Preparation of a Seed Specific Construct Containing Sequences OMEGA and SAR in Order to Increasing Gene Expression in Plant Seed

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Abstract

Low level expression of transgene is one of the most important factors limiting production of recombinant proteins in plants. Some ways that help to increasing gene expression are selecting of appropriate tissue and improving transgen expression. For this ,purpose, in this study we endeavorto obtain specifically gene expressionin plant seeds as well as improving desired gene expression by design and preparation of seed specific construct containing effective sequences in expression (OMEGA and SAR).Omega sequence acts as translation regulator and enhances the translation efficiency. It is believed that SAR fragment inhibits the silence of transgene by different mechanisms.In the first step, omega sequence joined to upstream of GUS gene by specific primers and PCR reaction. The SAR sequence (pS206-1), isolated from tobacco genome and then spliced to downstream of GUS gene by SOEingPCR reaction. The resulting fusion fragment with the expected size cloned in TA cloning vector. Cloning in TA vector was confirmed by colony-PCR, restriction enzyme digestion and sequencing. To obtain seed specific cassette we subcloned fusion fragment in plant expression vector pBI121 in which CaMV35s promoter has been replaced with seed specific promoter. Cloning was confirmed by colony-PCR and digestion.

References

Allen, G. C., Spiker, S. and Thompson, W. F. 2000. Use of matrix attachment regions (MARs) to minimize transgene  silencing. Plant Molecular Biology, 43: 361-376.
Bagheri, Kh., Jalali Javaran, M., Mahboudi, F., Moeini, A. and Zebarjadi, A. 2010.  Designing and development of Gama Interfron construct and expression of this protein in Brassica napus seeds. Iranian Journal of Biology. 23 (2): 151-160. (In Persian).
Boothe, J. G., Parmenter, D. L. and Saponja, J. A. 1997. Molecular farming in plants: Oilseeds as vehicles for the production of pharmaceutical proteins. Drug Development Research, 42(3-4): 172-181.
Dellaporta, S. L., Wood, J. and Hicks, J. D. 1983. A plant DNA mini preparation.Version II. Plant Molecular. Biology. Reports, 1: 19-21.
Droogenbroeck, B. V., Cao, J., Stadlmann, J., Altmann, F., Colanesi, S., Hillmer, S., Robinson, D. G., Lerberge, E. V., Terryn, N., Montagu, M. V., Liang, M., Depicker, A. and Jaeger, G. D. 2007. Aberrant localization and under glycosylation of high accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proceedings of the National Academy of Sciences of the United States of America, 104: 1430-1435.
Gallie, D. R. and Walbot, V. 1992. Identification of the motifs within the tobacco Mosaic virus 5' –leader responsible for enhancing translation. Nucleic Acids Research, 20(17): 4631-4638.
Gallie, D. R., Feder, N., Schimke, R. T. and Walbot, V. 1991. Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression. Molecular and General Genetics, 228: 258-264.
Leelavathi, S. and Reddy, V. S. 2003. Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Molecular Breeding, 11: 49-58.
Mandeles, S. 1968. Location of unique sequences in tobacco mosaic virus ribonucleic acid. The Journal of Biological Chemistry, 243(13): 3671-3674.
Mohsenpour, M., Tohidfar, M., Babaeian, N. A., and Habashi, A. A. 2007. Design and construction of a recombinant plasmid pBI121-Glu in order to Agrobacterium mediated transformation of cotton. Journal of Agricultural Sciences and Natural Resources 14(4): 112-124. (In Persian).
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiology, 15: 473- 497.
Swarnalatha, D. I., Dinesh kumar, V., Ansari, N. A. and Sivasankar, A. 2010. Studies on the expression pattern of seed-specific napin promoter (BcNAI) in transgenic (Nicotiana tabacum L.) tobacco seeds. International Journal of Environmental Science and Development, 1: 20-23.
Thompson, W. F., Spiker, S. and Allen, G. C. 2006. Matrix attachment regions and transcriptional gene silencing. BLUK050-Grasser, 136-161.
Twyman, R. M., Stoger, E., Schillberg, S., Christou, P. and Fischer, R. 2003. Molecular farming in plants: Host systems and expression technology. Trends in Biotechnology, 21: 570-578.
Xue, H., Yang, Y. T., Wu, C. A., Yang, G. D., Zhang, M. M. and Zheng, C. C. 2005. TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants. Theoretical and Applied Genetics, 110: 620-627.
 

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