Molecular Characterization and Cloning of the enzymatic portion of a chitin synthase from Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora

Abstract

Moniliophthora perniciosa is a phytopathogenic basidiomycosis that affects cacao; alternatives for its control and resistance for its development have not given significant results. A new control alternative is the use of enzyme inhibitors from the chitin synthesis route. Therefore, this work aims to characterize and clone the enzymatic portion of Moniliophthora perniciosa chitin synthase as a tool to enable in vitro tests of the potential inhibitors of this phytopathogen. The complete chitin synthase sequence was analyzed; the transmembrane region, introns and UTRs were removed to design specific primers. PCR, sequencing, plasmid linearization (pET28a and pET42a) and their digestion of inserts for ligation were performed. The transformation was carried out by electroporation and thermal shock into E.coli DH5α and BL21 strains. Colony PCR, plasmid extraction and PCR were performed to confirm the transformed ones. There was positive amplification of the interest region with the designed primers, confirmed by sequencing and alignment with the chitin synthase sequence from GenBank. Digestion and binding to the pET42a vector were efficient. Despite the success in amplification, digestion and ligation, the colonies grown and transformed only contained empty vectors. Testing new vectors and new proportions with the already validated protocols for amplification, digestion and binding of the nucleotide sequence of M. perniciosa chitin synthase will allow an efficient transformation.

DOI:https://doi.org/10.56238/uniknowindevolp-142