This application relates to aryl propenal double bond reductase proteins, to nucleic acid molecules and vectors encoding aryl propenal double bond reductase proteins, and to methods of altering the composition of plant material, such as wood, by altering the level of expression of one or more aryl propenal double bond reductase proteins within a plant.
Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990); Lewis et al., in Chemistry of the Amazon, Biodiversity Natural Products, and Environmental Issues, 588, (P. R. Seidl, O. R. Gottlieb and M. A. C. Kaplan) 135-167, ACS Symposium Series, Washington D.C. (1995)). Because of their pronounced antibiotic properties (Markkanen, T. et al., Drugs Exptl. Clin. Res. 7:711-718 (1981)), antioxidant properties (Faurxc3xa9, M. et al., Phytochemistry 29:3773-3775 (1990); Osawa, T. et al., Agric. Biol. Chem. 49:3351-3352 (1985)) and antifeedant properties (Harmatha, J., and Nawrot, J., Biochem. Syst. Ecol. 12:95-98 (1984)), a major role of lignans in vascular plants is to help confer resistance against various opportunistic biological pathogens and predators. Lignans have also been proposed as cytokinins (Binns, A. N. et al., Proc. Natl. Acad. Sci. USA 84:980-984 (1987)) and as intermediates in lignification (Rahman, M. M. A. et al., Phytochemistry 29:1861-1866 (1990)), suggesting a critical role in plant growth and development. Lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.
In addition to their functions in plants, lignans also have important pharmacological roles. For example, podophyllotoxin, as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990)). Antiviral properties have also been reported for selected lignans. For example, (xe2x88x92)-arctigenin (Schrxc3x6der, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)), (xe2x88x92)-trachelogenin (Schrxc3x6der, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)) and nordihydroguaiaretic acid (Gnabre, J. N. et al., Proc. Natl. Acad. Sci. USA 92:11239-11243 (1995)) are each effective against HIV due to their pronounced reverse transcriptase inhibitory activities. Some lignans, e.g., matairesinol (Nikaido, T. et al., Chem. Pharm. Bull. 29:3586-3592 (1981)), inhibit cAMP-phosphodiesterase, whereas others enhance cardiovascular activity, e.g., syringaresinol xcex2-D-glucoside (Nishibe, S. et al., Chem. Pharm. Bull. 38:1763-1765 (1990)). There is also a high correlation between the presence, in the diet, of the xe2x80x9cmammalianxe2x80x9d lignans or xe2x80x9cphytoestrogensxe2x80x9d, enterolactone and enterodiol, formed following digestion of high fiber diets, and reduced incidence rates of breast and prostate cancers (so-called chemoprevention) (Axelson, M., and Setchell, K. D. R., FEBS Lett. 123:337-342 (1981); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 41:3-8 (1992); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 52:97-103 (1995)). The xe2x80x9cmammalian lignans,xe2x80x9d in turn, are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al., J. Applied Bacteriol., 58:37-43 (1985)).
The biosynthetic pathways to the lignans are only now being defined. In this regard, loblolly pine (Pinus taeda) is a popular and commercially important softwood species in the United States, and has been used as a model for studies on lignin biosynthesis and monolignol regulation. Moreover, its cell suspension cultures accumulate various 8-5xe2x80x2 linked lignans, namely dehydrodiconiferyl alcohol (DDC) and dihydrodehydrodiconiferyl alcohol (DDDC), as well as the 8-8xe2x80x2 linked lignan, pinoresinol, when placed in a culturing medium containing 8% sucrose and 20 mM KI solution. The present inventors have utilized these cell cultures to characterize enzymes involved in the double bond hydrogenation system of DDC and coniferyl alcohol since the resulting metabolites help determine the color, durability and resistance of the wood, particularly the heartwood.
Thus, in one aspect, the present invention provides a cDNA molecule (SEQ ID NO:1) isolated from loblolly pine (Pinus taeda) cells, and the encoded aryl propenal double-bond reductase (SEQ ID NO:2). Aryl propenal double-bond reductase (SEQ ID NO:2) expressed from the isolated cDNA molecule (SEQ ID NO:1) was found to regiospecifically reduce the double-bond of dehydrodiconiferyl aldehyde (DDCAL) to afford dihydrodehydrodiconiferyl aldehyde (DDDCAL) in the presence of [4R]-NADPH. The recombinant aryl propenal double-bond reductase (SEQ ID NO:2) was also capable of reducing coniferyl aldehyde to dihydroconiferyl aldehyde, and P. taeda soluble enzyme preparations also catalyzed the reduction of dihydroconiferyl aldehyde to give dihydroconiferyl alcohol, i.e., the aryl propenal double-bond reductase (SEQ ID NO:2) acts on both monomeric and dimeric aryl propenals.
In one aspect, the present invention provides isolated nucleic acid molecules that each: (a) encode an aryl propenal double bond reductase; and (b) hybridize to a nucleic acid molecule consisting of the complement of the nucleic acid sequence set forth in SEQ ID NO:1 under conditions of 1xc3x97SSC at 55xc2x0 C. for one hour. Some nucleic acid molecules of this aspect of the invention are cDNA molecules. An exemplary nucleic acid molecule of the invention is the cDNA molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:1 that encodes the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
In another aspect, the present invention provides isolated nucleic acid molecules that each encode an aryl propenal double bond reductase that is at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2. The present invention also provides vectors that include a nucleic acid molecule of the invention, and host cells (such as plant cells) that include a vector of the invention. Thus, in one embodiment, the present invention provides vectors that each comprise a nucleic acid molecule that: (a) encodes an aryl propenal double bond reductase; and (b) hybridizes to the complement of SEQ ID NO:1 under conditions of 1xc3x97SSC, 55xc2x0 C. for one hour.
In yet another aspect, the present invention provides isolated aryl propenal double bond reductase proteins that are at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
In a further aspect, the present invention provides methods of enhancing the level of aryl propenal double bond reductase in a plant, the methods each comprising the steps of: (a) introducing into a plant an expression vector comprising a nucleic acid molecule that: (1) encodes an aryl propenal double bond reductase; (2) hybridizes to the complement of SEQ ID NO:1 under conditions of 1xc3x97SSC at 55xc2x0 C. for one hour; and (b) expressing the aryl propenal double bond reductase within the plant.
The present invention also provides methods of inhibiting the expression of aryl propenal double bond reductase in a plant, the methods comprising the steps of: (a) introducing into a plant an expression vector that comprises a nucleic acid molecule that is in antisense orientation relative to a promoter, the nucleic acid molecule hybridizing to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:1 under conditions of 1xc3x97SSC, 55xc2x0 C. for one hour; and (b) transcriptionally expressing the nucleic acid molecule in the plant.
The nucleic acid molecules, proteins and methods of the invention are useful for a variety of purposes, including altering the amount and/or type of lignans produced by plant cells. For example, enhancing lignan production by expressing a nucleic acid molecule or protein of the invention in a plant can enhance plant defense against predators (e.g., herbivores) and pathogens (e.g., fungal, bacterial, and viruses), as well as enhance desirable qualities of color, durability and integrity of woody plant tissues (such as heartwood and sapwood). Again by way of example, expression in plants of a nucleic acid molecule of the invention in antisense orientation can inhibit the production of aryl propenal double bond reductase and therefore of one or more plant lignans.
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, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley and Sons, New York (1999), for definitions and terms of the art.
The term xe2x80x9cisolatedxe2x80x9d used with respect to a nucleic acid molecule or polypeptide of the invention means a molecule that is substantially free from cellular components that are associated with the nucleic acid molecule or polypeptide as it is found in nature. As used in this context, the term xe2x80x9csubstantially free from cellular componentsxe2x80x9d means that the nucleic acid molecule or polypeptide is purified to a purity level of greater than 80% (such as greater than 90%, greater than 95%, or greater than 99%). Moreover, the terms xe2x80x9cisolated nucleic acid moleculexe2x80x9d and xe2x80x9cisolated aryl propenal double bond reductasexe2x80x9d include nucleic acid molecules and polypeptides which do not naturally occur, and have been produced by synthetic means. An isolated nucleic acid molecule or polypeptide generally resolves as a single, predominant, band by gel electrophoresis, and yields a nucleic acid or amino acid sequence profile consistent with the presence of a predominant nucleic acid molecule or polypeptide.
The term xe2x80x9caryl propenal double bond reductasexe2x80x9d refers to an enzyme that catalyzes the reduction of the propenal side chain of one or more phenylpropanoid molecule.
The term xe2x80x9cpercent identityxe2x80x9d or xe2x80x9cpercent identicalxe2x80x9d when used in connection with the aryl propenal double bond reductases of the present invention, is defined as the percentage of amino acid residues in a candidate polypeptide sequence that are identical with a subject polypeptide molecule sequence (such as the polypeptide amino acid sequence of SEQ ID NO:2), after aligning the candidate and subject sequences to achieve the maximum percent identity. When making the comparison, no gaps are introduced into the candidate polypeptide sequence in order to achieve the best alignment.
Amino acid sequence identity can be determined in the following manner. The subject polypeptide 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 utilizes the SEG program.
The term xe2x80x9cvectorxe2x80x9d 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 cDNA molecule. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide. 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 xe2x80x9cvectorxe2x80x9d includes the T-DNA of a Ti plasmid.
The term xe2x80x9cexpression vectorxe2x80x9d refers to a vector that includes the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide.
The term xe2x80x9cgymnospermxe2x80x9d refers to a class of plants that produce seeds that are not enclosed in an ovary.
As used herein, the abbreviation xe2x80x9cSSCxe2x80x9d refers to a buffer used in nucleic acid hybridization solutions. One liter of the 20xc3x97 (twenty times concentrate) stock SSC buffer solution (pH 7.0) contains 175.3 g sodium chloride and 88.2 g sodium citrate.
Denhardt""s reagent is utilized in nucleic acid hybridization solutions. 500 ml of 50xc3x97 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 500 ml.
As described in Example 2, the inventors isolated aryl propenal double bond reductase protein from cultured Pinus taeda cells. The purified protein was cleaved with trypsin to yield three peptide sequences ELILVAYANEGPVTDSHLNIR (SEQ ID NO:3), DGSSGDVAVQNLWISVDPYLR (SEQ ID NO:4), and ESDDGLYLPSFPLNQAIR (SEQ ID NO:5) which were used to screen the GenBank database. An expressed sequence tag (EST) (GenBank accession No. AA556927) was identified that encoded all three peptides (SEQ ID NOS:3, 4, 5).
As set forth in Example 3 herein, PCR amplification was utilized to obtain a longer cDNA that corresponded to the partial-length cDNA set forth in GenBank accession No. AA556927 and that encoded a complete aryl propenal double bond reductase protein. The resulting cDNA molecule is set forth in SEQ ID NO:1, and encodes the aryl propenal double bond reductase protein having the amino acid sequence set forth in SEQ ID NO:2. As set forth in Example 3, the aryl propenal double bond reductase cDNA (SEQ ID NO:1) was expressed in E. coli and shown to be enzymatically active.
Thus, in one aspect, the present invention provides isolated nucleic acid molecules that each: (a) encode an aryl propenal double bond reductase; and (b) hybridize to a nucleic acid molecule consisting of the complement of the nucleic acid sequence set forth in SEQ ID NO:1 under conditions of 1xc3x97SSC at 55xc2x0 C. for one hour. Hybridization can be conducted, for example, by utilizing the technique of hybridizing radiolabelled nucleic acid probes to nucleic acids immobilized on nitrocellulose filters or nylon membranes as set forth at pages 9.52 to 9.55 of Molecular Cloning, A Laboratory Manual (2nd edition), J. Sambrook, E. F. Fritsch and T. Maniatis eds, the cited pages of which are incorporated herein by reference. An exemplary hybridization protocol is set forth in Example 4 herein. Some nucleic acid molecules of this aspect of the invention hybridize to the complement of the nucleic acid sequence set forth in SEQ ID NO:1 under conditions of 1xc3x97SSC at 65xc2x0 C.
The present invention also provides isolated nucleic acid molecules that each encode an aryl propenal double bond reductase that is at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
The nucleic acid molecules of the invention that encode an aryl propenal double bond reductase can be isolated by using a variety of cloning techniques known to those of ordinary skill in the art: For example, all, or portions of, the cDNA molecule having the sequence set forth in SEQ ID NO:1 can be used as a hybridization probe to screen a plant genomic or cDNA 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 or cDNA library. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65xc2x0 C. in 5.0xc3x97SSC, 0.5% sodium dodecyl sulfate, 1xc3x97 Denhardt""s solution; washing (three washes of twenty minutes each at 55xc2x0 C.) in 1.0xc3x97SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5xc3x97SSC, 1% (w/v) sodium dodecyl sulfate, at 60xc2x0 C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1xc3x97SSC, 1% (w/v) sodium dodecyl sulfate, at 60xc2x0 C.
Again, by way of example, nucleic acid molecules of the invention that encode an aryl propenal double bond reductase can be isolated by the polymerase chain reaction (PCR) described in The Polymerase Chain Reaction (K. B. Mullis et al., eds. 1994), incorporated herein by reference. Gobinda et al. (PCR Methods Applic. 2:318-22 (1993)), incorporated herein by reference, disclose xe2x80x9crestriction-site PCRxe2x80x9d 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 are 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), incorporated herein by reference). 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.
Typically, the nucleic acid sequence of a primer useful to amplify nucleic acid molecules of the invention by PCR is based on a conserved region of amino acid sequence of the aryl propenal double bond reductase polypeptides of the invention (such as the aryl propenal double bond reductase polypeptide having the amino acid sequence set forth in SEQ ID NO:2).
In another aspect, the present invention provides vectors that comprise a nucleic acid molecule of the invention. Vectors of the invention include the elements necessary for replication in a target host cell, such as a prokaryotic cell, or a eukaryotic cell. Vectors that are functional in plants are preferably binary plasmids derived from Agrobacterium plasmids. Such vectors are capable of transforming plant cells. Briefly, these vectors typically contain left and right border sequences that are required for integration into the host (plant) chromosome. Typically, between these border sequences is the nucleic acid molecule (such as a cDNA) to be expressed under control of a promoter. In some embodiments, a selectable marker and a reporter gene are also included. The vector also may contain a bacterial origin of replication.
In another aspect, the present invention provides host cells comprising a vector of the invention. Host cells can be prokaryotic or eukaryotic, such as plant cells. For example, one or more vectors of the invention can be introduced into yeast host cells using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA, 75:1929 [1978]). Additional yeast transformation protocols are set forth in Gietz et al., N.A.R., 20(17):1425(1992); Reeves et al., FEMS, 99(2-3):193-197, (1992), both of which publications are incorporated herein by reference.
The 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, (1) direct DNA uptake, such as particle bombardment or electroporation (see, Klein et al., Nature 327:70-73 (1987); U.S. Pat. No. 4,945,050), and (2) Agrobacterium-mediated transformation (see, e.g., U.S. Pat. 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. The skilled artisan will recognize that different independent insertion events may result in different levels and patterns of gene expression (Jones et al., EMBO J. 4:2411-2418 (1985); De Almeida et al., MGG 218:78-86 (1989)), and thus that multiple events may have to be screened in order to obtain lines displaying the desired expression level and pattern.
Transgenic plants can be obtained, for example, by transferring vectors that include a selectable marker gene, e.g., the kan 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). Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens. 
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 cloned DNA and vectors 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, Fla. (1993), incorporated by reference herein). Representative examples include electroporation-facilitated DNA uptake by protoplasts in which an electrical pulse transiently permeabilizes cell membranes, permitting the uptake of a variety of biological molecules, including recombinant DNA (see, e.g., Rhodes et al., Science, 240:204-207 (1988)); treatment of protoplasts with polyethylene glycol (see, e.g., Lyznik et al., Plant Molecular Biology, 13:151-161 (1989)); and bombardment of cells with DNA-laden microprojectiles which are propelled by explosive force or compressed gas to penetrate the cell wall (see, e.g., Klein et al., Plant Physiol. 91:440-444 (1989) and Boynton et al., Science, 240(4858):1534-1538 (1988)). A method that has been applied to Rye plants (Secale cereale) is to directly inject plasmid DNA, including a selectable marker gene, into developing floral tillers (de la Pena et al., Nature 325:274-276 (1987)). 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. Pat. No. 5,635,381); and insertion into a target sequence by homologous recombination (see, U.S. Pat. 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); 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. Pat. Nos. 5,994,629, 5,767,378, and 5,599,670, describe the use of a beta-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 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. Pat. 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. Pat. 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. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073 and 6,020,539); peppermint (X. Niu et al., Plant Cell Rep. 17:165-171 (1998)); citrus plants (Pena, L. et al., Plant Sci. 104:183-191 (1995)); caraway (F. A. Krens, et al., Plant Cell Rep., 17:39-43 (1997)); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); and cereals (U.S. Pat. No. 6,074,877).
Cultures of mammalian host cells and other host cells that do not have rigid cell membrane barriers are usually transformed 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 DNA 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, incorporated herein by reference.
Prokaryotic host cells are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. 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 isolated aryl propenal double bond reductase proteins that are at least 50% identical (such as at least 70% identical, or at least 80% identical, or at least 90% identical) to the aryl propenal double bond reductase consisting of the amino acid sequence set forth in SEQ ID NO:2.
Aryl propenal double bond reductases of the invention can be prepared, for example, by expressing a nucleic acid molecule encoding an aryl propenal double bond reductase in a suitable host cell, such as E. coli. By way of representative example, a nucleic acid molecule (such as a cDNA molecule) encoding an aryl propenal double bond reductases is cloned into a plasmid vector, such as a Bluescript plasmid (available from Stratagene, Inc., La Jolla, Calif.). The recombinant vector is then introduced into an E. coli strain (such as E. coli XL1-Blue, also available from Stratagene, Inc.) and the aryl propenal double bond reductase is expressed in E. coli and then purified. For example, E. coli XL 1-Blue harboring a Bluescript vector including a cDNA molecule encoding an aryl propenal double bond reductase is grown overnight at 37xc2x0 C. in LB medium containing 100 xcexcg ampicillin/ml. A 50 xcexcl aliquot of the overnight culture is used to inoculate 5 ml of fresh LB medium containing ampicillin, and the culture grown at 37xc2x0 C. with vigorous agitation to A600=0.5 before induction with 1 mM IPTG. After an additional two hours of growth, the suspension is centrifuged (1000xc3x97 g, 15 min, 4xc2x0 C.), the media removed, and the pelleted cells resuspended in 1 ml of cold buffer that preferably contains 1 mM EDTA and one or more proteinase inhibitors. The cells can be disrupted by sonication with a microprobe. The chilled sonicate is cleared by centrifugation and the expressed, recombinant, aryl propenal double bond reductase purified from the supernatant by art-recognized protein purification techniques, such as those described herein.
Again by way of example, aryl propenal double bond reductases of the invention can be prepared by expressing a nucleic acid molecule encoding an aryl propenal double bond reductase in insect cells using the recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV; M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures [1986]; Luckow et al., Bio-technology, 6:47-55 [1987]). Infection of insect cells (such as cells of the species Spodoptera frugiperda) with the recombinant baculoviruses allows for the production of large amounts of aryl propenal double bond reductase proteins. In addition, the baculovirus system has other important advantages for the production of recombinant aryl propenal double bond reductase proteins. For example, baculoviruses do not infect humans and can therefore be safely handled in large quantities. In the baculovirus system, a DNA construct is prepared including a DNA segment encoding aryl propenal double bond reductase and a vector. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper cross-over during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. The vector is constructed so that (i) the DNA segment is placed adjacent (or operably linked or xe2x80x9cdownstreamxe2x80x9d or xe2x80x9cunder the control ofxe2x80x9d) to the polyhedron gene promoter and (ii) the promoter/aryl propenal double bond reductase combination is flanked on both sides by 200-300 base pairs of baculovirus DNA (the flanking sequences).
To produce the aryl propenal double bond reductase DNA construct, a cDNA clone encoding the full length aryl propenal double bond reductase is obtained using methods such as those described herein. The DNA construct is contacted in a host cell with baculovirus DNA of an appropriate baculovirus (that is, of the same species of baculovirus as the promoter encoded in the construct) under conditions such that recombination is effected. The resulting recombinant baculoviruses encode the full aryl propenal double bond reductase. For example, an insect host cell can be cotransfected or transfected separately with the DNA construct and a functional baculovirus. Resulting recombinant baculoviruses can then be isolated and used to infect cells to effect production of the aryl propenal double bond reductase. Host insect cells include, for example, Spodoptera frugiperda cells, that are capable of producing a baculovirus-expressed aryl propenal double bond reductase. Insect host cells infected with a recombinant baculovirus of the present invention are then cultured under conditions allowing expression of the baculovirus-encoded aryl propenal double bond reductase.
Again by way of example, aryl propenal double bond reductases of the invention can be prepared by expressing a nucleic acid molecule encoding an aryl propenal double bond reductase in yeasts. The baker""s yeast Saccharomyces cerevisiae, is a commonly used yeast, although several other strains are available. The plasmid YRp7 (Stinchcomb et al., Nature, 282:39 [1979]; Kingsman et al., Gene 7:141 [1979]; Tschemper et al., Gene, 10:157 [1980]) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trp1 gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics, 85:12 [1977]). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Yeast host cells are generally transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA, 75:1929 [1978]). Additional yeast transformation protocols are set forth in Gietz et al., N.A.R., 20(17):1425(1992); Reeves et al., FEMS, 99(2-3):193-197, (1992), both of which publications are incorporated herein by reference.
Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 [1980]) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 [1968]; Holland et al., Biochemistry, 17:4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3xe2x80x2 of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.
Prokaryotes may also be used as host cells for expression of aryl propenal double bond reductases of the invention. Suitable prokaryotic host cells include E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110 (ATCC No. 27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. 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).
Plants may also be used as host cells for expression of aryl propenal double bond reductases of the invention. Typically, a vector including a nucleic acid molecule encoding an aryl propenal double bond reductase is introduced into a plant cell by any art-recognized means, such as are set forth herein. One or more plants are regenerated from the cells and propagated. The aryl propenal double bond reductase is expressed within the plants (or within plant cell cultures containing the vector) and can be purified therefrom. In some embodiments, the nucleic acid molecule encoding an aryl propenal double bond reductase is under the control of an inducible promoter within the vector. Thus, the nucleic acid molecule will not be transcribed except in response to the specific stimulus.
Representative examples of art-recognized techniques for purifying, or partially purifying, aryl propenal double bond reductase from biological material, such as from prokaryotic cells or eukaryotic cells that express aryl propenal double bond reductase, are: exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography.
Hydrophobic interaction chromatography and reversed-phase chromatography are two separation methods based on the interactions between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group present on the chromatography matrix. In hydrophobic interaction chromatography the matrix is hydrophilic and is substituted with short-chain phenyl or octyl nonpolar groups. The mobile phase is usually an aqueous salt solution. In reversed phase chromatography the matrix is silica that has been substituted with longer n-alkyl chains, usually C8 (octylsilyl) or C18 (octadecylsilyl). The matrix is less polar than the mobile phase. The mobile phase is usually a mixture of water and a less polar organic modifier.
Separations on hydrophobic interaction chromatography matrices are usually done in aqueous salt solutions, which generally are nondenaturing conditions. Samples are loaded onto the matrix in a high-salt buffer and elution is by a descending salt gradient. Separations on reversed-phase media are usually done in mixtures of aqueous and organic solvents, which are often denaturing conditions. In the case of polypeptide and/or peptide purification, hydrophobic interaction chromatography depends on surface hydrophobic groups and is carried out under conditions which maintain the integrity of the polypeptide molecule. Reversed-phase chromatography depends on the native hydrophobicity of the polypeptide and is carried out under conditions which expose nearly all hydrophobic groups to the matrix, i.e., denaturing conditions.
Ion-exchange chromatography is designed specifically for the separation of ionic or ionizable compounds. The stationary phase (column matrix material) carries ionizable functional groups, fixed by chemical bonding to the stationary phase. These fixed charges carry a counterion of opposite sign. This counterion is not fixed and can be displaced. Ion-exchange chromatography is named on the basis of the sign of the displaceable charges. Thus, in anion ion-exchange chromatography the fixed charges are positive and in cation ion-exchange chromatography the fixed charges are negative.
Retention of a molecule on an ion-exchange chromatography column involves an electrostatic interaction between the fixed charges and those of the molecule, binding involves replacement of the nonfixed ions by the molecule. Elution, in turn, involves displacement of the molecule from the fixed charges by a new counterion with a greater affinity for the fixed charges than the molecule, and which then becomes the new, nonfixed ion.
The ability of counterions (salts) to displace molecules bound to fixed charges is a function of the difference in affinities between the fixed charges and the nonfixed charges of both the molecule and the salt. Affinities in turn are affected by several variables, including the magnitude of the net charge of the molecule and the concentration and type of salt used for displacement.
Solid-phase packings used in ion-exchange chromatography include cellulose, dextrans, agarose, and polystyrene. The exchange groups used include DEAE (diethylaminoethyl), a weak base, that will have a net positive charge when ionized and will therefore bind and exchange anions; and CM (carboxymethyl), a weak acid, with a negative charge when ionized that will bind and exchange cations. Another form of weak anion exchanger contains the PEI (polyethyleneimine) functional group. This material, most usually found on thin layer sheets, is useful for binding polypeptides at pH values above their pI. The polystyrene matrix can be obtained with quaternary ammonium functional groups for strong base anion exchange or with sulfonic acid functional groups for strong acid cation exchange. Intermediate and weak ion-exchange materials are also available. Ion-exchange chromatography need not be performed using a column, and can be performed as batch ion-exchange chromatography with the slurry of the stationary phase in a vessel such as a beaker.
Gel filtration is performed using porous beads as the chromatographic support. A column constructed from such beads will have two measurable liquid volumes, the external volume, consisting of the liquid between the beads, and the internal volume, consisting of the liquid within the pores of the beads. Large molecules will equilibrate only with the external volume while small molecules will equilibrate with both the external and internal volumes. A mixture of molecules (such as proteins) is applied in a discrete volume or zone at the top of a gel filtration column and allowed to percolate through the column. The large molecules are excluded from the internal volume and therefore emerge first from the column while the smaller molecules, which can access the internal volume, emerge later. The volume of a conventional matrix used for protein purification is typically 30 to 100 times the volume of the sample to be fractionated. The absorbance of the column effluent can be continuously monitored at a desired wavelength using a flow monitor.
A technique that is often applied to the purification of polypeptides is High Performance Liquid Chromatography (HPLC). HPLC is an advancement in both the operational theory and fabrication of traditional chromatographic systems. HPLC systems for the separation of biological macromolecules vary from the traditional column chromatographic systems in three ways; (1) the column packing materials are of much greater mechanical strength, (2) the particle size of the column packing materials has been decreased 5- to 10-fold to enhance adsorption-desorption kinetics and diminish bandspreading, and (3) the columns are operated at 10-60 times higher mobile-phase velocity. Thus, by way of non-limiting example, HPLC can utilize exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and immobilized metal affinity chromatography. 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).
In yet another aspect, the present invention provides methods of enhancing the level of aryl propenal double bond reductase in a plant cell, the methods comprising the steps of: (a) introducing into a plant an expression vector comprising a nucleic acid molecule that: (1) encodes an aryl propenal double bond reductase; (2) hybridizes to the complement of SEQ ID NO:1 under conditions of 1xc3x97SSC, 55xc2x0 C. for one hour; and (b) expressing the aryl propenal double bond reductase within the plant. In some embodiment of the methods of this aspect of the invention the nucleic acid molecule hybridizes to the complement of SEQ ID NO:1 under conditions of 1xc3x97SSC at 65xc2x0 C. for one hour.
In a further aspect, the present invention provides methods for inhibiting the expression of aryl propenal double bond reductase in a plant, the methods comprising the steps of: (a) introducing into a plant an expression vector that comprises a nucleic acid molecule that is in antisense orientation relative to a promoter, the nucleic acid molecule hybridizing to the nucleic acid molecule consisting of the nucleic acid sequence set forth in SEQ ID NO:1 under hybridization conditions of 1xc3x97SSC, 55xc2x0 C. for one hour; and (b) transcriptionally expressing the nucleic acid molecule in the plant. In some embodiments of the methods of this aspect of the invention, the nucleic acid molecule hydribizes to the nucleic acid molecule of SEQ ID NO:1 under conditions of 1xc3x97SSC at 65xc2x0 C. for one hour.
The successful implementation of antisense RNA in plants to inhibit the expression of specific genes has previously been demonstrated (Van der Krol et al., 1990 Plant Mol. Biol. 14:457; Visser et al., 1991, Mol. Gen. Genet. 225:289; Hamilton et al., 1990, Nature 346:284; Stockhaus et al., 1990, EMBO J. 9:3013; Hudson et al., 1992, Plant Physiol. 98:294; U.S. Pat. Nos. 4,801,340, 5,773,692, 5,723,761, and 5,959,180). For example, polygalacturonase is responsible for fruit softening during the latter stages of ripening in tomato (Hiatt et al., 1989 in Genetic Engineering, Setlow, ed. p. 49; Sheehy et al., 1988, Proc. Natl. Acad. Sci. USA 85:8805; Smith et al., 1988, Nature 334:724). The integration of antisense constructs into the genome, under the control of the CaMV 35S promoter, has inhibited this softening. Examination of the polygalacturonase mRNA levels showed a 90% suppression of gene expression.
An antisense gene is a DNA sequence produced when a sense gene is inverted relative to its normal presentation for transcription. The xe2x80x9csensexe2x80x9d gene refers to the gene which is being targeted for control using the antisense technology, in its normal orientation. An antisense gene may be constructed in a number of different ways provided that it is capable of interfering with the expression of a sense gene. Preferably, the antisense gene is constructed by inverting the coding region of the sense gene relative to its normal presentation for transcription to allow the transcription of its complement, hence the RNAs encoded by the antisense and sense gene are complementary. It is understood that a portion of the antisense gene incorporated into an antisense construct, of the present invention, may be sufficient to effectively interfere with the expression of a sense gene and thus the term xe2x80x9cantisense genexe2x80x9d used herein encompasses any functional portion of the full length antisense gene. By the term xe2x80x9cfunctionalxe2x80x9d it is meant to include a portion of the antisense gene which is effective in interfering with the expression of the sense gene.
The antisense gene need not be perfectly identical to the target gene to inhibit expression. Generally, higher homology can be used to compensate for the use of a shorter antisense gene sequence. Furthermore, the introduced antisense gene sequence need not have the same intron or exon pattern as the target gene, and homology of non-coding segments may be equally effective. Normally, a sequence of between about 25 or 40 nucleotides and about the full length of the target gene sequence should be used, though a sequence of at least about 100 nucleotides is preferred, a sequence of at least about 200 nucleotides is more preferred, and a sequence of at least about 500 nucleotides is especially preferred. The construct is then transformed into plants and the antisense strand of RNA is produced.
Representative plant expression vectors are set forth in F. Guerineau and P. Mullineaux, Plant Transformation and Expression Vectors, in Plant Molecular Biology LABFax, eds B. D. Hames and D. Rickwood, ps. 121-147, BIOS Scientific Publishers Limited (1993). Representative methods for introducing an expression vector into a plant are set forth supra, and include Agrobacterium-mediated plant transformation.
Any plant species can be treated in accordance with the methods of the invention, in particular gymnosperm plant species, such as plants of the genus Picea. 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.
All literature citations herein are incorporated by reference.
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