Manipulation of the napin primary structure alters its packaging and deposition in transgenic tobacco (Nicotiana tabacum L.) seeds

Napin is a 2S storage protein found in the seeds of oilseed rape (Brassica napus L.) and related species. Using protein structural prediction programs we have identified a region in the napin protein sequence which forms a `h ydrophilic loop' composed of amino acid residues located at the protein sur face. Targeting this region, we have constructed two napin chimeric genes c ontaining the coding sequence for the peptide hormone leucine-enkephalin as a topological marker. One version has a single enkephalin sequence of 11 a mino acids including linkers and the second contains a tandem repeat of thi s peptide comprising 22 amino acids, inserted into the napin large subunit. The inserted peptide sequences alter the balance of hydrophilic to hydroph obic amino acids and introduce flexibility into this region of the polypept ide chain. The chimeric genes have been expressed in tobacco plants under t he control of the seed-specific napA gene promoter. Analyses indicate that the engineered napin proteins are expressed, transported, post-translationa lly modified and deposited inside the protein bodies of the transgenic seed s demonstrating that the altered napin proteins behave in a similar fashion to the authentic napin protein. Detailed immunolocalisation studies indica te that the insertion of the peptide sequences has a significant effect on the distribution of the napin proteins within the tobacco seed protein bodi es.