manalysis of transgenic tobaccco plants for the
integration of Pkg::GUS. '+': Positive control (pPLeg::GUS
vector); '-':Negative control (DNA fron non transgenic tobacco
plant); '1-12' DNA samples from PLeg::GUS transgenic tobacco
plants.
Fig. 4--GUS expression in different tissues of PLeg::GUS
transgenic tobacco: A. Endosperm; B. Embryo; C, 1-wk-old
ge-nating
seed; and D. 2-wk-old s w g with cotyledonary
leaves.
Fig. M U S expression analysis in different parts of PLeg::GUS transgenic tobacco plants: A. Whole plant; B. Petal; C. Sepal;
D. Androecium; E. Gynoecium; and F. Mature seeds.
JAISWAL et al.: PIGEONPEA LEGUMIN GENE PROMOTER
Supplementary material: Multiple sequence alignment of 1eguxn.b promatem used in the study, revealing conserved regulatory elements.
INDIAN J BIOTECHNOL, OCTOBER 2007
502
The expression pattern controlled by the
pPLeg::GUS construct in transgenic tobacco was
confined to the seeds. It shows that the expression of
this promoter is tissue specific and developmentally
regulated. Tissue specificity of the gene suggests
the presence of embryo and endosperm regulatory
elements in the promoter. At further developmental
stages, the GUS expression was localized only in
the cotyledonary leaves and not in the other leaves
suggesting strong seed specificity activity of this
promoter. There is relatively low overall sequence
identity among promoter sequences from storage
protein genes as compared with their coding
regions. Several expression analysis experiments
involving promoter reporter gene constructs have
shown that there is high conservation in the pattern of
gene expression among orthologous genes from
different species4. Thus, conserved pattern of gene
expression might be programmed by regulatory
sequences associated with conserved, non-coding
sequences. Since the expression of the GUS gene
under the control of pigeonpea seed specific promoter
was observed only in mature seeds, this promoter will
be useful in genetic modification of seed properties
during the latter stages of the seed maturation, such
as protein quality and fatty acid composition. This
promoter can also be used to express the insecticidal
gene only in the seed to control the storage pests.
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