Bowman-birk inhibitor (BBI) genes in wild Phaseolus species: biochemical and molecular analyses

The study of BBI family in different Phaseolus wild species may lead to the identification of new variants potentially active against proteinases from different insect pests or to new variants at chymotrypsin inhibitory site, having anti-carcinogenic properties. With the exception of P. leptostachyus, where no BBI genes were amplified by PCR, all the remaining species analysed showed two or more BBI genes. Southern blot hybridisation confirms this result. Indeed, no hybridisation was detected in P. leptostachyus while four to six hybridisation fragments were observed in P. coccineus, P. glabellus, and P. maculatus. In these taxa an internal EcoRI restriction site is present in the isolated BBI gene, suggesting that at least two of the fragments can be the result of the cleavage of a single gene. Analysis on the primary structure of the gene isolated from all the wild Phaseolus species showed that these genes are strictly conserved and nucleotide identities among the different species ranged between 90-99%. Among the analysed species only P. coccineus and P. costaricensis, similarly to P. vulgaris, showed two types of trypsin binding loop that differed each other for the residue at P'2 position: the most active P'2-Ile and the weaker P'2-Arg. This finding confirms that P. costaricensis is more closely related to the domesticated Phaseolus group than the other wild species.