Seed proteins are an extremely and increasingly important component of nutrition for both humans and animals. Plant proteins are the cheapest available sources of protein in many countries where meat and/or dairy industries are not well developed or a distribution network of meat or dairy products is lacking. Besides, grain legumes or pulses they are also an important source of protein, particularly in countries where the diet is predominantly vegetarian, hence an important part of human diet. Plant protein is considered an ideal source of protein to humans without the concern of cholesterol and legumes have been well documented for their high seed storage protein contents.

Peanut, Arachis hypogaea, a legume, is commercially grown in many subtropical and tropical regions of the world and is endemic to south-eastern regions of the United States. Peanut is rich in oil (50-60%) and protein (24%) and is a major source of plant protein in most tropical and subtropical regions of the world. Peanuts are the third most important source of plant protein and provide approximately 11% of the world's protein supply.  The protein fraction is classically divided into water-soluble albumins and the water-insoluble globulins. Of these two, globulin is the major storage protein in various seeds. The globulin fraction of peanut seed consists of arachin (glycinin or legumin) and con-arachin (vicilin or con-glycinin). Of the globulins, glycinin (arachin) accounts for more than 50% of the total protein. Glycinin is superior to con-glycinin from the viewpoint of nutritional value as well as functional properties. Immunological studies also demonstrate that glycinin type seed storage peanut proteins (such as Ara h3 and Ara h4) are less severe compared to their vicilin (Ara h1) and con-glycinin (Ara h2) type seed storage proteins.

Recently, peanut allergy is a major health concern because of the severity of the allergic reaction. Therefore, one of the goals is to tailor peanuts to make them allergy free or reduce severity of allergic reaction through genetic engineering without changing the biological characteristics or nutritional value. It is obvious, that it is important to understand the biosynthesis, targeting and biological functions of seed proteins as a prerequisite to their rational manipulation in improving nutritional value. However, such information is still scarce for the seed storage proteins of peanut. Therefore, molecular studies are needed to characterize peanut seed storage proteins.

To this end, peanuts, cultivar Florunner, were grown in experimental plots, which normally start pegging about 80 days after planting. Pods of different maturity were collected from these plants by digging at weekly intervals for 9 weeks between 90 and 160 days. Pods were harvested and separated into six maturity categories. Pod maturity can be determined by scrapping the pods shell and hull colour. White shell colour indicates immature pods whereas colour changes as pods matures to yellow-1, yellow-2, orange, brown and black, which represents fully mature pods. Pods with similar maturity stage were pooled together, thoroughly washed with sterile water after surface sterilized with 2% bleach and dried with paper towels. Pods were shelled, seed coats removed and seeds from similar maturity were frozen immediately in liquid nitrogen and stored at -80ºC until used. Other vegetative tissues such as developed leaf, stem, root, flowers and pegs were collected between 75-85 days after germination. For post-germinating seedlings, mature peanuts were sterilized with detergent and bleach followed by washing the seeds several times with distilled water. Sterilized seeds were germinated at room temperature 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.

Forward and reverse degenerate oligonucleotide primers were designed and synthesized based on the conserved region of glycinin protein sequences of soybean and broad bean. Glycinin protein sequences from peanut were amplified using Polymerase Chain Reaction (PCR) method. The resulting PCR product of 350 base pair was of expected size based on the position of forward and reverse primers on amino acid sequence of glycinin protein. The PCR fragment was purified from the gel, cloned and sequenced. Sequence information revealed that it shares a high degree of nucleotide homology with glycinin and legumin seed storage protein.

To isolate the full-length gene from peanut for the amplified PCR fragment, complimentary DNA (cDNA) library was synthesized using RNA extracted from seeds of yellow-1 pod maturity. The PCR fragment was radiolabelled and used as a probe to screen cDNA library by plaque hybridization. Three cDNA 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 1.05 and 0.75 kb cDNA clones were truncated versions of 1.8 kb cDNA clone. The isolation of multiple independent cDNAs that represent the same mRNA suggests that the corresponding mRNA is abundant in immature seeds. The 1.8 kb clone was found to contain a complete gene as compared to other genes and designated as peanut Gly-1 (peanut glycinin-1) clone. Peanut Gly-1 clone was characterized for several characteristics and structural similarity with conserved domains of other glycinin genes. The predicted protein has 529 amino acid residues, with a calculated molecular weight of 60,447.61 Daltons and an isoelectric point of 5.489. The hydropathy profile of the peanut Gly-1 protein shows the presence of two hydrophobic regions which is long enough to span a membrane bilayer.

Peanut Gly-1 shows high homology with two other peanut glycinin clones - Ara h3 and Ara h4. Peanut Gly-1 nucleotide sequence shows 67 and 69% identity with soybean glycinin gene (Gly-3 and Gly-2 respectively). At present, the primary sequence of glycinin gene is known for a variety of legumes including soybean, broad bean and pea. 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. 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 central region of peanut glycinin protein shows three regions of variability like other legumin/glycinin proteins. 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 hyper-variable region (HVR). The second region of variability was referred to as variable region 1 (VR1), which has extended runs of glutamine and arginine that are not observed in other legumin-like proteins. The third region of internal heterogeneity, termed as variable region 2 (VR2) that tends to be rich in arginine and glutamine, a characteristic of the other variable regions.

The conserved domain analysis reveals that peanut Gly-1 has a bi-cupin domain that comprises two conserved motifs, each corresponding to two β-strands separated by a less conserved region. The N-terminal motif consists of 169 residues while the C-terminal motif consists of 145 residues. Both the N-terminal and C-terminal motifs of peanut gly-1 have high levels of similarity with cupin domain. Peanut Gly-1 clone belongs to 00190 Pfam family of 11S plant seed storage protein and contains a single metal-binding site. Structural similarity of glycinin protein 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.

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 where the total RNA was separated on a denaturing agarose gel and transferred onto a nitrocellulose membrane. Membrane pieces were hybridized with the radiolabelled cDNA clone as probe. After overnight hybridization blots were washed and exposed to X-ray film. High level of Gly-1 expression 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). Peanut Gly-1 expression was not detected in mature plant leaves, roots, stems, flower, and pegs and during seed germination. DNA gel blot analysis of peanut Gly-1 as probe suggests that the peanut genome contains additional glycinin (arachin) gene(s). 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. Hence, a small gene family encodes the 11S seed storage proteins. 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. Three cDNA clones for peanut glycinin protein (Gly-1, Ara h3 and Ara h4) have been isolated and known so far. Characterization of peanut Gly-1 seed storage protein will facilitate genetic engineering attempts to improve quality because manipulations in the three regions of variability (VR1 or VR2 or HVR) might not adversely affect the biological property or health of the peanut plant as these regions are diverge among the glycinin protein among legumes. 

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