Bt Research 2025, Vol.16, No.4, 157-167 http://microbescipublisher.com/index.php/bt 160 effectively bind toxins (Guo et al., 2015). For example, some Lepidopteran pests are believed to confer resistance to Cry1Fa by upregulating ABCC2 transporter expression to compensate for Cad receptor deletion. Receptor-mediated resistance often has the characteristics of single gene main effect, and its genetic mechanism and detection methods are relatively clear. Changes in toxin activation process: For example, changes in the activity of the midgut protease in insects will affect the activation of Cry protoxin. Resistant insects may change the isoform of midgut proteases by expressing them, reducing the efficiency of protoxin cleavage, resulting in the inability to form active fragments. Signaling pathways and cellular immune responses: Some studies suggest that resistant insects may activate the immune response pathways of midgut cells, promptly repair cellular damage caused by Bt toxin, or induce apoptosis pathways to avoid necrosis (Yu et al., 2022). In addition, epigenetic mechanisms and alterations in symbiotic intestinal flora have also been found to affect Bt virulence. Detoxification and metabolism enhancement: Insects may increase the activity of detoxification enzymes in the body and accelerate the degradation and removal of Bt toxins, thereby showing resistance. 3.3 Engineering transformation direction of toxin functional areas The surface loop structure of the toxin receptor binding region, namely the Cry toxin Domain II and III, is an important focus of engineering transformation. Through site-directed mutations or regionally directed evolution, the amino acid composition of these loop regions can be changed, thereby adjusting the toxin's recognition preference for different receptors. The researchers screened the saturation mutation of the third loop of the Cry1Ab toxin and successfully obtained variants that increased virility against bollworms and reduced effects on certain non-targets. The modification of toxin penetrating the membrane area (domain I) helps to improve pore-generating efficiency and virulence. Studies have shown that by changing the hydrophobic amino acids of the Cry toxin alpha helix, it can affect its insertion and channel stability on the membrane. A variant (L183I) was screened by saturation mutagenesis of Cry2Ab toxin, which was reduced by half on the target rhodopsia moth, due to the higher toxin aggregation and membrane pore formation ability of this variant (Khurshid and Zaheer, 2023). Chimeric toxin design has been a popular direction in recent years, namely toxins that create completely new functions through domain recombination. Improving toxin stability is also one of the transformation directions (Muralimohan et al., 2024). For example, by mutations to reduce protease-sensitive sites or improve thermal stability, toxins can remain active for longer in the field environment (UV, high temperature). There are also studies that use glycosylation site mutations to increase the retention time of Bt toxins in the insect midgut. 4 Synthetic Biology-Driven Bt Transformation Strategy 4.1 Design and synthesis of newcry toxin genes Applying synthetic biology ideas can transcend the limitations of natural Bt toxins and create new and efficient insecticidal protein genes through rational design or artificial evolution. Combination of directional evolution and rational design: Researchers can select key sites for saturation mutations or computer-aided design based on the aforementioned toxin structure-function relationship, and then use high-throughput screening to determine beneficial variants (Flórez et al., 2018). Chimeric toxins and modular splicing: By modularly combining the domains of different Cry toxins, new toxins with multiple parental advantages can be produced. Artificial gene synthesis and codon optimization: Through DNA synthesis, ideal mutations can be combined in one gene, while optimizing codons to enhance expression in Bt. In addition, other functional fragments can be grafted into synthetic genes, such as adding toxins to efficiently secrete signal peptides or protein tags, to confer new functions. Discovering and modifying new toxin genes: The popularity of high-throughput sequencing allows us to mine new toxin genes of Bt from the environment and quickly characterize and modify them through synthetic biology (Panwar et al., 2018). Fengshuo et al. (2024) discovered two newBt insecticidal genes cry39A-like and cry40-like through whole genome sequencing, and used PCR cloning and expression analysis to confirm that their encoding protein is virulent to pine caterpillars and slime larvae.
RkJQdWJsaXNoZXIy MjQ4ODYzNA==