[Relationship between structure and function of loops from Bacillus thuringiensis insecticidal crystal protein...

Area of Science:

• Molecular Biology
• Insect Toxicology
• Protein Engineering

Background:

• Bacillus thuringiensis insecticidal crystal proteins (Cry proteins) are vital for pest control.
• Understanding the structure-function relationship of Cry proteins, like Cry1Ba, is key to optimizing their efficacy.
• The role of specific protein domains, such as loops, in Cry1Ba's toxicity against Plutella xylostella remains to be fully elucidated.

Purpose of the Study:

• To investigate the relationship between the structure and function of loops in Bacillus thuringiensis insecticidal crystal protein Cry1Ba.
• To determine the influence of amino acid mutations within Cry1Ba loops on its toxicity against the diamondback moth, Plutella xylostella.
• To identify critical loop regions responsible for Cry1Ba's insecticidal activity.

Main Methods:

• Five site-directed mutations were introduced into the loops of the Cry1Ba gene using overlapping primer PCR.
• Mutated Cry1Ba proteins were expressed in E. coli BL21 (DE3).
• Toxicity assays were performed against Plutella xylostella larvae, and LC50 values were determined. Protein structures were predicted using Swiss-Model.

Main Results:

• A deletion mutation in loop1 (M1) significantly reduced Cry1Ba toxicity (LC50 35.51 μg/mL) compared to wild-type (LC50 0.96 μg/mL).
• Mutations in loop2 (M2, M3, M4) also decreased Cry1Ba toxicity to varying extents.
• A mutation in loop3 (M5) showed a slight, non-significant increase in activity against Plutella xylostella (LC50 0.81 μg/mL).

Conclusions:

• Loop1 and Loop2 of Cry1Ba are critical determinants of its insecticidal activity against Plutella xylostella.
• Amino acid mutations in these loops can significantly alter the protein's toxicity profile.
• Targeted mutagenesis of specific loops offers a strategy for potentially enhancing or modifying Cry1Ba efficacy.