Abstract
A mutated rice acetolactate synthase (mALS) gene expressed under the control of the rice callus-specific promoter (CSP) (CSP:mALS) becomes a useful selectable marker for producing transgenic rice seed with higher accumulation of recombinant protein. When amounts of transgene products in mature seeds are compared between transgenic rice lines containing the CSP:mALS selection gene cassette and those with the hygromycin phosphotransferase (HPT) gene under the control of the CaMV 35S promoter (35S:HPT), the former transgenic rice seeds usually resulted in 1.2- to 2-fold higher accumulation of transgene products than in the latter. It is considered that specific expression of a selection marker gene at the selection stage may allow enhanced transgene products in seeds.
This chapter represents a highly efficient Agrobacterium-mediated rice transformation system using the CSP:mALS gene cassette in place of the conventional constitutive selection using bacterial antibiotics. This selection stage specific expression using a rice-derived selection marker mALS will be especially beneficial for developing transgenic rice seeds accumulating bioactive proteins or peptides contributing to human health promotion; pharmaceutical products such as vaccine, antibodies, and biopharmaceuticals; and industrial enzymes.
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Notes
- 1.
mL AA-1 (1 g MnSO4∙4H2O, 0.3 g H3BO4, 0.2 g ZnSO4∙7H2O, 25 mg Na2MoO4∙2H2O, 2.5 mg CuSO4∙5H2O, 2.5 mg CoCl2∙6H2O, 75 mg KI), 100 mL AA-2 (15 g CaCl2∙2H2O), 100 mL AA-3 (25 g MgSO4∙7H2O), 100 mL AA-4 (4 g Fe-EDTA), 100 mL AA-5 (15 g NaH2PO4∙2H2O), 100 mL AA-6 (20 mg nicotinic acid, 200 mg thiamine hydrochloride, 20 mg pyridoxine monohydrochloride, 2 g myo-inositol), 100 mL AA-7 (1.77 g  l-arginine, 95 mg glycine).
- 2.
L DKN-macro (20× stock) (5.46 g NaH2PO4, 16 g KNO3, 1.34 g (NH4)2SO4, 5 g MgSO4, 3 g CaCl2∙2H2O), 100 mL DKN-micro (1,000× stock) (0.16 g MnSO4∙4H2O, 0.22 g ZnSO4∙7H2O, 0.3 g H3BO3, 12.5 mg CuSO4∙5H2O, 12.5 mg Na2MoO4∙2H2O), 100 mL modified B5-vitamins (1,000× stock) (100 mg nicotinic acid, 1 g thiamine hydrochloride, 100 mg pyridoxine monohydrochloride, 10 g myo-inositol, 200 mg glycine), 100 mL R2-iron (1,000× stock) (750 mg Na2∙EDTA, 550 mg FeSO4∙7H2O).
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Acknowledgments
This work was partly supported by research grants from the Ministry of Agriculture, Forestry, and Fishery of Japan (Genomics and Agricultural Innovation: GMC0003) to F. T. and Grant-in-Aid for JSPS fellows to Y. W. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Wakasa, Y., Takaiwa, F. (2012). Use of a Callus-Specific Selection System to Develop Transgenic Rice Seed Accumulating a High Level of Recombinant Protein. In: Dunwell, J., Wetten, A. (eds) Transgenic Plants. Methods in Molecular Biology, vol 847. Humana Press. https://doi.org/10.1007/978-1-61779-558-9_36
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DOI: https://doi.org/10.1007/978-1-61779-558-9_36
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