Bt Research 2025, Vol.16, No.5, 224-233 http://microbescipublisher.com/index.php/bt 231 7.2 Exogenous vectors and genome integration technology Another type of coping strategy is to not rely on plasmids to maintain virulence, but to transfer key virulence genes to more stable vectors or directly integrate them into chromosomes through molecular biology. One approach is to construct exogenous vectors and introduce Bt strains: clone the main cry gene on the easily lost plasmid onto a stable wide host-range plasmid vector (such as the pBE series), and transform it back into the plasmid-free mutant strain of the original strain. In this way, the new plasmid has the selection and is more stable, and can replace the proplasmid in the strain to express toxins. Further, genome integration technology: through homologous recombination or transposal vectors, the plasmid virulence gene is directly integrated into the Bt chromosome and survives stably. Modern genome editing technologies such as Cre-lox, CRISPR/Cas, etc. can also be used to site-direct the target gene on the plasmid to chromosomal safe sites to avoid plasmid dependence. In addition to integrating virulence genes, there are also attempts to integrate the origin of plasmid replication into chromosomes to form chromosome-derived "mini plasmids" to stabilize the carrying of exogenous genes. 7.3 Engineering strain construction and virulence recovery strategy For Bt strains that have already experienced plasmid loss, engineering strategies can be adopted to reconstruct their virulence. A direct idea is to plasmid recompense, extract the plasmids missing from the original strain from the stored strain, and then introduce the lost strains of the plasmid through electroshock transformation and other methods to restore virility (Chen and Zhan, 2025). Another strategy is to build a functionally complementary engineering strain complex. For example, if a strain loses a Cry1 plasmid, it causes ineffective against Lepidoptera but still retains virulence against Diptera, another Bt strain only targets Lepidoptera and its mixture can be used to make up for the virulence spectrum gap (Zheng et al., 2017). From the perspective of strain breeding, plasmid-stable variant strains can be screened. For example, by repeatedly culturing in an environment containing target insects, the plasmid-free subpopulations are gradually eliminated, thereby enriching individuals that still carry plasmids, and then isolating stable virulence strains. This method of “domesticizing” strains under selective pressure restores plasmids and virulence while maintaining the strain’s natural background (Zhu et al., 2015). Acknowledgements I would like to thank all colleagues involved in this study for their collaboration and contributions with Cuixi Biotechnology Institute, and we would like to thank the peer review for their anonymous revisions. Conflict of Interest Disclosure The author confirms that the study was conducted without any commercial or financial relationships and could be interpreted as a potential conflict of interest. References Aylett C., and Löwe J., 2012, Superstructure of the centromeric complex of TubZRC plasmid partitioning systems, Proceedings of the National Academy of Sciences, 109: 16522-16527. https://doi.org/10.1073/pnas.1210899109 Benfarhat-Touzri D., Jemli S., Driss F., and Tounsi S., 2019, Molecular and structural characterization of a novel Cry1D toxin fromBacillus thuringiensis with high toxicity to Spodoptera littoralis (Lepidoptera: Noctuidae), International Journal of Biological Macromolecules, 126: 969-976. https://doi.org/10.1016/j.ijbiomac.2018.12.175 Bravo A., Pacheco S., Gómez I., Garcia-Gómez B., Onofre J., and Soberón M., 2017, Insecticidal proteins fromBacillus thuringiensis and their mechanism of action, Cham: Springer International Publishing, 2017: 53-66. https://doi.org/10.1007/978-3-319-56678-8_4 Chelliah R., Wei S., Park B., Park J., Park Y., Kim S., Jin Y., and Oh D., 2019, New perspectives on Mega plasmid sequence (poh1) in Bacillus thuringiensis ATCC 10792 harbouring antimicrobial insecticidal and antibiotic resistance genes, Microbial Pathogenesis, 126: 14-18. https://doi.org/10.1016/j.micpath.2018.10.013 Chen H., Verplaetse E., Slamti L., and Lereclus D., 2022, Expression of the Bacillus thuringiensis vip3A insecticidal toxin gene is activated at the onset of stationary phase by VipR an autoregulated transcription factor, Microbiology Spectrum, 10(4): e01205-22. https://doi.org/10.1128/spectrum.01205-22 Chen X.Y., and Zhan C.Y., 2025, Future directions in Bt toxin engineering for enhanced efficacy, Bt Research, 16(2): 55-62. Daffrose S.K., and Prakash S., 2024, Enzyme-linked immunoassay for the detection and quantification of Cry protein in Bacillus thuringiensis strains, Biopesticides International, 20: 1. https://doi.org/10.59467/bi.2024.20.97
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