The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5′ control region of the napA gene were fused to the reporter gene β-glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between −1101 and −309 resulted in increased GUS activity. In contrast, deletion of sequences between −309 and −211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between −152 and +44. Further 5′ deletion of the fragment to −126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between −148 to −120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to −152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
An, G: Binary Ti vectors for transformation and promoter analysis. Meth Enzymol 153: 292–319 (1987).
Baszczynski, CL, Fallis, L: Isolation and nucleotide sequence of a genomic clone encoding a new Brassica napus napin gene. Plant Mol Biol 14: 633–635 (1990).
Blundy, KS, Blundy, MAC, Crouch, ML: Differential expression of members of the napin storage protein gene family during embryogenesis in Brassica napus. Plant Mol Biol 17: 1099–1104 (1991).
Bradford, MM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72: 248–254 (1976).
Burow, MD, Sen, P, Chlan, CA, Murai, N: Developmental control of the β-phaseolin gene requires positive, negative and temporal seed-specific transcriptional regulatory elements and a negative element for stem and root expression. Plant J 2: 537–548 (1992).
Bustos, MM, Begum, D, Kalkan, F, Battraw, MJ, Hall, TC: Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter. EMBO J 10: 1469–1479 (1991).
Bäumlein, H, Nagy, I, Villarroel, R, Inze, D, Wobus, U: Cis-Analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within the legumin box is essential for tissue specific expression of a legumin gene. Plant J 2: 233–239 (1992).
Chamberland, S, Daigle, N, Bernier, F: The legumin boxes and the 3′ part of soybean β-conglycinin promoter are involved in seed gene expression in transgenic tobacco plants. Plant Mol Biol 19: 937–949 (1992).
Crouch, ML, Sussex, IM: Development and storage-protein synthesis in Brassica napus L embryos in vivo and in vitro. Planta 153: 64–74 (1985).
DeClercq, A, Vandewiele, M, DeRycke, R, VanDamme, J, VanMontagu, M, Krebbers, E, Vandekerckhove, J: Expression and processing of an Arabidopsis 2S albumin in transgenic tobacco. Plant Physiol 92: 899–907 (1989).
DeLisle, AJ, Crouch, ML: Seed storage protein transcription and mRNA levels in Brassica napus during development and in response to exogenous abscisic acid. Plant Physiol 91: 617–623 (1989).
Dickinsson, DC, Evans, PR, Nielsen, NC: RY repeats are conserved in 5′-flanking of legume seed-protein genes. Nucl Acids Res 16: 371 (1988).
Ericson, ML, Murén, E, Gustavsson, H-O, Josefsson, L-G, Rask, L: Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear protein in vitro to a conserved sequence motif. Eur J Biochem 197: 741–746 (1991).
Fernandez, DE, Turner, RF, Crouch, ML: In situ localization of storage protein mRNA in developing meristems of Brassica napus embryos. Development 111: 299–313 (1991).
Giuliano, G, Pichersky, E, Malik, VS, Timko, MP, Scolnik, PA, Cashmore, AR: An evolutionary conserved protein binding sequence upstream of a plant light-regulated gene. Proc Natl Acad Sci USA 85: 7089–7093 (1988).
Gomez, J, Sánchez-Martínez, D, Stiefel, V, Rigau, J, Puigdomènech, P, Pages, MA: A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycine-rich protein. Nature 334: 262–264 (1988).
Guerche, P, Tire, C, Grosside, Sa F, DeClercq, A, VanMontagu, M, Krebbers, E: Differential expression of the Arabidopsis 2S albumin genes and the effect of increasing gene family size. Plant Cell: 2: 469–478 (1990).
Gustavsson, H-O, Ellerström, M, Stålberg, K, Escurra, I, Koman, A, Höglund, A-S, Rask, L, Josefsson, L-G: Distinct sequence elements in a napin promoter interact in vitro with DNA-binding proteins from Brassica napus. Physiol Plant 82: 205–212 (1991).
Höfgren, R, Willmitzer, L: Storage of competent cells for Agrobacterium transformation. Nucl Acids Res 16: 9877 (1988).
Höglund, A-S, Rödin, J, Larsson, E, Rask, L: The distribution of napin and cruciferin in developing rape seed embryos. Plant Physiol 98: 509–515 (1991).
Jefferson, RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Jefferson, RA, Kavanagh, TA, Bevan, MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).
Jofuku, KD, Okamuro, JK, Goldberg, RB: Interaction of an embryo DNA binding protein with a soybean lectin gene upstream region. Nature 328: 734–737 (1987).
Jones, JDG, Gilbert, DE, Grady, KL, Jorgensen, RA: T-DNA structure and gene expression in petunia plants transformed by Agrobacterium tumefaciens C58 derivates. Mol Gen Genet 207: 478–485 (1987).
Jordano, J, Almoguera, C, Thomas, T: A sunflower gene upstream sequence ensemble contains an enhancer and sites of nuclear protein interaction. Plant Cell 1: 855–866 (1989).
Josefsson, L-G, Lenman, M, Ericson, ML, Rask, L: Structure of a gene encoding the 1.7S storage protein, napin, from Brassica napus. J Biol Chem 262: 12196–12201 (1987).
Krebbers, E, Herdies, L, DeClercq, A, Seurinick, J, Leemans, J, VanDamme, J, Segura, M, Geysen, G, VanMontagu, M, Vandekerckhove, J: Determination of the processing sites of an Arabidopsis 2S albumin and characterization of the complete gene family. Plant Physiol 87: 859–866 (1988).
Krebbers, E, Seurinck, J, Herdies, L, Cashmore, AR, Timko, MP: Four genes in 2 diverged subfamilies encode the ribulose-1,5-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana. Plant Mol Biol 11: 745–759 (1988).
Leleivre, JM, Oliveira, LO, Nielsen, N: 5′-CATGCAT-3′ elements modulate the expression of glycinin genes. Plant Physiol 98: 387–391 (1992).
Leisy, DJ, Hnilo, J, Zhao, Y, Okita, TW: Expression of a rice glutelin promoter in transgenic tobacco. Plant Mol Biol 14: 41–50 (1989).
Lessard, PA, Allen, RD, Bernier, F, Crispino, JD, TFujiwara, Beachy, RN: Multiple nuclear factors interact with upstream sequences of differentially regulated soybean β-Conglycinin genes. Plant Mol Biol 16: 397–419 (1991).
Lohmer, S, Maddaloni, M, Motto, M, DiFiozo, N, Hartings, H, Salamini, F, Thompson, RD: The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene. EMBO J 10: 617–624 (1991).
MarcotteJr, WR, Russel, SH, Quatrano, RS: Abscisic acid-responsive sequence from the Em gene of wheat. Plant Cell 1: 969–976 (1989).
Matzke, MA, Stöger, EM, Schernthaner, JP, Matzke, AJM: Deletion analysis of zein gene promoter in transgenic tobacco plants. Plant Mol Biol 14: 323–332 (1990).
Matzke, MA, Matzke, AJM: Differential inactivation and methylation of a transgene in plants by two suppressor loci containing homologous sequences. Plant Mol Biol 16: 821–830 (1991).
Mettler, IJ: A simple and rapid method for minipreparation of DNA from tissue cultured plant cells. Plant Mol Biol Rep 5: 346–349 (1987).
Murphy, DJ, Cummins, I, Ryan, AJ: Immunocytochemical and biochemical study of the biosynthesis and mobilisation of the major seed storage proteins of Brassica napus. Plant Physiol Biochem 27: 647–657 (1989).
Peach, C, Velten, J: Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked T-DNA promoters. Plant Mol Biol 17: 49–60 (1991).
Radke, SE, Andrews, BM, Moloney, MM, Crouch, ML, Kridl, JL, Knauf, VC: Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75: 685–694 (1988).
Reire, WG, Whitecross, M, Higgins, TJV: Developmental and environmental regulation of pea legumin genes in transgenic tobacco. Mol Gen Genet 225: 148–157 (1991).
Sambrook, J, Fritsch, EF, Maniatis, T: Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Schindler, U, Terzaghi, W, Beckmann, H, Kadesch, T, Cashmore, AR: DNA binding site preferences and trancriptional activation properties of the Arabidopsis transcription factor GBF1. EMBO J 11: 1275–1289 (1992).
Scofield, SR, Crouch, ML: Nucleotide sequence of a member of the napin storage protein family from Brassica napus. J Biol Chem 262: 12202–12208 (1987).
Shaun, LA, Hobbs, PK, DeLong, CMO: The effect of T-DNA copy number, position and methylation on reporter gene expression in tobacco transformants. Plant Mol Biol 15: 851–864 (1990).
Shirsat, AH, Wilford, N, Croy, RD: Gene copy number and levels of expression in transgenic plants of a seed-specific gene. Plant Science 61: 75–80 (1989).
Shirsat, AH, Meakin, PJ, Gatehouse, JA: Sequences 5′ to the conserved 28 bp Leg box element regulate the expression of pea seed storage protein gene legA. Plant Mol Biol 15: 685–693 (1990).
Sokal, RR, Rohlf, FJ: Biometry, Second edition. W.H. Freman, San Francisco (1981).
Wilen, RW, VanRooijen, GJH, Pearch, DW, Pharis, RP, Holbrook, LA, Moloney, MM: Effect of jasmonic acid on embryo-specific processes in Brassica and Linum oilseeds. Plant Physiol 95: 399–405 (1991).
Williams, ME, Foster, R, Chua, N-H: Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell 4: 485–496 (1992).
About this article
Cite this article
Stålberg, K., Ellerström, M., Josefsson, LG. et al. Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco. Plant Mol Biol 23, 671–683 (1993). https://doi.org/10.1007/BF00021523
- Brassica napus
- deletion analysis
- storage protein