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Comptes Rendus

Molecular biology and genetics/Biologie et génétique moléculaires
Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset
Comptes Rendus. Biologies, Volume 337 (2014) no. 2, pp. 86-94.

Résumé

The wild plant species Calotropis procera (C. procera) has many potential applications and beneficial uses in medicine, industry and ornamental field. It also represents an excellent source of genes for drought and salt tolerance. Genes encoding proteins that contain the conserved universal stress protein (USP) domain are known to provide organisms like bacteria, archaea, fungi, protozoa and plants with the ability to respond to a plethora of environmental stresses. However, information on the possible occurrence of Usp in C. procera is not available. In this study, we uncovered and characterized a one-class A Usp-like (UspA-like, NCBI accession No. KC954274) gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. A number of GenBank accessions for Usp sequences were blasted with the recovered de novo assembled contigs. Homology modelling of the deduced amino acids (NCBI accession No. AGT02387) was further carried out using Swiss-Model, accessible via the EXPASY. Superimposition of C. procera USPA-like full sequence model on Thermus thermophilus USP UniProt protein (PDB accession No. Q5SJV7) was constructed using RasMol and Deep-View programs. The functional domains of the novel USPA-like amino acids sequence were identified from the NCBI conserved domain database (CDD) that provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).

Métadonnées
Reçu le :
Accepté le :
Publié le :
DOI : 10.1016/j.crvi.2013.12.008
Mots clés : BLAST, 3D Modeling, GTP-EFTU, Abiotic stress

Ahmed M. Shokry 1, 2 ; Saleh Al-Karim 1 ; Ahmed Ramadan 1, 2 ; Nour Gadallah 1, 3 ; Sanaa G. Al Attas 1 ; Jamal S.M. Sabir 1 ; Sabah M. Hassan 1, 4 ; Magdy A. Madkour 5 ; Ray Bressan 6 ; Magdy Mahfouz 7 ; Ahmed Bahieldin 1, 4

1 Department of Biological Sciences, Faculty of Science, King Abdulaziz University (KAU), P.O. Box 80141, Jeddah 21589, Saudi Arabia
2 Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, Egypt
3 Genetics and Cytology Department, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Egypt
4 Department of Genetics, Faculty of Agriculture, Ain Shams University, Cairo, Egypt
5 Arid Lands Agricultural Research Institute, Ain Shams University, Cairo, Egypt
6 School of Agriculture, Purdue University, West Lafayette, Indiana, USA
7 Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
@article{CRBIOL_2014__337_2_86_0,
     author = {Ahmed M. Shokry and Saleh Al-Karim and Ahmed Ramadan and Nour Gadallah and Sanaa G. Al Attas and Jamal S.M. Sabir and Sabah M. Hassan and Magdy A. Madkour and Ray Bressan and Magdy Mahfouz and Ahmed Bahieldin},
     title = {Detection of a {\protect\emph{Usp}-like} gene in {\protect\emph{Calotropis} procera} plant from the \protect\emph{de novo} assembled genome contigs of the high-throughput sequencing dataset},
     journal = {Comptes Rendus. Biologies},
     pages = {86--94},
     publisher = {Elsevier},
     volume = {337},
     number = {2},
     year = {2014},
     doi = {10.1016/j.crvi.2013.12.008},
     language = {en},
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%0 Journal Article
%A Ahmed M. Shokry
%A Saleh Al-Karim
%A Ahmed Ramadan
%A Nour Gadallah
%A Sanaa G. Al Attas
%A Jamal S.M. Sabir
%A Sabah M. Hassan
%A Magdy A. Madkour
%A Ray Bressan
%A Magdy Mahfouz
%A Ahmed Bahieldin
%T Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset
%J Comptes Rendus. Biologies
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Ahmed M. Shokry; Saleh Al-Karim; Ahmed Ramadan; Nour Gadallah; Sanaa G. Al Attas; Jamal S.M. Sabir; Sabah M. Hassan; Magdy A. Madkour; Ray Bressan; Magdy Mahfouz; Ahmed Bahieldin. Detection of a Usp-like gene in Calotropis procera plant from the de novo assembled genome contigs of the high-throughput sequencing dataset. Comptes Rendus. Biologies, Volume 337 (2014) no. 2, pp. 86-94. doi : 10.1016/j.crvi.2013.12.008. https://comptes-rendus.academie-sciences.fr/biologies/articles/10.1016/j.crvi.2013.12.008/

Version originale du texte intégral

1 Introduction

Plants have developed sophisticated defence mechanisms to deal with diverse unfavourable environmental factors [1,2]. Environmental stresses such as nutrient starvation, drought, high salinity, extreme temperatures and exposure to toxic chemicals can negatively impact agricultural crop yield and quality [3,4]. As an adaptive strategy, plant genomes harbour genes encoding proteins functioning as a response to stress [5–7]. Despite substantial research on responses to abiotic and biotic stresses in plants, there are still knowledge gaps regarding the molecular mechanisms underlying the diverse functions of environmental stress-associated plant genes and proteins [6]. The increasing availability of genomic sequences of members of the Viridiplantae (green algae and land plants) in conjunction with high-throughput bioinformatics tools and datasets provides new insights for examining gene families that could be central to stress responses in plants [5,7]. Genes encoding proteins with the conserved 140–160 residues USP domain (Pfam Accession: PF00582) are known to provide bacteria, archaea, fungi, protozoa, and plants with the ability to respond to different environmental stimuli [8–11]. In Escherichia coli, the Usp genes have been grouped into four classes according to their structure and amino acids sequences. They are Class I (UspA, UspC, UspD), Class II (UspF and UspG), Class III and Class IV (two USP domains of UspE) [12]. The USPA protein domain of MJ0577 (also called 1MJH) from Methanocaldococcus jannaschii crystallizes with a bound ATP, while the USPA domain of Haemophilus influenzae lacks both ATP-binding activity and residues [13]. However, the occurrence and biological significance of USPs in C. procera have not been described.

The Calotropis procera (C. procera) of the family Ascelpiadaceae is a drought-resistant, salt-tolerant wild plant species locally known as “Oshar”, which means “Giant” in English. It is an evergreen poisonous shrub naturally grown in KSA. Through its wind- and animal-dispersed seeds, it quickly becomes established as a weed along degraded roadsides, lagoon edges and in overgrazed native pastures. It has a preference for areas of abandoned cultivation, especially sandy soils with low rainfall [14,15]. It is native to west and east Africa, and south Asia, while naturalized in Australia, Central and Southern America, and the Caribbean island [15–17].

Although C. procera plant is toxic, it has many potential applications and beneficial uses. In medicine, it is both poisonous and health-giver in the same way as digitalis. The aqueous extract of C. procera (latex) inhibits cellular infiltration and affords protection against development of neoplastic changes in transgenic mouse model of hepatocellular carcinoma [18]. The root extract of C. procera has protective activity against carbon tetrachloride-induced liver damage [19]. C. procera latex is also reported to possess interesting activities such as the ability to combat diarrhoea or retard insect larval development [20,21]. Chloroform extract of roots has been reported to possess anti-inflammatory activity [22,23]. Aqueous extract of the flowers was found to exhibit analgesic, antipyretic and anti-inflammatory activity [24]. The alcoholic extracts from different parts were found to possess antimicrobial and spermicidal activities [25,26]. It has also been proven to have anti-fungal properties and can be used effectively in curing fungal diseases of the skin such as athlete's foot and ringworm [27]. Laticifer proteins (LP), recovered from the latex of this medicinal plant, are targets for DNA topoisomerase I that triggers apoptosis in cancer cell lines [28]. Also, C. procera has tannins, latex, rubber and a dye that are used in industrial practices [15]. C. procera has potentiality for bioenergy and biofuel production in semi-arid regions [29]. In ornamental field, C. procera is occasionally grown as an ornamental in dry or coastal areas because of being handsome, of a convenient size, and is easy to propagate and manage. This plant species is also recommended as a host for butterflies.

In this study, we uncovered and characterized a UspA-like gene in this medicinal plant from the de novo assembled genome contigs of the high-throughput sequencing dataset.

2 Materials and methods

2.1 Isolation of total RNA and RNAseq

RNA was the target for sequencing to avoid the presence of intron(s) in assembled sequences. Detection of exact intron sequences generally requires sophisticated problematic approaches. Three samples (reps) of leaf discs of C. procera were frozen in liquid nitrogen (approximately 50 mg tissue each) collected from upper leaves of three independent plants in the Mekka region, KSA, using Trizol (Invitrogen) and treated with RNase-free DNase (Promega). Sampling was done in the midday, as the plants are under heat and drought stresses that might help in the recovery of high-coverage of Usp-responsive transcripts. RNA samples were sent to Beijing Genomics Institute (BGI), Shenzhen, China for deep sequencing and datasets were provided for de novo assembly and bioinformatic analysis.

2.2 Sequence de novo assembly

The raw sequence data were obtained using the Illumina python pipeline v. 1.3. For obtained libraries, only high-quality reads (>20) were retained. Then, de novo assembly of the obtained short pair-end read datasets was performed using assembler Velvet [30] followed by creation of putative unique transcript (PUTs) with a combination of different k-mer lengths and expected coverage. In total, the yielded expressed sequence tag (EST) assemblies were merged into Solanum lycopersicum cDNA, clone: LEFL1037AD03, HTC in leaf (accession No. AK322367.1), where the identity of sequences was over 95% and 40 bp overlapping. This clone is identified as a Usp-like cDNA sequence in which the USP domain is located between bases 200 and 650 [31].

2.3 Basic local alignment search tool (BLAST)

BLAST was used to find regions of local similarity among Usp related sequences. The program was used to compare the recovered nucleotide and deduced amino acids sequences to sequence in the NCBI database, and calculates the statistical significance of matches based on pair-wise alignment method. BLAST was also used to infer functional and evolutionary relationships among the resulted sequences as well as help identifying members of UspA-like gene families (http://www.ncbi.nlm.nih.gov/BLAST).

2.4 AlignX and ClastalW

CLUSTAL [32] is a general purpose multiple sequence alignment program for DNA, RNA or protein. It produces biologically meaningful multiple sequence alignments of divergent sequences and calculates the best match for the selected sequences. Then, it aligns them up so that the identities, similarities and differences can be distinguished. Evolutionary relationships can be viewed via Cladograms or Phylograms. AlignX® Module (rapid multiple sequence alignment with minimal preparation AlignX®) uses a modified CLUSTAL algorithm to generate multiple sequence alignments of either protein or nucleic acid sequences for similarity comparisons and for annotation. The power of AlignX® is in maintaining annotated features within the alignment for easy visualization and localization of regions of interest.

2.5 Determination of phylogenetic relationships

The unweighted pair group method with arithmetic mean (UPGMA) method was used to build a tree where the evolutionary rates are free to differ in different lineages. To evaluate the reliability of the inferred trees, CLC Genomics Workbench (version 3.0) was used to allow the option of doing a bootstrap analysis. A bootstrap value is attached to each branch as a measure of confidence. The GenBank accession numbers for Usp cDNA sequences utilized in this work are shown in Table 1.

Table 1

Accession numbers, description of the genes and organisms with highest similarities to C. procera UspA-like cDNA sequence (accession No. KC954274).

Accession No. Description Organism (Latin name)
NP_566564 Universal stress protein (USP) family Arabidopsis thaliana
NP_566991 Universal stress protein (USP) family Arabidopsis thaliana
XP_002271154 PREDICTED: universal stress A-like protein Vitis vinifera
XP_002275745 PREDICTED: universal stress A-like protein Vitis vinifera
XP_002277653 PREDICTED: universal stress A-like protein isoform 1 Vitis vinifera
XP_002281607 PREDICTED: universal stress A-like protein Vitis vinifera
XP_002316036 Predicted protein Populus trichocarpa
XP_002519217 Conserved hypothetical protein Ricinus communis
XP_002877951 Universal stress protein family Arabidopsis lyrata subsp. Lyrata
XP_002885183 Universal stress protein family Arabidopsis lyrata subsp. Lyrata
XP_003529116 PREDICTED: universal stress A-like protein Glycine max
XP_003547361 PREDICTED: universal stress A-like protein Glycine max
XP_003567271 PREDICTED: universal stress A-like protein Brachypodium distachyon
XP_003568332 PREDICTED: universal stress A-like protein Brachypodium distachyon
XP_003568882 PREDICTED: universal stress A-like protein Brachypodium distachyon
XP_003569311 PREDICTED: universal stress A-like protein Brachypodium distachyon
XP_003594749 Universal stress A-like protein Medicago truncatula
XP_003616191 Universal stress A-like protein Medicago truncatula

2.6 The 3D homology modelling

The 3D homology protein modelling was carried out using Swiss-Model, a protein-modelling server, accessible via the EXPASY (http://www.expasy.org/). Superimposition of C. procera USPA-like predicted protein model on Thermus thermophilus USP UniProt accession No. Q5SJV7 (http://www.uniprot.org/) was constructed using RasMol (http://www.umass.edu/microbio/rasmol/) and Deep-View program (http://spdbv.vital-it.ch/). The functional domains were identified from the NCBI conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml/) and UniProtKB protein knowledge base (http://www.uniprot.org/). Software uses 3D structure information to explicitly define domain boundaries and provide insights into sequence structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).

2.7 Structure alignment

DaliLite was used as the structural alignment method on representative datasets [34–36]. Protein 3D structure comparison depends on the alignment algorithm, the similarity measure, and the fractions of the protein structures considered for the pairwise structure alignment [33]. Model of C. procera USPA-like deduced amino acids sequences was built on the Thermus thermophilus USP UniProt accession No. Q5SJV7. The protein model and its 3D structure were used in pairwise comparison of protein structures using DaliLite program server at EBI (http://www.ebi.ac.uk/Tools/dalilite/) [37] and protein alignment was done to identify conserved and diverse structure domains. Root mean square deviation (RMSD), measuring the average distance between the backbone of superimposed proteins, was detected using DaliLite according to the following formula:

=1ni=1nvixwix2+viywiy2+vizwiz2

3 Results

3.1 Nuclear genome sequence (NGS)

Nuclear genome paired-end short-sequence reads of C. procera cDNAs were generated using the Illumina Genome Analyser IIx (GAIIx) according to manufacturer's instructions (Illumina, San Diego, CA). Assemblies were mapped to the available Arabidopsis thaliana Usp sequence (accession No. NP_112578.4). The resulted C. procera contigs were blasted and the best hit (Vitis vinifera, accession No. XP_002277653.1) was used in a second iteration of mapping. BLAST analysis for the resulted contig was done and a third iteration of mapping was done against the next best hit (Catharanthus roseus accession No. XFD420803) using SAOP [38]. The number of reads aligned was 17 551, with an average coverage of ∼2933. The length of consensus sequence equals 542 nt. ORF analysis showed a full-length ORF with a start codon at bases 48–46 and a stop codon at bases 537–539 (Fig. 1a). The recovered UspA-like cDNA and deduced amino acids sequences were deposited in the NCBI and received the accession Nos. KC954274 and AGT02387, respectively.

Fig. 1

(Colour online) a: ORF restriction map for the obtained Usp-like cDNA sequence (accession No. KC954274). This sequence was characterized by the presence of BglII site (at 340-345 nt) and XhoI (at 376-381 nt); b: USP and GTP-EFTU domains of the deduced amino acids sequence (accession No. AGT02387) (with a length of 489 aa) of the obtained USP-like deduced amino acids sequence as analyzed by Pfam database.

The deduced amino acids sequence (with a length of 489) obtained from ORF analysis was analyzed against the Pfam database conserved domain database and UniProtKB protein knowledge base to locate protein domains. Domain analysis revealed the presence of a universal stress protein domain (conserved domain database accession No. CD00293 and Pfam database accession No. PF00582) (Fig. 1b). Function of this domain was evolutionarily conserved in numerous prokaryotic as well as eukaryotic organisms [9,39]. Generally, Usp genes play an active role in the abiotic stress response, but their function in plant remain largely unknown [40]. Conserved domain analysis also revealed a second domain belonging to elongation factor Tu GTP binding domain (Pfam database accession No. PF00009). The GTP–EFTU binding domain (Fig. 1b) is involved in a conformational change mediated by the hydrolysis of GTP to GDP [41].

3.2 BLAST analysis

BLAST (either protein-protein BLAST or BLASTp) was performed to identify sequence similarity with homologous USP-like amino acids sequences from other organisms. The interpretation of the score and sequence similarity from BLAST searching eventually led to the identification of putative homologous protein sequences. Results for the most closely-related amino acids sequence to C. procera USP-like predicted protein indicated that the PREDICTED: USPA-isoform 1 of Vitis vinifera has the lowest e-value (1 × e−87). These results indicate that the speculated C. procera USP can be a member of USPA subfamily (Table 2).

Table 2

Accession numbers, description (organism latin name) and the calculated e-value of homologous proteins to C. procera USP-like deduced amino acids sequence (accession No. AGT02387) identified using BLASTp search.

Accession Description Total score Query cover (%) E value Max identity (%)
XP_002277653.1 PREDICTED: universal stress A-like protein isoform 1 [Vitis vinifera] 264 99 1.00E-87 79
XP_002519217.1 Conserved hypothetical protein [Ricinus communis] 263 100 2.00E-87 78
XP_002316036.1 Predicted protein [Populus trichocarpa] 261 99 7.00E-87 75
XP_002885183.1 Universal stress protein family [Arabidopsis lyrata subsp. lyrata] 238 98 1.00E-77 72
NP_566564.1 Universal stress protein family [Arabidopsis thaliana] 236 98 7.00E-77 71
XP_003547361.1 PREDICTED: universal stress A-like protein [Glycine max] 233 98 8.00E-76 70
XP_003568882.1 PREDICTED: universal stress A-like protein [Brachypodium distachyon] 215 98 2.00E-68 64
XP_003594749.1 Universal stress A-like protein [Medicago truncatula] 207 98 1.00E-65 63
XP_003569311.1 PREDICTED: universal stress A-like protein [Brachypodium distachyon] 196 100 2.00E-61 59
XP_003568332.1 PREDICTED: universal stress A-like protein [Brachypodium distachyon] 190 98 1.00E-58 59
XP_002275745.1 PREDICTED: universal stress A-like protein [Vitis vinifera] 184 98 2.00E-56 56
XP_002271154.1 PREDICTED: universal stress A-like protein [Vitis vinifera] 184 98 2.00E-56 59
XP_003567271.1 PREDICTED: universal stress A-like protein [Brachypodium distachyon] 180 98 6.00E-55 55
XP_002281607.1 PREDICTED: universal stress A-like protein [Vitis vinifera] 178 98 3.00E-54 56
XP_003529116.1 PREDICTED: universal stress A-like protein [Glycine max] 172 98 8.00E-52 53
XP_002877951.1 Universal stress protein family [Arabidopsis lyrata subsp. lyrata] 171 98 2.00E-51 54
NP_566991.2 Universal stress protein (USP) family [Arabidopsis thaliana] 171 100 2.00E-51 53
XP_003616191.1 Universal stress A-like protein [Medicago truncatula] 171 98 2.00E-51 52

3.3 Multi-sequence alignment (MSA) and phylogenetic analysis

The best BLAST search hits were used to perform multi-sequence alignment. This resulted in 18 sequences originating from seven different plant species. Alignment of these sequences to the newly recovered sequence (accession No. KC954274) was obtained by a gap open penalty of 10 and a gap extension penalty of one. Sequences with more than 50% identity with the obtained C. procera USPA-like deduced amino acids sequence were used (Table 3 and Fig. 2). The results also showed that the closest sequence to that of C. procera USPA-like deduced amino acids sequence is that of Vitis vinifera PREDICTED: universal stress A-like protein isoform 1 (accession No. XP_002277653.1). These results support the obtained BLAST results. MSA results were used to perform phylogenetic tree for the 19 proteins and results (Fig. 3) were similar to those of previous analyses.

Table 3

Pairwise alignment between each USP hit sequences as compared to the obtained sequence of C. procera USPA-like deduced amino acids sequence (accession No. AGT02387).

Accession No. Gaps Differences Differences Identity % Identities
XP_002277653.1 7 47 0.33 71.86 120
XP_002519217.1 6 46 0.33 72.29 120
XP_002316036.1 6 54 0.4 67.47 112
XP_002885183.1 1 53 0.4 67.48 110
NP_566564.1 1 53 0.4 67.48 110
XP_003547361.1 6 56 0.42 66.27 110
XP_003594749.1 13 67 0.53 59.64 99
XP_003569311.1 4 73 0.61 55.21 90
XP_003568882.1 9 73 0.57 57.31 98
XP_002877951.1 8 79 0.66 52.41 87
NP_566991.2 6 79 0.67 51.83 85
XP_002275745.1 10 81 0.67 51.79 87
XP_002281607.1 10 89 0.77 47.02 79
XP_003529116.1 5 79 0.68 51.23 83
XP_003568332.1 7 77 0.63 54.17 91
XP_003567271.1 10 82 0.69 51.19 86
XP_002271154.1 8 81 0.68 51.5 86
XP_003616191.1 10 86 0.73 49.11 83
Fig. 2

(Colour online) Multi-sequence alignment of the 18 USP sequences with the obtained C. procera USPA-like deduced amino acids sequence (accession No. AGT02387).

Fig. 3

(Colour online) Phylogenetic analysis of 18 USP proteins and C. procera USPA-like deduced amino acids sequence (accession No. AGT02387).

3.4 3D structure modelling

USPs exist as homodimers, and genetic studies showed that their cellular assignments are extensive, including functions relating to stress resistance, carbon metabolism, cellular adhesion, motility, and bacterial virulence. Some recent articles suggest that USPs present as heterodimers [42]. Using multi-sequence alignment, we were able to identify important functional domains and motifs within C. procera USPA-like predicted protein sequence (Fig. 4a, b). USP proteins are characterized by the presence of ATP/AMP-binding motif residues indicating that they may be regulated by ATP. Ten ligand-binding sites were predicted based on multi-sequence alignment to the ATP-binding universal stress protein [43]. Table 4 shows a list of predicted proteins that bind to AMP, along with their position and binding sites. Based on structural alignment, a theoretical 3D model for C. procera USPA-like deduced amino acids sequence was created (Fig. 5), corresponding to residues 6-160 of the primary structure. The predicted model was created using the Swiss-Model, protein-modelling server. The overall dimensions of the predicted model are 58.547 Å × 45.719 Å × 30.844 Å.

Fig. 4

(Colour online) Identified cDNA sequence (accession No. KC95274): a: and protein (accession No. AGT02387) motifs (a,b) in the obtained Calotropis USPA-like cDNA and deduced amino acids sequences. USP domain = blue, ATP binding domain = turquoise, ATP/AMP binding residues = underlined, GTP-EFTUD domain = brown, G2XG9XG(S/T) domain = violet box, regions with no superimposition to other closely related USP 3D models = red.

Table 4

Predicted deduced amino acids of the C. procera USPA protein (accession No. AGT02387) that binds to AMP along with their positions and binding sites.

Amino acid Position Binding site
Ala 12 AMP, via carbonyl oxygen
Val 13 AMP, via carbonyl oxygen
Cys 18 AMP, via carbonyl oxygen
Ser 19 AMP
Val 41 AMP, via amide nitrogen and carbonyl oxygen
Gly 129 AMP, via amide nitrogen and carbonyl oxygen
Arg 131 AMP
Ser 143 AMP
Val 144 AMP, via amide nitrogen
Ser 145 AMP
Fig. 5

(Colour online) Theoretical 3D model for C. procera USPA-like deduced amino acids sequence (accession No. AGT02387): a: α-helix, β-sheets and turns; b: calculated surface of Calotropis USPA-like deduced amino acids sequence.

Multi-sequence alignment also showed that residues making contacts with ATP/AMP exist in five domains. The first domain (Ala (A), Val (V) and Asp (D)) is involved in coordinating a Mn2+ ion, which in turn binds to the phosphate groups. This residue is conserved in most of the bacterial and Arabidopsis sequences. The Ile (I) at position 41 of the second domain (Ile (I), Leu (L), Val (V), Val (V) and Ile (I)) binds to adenine with a hydrogen bond. This position is conserved in many of the aligned sequences. The G residue at position 129 of the third domain (Leu (L), Gly (G), ASN (N), Arg (R), Gly (G), Leu (L) and Gly (G)) binds to ribose with a hydrogen bond. This performance exists in nearly all the USPA sequences. Position Arg (R) at position 130 of the same domain binds with a hydrogen bond at the beta phosphate group, which is conserved in all aligned USPA sequences. Ser (S) at position 143 of the fourth domain (Gly (G), Ser (S), Val (V), and Ser (S)) binds to the gamma phosphate group with a hydrogen bond, which is conserved in all the aligned USPA sequences. Also, Ser (S) at position 145 of the same domain binds to the alpha phosphate with a hydrogen bond. This residue is also conserved in all aligned USPA sequences [44]. The G2XG9XG(S/T) domain is also identified in C. procera USPA-like deduced amino acids sequence from bases 129 to 143. All Identified residues and domains are shown in Fig. 4a, b.

3.5 Structure alignment

We used DaliLite to superimpose C. procera USPA-like model on Thermus thermophiles USP protein 3D structures (protein database accession No. 2Z3 V), which was downloaded from protein database. Accession No. 2Z3V is the closest homologous protein sequence with the obtained C. procera USP 3D predicted structure (Fig. 5). To proof the accuracy of our theoretical 3D model of C. procera USPA-like deduced amino acids sequence, we used DaliLite to compute optimal and suboptimal structural alignments between T. thermophiles USP protein and the theoretical 3D model of C. procera USPA-like amino acids sequence. The resulting superimposition is shown in Fig. 6 with Z-score of 26.7, and a number of equivalent residues of 135 and RMSD of 0.3. The figure also shows that 3D model of C. procera USPA deduced amino acids (yellow) has almost the same coordinates of T. thermophiles USP protein (grey) except at two positions, i.e., A (residues 53-70) and B (residues 95–97), which are hyper-variant regions. Regions A and B are located upstream the GTP-EFTU domain in an area that is not involved in any important identified domain. These results support our finding of obtaining a C. procera sequence belonging to USPA subfamily. Also, the results proved the accuracy of our theoretical 3D modelling for C. procera USPA-like deduced amino acids sequence.

Fig. 6

(Colour online) Superimposition between T. thermophiles USP amino acids sequence and the C. procera USPA deduced amino acids sequence (accession No. AGT02387). A & B shows first and second hyper-variant regions. Superimposition of C. procera USPA-like model was constructed on Thermus thermophilus USP UniProt accession No. Q5SJV7 (http://www.uniprot.org/) using RasMol (http://www.umass.edu/microbio/rasmol/) and Deep-View program (http://spdbv.vital-it.ch/).

4 Discussion

The obtained C. procera sequence showed features for a universal stress protein class A (USPA) domain. USPA protein domain whose features determine its function has been evolutionarily conserved in numerous prokaryotic as well as eukaryotic organisms [9,39]. Proteins with the USP domain are known to provide plants with the ability to overexpress and respond to environmental stresses such as nutrient starvation, drought, high salinity, extreme temperatures and exposure to toxic chemicals [45]. These conditions are normal for a desert wild plant, like C. procera. Conserved domain analysis revealed a second domain belonging to elongation factor Tu GTP binding domain (Pfam database accession No. PF00009) [41]. Some evidence showed that a number of EF-Tu protein group members are interacting with some proteins belongs to USP protein family in E. coli [46]. This is the first evidence to show that some USP proteins have EF-Tu activity.

To assign the identified USP protein to its appropriate protein subfamily, several tests were conducted. First, we used BLAST search against Genbank protein database. The interpretation of the score and sequence similarity led to the identification of putative homologous protein sequences. Results showed that the most closely-related gene was USPA-isoform 1 of Vitis vinifera, which showed the lowest e-value (1 × e−87). These results indicate that the speculated C. procera USP protein can be a member of USPA subfamily. The best BLAST search hits were used to perform multi-sequence alignment and 18 sequences were resulted. Alignment of the 19 sequences (Table 3 and Fig. 2) showed that the closest sequence to the obtained C. procera USPA-like amino acids sequence is Vitis vinifera PREDICTED: universal stress A-like protein isoform 1 of accession No. XP_002277653.1. This result also supports the hypothesis that C. procera USP can be a member of USPA subfamily. MSA results were used to perform phylogenetic tree for the 19 USP-like proteins and results (Fig. 3) were similar to those of previous analyses. Multi-sequence alignment also proved that all important functional domains and motifs belong to USP protein family are located within C. procera USPA-like deduced amino acids sequence (Fig. 4a,b). USPs show the presence of ATP/AMP-binding motif residues indicating that they may be regulated by ATP. Table 4 shows a list of predicted amino acids that bind to AMP, along with their positions and binding sites. Multi-sequence alignment also showed that residues making contact with ATP/AMP exist in five domains within C. procera USP-like sequence. These residues are conserved in all aligned sequences [44].

Finally, the 3D Superimposition analysis between T. thermophiles USP amino acids sequence and the constructed C. procera USPA predicted protein has almost the same coordinates except in two positions, i.e., A (residues 53-70) and B (residues 95-97), which are hyper-variant positions. Regions A and B are located upstream the GTP-EFTU domain in an area that is not involved in any important identified domain. These results support our finding of obtaining a C. procera protein sequences belonging to USPA subfamily. Also, the results prove the accuracy of our theoretical 3D modelling for the obtained C. procera USPA-like deduced amino acids sequence. Further study to detect the regulation of this gene under abiotic stress conditions is underway. The gene will also be overexpressed in a model transgenic plant, like tobacco or Arabidopsis, through transgenesis in order to detect the efficacy of introducing this gene in conferring abiotic stress tolerance in plant.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant No. 1432-3-15/HiCi. The authors, therefore, acknowledge with thanks DSR technical and financial support.


Bibliographie

[1] I.E. Somssich MAP kinases and plant defense, Trends Plant Sci., Volume 2 (1997), pp. 406-408

[2] C. Widmann; S. Gibson; M.B. Jarpe; G.L. Johnson Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human, Physiol. Rev., Volume 79 (1999), pp. 143-180

[3] J.R. Porter; M.A. Semenov Crop responses to climatic variation, Philos. Trans. Roy. Soc. Lond. Ser. B. Biol. Sci., Volume 360 (2005), pp. 2021-2035

[4] B. Valliyodan; H.T. Nguyen Understanding regulatory networks and engineering for enhanced drought tolerance in plants, Curr. Opin. Plant Biol., Volume 9 (2006), pp. 189-195

[5] T. Tatusova; B. Smith-White; J. Ostell A collection of plant-specific genomic data and resources at NCBI, Methods Mol. Biol., Volume 406 (2007), pp. 61-87

[6] T. Hirayama; K. Shinozaki Research on plant abiotic stress responses in the post-genome era: past, present and future, Plant J., Volume 61 (2010), pp. 1041-1052

[7] K. Mochida; K. Shinozaki Genomics and bioinformatics resources for crop improvement, Plant Cell Physiol., Volume 51 (2010), pp. 497-523

[8] T. Nystrom; F.C. Neidhardt Cloning, mapping and nucleotide sequencing of a gene encoding a universal stress protein in Escherichia coli, Mol. Microbiol., Volume 6 (1992), pp. 3187-3198

[9] K. Kvint; L. Nachin; A. Diez; T. Nystrom The bacterial universal stress protein: function and regulation, Curr. Opin. Microbiol., Volume 6 (2003), pp. 140-145

[10] D.A. Siegele Universal stress proteins in Escherichia coli, J. Bacteriol., Volume 187 (2005), pp. 6253-6254

[11] O. Persson; A. Valadi; T. Nystrom; A. Farewell Metabolic control of the Escherichia coli universal stress protein response through fructose-6-phosphate, Mol. Microbiol., Volume 65 (2007), pp. 968-978

[12] L. Nachin; U. Nannmark; T. Nystrom Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility, J. Bacteriol., Volume 187 (2005), pp. 6265-6272

[13] M.C. Sousa; D.B. McKay Structure of the universal stress protein of Haemophilus influenza, Structure, Volume 9 (2001), pp. 1135-1141

[14] J.K. Francis Calotropis proceraxxx, U.S. Department of Agriculture, Forest Service, International Institute of Tropical Forestry, Puerto Rico, 2003

[15] C. Orwa; A. Mutua; R. Kindt; R. Jamnadass; A. Simons Agroforestree Database: a tree reference and selection guide version 4. 0, 2009 (http://www.worldagroforestry.org/af/treedb/)

[16] M.A. Rahman; C.C. Wilcock A taxonomic revision of C. procera (Asclepiadaceae), Nord. J. Bot., Volume 11 (1991), pp. 301-308

[17] D. Brandes C. procera procera on Fuerteventura, Working Group for Vegetation Ecology, Institute of Plant Biology, Technical University Braunschweig, Germany, 2005 (Available at: http://www.biblio.tu-bs.de/geobot/fuerte.html)

[18] T. Choedon; G. Mathan; S. Arya; V.L. Kumar; V. Kumar Anticancer and cytotoxic properties of the latex of C. procera procera in a transgenic mouse model of hepatocellular carcinoma, World J. Gastroenterol., Volume 12 (2006), pp. 2517-2522

[19] A. Basu; T. Sen; R.N. Ray; A.K. Nag; Chaudhuri Hepatoprotective effects of C. procera procera root extract on experimental liver damage in animals, Fitoterapia, Volume 63 (1992), pp. 507-514

[20] S. Kumar; S. Dewan; H. Sangraula; V.L. Kumar Anti-diarrhoeal activity of the latex of C. procera procera, J. Ethnopharmacol., Volume 76 (2001), pp. 115-118

[21] T.A. Morsy; M.A. Rahem; K.A. Allam Control of Musa domestica third instar larvae by the latex of C. procera procera (Family: Asclepiadaceae), J. Egypt. Soc. Parasitol., Volume 31 (2001), pp. 107-110

[22] A. Basu; A.K. Chaudhuri Preliminary studies on the anti-inflammatory andanalgesic activities of Calotropis procera root extract, J. Ethnopharmacol., Volume 31 (1991), pp. 319-324

[23] V.L. Kumar; N. Basu Anti-inflammatory activity of the latex of C. procera procera, J. Ethnopharmacol., Volume 44 (1994), pp. 123-125

[24] N. Mascolo; R. Sharma; S.C. Jain; F. Capasso Ethnopharmacology of Calotropis procera flowers, J. Ethnopharmacol., Volume 22 (1988), pp. 211-221

[25] M.A. Qureshi; N.M. Qureshi; R. Arshad; R. Begum A study on the antisperm activity in extracts from different parts of C. procera, Pak. J. Zool., Volume 23 (1991), pp. 161-166

[26] N. Kishore; A.K. Chopra; O. Khan Antimicrobial properties of C. procera procera Ait. in different seasons: a study in vitro, Biol. Mem., Volume 23 (1997), pp. 53-57

[27] F.A. Kuta Anti-fungal effect of C. procera procera stem bark on Epidermophyton flocosum and Trichphyton gypseum, Afr. J. Biotechnol., Volume 7 (2008), pp. 2116-2118

[28] J.S. de Oliveira; D.P. Bezerra; C.D.T. de Freitas; J. Delano; B.M. Filho; M.O. de Moraes; C. Pessoa; L.V. Costa-Lotufo; M.V. Ramos In vitro cytotoxicity against different human cancer cell lines of laticifer proteins of Calotropis procera (Ait.) R. Br., Toxicol, In Vitro, Volume 21 (2007), pp. 1563-1573

[29] J. Garg; A. Kumar Hydrocarbon from plants as renewable source of energy, Bioherald, Volume 1 (2011), pp. 31-35

[30] D.R. Zerbino; E. Birney Velvet: algorithms for de novo short read assembly using de Bruijn graphs, Genome Res., Volume 18 (2008), pp. 821-829

[31] K. Aoki; K. Yano; A. Suzuki; K. Suda; T. Suzuki et al. Large-scale analysis of full-length cDNAs from the tomato (Solanum lycopersicum) cultivar icro-Tom, a reference system for the Solanaceae genomics, BMC Genomics, Volume 11 (2010), p. 210

[32] D.G. Higgins; P.M. Sharp CLUSTAL: a package for performing multiple sequence alignment on a microcomputer, Gene, Volume 73 (1988), pp. 237-244

[33] R. Kolodny; P. Koehl; M. Levitt Comprehensive evaluation of protein structure alignment methods, J. Mol. Biol., Volume 346 (2005), pp. 1173-1188

[34] J. Hou; G.E. Sims; C. Zhang; S.H. Kim A global representation of the protein fold space, Proc. Natl. Acad. Sci. USA, Volume 100 (2002), pp. 2386-2390

[35] R. Day; D.A.C. Beck; R.S. Armen; V. Daggett A consensus view of fold space: Combining SCOP, CATH, and the Dali Domain Dictionary, Protein Sci., Volume 12 (2003), pp. 2150-2160

[36] D. Barthel; J.D. Hirst; J. Blazewicz; E.K. Burke; N. Krasnogor A decision support system for protein (structure) comparison, knowledge, similarity and information, BMC Bioinformatics, Volume 8 (2007), pp. 416-437

[37] L. Holm; S. Kääriäinen; P. Rosenström; A. Schenkel Searching protein structure databases with DaliLite v.3, Bioinformatics, Volume 24 (2008), pp. 2780-2781

[38] R. Li; C. Yu; Y. Li; T.W. Lam; S.M. Yiu; K. Kristiansen; J. Wang SOAP2: an improved ultrafast tool for short read alignment, Bioinformatics, Volume 25 (2009), pp. 1966-1967

[39] R. O’Toole; H.D. Williams Universal stress proteins and Mycobacterium tuberculosis, Res. Microbiol., Volume 154 (2003), pp. 387-392

[40] R. Loukehaich; T. Wang; B. Ouyang; K. Ziaf; H. Li; J. Zhang; Y. Lu; Z. Ye SpUSP, an annexin-interacting universal stress protein, enhances drought tolerance in tomato, J. Exp. Bot., Volume 63 (2012), pp. 5593-5606

[41] W. Moller; A. Schipper; R. Amons A conserved amino acid sequence around Arg-68 of Artemia elongation factor 1 alpha is involved in the binding of guanine nucleotides and aminoacyl transfer RNAs, Biochimie, Volume 69 (1987), pp. 983-989

[42] L. Nachin; L. Brive; K.C. Persson; P. Svensson; T. Nyström Heterodimer formation within universal stress protein classes revealed by an in silico and experimental approach, J. Mol. Biol., Volume 380 (2008), pp. 340-350

[43] R.D. Isokpehi; O. Mahmud; A.N. Mbah; S.S. Simmons; L. Avelar; R.V. Rajnarayanan; U.K. Udensi; W.K. Ayensu; H.H. Cohly; S.D. Brown; C.R. Dates; S.D. Hentz; S.J. Hughes; D.R. Smith-McInnis; C.O. Patterson; J.N. Sims; K.T. Turner; B.S. Williams; M.O. Johnson; T. Adubi; J.V. Mbuh; C.I. Anumudu; G.O. Adeoye; B.N. Thomas; O. Nashiru; G. Oliveira Developmental regulation of genes encoding universal stress proteins in Schistosoma mansoni, Gene Regul. Syst. Biol., Volume 5 (2011), pp. 61-74

[44] D. Kerk; J. Bulgrien; D.W. Smith; M. Gribskov Arabidopsis proteins containing similarity to the universal stress protein domain of bacteria, Plant Physiol., Volume 131 (2003), pp. 1209-1219

[45] R.D. Isokpehi; S.S. Simmons; H.H. Cohly; S.I. Ekunwe; G.B. Begonia; W.K. Ayensu Identification of drought-responsive universal stress proteins in viridiplantae, Bioinform. Biol. Insights, Volume 5 (2011), pp. 41-58

[46] A. Weber; K. Jung Biochemical Properties of UspG, a universal stress protein of Escherichia coli, Biochemistry, Volume 45 (2006), pp. 1620-1628


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