Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-03-10
2002-03-26
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C435S419000, C536S024100
Reexamination Certificate
active
06362398
ABSTRACT:
FIELD OF THE INVENTION
The present invention generally pertains to plant molecular biology and more particularly pertains to a novel plastid promoter isolated from Arabidopsis thaliana and methods of use therefor. The present invention also pertains to a novel method for utilizing protein-coding regions of plastid genes to isolate intervening regulatory sequences. The present invention further pertains to the use of novel plastid promoter sequences to improve plastid transformation efficiency.
BACKGROUND OF THE INVENTION
Plastid transformation, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that may exceed 10% of the total soluble plant protein. In addition, plastid transformation is desirable because plastid-encoded traits are not pollen transmissable; hence, potential risks of inadvertent transgene escape to wild relatives of transgenic plants are obviated. Other advantages of plastid transformation include the feasibility of simultaneous expression of multiple genes as a polycistronic unit and the elimination of positional effects and gene silencing that may result following nuclear transformation. Plastid transformation technology is extensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818, and 5,576,198; in Intl. Application No. WO 95/16783; and in Boynton et al.,
Methods in Enzymology
217: 510-536 (1993), Svab et al.,
Proc. Natl. Acad. Sci. USA
90: 913-917 (1993), and McBride et al.,
Proc. Nati. Acad. Sci. USA
91: 7301-7305 (1994); all of which are incorporated herein by reference.
The basic technique for tobacco plastid transformation involves the particle bombardment of leaf tissue with regions of cloned plastid DNA flanking a selectable marker, such as an antibiotic resistance gene. The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the 156 kb tobacco plastome. Initially, point mutations in the chloroplast 16S rRNA and rpsl 2 genes conferring resistance to spectinomycin and/or streptomycin were utilized as selectable markers for transformation (Svab et al.,
Proc. Natl. Acad. Sci. USA
87: 8526-8530 (1990); Staub, J. M., and Maliga, P.,
Plant Cell
4: 39-45 (1992); both of which are incorporated herein by reference). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J. M., and Maliga, P.,
EMBO J
. 12: 601-606 (1993), incorporated herein by reference). Substantial increases in transformation frequency were obtained by replacement of the recessive rDNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al.,1993). Previously, this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga
Chiamydomonas reinhardtii
(Goldschmidt-Clermont, M.,
Nucl. Acids Res
. 19: 4083-4089 (1991), incorporated herein by reference). Techniques have also been described for the transfection of plastids in plant protoplasts (O'Neill et al.,
Plant Journal
3(5): 729-738 (1993) and Koop et al.,
Planta
199: 193-201 (1996), both of which are incorporated herein by reference).
An especially preferred plant plastid promoter for use in plastid targeting vectors to express foreign genes in the plant plastid is the clpP gene promoter. The clpP gene encodes the proteolytic subunit of the Clp ATP-dependent protease, which in Arabidopsis is constitutively expressed in the plastids of photosynthetic and nonphotosynthetic plant tissues (Shanklin et al.,
The Plant Cell
7: 1713-1722 (1995)), incorporated herein by reference. clpP is also one of the few plant plastid genes that is retained in the genomes of non-photosynthetic plants (e.g.
Epifagus virginiana
; Morden et al.
EMBO J
. 10: 3281-3288 (1991)) and the clpP message is known to be expressed in the plastids of the barley mutant
albostrians
, which lacks detectable plastid translational activity (Hübschmann and Börner, Plant Mol. Biol. 36: 493-496 (1998)). Hence, the clpP promoter is likely to be active transcriptionally even in non-green plastids. The characterization of the promoter region from the tobacco clpP gene is described in WO 97/06250, incorporated herein by reference. In this reference, the tobacco clpP gene is characterized as having 5′ promoter sequences that are recognized by both a nuclear encoded plastid (NEP) RNA polymerase and a plastid encoded plastid (PEP) RNA polymerase. A primary transcript arising from the tobacco clpP promoter sequence mapping to the −53 nucleotide position (upstream from the ATG translation initiation codon) is characterized in WO 97/06250 as being highly expressed in the bleached plastids of tobacco mutants lacking a plastid-encoded RNA polymerase by virtue of deletion of the rpoB gene.
A tobacco clpP promoter sequence has been used to drive expression of a herbicide-resistant form of the Arabidopsis Protoporphyrinogen IX (“PROTOX”) gene in the plastids of tobacco (WO 97/32011, incorporated herein by reference). Identical constructs substituting a GUS reporter gene have been introduced into tobacco plastids, demonstrating that clpP-driven expression is not restricted to green plastids but is also found in root plastids (leucoplasts, amyloplasts) and flower plastids (chromoplasts).
Despite the promise shown by plastid transformation, only recently has this technology been applied to plants other than tobacco. International Application No. WO 97/32977, incorporated herein by reference, describes methods and compositions for creating transplastomic plants in the Cruciferae family, such as Brassica and Arabidopsis, using leaf and cotyledon cells. However, what is also needed are novel plastid promoter sequences from plants other than tobacco, particularly Arabidopsis, which can be used to drive the expression of transgenes in green and non-green plastids of Arabidopsis and any other plant species.
SUMMARY OF THE INVENTION
In view of the above, one object of the invention is to provide a novel plastid promoter from
Arabidopsis thaliana
that is functional in all plastid types. Another object of the invention is to provide a method for utilizing protein-coding regions of plastid genes to isolate novel intervening regulatory sequences, such as novel promoter sequences or untranslated 3′ or 5′ RNA sequences. Still another object of the invention is to use novel plastid promoter sequences to improve plastid transformation efficiency by reducing undesired homologous recombination between native DNA sequences in the plastid genome and exogenous DNA sequences contained in chimeric DNA fragments incorporated into plastid transformation vectors.
In furtherance of these and other objects, the present invention provides a nucleic acid promoter isolated from the 5′ flanking region upstream of the coding sequence of the Arabidopsis plastid clpP gene. In a preferred embodiment, the nucleic acid promoter of the invention is substantially similar to a promoter sequence downstream of nucleotide number 263 of SEQ ID NO:1. In a more preferred embodiment, the nucleic acid promoter of the invention has sequence identity with a promoter sequence downstream of nucleotide number 263 of SEQ ID NO:1. In still another embodiment, the nucleic acid promoter of the invention is substantially similar to SEQ ID NO:1. In yet another embodiment, the nucleic acid promoter of the invention is comprised within SEQ ID NO:1. In still another embodiment, the nucleic acid promoter of the invention comprises a 20 base pair nucleo
Meigs J. Timothy
Morton Marcia R.
Nelson Amy J.
Syngenta Participations (AG)
LandOfFree
ClpP plastid promoter sequence does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with ClpP plastid promoter sequence, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and ClpP plastid promoter sequence will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2830962