Trypsin inhibitor gene sequence analysis in the genus Lens Mill.

Proteinase inhibitors are ubiquitous in legumes and other plant species. They are considered to be natural defence compounds that help to protect plants from attacks by insects, pathogens and other predators. These inhibitors are interesting for the study of host-pathogen co-evolution, as regulators of endogenous enzymes and as markers in studies on plant evolution and diversity. In the context of a wider study to isolate and characterise trypsin inhibitors in lentil, an analysis was started in taxa of the genus Lens. Three forward and one reverse specific nucleotide primers were designed on the conserved aminaocid sequence of the trypsin inhibitor from Pisum sativum L. taken from EMBL database. First, these primers were tested for DNA amplification in lentil (Lens culinaris Medik. subsp. culinaris). The amplification with each of the forward primers and the reverse produced one band of about 300, 250, and 200 bp respectively. The first combination of primers was used to amplify other wild Lens taxa and produced one band in all samples, except for L. nigricans (M. Bieb.) Godr., for which a good amplification product was obtained using the third forward primer. Possibly, in this species, the upstream region is not as well conserved as in the other related taxa. As a matter of fact, based also on other molecular evidence, L. nigricans is considered the most divergent species within the genus. On the other hand, partial TI gene sequence from the other Lens species showed very few polymorphisms. Phylogenetic inferences are deducted and comparisons between the phylogenetic behaviour of these sequences and the previously analysed Internal Transcribed Spacers (ITS) in the genus Lens are produced.