Germination-specific plant promoters

Details Germination-specific plant promoters Germination-specific plant promoters

1999-09-23

2002-03-19

Bui, Phuong T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C435S006120, C435S419000, C435S320100, C536S023100, C536S024100, C536S024300, C800S288000

Reexamination Certificate

active

06359196

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to regulatory sequences directing tissue-specific expression of a heterologous gene in a plant, and more particularly to DNA promoter sequence capable of conferring germination-specific expression of a gene in plant tissue.
BACKGROUND OF THE INVENTION
An important goal of plant biotechnology is to genetically engineer plants so they have a new or improved trait or characteristic. Initially, transformation was developed in model dicot plants. Monocot plants, which include all the major cereal crops, were more difficult, and the first successful transformations, in rice and maize, were not reported until the late 1980s. Although consistent transformation of the more recalcitrant cereals such as wheat and barley have only been achieved very recently, it has been shown that a homozygous transgenic barley line can transmit a heterologous gene over three generations to all progeny plants (Jensen et al., 1998
, Hereditas
129:215-225).
While tissue-specific, heterologous gene expression in plants can be achieved, the current stage of genetic engineering methodology does not offer the means of targeting where introduced DNA sequences are integrated into the chromosome; integration into plant chromosomes appears to be more or less random.
This invention relates to transgenic plants and involves a method of generating transgenic plants with controllable gene expression. Particularly, the invention relates to transgenic plants that have been modified such that expression of a heterologous introduced gene can be limited to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location, or a combination of these situations. More particularly, attention has been given to produce transgenic cereal plants of the grass family; common cereal plants include barley (Hordeum), wheat (Triticum), rice (Oryza), maize (Zea), rye (Secale) and sorghum (Sorghum).
A desired trait or characteristic is introduced into the plant by incorporating into the plant's genome a gene that encodes the polypeptide that confers the desired trait or characteristic. DNA sequences that regulate the expression of the gene must also be introduced into the plant in conjunction with the desired gene. For heterologous expression, the regulatory sequence—such as a sequence often called a gene promoter, or simply a promoter—directs transcription of a large number of RNA molecules from the operably linked heterologous DNA sequence, which serves as a template. Each plant gene comprises a promoter sequence to which specialized proteins bind and activate the gene. For example, specific nucleotide sequences within the promoter are recognized by RNA polymerase molecules that start RNA synthesis. After primary transcription, a second class of signals leads to the termination of RNA synthesis and the detachment of RNA polymerase molecules from their respective DNA templates. The RNA chains, which may undergo further processing, e.g. removal of intron sequences and attachment of poly(A) tails, can in turn serve as templates for the synthesis of specific polypeptide chains.
The selection of a promoter is often a critical factor in obtaining expression of a heterologous gene. A promoter can function as a constitutive promoter or as an inducible promoter. Constitutive promoters are those which are capable of expressing operably linked DNA sequences in all tissues of a plant throughout development. Even though providing constitutive expression of a gene in plants is often desirable, it is also desirable in some instances to direct expression of a gene to particular tissues and/or time of development in a plant. Tissue specific promoters are capable of selectively expressing heterologous DNA sequences in certain plant tissues. Tissue specific promoters may also be inducible, e.g. activated by application of external or internal inducing agent, such as gibberellic acid and abscisic acid which are known to exercise important control at the transcriptional level over a-amylase gene expression in aleurone cells (Skriver et al., 1991
, Proc. Natl. Acad. Sci. USA
88:7266-7270; Gubler and Jacobsen, 1992
, Plant Cell
4:1435-1441).
Of particular interest to the present invention are tissue specific promoters. These promoters can be fused with a heterologous DNA sequence and used to transform a plant cell to create a transgenic plant that selectively expresses the heterologous DNA in a specific tissue. Several promoters are currently being used for tissue-specific, heterologous gene expression in monocot cells. For example, the promoter regions from genes coding for hydrolases have been used to direct germination-specific expression of a heterologous DNA sequence in transgenic monocot cells (see Skriver et al., 1991, supra); Wolf, 1992
, Mol. Gen. Genet
. 234:33-42; Mikkonen et al., 1996
, Plant Mol. Biol
. 31:239-254; Jensen et al., 1996
, Proc. Natl. Acad. Sci. USA
93:3487-3491; Jensen et al., 1998, supra; U.S. Pat. No. 5,712,112).
A variety of plant promoters with different characteristics and which are effective in different plant species and/or organs is desirable in order to bring potential applications of transgenic plants into practice. Since the task of successfully cloning and demonstrating the utility of a highly expressed promoter is formidable, the use of systems for transient expression of heterologous genes in protoplasts has proven ideal to predict whether a gene construct will function during plant germination (Jensen et al., 1996, supra).
Since the location of the heterologous gene in the host's DNA can affect the efficiency with which it is expressed, it is often necessary to produce many individual transgenic plants to ensure that an effective line with the desired characteristics can be selected from them. These plants are then bred conventionally.
In view of the widespread occurrence of &agr;-glucosidases in higher plants and their potential importance in carbohydrate metabolism, it is surprising that there are only a few reports on &agr;-glucosidase genes. These include cDNA sequences for &agr;-glucosidases of barley (Tibbot and Skadsen, 1996
, Plant Mol. Biol
. 30:229-241), spinach (Sugimoto et al., 1997
, Plant Mol. Biol
. 33:765-768), and potato (Taylor et al., 1998
, Plant J
. 13:419-425), as well as a genomic sequence of an &agr;-glucosidase gene from
Arabidopsis thaliana
(Monroe et al., 1997
, Plant Physiol
. 115:863; GenBank Accession No. AF014806).
Only a few genomic DNA sequences encoding plant cystatins have been described, including the genomic sequence for rice cystatin (Kondo et al., 1989
, Gene
81:259-265), the sequence for potato cystatin (Waldron et al., 1993
, Plant Mol. Biol
. 23:801-812), and the sequence for maize cystatin (Abe et al., 1996
, Biosci. Biotech. Biochem
. 60:1173-1175).
In no previously reported case has an &agr;-glucosidase gene promoter or a cystatin gene promoter been used to direct heterologous expression in plants. One object of the present invention is therefore to provide an &agr;-glucosidase gene promoter or a cystatin gene promoter from an industrially important organism, and utilize the promoter to direct expression of heterologous protein in monocotyledonous grass plants, including the cereals.
SUMMARY OF THE INVENTION
Novel germination-specific promoters have now been identified and isolated from the barley genome. Two such useful regulatory sequences described and claimed herein are the promoters of the &agr;-glucosidase gene and the cystatin-1 genes from barley, which are useful to express a desired heterologous gene at high levels in the aleurone tissue of germinating kernels, particularly barley. In a preferred embodiment, these promoters are used to induce expression of heterologous genes in the aleurone tissue of kernels during germination, including the process of malting, for example, in the production of a brewed product such as beer.
An isolated nucleotide sequence comprising at least 1930 base pairs (hereafter abbreviated bp) upstream of th

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