Bt Research 2025, Vol.16, No.4, 136-146 http://microbescipublisher.com/index.php/bt 141 have also searched for regulatory elements by comparing genomes. Bioinformatics can also help identify binding sites for global regulatory factors. For example, using the information matrix scanning tool to scan the recognition sequence patterns of known regulatory proteins such as PlcR and Spo0A within the entire genome of Bt, it can predict the collection of target genes that these regulators may regulate. A study by Peng et al. found through comparative analysis that the PlcR regulatory cassette exists in various exotoxin and enzyme gene promoter regions, thus confirming that PlcR is an important global regulator of the Bt virulence gene. 5.3 Prediction of the role of non-coding RNA in gene regulation In addition to traditional protein regulators, the Bt genome also encodes various non-coding RNAs, which may play an important role in gene expression regulation. Bioinformatics provides means to predict and analyze the functions of these non-coding RNAs. First, the identification of small RNAs (sRNAs) can be performed by combining transcriptome data with algorithmic tools. For example, using tools such as Rockhopper to analyze the transcriptome of Bt, it is possible to find significantly expressed unannotated transcripts between two coding genes or on the antisense strands, thereby predicting new sRNAs (Gonçalves et al., 2021). Secondly, nuclear switches or ribozyme RNAs in known modes can also be identified by sequence search. In the genome of the Bt BMB171 strain, two types of switching RNA elements that regulate c-di-GMP and c-di-AMP metabolism were found by searching for nuclear switches of dicyclic nucleotide signal molecules. These non-coding elements can sense the concentration of the second messenger molecule in the cell and affect downstream gene transcription, and are speculated to be involved in the regulation of Bt population sensing and spore formation. In addition, bioinformatics can also predict the target mRNA of sRNA. A common method is to use software such as IntaRNA or TargetRNA to predict whether the two may bind and bind sites based on the complementary pairing energy of the sRNA sequence and the potential target mRNA sequence (Deng et al., 2018). 6 Genome Evolution and Comparative Genomics Tools 6.1 Multi-sequence alignment and phylogenetic construction (Clustal Omega, MEGA) Comparative genomics is an important means to understand the evolutionary status of Bt and the relationship between strains. One of the commonly used methods is to conduct multi-sequence alignment and phylogenetic analysis of the whole genome sequences of multiple strains of Bt and their neighbors. In the specific implementation, the whole genome alignment tools such as Mauve can be used to identify the collinear regions and rearrangements of the genomes of different strains. Then select the conservative single-copy core gene set, use Clustal Omega and other to perform multiple alignments on these gene sequences, and use software such as MEGA to build a phylogenetic tree. Within Bt strains, phylogenetic analysis can reveal the evolutionary relationships of different serotypes or virulence spectrum strains. For example, comparing the genomes of the Bt Israeli subspecies (highly toxic for Diptera mosquitoes) and the Ladybug subspecies of the Twenty-Eight Stars (highly toxic for Coleoptera), it was found that the two were highly similar in core genes, with different compositions of toxin genes on the plasmids. Systematic tree analysis also divided them into similar branches, indicating that Bt strains with different target spectrums may have common ancestors, and different insecticidal spectrums were obtained by obtaining specific plasmids. 6.2 Comparison of genomes of Bt and other Bacillus Bt belongs to the Bacillus cereus group bacteria and is closely related to anthrax, Bacillus cereus, etc. Comparing the genomes of Bt with these relative species can reveal genetic factors that determine their respective pathogenicity and ecological adaptability. In bioinformatics, genome alignment and pan-genome analysis are often used. The protein sequences of multiple Bt, B. cereus and B. anthracis were clustered by Orthovenn and other tools, and their pan-genome collections and unique gene sets were obtained (Fang et al., 2011). Analysis found that Bt and Bacillus cereus share a huge core genome, and it is difficult to distinguish it through single genes such as 16S, but the plasmids and toxin genes carried by Bt give it a unique insecticidal function. Meric et al.'s research shows that Bt and B. cereus are intertwined on evolutionary trees and are not single-line groups, which further supports the idea that the two are essentially different phenotypes of the same species. The difference is mainly caused by the virulence islands obtained on the plasmid: the Bt plasmid encodes a large number of
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