Abstract
The function of the 28 bp conserved Leg box element found in the 11S legumin-like genes of Vicia, Pisum, Glycine and Helianthus in controlling gene expression has been examined. Transgenic tobacco plants containing 124 bp of 5′ legA flanking sequence including the Leg box and all sequences downstream to the start of transcription failed to activate the gene, as judged by ELISA and northern assays. Expression was first detected in a promoter truncation construct which contained 549 bp of 5′ flanking sequence. These two promoter truncation constructs were additionally examined for their ability to interact with pea seed nuclear proteins. The use of DNA-protein binding and mobility shift assayshowed that the −124 bp fragment which included the Leg box did not bind nuclear proteins. In constrast, nuclear proteins were shown to strongly bind to the −549 bp flanking sequence fragment. The correlation of transgenic plant analysis with DNA binding assays indicates that the highly conserved 28 bp Leg box element is not the sole promoter determinant in legumin gene expression.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barker SJ, Harada JJ, Goldberg RB: Cellular localisation of soybean storage protein mRNA in transformed tobacco seeds. Proc Natl Acad Sci USA 85: 458–462 (1988).
Bevan M: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Boulter D, Evans IM, Ellis JR, Shirsat AH, Gatehouse JA, Croy RRD: Differential gene expression in the development of Pisum sativum. Pl Physiol Biochem 25: 283–289 (1987).
Bradford M: A rapid and sensitive method for the quantitation of microgramme quantities of protein utilising the principle of protein dye binding. Anal Biochem 72: 248–254 (1976).
Bustos MM, Guiltnan MJ, Jordano J, Beguin G, Kalkan F, Hall TC: Regulation of B glucuronidase expression in transgenic plants by an A/T rich cis acting sequence found upstream of a french bean B phaseolin gene. Plant Cell 1: 839–853 (1989).
Chen ZL, Schuler MA, Beachy RN: Functional analysis of the regulatory elements in a plant embryo specific gene. Proc Natl Acad Sci USA 93: 8560–8564 (1986).
Ditta G, Stanfield S, Corbin D, Helinski DR: Broad host range cloning system for gram negative bacteria. Proc Natl Acad Sci USA 77: 7347–7351 (1980).
Ellis JR, Shirsat AH, Hepher A, Yarwood JN, Gatehouse JA, Croy RRD, Boulter D: Tissue-specific expression of a pea legumin gene in seeds of Nicotiana plumbaginifolia. Plant Mol Biol 10: 203–214 (1988).
Evans IM, Gatehouse JA, Croy RRD, Boulter D: Regulation of the transcription of storage protein mRNA in nuclei isolated from developing pea (Pisum sativum) cotyledons. Planta 160: 559–568 (1984).
Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Forde BG, Heyworth A, Pywell J, Kreis M: Nucleotide sequence of a B1 hordein gene and the identification of possible upstream regulatory elements in endosperm storage protein genes from barley, wheat and maize. Nucl Acids Res 13: 7327–7339 (1985).
Galfre' G, Milstein C: Preparation of monoclonal antibodies-strategies and procedures. Met Enzymol 73: 3–46 (1981).
Gatehouse JA, Croy RRD, Boulter D: Isoelectric focussing properties and carbohydrate content of pea (Pisum sativum) legumin. Biochem J 185: 497–503 (1980).
Gatehouse JA, Evans IM, Croy RRD, Boulter D: Differential expression of genes during legume seed development. Philos Trans Roy Soc Lond 314: 367–384 (1986).
Goldberg BB: Regulation of plant gene expression. Philos Trans Roy Soc Lond 314: 343–353 (1986).
Graham DE: The isolation of high molecular weight DNA from whole organisms or large tissue masses. Anal Biochem 85: 609–613 (1978).
Harada JJ, Barker SJ, Goldberg RB: Soybean B conglycinin genes are clustered in several DNA regions and are regulated by transcriptional and post transcriptional processes. Plant Cell 1: 415–425 (1989).
Harris N, Mulcrone J, Grindley H: Tissue preparation techniques for in situ hybridisation studies of storage protein gene expression during pea seed development. In: Harris N, Wilkinson DJ (eds) In situ hybridisation: applications in developmental biology and medicine. Cambridge University Press (1990).
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303: 179–180 (1983).
Holdsworth MJ, Laties GG: Site specific binding of a nuclear factor to the carrot extensin gene is influenced by both ethylene and wounding. Planta 179: 17–23 (1989).
Jordano J, Almoguerra C, Thomas TL: A sunflower helianthin gene upstream sequence ensemble contains an enhancer and sites of nuclear protein interaction. Plant Cell 1: 855–866 (1989).
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).
Kang AS, Abbot SJ, Harris N: Assay of gene expression by immunodetection. In: Robbins RJ, Rhodes MJC (eds) Manipulating secondary culture. Cambridge University Press (1989).
Maier UG, Brown JWS, Toloczyki C, Feix G: Binding of a nuclear factor to a consensus sequence in the 5′ flanking region of zein genes from maize. EMBO J 6: 17–22 (1987).
Maniatis T, Fritsch EF, Sambrook J: Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).
Murashige T, Skoog F: A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Ptashne M: Gene regulation by proteins acting nearby and at a distance. Nature 322: 697–701 (1986).
Robert LS, Thompson RD, Flavell RB: Tissue specific expression of a wheat high molecular weight glutenin gene in transgenic tobacco. Plant Cell 1: 569–578 (1989).
Roussell DJ, Bostan RS, Goldsborough PB, Larkins BA: Delation of DNA sequences flanking a Mr. 19000 zein gene reduces its transcriptional activity in heterologous plant tissues. Mol Gen Genet 211: 202–209 (1989).
Scofield SR, Crouch ML: Nucleotide sequence of a member of the napin storage protein gene family from Brassica napus. J Biol Chem 262: 12202–12208 (1987).
Shirsat AH, Wilford NW, Croy R, Boulter D: Sequences responsible for the tissue specific promoter activity of a pea legumin gene in tobacco. Mol Gen Genet: 326–331 (1989).
Singh H, Sen R, Baltimore D, Sharp PA: A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature 319: 154–158 (1986).
Talbot DR, Adang MJ, Slightom JL, Hall T: Size and organisation of a multigene family encoding phaseolin, the major seed storage protein of Phaseolus vulgaris. Mol Gen Genet 198: 42–49 (1984).
Thompson AJ, Evans IM, Boulter D, Croy RRD, Gatehouse JA: Transcriptional and post transcriptional regulation of seed storage protein gene regulation in pea (Pisum sativum L.). Planta 179: 279–287 (1989).
Vieira J, Messing J: The pUC plasmids, an M13mp7 derived system for insertion mutagenesis and sequencing with universal primers. Gene 19: 259–268 (1982).
Willmitzer L, Wagner KG: The isolation of nuclei from tissue cultured plant cells. Exp Cell Res 135: 69–77 (1981).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shirsat, A.H., Meakin, P.J. & Gatehouse, J.A. 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). https://doi.org/10.1007/BF00016119
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00016119