Abstract
Colaphellus bowringi Baly mainly damages cruciferous vegetables, leading to huge economic losses. The secretory insecticidal protein (Sip) of Bacillus thuringiensis (Bt) has high insecticidal activity against C. bowringi Baly. The tertiary structure of Sip1Aa protein was analyzed by homologous modeling and other bioinformatics methods to predict the conserved domain of Sip1Aa protein. Acidic and basic amino acids in the conserved domain were selected, and alanine was used to replace these amino acids by site-directed mutation. The difference between the insecticidal activities of mutant protein and Sip1Aa protein was analyzed. The insecticidal activities of H99A, K109A, K128A, and E130A against C. bowringi Baly were significantly increased, among which that of K128A was the most obviously changed, and the LC50 value was decreased by about 10 times compared with that of Sip1Aa protein. The LC50 value of mutant E130A was 0.286 μg/mL, which was about six times less than that of Sip1Aa. K128 and E130 were both in the β9–β10 loop. The toxicity of D290A, H242A, and H303A to C. bowringi Baly was significantly reduced, and their LC50 value increased by about six, eight, and three times compared with that of Sip1Aa protein, respectively. This study showed that acidic and basic amino acid residues played a certain role in the toxicity of Sip1Aa protein, and the loss of side chains in key residues had a significant impact on the insecticidal activity of the protein. This study provides the theoretical basis for revealing the relationship between the structure and function of Sip1Aa protein and also provides a new method for the subsequent study of sip gene.
Highlights
Bacillus thuringiensis (Bt) is a gram-positive Bacterium that has been widely used in the control of agricultural and forestry pests such as Lepidoptera, Diptera, and Coleoptera due to its high specificity to target pests and non-target biosecurity (Schnepf et al, 1998)
Donovan studied the insecticidal activity of Bt strains on Coleoptera and found that the culture supernatant of some strains has insecticidal activity on the larvae of Colorado potato beetle (CPB) at the lethal dose 50 (LD50) of 0.12 (0.09∼0.15) μg/mL
This study provides the theoretical basis for explaining the relationship between the structure and function of Sip1Aa and provides the genetic resources for the prevention and control of Coleoptera (Aronson and Shai, 2001; Hu and Aroian, 2012; Fuente-Salcido et al, 2013), providing guidance for the study of the structure and activity mechanism of Sip1Aa protein
Summary
Bacillus thuringiensis (Bt) is a gram-positive Bacterium that has been widely used in the control of agricultural and forestry pests such as Lepidoptera, Diptera, and Coleoptera due to its high specificity to target pests and non-target biosecurity (Schnepf et al, 1998) It is the most successful biological insecticide for major agricultural pests at present (Xu et al, 2018). In 2012, the sip gene containing 1038 bp and encoding 345 amino acid sequences was cloned and identified from another Bt strain QZL26 in our laboratory, but the insecticidal activity was not reported (Liu et al, 2012). Our laboratory cloned a sip gene that contains 1188 bp and encodes 395 amino acid sequences from Bt strain DQ89 in 2015, which showed high toxicity to Colaphellus bowringi Baly, with LC50 of 1.542 mug/mL (Jinbo et al, 2015)
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