Cloning And Characterization Of Rice Stress-Responsive Gene OsMsr15

Rice is an important grain crop in China, as well as a model plant for functional genomics research. Abiotic stresses such as drought, cold and heat influence rice growth and development, result in rice yield decrease. Dissecting the mechanisms of rice responding to abiotic stress and improving rice abiotic stress tolerence are significant to increase rice yield potential. Identifying new rice functional genes will contribute to improving rice abiotic stress tolerence by genetic engineering. GeneChip Rice Genome Array (Affymetrix) was used to analyse the global genome expression profiles of rice Pei’ai64S under heat, drought and cold stresses. A number of stress responsive genes highly up-regulatied and down-regulated were identified. In order to obtain candidate functional genes for practical application, we selected an up-regulated gene OsMsrl5for further analysis. The main results are listed as following.1. Microarray data revealed that OsMsr15was increased9.38times in the leaf of seeding stage under cold stress. OsMsr15in the leaf was increased10.85、3.70and12.63times in booting stage under cold, heat and drought stresses, respectively, with an average of9.06times; under cold, and drought stresses OsMsrl5in the panicle was increased7.37and5.37times, with an average of6.37times. The increasing expression level of OsMsrl5in different stages and different tissues suggests that OsMsrl5may be a multiple stresses responsive gene.2. Quantitative real time PCR data confirmed the results from the microarray.3. Sequence analysis indicated that the902bp OsMsrl5cDNA contains an ORF of717bp, which encoded a protein of238amino acids with M.W≈24.6KD and pI≈8.90. Analysis of the putative promoter region found that several regulatory elements related to stress responses. The encoded protein contained typical conserved C2H2domain. A putative nuclear location signal (B-box) and an ERF-associated amphiphilic repression motif (EAR-motif) were found in the N-terminal and C-terminal regions of the protein, respectively. Proteins with high similarity to OsMsr15were found among other species. Thus, Oshtsr15might be a conserved candidate gene involved in plant stress tolerance.4. The overexpression vector of OsMsrl5was transformed to rice cultivar93-11and Arabidopsis ecotype Col-0by Agrobacterium-mediation method. The transgentic plantlets were analyzed by PCR with Hyg primers. The result showed that12transgenic rice plants with pCAMBIA1300-163-MSR15were confirmed by preliminary identification. Pure lines of transgenic Arabidopsis with pCAMBIA1300-163-MSR15were obtained.5. pCAMBIA1300-MSR15-GFP and pHB-OsMSR15-RNAi vectors were constructed and transformed into Agrobacterium EHA105. The constructed vectors lay the foundation for further research on OsMsrl5function.