Bt Research 2025, Vol.16, No.4, 136-146 http://microbescipublisher.com/index.php/bt 140 5 Bioinformatics Mining of Toxin Genes and Their Regulatory Mechanisms 5.1 Sequence alignment and phylogenetic analysis tools for the Cry gene family The most eye-catching gene family of Bt is the insecticidal crystal protein gene (cry gene) family. As more and more Bt genomes are sequenced, it is found that the cry gene family is very large, and hundreds of different types of Cry toxins have been identified so far. In order to clarify the evolutionary relationship of the cry gene family, it is often necessary to use sequence alignment and phylogenetic analysis tools. The general process is to first use multiple sequence alignment software (such as Clustal Omega, Muscle, etc.) to alignate the amino acid sequences of different Cry toxins to find conserved regions and mutation sites. Then, phylogenetic tree construction software (such as MEGA, IQ-TREE, etc.) is used to construct a phylogenetic tree of Cry protein based on the alignment results (Figure 1) (Lechuga et al., 2020). Under the new classification nomenclature system, toxin proteins are classified according to differences in three-dimensional domains. Through structural analysis and phylogenetic research, Crickmore et al. proposed a new nomenclature of Bt and other bacterial source insecticidal proteins, retaining the "Cry" name of traditional crystal proteins with three domain structure, and retaining those similar to the Cyt family as "Cyt", while other types of insecticidal proteins are named "Mpp", "Tpp", "Vip", etc. according to homologous families. The proposal of this structural classification requires a large number of sequence phylogenetic analysis as support. Therefore, bioinformatics analysis plays a key role in determining internal relationships of cry gene families and guiding naming. In practical applications, researchers can use models such as adjacency method (NJ) and maximum likelihood (ML) provided by MEGA software to quickly build a system tree. For example, aligning a set of newly discovered Cry protein sequences with a known Cry family and making achievements can determine which subclass the new sequence belongs to or whether it forms an independent branch (Zhou et al., 2020). Figure 1 HER1410 chromosomal cryBa4-containing genomic island analysis (Adopted from Lechuga et al., 2020) 5.2 Regulatory element identification and promoter analysis tools The expression regulation of Bt virulence gene involves a variety of cis-acting elements and trans-acting factors. To reveal these regulatory elements, commonly used bioinformatics tools look for conserved sequence patterns or known binding sites in upstream promoter regions of the gene. The MEME kit is a commonly used sequence pattern discovery tool that can search for enriched short sequence motifs in a given set of sequences, which is ideal for finding common regulatory elements. Another type of tool, such as the PlantCARE database, contains rich information on plant promoter cis-components. Although it is mainly aimed at plants, the identification of some basic components (such as TATA-box and CAAT-box) is also of reference value for Bt promoter analysis. For bacteria-specific promoter elements, prokaryotic promoter prediction tools such as BPROM can predict potential Sigma factor binding sites based on the typical sequence of -35/-10 boxes (Wang et al., 2013; Zhang et al., 2022). For example, BPROM analysis of the upstream sequence of the two-component system gene or toxin gene of Bt can predict the recognition sequence position of Sigma factors such as SigA or SigH. Recent studies
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