PCR cloning and nucleotide sequence analysis of cDNAs corresponding to an antifungal protein from larvae of tenebrio molitor

Abstract

Previously known antifungal protein, tenecin 3, secreted by larvae of Tenebrio molitor was isolated and its N-terminal 25 amino aci sequences were determined. In order to isolate cDNA clones of tenecin 3, mRNA was prepared from larvae of Tenebrio molitor an cDNA libraries were constructed using $\lambda$gt11 as a cloning vector. PCR experiments were carried out to screen the cDNA libraries. PCR primers were designed by deducing nucleotides from the N-terminal amino acid sequences and modifying a $\lambda$gt11 sequencing primer. The PCR-amplified DNA fragments with appropriate combination of the primers on the cDNA libraries as templates were cloned and their identity as cDNAs corresponding to tenecin 3 was confirmed by DNA sequencing. Arrangement of the DNA sequences of overlapping cDNAs relative to one another identified 326 nucleotides for mRNA encoding tenecins 3. The analysis of the nucleotide suquences indicates that tenecin 3 is composed of 78 amino acids and synthesized as a prepeptide which is matured by cleavage of a signal peptide. Tenecin 3 is rich in glycine (44%) and has a characteristic tetrapeptide unit which is 14 times repeated.