Characterization Of A Wheat (triticum Aestivum L.) Gene Tasnrk2.8 Involed In Abiotic Stress Responses

Drought, salinity and extreme temperatures are major factors limiting the yield and quality of wheat (Triticum aestivum L.). To survive adverse stresses, plants have developed complex signaling networks to perceive external stimuli, and then manifest adaptive responses at molecular and physiological levels. Reversible protein phosphorylation is central to the perception of, and response to, stress conditions. Sucrose non-fermenting1-related protein kinase 2 (SnRK2) plays a key role in abiotic stress signaling via phosphorylation in plants.To discover and utilize the genetic resources involved in abiotic stress tolerance, we constructed the cDNA library responsed to water defict from wheat seedlings using suppressive subtractive hybridization. In the study, one EST of SnRK2.8 was screened from the cDNA library. Based on the EST sequence, we isolated TaSnRK2.8 from wheat and characterized the functions in abiotic stress responses. The results were as follows:1. The full length cDNA of TaSnRK2.8 was cloned from wheat by in silico cloning. The TaSnRK2.8 cDNA was 1431 bp in length, which contained an ORF of 1101 bp and encoded a protein of 367 amino acids. The putative protein had a calculated molecular mass of 42 kDa and isoelectric point of 4.87. Moreover, TaSnRK2.8 might have both activities of serine/threonine and tyrosine kinases. Amino acid sequences alignment analysis showed that TaSnRK2.8 showed homology with counterpart SnRK2 family members from other plant species, viz. Oriza sativa, Zea mays and Arabidopsis thaliana. TaSnRK2.8 has 94.8% identity to OsSAPK8, 94% to ZmSAPK8, and 76.5% to AtSnRK2.2, respectively. Phylogenetic analysis indicated that TaSnRK2.8 belonged to subclass III subfamily of SnRK2.2. Quantitative real-time PCR were used to analyze the expression patterns of TaSnRK2.8 in wheat. TaSnRK2.8 was constitutively expressed in wheat, strongly in roots, weakly in stems, and marginally in leaves and spikes. Abiotic stress responses analyses revealed that TaSnRK2.8 was involved in response to PEG, NaCl and cold stresses, and possibly participates in ABA-dependent signal transduction pathways. The sensitivity degrees of TaSnRK2.8 responsing to abiotic stresses was in the order of hyperosmolality > low temperature > ABA > high salinity. 3. To investigate the role under various abiotic stresses, TaSnRK2.8 was transferred to Arabidopsis under control of the CaMV-35S promoter. Overexpression of TaSnRK2.8 resulted in enhanced tolerance to drought, salt and cold stresses, further confirmed by stronger roots and various physiological characteristics, including lower osmotic potential, higher relative water content, strengthened cell membrane stability, increased chlorophyll content and enhanced PSII activity. Meanwhile, significantly decreased total soluble sugar was detected in TaSnRK2.8 overexpressing plants, suggesting that TaSnRK2.8 might be involved in carbohydrate metabolism. Moreover, the expression levels of ABA biosynthesis (ABA1, ABA2), ABA signaling (ABI3, ABI4, ABI5), stress-responsive genes, including two ABA-dependent genes (RD20A, RD29B) and three ABA-independent genes (CBF1, CBF2, CBF3), were generally higher in TaSnRK2.8 plants than in WT/GFP control plants.4. To address the subcellular localization of TaSnRK2.8 in living cells, a construct containing TaSnRK2.8 fused in-frame with GFP driven by the CaMV 35S promoter was expressed in living onion epidermal cells and Arabidodsis. Both results showed the presence of TaSnRK2.8 in the cell membrane, cytoplasm and nucleus.5. TaSnRK2.8 in genome was about 6.1 kb, which contained 9 extrons and 8 introns. Sequencing results showed that two types of sequences were detected from common wheat, named TaSnRK2.8-A and TaSnRK2.7-B. TaSnRK2.8-A were mapped on chromosome 5A with nullisomic lines of Chinese Spring. One hundred and sixty five common wheat accessions were used to detect SNP of TaSnRK2.8-A-C in genomic sequence through sequencing. Three SNPs were detected in 165 TaSnRK2.8-A-C sequences. All the SNPs belong to transitons, and the frequency of SNP was 1 SNP/250 bp. There was no insertion/deletion. The nucleotide diversity (π), i.e., the average pairwise sequence differences between two random sequences in a sample, was 0.00068 per site. The average estimate ofθw, which is based on the observed number of polymorphic sites in a sample, was 0.00070 per site. The nucleotide diversity value in the 3′UTR was highest, lower in intron, the lowest in extron. Neutrality test indicated that there was no selection in TaSnRK2.8-A-C region. Association analysis of traits between A and G genotype (5917 bp, A→G) indicated that TaSnRK2.8 was involved in water-soluble carbohydrate, plant height, flag leaf width, seedling biomass, heading stage and drought resistance index. The A-allele was a preponderant allele in the 165 wheat varieties.6. In order to analyze transcriptional regulative mechanism of TaSnRK2.8 gene, the TaSnRK2.8 promoter (1797 bp) was isolated from wheat. The sequence is abundant in A/T base and predicted to contain a lot of putative cis-elements, such as TATA-box, CAAT-box, ABRE, HSE, TC-rich repeats, LTR and C-repeat/DRE. To identify the key promoter regulative region controlling gene expression, TaSnRK2.8 promoter was truncated according to those putative cis-elements, and inserted into the site upstream of GUS reporter gene. Then, vectors containing different length TaSnRK2.8 promoters were transferred into Arabidopsis. Histochemistry staining analysis showed that only the longest promoter fragment (Dp1870) could activate the expression of GUS gene under normal conditions. Meanwhile, GUS was only expressed in petioles and stems, while not observable in leaves and roots. GUS fluorescence intensity analysis indicated that GUS activity under control of the Dp1870 promoter was lower (0.16~0.35 times) than that under control of the CaMV-35S promoter.In summary, TaSnRK2.8 is a multifunctional regulatory factor, which could participate in sugar metabolic and stress signaling in wheat. It may be possible to utilize TaSnRK2.8 in the improvement of abiotic-stress tolerance in crop species.