MATERIALS AND METHODS

Materials

Peanuts (Arachis hypogaea L. cv. Florunner) were grown in experimental plots at the University of Florida, Experimental Station, Quincy, FL following the recommended cultural practices. Randomly selected plants were dug at weekly intervals for 9 weeks between 90 and 160 days after planting; pods were harvested, washed with tap water and separated into six maturity categories (white, yellow-1, yellow-2, orange, brown and black) following the Hull-scrap method of Williams and Drexler (1981). The pods were subsequently washed with distilled water, surface sterilized with 2% NaOCl (without any wetting agent) for 5 min, thoroughly washed (six times) with sterile water and excess water was removed with dry paper towels. Pods were shelled, seed coats removed and frozen immediately in liquid nitrogen and stored at -80ºC until used.

Tissues such as fully developed leaf, stem, root, flowers and pegs were collected between 75-85 days after germination. For post-germinating seedlings, seeds were sterilized first by washing with detergent (2% Tween 20) for 5 min; detergent was removed by washing the seeds 5-6 times with distilled water. Later seeds were treated with Clorox (0.15%) for 5 min and washed with 7-8 times with autoclaved distilled eater. Sterilized seeds were germinated in lab (at 25ºC) in Petri dishes on moist filter paper. Seedlings at 0, 2, 4, 6, 8, 10, 12, and 14 days after imbibitions were sampled, frozen immediately in
liquid nitrogen and stored at -80ºC until used.

Nucleic acid isolation

Total RNA was isolated from the seeds of different maturity pods (white to black maturity category), leaf, stem, root, flowers, pegs and post-germinating seedlings of 0 to 14 days. Tissues were homogenized in 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, 0.1 M ß-mercaptoethanol, and then extracted with an equal volume of phenol: chloroform (1:1). To the aqueous phase equal volume of isopropanol was added and chilled on ice for an hour. Total RNA was recovered by centrifugation (5000 g) and was further purified at least twice using LiCl (final concentration 2 M) precipitation and dissolved in sterile distilled water. Finally, the total RNA was precipitated with 2.5 volumes of 95% EtOH in the presence of 0.1% 3M NaOAc (pH 7.0) and stored at -80ºC until used for northern blot hybridization studies.

PCR amplification

Two forward (GlyUP.1 coding for DRHQKIYNFRE; and GlyUP.2 coding for ENEGEDKAI) and one reverse (GlyDN.1 coding for FVPHYNLNA) degenerate oligonucleotide primers were designed and synthesized (Genosys, Biotechnologies, Inc., Texas) based on the conserved region among soybean (Glycine max) (Nielsen et al. 1989), and broad bean (Vicia faba) (Panitz et al. 1997) glycinin protein sequences. PCR amplification was performed in 50 ml reaction volume with 1 mM for each of the forward and reverse primers, 1 ml of peanut seed (yellow-1 pod maturity) cDNA library (titer 1.2 X 10⁹ pfu per ml), 5 mM dNTP's, 1X PCR buffer [10 mM Tris HCl (pH 9.0), 50 mM KCl, 25 mM MgCl₂ and 0.1% Triton X100] and 10 U of Taq DNA Polymerase (Promega). The template was denatured by heating to 94ºC for 3 min, followed by 30 cycles of 30 seconds at 94ºC, followed by combinations of annealing temperatures for 1 min (37ºC or 45ºC) and polymerization for 1 min 30 seconds (at 68ºC or 72ºC), with a single final extension of 5 min at 72ºC. Total of eight combinations (including primer combination's, annealing and polymerization temperature) were used to amplify DNA sequences for peanut arachin clone. The PCR amplified product (25 µl) was separated by electrophoresis on a 1.0% agarose gel.

DNA sequencing and sequence analysis

The resulting PCR product was purified from the gel using GENECLEAN Kit (BIO 101, Vista, CA) and cloned into pBlueScript II KS⁺ (Stratagene, La Jolla, CA) at Eco RV site and designated as pKS Gly1-350. The cloned PCR fragment and purified cDNA clone(s) were sequenced on double stranded DNA using a dye-terminator cycle sequencing kit (PE Applied Biosystems) and an automated capillary DNA sequencer (Genetic Analyzer ABI 310, PE Applied Biosystems).

All sequencing data was analyzed using LASERGENE software (DNASTAR, Madison, WI). Searches for information and homology of nucleotide and amino acid sequences were carried out using homology search with BLAST against the sequences in the NCBI GenBank and EMBL DNA databases.

Construction of cDNA Library

Poly (A)+ RNA was obtained from total RNA of seeds from yellow-1 pod maturity, by two successive runs on oligo (dT)-cellulose spin column following the manufacturer's protocol (Clontech, Palo Alto, CA). First strand synthesis was performed in a total volume of 10 ml. The reaction mixture consisted of 2 m mRNA, 1 ml of 1 mM oligo dT (dT15) primer, 200 units of MMLV reverse transcriptase, 2 ml of 5 mM dNTP and 2 ml of 5X first strand buffer [250 mM Tris (pH 8.3), 30 mM MgCl₂, 375 mM KCl]. Double stranded cDNA was synthesized in the presence of DNA polymerase I (9.0 U/ml). Following the addition of Eco RI linkers the cDNA was ligated into the Uni-ZAP XR vector and packed in vitro with GigapackIII Gold Cloning Kit as recommended by the manufacturer (Stratagene, La Jolla, CA). The primary library was amplified in SM buffer [0.05 M Tris-HCl (pH 7.5), 0.58% NaCl, 0.2% MgSO₄ and 0.01% gelatin] according to the manufacturer's instructions and the aliquots of the amplified library was stored in 7% DMSO (v/v) at -80ºC.

Amplified PCR fragment was excised from pKS Gly1-350 using Sal I and Eco RI restriction enzymes. Fragment was purified using GENECLEAN Kit (BIO 101, Vista, CA). The double strand DNA (100 ng) was denatured by heating at 95ºC for 5 min and radiolabelled with a³²P dATP following the Prime-a-Gene Labeling System (Promega, Madison, WI). The unincorporated nucleotides were removed using Spin Column 30 (Sigma Chemical Co., St. Louis, MO) and radiolabelled probe was as a probe to screen cDNA library. For northern and southern blot hybridization, 1.6 kb Eco RI fragment was excised from the cDNA clone, radiolabelled (as described above) and used as probe.

Isolation of peanut glycinin DNA sequences

Amino acid sequence of glycinin seed storage protein of soybean and broad bean was used to design and synthesize degenerate oligonucleotide primers. PCR was performed using combinations of degenerate oligonucleotide primers (GlyUP.1, GlyUP.2 and GlyDN.1), annealing and polymerization temperatures and cDNA library of immature peanut seeds as template. No PCR product was detected from the primer combination of GlyUP.1 and GlyDN.1 One distinct PCR product (350 bp) was detected from the primer combination GlyUP.2 and GlyDN.1 at 45ºCannealing and 72ºC polymerization temperature. PCR was repeated three times, no other product was amplified. The PCR amplified fragment of 350 bp was of expected size based on the position of GlyUP.2 and GlyDN.1 primers on amino acid sequence of glycinin protein. As such no other product was amplified, consequently 350 bp PCR fragment was cloned and sequenced. BLAST information revealed that it shares a high degree of nucleotide homology with glycinin and legumin 11S seed storage protein.

Three clones were isolated and purified from the immature seed cDNA library. These clones contain inserts of 1.8, 1.05 and 0.75 kb. All cDNA clones were sequenced and compared for the homology. On sequence analysis it was found that they do not differ, the 1.8 kb clone was found to contain a complete gene as judged by comparison to other genes and designated as peanut Gly-1 (peanut glycinin-1; NCBI accessation # AF125192, gi|9864776) clone. Figure 1 shows the restriction map of the peanut glycinin clone spanning a total of 1836 bp. The cDNA lacks unique restriction sites for Bam HI, Pst I, Sac I, Sal I, Sma I and Xba I. The unique restriction sites present are Cla I, Eco RI, Xho I and Xho II (Figure 1). The predicted protein has 529 amino acid residues, with a calculated Mr of 60,447.61 Da and an isoelectric point of 5.489. The hydropathy profile of the protein, deducted by the algorithm of Kyte and Doolittle(1982), shows the presence of two hydrophobic regions located within the first 76 residues (residues 6 to 21 and 67 to 76), each of which is long enough to span a membrane bilayer (regions 1 and 2 in Figure 2). The protein contains two potential asparagines-linked glycosylation sites (Asn-X-Ser/Thr) at position 289 and 362. Nevertheless, no data is presently available on the glycosylation of other known plant glycinin genes. The 1.05 and0.75 kb cDNA clones that were initially selected and were found to represent shorter versions of peanut Gly1 cDNA clone. The isolation of multiple independent cDNAs that represent the same mRNA suggests that the corresponding mRNA is abundant.

The BLAST results of peanut arachin cDNA sequences show that sequences have high levels of similarity with other homologous glycinin or legumin seed storage genes. Peanut Gly-1 shows high homology with two other peanut glycinin clones - Ara h3 (95%) and Ara h4 (94%). Peanut Gly-1 nucleotide sequence shows 67 and 69% identity with soybean glycinin gene (Gly 3 and Gly 2 respectively), whereas it has 60 to 63% identical and 34 to 48% conserved residues using Hein with weighted method (Hein, 1990). At present, the primary sequence of glycinin gene is known for a variety of legumes including soybean (Nielsen et al. 1989), broad bean (Panitz et al. 1997) and Pea (Casey and Domoney, 1999). From these data it becomes evident that all glycinin or glycinin like protein genes, characterized so far, share a high level of similarity in the N-terminal and C-terminal region compared to the central region of the protein. In this respect, the amino and C-terminal part of the peanut glycinin gene (residue 1-120 and 369-553) also have high level of similarity to the previously reported glycinin sequences (Figure 3).

The alignment of peanut glycinin-1 sequence with those of legumin and glycinin proteins from other legumes shows that there is close similarity between these sequences (Figure 3). The sequence comparison and homology shows presence of a conserved a-subunit region in the N-terminal region, which infer that peanut Gly-1 decode type-A legumin or glycinin protein and deciphers 11S globulin seed storage protein. The amino acids located in the identical position reveal interesting features concerning the similarities among the protein (Figure 3). The alignments shown in the Figure 3 reveal three regions of variability in the legumin-like subunits. One occurs at the carboxyl ends of the acidic chains in the subunits, and is especially variable. This region has been referred to as the hypervariable region (HVR) (Argos et al. 1985). The second region of variability was first observed by Simon et al. (1985), and referred to as variable region 1 (VR1). It is located between positions 118 to 144 in Figure 3. The VR1 region of peanut Gly-1 has extended runs of glutamine and arginine that are not observed in other legumin-like proteins. Finally, the third region of internal heterogeneity, variable region 2 (VR2), spans amino acid position 212 to 251 of the alignments shown in Figure 3. The composition of VR2 also tends to be rich in arginine and glutamine, a characteristic of the other variable regions (Figure 3).

The predicted amino acid sequence was searched for conserved domain that aligned peanut Gly-1 sequence with that of recently designated cupin superfamily of proteins (Dunwell, 1998), which is among the most functionally diverse proteins. These proteins are named on the basis of the conserved ß-barrel fold ('cupa' is the Latin term for a small barrel), peanut Gly-1 have bi-cupin domains that comprises two conserved motifs (Figure 4), each corresponding to two ß-strands separated by a less conserved region. The N-terminal motif consists of 169 residues (38 through 206) while the C-terminal motif consist of 145 residues (362 through 506). Both the N-terminal and C-terminal motifs of peanut Gly-1 have high levels of similarity with cupin domain. According to the list of conserved domain database (CDD) peanut Gly-1 clone belongs to 00190 Pfam family of 11S plant seed storage protein. The phylogenetic and functional studies based on sequence alignment and conserved domains of known sequences reveal that most of them contain a single metal-binding site, which has been characterized by two conserved histidines and a glutamate in the first motif, together with a third conserved histidine in the second motif (Dunwell et al. 2001; Khuri et al. 2001). It is known that these conserved residues act as ligands for active-site metal, which has been shown to be a single manganese atom for barley germin (Requena and Bornemann, 1999; Woo et al. 2000) and for plant germin-like proteins (GLPs, Carter and Thornburg, 1999). Peanut Gly-1 does not contain the second histidine residues in the conserved active site of first motif instead it has a lysine. The presence of positively charged lysine in first motif region might provide the same function as histidine except altering the binding characteristics of the active site. Multiple alignment of the protein sequence presented in this paper (Figure 3) reveal the presence of a lysine in place of second histidine in the first motif that confirms the conservation of lysine with other legumin/glycinin genes. Such sequence variations have also been observed in some Glycine max ESTs clones (gi|5759981, gi|6725711, and gi|6566187). This infers that these proteins may change its function and may not bind a manganese atom, or that they may bind it loosely or strongly and only under certain pH condition. However, the conservation in lysine residue in the legumin/glyciningenes among legumes like soybean, field bean and broad bean indicates close phylogenetic relationship among these legumes and evolution of functional diversity in the cupin motif from other organisms. However, the evolutionary origin of bi-cupins, has yet to be resolved (Dunwell et al. 2001; Khuri et al. 2001).

In order to examine the expression of Gly-1 transcripts in plants, total RNA from leaf, stem, root, flower, developing peg, developing seeds and post germinating seeds was analyzed by northern blot using labeled probe derived from the Eco RI fragment of peanut Gly1 clone. As expected, the northern blotting detected Gly-1 mRNA band of approximately 1.6 kb. Northern blot results show high level of Gly-1 expression in seeds at immature pod stage (white) and decayed at the brown pod maturity stage (Figure 5). High levels of Gly-1 mRNAs transcripts were detected in immature embryos at white pod maturity stage (15 to 20 days after pod formation), which started declining at yellow-1 pod maturity stage (25 to 30 days old pods). Gly-1 expression continues to decline during yellow-2 (about 35 days old pods) and orange pod maturity stage (40 days old pods). Finally, Gly-1 expression decayed to non-detectable levels at brown and black pod maturity stage (48 days or older pods) (Figure 5). Peanut Gly-1 expression was not detected in mature plant leaves, roots, stems, flower and pegs (Figure 5). Northern blots of post germinating seedlings (0 to 14 days) also demonstrate that peanut Gly-1 does not express.

The cDNA clone of peanut Gly-1 does not have recognition sites for Bam HI, Pst I, Sal I and Xba I restriction enzymes. Several unique restriction enzymes including Bam HI, Pst I, Sal I and Xba I were used to digest the genomic DNA in DNA gel blot analysis study. It is possible that genomic DNA region that transcribes peanut Gly-1 mRNA might be interrupted by one or more intron because at least three introns are present in the soybean glycinin gene family (Nielsen et al. 1989). If introns are present in the genomic region of peanut Gly-1, it is possible that these interrupted regions might have additional restriction sites, and it might generate more than one band upon digestion with Bam HI, Pst I, Sal I or Xba I restriction enzyme. The blot showed one or two dark bands and presence of some lighter bands under high stringency wash conditions. Presence of several lighter bands in the DNA gel blot under high stringency wash condition suggests that the peanut genome contains additional glycinin (arachin) gene(s). Earlier studies with peanut proteins have demonstrated that peanut glycinin is composed of multiple subunits (Krishna and Mitra, 1987). Two other glycinin genes Ara h3 (Rabjohn et al. 1999) and Ara h4 (Kleber-Janke et al. 1999) from peanut have been isolated and characterized for their allergic property. In legumes, the 11S seed storage (legumins/glycinins) genes are the major storage proteins, presence of multiple subunits for these proteins is a common feature (Argos et al. 1985; Shewry et al. 1995) and an individual gene encodes for one subunit (Nielsen et al. 1989). Hence, a small gene family encodes the11S seed storage proteins (Cho et al. 1989; Nielsen et al. 1989). Lighter bands in the peanut gel blot analysis suggest the presence of additional gene/s in the peanut genome with some degree of homology with peanut Gly-1. Isolation and characterization of peanut genomic clones for other glycinin (arachin) genes will be helpful in understanding the complexity of the gene family, peanut genome and specific role of each glycinin subunit.

Comparison of 11S globulin (glycinin or legumin type) seed storage protein genes suggests that there are at least two protein subfamilies, A and B, which are probably of widespread occurrence (Wobus et al. 1986). Both subfamilies share sequence homology and some structural similarities; however, they have divergence that gave rise to two groups that occurred prior to speciation of soybean, pea and broad bean (Nielsen et al. 1989). Three cDNA clones for peanut glycinin protein (Gly-1, Ara h3 and Ara h4) were isolated and submitted to the NCBI gene bank around the same time. Designation this gene as Gly-1 was due to the fact that the PCR product isolated earlier showed high sequence homology with only other glycinin sequences.

The author is grateful to NCBI for creating an invaluable BLAST resource and North Florida Research and Education Center, University of Florida, Quincy for peanut material.

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