Characterization of Brassica rapa S-adenosyl-L-methionine synthetase gene including its roles in biosynthesis pathway

Abstract

A full length cDNA encoding the SAM synthetase gene was isolated from Chinese cabbage by RT-PCR and contains a 1,182 bp open reading frame encoding a putative 393 amino acid protein. This cDNA insert was subcloned into the pET15b vector to evaluate the expression and further characterize the SAMS gene. Recombinant SAMS was also generated in BL21 cells and showed a molecular weight of about 43 kDa. To elucidate the function of SAMS in the Chinese cabbage, overexpression and RNAi vectors for this gene were constructed and introduced into tobacco plants. For overexpression, the CaMV 35S promoter was introduced into the binary vector pBI121 and the full-length SAMS gene was subcloned into the resulting pCSAMS vector. To suppress SAMS, forward and reverse fragments from its ORF of 519 bp length was introduced into the RNAi vector, pJJSAMS. SAMS gene functions were subsequently evaluated by the phenotypic variation analysis and by observing the upregulation and/or downregulation of the ethylene and polyamine biosynthesis genes, 1-aminocyclopropane-1-carboxylate synthase (ACS) and spermidine synthase (SPDS) in transgenic tobacco plants. The results of these experiments suggest that SAMS regulates ethylene and polyamine biosynthesis, at least in part, at the transcriptional level.