ISSN: 2287-688X Annals of Plant Sciences Vol 9, Issue 5 (2020) pp. 3834-3842 Research Article *Corresponding Author: Anu Augustine, E-mail: anuaugus@rediffmail.com http://dx.doi.org/10.21746/aps.2020.9.5.1 Page | 3834 Construction of 6x myc tagged plant expression vector pBA 002 carrying Rhizophora mucronata Lam. specific glyoxalase I via homologous recombination Meera S. P. and Anu Augustine* Department of Biotechnology and Microbiology, Kannur University, Thalassery Campus, Palayad P.O., Kannur- 670661, Kerala, India. Abstract: 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. Keywords: Mangrove; Rhizophora mucronata; Salt tolerance; Glyoxalase; Expression vector; pBA Introduction Molecular cloning is an inevitable recombinant DNA technique in modern molecular biology research. In routine cloning reactions, the rest- riction digestion of vector and gene inserts are performed followed by their fusion using DNA ligase. Polymerase chain reaction (PCR) facile- tates the preparation of cloning gene inserts by selective amplification and addition of specific restriction sites. These gene fragments can be then inserted into any desired cloning vectors including those with expression capability in plants (Kelwick et al., 2014). The plant express- ion vectors utilize Agrobacterium based phyto pathogenicity to modify plant genome and thus explore functional aspects of plant proteins. These soil pathogens upon infection can trans- fer virulence genes of their genome into the plant cell. The afore mentioned peculiarity ena- bles researchers to transform model/crop plan- ts as per their assay design. The PCR amplified gene insert is incorporated first into a plasmid and then into an Agrobacterium carrying a seco- nd plasmid harboring virulence genes. This binary vector system is responsible for carrying, transferring and expressing the desired gene in the infected plant. It is difficult to culture and propagate Agrobacteria in vitro. Hence the initial sub cloning and the clone verification are usu- ally performed in E. coli (Glick and Thompson 1993).