High Level Accumulation of Recombinant Peptides In Marker-Free Transgenic Rice Seeds

Abstract:

We indicate here that the Hm/MAT-vector can generate marker-free transgenic rice seeds which requires a high level accumulation of recombinant peptide. The accumulation of a glucagon-like-peptides 1 (GLP-1), inserted to the variable region of rice seed storage protein globulin (Glb), was analyzed in marker-free transgenic rice seeds. The Hm/MAT-vector consists of the R/RS site-specific recombination system from Zygosaccharomyces rouxii into which the hygromycin phosphotransferase (hph) gene and the isopentenyltransferase (ipt) gene from Agrobacterium tumefaciens have been inserted. The presence of the GLP-1 gene was confirmed by PCR analyses, and the accumulation of modified globulin protein was confirmed by Western blot. GLP-1 concentrations in seeds were determined with a radioimmunoassay (RIA). We found that accumulation of GLP-1in marker-free transgenic rice mature seeds generated by the Hm/MAT-vector ranged from 3.5 to 50.9 ľg/20mg mature seed. These results represent that the Hm/MAT-vector is an effective marker-free system, but also is a booster system for high level accumulation of recombinant peptides. We also report that an extracted and trypsin-digested GLP-1 from marker-free transgenic rice seed is able to raise intracellular levels of cAMP in in vitro studies. These findings provide new pharmaceutical cereals that stimulate secretion of insulin from pancreatic ß-cells.