Abstract: In Arabidopsis thaliana expression of two Desiccation-Responsive rd29 genes are intensity induced under drought, salinity and temperature (heat and cold) conditions. These two genes called, rd29A and rd29B are located in 8-9 Kb of Arabidopsis genome. Also, transgenic plants containing these genes have more tolerant toward environmental stresses. Since rd29A has an important role in various non-biological stresses, in this study genetic and promoter analysis of this gene was performed. rd29A nucleotide sequence was obtained from NCBI gene bank, for bioinformatic analysis used the Map viewer, Plant care and Genevestigator software. In Arabidopsis rd29A has 8048 nucleotides and encode a linear mRNA with 2133bp. It located at AT5G52310 locus on chromosome 5 between nucleotides 21241898 and 21242457 and contains 4 exons and 3 introns. Promoter region analysis to determine the gene regulatory elements performed with using 1500 bp of the 5 'UTR sequence and the results indicated that the existence of diverse regulatory elements such as ABRE, DRE, Box I, C-repeat/DRE and TCA-element which represent involvement of this gene in different physiological pathways in plants. In addition the TATA-Box was identified as Core promoter in -30 position. Also, the pattern of gene expression was examined by Microarrays data and it was found that the expression levels were variable in different developmental stages, so that the lowest and highest expressions were in germination and mature stages. It seems that using of this gene promoter in expression cassettes can raise tolerance of transgenic plants to environmental stresses.
INTRODUCTION
Abiotic stresses conditions such as drought, high salinity and temperature stress (low or high) are the major environmental factors to limit growth and agricultural productivity (Modarresi et al., 2012, 2013). Plants have number of physiological and biochemical responses which can reduce stress corresponding damage. Molecular studies have been shown that the expression of nine genes associated with these stresses and responsive to dehydration induced (Yamaguchi-Shinozaki and Shinozaki, 1994). The rd29 (Responsive to Desiccation 29) is one of the nine RD genes of Arabidopsis thaliana, which isolated from this plant under drought stress condition. The rd29 is induced by dehydration stress very quickly (within 20 min) and strongly (within 3 hours), and this induction is a two-phase process Extensive restriction analysis has revealed that rd29 includes two genes, rd29A and rd29B (Yamaguchi-Shinozaki and Shinozaki 1993b). rd29 sequences are used as markers to monitor stress-response pathways in plants because its exquisitely sensitive to various abiotic stress and differently change in the response to drought, cold and freezing, salt stress, or exposure to ABA. Due to the unique elemental combinations, rd29A and rd29B are differentially induced under abiotic stress conditions; the promoter of rd29A was found to be more responsive to drought and temperature stresses, since the promoter of rd29B was highly responsive to salt stress (Msanne et al., 2011).
Arabidopsis thaliana is a model plant growing in Europe and Asia. Arabidopsis thaliana has a relatively small genome with approximately 135 Mega base pairs (Mbp) that widely used for genetically studies since it has short life cycle and small size (Miller et al., 2010).
Transgenic plants containing these genes (rd29A and rd29B) also are more resistant to environmental stresses. Transformation and expression of the rd29A promoter caused improving plant tolerance to drouht and salinity stresses, thus in this research investigated bioinformatics analysis of rd29A gene in Arabidopsis thaliana as a model plant.
MATERIALS AND METHODS
Gene location and locus number of rd29A gene from Arabidopsis thaliana obtained from PHYTOZOME site available at (www.phytozome.net). For determination gene regulatory elements, used 1500 bp of the 5' UTR for promoter region analysis by Place (http://www.dna. affrc.go.jp/PLACE/) and Plant care (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html) programs, then motifs extracting in the promoter region and examined their function. Also gene expression pattern analyzed by Genevestigator program (www.genevestigator.com) and Microarray data. rd29A protein domains determined by SMART (www.smart.embl-heidelberg.de) and STRING (http://string90.embl.de/), then genetic network designed. Protein Haplotype diversity analyzed by DnaSP software. Phylogenetic relationships of this protein performed by MEGA 5.0 software and the Neighbor-joining method.
RESULTS
The rd29A nucleotide sequence obtained from NCBI gene bank, also used Map viewer, Plant care and Genevestigator software for bioinformatics analysis. Arabidopsis thaliana rd29A gene has 8048 nucleotides, that encoding 2133 bp linear mRNA in locus AT5G52310 on chromosome 5 between nucleotides 21241898 and 21242457. This gene has 4 exons and 3 introns. Using 1500 bp of the 5' UTR promoter region analysis to determine the gene regulatory elements performed and the results indicate that existence of diverse regulatory elements such as ABRE (cis-acting element in the response to stress of various non-biological), DRE (Effective in response to drought, cold and salinity with (TACCGACAT) 9 bp sequence), Box I (element responds to light), C-repeat/DRE (in response to cold and drought stress) and TCA-element (in response to salicylic acid) which represents involvement of these genes in different physiological pathways in plants (Table 1). In addition identified TATA Box as Core promoter in -30 position. ProtParam (http://web.expasy.org/protparam/) analysis indicated that rd29A is a protein with 710 amino acids and 77.885 KDa molecular weight and pI = 4.45. Total number of negatively (Asp+Glu) and positively (Arg+Lys) charged residues are 168 and 78 respectively. This protein has 10650 atoms with C3342H5172N912O1215S9 formula. The Instability Index (II) is computed to be 53.32; this classifies the protein as unstable. Also rd29A aliphatic index is 48.82 and Grand average of hydropathicity (GRAVY) is -1.087. Uniprot (http://www.uniprot.org/) data indicated that this protein has repeated sequence of 2 X 14 AA repeats of P-[MV]-G-F-G-[DS]-E-S-G-A-E-L-E-K in region 303-370, 3 X 15 AA repeats of [DN]-[FS]-P-[STV]-R-S-H-[DE]-[FL]-D-[LM]-K-[NT]-E-[ST] in region 317-412, 5 X 5 AA repeats of [FV]-[ADT]-[EST]-[KM]-L in region 510-600, 2 X 23 AA repeats in region 648-696, 5 Asp and 4 Gly as compositional bias in position 63-67 and 638-341 respectively.
| Table 1: | Arabidopsis thaliana promoter region motifs |
| Fig. 1: | Codon usage for rd29A gene in Arabidopsis thaliana, Brassica oleracea, Thellungiella halophila, Craterostigma plantigineum and Glycine max |
DnaSP analysis for 5 rd29A from different species shown that for this gene singleton variable sites (two variants) is 135, Number of haplotypes is 5, Variance of haplotype diversity is 0.01600, average number of nucleotide differences is 706.600, nucleotide diversity is 0.69275 and conserved regions are ATGGADYHRMVRNDDVHHYVHHSWNNHVDBV in position 1-31 and KVRAVVHMKMWKKKSBKSMRMA SSDRARDCCYRKDDRHGDHGASRMRKYWSHWKM KRAKWWWWYTHCDGMVRVHDDWVATRWBRWR RARRHWGMRHCWSHKVWRRVDRR in position 1271-1379. Also codon usage for all amino acid indicated in Fig. 1.
Protein alignment shows similarity and homology among species. Results indicated that Arabidopsis thaliana rd29A has high identity with corresponding genes in Arabidopsis lyrata, Thellungiella halophila and Brassica oleracea (Fig. 2). Also phylogenetic tree shown that this gene has more relative with Arabidopsis lyrata, Thellungiella halophila and Cupressus sempervirens (Fig. 3). Protein interaction defined by SMART program and investigated that rd29A as LTI78 (low-temperature-induced 78); cold regulated gene, the 5' area of cor78 has cis-acting regulatory elements that can impart cold-regulated gene expression (Fig. 4).
DISCUSSION
Environmental factors such as drought, salinity and cold stresses reduce plant yield and production. The rd29A is stress inducible gene which not expressed in normal and optimal conditions. Therefore in this research promoter and CDs regions of this gene analyzed. Results indicated that some stress inducible transcription factors and cis-acting regulatory elements such as HSE, MBS, TC-rich repeats, etc exist in rd29A promoter region, this phenomenon explain this gene expression under stress conditions. The promoters of rd29A and rd29B, which are 60.93% identical and the 1640 bp spacer region between rd29B and rd29A is thought to contain cis-acting elements responsible for the induction of rd29A by environmental stress and ABA. These observations suggest that the rd29A promoter region include at least two types of independent cis-acting elements, one which is responsive to changes in osmotic potential (dehydration-responsive element) and is independent of ABA and another which is, perhaps, involved in ABA-responsive (ABA-responsive element) gene expression (Yamaguchi-Shinozaki and Shinozaki, 1993a). By contrast, the rd29B promoter does not appear to contain cis-acting elements that are involved in the initial rapid induction caused by changes in osmotic potential.
Protein sequence analysis performed for finding motifs and functional domains. Also, Jia et al. (2012) reported that the middle region of rd29A is a sequence of 112 amino acids, composed of two tandemly repeated sequences. A second tandemly repeated sequence of 21 amino acids is located near the C-terminal. Both of these repeats are absent in rd29B. Two acidic regions and one basic region can be found in the N-terminal regions of rd29A and rd29B.
| Fig. 2: | Multiple alignment of rd29A protein among Arabidopsis thaliana and other species |
| Fig. 3: | Phylogenetic tree of rd29A genes from various species |
| Fig. 4: | The rd29A (COR78) predicted protein interactions |
Our data analysis by STRING shown that rd24A has predicted protein interactions with KIN2; Encodes a gene that can be overexpressed by cold and abscisic acid and may be involved in temperature acclimation and salt tolerance, ERD10 (Early Responsive to Dehydration 10); actin binding; Encodes a gene induced by cold, freezing and dehydration, COR47 (Cold-Regulated 47); Belongs to the dehydrin protein family, amino acid homology with LEA II proteins (Late Embryogenesis Abundant proteins), cold regulated and responds gene to osmotic stress, abscisic acid, drought, etc. (Bihmidine et al., 2013; Narusaka et al., 2003).
CONCLUSION
Tolerant to environmental stress is important for preserving food production in 21th century. Finding and transformating genes to improve adaptability of major crops such as wheat and rice are critical to adverse tensions environments. Experiments demonstrated that rd29 genes expression increased in stress conditions. We analyzed rd29A gene and promoter and found some elementary regions and transcription factors related to stress condition. Generally, our results suggest that this gene is a good candidate for transformation into sensitive plants against different abiotic stress such as drought and temperature stresses.