The Molecular Characterization Of Wheat SnRK2 Family Member TaSRK2C1 And Its Roles In Mediating Plant Tolerance On Drought And Salt Stress

The osmotic stresses such as drought and salinity are important adverse environments, resulting in negative plant growth, development, and yield. For a long term evolution, plants have evolved the mechanisms to respond and withstand above stresses through transcriptional variations of large numbers of stress-responsive genes. Among them, protein kinase genes including calcium-dependent protein kinase genes （CDPKs）, motigen-activated protein kinase （MAPK） cascade genes, as well as distinct family members derived from SnRK, PTP, DsPTP, and PP2C play an important role on mediation of above signals transduction and on improvement of plant resistances for these stresses. In this study, one expressed sequence tag （EST） classified into SnRK2 family was further studied. The molecular characterization and expression patterns of this wheat SnRK2 family member （referred to TasRK2C1） were elucidated. Also, the effects of TaSRK2C1 on regulation of plant resistances to drought and salt stress were investigated in tobacco. The main results are as follows.1. Based on sequencing of clones in a wheat root subtratctive cDNA library that enriched genes to be drought resistance-related, one expresses sequence tag （EST） that shared high similarity to SRK2C1 （GenBank accession number NM₂02441.2）, a SnRK2 family member in Arabidopsis, was identified. Homology analysis figured out a tentative consensus （TC） （DFCI, http://compbio.dfci.harvard.edu/tgi/） harbored this EST and this TC contained an open reading frame （ORF）. Using reverse transcription RT-PCR, the cDNA sequence of this TC was amplified using the root total RNA-derived cDNA as the template. This wheat gene was designated as TaSRK2C1 due its high similarity to its Arabidopsis homologous conunterpart SRK2C1.2. The full length cDNA of TaSRK2C1 is 1331 bp, conataining an open reading frame of 1029 bp and encoding a polypeptide of 342 aa. The translated polypeptide has a molecular weight of 38.79 kD and an isoelectric point （pI） of 5.72. TaSRK2C1 harbors the consreved domains that are generally found in protein kinases, such as the active site, ATP binding site, substrate binding site, and activation loop （A-loop）. In addition, TaSRK2C1 also has the kinase domains including PKc, PKc_like superfamily, and S_TKc. This wheat kinase is targeted to cytoplasm after endoplasmic reticulum （ER） sorting. The three dimensional structure of TaSRK2C1 shares a high similarity to SnRK2.6, a SnRK2 fanmily member in Arabidopsis, based on homologous search analysis in protein bank （PPBD）.3. Using the polypeptide of TaSRK2C1 as the query, 5 proteins that derived from various plant species sharing high similarity to TaSRK2C1 were identified, including Arabidopsis SRK2C1 （GenBank accession number NP₉74170） that is classified into SnRK2 family, barley ABA-activated protein kinase HvPKABA1 （GenBank accession number BAB61735）, rice ABA-inducible protein kinase （GenBank accession number AAO65504）, wheat Ser/Threo protein kinase W55c （GenBank accession number ABD37624） and tobacco Ser/Threo kinase WAPK （GenBank accession number AAC69450）. Phylogenetic analysis suggests that TaSRK2C1 has derived from close progenitors similar to its conunterparts in diverse plant species.4. Under normal growth condition, the expression level of TaSRK2C1 was low. The transcripts of TaSRK2C1 in wheat roots were much more detected under stresses of drought and salinity, as well as under condition of exgenous ABA application, compared with those detected in normal growth condition. In a 9-h regime treatments, the TaSRK2C1 transcripts in roots were gradually increased with the progression of stresses of drought and salinity, as well as the ABA application. Thses resulsts suggest that the induction of TaSRK2C1 under stresses of drought and salinity was mediated based on ABA-dependent pathway. TaSRK2C1 did not show responses to deprivations of nitrogen （-N） and phosphorus （-P）.5. Using DNA reconbinant techniques, the binary expression pasmids referred to pCAMBIA3301-TaSRK2C1 and pCAMBIA3301-antiTaSRK2C1 that fused the TaSRK2C1 with sense- and antisense orientation mode, respectively, were constructed. The transgenic tobacco plants integrated above binary plasmids were generated via Agrobacterium-tumafeciens mediated trasformation approach. PCR analysis to specifically amplify distinct fragments of CaMV35S promoter that were inserted into transformed tobacco genome confirmed that the putative transgenic plants were all positive.6. After 3-week of drought and salt stress, there were significant variations on plant phenotypes in sense- and antisense-expressed tobacco plants compared with the control plants. It was clearly shown that the TaSRK2C1 sense-expressed plants behaved stronger growth and larger phenotypic, such as the transgenic lines of Sen 2 and Sen 6, whereas the TaSRK2C1 antisense-expressed plants showed weaker growth and smaller phenotypic, such as the transgenic lines of Ant 3 and Ant 5. Under above stresses, the plant dry weights and leaf physiological parameters such as cholorophyll contents, soluble protein contents, as well as the leaf soluble proline contents, soluble sugar contents and MDA contents, were all changed to accord to those of the plant phenotypes in the transgenic plants. Taken together, the results in this current study clearly demonstrated that TaSRK2C1 could improve the responding and withstanding capabilities on stresses of drought and salinity in plants.7. Under stresses of drought and salinity, the induction of TaSRK2C1 was performed possibly to be ABA-dependent manner, on which to mediate plant responses on above stress signals and improve plant resistances to these abiotic stresses. It seems that the plant species existed a conserved signaling pathway to be involved in transduction of the signals derived fron stresses of drought and salinity, in which TaSRK2C1 and its homologous counterparts are being acted as the important components. However, the downstream genes involved in responding and withstanding the stresses of drought and salinity that are activated by TaSRK2C1 as well as the molecular mechanism on improving these stresses mediated by TaSRK2C1 are necessary to be investigated further.