Cloning, characterization and expression analysis of a 7S globulin gene in mesocarp of oil palm (Elaeis guineensis Jacq.)

Scientia Horticulturae 143 (2012) 167–175

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Review

Cloning, characterization and expression analysis of a 7S globulin gene in mesocarp of oil palm (Elaeis guineensis Jacq.) Yijun Yuan, Yuanxue Liang, Yusheng Zheng, Li Dongdong ∗ Key Laboratory of Tropic Biological Resources of Ministry of Education, Hainan University, Renmin Road 58, 570228 Haikou, Hainan, China

a r t i c l e

i n f o

Article history: Received 8 March 2012 Received in revised form 28 May 2012 Accepted 29 May 2012 Keywords: Oil palm Mesocarp 7S globulin OPALM7SG1 Real-time PCR

a b s t r a c t Oil palm (Elaeis guineensis Jacq.) is one of the highest oil-yield crops in the world. Based on previous work on suppression subtracted hybridization library construction and analysis between mesocarp and kernel of oil palm fruits, the full-length cDNA of 7S globulin was obtained via rapid-amplification of cDNA ends. The gene, OPALM7SG1 (GenBank accession No. JN896330), encodes a polypeptide of 248 amino acids. The amino acid sequence of 7S globulin showed high homology with the sequence of 7S globulin from oil palm embryos in previous reports, and the amino acid composition of OPALM7SG1 revealed that it contained almost all kinds of amino acids, including high levels of glutamic acid, arginine and serine, and low levels of sulfur-containing amino acids cysteine and methionine. Real-time fluorescent quantitative PCR indicated that OPALM7SG1 was highly expressed in the fourth stage of oil palm mesocarp development (120–125 days after flowering), but remained at relatively low levels in the other four stages. This differential expression of OPALM7SG1 was essential for the accumulation of 7S globulin in the mesocarp of oil palm fruits. The full-length of 7S globulin identified in the present study and the results of its differential expression will be a good foundation for further study on functional analysis and genetic modification of 7S globulin during fruit development of oil palm. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Plant material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. RNA extraction and cDNA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cloning of OPALM7SG1 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Bioinformatics analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Fluorescence quantitative RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Molecular cloning, sequencing and characterization of OPALM7SG1 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Bioinformatic analysis of OPALM7SG1 gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Expression of OPALM7SG1 in mesocarp during fruit development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Oil palm (Elaeis guineensis Jacq.), a member of the monocotyledonous Palmaceae family, is a perennial crop growing in tropical areas, and has become one of the highest oil-yield oil crops in the

∗ Corresponding author. Fax: +86 0898 66279227. E-mail address: [email protected] (L. Dongdong).

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world (Yusof, 2007). Recently, there is an increasing attention on oil palm since the fruit can be widely used in daily life. The major product of oil palm fruit is palm oil, derived from both mesocarp and kernel, which is not only an important source of edible oil, but also a potential biofuel raw material (Corley, 2009). Initially, palm oil was popular as a healthy edible oil. In addition to providing humans with essential fatty acids, unrefined palm oil contains a high level of ␤-carotene which can serve as an important source of vitamin A, and the high content of vitamin E (tocopherols and tocotrienols)

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– a unique feature of palm oil – can act as an antioxidant and antithrombotic, providing several health benefits (Narasinga Rao, 1992). It has also been found that palm kernel oil contains mediumchain fatty acids, which apart from providing energy are effectively hydrolyzed by gastric and pancreatic lipases in the newborn and suckling young, allowing rapid provision of energy for both enterocytes and intermediary hepatic metabolism (Zentek et al., 2011). Now, with the development of the potential value of palm oil and the increasingly worsening global warming and other pollution problems, it had been verified that palm oil has great potential in manufacturing of renewable energy, and it is considered a suitable and attractive candidate as a source of biodiesel compared to other vegetable oils due to its lower cost (Shuit et al., 2009; Tan et al., 2009). Palm oil biodiesel is biodegradable, non-toxic and has significantly fewer emissions than petroleum-based diesel (petro-diesel) when burned (Sumathi et al., 2008). In addition, the palm oil fatty acid distillate, a byproduct of the production of edible palm oil, containing 96% (by weight) of free fatty acids, can be used to produce biodiesel by several different methods (Dos Santos Corrêa et al., 2011). In addition, oil palm is also well known for its other sources of renewable energy, e.g. huge quantities of biomass by-products are developed to produce value added products such as methane gas, bio-plastic, organic acids, bio-compost, plywood, activated carbon and animal feedstock (Sumathi et al., 2008). The pulp protein contains essential amino acids as an important part of a healthy diet, including leucine, valine, isoleucine and phenylalanine at optimal concentrations and kernel proteins contain abundant phenylalanine and valine (Bora et al., 2003). Considering the great potential value of oil palm, in particular its oil and fatty acids, many researchers have shown increasing interest in its fatty acid metabolism and oil synthesis. However, despite being most important storage protein, studies of 7S globulin of oil palm are very limited in comparison to other 7S globulins of other oil crops, which have been more widely reported and have made some advances. Recently, structures, basic properties and some functions of 7S globulins of several plants, e.g. soybean (Glycine ˜ and max; Shutov et al., 2010), sesame (Sesamum indicum L.; Orruno Morgan, 2007), Lupinus albus (Nadal et al., 2011), lima bean (Phaseolus lunatus L.; Chel-Guerrero et al., 2011) and red bean (P. angularis; Tang and Sun, 2011), have been clarified. The basic 7S globulin consists of 27 and 16 kDa chains linked by disulfide bonding, and under the combined temperature-high-pressure treatments, compared with 11S emulsions, 7S emulsions behaved very differently, that 7S globulin was mainly responsible for the global properties of soybean emulsions (Shutov et al., 2010; Puppo et al., 2010). The 7S globulins possess good physicochemical properties of solubility, water-holding capacity, oil-holding capacity and emulsion stability (Chel-Guerrero et al., 2011). In addition to being a storage protein hydrolyzed upon germination to produce a source of nitrogen (N) and carbon (C) for the early stages of seedling growth, 7S globulin can have physiological effects such as significantly reducing cholesterol and triglycerides in plasma and liver, as demonstrated in animal experiments (Duranti et al., 2008; Ferreira Ede et al., 2011; Higgins, 1984). However, 7S globulin is suspected of being an allergen for some people, as reported in the majority of oil crops (e.g. peanut, hazelnut, soy and pea) – probably induce antibodies via different antigen-binding characteristics (Kroghsbo et al., 2011). Moreover, researchers have discovered that a 7S globulin from E. guineensis serves as a novel coconut allergen, in patients allergic to this food, in an experiment for identifying coconut allergens (Benito et al., 2007). There is also some evidence that 7S globulin is involved in signal transduction (Shutov et al., 2010; Watanabe et al., 1994; Yamazaki et al., 2003). Despite previous work on 7S globulin, many critical issues have still not been clarified, especially in aspects of the precise role of 7S globulin in plant metabolism and its

activity. Although a study indicated that soybean genotypes enriched in ␤-conglycinin ␣- and ␣ -subunits were suitable sources of active peptides that inhibit fatty acid synthase activity and lipid accumulation (Gonzalez De Mejia et al., 2010), whether 7S globulin can effect fatty acid synthesis is still not known. It is likely that 7S globulin has other functions, and we are seeking clues for the basis of the former research productions, which will be of great help in further studies. Researchers have attempted to determine the allergen of 7S globulin to increase the use of edible oils (including palm oil) as healthy foods and to eliminate any possible danger to those allergic to it (Kroghsbo et al., 2011). Similarly, more attention is needed on palm oil if it is to be used to produce antihypertensive drugs (Wilson et al., 2005). Therefore, advanced studies are essential to solve these problems and to clarify the role of 7S globulin both at the molecular and cellular levels. In previous research, suppression subtracted hybridization (SSH) was used to identify differential gene expression in the mesocarp and kernel of oil palm fruits. The clones from SSH libraries were identified following differential screening and were sequenced (Xu et al., 2011). Sequence analysis preliminarily revealed the approximate biological features of these fragments – one of which, especially expressed in the mesocarp of oil palm, showed a high similarity to 7S globulin of other plants when BLASTed in the NCBI (National Center for Biotechnology Information) database. To further the study, we intended to obtain the full-length cDNA of the fragments via rapid-amplification of cDNA ends (3 - and 5 -RACE). Then, we aimed to perform bioinformatic analysis of sequences to preliminarily presume characterization and other important biological information. Next, to further peep at the function, we consequently used fluorescent quantitative RT-PCR to compare the expression of five different periods of development of oil palm mesocarp from juvenile stage to complete maturity. The cloning and expression analysis of 7S globulin in this report may provide some new clues for further study on functional analysis and genetic modification of 7S globulin in the development of oil palm.

2. Material and methods 2.1. Plant material Oil palm fruits (E. guineensis Jacq.), at five different developmental stage from juveniles to complete maturity, were collected from plants grown in the field at the Coconut Research Institute, Chinese Agricultural Academy of Tropical Crops, Hainan, PR China. Under field conditions, fruits completed their developmental and ripening process within about 155 days. The whole process can be divided into five periods: 30–35, 60–65, 90–95, 120–125 and 150–155 days after flowering (DAF). All the fruits were harvested at random and mesocarp tissues were physically isolated and immediately frozen in liquid nitrogen to form a sample pool. Each sample was then analyzed in duplicate for RNA concentration, cDNA synthesis, fulllength gene amplification and differential expression analysis.

2.2. RNA extraction and cDNA synthesis Frozen oil palm fruits were ground in liquid nitrogen and total RNAs were extracted from the mesocarp using CTAB methods following Li and Fan (2007). The quantity and quality of isolated total RNA was examined using spectrophotometry and gel electrophoresis, respectively. The total RNA from oil palm mesocarp was used as a template to synthesize cDNA using the TIANScript OneStep RTPCR Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions.

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previous work of our laboratory. Nested PCRs were carried out as follows: 5 -RACE PCR: the reverse-specific nested primer OPALM7SG1RR1 5 -GGCGAAGTCTTTTATTCCAAGG-3 in combination with the 5 -RACE Outer Primer, and OPALM7SG1RR2 5 -CTTTCCCTTCGTTCCATCTC-3 in combination with the 5 -RACE Inner Primer. 3 -RACE PCR: the forward-specific nested primer OPALM7SG1RF1 5 -TGAAGCAGATGGATAGGGTGAC-3 in combination with the 5 -RACE Outer Primer, and OPALM7SG1RF2 5 -TTAGGAGTTGTATGGACCCG-3 in combination with the 5 -RACE Inner Primer. All the RACE products were cloned into pMD18-T vector (TaKaRa, Dalian, China) and sequenced. 2.4. Bioinformatics analysis homology search was conducted based on A BLAST searches using the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST). Amino acid sequence analysis was obtained using ORF Finder (Open Reading Frame Finder) at http://www.ncbi.nlm.nih.gov/gorf/gorf.html. Multiple sequence alignment was done using ClustalW 1.8 (Chenna et al., 2003). Amino acid analysis was calculated using BioEdit 5.0.6. 2.5. Fluorescence quantitative RT-PCR Total RNA from oil palm mesocarp at five different developmental stages were isolated as above. First-stand cDNA was synthesized from 2 ␮g of total RNA using the TIANScript OneStep RT-PCR Kit. Reverse transcription was performed at 42 ◦ C for 60 min with a final denaturation at 70 ◦ C for 15 min. The cDNA was then subjected to real-time fluorescent quantitative RT-PCR using standard methods (Marone et al., 2001). The RT-PCR primers for OPALM7SG1 were designed by the Primer3 program according to the cDNA sequence. The ␤-actin gene was used as an internal control for expression. The primers used in this study were: ␤-actin F: 5 -TGGAAGCTGCTGGAATCCAT-3 , ␤-actin R: 5 -TCCTCCACTGAGCACAACGTT-3 , OPALM7SG1 F: 5 -AGTTGTATGGACCCGAGTGTGA-3 , OPALM7SG1 R: 5 -GGCGAAGTCTTTTATTCCAAGG-3 . RT-PCR amplification was performed using the SYBR® Premix Ex TaqTM II (TaKaRa) and a RT-PCR detector (TaKaRa Smart Cycler II system) by SYBR Green I chimeric fluorescence method following the manufacturer’s instruction. Expression was quantified in terms of comparative threshold cycle (Ct) using the 2–Ct method, and the results were expressed as log 2 of the relative quantity of the normalized gene (Livak and Schmittgen, 2001). Reactions in triplicate, including the no-template and no-reversetranscriptase controls, were monitored with Applied Biosystems 7300 RT-PCR instrumentation outfitted with SDS software version 1.3.1. 3. Results Fig. 1. Full-length of nucleotide sequence (1–831 bp) of the OPALM7SG1 gene and the deduced amino acid sequence of the coding region (ORF) from positions 85 to 831, encoding a polypeptide of 248 amino acids.

2.3. Cloning of OPALM7SG1 gene To obtain the full-length cDNA of oil palm mesocarp OPALM7SG1, 5 - and 3 -RACE PCR was performed using a 5 - and 3 -RACE Kit (TaKaRa, Dalian, China) following the manufacturer’s protocol. The gene-specific primers were designed based on the internal sequenced fragment obtained in

3.1. Molecular cloning, sequencing and characterization of OPALM7SG1 gene To isolate full-length cDNA, 3 - and 5 -RACE were performed using total RNA isolated from the mesocarp of oil palm fruits as template, and the PCR products from the 5 - and 3 -end of the OPALM7SG1 cDNA were cloned into the pMD18-T vector, and then sequenced as described in Section 2. The sequences, including the internally sequenced fragment obtained in previous work of our laboratory, from the overlapping OPALM7SG1 cDNA fragments were

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Fig. 2. Clustal W (Version 1.8) multiple sequence alignment of the amino acid sequences of 7S globulin proteins from different plants. Identities are indicated by asterisks below the sequence. Highly conserved residues are marked with two vertical dots, and less conserved residues with a single dot. Unaligned residues are shown as dashes within the sequence.

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Fig. 2. (Continued)

assembled into a contiguous sequence using the program SeqMan (DNAstar). Therefore, we obtained a 831-bp product containing the full-length cDNA, termed OPALM7SG1 (GenBank accession No. JN896330). Sequencing indicated that the cDNA contained an open reading frame of 747 nucleotides (nt) encoding a polypeptide of 248 amino acids (from positions 85 to 831; Fig. 1). The 7S globulin of oil palm contained a cupin superfamily region, which represents the conserved barrel domain of the cupin superfamily (Dunwell, 1998). 3.2. Bioinformatic analysis of OPALM7SG1 gene The deduced amino acid sequence of the OPALM7SG1 protein was compared with 7S globulins from E. guineensis (AAK28402.1), Ricinus communis (XP 002524752.1), Hordeum vulgare (AAA32936.1), Sorghum bicolor (XP 002464118.1), Pistacia vera (ABO36677.1), Vitis vinifera (CBI28721.3), Zea mays (ACN34339.1) and S. indicum (AAK15089.1) using ClustalW 1.8 (Fig. 2), and a phylogenetic tree (cladogram) of these sequences was generated by multiple alignments (Fig. 3). The results also showed that several residues were highly conserved in the different 7S globulin proteins, along with several differentially conserved substitutions (Fig. 2). In the cladogram, the amino acid sequence of OPALM7SG1 protein was clustered in the same branch as oil palm (E. guineensis) embryos 7S globulin (Morcillo et al., 2001; Fig. 3). The oil palm fruit OPALM7SG1 contained almost all kinds of amino acids (Table 1), including high levels of glutamic acid, arginine and serine, with relatively low level of sulfur (S)-containing amino acids cysteine and methionine.

3.3. Expression of OPALM7SG1 in mesocarp during fruit development RT-PCR analysis was carried out to detect the relative levels of transcription of the OPALM7SG1 gene at five different developmental stages of the mesocarp of oil palm fruit using ␤-actin as an internal control. The transcript levels of OPALM7SG1 in oil palm fruits changed during the five stages. Although OPALM7SG1

Table 1 Amino acid composition of oil palm fruit OPALM7SG1. Amino acid

Residues per 1000

Alanine Cysteine Aspartic acid Glutamic acid Phenylalanine Glycine Histidine Isoleucine Lysine Leucine Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine

40.2 16.1 24.1 128.85 40.2 84.3 28.1 60.2 36.1 56.2 16.1 56.2 36.1 68.3 92.4 88.4 32.1 52.2 8.0 36.1

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100 100

Sorghum bicolor Zea mays Hordeum vulgare OPALM7SG1

100 Elaeis guineensis

Sesamum indicum Vitis vinifera

98

Ricinus communis

65 90

Pistacia vera

0.1 Fig. 3. Cladogram generated by multiple alignments of the OPALM7SG1 protein and 7S globulins from other higher plants using Clustal W (Version 1.8).

transcripts were observed in mesocarp at all five stages, the transcription peaked in the mesocarp of fruits at 120–125 DAF (i.e. the fourth period of fruit development) and, despite having different expression levels, the other four stages were at significantly lower levels compared with the fourth stage (Fig. 4). This result was consistent with a former report about 7S globulin accumulation showing that their accumulation took place mostly at 14–17 weeks after pollination (Morcillo et al., 1998). The level of transcription gradually decreased during mesocarp development (Fig. 4), and the level approached zero during the third stage. 4. Discussion In this report, we described the cloning, nucleotide sequence, expression pattern of the OPALM7SG1 gene from the mesocarp of oil palm. Compared with other plant 7S globulins, high overall amino acid similarity and a number of different conserved regions in the OPALM7SG1 protein, reflected the likely identity of the clone. The derived polypeptide sequences of the OPALM7SG1 protein contains a cupin domain, belonging to the cupin superfamily, and the seed storage protein 7S globulin has previously been verified as a cupin protein, classified as a two-domain bicupin (Dunwell, 1998). The cupin superfamily, named on the basis of a conserved ␤-barrel fold (‘cupa’ is the Latin term for a small barrel), is a group of functionally diverse proteins found in all three kingdoms of life: Archaea, Eubacteria and Eukaryota (Khuri et al., 2001). The cupin superfamily of proteins was initially limited to several higher plant

Fig. 4. RT-PCR analysis of the OPALM7SG1 gene expressed in mesocarp at five different developmental stages of oil palm.

proteins such as seed storage proteins, germin (an oxalate oxidase), germin-like proteins and auxin-binding protein. Recent advances have shown that although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence (Stipanuk et al., 2011). One diagnostic feature for discriminating between the various classes of cupin is whether the conserved domain occurs singly, in proteins such as transcription regulators and phosphomannose isomerases or in a duplicated form within proteins such as oxalate decarboxylases and seed storage proteins (Khuri et al., 2001). Cupins have been identified in some 18 different functional classes that range from single-domain bacterial enzymes, such as isomerases and epimerases involved in the modification of cell wall carbohydrates, through to two-domain bicupins such as the desiccation-tolerant seed storage globulins, and multidomain transcription factors, including one linked to the nodulation response in legumes. Related research also revealed that the largest subset of the cupin superfamily is the 2-oxyglutarateFe2+ dependent dioxygenases, the substrates for which are many and varied and in total amount to probably 50–100 different biochemical reactions, including several involved in plant growth and development (Dunwell et al., 2001, 2004). Being a member of the cupin superfamily, 7S globulin may be involved in the growth and development of the plant and be important for further study, which should be further clarified. Moreover, the deduced amino acid sequence of the protein encoded by OPALM7SG1 showed significant homology to known or suspected 7S globulins from different plants, including that of E. guineensis oil palm embryos as previously reported (Morcillo et al., 2001). The storage proteins of oil palm fruits have been extracted, purified and characterized in previous work, which demonstrated that the predominant 7S globulins are heterogeneous oligomers (Morcillo et al., 1997). The results of the present study were identical with previous results and indicated that the OPALM7SG1 protein was another 7S globulin of oil palm fruits, and especially expressed in the mesocarp. The 7S globulins accumulate in large quantities as the main storage proteins in the seed or fruit of various plants, in particular oil crops, e.g. soybean, peanut and R. communis. Similarly, 7S globulins are the predominant storage protein group in oil palm zygotic embryos (Morcillo et al., 1997). Due to its high nutritional value in the human diet, 7S globulin has been widely analyzed biochemically, and that of soybean has had the most research of all plants (Shutov et al., 2010). The kinds and contents of various amino acids composing 7S globulin will become highly valued as they are increasingly recognized as beneficial to human health and nutrient requirements. Amino acid analysis of OPALM7SG1 showed

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that it contained high levels of glutamic acid, arginine and serine, which in addition to being essential amino acids are very important in the human body. Arginine functions in the human body as a free amino acid, a component of most proteins, and the substrate for several non-protein, N-containing compounds, many of which function in immunity (Nieves and Langkamp-Henken, 2002). Early on there was considerable evidence found suggesting that glutamic acid plays a significant role in cognitive behavior, and that it clearly affects intelligence (Vogel et al., 1966). Recently, the identification of glutamic acid residues with critical roles in IgE-binding to Ric c 3 and Ric c 1 support the potential use of free amino acids in allergy treatment (Deus-de-Oliveira et al., 2011). Nevertheless, OPALM7SG1 contained relatively low levels of sulfur (S)-containing amino acids cysteine and methionine. It has been reported that the amino acid composition of 7S globulins in embryos of oil palm is broadly similar to those of the 7S proteins of other monocotyledon embryos, but differs from those of the legume 7S vicilins (Morcillo et al., 1997). However, there is much evidence to suggest that both 7S globulins have several similarities, with one of the most outstanding characteristics being that they both contain limited amounts of methionine and cysteine, which are important nutritional components of protein meal (Khattab et al., 2009). The 7S globulins from pea, common bean (P. vulgaris L.), tepary bean (P. acutifolius var. lactifolius) and soybean are characterized by a relative deficiency in S amino acids (Carbonaro, 2006; Gatel, 1994; Panthee et al., 2004; Sathe et al., 1994). The S available in the human diet is derived almost exclusively from proteins, and yet only two of the 20 amino acids normally present in proteins contain methionine and cysteine (Nimni et al., 2007), which are the principal S-containing amino acids, and so have received increasing attention in recent times (Brosnan and Brosnan, 2006). S is an essential element for the entire biological kingdom because of its incorporation into amino acids, proteins and other biomolecules, and is also important in the iron-containing flavoenzymes. Unlike humans, plants can use inorganic S to synthesize S-containing amino acids, and therefore are an important source of S for humans (Atmaca, 2004). Some researchers have made progress in determining the functions and metabolic pathways of the two S-containing amino acids – they are considered to be metabolized by a variety of reactions and pathways to at least two dozen intermediates and products, and some of these metabolites serve functions essential for survival of the organism (Griffith, 1987). In addition, the S amino acids are increasingly critical and attractive due to the increased understanding of their relationship to chronic diseases (e.g. cardiovascular disease, dementia and cirrhosis), immunomodulation, DNA transcription and RNA translation (Fukagawa, 2006). In consideration of the importance and value of essential amino acids, many researchers aim to improve the content of some amino acids more essential to human health by modifying the structure and subunits of 7S globulin. One of these aims is improving the proportion of S-containing amino acids, because 7S globulin contains very high levels of essential amino acids but limited amounts of S amino acids (Carbonaro, 2006; Slightom and Chee, 1987). Fortunately, some advances in plant gene transfer technologies allow molecular biologists to correct the S amino acid deficiency inherent in storage proteins of oil palm and legumes (Slightom and Chee, 1987). Therefore, genetic engineering is highly suitable to change the molecular structure of 7S globulin, based on the explicit 7S globulin sequence and other important characterizations. Although neither the structure nor the function of 7S globulin has been fully elucidated, as a storage protein 7S globulin is a source of N and C for the early stages of seedling growth (Duranti et al., 2008; Higgins, 1984; Kim et al., 2011). Many types of seeds are adequate in one nutritional aspect but inadequate in others, and genetic engineering provides the opportunity to use the beneficial traits of certain seeds to ameliorate the negative aspects of others – and there has

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been some progress in transgenic expression of seed storage proteins to improve nutrient quality (Holding and Larkins, 2008). The 7S globulin structure also provides a basis for proposing engineered mutations of these proteins with the goal of enhancing their nutritional and functional properties (Lawrence et al., 1994). However, such studies of the 7S globulin of oil palm are few and this area needs more attention to further research. Oil palm fruits have a high nutritional value, and are extensively used in the food and pharmaceutical industries as a crude material. For instance, since 7S globulin plays an effective role in reducing cholesterol and triglycerides in the plasma and liver (Ferreira Ede et al., 2011), we should first confirm the role of 7S globulin in this effect, and then optimize the structure or modify the gene to attain a high yield of the active constituent – which could then be used to produce cholesterol-lowering medicines on a large scale. In addition, oil palm produces high yields of oil, the ingredients of palm oil is rich and specific, and these characteristics make it more healthy and beneficial to people compared to other oil crops. Recently there has been increasing attention on determining the allergen of 7S globulin and developing a clear understanding of the biological mechanisms. However, the 7S globulin of oil palm contains a novel allergen (Benito et al., 2007) and this will restrain broad application of oil palm in the food and pharmaceutical industries. With the rapid development of molecular biology, immunology and genetic engineering, increasing numbers of effective and suitable methods are being rapidly developed. Genetic engineering is one of the most popular means used by researchers in studying reduction of allergenic seed components (Holding and Larkins, 2008). Thus we may seek the allergen subunit and remove or denature it with the premise of no influence on the other important properties of oil palm 7S globulin. Similarly, molecular-level studies may benefit knowledge of other allergens of different plants. As a seed storage protein, 7S globulin will be hydrolyzed for the early stage of plant development and so a large amount of 7S globulin will accumulate in the fruit as it matures (Duranti et al., 2008; Higgins, 1984). There was a rapidly increasing expression of OPALM7SG1 in oil palm mesocarp from the third to fourth stages, which reverted to a low level at the fifth stage. This significant change of expression of OPALM7SG1 indicated that a mass of 7S globulin, encoded by OPALM7SG1, was synthesized and retained in the mesocarp of fruit in the fourth stage (120–125 DAF). Previous work suggested that 7S globulin plays roles in signal transduction because it is capable of binding bovine insulin and insulin-like growth factors, and is localized in the middle lamella of cell walls and plasma membranes (Watanabe et al., 1994; Yamazaki et al., 2003; Nishizawa et al., 1994). The 7S globulin was specifically expressed in mesocarp during fruit development and maturation because it is involved in metabolic mechanisms that occur only in the mesocarp, e.g. synthesis of oil peculiar to mesocarp or other pathways. It is well known that 7S globulin is a major storage protein that accumulates in significant quantity, and is rapidly hydrolyzed upon germination to produce a source of N and C for the early stages of seedling growth (Duranti et al., 2008; Higgins, 1984). Thus it seems that a significant augmentation of transcription occurred in the fourth stage, and that the change in expression of OPALM7SG1 was tightly correlated with the accumulation of 7S globulin during fruit development. Further studies will be required to ascertain whether the 7S globulin affects fatty acid metabolism or other pathways. Although many functions of 7S globulin have been suspected and presumed, no experiments have so far demonstrated this. How the protein accumulates and the regulation of the OPALM7SG1 gene is also not understood. Recently, many storage proteins of higher plants, especially oil crops, have been cloned and sequenced. To our knowledge, this is the first study on the OPALM7SG1 gene specifically expressed in the mesocarp of oil palm fruit. The protein produced differs to the 7S

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globulins of E. guineensis that have previously been reported, which were derived from both zygotic and somatic embryos (Morcillo et al., 2001), although they all have high similarity. The OPALM7SG1 gene may be involved in the process of mesocarp development and shows high research value for further studies of oil palm 7S globulins. The cloning and sequencing of other 7S globulin genes will provide new markers for studies on globulin biogenesis and fruit development of oil palm, although neither the structure nor the function of 7S globulin has yet been fully characterized. In summary, much more work dedicated to plant 7S globulins is needed in future – on both structure and function.

Acknowledgements This research was supported by the National Natural Science Foundation of China (NSFC) (No. 31160717 and 31060259), and partially supported by the State key subject of Botany at Hainan University (071001).

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