Transient Expression of Plant-Codon-Optimized Cry2ab39 Gene by Agroinfiltration in N. Benthamiana

Abstract

Objective: This study was performed to modify the coding sequence of a novel Cry2Ab39 gene derived from B. thuringiensis serovar canadensis strain SP142 in Vietnam and investigate the expression of this codon-optimized gene in Nicotiana benthamiana (N. benthamiana) leaves. Methods:The Cry2Ab39 gene sequence (Genebank No. MN319700.1) was modified based on codon bias of N. benthamiana. A sequence coding the legumin B4 signal peptide and a His-tag coding sequence followed by endoplasmic reticulum (ER) retention signal KDEL were added to the 5' and 3' ends of the codon optimized Cry2Ab39 gene, respectively. The binary vector pFGC5941 harboring optimized Cry2Ab39 under the control of either CaMV 35S or Arabidopsis rbcS1A promoters was constructed and used for transient gene expression in N. benthamiana via agroinfiltration. Results:The native Cry2Ab39 gene were optimized based on codon usage of N. benthamiana along with a preference of G/C-containing codons to increase the overall G-C content and the potential mRNA stability sequences. A significantly higher Cry2Ab39 protein quantity was produced from the construct driven by the Arabidopsis rbcS1A promoter as compared to the CaMV 35S promoter. In addition, the highest recombinant protein was accumulated in tobacco leaves at 6 days-post-infiltration (dpi) with bacterial density OD 600 of 0.5. The His-tagged Cry2Ab39 was successfully purified by affinity chromatography using Ni-NTA columns. Conclusion:The recombinant Cry2Ab39 protein was successfully produced and purified from N. benthamiana leaves in appropriate amounts using suitable promoters and optimal transformation parameters. These results suggest high production of codon-optimized Cry2Ab39 gene in the plant system and show its potential for further applications in creating insect resistant crops.