EP0877814A1 - Himbeerpromotoren zur transgenexpression in pflanzen - Google Patents

Himbeerpromotoren zur transgenexpression in pflanzen

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Publication number
EP0877814A1
EP0877814A1 EP97904883A EP97904883A EP0877814A1 EP 0877814 A1 EP0877814 A1 EP 0877814A1 EP 97904883 A EP97904883 A EP 97904883A EP 97904883 A EP97904883 A EP 97904883A EP 0877814 A1 EP0877814 A1 EP 0877814A1
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EP
European Patent Office
Prior art keywords
promoter
gene
plant
raspberry
dna
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EP97904883A
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English (en)
French (fr)
Inventor
Jill Anne Kellogg
Richard Keith Bestwick
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Exelixis Plant Sciences Inc
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Exelixis Plant Sciences Inc
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Priority claimed from US08/592,936 external-priority patent/US5783393A/en
Application filed by Exelixis Plant Sciences Inc filed Critical Exelixis Plant Sciences Inc
Publication of EP0877814A1 publication Critical patent/EP0877814A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8235—Fruit-specific

Definitions

  • the present invention relates to the identification of promoters from raspberry which are capable of providing constitutive expression of heterologous plant genes, and to chimeric genes, cassette vectors, kits, transgenic plants, and methods employing such promoters.
  • Promoters that regulate gene expression in plants are essential elements of plant genetic engineering.
  • Several examples of promoters useful for the expression of selected genes in plants are now available (Zhu, et al, 1995; Ni, et al, 1995).
  • a gene To be expressed in a cell, a gene must be operably linked to a promoter which is recognized by certain enzymes in the cell.
  • the 5' non-coding regions of a gene i.e., regions immediately 5' to the coding region, referred to as promoters or transcriptional regulatory regions, initiate transcription of the gene to produce a mRNA transcript.
  • the mRNA is then translated at the ribosomes of the cell to yield an encoded polypeptide.
  • Promoters typically contain from about 500-1500 bases, and can provide regulated expression of genes under their control.
  • a promoter used for expressing a heterologous gene in plant cells may be characterized as (i) a constitutive promoter, that is, a promoter capable of causing similar levels of gene expression in all or many plant tissues, or, (ii) a tissue selective promoter, that is, one which is capable of regulating gene expression to select tissues in a plant transformant (e.g., leaves or fruit).
  • a constitutive promoter that is, a promoter capable of causing similar levels of gene expression in all or many plant tissues
  • a tissue selective promoter that is, one which is capable of regulating gene expression to select tissues in a plant transformant (e.g., leaves or fruit).
  • Many such promoters have been characterized, including those derived from plant viruses, Agrobacterium genes, and a variety of plant genes. Considerable effort has gone into the isolation and characterization of constitutive promoters to drive the expression of a variety of heterolog
  • Viral promoters i.e., promoters from viral genes
  • CaMV Cauliflower Mosaic Virus
  • Promoters useful for regulating gene expression in plants and obtained from bacterial sources have been identified and isolated.
  • Such promoters include those derived from Agrobacterium T-DNA opine synthase genes, and include the nopaline synthase (nos) promoter (Rogers, 1991), the octopine synthase (ocs) promoter (Leisner and Gelvin, 1988) and mannopine synthase (mas) promoter.
  • Plant promoters (promoters derived from plant sources) effective to provide constitutive expression, are less well known, and include ⁇ sp80, Heat Shock Protein 80 from cauliflower, (Brunke and Wilson, 1993), and the tomato ubiquitin promoter (Picton, et al, 1993). These promoters can be used to direct the constitutive expression of heterologous nucleic acid sequences in transformed plant tissues. At present, a relatively small number of plant promoters, particularly constitutive plant promoters, has been identified. The use of such promoters in plant genetic engineering has been rather limited to date, since gene expression in plants is, for the most part, typically tissue, developmentally, or environmentally-regulated.
  • the present invention is directed to raspberry promoters which separately and in combination provide moderate-level, constitutive expression of nucleic acid sequences placed under their control.
  • the promoters of the invention can also confer constitutive expression on heterologous, non-constitutive promoters.
  • the present invention is directed to a promoter which, in a native raspberry genome, is operably linked to the coding region of a drul gene.
  • Chimeric genes of the present invention contain a DNA sequence encoding a product of interest under the transcriptional control of a raspberry drul promoter.
  • the DNA sequence is typically heterologous to the promoter and is operably linked to the promoter to enable constitutive expression of the product.
  • the product is a polypeptide that permits selection of transformed plant cells containing the chimeric gene by rendering such cells resistant to an amount of an antibiotic that would be toxic to non-transformed cells.
  • Exemplary products include, but are not limited to, aminoglycoside phosphotransf erases, such as neomycin phosphotransferase and hygromycin phosphotransferase.
  • a chimeric gene of the invention contains an hpt gene sequence encoding hygromycin phosphotransferase II under the transcriptional control of a drul promoter.
  • a chimeric gene of the invention contains an nptll gene sequence encoding neomycin phosphotransferase under the transcriptional control of a drul promoter.
  • the product is a polypeptide that confers herbicide-resistance to trans ⁇ formed plant cells expressing the polypeptide.
  • a chimeric gene of the present invention contains a bxn gene encoding a bromoxynil-specific nitrilase under the transcrip ⁇ tional control of a drul promoter. Transformed plants containing this chimeric gene express a bromoxynil-specific nitrilase and are resistant to the application of bromoxynil-containing herbi- cides.
  • genes conferring herbicide resistance include the EPSP synthase gene (encoding 5-enolpyruvylshikimate-3-phosphate synthase enzyme), which confers resistance to glyphosate; an acetolactate synthase gene, which confers resistance to the herbicide "GLEAN” ; a bialaphos resistance gene (the bar gene) coding for phosphinothricin acetyl- transferase (PAT), and the glyphosate-tolerant genes, CP4 and GOX.
  • Chimeric genes of the invention contain one or more of these herbicide-resistance genes, operationally linked to a drul promoter.
  • the DNA sequence or cDNA sequence encodes a viral coat protein, such as alfalfa mosaic virus coat protein, cucumber mosaic virus coat protein, tobacco streak virus coat protein, potato virus coat protein, tobacco rattle virus coat protein, and tobacco mosaic virus coat protein.
  • a chimeric gene of the invention contains a viral coat protein gene, such as ALMV, CMV, TSV, PVX, TRV, or TMV, under the transcriptional con ⁇ trol of a drul promoter.
  • the DNA sequence corresponds to a gene encoding a dominant defective protein, such as mutant forms of the ETR1 gene which confer ethylene insensitivity.
  • the DNA sequence corresponds to a gene capable of altering a plant biochemical pathway, such as such as the ACCD gene. The ACCD gene forms a product which degrades a precursor in the ethylene biosynthetic pathway.
  • the invention includes an isolated DNA molecule comprising a constitutive promoter from a raspberry drul gene.
  • a raspberry drul promoter is the drul 10 pro ⁇ moter, presented herein as SEQ ID NO:3.
  • Another exemplary constitutive raspberry promoter is the dru259 promoter, presented as SEQ ID NO:4. Additional fragments may be derived from the sequence representing the full-length drul promoter, SEQ ID NO:2, where the smaller fragments are effective to regulate constitutive expression of a DNA sequence under their control.
  • the present invention also includes the use of any of the above chimeric genes, DNA constructs, and isolated DNA sequences to generate a plant transformation vector.
  • Such vectors can be used in any plant cell transformation method, including Agrobacterium-based lemon ods, electroporation, microinjection, and microprojectile bombardment. These vectors may also form part of a plant transformation kit. Other components of the kit may include, but are not limited to, reagents useful for plant cell transformation.
  • the present invention includes a plant cell, plant tissue, transgenic plant, fruit cell, whole fruit, seeds or calli containing any of the above-described raspberry promoters, chimeric genes or DNA constructs.
  • the dru promoters described herein are employed in a method for providing moderate expression of a heterologous gene, such as a selectable marker gene, in transgenic plants.
  • a chimeric gene of the present invention containing a DNA sequence encoding a selectable marker product (e.g., a neomycin phosphotransferase or hygromycin phosphotransferase) is introduced into progenitor cells of a plant.
  • Transgenic plants containing the chimeric gene are selected by their ability to grow in the presence of an amount of selective agent (e.g., hygromycin, geneticin or kanamycin) that is toxic to non-transformed cells.
  • the transformed plant cells thus selected are then regenerated to provide a differentiated plant, followed by selection of a transformed plant which expresses the product.
  • the invention includes a method for producing a transgenic fruit-bearing plant.
  • the chimeric gene of the present invention typically carried in an expression vector allowing selection in plant cells, is introduced into progenitor cells of selected plant. These progenitor cells are then grown to produce a transgenic plant.
  • the method may further comprise isolation of a drul promoter (such as drul 10 or dru259) by the following steps:
  • chimeric genes, vectors, constructs, isolated DNA molecules, products and methods of the present invention can be produced using the raspberry drul promoter sequences essentially as described above.
  • Fig. 1 is a schematic diagram illustrating the creation of plasmid pAG-431 containing an exemplary raspberry drul promoter, referred to herein as dru259 pro, and the nptll gene;
  • Fig. 2 is a flow chart representing the steps followed in constructing vector pAG-421 containing a chimeric drul 10 pro- ⁇ ptll gene;
  • Fig. 3 outlines the steps involved in the construction of Agrobacterium binary vector pAG-
  • Fig. 4 is a flow chart depicting the creation of Agrobacterium binary vector pAG-7342 containing a chimeric dru259 pro-nptll gene
  • Fig. 5 is a graph representing relative levels of nptll gene expression across 10 transgenic events for three different promoter- ⁇ pt II chimeric gene combinations;
  • Figs. 6A and 6B present the genomic DNA sequence of the drul gene. Indicated in the figures are a CAAT box, TATA box, ATG start codon, two exons, an intron, splicing sites, a stop codon and poly-adenylation sites;
  • Figs. 7A and 7B present the DNA sequence of the full length drul promoter
  • Fig. 8 presents the DNA sequence of the drullO promoter
  • Fig. 9 presents the DNA sequence of the dru259 promoter
  • Fig. 10 presents representative results of polyacrylamide gel electrophoretic analysis of raspberry drupelet proteins
  • Figs. 11A and 11B schematically represent the reverse transcriptase-polymerase chain reaction (RT-PCR; Kawasaki, et al, 1989; Wang, et al, 1990) cloning of the raspberry drul gene;
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • Fig. 12 presents a schematic representation of the gene organization and protein structure of drul
  • Fig. 13 presents a Kyte-Doolittle hydrophilicity plot of the coding sequence of drul.
  • the hydrophilicity window size 7;
  • Fig. 14 shows the results of RNA dot blot analysis of drul RNA expression in raspberry leaf and receptacle. RNA was isolated from green, mature green, breaker & orange/ripe raspberries (corresponding to stages I, II, III, IV, respectively);
  • Fig. 15 shows the results of a RNA hybridization study evaluating the expression of drul RNA in raspberry leaf and fruit
  • Fig. 16 shows the results of polyacrylamide gel electrophoretic analysis of raspberry drupelet proteins obtained from drupelets at various stages of ripening
  • Figs. 17A and 17B depict a flow chart summarizing the construction of plasmid pAG-1542.
  • Chimeric gene refers to a non-naturally occurring gene which is composed of parts of different genes.
  • a chimeric gene is typically composed of a promoter sequence operably linked to a "heterologous" DNA sequence.
  • a typical chimeric gene of the present invention for transformation into a plant, will include a raspberry dru promoter (e.g., a drul 10 or dru259 promoter), a heterologous structural DNA coding sequence (e.g., the aminoglycoside phosphotransferase (nptll) gene) and a 3' non-translated polyadenylation site.
  • raspberry dru promoter e.g., a drul 10 or dru259 promoter
  • heterologous structural DNA coding sequence e.g., the aminoglycoside phosphotransferase (nptll) gene
  • nptll aminoglycoside phosphotransferase
  • a “constitutive” promoter refers to a promoter that directs RNA production in many or all tissues of a plant transformant, as opposed to a tissue-specific promoter, which directs RNA synthesis at higher levels in particular types of cells and tissues (e.g. , fruit specific promoters such as the tomato E4 or E8 promoter (Cordes, et al, 1989; Bestwick, et al, 1995).
  • promoter is meant a sequence of DNA that directs transcription of a downstream heterologous gene, and includes promoters derived by means of ligation with operator regions, random or controlled mutagenesis, addition or duplication of enhancer sequences, addition or modification with synthetic linkers, and the like.
  • plant promoter is meant a promoter (as defined above), which in its native form, is derived from plant genomic DNA.
  • Raspberry promoter refers to a promoter (as defined above) which, in its native form, is derived from a raspberry genome.
  • a drul promoter such as drullO or dru259
  • a raspberry promoter derived from a specified gene includes a promoter in which at least one or more regions of the promoter are derived from the specified raspberry gene.
  • An exemplary promoter of this type is one in which a region of the promoter (e.g., a dru259 promoter) is replaced by one or more sequences derived from a different gene, without substantially reducing the expression of the resulting chimeric gene in a host cell, or altering the function of the unaltered dru259 promoter.
  • a region of the promoter e.g., a dru259 promoter
  • Promoter strength refers to the level of promoter-regulated (e.g, drullO, dru259) expres ⁇ sion of a heterologous gene in a plant tissue or tissues, relative to a suitable standard (e.g., cauli- movirus cassava mottle vein virus promoter CAS or the hsp&O promoter). Expression levels can be measured by linking the promoter to a suitable reporter gene such as GUS (3-glucuronidase), dihydrofolate reductase, or nptll (neomycin phosphotransferase).
  • GUS 3-glucuronidase
  • dihydrofolate reductase dihydrofolate reductase
  • nptll neomycin phosphotransferase
  • a moderate promoter is one that drives expression of a reporter gene at about 10-90% of the level obtained with a promoter such as ⁇ sp80.
  • a "heterologous" DNA coding sequence is a structural coding sequence that is not native to the plant being transformed, or a coding sequence that has been engineered for improved characteristics of its protein product.
  • Heterologous refers to a coding sequence that does not exist in nature in the same gene with the promoter to which it is currently attached.
  • a gene considered to share sequence identity with the drul gene, or a particular region or regions thereof, has at least about 60% or preferably 80% global sequence identity over a length of polynucleotide sequence corresponding to the raspberry drul polynucleotide sequences disclosed herein (e.g. , SEQ ID NOs: 1-4).
  • Sequence identity is determined essentially as follows. Two polynucleotide sequences of the same length (preferably, corresponding to the coding sequences of the gene) are considered to be identical (i.e., homologous) to one another, if, when they are aligned using the ALIGN program, over 60% or preferably 80% of the nucleic acids in the highest scoring alignment are identically aligned using a ktup of 1, the default parameters and the default PAM matrix (Dayhoff, 1972).
  • Two nucleic acid fragments are considered to be "selectively hybridizable" to a polynucleo ⁇ tide derived from a drul gene if they are capable of specifically hybridizing to the coding sequences or a variants thereof or of specifically priming a polymerase chain amplification reaction: (i) under typical hybridization and wash conditions, as described, for example, in Maniatis, et al, 1982, pages 320-328, and 382-389; (ii) using reduced stringency wash conditions that allow at most about 25-30% basepair mismatches, for example: 2 x SSC (contains sodium 3.0 M NaCl and 0.3 M sodium citrate, at pH 7.0), 0.1% sodium dodecyl sulfate (SDS) solution
  • highly homologous nucleic acid strands contain less than 20-40% basepair mismatches, even more preferably less than 5-20% basepair mismatches.
  • degrees of homology i.e., sequence identity
  • sequence identity can be selected by using wash conditions of appropriate strin ⁇ gency for identification of clones from gene libraries (or other sources of genetic material), as is well known in the art.
  • a "drul encoded polypeptide” is defined herein as any polypeptide homologous to (i.e., having essentially the same sequence identity as) a drul encoded polypeptide.
  • a polypeptide is homologous to a drul encoded polypeptide if it is encoded by nucleic acid that selectively hybridizes to sequences of drul or its variants.
  • a polypeptide is homologous to a drul encoded polypeptide if it is encoded by drul or its variants, as defined above.
  • Polypeptides of this group are typically larger than 15, preferable 25, or more preferable 35, contiguous amino acids.
  • sequence comparisons for the purpose of determining "polypeptide homology" or "polypeptide sequence identity" are performed using the local alignment program LALIGN. The polypeptide sequence is compared against the drul amino acid sequence or any of its variants, as defined above, using the LALIGN program with a ktup of 1, default parameters and the default PAM.
  • Any polypeptide with an optimal alignment longer than 60 amino acids and greater than 55 % or preferably 80% of identically aligned amino acids is considered to be a "homologous polypeptide.”
  • the LALIGN program is found in the FASTA version 1.7 suite of sequence com ⁇ parison programs (Pearson and Lipman, 1988; Pearson, 1990; program available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, VA).
  • a polynucleotide is "derived from" drul if it has the same or substantially the same basepair sequence as a region of the drul protein coding sequence, cDNA of drul or complements thereof, or if it displays homology as defined above.
  • a polypeptide or polypeptide "fragment” is "derived from” drul if it is (i) encoded by a drul gene, or (ii) displays homology to drul encoded polypeptides as noted above.
  • nucleic acid sequences when referring to sequences which encode a protein, polypeptide, or peptide, is meant to include degenerative nucleic acid sequences which encode homologous protein, polypeptide or peptide sequences as well as the disclosed sequence.
  • a "plant cell” refers to any cell derived from a plant, including undifferen- tiated tissue (e.g., callus) as well as plant seeds, pollen, progagules and embryos.
  • the present invention relates, in one aspect, to a promoter which, in a native raspberry genome, (i) is operably linked to the coding region of a drul gene, and (ii) functions as a moderate strength, constitutive promoter.
  • This aspect of the invention is based upon the discovery of the drul gene in raspberries, which is expressed at very high levels in ripening fruit. Expres ⁇ sion directed by the full length drul promoter is fruit specific, and active during fruit ripening.
  • Fig. 10 A coomassie blue-stained SDS polyacrylamide gel of soluble drupelet proteins is shown in Fig. 10 (Examples 1A-B). As can be seen from Fig. 10, two highly abundant proteins isolable from raspberries are observed at approximately 17 and 15 kd, and are referred to herein as drupel and drupe2, respectively. The amount of drupel and drupe2 relative to the total amount of soluble protein can be determined, for example, by scanning densitometry.
  • drupel and drupe2 comprise approximately 23 and 37%, respectively, of the total soluble protein in raspberry drupelets.
  • purifica- tion and sequencing of drupel and drupe2 can be carried out, for example, by using a direct western blot approach.
  • total drupelet proteins are western blotted to PDVF membrane (Example IB) and the regions corresponding to drupel and drupe2 are subjected to N-terminal amino acid sequence analysis.
  • the drupel sample yields a thirty amino acid
  • N-terminal sequence (Example IB).
  • the amino terminal drupel sequence is presented herein as
  • mature green raspberry drupelet mRNA is prepared as described in Example 2A and 2B and used as template in a cDNA synthesis reaction.
  • the reaction is primed using the dTRANDOM primer (SEQ ID NO: 12) shown in Figures 11A and UB.
  • the resulting cDNA (Example 2C) is subjected to a standard PCR reaction using primers corresponding to a portion of the dTRANDOM primer and a 512-fold degenerate primer (Drupe
  • PCR amplification products are then analyzed.
  • Products from the above PCR reaction include a 710 bp product that is agarose gel purified and subcloned into vector pCRII (Example 3). Subsequent sequence analysis of several of these clones allows identification of those clones whose sequence encodes a protein matching the amino terminal sequence of drupel.
  • raspberry genomic DNA is digested with Nsil and ligated under dilute conditions to allow circularization of the restriction fragments.
  • the ligated DNA is then subjected to PCR amplification using primers internal to the drul coding sequence and oriented in opposite directions from each other. This produces a PCR reaction product containing part of the first exon and 1.35 kb of the promoter.
  • sequence analysis of this clone in combination with sequence information from the previously described clones produces the complete drul sequence.
  • the drul gene (Figs. 6A, 6B) encodes a protein with the predicted amino acid sequence presented as SEQ ID NO:20.
  • the predicted molecular weight for this protein is 17,088, which agrees closely with the 17kd molecular weight determined by gel electrophoresis (see Figure 10) of total drupelet protein.
  • the drul protein is relatively acidic with a predicted pi of 4.8. Nucleic acid and protein homology searches of d e current sequence databases can be carried out to look for significant matches. For drul, nucleic acid and protein homology searches of the current sequence databases produced no significant matches. This result supports the original observation made with the amino terminal sequence of the protein that drupel is a novel protein.
  • the gene expression pattern of drul can be also be evaluated at the RNA and protein levels to confirm the tissue specificity of the full length promoter.
  • Northern dot blots, Figs. 14 and 15, of total RNA from raspberry leaf and receptacles at different ripening stages indicate a tissue and stage specific gene expression pattern. This can be confirmed by comparison of northern blots of total RNA from various other plant tissues.
  • the tissue and stage specific gene expression pattern of drul was confirmed on northern blots of total RNA from leaf, receptacles, and drupelets (see Figs. 14 and 15). In both cases, no drul expression is observed in leaf RNA.
  • the RNA expression pattern in receptacles is temporally regulated while in drupelets it is fully expressed at the two stages (i.e., green and ripe) analyzed.
  • a protein gel of drupelet lysates from different ripening stages can also be carried out to further support stage specific expression of drul.
  • electrophoretic analysis of raspberry drupelet proteins obtained from drupelets at various stages of ripening i.e., green, mature green, breaker, orange, and ripe
  • stage specific expression pattern in drupelets Fig. 16
  • drul genomic clone Characterization of the drul genomic clone allows isolation of the drul promoter.
  • the nucleotide sequence of an exemplary full length drul promoter is presented as SEQ ID NO:2.
  • drullO and dru259 Two representative raspberry promoters of the invention, drullO and dru259, were isolated from the full length transcript of the drul promoter, which has been characterized as a stage and fruit-specific promoter. Surprisingly, these two new drul -derived promoters have been found to function as moderate level, constitutive promoters when fused to heterologous genes and evaluated for resultant patterns of expression in transformed plants.
  • the truncated dru promoters, drullO and dru259 can be obtained from the full length drul promoter as described in Examples 7 and 8.
  • a PCR reaction product containing part of the first exon and 1.35 kb of the drul promoter is ligated into plasmid pCRII (Invitrogen, Carlsbad, CA) to form a subclone, pAG-310, containing the full length drul pro ⁇ moter, as shown in Figs. 1 and 2.
  • pAG-310 containing the full length drul pro ⁇ moter, as shown in Figs. 1 and 2.
  • a 1.3 kb DNA fragment from pAG-310 is then PCR amplified under standard conditions using primers DrupeUp (5' primer, SEQ ID NO:7) and DrupeLow (3* primer, SEQ ID NO: 8).
  • Recovery of the amplified DNA is typically carried out by addition of solvent to the reaction mixture, followed by centrifugation, recovery of the aqueous phase, and precipitation with sodium acetate.
  • the recovered DNA is then typically purified by centrifugation and repeated washing, followed by drying of the recovered pellet.
  • the 1.3 kb DNA fragment is digested to completion with restriction enzymes Nsil and Xbal, followed by purification and ligation into plant expression vector, p35S-GFP (Clontech, Palo Alto, CA), which has been digested with Xbal and Pstl.
  • Restriction enzymes used to digest p35S- GFP and Nsil (used to digest the drul PCR product), both generate the same 3' overhanging cohesive ends (TGCA), so that upon ligation, neither restriction site is reconstructed.
  • Isolation of the raspberry drullO promoter is achieved by amplifying a 166 bp fragment of drul carried in plasmid pAG-155 using primers drul-118H3 (SEQ ID NO:9) and GFPStartR (SEQ ID NO: 10) under standard PCR reaction conditions. The amplified product is then recovered from the reaction mixture, and purified as described above, followed by digestion of the 166 bp product with Hind ⁇ ll and £c ⁇ RV to produce the 112 bp promoter referred to herein as drullO.
  • the raspberry promoters, drullO and dru259 can be used to regulate expression of heterologous genes.
  • Exemplary dru promoter, dru259 has the nucleotide sequence presented herein as SEQ ID NO:4.
  • Exemplary dru promoter, drullO has the nucleotide sequence presented as SEQ ID NO:3.
  • the present invention also provides a method for identifying and isolating a drul promoter, e.g. drullO and dru259, from a variety of plant sources, e.g. raspberry.
  • a drul promoter e.g. drullO and dru259
  • Such promoters are useful for the generation of vector constructs containing heterologous genes, such as selectable marker genes, or genes conferring herbicide resistance.
  • Southern blot experiments are used to demonstrate the presence of DNA molecules having significant sequence identity (i.e., typically greater than 55%, more preferably greater than 80% identity using standard sequence comparison programs) with the raspberry drul gene in, for example, strawberry, peach or plum. Similar Southern blot analyses may be performed on other fruit-bearing plants to identify additional drul genes.
  • Drul homologues are identified in a Southern blot (Ausubel, et al, 1992) of the plant genomic DNA, probed wi a labelled DNA fragment containing the coding sequence of the raspberry drul gene.
  • the probe is typically selected to contain the coding sequence of drul, ra er than the promoter sequence, because coding sequences are typically more conserved from species to species than are promoter sequences.
  • Probe molecules are generated from raspberry genomic DNA using primer-specific amplification (Mullis, 1987; Mullis, et al, 1987).
  • the oligonucleotide primers are selected such that the amplified region includes the entire coding sequence of the raspberry drul gene, as provided herein. Primers may also be selected to amplify only a selected region of the raspberry drul gene.
  • a probe can be made by isolating restriction-digest fragments containing the sequence of interest from plasmid DNA.
  • the probe is labeled with a detectable moiety to enable subsequent identification of homologous target molecules.
  • exemplary labeling moieties include radioactive nucleotides, such as 32 P-labeled nucleotides, digoxygenin-labeled nucleotides, biotinylated nucleotides, and the like, available from commercial sources.
  • labeled nucleotides may be directly incorporated into the probe during the amplification process.
  • Probe molecules derived from DNA that has already been isolated, such as restriction-digest fragments from plasmid DNA, are typically end-labeled (Ausubel, et al, 1992).
  • Target molecules such as Hind ⁇ ll DNA fragments from the genomes of the above-listed plants, are electrophoresed on a gel, blotted, and immobilized onto a nylon or nitrocellulose filter. Labeled probe molecules are then contacted with the target molecules under conditions favoring specific hybridization between die probe molecules and target molecules homologous to the probe molecules (Maniatis, et al, 1982; Sambrook, et al, 1989; Ausubel, et al, 1992).
  • the DNA containing the desired genes, including the promoter regions may be isolated from the respective species, by, for example, the methods described herein for the isolation of the raspberry drul gene.
  • Generation of truncated promoters may be accomplished by, for example, 5' deletions such as those described herein for the isolation of the drullO and dru259 promoters.
  • Variants of the drul promoter may be isolated from different raspberry cultivars and from other plants by the methods described above.
  • a reporter gene such as GUS (/ ⁇ -glucuronidase)
  • GUS / ⁇ -glucuronidase
  • GUS protein can be easily measured by fluorometric, spectrophotometric or histo- chemical assays (Jefferson, et al, 1987a; Jefferson, 1987b).
  • DNA sequences corresponding to regulatory domains can be identified using, for example, deletion analysis (Benfey, et al, 1990).
  • the dru259 promoter sequence presented as SEQ ID NO:4 can be functionally linked to the GUS reporter gene. Dele ⁇ tion analysis can then be carried out by standard methods (Ausubel, et al, 1992; Maniatis, et al., 1982; Sambrook, et al, 1989).
  • regions of the full length drul promoter sequence can be amplified using sequence-specific primers in PCR, as illustrated in Figs. 1 and 2. These amplified fragments can then be inserted 5' to the GUS coding sequences and the resulting expression patterns evaluated for moderate level, constitutive expression, which are features of the raspberry promoters of the invention.
  • exemplary chimeric genes containing a raspberry plant promoter sequence operably linked to a heterologous DNA sequence were constructed.
  • Exemplary chimeric gene constructs include drullOpto:nptll (Example 10) and dru259pvo:nptU (Example 9).
  • the protein expressed by the nptll gene, neomycin phosphotransferase is an aminoglycoside phosphotransferase, which confers kanamycin resistance to transgenic plants expressing the product.
  • This protein may function more efficiently if expressed (i) constitutively, and (ii) at moderate levels (rather than being overexpressed) in transgenic plants. Accordingly, exemplary promoters drullO and dru259 represent ideal promoters for satisfying this objective.
  • A. Construction of Agrobacterium Binary Plant Transformation Vectors Construction of Agrobacterium binary vectors, pAG-7242 and pAG-7342, containing the two representative chimeric genes described above, can be performed as described in Example 10 and
  • Example 19 (schematically represented in Figs. 3-4, drullOp ⁇ o:nptll, and dru259p ⁇ o:nptU, respectively). These binary vectors also contain a gene encoding SAMase, S-adenosylmethionine hydrolase (Ferro, etal, 1995; Hughes, et al, 1987), which is immaterial to the present invention.
  • Binary Plant Transformation Vector pAG-7342 Containing a dru259::np ⁇ l Chimeric Gene.
  • Binary plant transformation vector, pAG-7342 is constructed by excising a 13 kb nos pior.nptll fragment from subclone pAG-1542 by digestion with Hind ⁇ ll and BamHl, followed by ligation to a 1.1 kb Hindlll-BamUl fragment from subcloning vector, pAG- 431, to insert a dru259 pro:: nptll chimeric gene.
  • Plasmid pAG-1542 can be prepared using conventional cloning techniques known in the art (Sambrook, et al, 1989).
  • This illustrative subcloning binary vector contains a neomycin phosphotransferase II selectable marker gene (nptll) gene under the control of the nos promoter located near the left border, and the SAMase gene (Ferro, et al, 1995) driven by the tomato E8 promoter (Deikman, et al, 1988; Deikman, et al, 1992) located near the right border.
  • nptll neomycin phosphotransferase II selectable marker gene
  • Example 7 Construction of subclone pAG-431, containing the dru259:: nptll chimeric gene, is described in Example 7. Construction of binary plant transformation vector pAG-7342 is depicted schematically in Fig. 4 and detailed in Example 9.
  • FIG. 17 A flow chart summarizing the construction of plasmid pAG-1542 is presented in Fig. 17.
  • binary plant transformation vector Containing a drullOr.nptll Chimeric Gene.
  • binary plant transformation vector pAG-7242, is constructed by excising a 13 kb nos pro:: ⁇ ptII fragment from subclone pAG-1542 by digestion with Hindlll and BamUl, followed by ligation to a 0.95 kb dru259::npill fragment from pAG-421 to form the binary plant transformation vector pAG-7242.
  • chimeric genes can be inserted, for example, into plant cells.
  • nptll was selected as an exemplary marker gene to illustrate the ability of a raspberry plant promoter of the invention to regulate expression of a gene under its control, it will be understood that expression of any of a number of heterologous genes can be directed by the promoters of the present invention.
  • nptl and nptll are different and distinct enzymes, with differences in both their amino acid sequences and substrate specificities (Beck, et al, 1982).
  • the raspberry promoters of the invention are suitable for directing expression of either of these neomycin phosphotrans- f erases.
  • Plants suitable for transformation using the raspberry promoters of the invention include but are not limited to, raspberry, tomato, strawberry, banana, kiwi fruit, avocado, melon, mango, papaya, apple, peach, soybean, cotton, alfalfa, oilseed rape, flax, sugar beet, sunflower, potato, tobacco, maize, wheat, rice, and lettuce.
  • Chimeric genes containing a raspberry promoter can be transferred to plant cells by any of a number of plant transformation methodologies.
  • One such method, employed herein involves the insertion of a chimeric gene into a T-DNA-less Ti plasmid carried by A. tumefaciens, followed by co-cultivation of the A. tumefaciens cells with plant cells.
  • Agrobacterium binary plant transformation vectors, pAG-7242 and pAG-7342 are individually introduced into a disarmed strain of A.
  • tumefaciens by electroporation by electroporation (Nagel, etal, 1990), followed by co-cultivation with tomato plant cells, to transfer the chimeric genes into tomato plant cells.
  • alternative methodologies may be employed to elicit transformation of a plant host, such as leaf disk-based transformation, electroporation, microinjection, and microprojectile bombardment (particle gun transformation). These methods are well known in the art (Fry, et al, 1987; Comai and Coning, 1993; Klein, et al. , 1988; Miki, et al, 1987; Bellini, et al , 1989) and provide the means to introduce selected DNA into plant genomes.
  • DNA may include a DNA cassette which consists of a raspberry promoter (e.g. , drullO, dru259) functionally adjacent a heterologous coding sequence.
  • an iterative culture-selection methodology may be employed to generate plant transformants, and is particularly suited for transformation of woody species, such as raspberry. This method is described in detail in International Publication No. WO 95/35388, entitled “Plant Genetic Transformation Methods and Transgenic Plants", published on 28 December 1995.
  • a chimeric gene of interest is inserted into cells of a target plant tissue explant, such as by co-culturing a target explant in the presence of Agrobacterium containing the vector of interest.
  • the co- culturing is carried out in liquid for from about 1 to about 3 days.
  • the plant tissue explant can be obtained from a variety of plant tissues including, but not limited to, leaf, cotyledon, petiole and meristem.
  • Transformed explant cells are then screened for their ability to be cultured in selective media having a threshold concentration of selective agent. Explants that can grow on the selective media are typically transferred to a fresh supply of the same media and cultured again. The explants are then cultured under regeneration conditions to produce regenerated plant shoots. These regenerated shoots are used to generate explants. These explants from selected, regenerated plant shoots are then cultured on a higher concentration of selective agent. This iterative culture method is repeated until essentially pure transgenic explants are obtained.
  • transgenic explants are identified by dividing the regenerated plant shoots into explants, culturing the explants, and verifying that the growth of all explants is resistant to the highest concentration of selective agent used. That is, in the presence of selective agent there is no necrosis or significant bleaching of the explant tissue. Upon confirmation of production essentially pure transgenic explants, transgenic plants are produced by regenerating plants from the pure transgenic explants.
  • Transgenic plants are assayed for their ability to synthesize product mRNA, DNA, protein, and/or for their resistance to an aminoglycoside antibiotic, e.g., kanamycin.
  • the assays are typically conducted using various plant tissue sources, e.g., leaves, stem, or fruit.
  • Leaf-based assays are informative if the raspberry promoter driving the heterologous gene (transgene) is at least somewhat active in leaf tissue, as is the case for exemplary promoters drullO and dru259. In such cases, leaf-based assays are useful for initial screens of the expression level of a transgene, since they can be performed much earlier than fruit-based assays. Fruit-based assays, on the other hand, provide more accurate data on transgene expression in a target tissue itself such as fruit.
  • RNA-based assays can be carried out using, for example, an RNAase protection assay (RPA).
  • RPA RNAase protection assay
  • mRNA is typically extracted from plant cells derived from both transformed plants and wild-type plants.
  • RNAse Protection Assays RPA can be performed according to the manufacturer's instructions using an "RPAII" kit from Ambion, Inc. (Hialeah, FL), as previously described by Lee, et al, 1987.
  • Gene expression patterns for transgenic plants containing chimeric genes regulated by a raspberry promoter can also be evaluated by conducting Northern dot blots (e.g., Example 6).
  • Promoter function i.e., tissue and/or stage specific expression, or constitutive expression
  • Promoter function can be evaluated by comparing northern blots of total RNA from leaf and fruit tissues at different ripening stages to northern blots of total RNA from various other plant tissues.
  • a Western blot analysis can be carried out.
  • total soluble protein is extracted from frozen plant tissue and measured using, for example, the Coomassie Plus protein assay (Pierce, Rockford, IL).
  • Known quantities of soluble protein, or known quantities of purified protein product e.g., neomycin- phosphotransferase II, positive control
  • purified protein product e.g., neomycin- phosphotransferase II, positive control
  • the bound proteins were then probed with a monoclonal antibody specifically immunoreactive with the protein product.
  • a Southern hybridization analysis is performed. Typically, plant DNA is extracted by grinding frozen plant tissue in extraction buffer, followed by centrifugation, separation of the resulting supernatant, and precipitation with cesium chloride. The resulting CsCl gradients are then centrifuged for an extended period of time (e.g., 48 h), and the recovered DNA is dialyzed and precipitated with ethanol. Upon recovery of plant DNA, the DNA is digested with suitable restriction enzymes to obtain DNA fragments, followed by electrophoretic separation on agarose gel.
  • the resulting bands are transferred to nitrocellulose (Southern, 1975), and the blots are then probed with a labelled DNA fragment containing the nucleotide sequence of the transgene, to confirm the presence of DNA corresponding to a raspberry promoter-chimeric gene construct, as described above.
  • Tomato plants were transformed with plant transformation vectors, pAG-7242 and pAG- 7342, each containing a raspberry promoter operably linked to an nptll gene (Example 11).
  • plant transformation vector pAG-7242 contains the drullOv.nptll gene; and construct pAG-7342 contains the dru259::nptll gene.
  • Chimeric genes containing either the ⁇ sp80 promoter or the CAS promoter (caulimovirus cassava mottle vein virus promoter) fused to the nptll gene were also prepared and used to transform tomato plants, to provide a comparative basis for evaluating performance of the raspberry promoters of the invention.
  • Results from ten separate transgenic events employing the constructs described above are provided in Example 12.
  • protein extracts from leaf tissue of rooted plants available at the time of culture were assayed by ELISA. In some cases, only 1 plant was available for assay (e.g. , Table 1 , last two rows, column IV), while in other instances (e.g., Table 1, row 2, column IV), ten separate transgenic events were available for analysis.
  • transgenic plants containing a raspberry promoter of the invention e.g., drullO, dru259
  • a raspberry promoter of the invention e.g., drullO, dru259
  • Table 1 specifically, rows 3-7
  • nptll enzymatic activity was detected in a high percentage of the plants assayed, with values ranging from about 20-100%, depending upon the concentration of selection agent used and the number of rooted plants tested.
  • transformation frequency that is, the ratio of the number of tissue explants producing regenerated shoots that are capable of rooting in the presence of selection agent to the total number of initial explants, expressed as a percentage. Based on the results in column III, and referring to plants containing a raspberry promoter of the invention, on average, at least about half of the plants transformed with a raspberry promoter-con- taining construct survived selection with antibiotic, that is, they were capable of rooting in the pre ⁇ sence of an amount of selection agent that would otherwise be toxic to non-transformed plant cells.
  • the raspberry promoters of the invention provide constitutive expression of heterologous genes, as evidenced by the detection of nptll activity in all tissues obtained from transgenic plants transformed with exemplary plant transformation vectors pAG-7242 and pAG-7342.
  • nptll gene was evaluated by determining nptll enzyme levels in rransformants. The results are presented in Table 2 and in Fig. 5. Protein levels for leaf tissue obtained from transformants containing the CAS::nptH chimeric gene are not included in either the table or the figure, since values from two CAS:: ⁇ ptII events assayed were in excess of 6000 pg/ml, indicating the high level of gene expression regulated by the CAS promoter (i.e., a strong promoter).
  • drullO and dru259 are considered to function as moderate-level promoters.
  • the average nptll enzyme level for drullO (dru259)::nptll plants was about 5-9% that determined for CAS::nptII plants.
  • the average nptll enzyme activity determined for drullO (dru259)::nptll plants was about 40-60% of the nptll enzyme activity determined for hspiO :nptll plants.
  • promoters derived from the drul gene provide somewhat lower levels of gene expression than the ⁇ sp80 promoter, but are also considered to function as moderate strength promoters.
  • each of the exemplary raspberry promoters described herein is capable of directing constitutive expression of a transgene at sufficient levels to support its use in regulating expression of any of a number of heterologous gene products.
  • the transformation of tomato plants using the raspberry promoters of the present invention illustrates that a promoter region derived from raspberry can be used to promote expression of a gene within plant cells from a completely different genus, family, or species of plant.
  • the present invention provides vectors suitable for the transformation of plants.
  • the vectors, chimeric genes and DNA constructs of the present invention are also useful for the expression of heterologous genes.
  • Transgenic plants carrying the chimeric genes of the present invention may be a useful source of recombinantly-expressed material.
  • the chimeric genes of the present invention have two components: (i) a constitutive promoter derived from a raspberry drul gene, and (ii) a heterologous DNA sequence encoding a desirable product.
  • the vectors of the present invention may be constructed to carry an expression cassette containing an insertion site for DNA coding sequences of interest.
  • the transcription of such inserted DNA is then under the control of a suitable raspberry promoter (e.g., drullOpw or dru259p ⁇ o) of the present invention.
  • a suitable raspberry promoter e.g., drullOpw or dru259p ⁇ o
  • Such expression cassettes may have single or multiple transcription termination signals at the coding-3'-end of the DNA sequence being expressed.
  • the expression cassette may also include, for example, DNA sequences encoding (i) a leader sequence (e.g., to allow secretion or vacuolar targeting), and (ii) translation termination signals.
  • the vectors of the present invention may include selectable markers for use in plant cells (such as, a neomycin phosphotransferase II gene (nptll) or a neomycin phosphotransferase I gene).
  • selectable markers for use in plant cells such as, a neomycin phosphotransferase II gene (nptll) or a neomycin phosphotransferase I gene.
  • the presence of the nptll gene confers resistance to the antibiotic, kanamycin.
  • Another aminoglycoside resistance gene for use in vectors of the invention includes a gene encoding hygromycin phosphotransferase, i.e., an hpt gene (Gritz, et al, 1983). Plant cells containing the hpt gene are able to grow in the presence of the aminocyclitol antibiotic, hygromycin B.
  • selectable marker sequences for use in the present invention include glyphosate-tolerant CP4 and COX genes (Zhou, et al, 1995). Transgenic plants expressing either of these genes exhibit tolerance to glyphosate, which can be used in selection media to select for plant transformants.
  • the vectors may also include sequences mat allow their selection and propagation in a secondary host, such as, sequences containing an origin of replication and a selectable marker.
  • Typical secondary hosts include bacteria and yeast.
  • the secondary host is Escherichia coli
  • the origin of replication is a coIEl-type
  • the selectable marker is a gene encoding ampicillin resistance.
  • sequences are well known in the art and are also com ⁇ flashally available (e.g., Clontech, Palo Alto, CA; Stratagene, La Jolla, CA).
  • the vectors of the present invention may also be modified to intermediate plant transfor- mation plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T- DNA border region from Agrobacterium tumefaciens, and chimeric genes or expression cassettes (described above). Further, the vectors of the invention may comprise a disarmed plant tumor in ⁇ ducing plasmid of Agrobacterium tumefaciens.
  • the vectors of the present invention are useful for moderate level constitutive expression of nucleic acid coding sequences in plant cells. For example, a selected peptide or polypeptide coding sequence can be inserted in an expression cassette of a vector of the present invention.
  • the vector is then transformed into host cells, the host cells are cultured under conditions to allow the expression of the protein coding sequences, and the expressed peptide or polypeptide is isolated from the cells.
  • Transformed progenitor cells can also be used to produce transgenic plants bearing fruit.
  • vectors, chimeric genes and DNA constructs of the present invention can be sold individually or in kits for use in plant cell transformation and the subsequent generation of transgenic plants.
  • a raspberry promoter of the present invention includes a region of DNA that promotes transcription of the immediately adjacent (downstream) gene constitutively, in numerous plant tissues.
  • heterologous genes are operably linked to a raspberry promoter of the present invention.
  • heterologous genes for the transformation of plants include genes whose products are effective to confer antibiotic resistance. Some of these genes, including the nptll gene, are described above.
  • genes of interest that can be used in conjunction with a raspberry promoter of the invention include, but are not limited to, the following: genes capable of conferring fungal resistance, such as the polygalacturonase inhibiting protein (PGIP) gene from Phaseolus vulgaris (Toubart, et al, 1992) and modified forms of plant glucanase, chitinase (Jongedijk, et al, 1995) and other pathogenesis related (PR) genes (Melchers, et al, 1994; Ponstein, etal, 1994; Woloshuk, et al, 1991).
  • PGIP polygalacturonase inhibiting protein
  • PR pathogenesis related
  • These gene products can, for example, enhance resistance to fungi such as Fusarium, Sclerotinia sclerotiorum, and Rhizoctonia solani. Transformed plants expressing these products exhibit increased resistance to diseases such as seedling damping off, root rot disease, and the like.
  • Other representative genes for conferring both viral and fugal resistance to transgenic plants are described in "VIRUS / ND FUNGAL RESISTANCE: FROM LABORATORY TO FIELD" (Van Den Elzen, et al, 1994).
  • Additional exemplary heterologous genes for use with a raspberry promoter of the present invention include genes whose products are effective to confer herbicide-resistance to transformed plant cells.
  • Exemplary herbicide resistance genes include a bialaphos resistance gene (bar) which codes for phosphinothricin acetyltransferase (PAT) (Akama, etal, 1995). Transgenic plants con ⁇ taining this gene exhibit tolerance to the herbicide, "BASTA". This gene can also be used as a selectable marker gene, since explants carrying the bar gene are capable of growing on selective media containing phosphinothricin (PPT), which is an active component of bialaphos.
  • PPT phosphinothricin
  • Additional herbicide resistance genes include those conferring resistance to glyphosate- containing herbicides.
  • Glyphosate refers to N-phosphonomethyl glycine, in either its acidic or anionic forms.
  • Herbicides containing this active ingredient include "ROUNDUP” and "GLEAN”.
  • Exemplary genes for imparting glyphosate resistance include an EPSP synthase gene (5- enolpyruvyl-3-phosphosshikimate synthase) (Delanney, et al., 1995; Tinius, et al, 1995), or an acetolactate synthase gene (Yao, et al, 1995).
  • exemplary DNA coding sequences include a bxn gene encoding a bromoxynil-specific nitrilase (Stalker, et al, 1988), under the transcriptional control of a drul promoter. Transformed plants containing this chimeric gene express a bromoxynil-specific nitrilase and are resistant to the application of bromoxynil-containing herbicides.
  • genes encoding a viral coat protein to enhance coat-protein mediated virus- resistance in transgenic plants.
  • Exemplary genes include genes coding for alfalfa mosaic virus coat protein (A1MV), cucumber mosaic virus coat protein (CMV), tobacco streak virus coat protein (TSV), potato virus coat protein (PVY), tobacco rattle virus coat protein (TRV), and tobacco mosaic virus coat protein (TMV) (Beachy, et al, 1990).
  • A1MV alfalfa mosaic virus coat protein
  • CMV cucumber mosaic virus coat protein
  • TSV tobacco streak virus coat protein
  • PVY potato virus coat protein
  • TRV tobacco rattle virus coat protein
  • TMV tobacco mosaic virus coat protein
  • TMV tobacco mosaic virus coat protein
  • the vector constructs of the present invention can be used for transformation and expression of heterologous sequences in transgenic plants independent of the original plant source for the promoter sequence.
  • the drullOr.nptll and dru259::nptll chimeric genes were successfully introduced into tomato plant cells.
  • the raspberry promoters of the invention e.g. , drullO, dru259 are useful for promoting gene expression in heterologous plant systems, i.e., plant cells other than raspberry, such as tomato.
  • the expression mediated by the promoters appears to be constitutive even in heterologous plants.
  • raspberry -fritf -derived promoters of the invention can be cloned as described above employing sequence information described herein.
  • These raspberry promoters can be used to express any heterologous gene whose function would be enhanced or enabled by a moderate level, constitutive promoter. Exemplary genes are described above.
  • the use of these promoters cannot be considered limited to raspberries, particularly in view of the successful transformation of tomato using the raspberry promoters of the invention. Since raspberry is essentially a miniature drupe fruit, it is likely that the raspberry promoters will function in other drupe fruits.
  • the constructs and methods of the present invention are applicable to all higher plants including, but not limited to, the following: Berry-like fruits, for example, Vitis (grapes), Fragaria (strawberries), Rubus (raspberries, blackberries, loganberries), Ribes (cur ⁇ rants and gooseberries), Vaccinium, (blueberries, bilberries, whortleberries, cranberries), Actinida (kiwifruit and Chinese gooseberry).
  • other drupe fruits including, but not limited to, Malus (apple), Pyrus (pears), most members of the Prunus genera, sapota, mango, avocado, apricot, peaches, cherries, plums, and nectarines. Additional plant sources are described above.
  • the present invention provides compositions and methods to regulate plant cell expression of any gene in a constitutive manner.
  • the promoters of the present invention can be used to regulate expression of a selectable marker gene, such as nptll.
  • the raspberry promoters can be used to promote expression of a herbicide-resistance gene, or to regulate expression of a gene encoding a viral coat protein, to provide enhanced virus resistance.
  • the raspberry promoters of the invention can be used in chimeric genes, plant transformation vectors, expression cassettes, kits, and the like, to promote transformation of plant cells.
  • the raspberry promoters described herein may also be employed in a method for providing moderate level expression of a heterologous gene, such as a selectable marker gene, in a transgenic plant.
  • a heterologous gene such as a selectable marker gene
  • Bioreagents were typically obtained from the following vendors: 5' to 3' Prime, Boulder, CO; New England Biolabs, Beverly, MA; Gibco/BRL, Gaithersburg, MD; Promega, Madison, WI; Clontech, Palo Alto, CA; and Operon, Alameda, CA. Standard recombinant DNA techniques were employed in all constructions (Adams and Yang, 1977; Ausubel, et al, 1992;
  • a raspberry protein sample was prepared by grinding the frozen drupes of one whole berry into a fine powder.
  • Sample buffer 0.05 M Tris, pH 6.8, 1% SDS, 5% beta-mercaptoethanol, 10% glycerol; Laemelli, 1970
  • the sample was heated for 10 minutes at 90-95 °C and centrifiiged at 14K rpm, 4°C for 10 minutes. The supernatant was removed from the insoluble debris pellet and stored at -20°C.
  • Drupelet proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophore ⁇ sis (SDS PAGE) combined with coomassie blue staining using standard procedures.
  • a coomassie blue-stained SDS polyacrylamide gel of soluble drupelet proteins is shown in Figure 10.
  • lane 1 molecular weight markers (BioRad, Richmond, CA)
  • lanes 2, 3 and 5 each contain 9 ⁇ g of raspberry drupelet protein lysate prepared separately from individual fruit.
  • Lane 4 contained a higher amount of lysate.
  • drupel and drupe2 Two highly abundant proteins were observed at approximately 17 and 15 kd and were named drupel and drupe2, respectively. In Fig. 10 these two proteins are indicated by arrows. Scanning densitometry analysis of this gel indicated drupel and drupe2 comprise approximately 23 and 37%, respectively, of the total soluble protein in raspberry drupelets. As a result, a direct western blot approach to purification and sequencing of the protein was followed.
  • a protein blot (Applied Biosystems, Inc. User Bulletin Number 58; Ausubel, et al, 1992) was prepared using the raspberry protein lysate described above. Varying amounts of raspberry protein lysate (12-36 ⁇ g/well) were loaded on a 10 well 18% SDS-PAGE minigel (1.5 mm thick) with 4.5% stacker and electrophoresed at 100 volts in 25 mM Tris, 192 mM glycine, 0.1% SDS buffer for 2-2.5 hours.
  • Proteins were transblotted onto Applied BioSystem's "PROBLOTT" polyvinylidenedifluoride
  • PVDF PVDF membrane in a 25 mM Tris, 192 mM glycine, 10% methanol buffer at 90 volts for 2 hours at 4°C. After protein transfer, the blot was Coomassie blue stained and the 15 and 17 kilodalton (kd) protein bands were located on the blot and cut out. N-terminal sequencing of the proteins was carried out at the W.M. Keck Foundation, Biotechnology Resource Laboratory in New Haven, CT.
  • the drupel sample yielded a thirty amino acid N-terminal sequence.
  • the drupe2 sample did not yield useful sequence information likely due to a blocked amino terminus.
  • the amino terminal drupel sequence is presented as SEQ ID NO: 11. This 30 amino acid drupel sequence was compared to the protein database using BLAST searching; no significant matches were found indicating that drupel is a novel protein.
  • the estimated weight of the drupelets was 12 grams.
  • a cold mortar which contained liquid nitrogen, the whole berries were fractured by tapping them with a pestle.
  • the drupelets were separated from the receptacles.
  • the receptacles were removed from the mortar and discarded.
  • the drupelets were ground to a powder in the mortar, adding liquid nitrogen as necessary to keep the tissue frozen. The seeds were purposefully left intact. Homogenization buffer, 2 ml/gram of tissue, was used to extract the RNA.
  • the frozen powdered drupelet tissue was added to the buffer in 3 to 5 portions, vortexing between additions until all tissue was moistened.
  • the tissue plus buffer solution (referred to herein as the pulp) was diluted 1: 1 widi sterile water and 0.75 volumes of homogenization buffer were added to the diluted pulp.
  • the sample was incubated at 65°C for 10 to 15 minutes, followed by centrifugation in a swinging bucket rotor at 9000 g for 15 minutes at 4 G C. The supernatant was transferred to a clean tube.
  • Cesium chloride (CsCl) was added to the supernatant at 0.2 g/ml. The sample was mixed until the CsCl dissolved.
  • a 4 ml cushion was dispensed into a Beckman 1 x 3.5 inch polyallomer ultracentrifuge tube
  • the pellet was dissolved in 500 ⁇ SSTE and transferred to a microfuge tube (SSTE: 0.8 M NaCl, 0.4% SDS, 10 mM Tris-HCl, pH 8.0 and 1 mM Na ⁇ DTA, pH 8).
  • the sample was extracted twice with an equal volume of chloroform: isoamyl alcohol (24:1).
  • To precipitate the RNA 2.5 volumes ethanol were added to the aqueous phase.
  • the sample was collected by centrifugation, washed two times with 75% ethanol and resuspended in 100 ⁇ l TE. The yield was 1.6 mg.
  • the RNA was reprecipitated with 1/9 volume 3 M sodium acetate and 3 volumes ethanol for storage at -20°C.
  • mRNA from mature green raspberry drupelet total RNA was performed using the "STRAIGHT A'S" mRNA isolation system (Novagen, Madison, WI) according to the manufacturer's instructions. mRNA was isolated from the 1.6 mg of total RNA extracted from mature green raspberry drupelets described above. The yield of mRNA from this procedure was 6.6 ⁇ g.
  • reaction mixture was assembled for the cDNA synthesis reaction: H 2 0, 10.2 ⁇ l; 250 ng mRNA, 0.8 ⁇ l; 5 x BRL RT buffer (BRL, Bethesda, MD), 4.0 ⁇ l; 100 mM DTT (dithiothreitol - BRL, Bethesda, MD), 0.2 ⁇ l; "RNAguard" (23.4 U/ ⁇ l; an RNase inhibitor from Pharmacia, Piscataway, NJ), 0.5 ⁇ l; dNTP's (2.5 M each), 2.0 ⁇ l; 50 ⁇ M primer, 1.0 ⁇ l; [ 32 P]dCTP (3000 Ci/mmol; DuPont/NEN, Boston, MA), 1.0 ⁇ l; and AMV-reverse-transcriptase (38 U/ ⁇ l; Life Sciences, Inc., St.
  • the cDNA reaction was performed by combining mRNA and water for the reaction and heating to 65 °C for 3 minutes. The mixture was cooled on ice and micro uged (to collect condensation). The remaining reaction components were then added.
  • a degenerate PCR primer, Drupe20 was designed for the 5' end of the cDNA based on the reverse translation of the drul protein sequence.
  • a section of the known amino acid sequence of drul (SEQ ID NO: 13) was chosen for its proximity to the amino terminus and for the relatively low level of degeneracy in its reverse-translated sequence (SEQ ID NO: 14; Drupe20).
  • the Drupe20 primer (i) is the 512-fold degenerate nucleotide sequence corresponding to the amino acid sequence presented as SEQ ID NO: 13, and (ii) was used as the 3'-primer.
  • the 5' PCR primer (DrupeRANl ⁇ , SEQ ID NO:15, corresponding to the cDNA primer, dTRANDOM) was designed for the 3' end.
  • Polymerase chain reaction (PCR; Perkin-Elmer Cetus, Norwalk, CT; Mullis, 1987; Mullis, et al, 1987, was performed following the manu ⁇ facturer's procedure using "AMPLITAQ" (Perkin Elmer Cetus), PCR buffer II (50.0 mM KC1, 10 mM Tris-HCl, pH 8.3), 2 mM MgCl 2 , 0.2 mM of each dNTP, mature green drupelet cDNA and Drupe20 and DrupeRANl ⁇ primers under the following conditions: 1 cycle at 95 °C, 1 minute,
  • the 700 bp band was isolated from a 1 % "SEAPLAQUE" agarose gel using (3-agarase (New England Biolabs, Beverly,
  • the cDNA clones of the drul gene were identified by screening plasmid miniprep DNA prepared from 1.6 ml of culture using the alkaline lysis method (Ausubel, et al, 1992). The double-stranded DNA was sequenced by the dideoxy chain-termination method using the
  • the sequence was read from the autoradiograph and analyzed for its homology with the reverse translated N-terminal protein sequence from drupel. The actual DNA sequence was determined, as opposed to the degenerate DNA sequence obtained through reverse translation of the protein sequence. The correlation between the cDNA and the remainder of the N-terminal protein sequence was confirmed. A clone (designated pAG-301) was selected, following these criteria, for further characterization.
  • the nucleic acid sequence of the drul cDNA insert of pAG- 301 is presented as SEQ ID NO: 16.
  • CTAB hexadecyl-trimethyl-ammonium bromide
  • Doyle and Doyle 1990
  • PCR primers DruGen5 ⁇ SEQ ID NO: 17; DruGen3', SEQ ID NO: 18
  • OLIGO a multi-functional program from National Biosciences, Inc. (Plymouth, MN), was used to facilitate design of the primers.
  • PCR was performed following the manufacturer's procedure using "AMPLITAQ” (Perkin-Elmer Cetus), PCR buffer (50.0 mM KC1, 10 mM Tris-HCl pH 8.3, and 1.5 mM MgClj), 0.2 mM of each dNTP, raspberry genomic DNA and DruGen5' and DruGen3' primers under the following (“HOT START”) conditions:
  • This amplification reaction produced 3 major products: a predominant product of 710 bp and 2 less abundant products of 690 and 625 bp.
  • the PCR reaction products were then ligated to the vector pCRII, the TA cloning vector from Invitrogen (San Diego, CA), following the manufacturer's instructions.
  • a clone was selected with a 710 bp insert and designated pAG-302.
  • Plasmid DNA of pAG-302 was prepared from 1.6 ml of culture using the alkaline lysis method (Ausubel, et al, 1992) and sequenced by the dideoxy chain-termination method using "SEQUENASE” ver.2 enzyme and kit components (USB, Cleveland, Ohio) and [ ⁇ -35S]-dATP (DuPont/NEN). The sequencing reactions were primed with the M 13 universal forward and reverse primers (New England Biolabs, Beverly, MA). Further sequencing reactions were primed with 2 additional internal primers. Sequencing reactions were resolved on an acrylamide gel and detected through autoradiography.
  • Inverse PCR primers (designated DruInvUp, SEQ ID NO:5, and DruInvLow, SEQ ID NO:6) were designed based upon the genomic DNA sequence and optimized using OLIGO.
  • Genomic raspberry DNA was digested with restriction enzyme Nsil.
  • Nsil was chosen because, based on the cDNA sequence, Nsil was known to cut in the 3' -untranslated region of the gene.
  • a small portion of the Nsil digested DNA was run on an analytical agarose gel and a Southern transfer was performed (Ausubel, et al, 1992). The Southern blot was probed with the cDNA fragment contained in pAG-302.
  • the probe identified a Nsil fragment of about 2-2.3 kb: this fragment hybridized strongly with the genomic clone.
  • a second, smaller fragment hybridized to the probe as well but hybridized weakly with die genomic clone.
  • DNA in the range of 2-2.3 kb was excised from the gel.
  • the DNA was purified using /3-agarase
  • the 1.8 kb band was isolated from a 1% "SEAPLAQUE" agarose gel using ⁇ -agarase. This fragment was ligated to pCRII to give rise to pAG-310.
  • a schematic representation of the preparation of subclone pAG-310 is presented in Figs. 1 and 2.
  • the pAG-310 insert was sequenced in its entirety (SEQ ID NO:l) and the drul insert sequence was found to be identical to the cDNA clone (SEQ ID NO: 16) and the genomic clone (SEQ ID NO: 19) in the regions where sequence was shared.
  • the normal elements of plant genes and their regulatory components were identified (Figs. 6 A and 6B) including a CAAT box, TATA box, ATG start codon, two exons, an intron, splicing sites, a stop codon and poly-adenylation sites.
  • the gene organization and protein structure of drul is schematically displayed in Figure 12.
  • the gene encodes a protein having the predicted amino acid sequence presented as SEQ ID NO:20.
  • the predicted protein has a calculated molecular weight of 17,087.64 and an estimated pi of 4.80.
  • a Kyte-Doolittle hydrophobicity plot of the drul protein is presented as Fig. 13.
  • RNA dot blots were prepared using 5 ⁇ g of total raspberry leaf RNA and 5 ⁇ g each of total receptacle RNA from green, mature green, breaker, and orange/ripe raspberries (corresponding to stages I, II, III, IV, respectively, in Figure 14). The blots were probed with the drul cDNA fragment, labeled with [32-P]dCTP (> 3000 Ci/mmole) by the random primed method (Boeringer
  • the hybridizing probe was detected through standard autoradiographic methods.
  • the exposure of the blot to film was for 4 hours and 10 minutes with an intensifying screen at -80°C.
  • RNA dots are, respectively from left to right, leaf RNA and receptacle RNA from green (Fig. 14, “I”), mature green (Fig. 14, “II”), breaker (Fig. 14, “III”) and orange/ripe raspberries (Fig. 14, “IV”).
  • RNA extraction method (Chang, et al. , 1993) was used for receptacles and leaves.
  • the raspberry drupelet RNA extraction method described above was used for the drupelets and strawberry fruit.
  • a Northern blot was prepared using 5 ⁇ g/lane of each sample RNA.
  • the RNA samples were as follows: raspberry leaf (Fig. 15, lane 1), mature green raspberry receptacles (Fig. 15, lane 2), orange/ripe raspberry receptacles (Fig. 15, lane 3), mature green raspberry drupelets (Fig. 15, lane 4), and orange/ripe raspberry drupelets (Fig. 15, lane 5).
  • the blot was probed with the drul cDNA fragment, labeled with pPJdCTP (>3000 Ci/mmole) by random primed reactions. Hybridization was carried out overnight at 45°C in "HYBRISOL I" (Oncor, Gaithersburg, MD). A probe concentration of 4.2 x 10 6 DPM/ml was used. The blot was washed after the overnight hybridization with a final wash using 0.1 x SSC at 50°C for 30 minutes. The hybridizing probe was detected through standard autoradiographic methods. The exposure of the blot to film was for 1 hour at room temperature without an intensifying screen.
  • Protein lysates were prepared (as described in Example 1) from raspberry drupelets at various stages of ripening.
  • the lysates were size-fractionated by PAGE and the gel stained with Coomaise blue (50% MeOH, 10 mM Tris-HCl pH 8.3, 1.5 mM MgC12).
  • Coomaise blue 50% MeOH, 10 mM Tris-HCl pH 8.3, 1.5 mM MgC12.
  • the results are presented in Fig. 16.
  • the lysates in the lanes were as follows: lane 1, green drupelet; lane 2, mature green drupelet; lane 3, breaker drupelet; lane 4, orange drupelet; and lane 5, ripe drupelet.
  • the results of this analysis supports a stage specific expression pattern in drupelets.
  • a DNA fragment containing the drul promoter was PCR amplified from subclone pAG-310 using primers, 5' primer, DrupeUp (SEQ ID NO:7) and 3' primer, DrupeLow (SEQ ID NO:8) under standard PCR reaction conditions.
  • the PCR reaction mixture contained the following components: 79.0 ⁇ l water, 10.0 ⁇ l 10X Vent buffer, 1.0 ⁇ l DrupeUp primer (50 ⁇ M solution), 1.0 ⁇ l DrupeLow primer (50 ⁇ M solution), 8.0 ⁇ l dNPTs (2.5 M each), 1.0 ⁇ l template DNA (100 ng).
  • the PCR reaction conditions employed were as follows:
  • the amplification reaction produced a 1.3 kb fragment product as illustrated in the top portion of Figs. 1 and 2.
  • This fragment was purified from the reaction mixture as follows.
  • the PCR reaction mixture was transferred to a light Phase Lock Gel tube (5 Prime to 3 Prime, Boulder, CO.).
  • the tube was spun in a microcentrifuge following the manufacturer's instructions.
  • the upper aqueous phase was transferred to a Select, G-50 spin column (5 Prime to 3 Prime, Boulder, CO.) and the DNA was centrifuged through the column according to the manufacturer's instructions.
  • To the eluant was added 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol, in order to precipitate the DNA.
  • the sample was incubated on ice for a period of no less than 10 minutes, and then microcentrifuged at 4°C for 30 minutes at 14,000 rpm. The supernatant was decanted from the tube and the pellet washed twice with 75% ethanol. The pellet was allowed to dry, and then resuspended in 25 ⁇ l 1/2 strength TE (5 mM Tris-HCl, 0.5 mM EDTA, pH 8). The DNA fragment was digested to completion with restriction enzymes Nsil and Xbal to produce a drul promoter fragment. This fragment was puri ⁇ fied in the same manner as was the PCR product described above.
  • the non-integrating plant expression vector p35S-GFP (Clontech Laboratories, Palo Alto, CA) was digested with Xbal and ⁇ tl.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 3.7 kb fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using /5-agarase from New England Biolabs (Beverly, MA), following the manufacturer's instructions.
  • the gel region containing the 0.85 kb 35S promoter was discarded.
  • the 3.7 kb fragment from p35S-GFP2 and the 1.3 kb drul promoter fragment were combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following the manufacturer's instructions, to form the intermediate plasmid pAG-155.
  • the resulting plasmid containing the raspberry drul promoter was designated pAG-155, as illustrated in Figs. 1 and 2.
  • Plasmid pAG-155 was digested to completion with SnaBI and EcoRV, both blunt cutters, releasing the 259 bp drul promoter fragment, designated herein as dru259, where nucleotide number one is immediately 5' of the ATG start codon.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 259 bp drul promoter fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using /S-agarase (New England Biolabs, Beverly, MA), following the manufacturer's instructions. The gel region containing the remainder of the plasmid was discarded.
  • Subclone pAG-411 containing the nos:: ptll cassette was prepared as follows.
  • Cloning vector pGEM*3Zf(+) (Promega, Madison, WI) was digested with Xbal and Bam ⁇ l.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 3.2 kb fragment was cut from the body of the gel.
  • the plant binary transformation vector pGPTV-kan (Max-Planck Institut, Koln, Germany) was digested with Xbal and BamEl.
  • the digested plasmid was run on a 1% low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 1.48 kb nos::nptII fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using 3-agarase (New England Biolabs, Beverly, MA), following the manufacturer's instructions. The gel region containing the 13.3 kb fragment was discarded.
  • Plasmid pAG-411 was digested to completion with Hindi and PshAl, both blunt cutters, releasing the 636 bp nos promoter fragment.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 4 kb fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using /J-agarase (New England Biolabs, Beverly, MA), following the manufacturer's in ⁇ structions.
  • the gel region containing the 636 bp nos promoter fragment was discarded.
  • the 4 kb fragment from pAG-411 and the 259 bp drul promoter fragment from pAG-155 were combined in a ligation reaction, using Gibco/BRL's T4 DNA ligase, following manufac- turer's instructions, to form the intermediate vector pAG-431.
  • the nucleotide sequence for the truncated promoter dru259 is presented herein as SEQ ID NO:4.
  • a DNA fragment containing 166 bp of the drul promoter was PCR amplified from subclone pAG-155 using primers Drul-118 ⁇ 3 (SEQ ID NO:9) and GFPStartR (SEQ ID NO:10) under the following PCR reaction conditions.
  • the 166 bp of the drul promoter fragment was men purified as follows.
  • the PCR reaction mixture was transferred to a light Phase Lock Gel tube (5 Prime to 3 Prime, Boulder, CO.).
  • a mixed solvent system containing phenol :chloroform:isoamyl alcohol (25:24:1) was added to this tube at a volume equal to the PCR reaction volume.
  • the tube was then spun in a microcentrifiige following the manufacturer's instructions.
  • the upper, aqueous phase was transferred to a Select, G-50 spin column (5 Prime to 3 Prime, Boulder, CO.) and the was DNA centrifuged through the column following the manufacturer's instructions. To the eluant 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added, to precipitate the DNA. The sample was incubated on ice for a period of no less than 10 minutes. Following incubation, the sample was microcentrifuged at 4°C for 30 minutes at 14,000 rpm. The supernatant was decanted from the tube and the pellet washed twice with 75% ethanol. The pellet was allowed to dry, followed by resuspension in 31.6 ⁇ l H 2 0. This fragment was digested to completion with restriction enzymes Hind ⁇ l and £coRV to produce a 112 bp drul promoter fragment. This fragment was purified in the same manner as the PCR product described above.
  • Plasmid pAG-411 was digested to completion with Hind ⁇ ll and PshA , releasing a 620 bp nos promoter fragment.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 4 kb fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using ⁇ - agarase from New England Biolabs (Beverly, MA), following the manufacturer's instructions.
  • the gel region containing the 420 bp nos promoter fragment was discarded.
  • Plasmid pAG-1542 was constructed using conventional cloning techniques known in the art (Sambrook, et al, 1989).
  • Subcloning binary vector pAG-1542 contained the nptll marker gene under the control of the nos promoter located near the left border and the SAMase gene (Ferro, et al, 1995) driven by the tomato E8 promoter (Deikman, et al, 1988; Deikman, et al, 1992) located near the right border.
  • pAG-7342 Construction of subclone pAG-431, containing the dru259::nptll chimeric gene, is described in Example 7.
  • Plasmid pAG-1542 was digested with Hindill and BamHl.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 13 kb fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using ⁇ -agarase from New England Biolabs (Beverly, MA), following the manufacturer's instructions.
  • the gel region containing the 1.46 kb nos::npt ⁇ fragment was discarded.
  • Plasmid pAG-431 was digested with Hi ⁇ II and BamHl.
  • the digested plasmid was run on a 1% low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 1.1 kb dru259: :nptll fragment was cut from the body of the gel .
  • the DNA fragment was then purified away from the gel using 0-agarase from New England Biolabs
  • Example 9A Construction of plasmid pAG-1542 is described in Example 9A. Construction of subclone pAG-421 , containing the drullO: :npill chimeric gene, is described in Example 8.
  • Plasmid pAG-1542 was digested with Hindill and BamHl.
  • the digested plasmid was run on a 1% low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 13 kb fragment was cut from me body of the gel.
  • the DNA fragment was then purified away from the gel using 0-agarase from New England Biolabs (Beverly, MA), following the manufacture's instructions.
  • the gel region containing the 1.46 kb nos::nptII fragment was discarded.
  • Plasmid pAG-421 was digested with Hindill and BamHl.
  • the digested plasmid was run on a 1 % low melting point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME).
  • the gel region containing the 0.95 kb dru259: :npt ⁇ l fragment was cut from the body of the gel.
  • the DNA fragment was then purified away from the gel using ⁇ -agarase from New England Biolabs (Beverly, MA), following the manufacturer's instructions.
  • the gel region containing the remainder of the plasmid was discarded.
  • Example 11 Plant Transformation Using Binary Vectors pAG-7242 and pAG-7342 Agrobacterium-based plant transformation using binary vectors pAG-7242 and pAG-7342 containing chimeric drullO::nptll and dru254::nptll genes, respectively, was carried out using tomato cotyledons as described below for exemplary plasmid pAG-7242.
  • Agrobacterium tumefaciens strain C58 was used to introduce coding sequences into plants. This strain contains a T-DNA-less Ti plasmid. The pAG-7242 plasmid was transferred into EHA101 using electroporation essentially as described by Nagel, et al. (1990).
  • an Agrobacte ⁇ um tumefaciens culture was grown to mid-log phase (OD 600 0.5 to 1.0) in MG/L agar media containing tryptone (5 g/1), yeast extract (2.5 g/1), NaCl (5 g/1), mannitol (5 g/1), sodium glutamate (1.17 g/1), K 2 HP0 4 (0.25 g/1), MgS0 4 (0.1 g/1) and biotin (2 ⁇ g/1), adjusted to pH 7.2 by addition of sodium hydroxide.
  • Cotyledon explants were pre-conditioned overnight on tobacco feeder plates (Fillatti, et al, 1987). The pre-conditioned explants were innoculated by placing them in a 20 ml overnight culture of EHA105/pAG-7242 for 15 minutes. The explants were then co-cultivated with EHA105/pAG-7242 for 2 days on tobacco feeder plates as described by Fillatti, et al, (1987).
  • the explants were grown in tissue culture media containing 2Z media (Fillatti, et al. , 1987), Murisheegee and Skoog (MS) salts, Nitsch and Nitsch vitamins, 3% sucrose, 2 mg/1 seatin, 500 mg/1 carbenicillin, 60-200 mg/1 kanamycin, and 0.7% agar.
  • the explants were grown in tissue culture for 8 to 10 weeks.
  • the carbenicillin treatments were kept in place for 2 to 3 months in all media.
  • the explants and plants were kept on carbenicillin until they were potted in soil as a counter-selection to rid the plants of viable Agrobacterium tumefaciens cells.
  • Table 1 presents a summary of the plant transformation experiments, including concentrations of selection agent utilized, and transformation frequencies.
  • Results obtained for plant transformation experiments using the novel raspberry promoters of me present invention are compared to those obtained using binary vectors containing two different strong constitutive promoters, a caulimovirus promoter, the cassava mottle vein virus promoter (CAS) and the hspiO promoter.
  • the CAS promoter was obtained from The Scripps Research Institute (La Jolla, CA). Isolation of the hspiO promoter, its nucleotide sequence, as well as vector constructions and expression levels of transgenes containing the n_p80 promoter have been described (Brunke and Wilson, 1993).
  • nptll assay was carried out with a few samples using rooted plants which were available in culture at the time of testing. Thus, not all rooted plants were tested for nptll expression.
  • the results of the ELISA assay are presented in column (IV) of Table 1 below.
  • transformation frequency is defined as the ratio of the number of tissue explants producing regenerated shoots that are capable of rooting in the presence of selection agent (kanamycin) to the total number of initial tissue explants, expressed as a percentage.
  • Nptll expression level expressed as a percentage, is the ratio of nptll positive plants to the total number of rooted plants tested for nptll, based upon the results of the ELISA assay described in Example 12.
  • a positive nptll result is an ELISA value greater than background. For example, the first entry under column (IV) indicates that out of 10 events tested for nptll, 10 exhibited positive ELISA results.
  • Relative expression levels of nptll are presented in Table 2.
  • exemplary dru259 and drullO promoters direct lower level expression of genes placed under their control than does the hspiO promoter.
  • these two exemplary raspberry dru promoters are both capable of expressing sufficient levels of nptll to allow selection of transgenic plants.
  • ADDRESSEE Dehlinger & Associates
  • STREET 350 Cambridge Avenue, Suite 250
  • GGTGTACCGT GTACTAATCA AATATCTACC TGATCTTATT GATTTGAAAG ATCATAAAAA 240
  • CTCTCGGCGG TAAGAGGAGA TATCCTCAGT CGAATTATGA GCCGATCGAG GAAAGCTCGA 420
  • GACTCTTAGC AACTTAAGTT TCAAACCGTG ACGAACCAAT AAAATTTGAC AAATTAATCA 540
  • GGTGTACCGT GTACTAATCA AATATCTACC TGATCTTATT GATTTGAAAG ATCATAAAAA 240
  • GACTCTTAGC AACTTAAGTT TCAAACCGTG ACGAACCAAT AAAATTTGAC AAATTAATCA 540
  • Dru259 promoter sequence (minus 259 region from start codon)
  • CAATTCACTC AAAAGAATGA TGGCAGCAGC ATTGCCAAAG TGTCCATTGA ATATGAGAAA 360
  • Lys Ala lie lie Leu Asn Gly Leu Glu Gly Asp Val Phe Gin Tyr Tyr 85 90 95

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