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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. References 1 2 3 4 5 6 7 Evans I M, Gatehouse J A, Croy R D & Boulter D, Regulation of the transcription of storage protein mRNA in nuclei isolated from developing pea (Pisum sativum) cotyledons, Planta, 160 (1984) 559-568. 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