A trypsin inhibitor cDNA from a novel source, snail medic ( Medicago scutellata L.): cloning and functional expression in response to wounding, herbivore, jasmonic and salicylic acid

Abstract

A cDNA clone coding for the trypsin inhibitor MsTI from snail medic ( Medicago scutellata L.) was isolated by RT-PCR using poly(A) + mRNA purified from immature seeds. A partial cDNA fragment was produced using degenerated oligonucleotides designed from the available MsTI amino acid sequence. The full-length cDNA was obtained by RACE-PCR experiments. A putative signal peptide for subcellular localization is part of the open reading frame. This is the first example of a cloned cDNA sequence from the pasture legume M. scutellata. Southern analysis revealed the presence of a small size multigene family. When polyclonal antibodies raised against the purified seed protein were utilized in Western blot analyses, a 6.9 kDa protein from crude extract was recognized. Expression studies were carried out in order to define the role of the MsTI inhibitor in the natural defense system developed by plants against pathogen attacks. A constitutive expression pattern of the MsTI gene was observed in stems, while the transcript was absent in both root and leaf tissues. Local and systemic accumulation of MsTI mRNA was detected in leaves in response to mechanical wounding and exposure to Hypera postica larvae. The insect damage determined a faster response compared to mechanical treatment. In addition, induction of the MsTI gene expression after exogenous applications of salicylic acid and jasmonic acid was observed. An insect bioassay, performed with larvae of the insect pest Hyphantria cunea fed with the purified protein, was carried out. Results from this study suggest a role of MsTI protein in plant defense reactions, highlighting its possible use for biotechnological applications.