3.1. RWC
Generally, RWC is assumed as the most alternating measure of water shortage stress resistance in plants in terms of cellular dehydration. The abundance of RWC of all the three cultivars enhanced significantly upon varying in water contents (Fig. 1). The maximum value of RWC abundance (a mean of 96 %) were obtained under control condition (100 % field capacity). Unlike, under the three levels of water shortage stress, as expected, the lower amount of RWC were remarkably decreased (p<0.01), declining to its minimum level in severe drought stress (25 % FC). Regarding difference between control group and W1 level, no significant ratio was detected, but for W2 and W3 levels a number of reductions (p< 0.01) were detected (Fig. 1), indicating that W2 and W2 could be noticed as notable water stress levels. The highest ratio of RWC in cultivar 1 under the last level of water shortage stress (W3) as compared to the two other cultivars, represent that this cultivar is much more resistant to water deficit status than cultivar 2 and 3.
3.1. Cloning of the full-length cDNA of ObGAPDH
A 671 bp ObGAPDH fragment, as consensus sequence amplified was obtained using degenerate oligonucleotide PCR primers of a ObGAPDH cDNA, which alignment analysis exhibited most identity to the GAPDH disclosed in different plant species (data not shown). The cDNA 3′ end was obtained from the fragment of plant GAPDH genes with higher identity by RACE-PCR. The full length of ObGAPDH gene was amplified by PCR with different numbers of gene specific primers based on amplified conserved partial sequence and highly conserved sequence of 5′ end GAPDH gene. The full-length GAPDH cDNA sequence was acquired from O. basilicum (GenBank accession no. KU375117.1). It had a 1315 bp complete CDS with a poly (A), contains a 1200 bp ORF encoding a 399-amino acid protein.
3.2. Analysis of the predicted protein sequence and phylogenic analysis
Blast P online as homology tool (http://www.ncbi.nlm.nih.gov/BLAST) and Clustal W result showed considerable homology to other plant GAPDH. It was found that the deduced ObGAPDH amino acid sequence presented 95.49 %, 94.24 %, 93.73 %, 91.48 %, 90.07 %, and 89.78 % identity to Salvia splendens, Sesamum indicum, Paulownia fortune, Buddleja alternifolia, Nicotiana benthamiana, and Erythranthe guttata, respectively (Fig. 2).
A Neighbor-joining phylogenic tree was created for additional recognized the relationship between the protein sequence of ObGAPDH and known GAPDH amino acid sequences of other plants by MEGA 6.0 (Fig. 3). The phylogenetic tree was divided into two groups and was found ObGAPDH associated with Salvia splendens and then to Sesamum indicum with identities 95.42 %, and 94.24 %, respectively (Fig. 3).
For analyzing physicochemical properties such as pI, molecular weight, etc. of the ObGAPDH amino acid sequence, ScanSite and ProtParam tools (http://www.Expasy.ch/tools/protparam.html) was used. The ObGAPDH protein molecular formula, molecular mass, and molecular weight was predicted to be C1877H3012N522O581S11, 42.54 kDa, and 6.01 computed by the pI/MW online tool. Instability index of the protein has been calculated 24.35, indicating ObGAPDH is a stable protein (instability parameter <40 considered for stable proteins). Because of the presence of methionine at the N-terminal end of ObGAPDH, the protein half-life in in vitro condition was computed 30 h. Also, aliphatic index, as a main agent in distinguishing the protein stable to heat was obtained 94.01. The hydrophobicity pattern was illustrated by ProtScale with Kyteand Doolittle method (Kyte and Doolittle 1982).The highest score was 2.144 for valine in position 280 and the lowest score was -2.433 for glycine in position 382 that are associated with the strongest and weakest condition of hydrophilicity. The hydrophilic and hydrophobic domains were showed below and above the zero line, respectively (Fig. 4). Results of SOPMA tool for prediction of the protein secondary structure revealed that predicted ObGAPDH protein mainly consisted of 129 α-helix (32.33 %), 99 extended strand (24.81 %), 27 β-turn (6.77 %), and 144 random coil (36.09 %) (Fig. 5). The results of Plant-mPLoc server, Prosite, and Target P to predict of subcellular localization revealed at least one chloroplastic signal peptide in ObGAPDH protein. TMHMM tool showed no transmembrane helix at the ObGAPDH protein. The results of HMM scan showed ObGAPDH protein has functional domain contain glyceraldehyde 3-phosphate dehydrogenase with two accession numbers PF02800.20 and PF00044, which are C-terminal and NAD binding domains, respectively.
Moreover, the predicted amino acid sequence had conserved consensus active site, NADB_Rossmann domain (GXGXXG) to GAPDH activities conserved in different plant species. Also, the multiple alignment results showed that Gp_dh_C domain as the C-terminal area of ObGAPDH protein sequence revealed the maximum identity in length and composition of protein sequence. The results of MotifScan server showed that the protein sequence of ObGAPDH had 10 functional domains includes of AMIDATION (Amidation site), ASN_GLYCOSYLATION (N-glycosylation site), CAMP_PHOSPHO_SITE (cAMP- and cGMP-dependent protein kinase phosphorylation site), PKC_PHOSPHO_SITE (Protein kinase C phosphorylation site), E4PD_MF_01640 (D-erythrose-4-phosphate dehydrogenase), DapB_N (Dihydrodipicolinate reductase, N-terminus), GFO_IDH_MocA (Oxidoreductase family, NAD-binding Rossmann fold), Gp_dh_C (Glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain), Gp_dh_N (Glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain), and FARP (FMRFamide related peptide family).
3.3. Analysis of codon usage and miRNA-targeted ObGAPDH
The codon usage of cDNA as a marker in molecular characterization of ObGAPDH was performed. The results showed the ENC was 42.11 in ObGAPDH. However, in overall, the ENC range is between 21 to 61, which the lower the value, indicates the use of only one codon decoded the amino acid, and vice versa (De Mandal et al. 2020). The value of codon bias index (CBI) is reported to be between zero and one and for ObGAPDH was 0.49. The RSCU value was utilized to recognition overrepresented and underrepresented codons for each amino acid (Fig 6). The higher RSCU value exhibits a greater frequency. Schi2 is the discrepancy between the quantity of observed codons and the anticipated codon of the same amino acids. This value for ObGAPDH was 0.52 that the mean of value. The percentages of GC and GC in third position of codon were 0.57 and 70, respectively.
19 miRNA-targeted ObGAPDH that 18 of them regulated gene expression in cleavage site was found, but results showed only mtr-miR2089-5p can be a regulated agent of gene expression at translating level. So, these two actions can alter amounts of gene levels after that transcription. Also, the miRNA interaction to ObGAPDH was illustrated in Fig 7.
3.4. Predicted tertiary structure, molecular docking and gene ontology of ObGAPDH
Tertiary structure of the deduced ObGAPDH protein sequence was surveyed using I-TASSER online server using homology procedure. Results revealed that among models, the best model with estimated RMSD (8.6±4.5A○), S-core (-0.76), and estimated TM-Score (0.61±0.14) was selected (Fig. 8a). This model had 0.85 % coverage and 0.66 % identity with template (6ghrB), in which it contains 117 (29 %) strands, 193 (49 %) coils, 56 (14 %) turn, and 88 (21 %) helices. The selected model was measured according to the Ramachandran plot, in which 67.9 %, 23.1 %, and 4.3 % of residue were in the core, allowed, and generous areas, respectively and also 4.6 % of these were placed in the external area.
Molecular docking results showed the most appropriate protein interaction and pose is belonged to the first ligand binding pose with the lowest energy -31.6258 in docking procedure. Afterwards, this pose submitted to survey the detailed interaction between ligand and protein (Fig. 8b), which contains four amino acids with distinct positions Try 245, Glu 330, Pro 301 and Asn 302, with hydrogen inaction and ester bond.
Gene ontology results revealed that ObGAPDH had the phosphorylation activity (NADP-dependent glyceraldehyde-3-phosphate dehydrogenase), an important role, as high statically level of molecular function verified this feature (Fig. 9). This result confirms the consequences of experiments which derived from in silico and in vivo conditions. As previous studies showed, the protein with 0.5 statically level is sufficient to mediate glucose metabolic process, reductive pentose phosphate cycle, response to temperature stimulation, response to chemical material, responses to stress, response to abiotic condition and, oxidation–reduction process, as exactly mention in different resources.
3.5. Transcription profile of ObGAPDH
In order to discover a preliminary insight into the potential function of GAPDH in basil under drought stress, the expression ratio of ObGAPDH were examined at various levels of drought stress using qRT-PCR. The transcript level of actin gene as the reference gene was put for normalization the transcription level under water shortage stress. As Fig. 10 is demonstrated, the transcript ratio of ObGAPDH boosted at the level of water shortage stress (W1; 4.14-fold), and slightly enhanced 5.32- and 5.34 times in W2 level and increased sharply ~6.28- and 6.34-fold change upon severe shortage water treatment level in both Cultivar. 1 and 3, respectively. Regarding Cultivar 2, the transcription level of ObGAPDH was slowly increased during the first, second, and third levels of drought treatment (W1, W2, and W3; up to 5.52, 5.38, and 5.68-fold change).