Isolation and cloning of the Pseudomonas fluorescens chitinase gene – An ecofriendly approach for its use as a specific biopesticide

Insect pests are one of the major biotic factors limiting the yield of agricultural food crops. In routine agricultural practice, chemical pesticides are used to control the insect pests; however, repeated spraying leads to numerous environmental and health concerns. The search for ecofriendly and sustainable alternatives to chemical pesticides has led to the exploitation of biological control agents. Among these, chitinase plays a target-specific insecticidal activity by degrading the chitin in the insect exoskeleton and gut regions, making it a desirable alternative to chemical
pesticides. Therefore, the objective of the study was to isolate and clone the Pseudomonas fluorescens chitinase gene
for its use as a specific biopesticide. Full-length chitinase gene coming under family 18 Group D glycosyl hydrolases
was isolated from P. fluorescens genomic DNA using polymerase chain reaction amplification and cloned into an
expression vector pET32C+ and transformed into Escherichia coli. The sequencing results showed that the chi gene
containing 1800 bp long open reading frame encoding 353 amino acids. Deduced amino acid sequence showed that the protein consisted of chitin-binding domain, a catalytic domain, and a fibronectin Type III domain also an amino terminus signal peptide. This study allows the dentification of new, target-specific bacterial metabolite as a biopesticide for safe, environment-friendly pest management strategi