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
1999-01-08
2001-01-23
Hutzell, Paula K. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide contains a tissue, organ, or cell...
C435S069100, C435S320100, C435S414000, C435S415000, C435S416000, C536S024100, C800S312000, C800S314000, C800S317000, C800S317300, C800S322000
Reexamination Certificate
active
06177613
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
BACKGROUND OF THE INVENTION
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. In contrast, where gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. Where expression in specific tissues or organs are desired, tissue-specific promoters are used. That is, these promoters can drive expression in specific tissues or organs. Additional regulatory sequences upstream and/or downstream from the core promoter sequence can be included in expression cassettes of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Isolation and characterization of seed-preferred promoters that can serve as regulatory elements for expression of heterologous nucleotide sequences of interest in a seed-preferred manner are needed for improving seed traits in plants.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a novel nucleotide sequence for modulating gene expression in a plant.
It is a further object of the present invention to provide an isolated promoter capable of driving transcription in a seed-preferred manner.
It is a further object of the present invention to provide a method of improved control of an endogenous or exogenous product in the seed of a transformed plant.
It is a further object of the present invention to provide a method for providing useful changes in the phenotype of a seed of a transformed plant.
It is a further object of the present invention to provide a method for producing a novel product in the seed of a transformed plant.
It is a further object of the present invention to provide a method for producing a novel function in the seed of a transformed plant.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of:
a) nucleic acids driving expression of the polynucleotide encoding soybean albumin;
b) nucleic acids comprising a functional variant or fragment of at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO 1;
c) the nucleic acids set forth in SEQ ID NO 1;
d) nucleic acids that hybridize to any one of a), b), or c), under stringent conditions; wherein stringent conditions include: a hybridization at 42° C. in a solution of 50%(w/v) formamide, 6× SSC, 0.5% SDS, 100 ug/ml salmon sperm, washed with 0.5% SDS and 2×SSC at 65° C. for 30 minutes and repeated;
e) nucleic acids having at least 65% sequence identity to SEQ ID NO 1 wherein the % sequence identity is based on the entire sequence and is determined by BLAST analysis under default parameters.
In other aspects, the present invention relates to expression cassettes comprising the promoter operably linked to a nucleotide sequence, vectors containing the expression cassette, and plants stably transformed with at least one expression cassette.
In a further aspect, the present invention relates to a method for modulating expression in the seed of a stably transformed plant comprising the steps of (a) transforming a plant cell with an expression cassette comprising the promoter of the present invention operably linked to at least one nucleotide sequence; (b) growing the plant cell under plant growing conditions and (c) regenerating a stably transformed plant from the plant cell wherein expression of the nucleotide sequence alters the phenotype of the seed.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention, a nucleotide sequence is provided that allows initiation of transcription in seed. The sequence of the invention comprises transcriptional initiation regions associated with seed formation and seed tissues. Thus, the compositions of the present invention comprise a novel nucleotide sequence for a plant promoter, more particularly a seed-preferred promoter for the gene AL3 (soybean 2S albumin pre-propeptide).
By “seed-preferred” is intended favored expression in the seed, including at least one of embryo, kernel, pericarp, endosperm, nucellus, aleurone, pedicel, and the like.
By “heterologous nucleotide sequence” is intended a sequence that is not naturally occurring with the promoter sequence. While this nucleotide sequence is heterologous to the promoter sequence, it may be homologous (native) or heterologous (foreign) to the plant host.
By “promoter” is intended a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter can additionally comprise other recognition sequences generally positioned upstream or 5′ to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. It is recognized that having identified the nucleotide sequences for the promoter region disclosed herein, it is within the state of the art to isolate and identify further regulatory elements in the 5′ untranslated region upstream from the particular promoter region identified herein. Thus the promoter region disclosed herein is generally further defined by comprising upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers and the like. In the same manner, the promoter elements which enable expression in the desired tissue such as the seed can be identified, isolated, and used with other core promoters to confirm seed-preferred expression.
The isolated promoter sequence of the present invention can be modified to provide for a range of expression levels of the heterologous nucleotide sequence. Less than the entire promoter region can be utilized and the ability to drive seed-preferred expression retained. However, it is recognized that expression levels of mRNA can be decreased with deletions of portions of the promoter sequence. Thus, the promoter can be modified to be a weak or strong promoter. Generally, by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended levels of about 1/10,000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Conversely, a strong promoter drives expression of a coding sequence at a high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1,000 transcripts. Generally, at least about 20 nucleotides of an isolated promoter sequence will be used to drive expression of a nucleotide sequence.
It is recognized that to increase transcription levels enhancers can be utilized in combination with the promoter regions of the invention. Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like.
The promoter of the present invention can be isolated from the 5′ untranslated region flanking its respective transcription initiation site. The term “isolated” refers to material, such as a nucleic acid or protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in a cell other than the locus native to the material. Methods for isolation of promoter regions are well known in the art. One method is described in U.S. patent application Ser. No. 09/3
Coughlan Sean J.
Winfrey, Jr. Ronnie J.
Hutzell Paula K.
Mehta Ashwin D.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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