Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 127 on genetically modified plants has shown that allowing plants to simultaneously express two Bt toxin genes can effectively resist more types of pests, including Lepidoptera and Coleoptera insects. For example, there is a poplar variety called Populus x euramericana, which can express both Cry1Ac and Cry3Agenes simultaneously and has strong resistance to various insect larvae. Both of these two genes can undergo normal transcription and translation in plants (Ren et al., 2021). This method not only enhances the effectiveness of pest control but also slows down the rate at which the target pests develop resistance. However, due to the differences in structure and regulatory elements among different toxin genes, even when expressed together, their yields will be significantly different. In the above-mentioned study, the content of Cry3A protein was significantly higher than that of Cry1Ac, which might affect the overall insecticidal effect of transgenic organisms (Ren et al., 2021). Therefore, it is necessary to rationally design the structure of the recombinant gene, such as optimizing the codon and adjusting the regulatory elements, to make the expression more balanced. This is very important for achieving the ideal expression ratio and maximizing the synergy. 3.2 Optimize regulatory elements to enhance the expression of toxin genes To increase the expression level of Bt toxin genes, it largely depends on optimizing regulatory elements, such as promoters, ribosome binding sites (RBS), and transcriptional regulatory factors. The selection of different promoters and RBS will have a significant impact on the transcriptional and translation efficiency of each toxin gene. Just as observed in transgenic lines, different promoter types can lead to differences in the contents of Cry1Ac and Cry3A proteins (Ren et al., 2021). If the promoter used is suitable for the host's transcriptional mechanism, has high intensity, can continuously function or can be induced to activate, it can significantly promote the production of toxins. In addition, regulatory elements must be carefully matched with the host strain and specific toxin genes to avoid gene silencing or imposing additional metabolic burdens on the host. The stability and high-level expression of exogenous genes may be affected by environmental factors or changes in host growth and development, resulting in a decrease in insect resistance over time (Ren et al., 2021). Future genetic engineering work should focus on adjusting the details of regulatory sequences and utilize synthetic biology tools to ensure that multiple toxin genes can be stably, efficiently and continuously expressed. 3.3 Enhance plasmid stability and host adaptability The stability of the plasmid is the key for the continuous functioning of the Bt toxin gene, especially when there is no pressure from antibiotic screening. Research has found that adding a toxin-antitoxin (TA) system to the plasmid structure can effectively ensure the long-term existence of plasmids in the host microbiota. At present, different types of TA systems have been developed, such as those based on ccd modules or prcA/prcT modules. They can kill the progeny bacteria that did not obtain the plasmid, thus ensuring that the modified plasmid is stably passed on to the next generation and retained for a long time (Wright et al., 2015; Fraikin and Van Melderen, 2023; Liu, 2024). The stabilizing effects of these systems can be compared with the effect of integrating genes into chromosomes, and at the same time, they can maintain a high expression level of exogenous genes (Takashima et al., 2021; Ren et al., 2023). In addition, optimizing the replication origin (ori) of plasmids and adopting host-dependent replication systems can further enhance the stability and adaptability of plasmids. By using conditional replication starting points and modular integration systems, plasmids can also be restricted to transfer only within the specified host bacteria, thereby reducing the risk of gene-level transfer to other bacteria and diffusion to the environment. By combining these methods with the TA system, stable and safe Bt plasmid vectors suitable for agricultural and environmental applications can be designed. 4 Evaluation of the Efficacy after Enhanced Insecticidal Activity 4.1 Laboratory bioassay of target insects and expansion of the insecticidal range Conducting biological experiments in the laboratory is an important step in determining the insecticidal efficacy
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