Bt Research 2025, Vol.16, No.3, 110-117 http://microbescipublisher.com/index.php/bt 115 7 Knowledge Gaps and Future Directions 7.1 Need for time-series transcriptomics under combined stresses. Most current studies only look at transcriptome responses under a single pressure. There is still a lack of systematic time series analysis for the situation where multiple pressures coexist (such as the combined action of temperature, pH and oxidation). Studies have found that when Bt spores germinate and grow in the food environment, many genes are regulated in time (Bassi et al., 2016). However, dynamic transcriptome data under combined stress are still scarce. 7.2 Limited integration with proteomic/metabolomic data. Some studies have combined the transcriptome and proteome to analyze the regulatory mechanism of Bt in spore formation and toxin production. Overall, however, there are still not many integrated analyses of multi-omics. This leads to an insufficient understanding of the relationship among gene expression, protein function and metabolites, and also makes it difficult to comprehensively explain the mechanism by which Bt responds to stress. 7.3 Few studies on natural soil conditions versus laboratory stressors. Most transcriptome studies are conducted in laboratories where conditions are relatively limited. However, studies on the expression of the Bt gene in natural soil or real ecological environments are still scarce (Zhu et al., 2015). In the natural environment, there are various complex biological and abiotic pressures, which may cause the transcriptome response of Bt to be completely different from that in the laboratory environment, thereby affecting our judgment of its ecological adaptability. 7.4 Potential of CRISPR/dCas9-based regulation to validate transcriptomic insights. Although transcriptomics has discovered many genes related to stress responses, the functional verification of these genes is still insufficient. Nowadays, some new tools, such as CRISPR/dCas9, can regulate or verify the functions of these genes more directly (Zhao et al., 2024). Such methods are expected to help us more accurately analyze the stress adaptation mechanism of Bt and also provide new ideas for strain improvement. 8 Conclusion Bacillus thuringiensis (Bt) exhibits very complex transcriptome regulation when facing different environmental pressures. These abilities are not fixed but are related to specific conditions. Its tolerance not only affects its survival and competitiveness in the natural environment, but also its stability and efficiency in biopesticides and industrial fermentation. Under various pressures such as alkalinity, salinity, metals or oxidation, Bt activates more genes related to glycolysis, organic acid synthesis, antioxidant enzymes and stress proteins. This can help it adjust its metabolic network and adapt to the new environment. For instance, under alkaline conditions, the genes for glycolysis and organic acid synthesis are significantly upregulated, and malic acid and lactic acid will accumulate in cells. These acidic metabolites can balance the pH inside and outside the cell, making Bt more adaptable to the environment. Under salt stress and oxidative stress, it enhances viability by adjusting proteins and antioxidant systems, and also increases functional diversity. These transcriptome reactions are not only related to cellular homeostasis and stress tolerance, but also affect the expression of toxin proteins and signaling molecules. They interweave to form a multi-level regulatory network. Meanwhile, the long-term survival of Bt in the host or natural environment also depends on the dynamic perception and transcriptional regulation of pressure signals. Nowadays, with the development of new technologies such as high-throughput sequencing, multi-omics integration and gene editing, people will have a more comprehensive and in-depth understanding of the mechanism by which Bt responds to stress. These understandings will provide a molecular-level basis for improving Bt strains, enhancing ecological adaptability and industrial application potential. Acknowledgments Thank you to the reviewers for their rigorous academic approach in reviewing this study’s manuscript and offering many constructive suggestions.
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