Molecular Characterization and Physiological Function Analysis of OsSAPK6/OSRK1 Gene in Rice (Oryza sativa L.)
Date Issued
2010
Date
2010
Author(s)
Chen, Bor-Hong
Abstract
Abiotic stress can greatly affect plant growth and production, so it is important to identify the mechanism of abiotic stress tolerance. When plant encounter abiotic stress, plant accumulate ABA and reprogram gene and metabolites expression. SnRK2 (Sucrose Non-fermenting 1 Related protein Kinase 2) are specific present in plant and regulate ABA and abiotic stress signaling pathway. There are 10 OsSAPKs (Stress Activated Protein Kinase) in Oryza sativa L. In this study, we focus on OsSAPK6 to study the gene expression pattern under various abiotic stresses. We also use Tos17 OsSAPK6 knock-out mutant to dissect possible function of OsSAPK6 gene.
At first, we took bioinformatic approach to examine SnRK2 phylogenetic relationship from Arabidopsis and rice then use PLACE to find the putative cis-acting elements in promoter of OsSAPK6 gene. Different cis-acting elements, such as CRT, ABRE and WRKY binding site can be identified in the OsSAPK6 promoter region and indicated that OsSAPK6 expression may be induced under abiotic stress. Using RT-PCR and cDNA from TNG67 treated with salt, drought, high and low temperature, we found that OsSAPK6 expression can be induced by salt and drought stress. Next we monitored the OsSAPK6 expression in different developmental stages and tissues. OsSAPK6 gene was highly expressed in leaf blade. To monitor OsSAPK6 expression in different rice cultivars, we confirmed that OsSAPK6 gene expression was lower in TCN1 than TNG67 under salt and drought stress. Furthermore, to understand function of OsSAPK6 in various stress responses of rice, Tos17 mutants from RGRC were treated by salt and drought stress and the phenotypes and OsSAPK6 gene expression were compared with wild type. Also, to reveal role of OsSAPK6 in the molecular mechanism of stress response, we determined genes expression of two transcription factors (DREB1A & DREB2A) and two down stream stress response genes (DHN1 & SalT) in WT and Tos17 mutants . The expression of DREB1A, DREB2A, DHN1 and SalT was down-regulated in Tos17 mutants as compared with WT. Finally, to analysis the OsSAPK6 promoter activity, we made pSAPK6::GUS plasmid construct and transformed into rice callus. The GUS staining showed blue color on callus and indicated that the 1.5kb DNA fragment of OsSAPK6 promoter is functional. To address the subcellular localization of OsSAPK6 protein, the OsSAPK6::GFP plasmid was particle bombarded to onion epidermis cells and we observed that OsSAPK6 was specifically localized in nucleus. Above all, these results suggested that OsSAPK6 is a positive regulator that involve in the upstream of salt and drought stress tolerance in rice. OsSAPK6 can regulate expression of transcription factors and downstream stress response gene such as DREB1A, DREB2A, DHN1,SalT and affects rice salt and drought stress tolerance.
Subjects
protein kinase OsSAPK6
gene expression
salt and drought stress
promoter activity analysis
protein subcellular localization
Type
thesis
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