Bt Research 2025, Vol.16, No.4, 125-135 http://microbescipublisher.com/index.php/bt 129 4.3 The role of multiple toxin expressions in restoring insect sensitivity and delaying resistance Making Bt express multiple toxins with different modes of action simultaneously is a proven effective strategy - it can not only make insects that have already developed resistance sensitive to Bt again, but also delay the development of insect resistance. Both laboratory and field data show that Bt crops or Bt products that can produce multiple toxins can kill pests multiple times. Even if insects develop resistance to one toxin, they are still sensitive to the other, which reduces the probability of insect resistance being fixed (Tabashnik et al., 2013; Tabashnik et al., 2015). For instance, studies have found that the proprotein form of Cry protein is more effective against resistant insect strains than its activated form, which also demonstrates the value of allowing Bt to express multiple toxins. In addition, by using the modified toxins or the combined application of multiple toxins, not only can the control range of Bt over pests be expanded, but also the difficulty for pests to develop resistance can be increased. This comprehensive strategy is of great significance for ensuring the long-term effectiveness of BT-based agricultural pest control techniques (Deist et al., 2014). 5 Application Cases and Commercial Development 5.1 Characteristics of successfully engineered Bt strains and plasmid design The improvement of Bt strains is aimed at making up for the shortcomings of natural Bt strains, such as the limited variety of pests they can kill and the fact that pests often develop resistance very quickly. With the help of recombinant DNA technology, researchers can replicate genes that produce insecticidal proteins (such as the cry gene), and then put these genes into Bt strains or other hosts. In this way, strains with stronger insecticidal effects and better pest control capabilities can be obtained. For instance, there was a study that used electroporation technology to introduce the Cry3Aa7 gene into wild Bt strains, resulting in the recombinant strain G033A. This strain is highly toxic to lepidoptera pests and is also the first transgenic Bt strain registered as a pesticide in China (Li et al., 2022). These modified strains, usually with specially designed plasmids, can effectively express the target genes, remain stable, and transfer between different hosts, thus expanding their application possibilities in commercial biopesticide formulations (Figure 2) (Nakamura, 2020; Li et al., 2022). Figure 2 Environmental behaviors of Bt protein (A) and its three-dimensional structures (B). I, II, and III: domains I, II, and III (Adopted from Li et al., 2022) In the plasmid design of these strains, selectable markers, optimized promoters and regulatory sequences are usually included to ensure the stable production of large amounts of toxins. There are also some technological advancements, such as the construction of temperature-sensitive recombinant plasmids or the replacement of antibiotic resistance markers (for instance, using tetracycline resistance instead of erythromycin resistance), which have enriched the tools of genetic engineering and enabled the development of safer and more widely applicable strains for commercial use (Yanhua et al., 2008).
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