to environmental cues
to environmental cues. However it had been proved that the PA which resulting from the DGK pathway could be distinguished from the PA derived from PLD pathway, due to its differential 32Pi-labeling characteristics and the composition of their fatty acid, the PA production was amplified under osmotic stress 3. Even if several enzymes which involve in the PA pathway have been characterized, such as PLC, PIPLC, NPC, DGK, and PLD, the relationships between PA pathway and any stresses remains unclear. These researches prompted a curiosity the roles of DGK gene in Soybean. Whereas The 12 DGK gene in soybean were divided into three distinct clusters, the GmDGKs were slightly comparable in number to the DGK gene in Arabidopsis and rice, but less than the 10 DGK found in mammalian species which members of DGK families fall into five subtypes, I through V 32. GenBank numbers were described for DGK homologues from tomato (AW035995), maize (AY106320), wheat (BT009326), a Populous cross (BU828590), grape (CB981130), and apricot (Prunus armeniaca; CB821694) 10. These investigations confirm that, DGK were widely distributed in plants, and that a better understanding of their functions could for a better dissection of the PA pathway. We noted that the three genes in Cluster I, were most complex GmDGKs in Soybean, were very similar to other plants cluster I such as Rice, Apple, Maize, and Arabidopsis and in addition, the most complex DGKs in plants are closest to DGKs Type III, which are the most primitives DGKs in mammalian cells 3. Unlike plants DGK genes, many studies have been conducted on mammals DGK genes, and different result indicate that the mammalian DGK genes are more advanced and have diversified functions. In plants, all genes have two conserved domains; a conserved catalytic DGK kinase domain, and an accessory conserve DGK domain accessory domain. But in the cluster I genes, in addition to these two domains all genes in Cluster I harbor 2 proteins kinase C conserved region 1 (C1) domains that could be in charge of binding DAG, and a predicted trans-membrane helix that targets these DGKs to the membrane 34, the pivotal characteristics of some genes in cluster III is the presence of calmodulin-binding domain (CBD) 26-10. As other gene of the Cluster I of our phylogenetic tree, Soybean harbor two replicas of the DAG/PE-binding domain (C1), each containing a C6/H2- type core whose alignment sequences differ slightly from each other, and that the first C1 domain is bound by an upstream basic region and the second is directly followed by an extCRD-like sequence 31 by comparing the structures of the DGK genes between the Soybean and Arabidopsis in cluster II and III, we found that the GmDGKs in cluster II were very similar to those in cluster III, both in terms of conserved domains and in terms of their exon/intron organization. This similarity between genes of different species suggest that they would come from the same ancestor, and that the DGK have been strongly affected by the repetitive phenomenon of DNA duplication during the evolution process over time. Also the undeniable role of the DGK in the PA accumulation process under the cold stress had been updated by recent research 37-36 We monitored the expression patterns of 10 GmDGK genes, using qRT-PCR to identify their transcript levels in leaves and roots and we found that, except GmDGK1 which was smoothly affected after the stress induction, the expression transcript of the nine others GmDGKs were dramatically affected in the leaves. And that in the root all the GmDGKs expression level were smoothly affected under the drought stress, the salt treatments. Therefore, according to our results with these four stress treatments, we could provide the basics for further investigations about GmDGKs functioning by cluster members, stress responsive element of the DGKs genes in soybean. Finally, expression varied among genes in response to each stress treatment, even though all soybean DGKs could contain ABREs in their promoters. These modifications of the DGK genes transcript under the application of various stresses, reveal the activities of the DGK genes and their likely impact on the processes of adaptation and resistance to abiotic stress in soybeans. And the DGK gene expression could be modulated by complex mechanisms based on the discoveries that the promoter regions of those genes contained many cis-elements that are correlated with responses to abiotic stresses
4. MATERIALS AND METHODS
4.1. Localization and identification of SOYBEAN DGK genes on chromosomes
The census of the DGK gene was done with an online search. We used the full name of the “diacylglycerol kinase” to find soybean DGK sequences on SOYBASE (https://soybase.org). Then we blasted all the soybean DGK sequences on the database (http: //www.phytozome. org/) and we used genes ID found on Phytozomev9.1 as the keywords in browser tool on the NCBI website (http://www.ncbi.nlm.nih.gov/Structure/ cdd/wrpsb.cgi) to confirm their position on the chromosome. Furthermore the DGK gene sequences of Arabidopsis thaliana were collected on the online Arabidopsis platform Resource TAIR 10.0, Oryza sativa DGK sequences were found on RGAP (Rice Genome Annotation Project), and finally, we recovered the locational data of all putative genes from Computational Biology Web Resources (http://genomics.research.iasma.it/gb2/gbrowse/apple/). Then we used the Microsoft PowerPoint 2013 to sketch all DGK genes on their respective chromosomes
4.2- Domain analysis and the phylogenetic analysis of soybean DGK genes
The soybean DGK genes characterization were implemented by analyzing their structures and the construction of their phylogenetic tree. The multiple sequences alignments of DGK protein sequences were established with the DNAMAN program, on the default parameter mode. The evolutionary relationship amount plants DGK genes was found by collecting the proteins sequences of four plants; Arabidopsis, rice, soybean and apple from the NCBI protein database. Then we used the integrated MUSCLE alignment program in MEGA6.0 software to align all protein sequences, using the neighbor-joining (NJ) method and 1000 replications for the bootstrap test 27.
4.3. Functional domain analysis and Phylogenetic relationship of GmDGK proteins
To establish the multiple sequences alignment of all plant DGKs which was downloaded from the Phytozome protein database (https://phytozome.jgi.doe.gov), we used DNAMAN program to perform this work, and then we used the DOG 2.0 software to draw the schematic diagram of the functional motifs in all soybean DGKs gene. The alignment of DGK gene sequences of different plants species was also realized by MEGA6.0 software (Hall 2013). And then the neighbor-joining method of MEGA6.0 was used to create a phylogenetic tree, and the bootstrap test was applied with 1000 replicates for each node 31.
4.4. Predictions of subcellular localization and Promoter element analysis for soybean DGK genes
The DNA sequences of 1500 bp upstream of the start codon of all soybean DGK genes were downloaded from Phytozome V11.0, and were used to search the databases from PLACE 2 (http://www.dna.affrc.go.jp/PLACE/sig nalscan.html) 13 and PlantCare (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/) 19 website. The presence of various cis-elements in the promoter regions of GmDGKs were predicted. (Table 3)
4.5. Plant materials and stress treatments
Glycine max, Williams 82 were chosen as a model plant for Soybean. We selected good seeds, and we grow them in pots hydroponic to insure a better growth to our plants. For that, we made a nutrient Hoagland’s solution with the following composition; 235 ppm K, 200 ppm Ca, 0.5 ppm B 31 ppm P, 64 ppm S, 48 ppm Mg, 0.5 ppm Mn, 0.05 ppm Zn, 0.02 ppm Cu, 210 ppm N, 0.01 ppm Mo and 1 to 5 ppm Fe. We constantly kept plants under a 12 h light/12 h dark cycle, at 25°C, and on 70% of humidity, also, the nutrient solution was replaced every 48 hours. After 14 days, when our plants reached at the four leaves stage we divided them into 5 different groups, then we transferred 4 of them into the various stress solutions, PEG (8% PEG8000), alkali (100 mM NaHCO3), salt (110 mM NaCl), and saline+alkali (70 mM NaCl + 50 mM NaHCO3) for 0, 1, 3, 6, 9, 12 h, and 24h. The fifth group were kept growing in Hoagland’s solution without any stress and used as the plant control. Finally, the collection of the samples (roots and leaves) were done at about 11 AM and jealously preserved at ?80°C for further usages.
4.6. RNA extraction, cDNA synthesis, and expression analysis of selected SOYBEAN DGK genes
After the collection of different samples (roots and leaves) we separately extracted the total RNA from all the collected tissues. We used the Trizol reagent (Invitrogen, Carlsbad, CA, USA) with its following manufacturer’s protocols, then after each RNA extraction, we checked the concentration and quality of the using a NanoDrop 2000 (ThermoFisher Scientific, Beijing, China). And then each cDNA was synthesized from 2 ?g of the +corresponding RNA using the PrimeScript RT reagent kit (Takara, Japan). In total more than 320 samples of soybean RNA were extracted and jealously preserved at -80 C.
4.7. Quantitative real-time PCR and Statistical analysis
This surgery stated with the selection of the reference genes for the qRT-PCR; Actin11 for leaves and EF1A as the reference gene for the roots tissues. And then the primer designing for all GmDGKs, and a better illustration we used the Primer Premier 5 software as are shown in Table1. For expression analysis of the GmDGK gene under abiotic stresses, we performed an RT-PCR on a Stratagene Mx3000P thermocycler (Agilent) with the following settings: 95°C for 15 s; 40 cycles of 95°C for 15s; and annealing at 58°C for 30 s. Here emphases that each reaction was performed in triplicates. The expression level of each gene in different was calculated using the 2???Ct methods for abiotic treatments and the 2?Ct method for different tissues. Then we used the SPSS statistics 17.0 to make the statistical analysis. So to confirm the significance of our data, we used the Tukey’s student zed range (H SD) test, to compare variations of all stress treatments against their respective control and among the gene expression in different tissues. The significant level was ? = 0.05
In conclusion, at the end of this study, we identified 12 GmDGK genes from the soybean genome, which were distributed on 6 of the 20 soybean genome. The C conversed domain and phylogenetic analyses were carried out, and verified similarities and evolutionary relationships among all soybean DGK genes. Furthermore the collected information here will enhance our understanding of the roles of the DGK gene family in soybean, the expression profiles analysis of the soybean DGK genes under drought stress were inspected throughout the development and under various stresses, and based on qRT-PCR results, we bring out serious candidates for further exploration of their function activities such as GmDGK2, GmDGK4, GmDGK7 and GmDGK10. all these results reveal a possible rules of these DGK genes in different processes of soybean plants adaptation to abiotic stress, especially to drought stress, because des transcript levels varied following the exposure to drought, salt, alkali, and salt-alkali stresses. Thus for future research, we could update the fundamental roles of diacylglycerol kinase in the molecular mechanism of adaptive responses to drought stress in soybean, this will deliver more opportunities to use GmDGK genes for rootstock breeding and improving the resistance of some rootstocks. And together with cis-regulatory elements analysis, our results suggest that soybean DGK genes are involved in various cellular processes. Moreover, and subcellular localization