Construction of 6x myc tagged plant expression vector pBA 002 carrying Rhizophora mucronata Lam. specific glyoxalase I via homologous recombination

Plants are subjected to internal damage during stress conditions due to enhanced levels of methyl glyoxal (MG). Glyoxalase enzymes play the key role in MG detoxification and help the plant to survive. The glyoxalase system of Rhizophora mucronata Lam. was decoded; characterized and salt dependant increase in gene expression was analyzed in our previous studies ( GenBank Accessions GGEC01061405, GGEC01044968, and GGEC01022591) . In order to utilize these stress responsive genes in crop improvement, it is needed to monitor their methylglyoxal detoxification efficiency in vivo . For this, over expression of the glyoxalase enzyme(s) in a model/cop plant system can be done. Construction of a binary vector carrying coding region of glyoxalase gene(s) which can replicate both in E coli and Agrobacterium tumefaciens is the prime step in plant transformation research. In the present study in silico cloning of glyoxalase I, II and III specific to R. mucronata (Rm GLY I, Rm GLY II and Rm GLY III) were performed into pBA 002 plant expression vector carrying 6x myc insert. The binary vector is linearized with BSrG1 restriction enzyme. Cloning primers for all the three glyoxalase coding regions with 5’ end terminal homology to the linear myc pBA were synthesized and validated in vitro . To account for in silico cloning, the Rm GLY I insert was successfully cloned via homologous recombination into myc pBA. The presence of Rm GLYI insert in the final construct was confirmed by colony PCR and sequence analysis.