J Mod Agric Biotechnol 2022; 1(2): 11 1/9 Copyright © 2022 The Author(s). This open-access article is licensed under a Creative Commons Attribution 4.0 International License (https:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, sharing, adaptation, distribution, and reproduction in any medium, provided the original work is properly cited. htps://doi.org/10.53964/jmab.2022011 ISSN 2788-810X ( Online ) Journal of Modern Agriculture and Biotechnology Open Access https://www.innovationforever.com Research Article Transient Expression of Plant-Codon-Optimized Cry2ab39 Gene by Agroinfiltration in N. Benthamiana Ngoc Thu Le 1,2 , Huyen Thi Tran 1 , Vy Thai Trinh 1 , Tra Thi Nguyen 1 , Ha Hoang Chu 1,2 , Phat Tien Do 1,2* , Thanh Minh Thi Le 1 , Ngoc Bich Pham 1,2* 1 Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, Hanoi, Vietnam * Correspondence to: Phat Tien Do, PhD, Head, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cay Giay, Hanoi 13111, Vietnam; Email: dtphat@ibt.ac.vn Ngoc Bich Pham, Vietnam Academy of Science and Technology, 8 Hoang Quoc Viet, Cay Giay, Hanoi 13111, Vietnam; Email: pbngoc77@gmail.com Received: February 14, 2022 Accepted: March 14, 2022 Published: April 28, 2022 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 agroinfltration. 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.