Cloning and characterization of Thaumatin-like gene(TLP) from Chumphon Cacao.

Abstract

In this work, the Thaumatin-like protein (TLP) from Chumphon 1 Cacao (Theobroma cacao.L) was successfully cloned and based on bioinformatics analysis and prediction of antifungal functional properties, it was successfully induced by IPTG at a low temperature, transformed in Escherichia coli, and detected by SDS-PAGE. Antifungal activity was mainly revealed by functional analysis and prediction of protein structure bioinformatics, BLAST and Multi-Sequence Alignment analysis and phylogenetic tree construction were used to compare the relationship and similarity between homologous species, and molecular docking and molecular dynamic simulation evaluation were carried out using molecular bioinformation technology proteins and beta-1,3-glucan, beta-1,2-Glucan, beta-1,6-Glucan, and beta-1,4-Mannan, four important components of the fungal cell membrane structure, and the degree of tightness of TLP in this dynamic simulation process induces fungal cell death by binding to the cell membrane components and then causing them to lyse. For example, the dynamic values of RMSD, RMSF, and H-Bonds were calculated after the dynamic simulation to evaluate whether these glucans could bind to TLP proteins and the degree of binding. Based on the above data, it is concluded that beta-1,3-glucan has a strong and stable binding relationship with TLP protein, which provides strong evidence for subsequent antifungal testing of TLP in vitroThese discoveries offer a fundamental comprehension of the TLP gene in Chumphon Cacao and emphasize its significance in creating cacao cultivars that are resistant to illnesses.In this work, the Thaumatin-like protein (TLP) from Chumphon 1 Cacao (Theobroma cacao.L) was successfully cloned and based on bioinformatics analysis and prediction of antifungal functional properties, it was successfully induced by IPTG at a low temperature, transformed in Escherichia coli, and detected by SDS-PAGE. Antifungal activity was mainly revealed by functional analysis and prediction of protein structure bioinformatics, BLAST and Multi-Sequence Alignment analysis and phylogenetic tree construction were used to compare the relationship and similarity between homologous species, and molecular docking and molecular dynamic simulation evaluation were carried out using molecular bioinformation technology proteins and beta-1,3-glucan, beta-1,2-Glucan, beta-1,6-Glucan, and beta-1,4-Mannan, four important components of the fungal cell membrane structure, and the degree of tightness of TLP in this dynamic simulation process induces fungal cell death by binding to the cell membrane components and then causing them to lyse. For example, the dynamic values of RMSD, RMSF, and H-Bonds were calculated after the dynamic simulation to evaluate whether these glucans could bind to TLP proteins and the degree of binding. Based on the above data, it is concluded that beta-1,3-glucan has a strong and stable binding relationship with TLP protein, which provides strong evidence for subsequent antifungal testing of TLP in vitroThese discoveries offer a fundamental comprehension of the TLP gene in Chumphon Cacao and emphasize its significance in creating cacao cultivars that are resistant to illnesses.