Bt Research 2025, Vol.16, No.5, 194-203 http://microbescipublisher.com/index.php/bt 197 3.3 Research on the integration of proteomics and metabolomics While the transcriptome can reveal trends in gene expression, it is proteins and metabolites that really perform functions. Therefore, proteomics and metabolomics provide essential supplementary information for improving the Bt regulatory network. The application progress of proteomics in Bt research points out that the proteome has become one of the important methods for detecting and identifying Bt toxin proteins. Through proteomic analysis, it is possible to directly verify whether certain proteins predicted by transcriptional regulation are actually expressed and changes in expression levels. Caballero et al. (2020) used proteomics to analyze the crystal protein composition of Israeli subspecies strains. In addition to the main Cry toxin, a variety of auxiliary proteins were also detected, revealing the complex composition and potential interactions of toxin crystals. In terms of metabolomics, since Bt is significantly reprogrammed in the process of bud formation, metabolomic data can reflect the end effects of the regulatory network. Bt will produce different small molecule metabolites at different growth stages, such as certain secondary metabolites have a synergistic insecticidal or antibacterial effect in biocontrol. Integrating the proteome and metabolomic group can fully characterize the functional phenotype of Bt (Lazarte et al., 2021). 4 Signal Transduction Pathways and Regulatory Mechanisms 4.1 Regulatory pathways of toxin genes (Cry, Cyt, etc.) Bt is known for producing crystalline toxins, and the expression of these toxin genes is regulated by complex signaling pathways. From a time perspective, most cry genes are silent during the vegetative period, and are transcribed at the transitional stage/spore formation early, and are expressed in large quantities in the middle and late stages of spore formation. This process is mediated by a series of step-by-step activated σ factors: σ^H is usually upregulated at the end of the vegetation, driving the initial transcription of some cry genes; after entering spore formation, the specific σ^F/σ^G in the prespore and the specific σ^E/σ^K in the mother cells take over (Du et al., 2012). Research evidence shows that toxin genes such as cry4A, cry4B, and cry11A are transcribed at low levels during the trophic transition period and are initiated by σ^H-associated RNA polymerase, but their high-level expression after spore formation depends on σ^E activation. Spacely, different types of Cry toxin genes may be distributed on different plasmids or loci, and their expression also requires corresponding local regulatory elements. There are specific regulatory sequences upstream of the promoters of many toxin genes, which can bind to transcriptional activation proteins. 4.2 Regulation of gene expression by environmental response signals Bt will adjust its gene expression under different environmental conditions to adapt to external changes and maintain survival and insecticidal functions. This response is mediated by a variety of signal transduction pathways and can act on the gene regulatory network of Bt. Common environmental factors include nutrient sources, temperature, pH, oxidative pressure and population density. Taking carbon sources as an example, when carbon sources are abundant in the culture medium, Bt will inhibit secondary metabolism and the expression of certain virulence genes through the "carbon source repression" mechanism. This mechanism is implemented by the global regulator CcpA (Jiang et al., 2016). Looking at the temperature signal, the gene expression spectrum of Bt at different temperatures is also different. It has been reported that sublethal high temperatures can induce the expression of heat shock proteins and chaperone molecules, and temporarily reduce the transcription of certain crystal toxin genes. It is speculated that bacteria preferentially ensure their own survival and then restore their insecticidal function. pH is also an important signal, and Bt shows different regulatory modes in an alkaline environment. Some studies have found that upregulation of certain protease and toxin gene expression of Bt under alkaline conditions is conducive to playing a role in the insect midgut, while these genes are expressed at low levels in the neutral environment (Zhang et al., 2024). 4.3 Mutations and network changes in adaptive evolution The Bt gene regulatory network is not static. During the long-term evolution or artificial mutagenesis, the genome and regulatory network adjustments will occur, thereby obtaining adaptive advantages or phenotypic changes. A
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