Cloning And Expression Of Rice Stress-Responsive Induced Genes OsMsr12, OsPP2Cl, OsTMP14and Functional Analysis Of OsTMP14

To discover new stress-tolerance genes and understand molecular mechanism(s) underlying stress responses in rice (Oryza sativa L.), we conducted a global genome expression profiling of the indica cultivar Pei’ai64s subjected to cold, drought, or heat stresses. Expression profiles were obtained for both leaf and panicle tissues at seedling, booting and heading stages of rice using the GeneChip Rice Genome Array (Affymetrix). Based on the obtained expression profiles, three genes, designated OsPP2Cl (O. saliva L. protein phosphatase2C-1, Ak063334), OsMsr12(Oryza sativa L. multiple stresses responsive gene12, EAZ02952.1) and OsTMP14(O. sativa L. thylakoid membrane protein of14kDa, GenBank ID BAF27084.1) were selected and cloned for further analysis.As revealed by the microarray, all the three genes were highly induced in both leaf and panicle at all the developmental stages of rice analyzed, in response to cold and drought stresses tested. The microarray data obtained was confirmed by quantitative real-time RT-PCR analysis of OsPP2C1, the two sets of data matched very well. We cloned their cDNA from Pei’ai64s through qRT-PCR. included the compele ORF (open reading frame). Predicted the function of genes based on their sequence of ORF.Sequence analysis showed that the cDNA of OsPP2C1contains an open reading frame (ORF) encoding a protein of358amino acid residues with M.W. about37.6kDa and pI about6.16. The phylogenctic analysis of indica78OsPP2C genes showed that can be divided into11sub-families;OsPP2C1belong to subfamily A, the gene sequence encoded a protein phosphorylated2C protein associated with stresses tolerance. Analysis of the putative promoter region of OsPP2Cl for cis-regulatory elements using PlantCARE software identified16matches to known cis-elcments related to stress responses.OsMsrl2encodes a protein of375amino acid residues with M.W. about40.62kDa and pI about5.75. Searching sequence databases find that the ORF contained a leucine-rich repeats domain and was similar to proteins of the leucine-rich repeats receptor-like kinases family in indica and japonica, Zea mays, Arabidopsis thaliana. Analysis of the putative promoter region for candidate cis-regulatory elements identified8matches to cis-elements related to stress responses. Based on the above analyses and results, we propose that OsPP2C1and OsMsr12are two novel candidate genes involved in stress tolerance in rice.Sequence analysis showed that OsTMP14cDNA encodes a protein of172amino acid residues with theoretical M.W. about17.95kDa and pI about7.85. Through analysis of sequence alignments, the gene of rice (Nipponbare) cDNA identity of Os10g0536500is100%. TMP14is PsaP that the core protein of the photosystem I (PSI), a kind of nuclear genome-encoded chloroplast thylakoid membrane protein, natural phosphorylation in vivo. Analysis of its putative promoter regions for cis-regulatory elements identified9matches to known cis-elements related to stress responses. To know whether or not OsTMP14involved in stress tolerance, its cDNA was inserted into binary vector pCAMBIA1300under the direction of the2×CaMV35S promoter and delivered successfully into indica rice93-11by Agrobacterium-mediated method. Ten independent transgenic rice plants were obtained.