PLANT PROMOTERS, AND METHODS OF USE FIELD OF THE INVENTION The present application relates to plant promoter sequences and to methods of using plant promoter sequences to direct gene expression in plants. BACKGROUND OF THE INVENTION
There is a continuing need for promoters that direct gene expression exclusively, or predominantly, in one or more cell or tissue types within plant seeds, such as the grain of cereal crops. These promoters are useful for directing the expression of proteins or functional RNA molecules (such as antisense RNA molecules) within the seeds, thereby altering a characteristic of the seed, such as resistance to disease.
For example, in wet climates barley is most susceptible to infection by fungus of the genus Fusarium from the time the spike emerges from the leaf sheath to about the hard dough stage of kernel development. Few kernels are produced when barley is severely infected with Fusarium, and they are discolored with the mycotoxin deoxynivalenol. The hyphae of Fusarium enter into the developing aleurone and starchy endosperm of the grain during differentiation of the testa cells at 10 to 35 days after anthesis. Mutants with high sensitivity to Fusarium infection suffer uninhibited penetration of the testa layer by the infecting hyphae, while in less susceptible lines penetration is retarded or blocked (Skadhauge, B., et al., Hereditas 126: 147-160 (1997)). Thus, there is a need for isolated promoters, that are predominantly or exclusively active in the testa of barley seed, that can be used to express an antifungal agent in the barley testa, thereby preventing or reducing fungal infection of barley seed.
Representative antifungal agents include the enzyme oxalate oxidase, which catalyzes the production of hydrogen peroxide, and antisense nucleic acid molecules that inhibit the production of one or more catalases, and/or inhibit the production of one or more ascorbate peroxidases, that scavenge hydrogen peroxide in cells. Hydrogen peroxide is produced in plant cells in response to pathogen infection (such as fungal infection), and is implicated in pathogen toxicity, cross-linking of cell wall proteins, lignification, and signal transduction for defense gene activation. An example of a plant defense response against pathogen infection, that utilizes hydrogen peroxide as a signal, is the hypersensitive response in which rapid plant cell death occurs around the site of infection, thereby limiting the proliferation of the pathogen (see, Hammond-Kosack, K.E. & Jones, J.D.G., Plant Cell 8:1773-1791 (1996); Dangl, J.L., et al., Plant Cell 8: 1793- 1807 (1996).
In this regard, tobacco plants that express antisense nucleic acid molecules that inhibit production of hydrogen peroxide scavenging enzymes, including peroxisomal catalases and ascorbate peroxidase, initiate a hypersensitive response to low levels of bacterial pathogens that do not trigger a hypersensitive response in control tobacco plants that do not express the antisense nucleic acid molecules (see, e.g., Takahashi, H., et al., Plant J. 11: 993-1005 (1997).
Thus, enhancing the expression of one or more enzymes that produce hydrogen peroxide in plant cells, or inhibiting the expression of one or more enzymes that scavenge hydrogen peroxide in plant cells, will enhance the ability of the plant to mount a defense response to pathogen infection.
Again by way of example, the testa cells of barley seed synthesize proanthocyanidins. Proanthocyanidins are undesirable in malting barley for beverage production, as they precipitate proteins and cause haze formation. The first part of the pathway leading to these compounds is shared with the synthetic pathway for anthocyanins, and many other flavonoids occurring in many tissues of the plant, while the last steps of the conversion of (+) 2,3-trans-3,4-cis-leucocyanidin to catechin and procyanidin B3 takes place only in the testa cells. The removal of proanthocyanidins from malting barley by blocking the proanthocyanidin biosynthetic pathway is preferably limited to these last steps in the testa cells, because blocking earlier steps in the flavonoid pathway throughout the plant may lead to deficiencies in plant development and stress resistance (see, von Wettstein, D., Breeding of value added barley by mutation and protein engineering, in: Proc. Int. Sympos. on the Use of Induced Mutations and Molecular Techniques in Crop Improvement, International Atomic Energy Agency, Vienna, IAEA-SM-340/15, 1995, pp. 67-76). Thus, there is a need for promoters, that are predominantly or exclusively active in barley testa tissue, that can be used to express agents (such as antisense RNA molecules) that inhibit the production of enzymes involved in the last steps of the conversion of (+) 2,3-trans-3,4-cis-leucocyanidin to catechin and procyanidin B3.
In accordance with the foregoing, the present invention provides novel germin genes and novel germin promoters. Germins include one group of protein isoforms that possess oxalate oxidase activity (see, Lane, B.G., FASEB J. 8: 294-301 (1994); Lane, B.G., et al., J. Biol Chem. 266: 10461-10469 (1991); Lane, B.G., et al., J. Biol. Chem. 268: 12239-12242 (1993).), and a number of proteins in wheat, barley, rice and Arabidopsis thaliana with a primary structure highly homologous to oxalate oxidase but for which oxalate oxidase activity, or any other enzymatic activity, has not been demonstrated (see, Dunwell, J.M., Biotech. Genet. Engin. Rev. 15: 1-32 (1998); Membre, N., et al., Plant. Mol. Biol. 35: 459-469 (1997); Wei, Y., et al., Plant Mol. Biol. 36: 101- 112 (1998)).
Some promoters of the invention can be used, for example, to direct the expression of an open reading frame in the testa of plant seeds, including the seeds of cereal crops such as barley. Some promoters of the invention can be used, for example, to direct the expression of an open reading frame in plant leaves, including the leaves of cereal crops such as barley.
SUMMARY OF THE INVENTION In accordance with the foregoing, in one aspect the present invention provides five isolated barley germin genes. The nucleic acid sequences of the five isolated germin genes are set forth in SEQ ID NO:l (called Ger A and encoding the germin protein consisting of the amino acid sequence set forth in SEQ ID NO:2); SEQ ID NO:3 (called Ger B and encoding the germin protein consisting of the amino acid sequence set forth in SEQ ID NO:4); SEQ ID NO:5 (called GerD and encoding the germin protein consisting of the amino acid sequence set forth in SEQ ID NO:6); SEQ ID NO:7 (called Ger E and encoding the germin protein consisting of the amino acid sequence set forth in SEQ ID NO:8); and SEQ ID NO:9 (called Ger F and encoding the germin protein consisting of the amino acid sequence set forth in SEQ ID NO: 10). In another aspect, the present invention provides isolated germin gene promoters, such as the germin gene promoters set forth in SEQ ID NO: 11 (which is the promoter naturally associated with the barley germin gene Ger A set forth in SEQ ID NO:l), SEQ ID NO: 12 (which is the promoter naturally associated with the barley germin gene Ger B set forth in SEQ ED NO:3), SEQ ID NO: 13 (which is the promoter naturally associated with the barley germin gene Ger D set forth in SEQ ID NO:5), and SEQ ID NO: 14 (which is the promoter naturally associated with the barley germin gene GerF set forth in SEQ D NO:9). Based upon nucleic acid hybridization studies, and/or in vivo expression studies in barley seeds, the Ger A promoter set forth in SEQ ID NO: 11 is predominantly expressed in leaf tissue; the Ger B promoter set forth in SEQ ID NO: 12 is predominantly expressed in the seed testa; the Ger D promoter set forth in SEQ ED NO: 13 is predominantly expressed in seed tissue; and the Ger F promoter set forth in SEQ ED NO: 14 is predominantly expressed in the seed testa and pericarp.
Thus, in one aspect, the present invention provides isolated promoters that each hybridize under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO:14.
In another aspect, the present invention provides isolated promoters that are each at least 70% identical (such as at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical), to a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14.
In another aspect, the present invention provides vectors that each comprise a promoter that hybridizes under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO: 11, SEQ LD NO: 12, SEQ ED NO: 13, and SEQ ED NO: 14. The present invention also provides vectors that each comprise a promoter that is at least 70% identical (such as at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14. The invention also provides plant cells, such as barley seed cells, that include a vector of the invention.
In another aspect, the present invention provides methods of directing the expression of an open reading frame in a plant (such as a barley plant). The methods of this aspect of the invention each include the step of introducing into a plant cell a nucleic acid molecule comprising: (a) a promoter that hybridizes under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule selected from the group consisting of SEQ ED NO:ll; SEQ ED NO: 12; SEQ ED NO: 13; and SEQ ED NO: 14 and (b) an open reading frame operably linked to the promoter. The open reading frame is expressed within the plant cell. In some embodiments of this aspect of the invention, a promoter that hybridizes under conditions of 2 X SSC at 55°C for 30 minutes to the complement of the nucleic acid molecule of SEQ ED NO: 12 is utilized and more than 50% of the total amount of expression of the open reading frame in the plant occurs in the testa. In some embodiments of this aspect of the invention, a promoter that hybridizes under conditions of 2 X SSC at 55°C for 30 minutes to the complement of the nucleic acid molecule of SEQ ED NO: 14 is utilized and the combined amount of expression of the open reading frame in the testa and pericarp is more than 50% of the total amount of expression of the open reading frame in the plant. By way of example, the isolated germin genes of the invention can be introduced into plants and expressed therein to enhance the level of germin protein in the plants. By way of example, the isolated promoters of the invention can be operably linked to a nucleic acid molecule encoding a protein, or encoding a functional RNA molecule such as an antisense RNA molecule, and introduced into plants to direct the expression of the encoded protein or functional RNA molecule (for example in the testa of barley seed). Again by way of example, the isolated promoters and germin genes of the invention, or portions thereof, can be used as hybridization probes to physically and/or genetically map the barley genome. The methods of the invention are useful for directing the expression of an open reading frame in a plant (such as a barley plant), for example to enhance a plant defense response.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainsview, New York (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art.
As used herein, the term "isolated", when used with respect to a nucleic acid molecule of the invention (such as an isolated gene or promoter molecule), or with respect to an isolated protein of the invention, means a nucleic acid molecule, or protein, that is substantially free from cellular components that are associated with the nucleic acid molecule, or protein, as it is found in nature. As used in this context, the term "substantially free from cellular components" means that the nucleic acid molecule, or protein, is purified to a purity level of greater than 80% (such as greater than 90%, greater than 95%, or greater than 99%). Moreover, the term "isolated", when used with respect to a nucleic acid molecule, or protein, of the invention, includes nucleic acid molecules, or proteins, which do not naturally occur, and have been produced by synthetic means. An isolated nucleic acid molecule, or protein, generally resolves as a single, predominant, band by gel electrophoresis, and yields a nucleotide sequence profile, or amino acid sequence profile, consistent with the presence of a predominant nucleic acid molecule or protein. As used herein in connection with the isolated nucleic acid molecules of the invention, the term "complement" refers to a nucleic acid molecule that can hybridize to a specified nucleic acid molecule (such as a promoter molecule consisting of any one of the sequences set forth in SEQ ED NO: 11; SEQ ED NO: 12, SEQ ED NO: 13 or SEQ ED NO: 14) of the invention through Watson-Crick base pairing under defined conditions of temperature and salt concentration.
The term "vector" refers to a nucleic acid molecule, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a promoter molecule of the present invention. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated. The term "vector" includes the T-DNA of a Ti plasmid. Some vectors include the elements necessary to express RNA encoded by the insert nucleic acid molecule.
The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to an open reading frame when the promoter is capable of affecting the expression of that open reading frame (i.e., the open reading frame is under the transcriptional control of the promoter).
The term "open reading frame" refers to a DNA sequence that encodes a protein or functional RNA, such as an antisense RNA. The abbreviation "SSC" refers to a buffer used in nucleic acid hybridization solutions. One liter of the 20 X (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.
As described in Examples 1 and 2 herein, five germin genes were isolated from barley (Hordeum vulgare). The nucleic acid sequences of the five barley germin genes are set forth in SEQ ED NO:l (called the Ger A gene which encodes the protein set forth in SEQ ED NO:2), SEQ ED NO:3 (called the Ger B gene which encodes the protein set forth in SEQ ED NO:4), SEQ ED NO:5 (called the Ger D gene which encodes the protein set forth in SEQ ED NO:6), SEQ ED NO:7 (called the Ger E gene which encodes the protein set forth in SEQ ED NO:8), and SEQ ED NO:9 (called the Ger F gene which encodes the protein set forth in SEQ ED NO: 10).
Thus, in one aspect the present invention provides isolated germin genes which each consist of a nucleic acid sequence selected from the group of nucleic acid sequences consisting of SEQ ED NO:l; SEQ ED NO:3; SEQ ED NO:5; SEQ ED NO:7; and SEQ ED NO:9. In a related aspect, the present invention provides isolated germin genes that each hybridize under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a germin gene consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, and SEQ ED NO:9. Some genes of this aspect of the invention hybridize to the complement of any one of the nucleic acid sequences set forth in SEQ ED NO: 1, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, and SEQ ED NO:9 under conditions of 1 X SSC at 55°C for 30 minutes. Some genes of this aspect of the invention hybridize to the complement of any one of the nucleic acid sequences set forth in SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, and SEQ ED NO:9 under conditions of 0.2 X SSC at 55°C for 30 minutes. In another aspect, the present invention provides isolated promoters that each hybridize under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO:14. Some promoters of this aspect of the invention hybridize to the complement of any one of the nucleic acid sequences set forth in SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14 under conditions of 1 X SSC at 55°C for 30 minutes. Some promoters of this aspect of the invention hybridize to the complement of any one of the nucleic acid sequences set forth in SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14 under conditions of 0.2 X SSC at 55°C for 30 minutes.
Hybridization can be conducted, for example, by utilizing the technique of hybridizing labeled nucleic acid probes to nucleic acid molecules immobilized on nitrocellulose filters or nylon membranes. An exemplary hybridization protocol is set forth in Example 3 herein. For example, utilizing the exemplary hybridization protocol set forth in Example 3, promoter molecules of the invention, that hybridize under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO:14, can be identified by immobilizing the promoter molecule to a nylon membrane (or nitrocellulose filter). The membrane is incubated in aqueous solution in the presence of the probe nucleic acid molecule (such as the complement of any one of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO:14) under low stringency conditions (such as 6 X SSC at 50°C for 12 hours). The membrane is then washed under conditions of 2 X SSC at 55°C for 30 minutes. An isolated promoter molecule of the invention will remain hybridized to the immobilized target molecule under these wash conditions of 2 X SSC at 55°C for 30 minutes.
The present invention also provides isolated germin genes that are at least 70% identical (such as at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a germin gene selected from the group consisting of SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, and SEQ ED NO:9.
In another aspect, the present invention provides isolated promoters that are each at least 70% identical (such as at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical), to a promoter consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO: 14.
In another aspect, the present invention provides isolated germin proteins that are each at least 70% identical (such as at least 80% identical, at least 90% identical, at least 95% identical, or at least 99% identical) to a polypeptide sequence selected from the group consisting of SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:8, and SEQ ED NO: 10. Sequence identity is defined as the percentage of nucleic acid residues in a candidate nucleic acid sequence (such as the nucleic acid sequence of a candidate promoter molecule of the invention), or the percentage of amino acid residues in a candidate amino acid sequence, that are identical to the corresponding nucleic acid residues in a subject nucleic acid sequence (such as a nucleic acid sequence set forth in any one of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, or SEQ ED NO: 14), or to the corresponding amino acid residues in a subject amino acid sequence (such as an amino acid sequence set forth in any one of SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:8, or SEQ ED NO: 10), after aligning the sequences to achieve the maximum percent identity, and not considering any nucleic acid residue substitutions as part of the sequence identity. The candidate nucleic acid sequence or candidate amino acid sequence (which may be a portion of a larger nucleic acid sequence or amino acid sequence) is the same length as the subject nucleic acid sequence or amino acid sequence, and no gaps are introduced into the candidate nucleic acid sequence or amino acid sequence in order to achieve the best alignment.
Nucleic acid sequence identity can be determined, for example, in the following manner. The candidate nucleic acid sequence is used to search a nucleic acid sequence database, such as the Genbank database (accessible at Website http://www.ncbi.nlm.nih.gov/blast/), using the program BLASTN version 2.1 (based on Altschul et al., Nucleic Acids Research 25:3389-3402 (1997)). The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity as defined in Wootton, J.C. and S. Federhen, Methods in Enzymology 266:554-571 (1996). The default parameters of BLASTN are utilized.
Amino acid sequence identity can be determined in the following manner. The subject amino acid sequence is used to search a polypeptide sequence database, such as the GenBank database (accessible at web site http://www.ncbi.nln.nih.gov blast/), using the BLASTP program. The program is used in the ungapped mode. Default filtering is used to remove sequence homologies due to regions of low complexity. The default parameters of BLASTP are utilized. Filtering for sequences of low complexity utilize the SEG program.
Some isolated germin genes of the invention are present on a DNA fragment that is less than 50 kilobases long. Some isolated germin genes of the invention are present on a DNA fragment that is less than 20 kilobases long. Some isolated germin genes of the invention are present on a DNA fragment that is less than 10 kilobases long. Some isolated germin genes of the invention are present on a DNA fragment that is less than 5 kilobases long. Some isolated promoters of the invention are present on a DNA fragment that is less than 50 kilobases long. Some isolated promoters of the invention are present on a DNA fragment that is less than 20 kilobases long. Some isolated promoters of the invention are present on a DNA fragment that is less than 10 kilobases long. Some isolated promoters of the invention are present on a DNA fragment that is less than 5 kilobases long. Some isolated promoters of the invention are present on a DNA fragment that is less than 1 kilobase long.
The nucleic acid molecules of the invention can be isolated by using a variety of cloning techniques known to those of ordinary skill in the art. For example, all, or one or more portions, of the complement of a nucleic acid molecule having a nucleic acid sequence set forth in SEQ ED NO:l l, SEQ ED NO:12, SEQ ED NO:13, or SEQ ED NO:14 can be used as a hybridization probe to screen a plant genomic library. The technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes can be used to screen the genomic library. Exemplary hybridization and wash conditions for screening the genomic library are: hybridization for 20 hours at 50°C in 5.0 X SSC, 0.5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of twenty minutes each at 25°C) in 2 X SSC, 1% (w/v) sodium dodecyl sulfate, and one wash (for twenty minutes) in 2 X SSC, 1% (w/v) sodium dodecyl sulfate, at 50°C. A more stringent optional wash can be utilized if desired. Exemplary conditions for a more stringent wash are 0.5 X SSC, 1% (w/v) sodium dodecyl sulfate, at 50°C for twenty minutes.
Again, by way of example, nucleic acid molecules of the invention can be isolated by the polymerase chain reaction (PCR) described in The Polymerase Chain Reaction (K.B. Mullis et al., eds. 1994). For example, Gobinda et al. (PCR Methods Applic. 2:318-22 (1993)), incorporated herein by reference, disclose "restriction-site PCR" as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus. First, genomic DNA is amplified in the presence of a linker-primer, that is homologous to a linker sequence ligated to the ends of the genomic DNA fragments, and in the presence of a primer specific to the known region. The amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR can be transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase. Further, by way of example, inverse PCR permits acquisition of unknown sequences starting with primers based on a known region (Triglia, T., et al., Nucleic Acids Res 16:8186 (1988)). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region.
The isolated germin proteins of the invention can be produced, for example, by introducing a vector, comprising a nucleic acid molecule (such as a cDNA or genomic clone) that encodes a germin protein of the invention, into a host cell under conditions that enable expression of the encoded germin protein within the host cell. Useful host cells in this aspect of the invention include prokaryotic and eukaryotic host cells (including plant cells, such as barley plant cells). Thus, for example, a vector comprising a nucleic acid molecule that encodes a germin protein can be stably integrated into the genome of a plant (such as a barley plant). The genetically modified plant can be used to produce (such as by self-fertilization) a population of plants that each include the vector, and that each express a germin protein of the invention in some or all of their cells. The vector, comprising a nucleic acid molecule that encodes a germin protein, can be introduced into a plant by any art-recognized means, such as the methods described infra for introducing nucleic acid molecules into plants. The expressed germin proteins can be isolated from the plant tissue by any art-recognized means, such as exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography. Additionally, art-recognized techniques for the purification of proteins and peptides are set forth in Methods in Enzymology, Vol. 182, Guide to Protein Purification, Murray P. Deutscher, ed. (1990), which publication is incorporated herein by reference.
In another aspect, the present invention provides vectors that include a promoter of the invention. Vectors that are functional in plants include plasmids derived from Agrobacterium plasmids. Such vectors are capable of genetically transforming plant cells. Briefly, these vectors typically contain left and right border sequences that are required for integration into the host (plant) chromosome. A promoter molecule of the invention can be inserted between these border sequences. In some embodiments, a selectable marker gene is also included. The vector also may contain a bacterial origin of replication.
In another aspect, the present invention provides host cells including a vector of the invention. Host cells can be prokaryotic or eukaryotic, such as plant cells. Vectors of the invention can be introduced into plant cells using techniques well known to those skilled in the art. These methods include, but are not limited to, direct DNA uptake, such as particle bombardment or electroporation (see, Klein et al., Nature 327:70-73 (1987); U.S. Patent No. 4,945,050), and AgrobαcteriM/n-mediated transformation (see, e.g., U.S. Patent Nos: 6,051,757; 5,731,179; 4,693,976; 4,940,838; 5,464,763; and 5,149,645). Within the cell, the transgenic sequences may be incorporated within the chromosome. Transgenic plants can be obtained, for example, by transferring vectors that include a selectable marker gene, e.g., the lan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature, 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices, or other tissues or cells, of the plant to be transformed as described by An et al., Plant Physiology, 81:301-305 (1986).
Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, for example, kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.
In addition to the methods described above, several methods are known in the art for transferring nucleic acid molecules into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Florida (1993), incorporated by reference herein). A representative example is treatment of protoplasts with polyethylene glycol (see, e.g., Lyznik et al., Plant Molecular Biology, 13:151-161 (1989)). Further, plant viruses can be used as vectors to transfer genes to plant cells. Examples of plant viruses that can be used as vectors to transform plants include the Cauliflower Mosaic Virus (see, e.g., Brisson et al., Nature 310:511-514 (1984). Other useful techniques include: site-specific recombination using the Cre-lox system (see, U.S. Patent No. 5,635,381); and insertion into a target sequence by homologous recombination (see, U.S. Patent No. 5,501,967). Additionally, plant transformation strategies and techniques are reviewed in Birch, R.G., Ann Rev Plant Phys Plant Mol Biol, 48:297 (1997); and Forester et al., Exp. Agric, 33:15-33 (1997).
Positive selection markers may also be utilized to identify plant cells that include a vector of the invention. For example, U.S. Patent Nos. 5,994,629, 5,767,378, and 5,599,670, describe the use of a β-Glucuronidase transgene and application of cytokinin- glucuronide for selection, and use of mannophosphatase, or phosphmanno-isomerase, transgene and application of mannose for selection.
The cells which have been genetically transformed may be grown into plants by a variety of art-recognized means. See, for example, McConnick et al., Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either selfed or crossed with a different plant strain, and the resulting homozygotes or hybrids having the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that the subject phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure the desired phenotype or other property has been achieved. The following are representative plant species that are suitable for genetic manipulation in accordance with the present invention. The citations are to representative publications disclosing genetic transformation protocols that can be used to genetically transform the listed plant species. Rice (Alam, M.F., et al., Plant Cell Rep. 18:572-575 (1999)); maize (U.S. Patent Serial Nos. 5,177,010 and 5,981,840); wheat (Ortiz, J.P.A., et al., Plant Cell Rep. 15:877-881 (1996)); tomato (U.S. Patent Serial No. 5,159,135); potato (Kumar, A., et al., Plant J. 9:821-829 (1996)); cassava (Li, H.-Q., et al., Nat. Biotechnology 14:736-740 (1996)); lettuce (Michelmore, R., et al., Plant Cell Rep. 6:439- 442 (1987)); tobacco (Horsch, R.B., et al., Science 227:1229-1231 (1985)); cotton (U.S. Patent Serial Nos. 5,846,797 and 5,004,863); grasses (U.S. Patent Nos. 5,187,073 and 6,020,539); peppermint (Niu, X., et al., Plant Cell Rep. 17:165-171 (1998)); citrus plants (Pena, L., et al., Plant Sci. 104:183-191 (1995)); caraway (Krens, F.A., et al., Plant Cell Rep., 17:39-43 (1997)); banana (U.S. Patent Serial No. 5,792,935); soybean (U.S. Patent Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Patent Serial No. 5,952,543); poplar (U.S. Patent No. 4,795,855); monocots in general (U.S. Patent Nos. 5,591,616 and 6,037,522); brassica (U.S. Patent Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Patent No. 6,074,877). Representative transformation protocols for Picea species are set forth in D. H. Clapham et al., Molecular Biology of Woody Plants (S.M. Jain and S.C. Minocha, eds.) Vol. 2, 105-118 (2000), Kluwer Academic Publishers.
Cultures of mammalian host cells, and other host cells that do not have rigid cell membrane barriers, can be transformed, for example, using the calcium phosphate method as originally described by Graham and Van der Eb (Virology, 52:546 [1978]) and modified as described in sections 16.32-16.37 of Sambrook et al., supra. However, other methods for introducing nucleic acid molecules into cells, such as Polybrene (Kawai and Nishizawa, Mol. Cell. Biol, 4:1172 [1984]), protoplast fusion (Schaffner, Proc. Natl. Acad. Sci. USA, 77:2163 [1980]), electroporation (Neumann et al., EMBO J., 1:841 [1982]), and direct microinjection into nuclei (Capecchi, Cell, 22:479 [1980]), may also be used. Additionally, animal transformation strategies are reviewed in Monastersky, G.M. and Robl, J.M., Strategies in Transgenic Animal Science, ASM Press, Washington, D.C., 1995.
Prokaryotic host cells can be transformed, for example, using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Electroporation may also be used for transformation of these cells. Representative prokaryote transformation techniques are set forth in Dower, W.J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp., 1990; Hanahan et al., Meth. Enzymol., 204:63 (1991). In another aspect, the present invention provides methods for directing the expression of an open reading frame in a plant cell. The methods of this aspect of the invention comprise the step of introducing into a plant cell a nucleic acid molecule comprising (a) a promoter that hybridizes under conditions of 2 X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule selected from the group consisting of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO: 14; and (b) an open reading frame operably linked to the promoter. In some embodiments of the methods of this aspect of the invention, the promoter hybridizes under conditions of 1 X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:l l; SEQ ED NO: 12; SEQ ED NO: 13; and SEQ ED NO: 14. In some embodiments of the methods of this aspect of the invention, the promoter hybridizes under conditions of 0.2X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:ll; SEQ ED NO:12; SEQ ED NO:13; and SEQ ED NO:14.
In a related aspect, the present invention provides methods of directing the expression of an open reading frame in a plant cell, the methods comprise the step of introducing into a plant cell a nucleic acid molecule comprising (a) a promoter that is at least 70% identical (such as at least 80% identical, or at least 90% identical, or at least 95% identical, or at least 99% identical) to a nucleic acid molecule selected from the group consisting of SEQ ED NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14; and (b) an open reading frame operably linked to the promoter.
In some embodiments of the methods of the invention, the promoter is operably linked to an open reading frame that encodes a protein, such as an open reading frame that encodes a reporter protein. An example of a reporter protein that can be used in plant cells is β-Glucuronidase (GUS). Other exemplary proteins that can be expressed in plant cells in accordance with the methods of the invention are proteins that enhance one or more plant defense responses. For example, when some proteins are expressed in plant cells in accordance with the methods of the invention, the plant generates a defense response to a lower titer of infecting pathogen (and/or to a lesser amount of physical damage) compared to control plants that are not treated in accordance with the methods of the invention, and/or the magnitude of one or more plant defense responses is greater in plants expressing the protein in accordance with the methods of the invention compared to control plants.
Examples of proteins that enhance one or more plant defense responses include oxalate oxidases that produce hydrogen peroxide. Thus, for example, expressing an oxalate oxidase in plant cells (such as barley seed cells, including barley testa cells), under the control of a germin promoter, enhances the sensitivity of signal transduction pathways that utilize hydrogen peroxide as a signal, such as the signal transduction pathway that regulates the hypersensitive response. Thus, the modified cells generate a hypersensitive response to a lower innoculum of pathogen compared to unmodified cells. Another example of proteins that can be expressed in plant cells (such as barley seed cells, including barley testa cells) in accordance with the methods of the invention, to enhance one or more plant defense responses, are the cysteine-rich antifungal proteins disclosed in Terras et al., Plant Cell 7: 573-588 (1995), which publication is incorporated herein in its entirety.
Another example of a protein that can be expressed in plant cells (such as barley seed cells, including barley testa cells), in accordance with the methods of the invention, to enhance one or more plant defense responses, is the endochitinase from Trichoderma harzianum which provides enhanced resistance to such microbial pathogens as Alternaria alternata, A. solani, Botrytis cinerea, Rhizoctonia solani, and Fusarium solani. (see, e.g., Lorito, M., et al., Proc. Natl. Acad. Sci. USA 95: 7860-7865; Lorito, M., et al., Phytopathology 83: 302-307 (1993)). SEQ ED NO: 15 discloses the nucleic acid sequence of a representative cDNA molecule that encodes a representative endochitinase (SEQ ED NO: 16) from T. harzianum. SEQ ED NO: 15 is also disclosed in GenBank Ace. No. S78423 and its isolation is described by Garcia, L., et al., Curr. Genet. 27: 83-89 (1994).
Thus, in one embodiment, the present invention provides methods for expressing endochitinase in plant cells (such as barley testa cells), wherein the methods comprise the step of introducing into a plant cell a nucleic acid molecule comprising (a) a promoter that hybridizes under conditions selected from the group of conditions consisting of 2 X SSC at 55°C for 30 minutes, 1 X SSC at 55°C for 30 minutes, and 0.2 X SSC at 55°C for 30 minutes, to the complement of a nucleic acid molecule selected from the group consisting of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO: 14; and (b) an open reading frame operably linked to the promoter, wherein the open reading frame encodes an endochitanase and consists of a nucleic acid sequence that hybridizes to the complement of the nucleic acid sequence set forth in SEQ ED NO: 15 under conditions selected from the group of conditions consisting of 2 X SSC at 55°C for 30 minutes, 1 X SSC at 55°C for 30 minutes, and 0.2 X SSC at 55°C for 30 minutes. The endochitinase is expressed within the plant cell.
In some embodiments of the methods of this aspect of the invention, the promoter molecule is operably linked to an open reading frame that encodes a functional RNA molecule, such as an open reading frame that encodes an antisense RNA molecule or a ribozyme.
An open reading frame that encodes an antisense RNA molecule expresses an RNA transcript that is complementary to a target mRNA molecule expressed within the host cell (i.e., the RNA transcript can hybridize to the target mRNA molecule through Watson-Crick base pairing). An open reading frame that encodes an antisense RNA molecule may be constructed in a number of different ways provided that it is capable of interfering with the expression of a target gene. An open reading frame that encodes an antisense RNA molecule can be constructed, for example, by inverting the open reading frame (or a portion thereof) of the target gene relative to its normal orientation for transcription to allow the transcription of its complement.
The open reading frame that encodes an antisense RNA molecule generally will be substantially identical to at least a portion of the target gene or genes. The sequence, however, need not be perfectly identical to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter open reading frame that encodes an antisense RNA molecule. The open reading frame that encodes an antisense RNA molecule typically will be substantially identical (although in antisense orientation) to the target gene. The minimal identity will typically be greater than about 65%, but a higher identity might exert a more effective repression of expression of the endogenous sequences. Substantially greater identity of more than about 80% is preferred, though about 95% to absolute identity would be most preferred.
Ribozymes can also be utilized. Ribozymes are catalytic RNA molecules that can cleave nucleic acid molecules having a sequence that is completely or partially homologous to the sequence of the ribozyme. It is possible to design ribozyme transgenes that encode RNA ribozymes that specifically pair with a target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. In carrying out this cleavage, the ribozyme is not itself altered, and is thus capable of recycling and cleaving other molecules. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the antisense constructs.
Ribozymes useful in the practice of this aspect of the invention typically comprise a hybridizing region of at least about nine nucleotides, which is complementary in nucleotide sequence to at least part of the target RNA, and a catalytic region which is adapted to cleave the target RNA (see, e.g., EPA No. 0 321 201; WO88/04300; Haseloff & Gerlach, Nature 334:585-591 [1988]; Fedor & Uhlenbeck, Proc. Natl Acad. Sci., USA 87:1668-1672 [1990]; Cech & Bass, Ann. Rev. Biochem. 55:599-629 [1986]). By way of non-limiting example, the methods of this aspect of the invention can be used to express, in plant cells, such as barley testa cells, one or more antisense molecules, and/or ribozymes, that target mRNA molecules that encode one or more enzymes that scavenge hydrogen peroxide. These enzymes include ascorbate peroxidases and catalases. SEQ ED NO: 17 sets forth the nucleic acid sequence of a cDNA molecule that encodes a representative ascorbate peroxidase having the amino acid sequence set forth in SEQ ED NO: 18. This ascorbate peroxidase is disclosed in GenBank accession number AJ006358 and in Hess and Boerner, Plant Physiol. 118: 329 (1998)). SEQ ED NO: 19 sets forth the nucleic acid sequence of a cDNA molecule that encodes a representative catalase having the amino acid sequence set forth in SEQ ED NO:20). This catalase is disclosed in GenBank accession number U20777 and in Skadsen, R.W., et al., Plant Mol. Biol. 29: 1005-1014.
Thus, in one embodiment, the present invention provides methods for enhancing the ability of a plant to respond to pathogen infection, wherein the methods comprise the step of introducing into a plant cell a nucleic acid molecule comprising (a) a promoter that hybridizes under conditions selected from the group of conditions consisting of 2 X SSC at 55°C for 30 minutes, 1 X SSC at 55°C for 30 minutes and 0.2 X SSC at 55°C for 30 minutes to the complement of a nucleic acid molecule selected from the group consisting of SEQ ED NO: 11, SEQ ED NO: 12, SEQ ED NO: 13, and SEQ ED NO: 14; and (b) an open reading frame operably linked to the promoter, wherein the open reading frame consists of a nucleic acid sequence that hybridizes to a nucleic acid sequence set forth in SEQ ED NO: 17 or SEQ ED NO: 19 under conditions selected from the group of conditions consisting of 2 X SSC at 55°C for 30 minutes; I X SSC at 55°C for 30 minutes; and 0.2 X SSC at 55°C for 30 minutes. The open reading frame is expressed within the plant cell. In the practice of the methods of the present invention, the nucleic acid molecule comprising the promoter operably linked to the open reading frame is typically part of a vector, such as a T DNA vector. The nucleic acid molecule comprising the promoter operably linked to the open reading frame can be introduced into the plant cell by any art- recognized means, such as the methods described supra for introducing nucleic acid molecules into plant cells. The nucleic acid molecule can be introduced into the plant cell and one or more whole plants can be regenerated from the plant cell. A population of plants can be produced from the regenerated plant(s). In some embodiments of the methods of the invention, the nucleic acid molecule is expressed in seed cells, such as testa cells, pericarp cells and/or epicaφ cells. In some embodiments of the methods of the invention, the nucleic acid molecule is expressed in the seed of a cereal crop species, such as barley seed cells, such as barley testa cells, barley pericarp cells and/or barley epicarp cells.
By way of non-limiting example, the methods of the invention can be used to express, in barley testa cells, one or more antisense molecules, and/or ribozymes, that target mRNA molecules that encode one or more enzymes involved in the biosynthesis of proanthocyanidins. Proanthocyanidins are undesirable in malting barley for beverage production, as they precipitate proteins and cause haze formation.
The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.
EXAMPLE 1 This example describes the materials and methods used to isolate and characterize germin genes from Hordeum vulgare, as described in Example 2.
Materials: Hordeum vulgare cv Gula was grown in soil in a green house with light/dark cycles of 16-h light, 21°C and 8-h dark, 16°C. An average photon flux density of 200 μEinstein m'V1 was measured. The spikes were harvested about 15 days after anthesis for mRNA isolation and particle gun bombardment. [32P]α-dCTP with specific activity 10n Bq/mmole was obtained from New England Nuclear, Boston MA, USA. M- MLN reverse transcriptase, E. coli DΝA polymerase I, RΝAase H, restriction endonucleases and TRIZOL reagent were obtained from GIBCOBRL Life Technologies, MD, USA. PolyATract mRΝA isolation systems was from Promega, Madison, Wisconsin, USA. Isolation of cDΝA Clones: The SABRE procedure was performed as described in
Lavery et al. (Lavery, D.L., et al., Proc. Natl. Acad. Sci. USA 95: 6831-6836 (1997)). The tester cDΝA was prepared from mRΝA isolated from testa plus pericaφ tissues of the barley kernels at 10 to 25 days after anthesis. The mRNA isolated from leaf tissue was reverse transcribed and used as driver. The tissues (100-200 mg) were homogenized in liquid nitrogen and total RNA was extracted by the method described by Tesniere and Vayda (Plant Mol. Biol. Rep. 9: 242-251 (1991)), except that the purification by CsCl centrifugation was replaced by purification through a polyvinyl-polypyrrolidone column. Thereafter mRNA was isolated as instructed in the Technical Manual of PolyATract mRNA isolation systems. This involved annealing to biotinylated oligo-dT, binding to strepavidin-coated paramagnetic particles and capturing particles in a magnetic stand.
The cDNA was synthesized according to the Instruction Manual for cDNA Synthesis System from GIBCOBRL life technologies. This method uses the M-MLV reverse transcriptase for the first strand and E. coli DNA polymerase I with RNAase H for the second strand synthesis. Briefly, in the SABRΕ procedure both cDNA libraries were digested with Sα«3Al to give fragments of 150-1500 bp with 5'-GATC overhangs. An adaptor consisting of the two annealed oligonucleotides (5'-P- GATCGGTTGGATGGACCGT-3' (SΕQ ΕD NO:21) and 5'-GGTCCATCCAACC-3* (SΕQ ΕD NO:22)) was ligated to the two libraries. For PCR amplification of the tester (testa + pericaφ) library the oligonucleotide 5'-Biotin-CCAGGATCCAACCGATC-3' (SΕQ ΕD NO:23) with a BamHl site served as primer. Amplification of the driver (leaf) library was with the 5'-GGTCCATCCAACCGATC-3* (SΕQ ΕD NO:24) oligonucleotide lacking the biotinylated nucleotide and BamHl site. The amplified tester and driver libraries were combined in a ratio of 1:30 and subjected to the phenol emulsion denaturing and reassociation technique (Miller R.D., Riblet R., Nucleic Acids Res. 23: 2339-2340 (1995)) in a thermocycler for 24h followed by incubation with SI nuclease to remove single stranded DNA. Tester homohybrids and tester/driver heterohybrids were isolated by attachment to strepavidin coupled magnetic beads. The tester homohybrids were separated from the heterohybrids by digestion with BamHl. The purified homohybrids in the supernatant were PCR amplified, analyzed by agarose gel electrophoresis, and the amplified product was used in a second and third round of selective amplification. Control hybridization was carried out at each round of selection as follows: The tester and the driver libraries were amplified with the biotinylated primer and the driver library was also amplified with the non-biotinylated primer. In the control the driver library that was amplified with the biotinylated primer was hybridized with the driver library amplified with the non- biotinylated primer. The products of this control hybridization were used as the new source of the driver DNA population.
Screening the barley bacterial artificial chromosome (BAC) library: The BAC library of the cultivar Morex contained 313,344 clones. These clones were spotted in duplicate in a grid pattern on 17 (22.5x22.5 cm) nylon filters. A 379 bp germin cDNA fragment (SEQ ED NO:20) was labeled with [32P]α-d-CTP and the RTS RadPrimer DNA labeling System (GIBCOBRL) and hybridized to the BAC clones on the nylon filter.
Seed coat specific expression of β-Glucuronidase with germin gene promoters: The germin promoters (SEQ ED NO: 12 and SEQ ED NO: 15) were spliced to the uidA open reading frame in a pUC 19-derived plasmid as described by Knudsen S. and Miiller M., Planta 185: 330-336 (1991). The constructs were introduced into epicaφ, testa plus pericaφ and endosperm tissues isolated from developing kernels. Caryopses with husks were placed in modified B5 medium of amino acids supplemented with 1.4g L"1 ammonium nitrate and added cefotaxin and timentin, then sterilized with 70% ethanol for 5 min and air-dried (Knudsen S. and Miiller M., supra). The barley tissues were isolated and placed on a filter paper wetted with solution containing ammonium nitrate (1.4 mg/mL"1), cefotaxin (0.4 mg/mL"1) and timentin (0.36 mg/mL"1).
Gold particles (50 μL of 1.5-3.0 μM) were mixed with 10 μL of DNA solution (1 μg/μL'1), 50 μL of CaCl2 (2.5 M) and 20 μL of spermidine (0.1 mM). The particles were centrifuged down and washed in 100% ethanol by first suspending them in 50 μL ethanol followed by 550 μL ethanol. The gold particles with adsorbed DNA were collected by centrifugation and resuspended in 40 μl 100% ethanol, and 10 μL aliquots were loaded onto the center of the macrocarrier for the helium-driven particle delivery system (DuPont). The tissue was bombarded with one shot per plate (rupture disk of 1100 psi. at 26 mm in Hg). The tissues were incubated in the modified B5 medium for 48 h at 25°C. After incubation formation of β-Glucuronidase was analyzed by histochemical staining using 5-bromo-4-chloro-3-indoyl-β-glucuronic acid (X-glu) for 24 h as previously described by Knudsen, S. and Miiller, M., supra. Assay and extraction of oxalate oxidase: the activity of oxalate oxidase was determined in tissues, extracts and SDS gels according to the procedure of Zhang et al. (Plant Mol Biol. Rep. 14: 266-272 (1996)) with a solution containing 40 mM succinic acid-NaOH pH 3.8, 60% (v/v) ethanol, 2 mM oxalic acid, 5 units/ml horseradish peroxidase and 50 g -100 mL"1 4-chloro-l-naphtol. In the presence of oxalic acid oxidase a black deposit forms. For the extraction of oxalate oxidase, the epicaφ, pericaφ, testa and endosperm tissues of the developing caryopses from barley cv. Gula were dissected, washed in water by centrifugation for 5 minutes at 20,000 xg. The tissues were first frozen in liquid nitrogen, thawed, and then ground in 100 μl of water. After centrifugation for 10 minutes at 20,000 xg. the supernatants were collected and the protein content determined (epicaφ 12 μg/μl, pericaφ 8 μg/μl, testa 15 μg/μl and endosperm 45 μg/μl). The protein samples were prepared in loading buffer (see, Sambrook, J., Fritsch, E.F., Maniatis, T., Molecular cloning: A laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) without any reducing agent (β-mercaptoethanol or dithiothreitol) and loaded without heat treatment on a 10% SDS-PAGE gel. After electrophoresis the gel was shaken overnight in Zhang's solution to develop the zymogram. Other methods: Standard procedures described in Sambrook et al., supra, were used for Northern and Southern blot analysis, agarose gel electrophoresis, DNA sequencing, cloning and SDS-PAGE.
EXAMPLE 2 This example describes the isolation and characterization of germin genes and promoters from Hordeum vulgare.
Testa and pericaφ specific cDNAs obtained with the SABRE procedure: The Selective Amplification via Biotin and Restriction enzyme mediated Enrichment (SABRE) procedure was chosen for identifying differentially expressed mRNA species, as it is capable of detecting moderately rare mRNA species (Lavery, D.L., et al., supra). Using mRNA from testa plus pericaφ tissue of the cv. Gula as tester, and with mRNA of leaf tissue as driver, three cDNA fragments were obtained. These were cloned into pBluescript-KS II vector and sequenced. A 379 bp cDNA fragment (SEQ ED NO:25) consisted of an open reading frame with 125 codons translating into an amino acid sequence (SEQ ED NO:26) with 22.4% and 67.2% identity to residue positions 72-201 of the barley root oxalate oxidase and barley leaf specific germin (GenBank accession number X93171), respectively. No obvious homologies were found for the other two cDNA fragments in the data bases and they did not represent continuous open reading frames. The 379 bp fragment (SEQ ED NO:25) hybridized on Southern blots specifically to testa + pericaφ cDNA and to the amplified homodimers of the three rounds of selection.
Genes in the barley genome with sequence homologv to the 379 bp cDNA fragment (SEQ ED NO:25): The radioactively labeled 379 bp cDNA fragment (SEQ ED NO:25) hybridized to 53 clones in a Morex barley bacterial artificial chromosome (BAC) library. This library provides 6.3 haploid genome equivalents with an insert size range of 30-195 kbp. At least 11 different hybridization patterns were obtained when isolated BAC DNAs digested with HindEII were separated on agarose gels and analyzed with the 379 bp cDNA fragment probe (SEQ ED NO:25) in Southern blots. The BAC clones were grouped as A, B, C, D, E and F according to the hybridizing restriction fragment sizes and numbers, assuming that the size of the bands identify clones containing portions of the same chromosome segment with one or several germin genes. This holds for BAC clones B, D, E and F, in which only one fragment hybridized, and for group A with three hybridizing fragments. The heterogeneous 24 BAC clones of group C comprise clones with one to six HindUI fragments hybridizing with the 329 bp cDNA probe (SEQ ED NO:25). Five of the C group BAC clones contained three restriction fragments with the same three different sizes. The other five clones represented five different patterns with respect to number and sizes of the hybridizing fragments. The blots were re-hybridized to Hindlll-restricted and labeled total barley genomic DNA. The puφose of this fingeφrinting was to determine if a BAC clone containing a germin gene includes DNA sequences that are abundant in the barley genome. The two A clones displayed a strong and a weak hybridizing band and thus contain few abundant DNA sequences, in contrast to BAC clone B, that contains many common genomic sequences. The hybridization patterns of the five D BAC clones share several hybridizing fragments but are unique for others. These BAC clones likely come from the same chromosome region but covering different adjacent, overlapping segments. The same situation applies to the E, F and C clones. Some of the latter group showed identical patterns of abundant DNA fragments. These were considered redundant BAC clones. One C clone was unique.
Southern hybridization of Hindlfl-digested genomic DNA of the cultivars Harrington, Morex, Steptoe and Q21861 with the 379 bp probe (SEQ ED NO:25) identified genes on restriction fragments which were the same size as those hybridizing in the Southerns of the BAC clones A to E. An exception was cv. Q21861 in which the Hindfll fragment characteristic of the B clones was larger. This confirms that these fragments, which represent segments in the genome, contain germin genes A-F. In the Southern blot some Hindlll fragments hybridized strongly to the testa- pericaφ specific 379 bp probe (SEQ ED NO:25), e.g., BAC clones B, D, and E. These fragments hybridized weakly, when probed with the leaf specific HvOxOLP cDNA, which encodes a mildew-induced germin (Wei, Y., et al., Plant Mol Biol. 36: 101-112 (1998)). Fragments that hybridized strongly with the mildew induced germin also hybridized weakly with the testa-pericaφ specific probe.
Hybridization of the Hindlll-restricted BAC clones with testa cDNA revealed positive hybridization signals with the fragments from clones B, F and E, whereas hybridization with leaf cDNA occurred with fragments of clones A and C. The C-BAC clones contain both genes for leaf and testa specific germins. From the present analysis it is concluded that the barley genome contains at least six testa-pericaφ specific and eight leaf specific germin genes.
The deduced primary structure of five germin precursor proteins (SEQ ED NO:2, SEQ ED NO:4, SEQ ED NO:6, SEQ ED NO:8, and SEQ ED NO: 10) from barley:
The DNA of the BAC clones A, B, D and E were cut with Hindlll and the DNA of clone F was cut with Xbal, ligated into pBluescript KSII vector and transformed into Escherichia coli. Plasmids with subclones containing the desired genes were identified by hybridization with the 379 bp cDNA fragment (SEQ ED NO:25), the inserts were excised with Xbal or Hindlll and separated on an agarose gel to determine their size. The five germin genes were sequenced with identical results from two independent clones each by the dideoxy chain termination method with an ABI automated sequencer at the WSU sequencing facility. The nucleotide sequences are available in the EMBL, GenBank, and DDBJ Data Base libraries under the accession nos: GerA, AF250933 (SEQ ED NO:l)(exons extending from nucleic acid residue 214 through 334, and 471 through 1039); GerB, AF250934 (SEQ ED NO:3)(exons extending from nucleic acid residue 1591 through 1711, and 1945 through 2504); GerD, AF250936 (SEQ ED NO:5)(exons extending from nucleic acid residue 396 through 519, and 699 through 1252); GerE, AF 250937 (SEQ ED NO:7)(exon extending from nucleic acid residue 87 through 646) and GerF, AF 250935 (SEQ ED NO:9)(exons extending from nucleic acid residue 1480 through 1600 and 1834 through 2393).
A sequence alignment was assembled and a family tree based on the sequence distance method using the Neighbour Joining algorithm of Saitou and Nei (Mol. Biol. Evol. 4: 406-425 (1987)) was constructed. The alignment compares the deduced amino acid sequences (SEQ ED NOS:2, 4, 6, 8, 10) of the open reading frames of the five barley germin genes (SEQ ED NOS:l, 3, 5, 7, 9) with the authentic or deduced amino acid sequences established for the barley root oxalate oxidase (Lane, B.G., et al., J. Biol. Chem. 268: 12239-12242 (1993)), the mildew induced oxalate oxidase in the mesophyll of barley (Zhou, F., et al., Plant Physiol 117: 33-41 (1998)) (Ace. No. Y14203), the wheat embryo oxalate oxidase (Lane, B.G., et al., J. Biol. Chem. 268: 12239-12242 (1993)) (germin gf 2.8; Ace. No. M63223), the barley leaf specific germin (Wei, Y., et al., Plant Mol. Biol. 36: 101-112 (1998)) (Ace. No. X93171), the wheat germin 2b (Ace. No. AJ237943), two rice germins (germin 1 Ace. No. AF032971 and Ace. No. Afl41878), and the Arabidopsis germin 6 (Ace. No. AF141878). Barley germins B (SEQ ED NO:4), F (SEQ ED NO: 10), D (SEQ ED NO:6), and E (SEQ ED NO:8) expressed in the testa pericaφ tissues are closely related to the rice germin 1, but are distinct from oxalate oxidase of barley and wheat. A. thaliana germin 6 is more closely related to the testa- pericaφ specific germins than to oxalate oxidase. The leaf specific barley germins are related to the rice germin (AF141878) and wheat germin 2b.
The alignment illustrates the remarkable conservation of the manganese (II) binding His and Glu residues of oxalate oxidase (Requena, L., Bomemann, S., Biochem. J. 343: 185-190 (1999)) and the μ-barrel secondary structure (Woo, E-J., EE5S Letters 437: 87-90 (1998)) also in all germins, which so far have not been identified as an oxalate oxidase or as another oxidase enzyme. The N-terminal amino acid sequence of the mature barley oxalate oxidase has been determined to be SDPDPLQDFCVADLDGKA (SΕQ ΕD NO:27)(Lane, B.G., J. Biol. Chem. 268: 12239- 12242 (1993)). This sequence (SΕQ ΕD NO:27) is highly conserved in eleven of the twelve deduced amino sequences from genomic or cDNA clones. The alignment with introduced gaps contains 235 residue positions. With the exception of germin Ε (SΕQ ΕD NO:8) the N-terminus of the mature proteins can be assigned to position 25 consisting of a Thr, Ser, Phe or Tyr residue. The pre-sequences (except for Ε (SΕQ ΕD NO: 8)) of the proteins (SEQ ED NOS:2, 4, 6, 8, 10) thus consist of 23 or 24 amino acids displaying considerable sequence homology within families of the tree. This pre-sequence is considered to target the oxalate oxidase and the germins to the cell wall. The pentameric or hexameric proteins show high resistance to detergents and proteases, which is lost upon dissociation into monomeric subunits. Two conserved Cys residues in position 34 and 52 are candidates to form a disulfide linkage. Three non-conserved potential N- glycosylation sequons are present in the two oxalate oxidases from barley and wheat. They are Asn74-Thr75-Ser76; Asn80-Gly81-Ser82; and Asnl40-Aspl41-Serl42.
A closer analysis of the homologies among the germins (SEQ ED NOS:2, 4, 6, 8, 10) provides a more differentiated picture than the family tree. A comparison of the testa- pericaφ specific germin B (SEQ ED NO:4) and F (SEQ ED NO: 10) precursors (227 aa) reveal a single amino acid difference in position 20 of the presequence (99.7% identity). The mature proteins of germin B (SEQ ED NO:4) and D (SEQ ED NO:6) (201 aa) have 98.5% sequence identity (3 aa difference). Germin E (SEQ ED NO:8) shows 93.5% sequence identity with germin B (SEQ ED NO:4) at residue positions 43- 235 (12 aa differences). The DNA sequence upstream of codon 43 has not yet been unequivocally determined. The mature rice germin 1 (Ace. No. AF032971) has 81.6% sequence identity with the barley germin B (SEQ ED NO:4) (37 aa difference among 202 aa). It may represent an ortholog to the barley testa-pericaφ germins. The precursor protein (230 aa) of the mildew-induced oxalate oxidase in the mesophyll of barley (Ace. No. Y 14203) and that of the wheat germinating embryo (Ace. No. M 63223) have a sequence identity of 95.2% with 11 aa differences. Identity with the barley root oxalate oxidase is likewise 95%. The mature proteins of the barley oxalate oxidase share with barley germin B (SEQ ED NO:4) 41.7% amino acid sequence identity and an identity of 44.4% is registered with the Arabidopsis germin 6. The sequence identity of the mature Arabidopsis germin 6 with barley germin B (SEQ ED
NO:4) reaches 62.3%. The level of sequence identity between the mature proteins of oxalate oxidase and the leaf-specific barley germin A (SEQ ED NO:2) amounts to 52.4%.
The amino acid sequence comparison of the barley leaf specific germin A precursor (SEQ ED NO:2) with the mildew-induced, leaf epidermis papilla-specific germin precursor (X 93171) yields an identity of 93.9%. An identity at this level (95.7%) is also seen with the Triticum aestivum 2b precursor protein and the Oryza germin precursor (89.3%). The amino acid sequence identity between the testa-pericaφ specific germin B (SEQ ED NO:4) and the leaf-specific germin A (SEQ ED NO:2) amounts to 67.9%.
The protein sequence comparisons indicate that there are at least three groups of germins in the barley plant: the testa-pericaφ specific proteins, the germin in the leaf epidermis and the oxalate oxidases in the root and mesophyll. Each of these groups is present in several isoforms encoded by separate genes.
The barley germin genes B (SEQ ED NO:3). F (SEQ ED NO:9), A (SEQ ED NO:l h D (SEQ ED NO:5) and E (SEQ ED NO:7): The nucleotide sequence of the gene for the testa specific germin B is presented in SEQ ED NO:3. A sequence of 1,590 bp was determined upstream of the ATG start codon and 847 bp of the sequence (SEQ ED NO: 12) were used for transient expression analysis. The open reading frame is disrupted by an intron of 225 bp with the obligatory tg and ag dinucleotides bordering the 5μ and 3μ splice sites. The putative TATA and CAAT boxes are rather far upstream of the start codon. A Myb-like recognition sequence is located at position minus 1472. A comparison of the structure of the germin B (SEQ ED NO:3) and F (SEQ ED NO:9) genes shows a conservation of all restriction sites and the same size and location of the intron.
Eight hundred and ninety nine base pairs of the F gene promoter (SEQ ED NO: 14) were used for transient expression analysis. The two promoter sequences (SEQ ED NO: 12 and SEQ ED NO: 14) are similar (93.3% identity among 1352 comparable nucleotides), but distinguished by a number of deletions, insertions and base changes. The introns are also located at the same position of the open reading frames in the barley genes for germins D (SEQ ED NO:5) and E (SEQ ED NO:7) as well as in the leaf specific gene of germin A (SEQ ED NO:l). The genes for the barley germins B (SEQ ED NO:3), F (SEQ ED NO:9), D (SEQ ED NO:5) and A (SEQ ID NO:l) thus contain an intron that separates the third base of the Val triplet at position 44 (or 47 in germin D (SEQ ED NO:5)) from the first two bases.
Testa specificity of the barley germin B and F gene promoters (SEQ ED NOS. 12, 14): The B germin gene promoter (847 bp) (SEQ ED NO: 12) and the F germin gene promoter (899bp) (SEQ ED NO: 14) were fused to the uidA open reading frame for transient expression in barley tissues. Developing barley caryopses were harvested 15-20 d after fertilization, sterilized with ethanol and the epicaφ, pericaφ plus testa and endosperm tissues isolated and placed into the nutritional medium. The tissues were bombarded with gold particles coated with the promoter constructs and incubated for 48 hours at 25°C, whereafter they were stained with the chromogenic X-Glu solution for 24 hours. The number of blue spots within transformed cells and cell clusters were counted under a microscope. It was not practical to separate the testa tissue from the pericaφ prior to bombardment and the histochemical staining. However, dissection of the stained tissue revealed all blue stained cells to be in the testa tissue. The promoter of the barley germin F gene (SEQ ED NO: 14) elicited a highly significant expression of the reporter gene in the testa tissue and a more limited expression in the epicaφ tissue (Table 1). The germin B gene promoter (SEQ ED NO: 12) gave a weaker GUS expression in the testa tissue and none in the epicaφ. Only occasionally was GUS expression observed in the endosperm with either promoter.
Table 1 shows β-Glucuronidase activity in isolated epicaφ, testa and endosperm tissue of developing barley grains after bombardment of the tissues with plasmids containing the uidA open reading frame under the control of the barley germin F gene promoter (SEQ ED NO: 15) or B gene promoter (SEQ ED NO: 12). Dots refers to blue dots after staining with X-glu. The ratio of the number of blue dots, produced by expression of β-Glucuronidase under the control of the germin F gene promoter (SEQ ED NO: 14), in testa versus epicaφ was 2.15 (t=15.6; P< 0.001). The ratio of the number of blue dots, in the testa, produced by expression of β-Glucuronidase under the control of the germin F gene promoter (SEQ ED NO: 14) versus the number of blue dots, in the testa, produced by expression of β-Glucuronidase under the control of the germin B gene promoter (SEQ ED NO: 12) was 3.5 (t=26.0; P<0.001).
Table 1
Tissue Promoter Tissues Dots Mean Promoter Tissues Dots Mean n n n n
Epicaφ F 32 26 B 30 0
F 28 24 B 30 0
F 36 28 B 36 0
F 38 84 74±45 B 36 0 0 Testa F 32 98 B 32 28
F 48 158 B 32 35
F 30 94 B 36 35
F 38 284 159: ±73 B 38 89 47±14
Endosperm F 36 1 B 36 0
F 30 0 B 30 0
F 30 0 B 30 0
F 36 1 0.5±0.3 B 36 0 0
H^O? producing oxalate oxidase activities in the developing barley caryopsis:
Oxalate oxidase is one of the enzymes that scavenges reactive oxygen intermediates. It catalyzes the conversion of oxalate and O2 into H2O2 and CO2. Its activity can be determined in tissues, extracts and SDS gels according to the procedure of
Zhang, Z., et al., Plant Mol. Biol. Rep. 14: 266-272 (1996). In the presence of oxalic acid oxidase a black deposit forms. As control the solution without oxalic acid is applied.
With dissected tissues activity was demonstrated in root tips and root vascular tissue of barley seedlings. In the developing caryopses high activity was found in the apical hairs of the epicaφ, in the epicaφ and in the root primordium of the developing embryo.
Some small amounts of black deposits were found in the pericaφ and testa tissues. The presence of oxalate oxidase was also tested with tissue extracts. The bulk of the oxalate oxidase activity is found in the epicaφ of the developing barley grain. Obviously, the oxalate oxidase activity cannot be ascribed to the F-germin (SEQ ED NO: 10), as the activity is lacking in the testa, where expression of the F promoter (SEQ ED NO: 14) is strongest.
EXAMPLE 3
This example describes a representative hybridization protocol that can be used to identify genes and/or promoters of the invention that hybridize to the complement of a nucleic acid molecule consisting of a nucleic acid sequence selected from the group consisting of SEQ ED NO:l, SEQ ED NO:3, SEQ ED NO:5, SEQ ED NO:7, SEQ ED NO:9, SEQ ED NO:l l, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO:14, under defined conditions.
Hybridization solution should preferably be prepared and filtered through a 0.45- micron disposable cellulose acetate filter. The composition of the hybridization solution is 6 X SSC, 5 x Denhardt's reagent, 0.5% sodium dodecyl sulfate (SDS), lOO μg/ml denatured, fragmented salmon sperm DNA.
Denhardt's reagent is utilized in nucleic acid hybridization solutions. 500 ml of 50 X Denhardt's reagent (the 50-fold concentrate) includes 5 g Ficoll (Type 400, Pharmacia), 5 g polyvinylpyrrolidone, 5 g bovine serum albumin (Fraction V, Sigma) and water to a final volume of 500 ml.
The nitrocellulose filter or nylon membrane containing the target DNA (e.g., a putative promoter) is floated on the surface of a tray of 6 X SSC until it becomes thoroughly wetted from beneath. The filter is submerged for 2 minutes and then slipped into a heat-sealable bag. 0.2 ml of hybridization solution is added for each square centimeter of nitrocellulose filter or nylon membrane.
As much air as possible is squeezed from the bag, and the open end of the bag is sealed with a heat sealer. The bag is incubated for 1-2 hours submerged at the desired temperature (typically no higher than the hybridization temperature). It is desirable to agitate the bag. If the radiolabeled probe is double-stranded, it is denatured by heating for
5 minutes at 100°C. Single-stranded probe need not be denatured. The denatured probe is chilled rapidly in ice water. Ideally, probe having a specific activity of 109 cpm/μg, or greater, is used. Hybridization is carried out for the desired time period at 50°C, typically using 1-2 μg/ml radiolabeled probe. The probe can be, for example, a nucleic acid molecule consisting of the complement of a nucleic acid sequence selected from the group consisting of SEQ ID NO:ll, SEQ ED NO:12, SEQ ED NO:13, and SEQ ED NO: 14.
The bag containing the filter is quickly removed from the water bath and opened by cutting off one corner with scissors. The denatured probe is added to the hybridization solution, and then as much air as possible is squeezed from the bag which is then resealed with the heat sealer so that as few bubbles as possible are trapped in the bag. To avoid radioactive contamination of the water bath, the resealed bag should be sealed inside a second, noncontaminated bag.
The bag is submerged in a water bath for the required period of hybridization (for example, 16 hours) at 50°C. The bag is removed from the water bath and one corner is cut off. The hybridization solution is poured into a container suitable for disposal, and then the bag is cut along the length of three sides. The filter is removed and immediately submerged in a tray containing several hundred milliliters of 2 X SSC and 0.5% SDS at room temperature (no higher than 25°C). The filter should not be allowed to dry out at any stage during the washing procedure. After 5 minutes, the filter is transferred to a fresh tray containing several hundred milliliters of 2 X SSC and 0.1% SDS, and incubated for 15 minutes at room temperature (no higher than 25°C) with occasional gentle agitation. The filter should then be washed at the desired stringency, i.e., in the desired concentration of SSC and at the desired temperature. If, for example, nucleic acid molecules that hybridize to the probe at a temperature of 55°C in 2 X SSC are sought, then the filter is washed in 2 X SSC at 55°C, i.e., nucleic acid molecules that do not hybridize to the probe under conditions of 2 X SSC at 55°C are washed off. Washing can be done for any desired time period, such as one hour, with several changes of washing solution.
Most of the liquid is then removed from the filter by placing the filter on a pad of paper towels. The damp filter is then placed on a sheet of Saran Wrap. Adhesive dot labels marked with radioactive ink are applied to several asymmetric locations on the Saran Wrap. These markers serve to align the autoradiograph with the filter. The labels are covered with Scotch Tape which prevents contamination of the film holder or intensifying screen with the radioactive ink. Radioactive ink is made by mixing a small amount of32P with wateφroof black drawing ink. A fiber-tip pen can be used to apply ink to the adhesive labels.
The filter is covered with a second sheet of Saran Wrap, and exposed to X-ray film (Kodak XAR-2 or equivalent) to obtain an autoradiographic image. The exposure time should be determined empirically. While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.