Molecular Cloning Of Rice OsBISAMT1, Encoding S-adenosyl-L-methionine: Salicylic Acid Carboxyl Methyltransferase

In previous studies in our laboratory, hundreds of differentially expressed cDNA clones associated with rice (Oryza saliva) disease resistance response induced by benzothiadiazole (BTH) were isolated and identified through suppression subtractive hybridization. Of these rice cDNA clones, clone BIHI-w8 showed high level of similarity to genes encoding S-adenosyl -L-methionine: salicylic acid carboxyl methyltransferase (SAMT) in plants. Therefore, it should contain a fragment of a rice gene encoding SAMT. In this study, I cloned and identified the full-length cDNA of this putative SAMT gene and studied the possible role of the gene in rice disease resistance by analyzing expression patterns in disease resistance responses and phenotype of transgenic tobacco plants.Differentially expressed cDNA clone, BIHI-w8, contained an insert of 562 bp and showed high level of similarity to other plant SAMTs. The missing sequences at 5'- and 3'-ends were amplified by rapid amplification of cDNA ends (RACE) with phage DNA prepared from a rice cDNA library as template. Full-length cDNA was obtained by assembling the 5'- and 3'-sequences and the original sequence in clone BIHI-w8 and designated as OsBISAMTl (Oryza saliva L. benzothiadiazole -induced SAMT 1). The full-length cDNA of OsBISAMTl has 1371 bp and contain a predicted open reading frame (ORF) of 1128 bp, which encodes a protein of 375 amino acids with calculated molecular weight of 42-kD and pI of 5.0. The predicted amino acid sequence of OsBISAMTl protein shows 31-36% identity to SAMTs from other plants and contained the conserved characteristic methyltransferase domain of SAMT proteins and amino acid residues required for binding SA and SAM/SAH. The OsBISAMTl gene was mapped on chromosome 12 of the rice genome and composed of 4 extrons and 3 introns. Southern blotting analysis revealed that OsBISAMTl might be a member of a small gene family containing 2-4 members in rice genome.To explore the possible role of OsBISAMTl in rice disease resistance response, I analyzed the expression patterns of the gene in response to treatments with inducers of disease resistanceresponses and in compatible and incompatible interactions between rice and the blast fungus, Magnaporthe grisea. The results showed that expression of OsBISAMTl in rice leaves was activated by treatments with BTH and salicylic acid, which are capable of inducing rice disease resistance, and up-regulated rapidly and specifically during the incompatible interaction between M. grisea and a resistant rice genotype. Moreover, mechanical wounding also activated OsBISAMTl expression. The results suggest that OsBISAMTl may play a role in disease resistance responses as well as in wound response in rice.To better understand the function of OsBISAMTl in disease resistance response, the OsBISAMTl coding sequence was cloned into a plant binary vector, CHF3, under control of the CaMV 35S promoter and resulting construct was used for transforming tobacco leaf discs by Agrobacterium-medlated transformation. Eleven kanamycin-resistant OsBISAMTl transgneic tobacco lines were obtained and confirmed by PCR detection. Southern blotting confirmed the integration of the transfer OsBISAMTl gene into tobacco genome. Northern blotting showed exogenous OsBISAMTl gene was transcribed in the transformants. Five transformants bloomed one month earlier than CK, and showed stunted plants with fewer flowers in comparison with wild ones.