Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide contains a tissue – organ – or cell...
Reexamination Certificate
1999-07-28
2002-07-02
Yucel, Remy (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C800S278000, C435S468000, C435S419000
Reexamination Certificate
active
06414221
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to field of plant genetic engineering. In particular, it relates to promoters useful for inducible, transient expression of heterologous genes.
BACKGROUND OF THE INVENTION
In the production of transgenic plants it is extremely valuable to have a variety of plant promoters that can be induced in response to various environmental stimuli. Currently, most inducible plant promoters are induced by application of chemicals. For example, plant promoters induced by tetracyline (Caddick et al.
Nature Biotechnology
16:177-180 (1998)), dexamethasone (Gatz,
Annu. Rev. Plant Physiol. Plant Mol. Bio
. 48:89-108 (1997)) and ethanol (Aoyoma et al.
Plant Journal
11:605-612 (1997)) are known. Identification of plant promoters induced by non-chemical stimuli would be extremely useful.
One of the most common plant responses to wounding or flooding is the induction of ethylene synthesis. Ethylene plays an important role in plant growth and development, including the control of seed germination, leaf senescence, floral fading, senescence and fruit ripening. Besides the stimulation by internal factors, ethylene synthesis is also stimulated by external factors. These include the induction of ethylene by chemicals and stresses, such as the attack by pathogens and environmental stresses, such as pollution, drought, chilling, heating, wounding and water logging, i.e. the soil flooding of plants (Jackson, M. B.
Annu. Rev.Plant Physiol
., 36, 145-175 (1985); Abeles et al.,
Ethylene in Plant Biology
, Ed. 2, Academic Press, San Diego (1994)).
Ethylene is synthesized from S-adenosylmethionine via 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (Adams, D. Q. and Yang, S. F.,
Proc. Natl. Acad. Sci. USA
, 76, 170-174 (1979)). The reactions are catalyzed by ACC synthase (ACS), the key enzyme of ethylene synthesis and ACC oxidase (ACO) (Kende H,
Annu. Rev. Plant Physiol
., 44, 283-307 (1993)).
Flooded tomato roots synthesize the ethylene precursor ACC which is transported via the xylem to the leaves within 6 to 12 hours (Bradford K J. and Yang S. F.,
Plant Physiol
., 65, 322-326 (1980)). The ethylene produced by waterlogged plants can be interpreted as an early warning of deteriorating soil conditions, inducing changes above ground to increase stress tolerance. Anaerobiotic conditions caused by flooding induce transcription of ACC synthase mRNAs in roots (Zarembinski T. I. and Theologis A.,
Mol. Biol. Cell
4, 363-373 (1993); Olson et al.,
J. Biol. Chem
., 270, 14056-14061 (1995); Zarembinski, T. I. and Theologis, A.
Plant Mol. Biol
., 33, 71-77 (1997)). ACC synthase is encoded by a multigene family and in the tomato seven members of this gene family, LE-ACS1a, 1b, 2, 3, 4, 5 and 6 are known and differentially expressed (Van der Straeten et al.,
Proc.Natl.Acad.Sci. USA
, 87,4859-4863 (1990); Rottmann et al.,
J.Mol.Biol
., 222, 937-961 (1991); Olson et al.,
Proc. Nat. Acad. Sci. USA
88, 5340-5344 (1991); Yip et al.,
Proc. Natl. Acad. Sci. USA
, 89, 2475-2479 (1992); Oetiker et al.,
Plant Mol. Biol
., 34, 275-286 (1997)).
The gene LE-ACS2, commonly known as the ACC synthase that causes fruit ripening is induced in flooded roots (Van der Straeten et al.,
Proc.Natl.Acad.Sci. USA
, 87,4859-4863 (1990); Rottmann et al.,
J.Mol.Biol
., 222, 937-961 (1991); Parsons B. L. and Mattoo A. K.,
Plant Mol. Biol
., 17, 453-464 (1991); Oeller,
Science
, 254, 437-439 (1991); Olson et al.,
J. Biol. Chem
., 270, 14056-14061 (1995)). However, LE-ACS2 cannot be solely responsible for flooding induced ethylene because ACC is delivered from root to shoot within 6 to 12 hours after flooding but the LE-ACS2 transcript appears only 8 hours after flooding of the roots (Bradford K J. and Yang S. F.,
Plant Physiol
., 65, 322-326 (1980); English, et al.,
J. Exp. Botany
45, 33.-33. (1995); English et al.,
Plant Physiol
., 109, 1435-1440 (1995))(Olson et al.,
J. Biol. Chem
., 270, 14056-14061 (1995)). LE-ACS3 is also induced in flooded tomato roots, but half of its polyadenylated mRNA remains unspliced, a commonly observed phenomenon under anaerobiotic stress (Olson et al.,
J. Biol. Chem
., 270, 14056-14061 (1995)). Such stress-induced splicing failures will not lead to a functional ACS protein and therefore, we were looking for further ACS transcripts, potentially responsible for the initial burst of ethylene produced by leaves of waterlogged plants.
Identification of plant genes induced by flooding or other environmental stresses would be useful in isolation and preparation of inducible plant promoters. For example, identification of new inducible promoters that are induced quickly and provide transient expression of heterologous nucleic acids would extremely useful in controlling plant responses to various environmental stimuli. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides recombinant expression cassettes comprising an ACS7 promoter operably linked to a heterologous polynucleotide sequence. The promoter is at least about 70% identical to SEQ ID NO:1, the LE-ACS7 promoter from tomato. The heterologous polynucleotide sequence is not critical part of the invention. In some embodiments, the polynucleotide encodes a polypeptide, for example, one that confers resistance to a plant pathogen. Alternatively, the heterologous polynucleotide can be operably linked to the ACS7 promoter in the antisense orientation.
The recombinant expression cassettes of the invention are typically incorporated into vectors suitable for production of transgenic plants. The vector will usually further comprise an independent terminator sequence, replication sequences and a selection marker sequence. The expression cassettes of the invention can also be incorporated into the genome of transgenic plant. Thus, the invention also provides transgenic plants comprising the recombinant expression cassette of the invention. The transgenic plant can be any species, for example, tomato.
The invention also provides methods of expressing a nucleic acid in a plant. The methods comprise providing a transgenic plant comprising a recombinant expression cassette of the invention, and, subjecting the plant to an environmental stimulus which activates the ACS7 promoter, thereby transcribing the heterologous nucleic acid. The environmental stimulus is typically flooding or wounding.
Definitions
The term “stress” refers to harm or damage done to organs and/or tissues of a plant, including the roots, stems, leaves, buds, etc. The cause of stress can be exposure of the plant to anaerobic conditions (e.g., as a result of flooding), wounding, (e.g., cutting, scraping, freezing, and pinching), exposure to chemicals, (e.g. herbicides, fungicides, and insecticides), exposure to pathogens, (e.g., bacteria, viruses, and fungi), or exposure to pests, (e.g. insects, and nematodes), and the like.
The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
An “ACS7 promoter” refers to a plant promoter capable of initiating transcription from an operably linked heterologous sequence and which is at least substantially identical to the promoter sequence in SEQ ID NO:1 (LE-ACS7, from tomato). The promoters of the invention need not be full length, the promoters are typically from about 1000 to about 2500 nucleotides in length, usually from about 1500 to about 2000 nucleotides and often between about 2300 and about 2450 nucleotides.
A polynucleotide sequence is “heterologous to” a second polynucleotide sequence if it originates from a different gene, or, if from the same gene, is modified from its original form. For example, a promoter operably linked to a heterolo
Oetiker Juerg H.
Shiu Oy Yin
Yang Shang Fa
Yip Win Kin
Katcheves Konstantina
The Regents of the University of California
Townsend & Townsend & Crew LLP
Yucel Remy
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