Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants

• Published in: Plant molecular biology 1997-05, Vol.34 (1), p.15-29
• Main Authors: Keeler, S.J. (Experimental Station, E.I. Dupont de Nemours, Wilmington, DE (USA).), Maloney, C.L, Webber, P.Y, Patterson, C, Hirata, L.T, Falco, S.C, Rice, J.A
• Format: Article
• Language: English
• Subjects: ACTIVADOR GENICO; ACTIVATEUR GENIQUE; ALMACENAMIENTO DE SEMILLAS; Amino Acid Sequence; Base Sequence; CODE GENETIQUE; CODIGO GENETICO; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; Fabaceae - genetics; GENE ACTIVATORS; GENE EXPRESSION; GENETIC CODE; GENETIC ENGINEERING; Genetic Vectors; GENIE GENETIQUE; GRAINE; INGENIERIA GENETICA; LISINA; LYSINE; Lysine - biosynthesis; METHIONINE; METIONINA; Molecular Sequence Data; NICOTIANA; Nicotiana - chemistry; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; NUCLEOTIDE SEQUENCE; Plant Proteins - biosynthesis; Plant Proteins - chemistry; Plant Proteins - genetics; PLANTAS TRANSGENICAS; PLANTE TRANSGENIQUE; Plants, Genetically Modified; Plants, Medicinal; Plants, Toxic; Promoter Regions, Genetic; Protein Structure, Secondary; PROTEINAS; PROTEINE; PROTEINS; SECUENCIA NUCLEOTIDICA; SEED STORAGE; SEEDS; Seeds - growth & development; Seeds - metabolism; SEMILLA; SEQUENCE NUCLEOTIDIQUE; STOCKAGE DES SEMENCES; Transformation, Genetic; TRANSGENIC PLANTS
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1023/A:1005809900758

We have designed protein molecules based on an alpha-helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean beta-conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean beta-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.