Cloning And Expression Analysis Of Wheat (Triticum Aestivum L.) Stress Tolerance Genes TaUES And TaSTK

1 We chose a gene similar to Wali6 (Gene ID: TC248207) whose expression level increased dramatically after 24 h of NaCl treatment from the results of gene chip, isolated a gene from the leaf of the somatic hybrid Shanrong No.3 by RT-PCR and named as TaUES (Upregulated Expression under Saline-stress in wheat). Sequence analysis shows that the TaUES cDNA has a single open reading frame (ORF) of 291 bp encoding a protein of 97 amino acids with an estimated molecular mass of 10.3 kD and an isoelectric point of 7.52.The eatimated protein doesnot contain conserved domains, has a signal peptide and is possible to be a secreted protein. However, transient expression analysis in onion epidermal cells by genegun indicated TaUES-GFP fuse protein had no specific subcellular localization. Although we used PI to stain onion epidermal cells which had expressed TaUES-GFP fuse protein and the results showed it sulocated in the nucleus, we still needed to use a variety of dye staining to determine its precise location.The expression patterns of TaUES in the leaf and root of wheat were analyzed by semiquantitative RT-PCR. It was found that the expression level of TaUES was high effected by salt and drought stress, and we concluded that TaUES was involved in responses to salt and drought stress. The expression of TaUES in root was induced slightly after 24 h of NaCl treatment, and when the roots were removed from medium containing NaCl, the expression of TaUES decreased to nearly zero. The expression of TaUES in leaf increased dramatically after 12 h of NaCl treatment, and when the roots were removed from medium containing NaCl, the expression of TaUES returned to pretreatment levels, showing that TaUES in leaf belonged to"late"expression type.In contrast to above expression patterns, TaUES in the root which was treated by PEG had a complex pattern of induction, with a transient peak of expression after 12 h of PEG treatment and a second increase after 48 h. When the roots were removed from medium containing PEG, the expression of TaUES decreased to nearly zero. The expression level of TaUES in the leaf which was treated by PEG came to maximum and maintained at this higher level in the subsequent processing. When the roots were removed from medium containing PEG, the expression of TaUES returned to zero, showing that TaUES in leaf belonged to"early"expression type.To further study the function of TaUES gene, we constructed a constitutive expression vector. Over-expression vector was transformed into tobacco and Arabidopsis. Now we obtained T1 line of tobacco and T0 line of Arabidopsis.2 Wheat growth, development, signal conduction and stress responses form a complex and large network, in which serine/threonine protein kinase plays an important role in the regulation. We chose a gene (Gene ID: TC264050) whose expression level increased dramatically after 24 h of NaCl treatment from the results of gene chip. We isolated a gene from the leaf of the somatic hybrid Shanrong No.3 by RT-PCR and named as TaSTK. Sequence analysis shows that the TaSTK encodes a 50.8 kD protein with a calculated pI of 5.3 and has a S_TKc conserved domain from 19 to 279 amino acids. The eatimated protein has no signal peptide and sublocates in cytoplasm.The expression patterns of TaSTK in the leaf and root of wheat were analyzed by semiquantitative RT-PCR. It was found that the expression level of TaSTK was high effected by salt and drought stress, and we concluded that TaSTK was involved in responses to salt and drought stress. The expression of TaSTK in root was induced slightly after 3 h of NaCl treatment, and when the roots were removed from medium containing NaCl, the expression of TaSTK decreased to nearly basal levels, which indicated that TaSTK in root fit the category of"eraly"induction. TaSTK mRNA in leaf followed the expression pattern of that in root, with maximal induction 24 h after NaCl exposure.TaSTK in root and leaf showed simple induction, which the expression of TaSTK in root reached maximal level after 6h PEG exposure and that of TaSTK in root reached maximal level after 0.5 h PEG exposure, retumed to near basal levels when the roots were removed from medium containing PEG.Overexpression vector was constructed to examine the function of TaSTK. By using Agrobacterium mediated leaf-disk tobacco transformation, the recombinant was transformed to Nicotiana tabacum L. Over-expression vector was also transformed into Arabidopsis. The transgenetic strains are screening.In summary, we have isolated two wheat genes, TaUES and TaSTK, and analyzed their physiological functions. These results will provide practical evidence to understand the mechanism of wheat resistant to salt and drought stress and supply theoretical mechanism to improve wheat salt and drought resistance.