Expression of the Small Peptide GLP-1 in Transgenic Plants

Abstract

Glucagon-like peptide 1 (GLP-1) has great potential in diabetes therapy. In order to accumulate GLP-1 in endosperm tissue of rice, a codon-optimized GLP-1 (mGLP-1) synthetic gene was directly expressed under the control of rice storage protein glutelin GluB-1 promoter in transgenic rice plants. Unexpectedly, neither the transcripts nor the transgene products were detected in the seeds of regenerated plants. Furthermore, transcripts of GluB-1 gene in these transgenic plants were not detected, and small interfering RNAs (siRNAs) corresponding to the transgene were detected. These results indicated that the expression of mGLP-1 was silenced by co-suppression in rice transgenic seeds. To avoid silencing, mGLP-1 was fused to GFP with or without self-processing 2A sequence, and introduced into rice plants. Both chimeric genes were highly expressed in these transgenic rice seeds, indicating that gene silencing could be avoided by changing the transgene components. Furthermore, the fusion protein containing the 2A sequence were processed into GFP-2A and mGLP-1 peptides with the efficiency of more than 80%, but the processed mGLP-1 peptides were not detected. Lack of accumulation of mGLP-1 may be explained by proteolytic digestion in the cytoplasm.

Reducing Rice Seed Storage Protein Accumulation Leads to Changes in Nutrient Quality and Storage Organelle Formation

“…RM9). We generated transgenic rice in which the levels of SSPs were reduced (SSP-less mutants) using the modified Glucagon-like peptide-1 sequence as a silence-inducing sequence (Yasuda et al, 2005). Their target genes are as follows: GluA and GluB in Glu-less, GluB in GluB-less, GluB and Glb-1 in GluB·Glb-less, 13-kD prolamins in 13-kD Pro-less, RP10 in 10-kD Pro-less, and RP16 in 16-kD Pro-less (Table I).…”

“…We have identified a modified Glucagon-like peptide-1 sequence as an RNA silencing-inducible sequence (RSIS) in rice (Oryza sativa) and developed a new RNA-silencing system, which is induced by simply linking RSIS to a target gene sequence (Yasuda et al, 2005;H. Yasuda, Y. Wakasa, T. Kawakatsu, and F. Takaiwa, unpublished data).…”

RNA Silencing Induced by an Artificial Sequence That Prevents Proper Transcription Termination in Rice

“…RSIS-Mediated RNA Silencing Is Nuclear PTGS siRNAs were previously reported as being produced from the RSIS-expression cassette in GluB-less seed (Yasuda et al, 2005). To confirm whether specific siRNAs derived from the suppressed transcripts accumulate in transgenic plants, northern analysis was conducted to analyze small RNAs prepared from developing seeds of wild-type, GluB-less, GluB$Glb-less, and 13-kD Pro-less transgenic plants.…”

“…This expression system using the GluB-1 promoter and terminator is routinely used for the expression of functional proteins/ peptides in rice endosperm . However, cosuppression of endogenous GluB-1 as well as mGLP-1 was observed in the endosperm of transgenic rice plants (Yasuda et al, 2005;. When the terminator was substituted with the globulin Glb-1 terminator, GluB-1 and Glb-1 were suppressed .…”

“…In an attempt to accumulate GLP-1 peptides in rice (Oryza sativa) endosperm, a codon-optimized modified GLP-1 (mGLP-1) gene was expressed under the control of the endospermspecific glutelin GluB-1 promoter. This construct contained the GluB-1 59 untranslated region (UTR) and signal peptide, as well as the GluB-1 terminator containing the 39 UTR (Yasuda et al, 2005). This expression system using the GluB-1 promoter and terminator is routinely used for the expression of functional proteins/ peptides in rice endosperm .…”