Bioprospecting in both domestic and wild species of the genus Vigna Savi: the Bowman-Birk inhibitors

The Vigna Savi genus, of the Fabaceae family, includes different important crops, such as Vigna unguiculata (cowpea), Vigna angularis (adzuki bean), and others. They can grow in dry climates and poor soils, and their seeds are abundant both in nutrients and nutraceutical compounds. Variants of these plants are also present in the wild while the majority of the Vigna species were never domesticated. Among the useful compounds of these plants are the Bowman-Birk protease inhibitors (BBI), molecules produced as defensive mechanism against insect attacks. Various nutraceutical effects of these proteins on humans have been demonstrated, including chemopreventive e and anticarcinogenic ones. This study analysed the natural genetic variability of two BBI isoforms in different species of the genus Vigna. In particular, the two isoforms encode for a trypsin-trypsin BBI, characterized by two trypsin-specific domains (BBI-TT) and the second one, a trypsin-chymotrypsin BBI, characterized by one trypsin and one chymotrypsin-specific domain (BBI-TC). For this purpose, the sequences of both genes were first obtained from 12 published genomes. Then DNA was extracted from 44 Vigna species and different primers were used to amplify and then sequence both genes. Ultimately, the BBI-TT were obtained from 34 species, revealing 55 haplotypes corresponding to 40 isoforms and 32 mature proteins. Meanwhile the BBI-TC were obtained from 40 species, revealing 70 haplotypes corresponding to 56 isoforms and 39 mature proteins. A phylogenetic tree of both genes was constructed to study their evolution and to apply an ancestral sequence reconstruction analysis. Furthermore, sites under selective pressure were identified with different algorithm approaches. In conclusion, this work provided insights about the evolution of these genes in Vigna and allowed the detection of several natural forms of BBI. These natural proteins could potentially be used to improve the defences of cultivated plants or for pharmaceutical and nutritional purposes.