ScienceWeek

3. CURRENT USE OF GENETICALLY MODIFIED FOODS

THE PRODUCTION OF RECOMBINANT PROTEINS IN TRANSGENIC BARLEY GRAINS

The following points are made by H. Horvath et al (Proc. Nat. Acad. Sci. 2000 97:1914):

1) Barley cultivars have been and are being bred to store large amounts of carbohydrates, oil, and protein during grain development. In germination, the stored amino nitrogen and carbohydrates are mobilized to sustain the development of the embryo and seedling. The stored carbohydrates, oils, and proteins in the kernels are exploited for feed, food, and beverages, both in the form of mature grain and as malt produced in large scale by steeping, germination, and kiln drying (1,2). Advances in the molecular genetics of barley storage proteins (3,4), elucidation of mechanisms by which storage proteins are synthesized, targeted, and sequestered for long-term storage in the endosperm cells (5), and establishment of genetic transformation procedures provide the basis for studying the production of recombinant proteins in the developing barley grain. The unique capacity of the scutellum and aleurone cells to synthesize hydrolytic enzymes during germination/malting and secrete them into the starchy endosperm provides an additional avenue for the production of recombinant proteins in this cereal.

2) Barley grains of cultivars with a 1,000-grain weight of 30-45 g contain 8-16% protein on a dry-weight basis (1). A thousand grains of barley contain on the average 4.5 g of protein of which 2.3 g is distributed to the endosperm and the remainder to the embryo and aleurone tissue. The promoters and signal peptide codes of the genes encoding the barley prolamins (hordeins) are suitable candidates for deposition of recombinant proteins in the protein bodies of the developing endosperm cells. These isopropanol-soluble proteins account for approximately 50% of the total endosperm protein, comprising the high molecular weight D, the sulfur-poor C, and the sulfur-rich B and hordein polypeptides (3,4). The different hordein proteins are coordinately expressed during endosperm development.

3) Electron microscopic analyses of wild types and mutants deficient in groups or individual hordein polypeptides in combination with biochemical analyses have revealed the following picture of the synthesis and deposition of the hordein polypeptides: the mRNAs of the Hor1, Hor2, Hor3, and Hor5 loci on chromosome 5 encoding the hordein C, B, D, and precursor polypeptides (3,5) are translated on the polysomes of the endoplasmic reticulum (ER). The N-terminal signal peptides direct the nascent polypeptide chains into the lumen of the ER, where they are cleaved off during or immediately after translocation. Monoclonal and polyclonal antibodies recognizing individual hordein polypeptides or polypeptide families indicate that they are transported in small vesicles via the Golgi cisternae into the storage vacuoles of the endosperm cells (5). Surrounded by the tonoplast, the protein bodies consist of globules containing C, B, and hordein (5) and of a reticulate component, identified as D hordein. In certain genotypes and under not clearly defined conditions, hordein polypeptides also accumulate in the cisternae of the ER.

4) The authors describe the expression of a protein-engineered thermostable (1, 3-1, 4)--glucanase with the Hor3-1 D hordein gene promoter in transgenic barley plants obtained by Agrobacterium-mediated transformation. The characteristics of and requirements for the synthesis of the recombinant protein with the endosperm-specific promoter are compared with results obtained with transgenic barley plants expressing the same recombinant protein with an aleurone-specific amylase promoter.

References (abridged):

1. Newman, C. W. & Newman, R. K. (1992) in Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology, ed. Shewry, P. R. (C.A.B., Wallingford, Oxon, U.K.), pp. 351-368

2. Briggs, D. E. (1992) in Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology, ed. Shewry, P. R. (C.A.B., Wallingford), pp. 369-401

3. Kreis, M. & Shewry, P. R. (1992) in Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology,, ed. Shewry, P. R. (C.A.B., Wallingford), pp. 319-333

4. Rechinger, K. B., Bougri, O. V. & Cameron-Mills, V. (1993) Theor. Appl. Genet. 85, 829-840

5. Rechinger, K. B., Simpson, D. J., Svendsen, I. & Cameron-Mills, V. (1993) Plant J. 4, 841-853

ScienceWeek http://www.scienceweek.com