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

Meera S.P., Anu Augustine

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

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References


Altschul SF, TL Madden, AA Schaffer, ZJ Zhang, W Miller, DJ Lipman. “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Research. 25 (1997): 3389-3402.

Block MD, J Botterman, M Vandewiele et al. “Engineering herbicide resistance in plants by expression of a detoxifying enzyme.” EMBO Journal. 6. 9 (1987): 2513‐2518.

Figurski DH, DR Helinski. “Replication of an origin containing a derivative of plasmid RK2 dependent on a plasmid function provided in trans.” Proceedings of the National Academy of Sciences of the United States of America. 76 (1979): 1648-1652.

Geu-Flores F, HH Nour-Eldin, MT Nielsen, BA Halkier. “USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products.” Nucleic Acids Research. 35 (2007): e55.

Glick BR, JE Thompson. “Methods in plant molecular biology and biotechnology.” CRC Press, Inc. Boca Raton, Florida (1993).

Hartley JL, GF Temple, MA Brasch. “DNA cloning using in vitro site-specific recombination.” Genome Research. 10 (2000): 1788–1795.

Hillman MC, LS Yang, S Sun S et al. “A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide.” Protein Expression and Purification. 23. 2 (2001) 359–368.

Howard DH. “The preservation of bacteria by freezing in glycerol broth.” Journal of Bacteriology. 71 (1956): 625.

Jacobus AP, J Gross. “Optimal cloning of PCR fragments by homologous recombination in Esch-erichia coli.” PLoS ONE. 10.3 (2015) e0119221.

Jordens JZ. “Restriction enzyme analysis of chromosomal DNA its application in epidemiological studies. Journal of Hospital Infection. 18 (1991): 432–7.

Kelwick R, JT MacDonald, AJ Webb, P Freemont. “Developments in the tools and methodologies of synthetic biology.” Frontiers in Bioengineering and Biotechnology. 2 (2014): 60.

Li MZ, SJ Elledge. “Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.” Nature Methods. 4 (2007): 251–256.

Rubio-pina J, O Zapata-Perez. “Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants.” Electronic Journal of Biotechnology. 4 (2011): 5.

Sanger F, S Nicklen, AR Coulson. “DNA sequencing with chain-terminating inhibitors.” Proceedings of the National Academy of Sciences of the United States of America. 74 (1977): 5463–5467.

Woodman ME, CR Savage, WK Arnold, B Stevenson. “Direct PCR of intact bacteria (Colony PCR).” Current Protocols in Microbiology. 42. 1 (2016).

Zhang Y, U Werling, W Edelmann. “SLiCE: a novel bacterial cell extract-based DNA cloning method.”Nucleic Acids Research. 40. 8 (2012) e55




DOI: https://doi.org/10.5281/aps.2020.9.5.1



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