Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

Abstract

Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the Nand C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5 and 3 flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp 67 and Arg 68 , as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5-100 µg mL −1 for spore germination and 1-50 µg mL −1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.

Comparative study of the effects of Tartary buckwheat seed and sprout consumption on the physiological indices and gut microbiota of C57BL/6J mice

“…(Kanauchi et al, 2000). TIs are defence proteins in plants but toxic to mammals and humans(Ruan et al, 2018). TI addition to the diet for mice can increase the pancreas weight, decrease trypsin activity in the intestine and even cause colitis by affecting intestinal microflora(Pickert et al, 2020;Roy & Schneeman, 1981).…”