Abstract
Glutelin is a major seed storage protein, accounting for 60–80 % of the total endosperm protein content in rice. To test whether we could augment the expression of an introduced recombinant protein in rice by suppressing the glutelin gene, we generated transgenic glutelin RNAi (glu RNAi) rice seeds. RNA gel blot analyses confirmed that the endogenous glutelin gene was severely suppressed in these transgenic rice lines. RT-PCR analysis further revealed that all the members of glutelin multigene family were downregulated. Transgenic glu RNAi rice seeds expressing a recombinant red fluorescent protein (RFP) showed stronger fluorescence than seeds transformed with the RFP gene only. Western blot analysis further revealed that the relative accumulation of RFP in glu RNAi seeds was twofold higher than that in the RFP-only transgenic seeds. These results suggest that RNAi targeting of an endogenous storage protein could be of great utility in obtaining higher transgene expression in genetically engineered rice and other plant lines.
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Acknowledgments
We thank Dr. Tom Okita and Dr. Ju-Kon Kim for kindly providing the anti-AGPase antibody and pMJ202 vector, respectively. We also thank Dr. Tanaka-Katsube for technical assistance and helpful discussions. This work was supported by National Academy of Agricultural Science, Rural Development Administration (PJ006680 to Y.-M. Kim), Korea.
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11816_2012_230_MOESM1_ESM.pptx
Fig. S1. Genomic Southern blot analysis of Glu RNAi transgenic rice lines. Genomic DNAs from leaf tissues were digested with XhoI (Xh) and hybridized with the probes indicated in Figure 1. P, pGluRNAi vector positive control; WT, non-transgenic wild-type rice used as a negative control. (PPTX 276 kb)
11816_2012_230_MOESM2_ESM.pptx
Fig. S2. Analysis of the T-DNA flanking position in Glu RNAi rice plant line. The horizontal line represents rice chromosome 6. Filled arrows indicate the direction and position of the gene incorporated into the flanking region of the T-DNA in the rice genome. BL and BR indicate the borders of the pGluRNAi T-DNA. (PPTX 38 kb)
11816_2012_230_MOESM3_ESM.pptx
Fig. S3. Quantification of each band using the SCION image processing software. The arrows indicate each band’s corresponding pixel in the plot image. The bands analyzed are marked from 1 to 13. (PPTX 361 kb)
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Kim, YM., Lee, JY., Lee, T. et al. The suppression of the glutelin storage protein gene in transgenic rice seeds results in a higher yield of recombinant protein. Plant Biotechnol Rep 6, 347–353 (2012). https://doi.org/10.1007/s11816-012-0230-7
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DOI: https://doi.org/10.1007/s11816-012-0230-7