Bt Research 2025, Vol.16, No.4, 168-181 http://microbescipublisher.com/index.php/bt 170 2.2 Structure and mechanism of action of Cry and Vip proteins Cry protein is the most studied category of Bt toxins. Its prototoxin form is insoluble crystal protein. After feeding the insects, they dissolve under alkaline conditions in the midgut and are cleaved by proteases to activate toxin fragments of about 65kDa~70 kDa. Activated Cry toxin has three functional domains, which can bind to the specific receptors of the brush-like edge membrane of insect midgut epithelial cells, trigger conformational changes and insert them into the cell membrane to form pores, resulting in osmotic pressure imbalance of midgut epithelial cells, swelling and rupture, thus causing insects to die quickly. The types of receptors recognized by different Cry proteins vary, generally including glycoprotein receptors, alkaline aminopeptidases, cadherin and other membrane proteins. Vip3A protein is a single component toxin secreted by Bt bacteria during the nutritional period, and it does not share receptors with Cry protein. Vip3A protoxin is about 88 kDa and needs to be activated by partial cleavage and activation by insect midgut protease (Infante et al., 2024). The activated Vip3A can also penetrate the midgut cell membrane, causing cell lysis and death. Since Vip3A and Cry protein act sites, combining Cry and Vip genes into the same crop can provide a double blow to the pest and reduce the probability that the pest will develop resistance to both at the same time (Wang et al., 2023). 2.3 Cloning and transformation technology of Bt gene Since the first cloning of the Bt insecticidal protein gene in the 1980s, scientists have cloned a variety of Cry and Vip genes and used for crop transgenic breeding. However, introducing bacterial genes into plants faces the problem of inefficient expression. To this end, it is usually necessary to modify the Bt gene when it is used for plant transformation, including codon optimization, removal of sequence and structural elements that are unfavorable to expression, addition of plant promoters and enhancers, etc. When China developed Bt insect-resistant rice, the GC content of the Cry1Ab/Ac fusion gene was increased through codon optimization, thereby significantly improving the expression level in rice. The cloned and modified Bt gene is often transferred to plant cells through Agrobacterium mediation or gene marks method to obtain stable transgenic plants. Since the 1990s, the Bt gene has been successfully introduced into cotton, corn, potato, rice, soybean and other crops, and a series of new insect-resistant varieties have been cultivated. In 1996, the first commercial insect-resistant cotton in the United States was the introduction of the Cry1Ac gene; then there were subsequent insect-resistant corn introduced with Cry1Ab. The insect-resistant hybrid rice "Huahui No. 1" and "Bt Shanyou 63" independently cultivated in 1999 introduced the Cry1Ab/Ac fusion gene and obtained the production and application safety certificate in 2009. Advances in plant transformation technology (such as efficient promoter screening, multigene co-transformation, etc.) continuously improve the expression stability and level of Bt genes in crops, allowing transgenic plants to continuously produce sufficient insecticidal proteins throughout the growing season (Latif et al., 2015). 2.4 Pest target action pathway and resistance risks The Bt toxin needs to work with insect midgut specific receptors and destroy cellular function, and this precise mechanism is also accompanied by the risk of pest resistance. The main mechanisms for field pests to develop resistance to Bt crops include: receptor mutation or deletion, altered midgut protease activity, enhanced toxin isolation and degradation mechanisms, etc. Among them, receptor mutations are common reasons. Laboratory strains that are resistant to Cry1Ac by pests such as vermithiasis often have functional mutations in the cadherin receptor gene, resulting in the inability of Cry toxin to bind effectively. Changes in protease activity can also affect toxin activation and action, such as downregulation of the expression of midgut protease in some resistant bollworms, which prevents the Cry1Ac prototoxin from being able to be activated smoothly (Zhang et al., 2022). In addition, factors such as pest intestinal flora and symbiotic viruses may also affect the action pathway of Bt toxin. Studies have found that the surviving cotton bollworms in China carry a symbiotic virus (HaDV2) in their bodies, which can improve the tolerance of cotton bollworms to Bt by regulating the host immune pathway (Lawrie et al., 2020). With the large-scale cultivation of Bt crops and continuous selection pressures, multiple resistance mechanisms may evolve and accumulate in pest populations. This presents challenges the sustainability of Bt crops. If resistance management strategies are not adopted, theoretically, pests will sooner or later have broad resistance to Bt toxins.
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