Isolation and semi quantitative PCR of Na+/H+ antiporter (SOS1& NHX) genes under salinity stress in Kochia scoparia

Kochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread. environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na+ concentrations. Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na+/H+ antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na+ concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts. Coding sequence of plasma membrane Na +/H + antiporter (SOS1) and vacuole membrane Na /+H + antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologue in salicornia brachiata and suadea maritima, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control. The results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops.


Introduction
Most of studies have revealed that the greatest lost in various crop production is due to abiotic stresses, such as, salinity, water deficit, low temperature and heavy metals adversely affect the growth and several physiological processes such as leaf cell growth and biomass production of plants. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Processes such as seed germination, seedling growth and vigor, vegetative growth, flowering and fruit set are adversely affected by high salt concentration, ultimately causing yield lost. Salinity stress can reduce the productivity of glycophytes, which are the majority of agricultural products. High salt concentrations cause hyper osmotic stress and ion imbalance in plants which often as a secondary effect leads to oxidative damage in cellular components (Qadir, Tubeileh et al. 2008). Plants adapt to environmental stresses via responses, including the activation of molecular networks that regulate stress perception, signal transduction and the expression of both stress related genes and metabolites (Huang, Ma et al. 2012). Plants have stress specific adaptive responses as well as responses which protect the plants from more than one environmental stress (Huang, Ma et al. 2012). Plants employ three different strategies to prevent and adapt to high Na + concentrations: 1) active Na + efflux, 2) Na + compartmentalization in vacuoles, and 3) Na+ influx prevention (Niu, Bressan et al. 1995, Rajendran, Tester et al. 2009). Antiporters are important groups of genes that have a key role in ion homeostasis in plants. Na + /H + antiporters (NHX1 and SOS1) maintain the appropriate concentration of ions in the cytosol, thereby minimizing cytotoxicity. NHX1 are located in tonoplasts and reduce cytosolic Na + concentration by pumping it in the vacuole (Gaxiola, Rao et al. 1999), whereas SOS1 is localized at the plasma membrane and extrudes Na+ in apoplasts (Shi, Quintero et al. 2002). Both of these antiporters are driven by a motive proton force generated by the H + -ATPase (Blumwald, Aharon et al. 2000). The SOS signaling pathway consists of three major proteins including: SOS1, SOS2, and SOS3. SOS1, which encodes a plasma membrane Na + /H + antiporter, is essential in regulating Na + efflux at the cellular level. It also facilitates long distance transportation of Na + from root to shoot. Over expression of this protein leads to salt tolerance in plants (Shi, Ishitani et al. 2000). Activation of SOS1 by direct phosphorylation of the self-regulation scope is possible by serine/threonine protein kinas or SOS2 that requires calcium binding protein or SOS3 ( K.scoparia, a dicotyledonous erect annual herb belongs to Amaranthaceae family with high genetic diversity and great foliage potential (Mullinex 1998 Borrelli and Izzo, 2000). The aim of this study was to investigate the presence of SOS1 and NHX1 genes and trace it using by induced salt stress in Kochia scoparia, Futures of these genes in protein structure characterized with in silico tools. Furthermore profiling gene expression for two gene charecterized. K.scoparia is an attractive plant model for study the mechanism of salt tolerance. This work to gain insights into the role played by this transporter in K.scoparia halophyte.  all specific primers designed based on the most conservative parts of the alignments. specific forward and reverse primers were designed ( Table 2).

RNA isolation and cDNA amplification in K.scoparia
Total

Results and Discussions
In this research, isolation of the coding sequence of plasma membrane Na + /H + antiporter (SOS1) and vacuolar membrane Na + /H + exchanger (NHX) in Kochia scparia was performed and, the consequence of salinity stress was studied on the expression profile of this gene. We focused on SOS1 and NHX the critical genes in the SOS pathway and vacuolar membrane for the resistant to salt stress (Fig. 1). The SOS pathway and vacuolar membrane Na + /H + exchanger (NHX) are currently the most extensively studied mechanisms in controlling the salt stress response in plants. The SOS and vacuolar membrane Na + /H + exchanger (NHX) pathway is responsible for ion homeostasis and salt tolerance in plants.

K.scoparia
After sequencing the coding SOS1 and NHX genes sequences in K.scoparia, Conserved domain specified using of NCBI revealed that putative protein SOS1 belongs to the Maximum identity up to90% with Suadeamaritimaof NHX gene and for SOS1 gene is 84% by Salicorniabrachiata.

Effects of salinity stress on SOS1 and NHX genes expression profiles
Studies have identified salt tolerance determinants in organisms ranging from cyanobacteria to fungi and from algae to higher plants. Research with halophytic species has provided information on adaptive behavior but information on the molecular level is still Insufficient.
Furthermore, information related to salt tolerance of K.scoparia at molecular level is insufficient. In this study we tried to be focused on the analysis of isolation, characterization and gene expression pattern of key genes involved in salinity tolerance in halophytes species such as K.scoparia. Gene expression profile for SOS1 and NHX were checked in 48hours after treatment with 0, 150, 300 mMNaCl. In this study, we found a basal level of SOS1 and NHX in K.scoparia without salt stress, which is regulated with salt treatments. Gene expression Profile for SOS1 and NHX in K.scoparia shoot parts showed that salinization was affected SOS1 and NHX levels positively and positive correlation with salinity levels. In other words K.scoparia compared to control like most halophytes leaves are progressively increased under all salinity stress. Amounts of mRNA increased for SOS1 gene: 1.5 and 2.5 and NHX gene: 1 and 2 times higher than the control (0mM) in 150 and 300mM stressed plants after 48 hours of exposure respectively (Fig.3). While amounts of mRNA increased for SOS1 gene and NHX in root plant but less than the increase in leaves, 1 and 2 times for SOS1 , 0.5 and 1 fold higher than the control in 150 and 300mM treated plants (Fig.3). Samplings were carried out at 24 hours after treatments with 0, 150, 300 mM salt stress.

Prediction of SOS1 antiporter using Bioinformatic tools
For prediction binding of Cyclic nucleotide to cyclic nucleotide-binding domain to SOS1 protein isolated from K.scoparia: First, the tertiary structure of the desired protein domain was predicted (Automated Mode, The pipeline will automatically identify suitable templates based on Blast (Altschul, Gish et al. 1990) and HHblits (Remmert, Biegert et al. 2012).
Cyclic nucleotide-binding domain in these proteins has a For prediction binding of Cyclic nucleotide to cyclic nucleotide-binding domain to SOS1 protein isolated from K.scoparia: First, the tertiary structure of the desired protein domain was predicted (Automated Mode, The pipeline will automatically identify suitable templates based on Blast and HHblits.
Cyclic nucleotide-binding domain in these proteins has a common structure with 120 amino acids. The domain consists of three alpha helixes and eight turn structures that form a porelike structure. Three protected glycine amino acids seem important to maintain the barrel-

Discussion
Studies have identified salt tolerance determinants in organisms ranging from cyanobacteria to fungi and from algae to higher plants. In plant cell maintain a high K + /Na + in the cytoplasm, under normal conditions. Under salt stress conditions plants have several strategies and adaptive mechanisms for tolerant to these conditions. In these mechanisms, to be launched sensing, signal transduction, gene expression and metabolic pathways. Evidence may be these tolerance progarms slow and steady adapatation in the sensitives plants.
Therefore, understanding the components of these mechanisms in halophytes can do contribute substantially to improving retrofitting sensitive plants.We focused on isolation, characterization and gene expression pattern analysis of main genes involved in salinity tolerance in halophytes from K.scoparia. In the present study, the relatively high basal expression level of V-NHX indicated the important physiological function of NHX in K.scoparia, even in the absence of stress NHX levels positively have correlation with salinity levels. In other words, compared to control K.scoparia like most halophytes leaves progressively increases under salinity stress. Amounts of mRNA increased for NHX gene: 1 and 2 times higher than the control (0mM) in 150 and 300mM stressed plants after 48 h of exposure respectively (Fig.4) ,The higher NHX1 expression in the leaves was a prompt response to NaCl treatment which could have helped decrease the Na + content in the . Furthermore SOS1 has been demonstrated to be a target of SOS pathway, releationships between SOS1 and SOS2/SOS3 can be a way of regulating the activity of SOS1. Ceratin domains of SOS1 reacted with SOS2/SOS3 that charectrization of these domains can be helped to the use of this protein in process will creat resistance plants. To provide factors will be affected the SOS1 activity and to determine suitable methods to activate these proteins in Glycophyt plant. On the other NHX exchanger acts as a mediator of K + taransport between cytosol and vacoule, SOS2 also activates the vacuolar-ATPase and vacuolar Na + /K + antiporter NHX exchangers, which compartmentalizations Na + /K + into vacuoles. K + compartmentalization in the vacuole coulad result in a cytosolic K + deficiency (Zhang et al., 2014). So determine how to communicate this antiporters can be controlled out ways of increasing salt tolerance to be properly.
Sequencing of SOS2 and prediction of interact with NHX can be used for controling of SOS pathway.

Conclusion
In the my study SOS1 and NHX genes sequenced and determind Proteins characteristics with insilico tools. Charecterization of other genes involved these pathways and signaling pathway and investigation invitro of proteins are apromising area of research that may lead to improvments in the biomass production of crop with external applications materials and genetic manipulation.