Bt Research 2025, Vol.16, No.3, 118-124 http://microbescipublisher.com/index.php/bt 121 4.2 Horizontal gene transfer and plasmid regulation Most of the toxin genes of Bt exist on plasmids, and epigenetic modifications affect the stability of plasmids and the activity of genes. Huo et al. (2021) and Gu et al. (2024) both found through research that changes in DNA methylation levels may alter plasmid replication and gene transcription, thereby affecting the expression of toxin genes such as Cry and Cyt. Gu's team (2024) pointed out that these modifications might also alter the efficiency of horizontal gene transfer, promoting or preventing the spread of toxin genes among different bacteria. 4.3 Phenotypic heterogeneity Epigenetic modifications can also induce different phenotypes within the Bt population by regulating the variability of gene expression (Gu et al., 2024). In some environments, some cells produce more toxins, while others produce less. This difference enables the entire group to better cope with environmental changes and survival pressures. The flexibility of the epigenetic mechanism also enables Bt to respond more quickly to external changes. 5 Case Study: Epigenetic Regulation of Cry Genes in Bt subsp. kurstaki 5.1 Background The kurstaki subspecies of Bacillus thuringiensis (Bt) is famous for its ability to produce insecticidal crystalline protein (Cry protein). These proteins are highly toxic to many pests. The expression of the Cry gene is controlled by multiple aspects, such as the promoter, terminator, sigma factor, and the stability of mRNA (Zhang et al., 2018; Singh et al., 2021). It is important to understand these regulatory methods, as this can increase the production of Cry protein and also slow down the rate at which pests develop resistance. 5.2 Study findings The expression of the Cry gene mainly relies on promoters and terminators. For instance, in Bt kurstaki HD73, the expression of the cry1Ac gene is influenced by the σ^E and σ^K factors. Moreover, some strong promoters of non-CRY genes (such as HD73_5014) can also drive the expression of cry1Ac very well, and the effect is similar to that of the known strongest Cry promoter (Zhang et al., 2018). In Bt kurstaki HD-1, the cry gene has multiple overlapping promoters (BtI and BtII). They will be activated at different stages of spore formation. The terminator at the 3' end of the gene can also form a stem-loop structure, making mRNA more stable and thereby increasing protein production. The research also found that the expression of the Cry gene is not solely dependent on its own promoter. Sometimes, strong promoters of non-CRYgenes screened out can also be used to enhance expression (Zhang et al., 2018). In addition, the structures of the terminator and the 5' non-coding region also affect the stability of mRNA and the expression level of protein (Singh et al., 2021). 5.3 Implications From the above research results, it can be concluded that in Bt kurstaki, the regulation mode of the Cry gene is diverse and flexible. The expression efficiency of Cry proteins can be significantly improved by utilizing the strong promoters of non-CRY genes, optimizing the terminator structure or enhancing the stability of mRNA. These methods provide new molecular tools for the development of Bt formulations and transgenic crops (Zhang et al., 2018; Singh et al., 2021). A deeper understanding of these mechanisms can also help us slow down the development of pest resistance in our research and enhance the long-term application value of Bt biopesticides. 6 Applications and Future Perspectives 6.1 Biotechnological applications Epigenetic modifications include DNA methylation, histone modification and non-coding RNA regulation (Figure 2) (Chen et al., 2023). These modifications are reversible and can also delicately affect gene expression, so they are very useful in biotechnology. In medicine and agriculture, epigenetic markers are often used for the early diagnosis of diseases, prognosis analysis, and the design of individualized treatment plans. For instance, abnormal DNA methylation and histone modification can serve as markers of tumors, helping to predict diseases and make
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