Abstract

Plant transformation vectors for constitutive gene expression were prepared to study the role of Esi47, a gene that encodes a protein kinase, which normally is induced as part of the salt-stress response in Lophopyrum elongatum. Agrobacterium-mediated transformation of A. thaliana, by tissue-culture and vacuum infiltration, were both attempted. A number of plant transformation constructs were made for gene promoter analysis. Expression constructs were also made and tested for the production of Esi47 protein in E. coli using the pATH11 trpE fusion vector. The tissue-culture method was partially successful. Callus cultures were established for the Arabidopsis Col-2 genotype, embryogenesis was observed in Col-2, and ten regenerated seedlings arose directly from RLD-1 genotype explants grown on shoot inducing medium (SIM) containing 100 $\mu$g/ml kanamycin. Col-2 callus was maintained for three passages on SIM with antibiotics, while the RLD-1 regenerated seedlings were plated directly on hormone free growth medium (GM) with 100 $\mu$g/ml kanamycin for three weeks. All developed roots and were transferred to the greenhouse, but rapidly wilted and died upon transfer. Attempts were made to regenerate from de novo meristem with Col-2 callus tissue by subculturing on B5 medium with various combinations of benzylaminopurine (BAP) and naphthalene acetic acid (NAA), but the callus remained unresponsive. Five series of transformations by vacuum infiltration were attempted, but proved unsuccessful. The trpE::Esi47 fusion gene constructs, one with the full protein kinase open reading frame, and the other with the carboxylic-terminus of Esi47, were successfully expressed in E. coli. The TrpE fusion proteins were found in the insoluble fraction of lysed cells. Resolubilized fusion proteins derived from the two expression constructs had apparent molecular masses of 75 and 45 kDa, respectively.