Bt Research 2025, Vol.16, No.3, 110-117 http://microbescipublisher.com/index.php/bt 112 3 Transcriptomic Approaches in Bt Stress Studies 3.1 High-throughput sequencing (RNA-Seq) RNA-Seq is a common transcriptome analysis method at present, which can comprehensively record the changes in gene expression of Bt under different pressures. For instance, in an alkaline environment, researchers found that the genes related to glycolysis and organic acid synthesis of Bt were significantly upregulated through methods such as RNA extraction, chip hybridization and qRT-PCR. This indicates that Bt can adapt to alkaline conditions by adjusting metabolism. In salt stress studies, RNA-Seq was also used to analyze the expression changes of antioxidant enzymes (APX, SOD) and some stress genes, thereby helping to understand the salt tolerance of Bt and its role in promoting plant growth (Ali et al., 2022). 3.2 Bioinformatic pipelines The analysis of transcriptome data usually requires a complete set of bioinformatics steps. First, quality control should be carried out, such as using FastQC to check data quality. The next step is to align the sequence to the reference genome. Commonly used software includes HISAT2 and STAR. Then the expression of the gene needs to be quantified. HTSeq or featureCounts can do this. Next comes differential expression analysis, with commonly used methods including DESeq2 and edgeR. The last step is functional annotation and pathway analysis, such as understanding gene functions and related pathways through GO and KEGG. These steps combined can systematically reveal the metabolic pathways, signal transduction and stress response mechanisms of Bt under different stresses. 3.3 Emerging methods In addition to the traditional RNA-Seq, some new methods are now gradually being applied to the stress research of Bt. Proteomics, such as LC-MS/MS, can be used to analyze the changes of Bt secreted proteins under salt stress, which can help reveal the regulatory mechanisms at the protein level (Subramanian et al., 2021). Whole-genome resequencing has also been used to search for and screen for important gene mutations related to UV resistance (Ma et al., 2023) (Figure 2). In addition, some new technologies, such as single-cell transcriptomics and spatiotemporal dynamic transcriptomics, are also expected to further refine the molecular reaction network of Bt under stress in the future. Figure 2 Classification of gene variations compared with KEGG database by blast (Adopted from Ma et al., 2023) 4 Transcriptomic Responses to Specific Stress Conditions 4.1 Heat and cold stress In high-temperature environments, Bt activates related genes such as heat shock proteins (HSPs). These proteins help fold and repair damaged proteins, thereby enhancing cell tolerance. Proteomics research has found that at high temperatures, genes related to DNA synthesis and heat shock proteins in Bt are upregulated, while proteins related to carbon metabolism and energy metabolism are downregulated. This indicates that cells devote more
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