Rice ubiquitin-derived promoters

Details Rice ubiquitin-derived promoters Rice ubiquitin-derived promoters

2000-02-29

2003-03-04

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S320000, C800S320200, C800S279000, C435S419000, C435S468000, C435S320100, C536S023600, C536S024100

Reexamination Certificate

active

06528701

ABSTRACT:

This invention pertains to four novel ubiquitin promoters derived from ubiquitin genes isolated from rice (
Oryza sativa L
.), promoters that efficiently drive constitutive gene expression in transgenic plants.
Significant advances in cell biology and gene delivery techniques have allowed incorporation of foreign genes into many crop plants. Foreign genes are transferred into plants, named “transgenic plants,” primarily to express proteins that will confer a beneficial trait, such as resistance to pathogenic micro-organisms or insects, resistance to herbicides, or tolerance to drought or other adverse environrments. Usually, the DNA coding region for the foreign gene is linked to a strong and constitutive DNA promoter region to ensure the efficient expression of the foreign gene in the transgenic cell.
A number of common promoters are used to drive foreign gene expression in transgenic plants. The cauliflower mosaic virus (CaMV) 35S promoter has been widely used in both dicots and monocots, but its effectiveness in monocots was found to be substantially less than in dicots. CaMV 35S was even inactive in certain cell types, such as pollen. See Guerrero et al., “Promoter sequences from a maize pollen-specific gene direct tissue-specific transcription in tobacco,” Mol. Gen. Genet, vol. 224, pp. 161-168 (1990).
The maize alcohol dehydrogenase (Adh1) promoter has also been used in monocot transformation studies. This promoter has been shown to be 10 to 20 times more active than the CaMV 35S promoter in transformed rice protoplasts and cultured cells; however, the maize Adh1 promoter was not consitutively active in all transformed tissues. This promoter was induced by anaerobic stress in the transformed rice protoplasts. See Zhang et al., “Efficient regeneration of transgenic plants from rice protoplasts and correctly regulated expression of the foreign gene in the plants,” Theor. App. Genet., vol. 76, pp. 835-840 (1988).
Promoters can be more effective if isolated from the same species as the transgenic plant. &bgr;-glucuronidase (GUS) expression under the control of a rice actin promoter (Act1) in transformed rice protoplasts was approximately 6-fold greater than expression under control of the maize Adh1 promoter. Activity of the rice actin promoter is dependent on the presence of an intact Act1 5′ intron; i.e., removal of the intron resulted in no gene expression. See McElroy et al., “Isolation of an Efficient Actin Promoter for Use in Rice Transformation,” The Plant Cell, vol. 2, pp. 163-171 (1990).
Ubiquitin is one of the most highly conserved proteins in eukaryotes. See Callis et al., “Ubiquitin and Ubiquitin Genes in Higher Plants,” Oxford Surveys of Plant Molecular & Cell Biology, vol. 6, pp. 1-30 (1989). One physiological role for ubiquitin is to conjugate with a target protein as a recognition signal for protein degradation. See Viersta, R.D., “Proteolysis in plants: mechanisms and functions,” Plant Molecular Biology, vol. 32, pp. 275-302 (1996). In higher organisms, ubiquitin has been shown to be encoded by two small gene families, named “polyubiquitin genes” and “ubiquitin fusion genes.” Polyubiquitin genes comprise tandem head-to-tail repeats of 228 bp, with each repeat encoding 76 amino acids of a ubiquitin monomer. The number of tandem repeats reported varies between genes within genomes and between organisms, from 3 in Dictostylium to approximately 50 in
Trypanosoma cruzi
. On the other hand, the ubiquitin fusion gene family encodes a single repeat fused to one of two other polypeptides of either 52 or 76-80 amino acids. See Callis et al., “Ubiquitin and Ubiquitin Genes in Higher Plants,” Oxford Surveys of Plant Molecular & Cell Biology, vol. 6, pp. 1-30 (1989). Studies of ubiquitin genes in a number of plants indicate that ubiquitin genes are expressed in all tissues; however, differential expression of the ubiquitin genes is also indicated among the ubiquitin gene family. Each tandem repeat or ubiquitin gene may be expressed differently over time and in different cells or tissues. Examples are given below.
The conditions that cause genetic expression of four ubiquitin-encoding cDNAs, including one ubiquitin fusion cDNA and three polyubiquitin cDNAs with 6 or 7 repeats, have been characterized in potato tuber. See Garbarino et al., “Expression of stress-responsive ubiquitin genes in potato tubers,” Plant Molecular Biology, vol. 20, pp. 235-244 (1992). The ubiquitin fusion cDNA encoded a single ubiquitin unit fused to an 80 amino acid ribosomal extension protein. Expression of the ubiquitin fusion gene was induced by injury or ethylene, but not by heat. Expression of the three polyubiquitin genes differed: one was induced by injury, heat, or ethylene treatment; another was induced by injury or heat, but not by ethylene treatment; and the remaining gene was expressed at the highest level, but its expression decreased in response to injury, heat, or ethylene treatment.
Expression of the ubiquitin gene families may be dependent on the type and age of the plant tissue, as well as certain environmental factors. A polyubiquitin gene from
Nicotiana tabacum
, Ubi.U4, was expressed throughout the plant, except in just-fully-expanded leaves. See Genschik et al., “Sturcture and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of
Nicotiana tabacum
,” Gene, vol. 148, pp. 195B-202 (1994). In tomato, expression of a ubiquitin fusion gene, ubi3, was highest in young leaves and immature green fruits and lowest in mature leaves and petals; however, expression was reduced by heat or light deprivation. See Hoffman et al., “Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3,” Plant Molecular Biology, vol. 17, pp. 1189-1201 (1991). In parsley, expression of one polyubiquitin gene, ubi4, was predominant and was at comparable levels in all plant organs tested. See Kawalleck et al., “Polyubiquitin gene expression and structural properties of the ubi
4-2
gene in
Petroselinum crispum
,” Plant Molecular Biology, vol. 21, pp. 673-684 (1993).
Promoters from ubiquitin genes have been shown to drive reporter gene expression, usually GUS or chloramphenicol acetyl transferase (CAT), in transformed cells or plants. Such promoters have been isolated from Arabidopsis (Callis et al., “Ubiquitin Extension Proteins of
Arabidopsis thaliana
,” The Journal of Biological Chemistry, vol. 265, no. 21, pp. 12486-12493 (1990)); sunflower (Binet et al., “Analysis of a sunflower polyubiquitin promoter by transient expression,” Plant Science, vol. 79, pp. 87-94 (1991)); tobacco (Genschick et al., “Structure and promoter activity of a stress and developmentally regulated polyubiquitin-encoding gene of Nicotiana tabacum,” Gene, vol. 148, pp. 195-202 (1994)); and maize (Christensen et al., “Maize polyubiguitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation,” Plant Molecular Biology, vol. 18, pp. 675-689 (1992)).
The ubiquitin promoter ubil isolated from a maize polyubiquitin gene was shown to drive the expression of the CAT reporter gene more efficiently than the CaMV 35S promoter in maize protoplasts. See Christensen et al., “Maize polyubiguitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation,” Plant Molecular Biology, vol. 18, pp. 675-689 (1992). The maize ubil-promoter has been used to express a herbicide resistance gene in rice. See Toki et al., “Expression of a Maize Ubiquitin Gene Promoter-bar Chimeric Gene in Transgenic Rice Plants,” Plant Physiol, vol. 100, pp. 1503-1507 (1992).
U.S. Pat. Nos. 5,614,399 and 5,510,474 describe a promoter from a maize polyubiquitin gene. The promoter regulates expression of a maize polyubiquitin gene containing 7 tandem repeats. Expression of this maize ubiquitin gene was constitutive at 25° C., and was induced by heat shock at 42° C. The promoter was successfully tranformed

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