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 Nain

1, M Z Abdin

2 and P A Kumar

1*

1NRC on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012, India 2Centre for Transgenic Plants Development, Department of Biotechnology, Jamia Hamdard, New Delhi 110 062, India

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,

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 sequences10,11

. 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 promoters13

.

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 cis-

acting regulatory databases and tools of

bioinformatics help to predict the transcriptional

___________

*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

INDIAN J BIOTECHNOL, OCTOBER 2007

496

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 method21

.

Polymerase chain reaction (PCR) was perfor-

med 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. Amplifi-

cation 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 ex-

pected 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 WU-

BLAST 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 sequ-

ences, 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 Agrobac-

terium tumefaciens strain LBA4404 by freeze thaw

method30

. 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 self-

pollinate. 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

497

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

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 apparent33

.

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 promoters12

,

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

CAP site ctccgcAt +1 Transcription start site

TATA box tcccTATAaataa -33 Important for recognition by RNA polymerase II

CAT box gCCAAc -49 Common cis-acting element in promoter and enhancer regions

G-box tgACGTgt -66

-158

Conserverd element in promoters of genes inducible by various

stress and physiological cues

TGA-box TGACgtgt -66 Part of an auxin-responsive element

TGACG- TGACg -66 cis-acting regulatory element involved in the MeJA-responsiveness

W Box cttctTTGAcgtgtcca -72 WRKY plant specific zinc-finger-type factor associated with

pathogen defense

ABRE acaccttctttgACGTGtccatccttc -76 cis-acting element involved in the abscisic acid responsiveness

Legumin box tccatacCCATgcaagctgaagaatgtc -118 Highly conserved sequence element about 100bp upstream of TSS

in legumin gene promoters

ERE ATTTcaac -240 Ethylene-responsive element

ABI4 CACCg -245 ABA insensitive protein 4

AAGAA-

motif

agaAAGAa -294 Cis-element involved in seed specific expression

I-box gATATga -302 Covered in light inducible gene promoters

WUN-motif tAATTacac -348 Wound-responsive element

Opaque-2 TAATtacacatatttta -348 Cis-element involved in seed specific expression

A box TATCaagcact -362 Sequence conserved in alpha-amylase promoters

Prolamin box TTaaaTGTAAAAAgtAa -385 Conserved in cereal seed storage protein gene promoters

TCA-element gAGAAgagaa -646 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

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 DNA17,18

. 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/prog-

rams/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 nucleosome-

formation. However, both the linker DNA regions

have at least one conserved motif (Fig. 1C), which

indicates that linker DNA may not be entirely


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 protein- binding 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 nucleosome- formation 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


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

I- rn

Fig. 4--GUS expression in different tissues of PLeg::GUS Fig. 2--South~m analysis of transgenic tobaccco plants for the transgenic tobacco: A. Endosperm; B. Embryo; C, 1-wk-old integration of Pkg::GUS. '+': Positive control (pPLeg::GUS ge-nating seed; and D. 2-wk-old s w g with cotyledonary vector); '-': Negative control (DNA fron non transgenic tobacco leaves. plant); '1-12' DNA samples from PLeg::GUS transgenic tobacco plants.

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.


Supplementary material: Multiple sequence alignment of 1eguxn.b promatem used in the study, revealing conserved regulatory elements.


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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