Determination of TaGSK1 gene expression in selected wheat under different salt stresses

TaGSK1 gene was shown to be differentially expressed in salt tolerant wheat plants. This study was conducted to reveal the expression of this gene in four selected wheat cultivated in Iraq. Two wheat cultivars (Dijila and Furat) and one genotype (2H) were grown in three salt levels, 0, 15, 25 ds/m. Meanwhile, cultivar (Tamooze 2) was grown only at zero level salt. Total RNA of the four plants was isolated, cDNA was synthesyzed and PCR amplification was tried. The results showed that a fragment of 189 bp was amplified in the selected salt tolerant wheat but not amplified in control one. In addition, TaGSK1 gene was induced in salt tolerant wheat only and its expression level increased with increasing salt concentration as determined by SYBR green real time PCR. These results indicated the suitablility of TaGSK1 gene as salt-tolerance marker in wheat under study.


INTRODUCTION
Salt stress is a serious environmental factor limiting crop productivity and affects about 20％ of irrigated agricultural land (Flowers and Yeo, 1995). Genes that limit the rate of salt uptake from the soil and the transport of salt throughout the plant, that adjust the ionic and osmotic balance of cells in roots and shoots, and that regulate leaf development and the onset of senescence are considered resbonsible of salinity tolerance (Munns, 2005). RNA finger-printing technique, cDNA-amplified fragment length polymorphism (AFLP) were used to analyze genes that are differentially expressed in both salt tolerance and sensitive with or without salt-stress (Lui et al., 2000). Many genes encoding protein kinases have been shown to be induced under high NaCl conditions (Mizoguchi et al., 1996). TaGSK1 (Triticum asetium L. glycogen synthase kinase-shaggy kinase (TaGSK1)) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was studied. Its was induced by NaCl stress and its expression level was more strongly in salt-tolerant mutant than salt-sensitive genotypes as indicated by Northern blot, suggesting it may be involved in signal transduction of salt-stress in wheat (Chen et al., 2003). In addition, TaGSK1 expression was used as salt tolerance marker in wheat (Bahrami et al., 2009). The aim of this study is estimate the TaGSK1 gene expression using SYBR real time PCR for some selected wheat cultivated in Iraq.

Wheat genotypes cultivation:
Three wheat cultivars and one genotype cultivated in Iraq were used in this research. Seeds of the four wheat plants were washed with tap water for 30 min, immersed in 50% of sodium hypochlorite then treated with 2-3 drops of Tween 20 for 10 min, finally these seeds were washed once with 70% ethanol and several times with distilled sterile water. From each plant, five sterilized seeds were placed in culture bottle containing 15 ml of solidified MS medium with each of 0 ds/m, 15 ds/m and 25 ds/m of salt concentrations (NaCl) without any growth regulators. Each treatment for wheat was replicated thrice. All cultures were performed in 16: 8 (L: D) photoperiod at 25 o C. Growth results were recorded after 15 days and leaves were taken for next steps.

RNA isolation and cDNA synthesis
Total RNA were isolated using Geneaid total RNA purification mini kit (Taiwan) accordingto the manufacturer's instructions. Isolated RNA was treated with RNase-free DNase I (Biobasic, Canada) for 20 min at 37°C, DNase I was inactivated at 65°C for 10 min. The integrity of the RNA was verified after separation by electrophoresis on a 1.5% agarose gel containing 0.5% (v/v) ethidium bromide (How do you validate RNA integrity and purity? PLZ specify). First-strand cDNA was synthesized from 500 ng of total RNA using Reverse Transcription System (Bioneer, Korea) with an oligo-dT 15 primer. Reaction solution was used as template for reverse transcriptase polymerase chain reaction (RT-PCR) of the four wheat plants.

TaGSK1 gene amplification
TaGSK1 (target gene) and wheat housekeeping B-actin (reference gene) cDNA were amplified using primers in Table (1). Polymerase chain reaction was initiated with hot start method using the cDNA template on Labnet Thermo cycler (USA). The PCR reaction was carried out at 95°C for 5 min and 40 cycles at 95°C for 1 min, 60 °C for 45 s and 72°C for 1 min.

Gene expression analysis by SYBR green real-time RT-PCR
The expression of TaGSK1 gene was examined by SYBR real-time RT-PCR using Exicycler real time PCR (Bioneer, Korea). One step RT-PCR was performed using premix RT-PCR qPCR kit (Bioneer, Korea), following the manufacturer's protocol. The thermal cycling profile consisted of initial denaturation at 95°C for 5 min and 40 cycles at 95°C for 1 min, 60°C for 45 s and 72 °C for 1 min, followed with melting curve analysis at 60-95°C. To confirm amplification specificity, the amplified fragments were analyzed by 1.5% agarose gel electrophoresis containing ethidium bromide. Quantitation of relative expression was determined by the 2 −(∆∆CT) method (Livak and Schmittgen, 2001). Each sample was run in triplicate. For estimation of standard curve, online software was used to convert DNA concentration to log copy number (http://www.uri.edu/research/gsc/resources/cndna.html).

Statistical analyses:
Statistical analysis was carried out using MINITAB 11 (Minitab, Inc., Pennsylvania, USA).Analysis of variance (ANOVA) as shown in Tables (6 and 7) was employed to investigate significant differences in the TaGSK1 gene expressions under different NaCl concentrations treatment. Paired t test was calculated to determine the significant differences in the gene expression profile between wheat cultivars (Ramezani 2012).

RESULTS
The results outlined in Table (2) showed that salinity led to a significant decrease in the percentage of germination in Tamooze 2 cultivar. Results also indicated that there was a clear difference among wheat in the percentage of germination and particularly in the second and third levels (Clarify). This indicated that the (Furat and Dijila) cultivars and 2H genotype were more salt-tolerant than the (Tamooze 2) cultivar. Total RNA was isolated, genomic DNA digested with DNaseI and the integrity were estimated by agarose electrophoresis (Fig. 1). RT-PCR results showed that the size of B-actin band was 118 bp and for TaGSK1 was 189 bp (Figs. 2 and 3). Conventional and SYBR green real time PCR were used for amplification of both genes, TaGSK1 and B-actin. Melting curve analysis was used to indicate the presence of target genes and lack of Primer dimer or other non-specific DNA amplifications (Fig. 3). Absolute and relative gene expression of the TaGSK1 gene was estimated using standard curve and reference gene (B-actin) (Figs. 4 and 5).
Standard curve shows slope and R 2 values to determine the efficiency of PCR reaction for TaGSK1 gene amplification (Figs.6 and 7).    Tables (3 and 5) listed the values of CT for B-actin and TaGSK1 genes and referred to the rate of gene expression for each concentration of saline and each sample of wheat studied.
In order to study gene expression, the efficiency of RT-qPCR was determined by using several serial decimal dilutions of eluted PCR bands. The logarithm of gene copy numbers were estimated according to DNA concentrations using the online software DNA copy number (Table, 4): Table 4: CT values and the number of copies of the DNA template of the gene TaGSK1 ------------25

DISCUSSION
There is an extensive evidence for the importance of phosphorylation and dephosphorylation in plant response to salinity stress (Xiong et al., 2002). TaGSK1, a salt-inducible gene was previously shown by several researchers to involve in salt tolerance. Recently, this gene was cloned into Arabidopsis plants which enhanced salt tolerance . The results of this study showed that TaGSK1 gene was expressed in the salt-tolerant cultivars (Dijila and Furat) and the genotype (2H) but not expressed in salt-sensitive cultivar (Tamooze 2). These results are in agreement with the results of (Chen et al., 2003, Bahrami et al., 2009. They indicated the importance of this gene as wheat salt-resistance marker.
The results also showed that TaGSK1 gene expression increased with increase of salt concentration. These results indicated that TaGSK1 gene is very important in salt tolerance as its expression incearesed with salt stress increase (Chen et al., 2003: Bahrami et al., 2009Guang et al., 2011;. The result also indicated that B-actin gene was suitable as a reference gene for the used plant cultivars due to its stablity at specific CT value during SYBR green real time PCR in salt resistance and sensitive wheat plants.
In conclusion, TaGSK1 gene can be used as a salt tolerance marker for Iraq local wheat plants.