Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 126 2 The Structure and Function of the Bt Plasmid 2.1 Bt plasmid types, replication mechanisms and their stability in host strains Bt strains usually contain multiple plasmids, including large plasmids, which vary in size and carry different genetic information. People can classify plasmids based on their size, the genes they contain, and whether they can coexist in the same bacterium. For instance, through genome-wide analysis of Bt strains, researchers discovered several large plasmids (also known as giant plasmids), each carrying different insecticidal genes and genes that make bacteria toxic (Li, 2024). The rich plasmid types enable Bt strains to adapt to different living environments and parasitic insects by obtaining or losing specific plasmids (Pacheco et al., 2021). The replication mode of Bt plasmids is crucial for their retention and stability in bacteria. The presence of Rep protein and the biased analysis of GC content indicated that many Bt giant plasmids were replicated through the bidirectional theta mode. The stability of these plasmids in the host strain is also assisted by some genes-these genes are involved in the binding and division processes of plasmids, ensuring that when bacteria divide, the plasmids can be accurately passed on to the next generation of bacteria. In addition, some plasmids have the CRISPR-Cas system, which may further stabilize the plasmids by resisting foreign DNA (Navas et al., 2017). 2.2 Distribution and regulation of insecticidal genes (Cry, Cyt, Vip, etc.) on plasmids Insecticidal genes such as cry, cyt and vip are mainly located on Bt plasmids and are usually concentrated in large pathogenic gene regions. These concentrated gene groups may contain multiple toxin genes. For instance, in some Bt strains, as many as 13 genes capable of producing insecticidal proteins are aggregated on a single plasmid, forming "toxin islands", which are similar to the pathogenic gene regions in other bacteria (Zhou et al., 2024). These genes are arranged in such a way that it is conducive to the mutual coordination of the expressions of different toxins and may also produce a synergistic effect among them (Wang et al., 2020). The control process of these insecticidal genes is rather complex and involves the promoter and regulatory components of the plasmid itself. The function of genes is also influenced by environmental conditions and the physiological state of bacteria. For instance, researchers discovered through proteomic analysis that multiple toxin genes in the plasmid gene population can undergo transcription and translation, indicating that these genes are being actively controlled at specific stages of bacterial growth (Pacheco et al., 2021; Zhou et al., 2024). In addition, genetic recombination can also place new toxin genes at the existing positions of plasmids, thus enabling Bt to have the ability to kill more types of pests (Wang et al., 2020). 2.3 The contribution of plasmids to Bt toxicity and the co-expression of multiple toxin genes Plasmids play a core role in the toxicity of Bt, as most of the insecticidal proteins that make Bt effective against pests are synthesized under the guidance of genes on plasmids. If there are multiple toxin genes on a plasmid, it can produce various toxins, enabling Bt to target more types of insects and enhancing its overall toxicity (Navas et al., 2017). For instance, studies have found that when the cry gene and the vip gene are located on the same plasmid, they can produce a synergistic insecticidal effect, making the killing power of Bt strains against resistant pests stronger (Pacheco et al., 2021). Simultaneous expression of multiple toxin genes is a key method to delay the development of resistance in target insects. Proteomic analysis and functional studies have shown that Bt strains that can express multiple Cry proteins, or simultaneously express Cry proteins and Vip proteins, have stronger toxicity and a wider range of parasitic insects (Wang et al., 2020; Pacheco et al., 2021). This mode of simultaneous expression of multiple genes not only enables better pest control but also reduces the probability of insects developing resistance, thus allowing BT-based biopesticides to exert their effects for a long time and continuously. 3 The Key Strategies for Bt Plasmid Engineering 3.1 Design of recombinant toxin genes and co-expression of multiple toxins Simultaneously activating multiple Bt toxin genes such as Cry1Ac and Cry3A in the same host bacteria or the same plasmid is an effective approach to expand the insecticidal range and better address pest resistance. Research
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