Bt Research 2025, Vol.16, No.2, 79-85 http://microbescipublisher.com/index.php/bt 80 needs to be capable of accurately identifying target molecules with low abundance or weak activity to avoid false negatives (Lloyd, 2020; Ayon, 2023). The second one is flux. That is, the number of samples that can be screened within a unit of time. HTS is usually achieved through miniaturization techniques (such as 96/384/1536-well plates), automated operation and parallel detection, and can complete the screening of tens of thousands of samples within one day (Leavell et al., 2020). The third one is repeatability. The experimental results must be stable and reliable, which requires standardized procedures, automated equipment and strict quality control to ensure (Szymański et al., 2011; Zeng et al., 2020). 2.3 Comparison of HTS with conventional screening methods Compared with traditional low-throughput methods, HTS has obvious advantages in terms of efficiency, scale and data quality. Traditional methods mainly rely on manual operation and single-sample testing, which are time-consuming and require a small sample size, making it difficult to meet the demands of large-scale screening. By using automation and miniaturization technologies, HTS has significantly increased the screening speed and sample coverage, and also reduced the cost of individual samples and the demand for manpower (Szymański et al., 2011; Leavell et al., 2020). In addition, HTS can also be combined with modern bioinformatics, synthetic biology and other technologies to facilitate data-driven efficient screening and functional mining (Sarnaik et al., 2020; Zeng et al., 2020; O’Connor et al., 2024). 3 HTS Technologies Applied to Bt Toxin Discovery 3.1 Genomic and metagenomic approaches for toxin gene identification Genomics and metagenomics have provided important tools for the discovery of Bt toxin genes. Mendoza-Almanza et al. (2020) identified a variety of new toxin genes, including Cry, Cyt, Vip and Sip, by conducting whole-genome sequencing on Bt strains and environmental samples. Using gene editing techniques such as CRISPR/Cas9, functional verification of candidate toxin genes can also be conducted to clarify their mechanisms of action in target insects (Wang et al., 2016; Zhang, 2024). These molecular biological methods have greatly accelerated the discovery and functional research of newBt toxin genes. 3.2 Automated protein expression and activity screening platforms The automated protein expression system, combined with a high-throughput screening platform, can express and purify many candidate Bt toxin proteins in a short period of time. Subsequently, through automated activity screening processes (such as phage-assisted continuous evolution systems), toxin variants with strong binding ability to specific insect receptors can be rapidly identified (Badran et al., 2016). This method not only improves the screening efficiency, but also enables the targeted evolution of new and highly efficient toxins for resistant pests, providing a new idea for solving the resistance problem. 3.3 High-throughput bioassays for insecticidal activity evaluation High-throughput bioassay is the key to evaluating the insecticidal activity of Bt toxin. Through automatic sample allocation and standardized feeding conditions, the lethal or inhibitory effects of a large number of toxin samples on different pests can be simultaneously tested in multi-well plates (Deans et al., 2017). Furthermore, field monitoring networks (such as Sentinel cornfield trials) combined with laboratory bioassays can dynamically assess the sensitivity changes of pests to Bt toxins and promptly detect the trend of resistance development (Dively et al., 2020). These high-throughput bioassay methods provide reliable data support for the screening and resistance management of new Bt toxins. 4 Integration with Omics and Computational Tools 4.1 Transcriptomics and proteomics for functional characterization Transcriptomics can reveal the expression patterns of toxin genes under different conditions, which is helpful for identifying candidate genes that are highly expressed in target insects or specific environments. Proteomics is the direct detection of the expression, modification of toxin proteins and their interaction with target molecules by methods such as high-resolution mass spectrometry, thereby conducting more in-depth research on the functions of toxins. A single omics can discover some biomarkers, but combining multiple omics can more comprehensively
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