Classifications

• • 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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • 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)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
• a selectable marker is used to recover transformed cells.
• Traditional selection schemes expose all cells to a phytotoxic agent and rely on the introduction of a resistance gene to recover transformants. Unfortunately, the presence of dying cells may reduce the efficiency of stable transformation. It would therefore be useful to provide a positive selection system for recovering transformants.
• the present invention relates to a HAP3-type CCAAT-box binding transcriptional activator polynucleotides and polypeptides, and in particular, the leafy cotyledon 1 transcriptional activator (LEC1) polynucleotides and polypeptides.
• the present invention relates to expression cassettes optionally linked in antisense orientation, host cells transfected with at least one expression cassette, and transgenic plants and seeds comprising the expression cassettes. Further aspects of the invention include methods of using the polynucleotides and polypeptides.
• the present invention relates to a method of modulating expression of the polynucleotides encoding the polypeptides of the present invention in a plant. Expression of the polynucleotides encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
• Figure 1 depicts the comparison of various sequences and the alignment of the conserved regions.
• isolated refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
• nucleic acid means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not alter the expression of a polypeptide encoded by the polynucleotide.
• LEC1 nucleic acid means a nucleic acid or polynucleotide that codes for a LEC1 polypeptide.
• polypeptide means proteins, protein fragments, modified proteins, amino acid sequences and synthetic amino acid sequences. The polypeptide can be glycosylated or not.
• LEC1 polypeptide means a HAP3 family member, CCAAT-box binding transcriptional activator polypeptide that regulates gene expression during embryo development, and that contains the conserved sequence set out in SEQ ID NO: 23.
• plant includes plants and plant parts including but not limited to plant cells, plant tissue such as leaves, stems, roots, flowers, and seeds.
• promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
• fragment is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby.
• Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native nucleic acid.
• fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity.
• fragments of a nucleotide sequence are generally greater than 20, 30, 50, 100, 150, 200 or 300 nucleotides and up to the entire nucleotide sequence encoding the proteins of the invention.
• the probes are less than 1000 nucleotides and preferably less than 500 nucleotides.
• Fragments of the invention include antisense sequences used to decrease expression of the inventive polynucleotides. Such antisense fragments may vary in length ranging from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to and including the entire coding sequence.
• “functional equivalent” as applied to a polynucleotide or a protein is intended a polynucleotide or a protein of sufficient length to modulate the level of LEC1 protein activity in a plant cell.
• a polynucleotide functional equivalent can be in sense or antisense orientation.
• nucleic acid sequence variants of the invention will have at least 60%, 65%, or 70%, preferably 75%, 80% or 90%, more preferably at least 95% and most preferably at least 98% sequence identity to the native nucleotide sequence, wherein the % sequence identity is based on the entire sequence and is determined by GAP analysis using Gap Weight of 50 and Length Weight of 3.
• polypeptide sequence variants of the invention will have at least about 50%, 55%, 60%, 65%, 70%, 75% or 80%, preferably at least about 85% or 90%, and more preferably at least about 95% sequence identity to the native protein, wherein the % sequence identity is based on the entire sequence and is determined by GAP analysis using Gap Weight of 12 and Length Weight of 4.
• a “responsive plant cell” or “responsive host cell” refers to a cell that exhibits a positive response to the introduction of LEC1 polypeptide or LEC1 polynucleotide compared to a cell that has not been introduced with LEC1 polypeptide or LEC1 polynucleotide.
• the response can be to enhance tissue culture response, induce somatic embryogenesis, induce apomlxis, increase transformation efficiency or increase recovery of regenerated plants.
• a "recalcitrant plant cell” is a responsive plant cell that generally does not exhibit a positive response such as tissue culture response, transformation efficiency or recovery of regenerated plants.
• the present invention relates to a HAP3-type CCAAT-box binding transcriptional activators, and in particular, the leafy cotyledon 1 transcriptional activator (LEC1). Expression of the LEC1 polynucleotide initiates formation of embryo-like structures and improves growth and recovery of transformants.
• LEC1 leafy cotyledon 1 transcriptional activator
• apomixis is used to describe asexual reproduction that replaces or substitutes sexual methods of reproduction. When apomixis occurs, embryos are produced from maternal tissue and use only the maternal genome.
• the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of:
• a polynucleotide comprising at least 20 contiguous bases of SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, or 21;
• a polynucleotide encoding a plant HAP3-type ccaat-box transcriptional activator with the conserved motif of SEQ ID NO: 23, wherein the polynucleotide is from a plant other than Arabidopsis;
• a polynucleotide comprising at least 25 nucleotides in length which hybridizes under high stringency conditions to a polynucleotide having the sequence set forth in SEQ ID NO: 1 , 7, 9, 11 , 13, 15, 17, 19, or 21 ;
• apomixis maternal tissues such as the nucellus or inner integument "bud off' producing somatic embryos. These embryos then develop normally into seed. Since meiosis and fertilization are circumvented, the plants developing from such seed are genetically identical to the maternal plant. Expression of the leafy cotyledon 1 gene in the nucellus integument, or cell specific expression in the megaspore mother cell would trigger embryo formation from maternal tissues.
• Producing a seed identical to the parent has many advantages. For example high yielding hybrids could be used in seed production to multiply identical copies of high yielding hybrid seed. This would greatly reduce seed cost as well as increase the number of genotypes which are commercially available. Genes can be evaluated directly in commercial hybrids since the progeny would not segregate. This would save years of back crossing. Apomixis would also provide a method of containment of transgenes when coupled with male sterility. The construction of male sterile autonomous agamospermy would prevent genetically engineered traits from hybridizing with weedy relatives. Gene stacking would be relatively easy with apomixis. Hybrids could be successively re-transformed with various new traits and propagated via apomixis. The traits would not need to be linked since apomixis avoids the problems associated with segregation.
• Apomixis can provide a reduction in gene silencing. Gene silencing is frequently seen following meiotic divisions. Since meiotic divisions never occur, it may be possible to eliminate or reduce the frequency of gene silencing. Apomixis can also be used stabilize desirable phenotypes with complex traits such as hybrid vigor. Such traits could easily be maintained and multiplied indefinitely via apomixis.
• the Cauliflower Mosaic Virus 35S promoter has been used to overexpress
• ectopic expression of the LEC1 gene under the appropriate control elements can be used to stimulate embryo formation in tissues/genotypes normally not amenable to culture.
• ectopic expression in genotypes amenable to culture can increase the number of embryo precursor cells (or increase the number that develop into embryos) leading to an increase in transformation frequency.
• Transient expression using RNA or protein may be sufficient to initiate the cascade of events leading to embryo formation. This would be valuable in such target tissues as maize scutella, immature leaf bases, immature tassels, etc.
• the LEC1 gene could be used as a positive selectable marker, i.e.
• Arabidopsis LEC1 polypeptide is homologous to the HAP3 subunit of the "CCAAT-box binding factor” class of eukaryotic transcriptional activators (Lotan et al, 1998, Cell 93:1195-1205). This class of proteins, which consist of Hap2/3/4 and 5, form a heteroligomeric transcriptional complex, that appears to activate specific gene sets in eukaryotes.
• HAP3 polypeptides can be recognized by a high degree of sequence identity to other HAP3 homologs in the "B domain" of the protein.
• the B domain for the Arabidopsis LEC1 shares between 55% and 63% identity (75-85% similarity) to other members of the HAP3 family, including maize (HAP3), chicken, lamprey, Xenopus, human, mouse, Emericella nidulens, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Kluuyveromyces lactis (Lotan et al, 1998).
• HAP3 maize
• Xenopus human
• mouse Emericella nidulens
• Schizosaccharomyces pombe Schizosaccharomyces pombe
• Saccharomyces cerevisiae Saccharomyces cerevisiae
• Kluuyveromyces lactis Kluuyveromyces lactis
• LEC1 expression is necessary for proper embryo maturation in the latter stages of embryo development, and LEC1 transgene expression thus may also promote these processes.
• the combined effect of these impacts on somatic embryogenesis is not only to stimulate growth of transformed cells, but also to insure that transformed somatic embryos develop in a normal, viable fashion (increasing the capacity of transformed somatic embryos to germinate vigorously).
• Continued ectopic overexpression beyond embryo maturation may negatively impact germination and vegetative plant growth (which may necessitate down- regulation of the LEC1 transgene during these stages of development.
• LEC1 gene will stimulate growth in cells with the potential to initiate or maintain embryogenic growth.
• Cells in established meristems or meristem-derive cell lineages may be less prone to undergo the transition to embryos.
• transformation methods that target certain reproductive tissues (or cells) such as vacuum-infiltration of Agrobacterium into Arabidopsis may have detrimental effects on recovery of transformants (triggering genes associated with embryogenesis may disrupt the proper functioning of these cells).
• polypeptides encoded by the present plant LEC1 genes can be distinguished from non-LEC HAP3 proteins by using the diagnostic motif shown in SEQ ID NO: 23.
• the isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques, or combinations thereof.
• the polynucleotides of the present invention will be cloned, amplified, or otherwise constructed from a monocot or dicot.
• the monocot is corn, sorghum, barley, wheat, millet, or rice.
• Preferred dicots include soybeans, sunflower, canola, alfalfa, potato, or cassava.
• Functional fragments included in the invention can be obtained using primers which selectively hybridize under stringent conditions. Primers are generally at least 12 bases in length and can be as high as 200 bases, but will generally be from 15 to 75, preferably from 15 to 50. Functional fragments can be identified using a variety of techniques such as restriction analysis, Southern analysis, primer extension analysis, and DNA sequence analysis.
• the present invention includes a plurality of polynucleotides that encode for the identical amino acid sequence.
• the degeneracy of the genetic code allows for such "silent variations" which can be used, for example, to selectively hybridize and detect allelic variants of polynucleotides of the present invention.
• the present invention includes isolated nucleic acids comprising allelic variants.
• allele refers to a related nucleic acid of the same gene.
• Variants of nucleic acids included in the invention can be obtained, for example, by oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like. See, for example, Ausubel, pages 8.0.3 - 8.5.9. Also, see generally, McPherson (ed.), DIRECTED MUTAGENESIS: A Practical Approach, (IRL Press, 1991 ). Thus, the present invention also encompasses DNA molecules comprising nucleotide sequences that have substantial sequence similarity with the inventive sequences.
• Variants included in the invention may contain individual substitutions, deletions or additions to the nucleic acid or polypeptide sequences which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
• nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host.
• the present invention also includes "shufflents" produced by sequence shuffling of the inventive polynucleotides to obtain a desired characteristic. Sequence shuffling is described in PCT publication No. 96/19256. See also, Zhang, J. H., et ai, Proc. Natl. Acad. Sci. USA 94:4504-4509 (1997).
• the present invention also includes the use of 5' and/or 3' UTR regions for modulation of translation of heterologous coding sequences.
• Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res. 13:7375 (1985)).
• Negative elements include stable intramolecular 5' UTR stem-loop structures (Muesing et ai, Cell 48:691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol.
• polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as "Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
• inventive nucleic acids can be optimized for enhanced expression in plants of interest.
• the present invention provides subsequences comprising isolated nucleic acids containing at least 16 contiguous bases of the inventive sequences.
• the isolated nucleic acid includes those comprising at least 16, 20, 25, 30, 40, 50, 60, 75 or 100 contiguous nucleotides of the inventive sequences.
• Subsequences of the isolated nucleic acid can be used to modulate or detect gene expression by introducing into the subsequences compounds which bind, intercalate, cleave and/or crosslink to nucleic acids.
• the nucleic acids of the invention may conveniently comprise a multi- cloning site comprising one or more endonuclease restriction sites inserted into the nucleic acid to aid in isolation of the polynucleotide.
• translatable sequences may be inserted to aid in the isolation of the translated polynucleotide of the present invention.
• a hexa-histidine marker sequence provides a convenient means to purify the proteins of the present invention.
• a polynucleotide of the present invention can be attached to a vector, adapter, promoter, transit peptide or linker for cloning and/or expression of a polynucleotide of the present invention.
• Additional sequences may be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
• Use of cloning vectors, expression vectors, adapters, and linkers is well known and extensively described in the art. For a description of such nucleic acids see, for example, Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, CA); and, Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, IL).
• RNA, cDNA, genomic DNA, or a hybrid thereof can be obtained from plant biological sources using any number of cloning methodologies known to those of skill in the art.
• oligonucleotide probes which selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
• RNA and mRNA isolation protocols are described in Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et ai., Eds., Greene Publishing and Wiley-lnterscience, New York (1995).
• Total RNA and mRNA isolation kits are commercially available from vendors such as Stratagene (La Jolla, CA), Clonetech (Palo Alto, CA), Pharmacia (Piscataway, NJ), and 5'-3' (Paoli, PA). See also, U.S. Patent Nos. 5,614,391 ; and, 5,459,253.
• cDNA synthesis protocols are well known to the skilled artisan and are described in such standard references as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, et ai., Eds., Greene Publishing and Wiley-lnterscience, New York (1995).
• cDNA synthesis kits are available from a variety of commercial vendors such as Stratagene or Pharmacia.
• Subtracted cDNA libraries are another means to increase the proportion of less abundant cDNA species. See, Foote et al. in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique 3(2):58-63 (1991); Sive and St. John, Nucl. Acids Res. 16(22): 10937 (1988); Current Protocols in Molecular Biology, Ausubel, et ai., Eds., Greene Publishing and Wiley-lnterscience, New York (1995); and, Swaroop et al., Nucl. Acids Res. 19(8): 1954 (1991 ).
• cDNA subtraction kits are commercially available. See, e.g., PCR-Select (Clontech).
• genomic libraries large segments of genomic DNA are generated by random fragmentation. Examples of appropriate molecular biological techniques and instructions are found in Sambrook, et ai., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits for construction of genomic libraries are also commercially available.
• the cDNA or genomic library can be screened using a probe based upon the sequence of a nucleic acid of the present invention such as those disclosed herein. Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous polynucleotides in the same or different plant species.
• Probes may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous polynucleotides in the same or different plant species.
• degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent.
• the degree of stringency can be controlled by temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
• stringent hybridization conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides).
• Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For purposes of defining the invention the following conditions are provided.
• Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1 % SDS at 37°C, and a wash in 0.5X to 1X SSC at 55°C.
• Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1 % SDS at 37°C, and a wash in 0.1 X SSC at 60°C.
• the time of hybridization is from 4 to 16 hours.
• An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). Often, cDNA libraries will be normalized to increase the representation of relatively rare cDNAs.
• the nucleic acids of the invention can be amplified from nucleic acid samples using amplification techniques. For instance, polymerase chain reaction (PCR) technology can be used to amplify the sequences of polynucleotides of the present invention and related polynucleotides directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
• PCR polymerase chain reaction
• nucleic acids can be amplified from a plant nucleic acid library.
• the nucleic acid library may be a cDNA library, a genomic library, or a library generally constructed from nuclear transcripts at any stage of intron processing. Libraries can be made from a variety of plant tissues. Good results have been obtained using mitotically active tissues such as shoot meristems, shoot meristem cultures, embryos, callus and suspension cultures, immature ears and tassels, and young seedlings.
• the cDNAs of the present invention were obtained from immature zygotic embryo and regenerating callus libraries.
• sequences of the invention can be used to isolate corresponding sequences in other organisms, particularly other plants, more particularly, other monocots.
• methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial sequence similarity to the sequences of the invention.
• PCR Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York)
• Innis et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York). Coding sequences isolated based on their sequence identity to the entire inventive coding sequences set forth herein or to fragments thereof are encompassed by the present invention.
• the isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang et ai., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown et ai., Meth. Enzymol. 68:109-151 (1979); the diethylphosphoramidite method of Beaucage et ai., Tetra. Lett. 22:1859-1862 (1981 ); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts.
• nucleic acids of the present invention include those amplified using the following primer pairs: SEQ ID NOS: 3 and 4, 5 and 6, 9 and 10, or 11 and 12 or primers determined by using Vector nti Suite, InforMax Version 5.
• expression cassettes comprising isolated nucleic acids of the present invention are provided.
• An expression cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences which will direct the transcription of the polynucleotide in the intended host cell, such as tissues of a transformed plant.
• plant expression vectors may include (1 ) a cloned plant gene under the transcriptional control of 5' and 3' regulatory sequences and (2) a dominant selectable marker.
• Such plant expression vectors may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
• a promoter regulatory region e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression
• a transcription initiation start site e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression
• a transcription initiation start site e.g., one conferring inducible,
• constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumefaciens, the actin promoter, the ubiquitin promoter, the histone H2B promoter (Nakayama et al., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known in the art.
• CaMV cauliflower mosaic virus
• 1'- or 2'- promoter derived from T-DNA of Agrobacterium tumefaciens the actin promoter
• the ubiquitin promoter the histone H2B promoter
• the Smas promoter the cinnamyl alcohol dehydr
• inducible promoters examples include the Adh1 promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, the PPDK promoter which is inducible by light, the In2 promoter which is safener induced, the ERE promoter which is estrogen induced and the Pepcarboxylase promoter which is light induced.
• promoters under developmental control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, fruit, seeds, or flowers.
• An exemplary promoter is the anther specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051).
• seed-preferred promoters include, but are not limited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A., Martinez, M.C., Reina, M., Puigdomenech, P. and Palau, J.; Isolation and sequencing of a 28 kD glutelin-2 gene from maize: Common elements in the 5' flanking regions among zein and glutelin genes; Plant Sci.
• a weak constitutive promoter such as the Nos promoter
• an inducible promoter such as In2
• a nucellus-preferred or integument-preferred promoter are used to induce apospory.
• the barley or maize Nucl promoter the maize Cim 1 promoter or the maize LTP2 promoter can be used to preferentially express in the nucellus. See for example US Serial No. 60/097,233 filed August 20, 1998 the disclosure of which is incorporated herein by reference.
• Either heterologous or non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
• promoters can also be used, for example, in expression cassettes to drive expression of antisense nucleic acids to reduce, increase, or alter concentration and/or composition of the proteins of the present invention in a desired tissue.
• polypeptide expression it is generally desirable to include a polyadenylation region at the 3'-end of a polynucleotide coding region.
• the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA.
• the 3' end sequence to be added can be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
• An intron sequence can be added to the 5' untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates. See for example Buchman and Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis ef a/., Genes Dev. 1 :1183-1200 (1987).
• Use of maize introns Adh1-S intron 1 , 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994).
• the vector comprising the sequences from a polynucleotide of the present invention will typically comprise a marker gene which confers a selectable phenotype on plant cells.
• the selectable marker gene will encode antibiotic or herbicide resistance.
• Suitable genes include those coding for resistance to the antibiotics spectinomycin and streptomycin (e.g., the aada gene), the streptomycin phosphotransferase (SPT) gene coding for streptomycin resistance, the neomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (HPT) gene coding for hygromycin resistance.
• SPT streptomycin phosphotransferase
• NPTII neomycin phosphotransferase
• HPT hygromycin phosphotransferase
• Suitable genes coding for resistance to herbicides include those which act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonyl urea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance in particular the S4 and/or Hra mutations), those which act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art.
• the bar gene encodes resistance to the herbicide basta and the ALS gene encodes resistance to the herbicide chlorsulfuron.
• LEC1 transformants are recovered based solely on their differential growth advantage.
• Typical vectors useful for expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described by Rogers et al., Meth. In Enzymol. 153:253-277 (1987).
• Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl. Acad. Sci. USA 86:8402-8406 (1989).
• Another useful vector herein is plasmid pBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto, CA).
• a variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
• CaMV cauliflower mosaic virus
• geminivirus geminivirus
• brome mosaic virus brome mosaic virus
• tobacco mosaic virus a variety of plant viruses that can be employed as vectors.
• a polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired.
• antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the enzyme of interest, see, e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); and Hiatt et ai., U.S. Patent No. 4,801,340.
• Another method of suppression is sense suppression.
• Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes.
• RNA molecules or ribozymes can also be used to inhibit expression of plant genes.
• the inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs.
• the design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591 (1988).
• cross-linking agents, alkylating agents and radical generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids.
• Vlassov, V. V., et al., Nucleic Acids Res (1986) 14:4065-4076 describe covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences.
• a report of similar work by the same group is that by Knorre, D. G., et al., Biochimie (1985) 67:785-789.
• the invention relates to an isolated protein comprising a member selected from the group consisting of: (a) a polypeptide comprising at least 25 contiguous amino acids of SEQ
• polypeptide which is a plant HAP3-type CCATT-box binding transcriptional activator that regulates gene expression during embryo development and maturation;
• polypeptide comprising at least 60% sequence identity to SEQ ID NO: 2, 8, 10, 12, 14, 16, 18, 20, or 22;
• Proteins of the present invention include proteins derived from the native protein by deletion (so-called truncation), addition or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
• amino acid sequence variants of the polypeptide can be prepared by mutations in the cloned DNA sequence encoding the native protein of interest. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et ai. (1987) Methods Enzymol. 154:367-382; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York); U.S. Patent No.
• the isolated proteins of the present invention include a polypeptide comprising at least 23 contiguous amino acids encoded by any one of the nucleic acids of the present invention, or polypeptides which are conservatively modified variants thereof.
• the proteins of the present invention or variants thereof can comprise any number of contiguous amino acid residues from a polypeptide of the present invention, wherein that number is selected from the group of integers consisting of from 23 to the number of residues in a full-length polypeptide of the present invention.
• this subsequence of contiguous amino acids is at least 25, 30, 35, or 40 amino acids in length, often at least 50, 60, 70, 80, or 90 amino acids in length.
• the present invention includes catalytically active polypeptides (i.e., enzymes).
• Catalytically active polypeptides will generally have a specific activity of at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% that of the native (non-synthetic), endogenous polypeptide.
• the substrate specificity (kc a t/K m ) is optionally substantially similar to the native (non-synthetic), endogenous polypeptide.
• the K m will be at least 30%, 40%, or 50%, that of the native (non- synthetic), endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or 90%.
• the present invention includes modifications that can be made to an inventive protein. In particular, it may be desirable to diminish the activity of the LEC1 gene. Other modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
• modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.
• nucleic acids of the present invention may express a protein of the present invention in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or preferably plant cells.
• the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
• an intermediate host cell will be used in the practice of this invention to increase the copy number of the cloning vector. With an increased copy number, the vector containing the gene of interest can be isolated in significant quantities for introduction into the desired plant cells.
• Host cells that can be used in the practice of this invention include prokaryotes, including bacterial hosts such as Eschericia coli, Salmonella typhimurium, and Serratia marcescens. Eukaryotic hosts such as yeast or filamentous fungi may also be used in this invention. Since these hosts are also microorganisms, it will be essential to ensure that plant promoters which do not cause expression of the polypeptide in bacteria are used in the vector.
• prokaryotic control sequences include such commonly used promoters as the beta lactamase (penicillinase) and lactose (lac) promoter systems (Chang et ai., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel et ai., Nucleic Acids Res. 8:4057 (1980)) and the lambda derived P L promoter and N-gene ribosome binding site (Shimatake et al., Nature 292:128 (1981)).
• the inclusion of selection markers in DNA vectors transfected in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.
• Bacterial vectors are typically of plasmid or phage origin. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., Gene 22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)).
• yeast Synthesis of heterologous proteins in yeast is well known. See Sherman, F., et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982). Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen). Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
• promoters including 3-phosphoglycerate kinase or alcohol oxidase
• a protein of the present invention once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation techniques to the lysates.
• the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay of other standard immunoassay techniques.
• the proteins of the present invention can also be constructed using non- cellular synthetic methods.
• Solid phase synthesis of proteins of less than about 50 amino acids in length may be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence.
• Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield, et ai., J. Am. Chem. Soc. 85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.
• Proteins of greater length may be synthesized by condensation of the amino and carboxy termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxy terminal end (e.g., by the use of the coupling reagent N,N'-dicycylohexylcarbodiimide)) is known to those of skill.
• the proteins of this invention, recombinant or synthetic may be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. See, for instance, R.
• the present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the present invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the present invention) in a plant.
• the method comprises transforming a plant cell with an expression cassette comprising a polynucleotide of the present invention to obtain a transformed plant cell, growing the transformed plant cell under conditions allowing expression of the polynucleotide in the plant cell in an amount sufficient to modulate concentration and/or composition in the plant cell.
• the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down- regulate gene expression.
• the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Patent 5,565,350; Zarling et al., PCT/US93/03868.
• One method of down- regulation of the protein involves using PEST sequences that provide a target for degradation of the protein. It has been observed that high levels of LEC1 prevent germination. See Lotan et ai., Cell 1998 June 26; 93(7): 1195-1205.
• temporal regulation of LEC1 expression may be desirable in certain species to permit proper germination, vegetative growth, flowering and reproduction.
• an isolated nucleic acid e.g., a vector
• a plant cell comprising the promoter operably linked to a polynucleotide of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting ampl icons produced therefrom.
• a plant or plant part altered or modified by the foregoing embodiments is grown under plant forming conditions for a time sufficient to modulate the concentration and/or composition of polypeptides of the present invention in the plant. Plant forming conditions are well known in the art.
• concentration or composition is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative to a native control plant, plant part, or cell lacking the aforementioned expression cassette.
• Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development.
• Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation as discussed in greater detail, supra.
• Induction of expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of inducing compound.
• polypeptides of the present invention are modulated in monocots or dicots, preferably maize, soybeans, sunflower, sorghum, canola, wheat, alfalfa, rice, barley and millet.
• Means of detecting the proteins of the present invention are not critical aspects of the present invention.
• the proteins are detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
• immunological binding assays see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
• the immunoassays of the present invention can be performed in any of several configurations, e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Florida (1980); Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, FL (1987); Principles and Practice of Immunoassays, Price and Newman Eds., Stockton Press, NY (1991); and Non- isotopic Immunoassays, Ngo, Ed., Plenum Press, NY (1988).
• Typical methods include Western blot (immunoblot) analysis, analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, and various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, and the like.
• Western blot immunoblot
• analytic biochemical methods such as electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like
• various immunological methods such as fluid or gel precipitin reactions, immunodiffusion (single or double), immunoelectrophoresis, radioimmunoassays (RIAs), enzyme
• Non-radioactive labels are often attached by indirect means.
• a ligand molecule e.g., biotin
• the ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound.
• an anti-ligand e.g., streptavidin
• a number of ligands and anti-ligands can be used.
• a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands.
• any haptenic or antigenic compound can be used in combination with an antibody.
• the molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore.
• Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases.
• Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.
• Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
• agglutination assays can be used to detect the presence of the target antibodies.
• antigen-coated particles are agglutinated by samples comprising the target antibodies.
• none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.
• the proteins of the present invention can be used for identifying compounds that bind to (e.g., substrates), and/or increase or decrease (i.e., modulate) the enzymatic activity of, catalytically active polypeptides of the present invention.
• the method comprises contacting a polypeptide of the present invention with a compound whose ability to bind to or modulate enzyme activity is to be determined.
• the polypeptide employed will have at least 20%, preferably at least 30% or 40%, more preferably at least 50% or 60%, and most preferably at least 70% or 80% of the specific activity of the native, full-length polypeptide of the present invention (e.g., enzyme). Methods of measuring enzyme kinetics are well known in the art.
• Antibodies can be raised to a protein of the present invention, including individual, allelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill.
• monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc.
• Description of techniques for preparing such monoclonal antibodies are found in, e.g., Basic and Clinical Immunology, 4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane, Supra; Goding, Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press, New York, NY (1986); and Kohler and Milstein, Nature 256:495- 497 (1975).
• the antibodies of this invention can be used for affinity chromatography in isolating proteins of the present invention, for screening expression libraries for particular expression products such as normal or abnormal protein or for raising anti-idiotypic antibodies which are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.
• the proteins and antibodies of the present invention will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
• labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
• Transfectlon/Transformation of Cells The method of transformation/transfection is not critical to the instant invention; various methods of transformation or transfection are currently available. As newer methods are available to transform crops or other host cells they may be directly applied. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a host cell to obtain the transcription and/or translation of the sequence to effect phenotypic changes in the organism. Thus, any method which provides for efficient transformation/transfection may be employed.
• a DNA sequence coding for the desired polynucleotide of the present invention can be used to construct an expression cassette which can be introduced into the desired plant.
• Isolated nucleic acid acids of the present invention can be introduced into plants according techniques known in the art.
• expression cassettes as described above and suitable for transformation of plant cells are prepared. Techniques for transforming a wide variety of higher plant species are well known and described in the technical, scientific, and patent literature. See, for example, Weising et ai., Ann. Rev. Genet. 22:421-477 (1988).
• the DNA construct may be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, PEG poration, particle bombardment, silicon fiber delivery, or microinjection of plant cell protoplasts or embryogenic callus. See, e.g., Tomes, et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment, pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods, eds. O. L. Gamborg and G.C. Phillips. Springer- Verlag Berlin Heidelberg New York, 1995.
• the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Patent No. 5,591,616.
• Agrobacterium tumefaciens-med ' tiated transformation techniques are well described in the scientific literature. See, for example Horsch et al., Science 233:496-498 (1984), and Fraley et ai., Proc. Natl. Acad. Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maize is described in WO 98/32326. Agrobacterium transformation of soybean is described in US Pat. No. 5,563,055. Other methods of transfection or transformation include (1 ) Agrobacterium rhizogenes-med ⁇ a ⁇ ed transformation (see, e.g., Lichtenstein and Fuller In: Genetic Engineering, Vol.
• DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et ai., Methods in Enzymology, 101:433 (1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et ai., Plane Mol. Biol. Reporter, 6:165 (1988).
• Expression of polypeptide coding polynucleotides can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al., Nature, 325:274 (1987).
• DNA can also be injected directly into the cells of immature embryos and the rehydration of desiccated embryos as described by Neuhaus et ai., Theor. Appl. Genet., 75:30 (1987); and Benbrook et ai., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986).
• Animal and lower eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
• eukaryotic (e.g., yeast) host cells are competent or rendered competent for transfection by various means.
• methods of introducing DNA into animal cells include: calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, DEAE dextran, electroporation, biolistics, and micro-injection of the DNA directly into the cells.
• the transfected cells are cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977).
• somatic embryogenesis it is possible to alter plant tissue culture media components to suppress somatic embryogenesis in a plant species of interest (often having multiple components that potentially could be adjusted to impart this effect). Such conditions would not impart a negative or toxic in vitro environment for wild-type tissue, but instead would simply not produce a somatic embryogenic growth form.
• Introducing a transgene such as LEC1 will stimulate somatic embryogenesis and growth in the transformed cells or tissue, providing a clear differential growth screen useful for identifying transformants.
• Altering a wide variety of media components can modulate somatic embryogenesis (either stimulating or suppressing embryogenesis depending on the species and particular media component). Examples of media components which, when altered, can stimulate or suppress somatic embryogenesis include;
• auxins indole acetic acid, indole butyric acid, 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid, picloram, dicamba and other functional analogues
• cytokinins zeatin, kinetin, benzyl amino purine, 2-isopentyl adenine and functionally-related compounds
• abscisic acid adenine, and gibberellic acid
• Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype.
• Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with a polynucleotide of the present invention.
• a tissue culture growth medium typically relying on a biocide and/or herbicide marker that has been introduced together with a polynucleotide of the present invention.
• For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell, 2:603-618 (1990).
• Plants cells transformed with a plant expression vector can be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. It is well known in the art that various cells, tissues, and organs from almost any plant can be successfully cultured to regenerate an entire plant. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding, Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp. 21-73 (1985).
• Transgenic plants of the present invention may be fertile or sterile.
• Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987). The regeneration of plants from either single plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H.
• transgenic plants can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
• vegetatively propagated crops mature transgenic plants can be propagated by the taking of cuttings, via production of apomictic seed, or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.
• seed propagated crops mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.
• Parts obtained from the regenerated plant such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences.
• Transgenic plants expressing a selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Transgenic lines are also typically evaluated on levels of expression of the heterologous nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression- positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the heterologous RNA templates and solution hybridization assays using heterologous nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention.
• in situ hybridization and immunocytochemistry can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue.
• a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
• a preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; i.e., a transgenic plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair.
• a homozygous transgenic plant can be obtained by sexually mating (selfing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-transgenic). Back- crossing to a parental plant and out-crossing with a non- transgenic plant are also contemplated. Alternatively, propagation of heterozygous transgenic plants could be accomplished through apomixis.
• the present invention provides a method of genotyping a plant comprising a polynucleotide of the present invention.
• Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
• Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7, Clark, Ed., Springer-Verlag, Berlin (1997). For molecular marker methods, see generally, The DNA Revolution by Andrew H.
• RFLPs restriction fragment length polymorphisms
• the particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs).
• RFLPs are the product of allelic differences between DNA restriction fragments caused by nucleotide sequence variability.
• the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.
• Plants which can be used in the method of the invention include monocotyledonous and dicotyledonous plants.
• Preferred plants include maize, wheat, rice, barley, oats, sorghum, millet, rye, soybean, sunflower, alfalfa, canola and cotton.
• Seeds derived from plants regenerated from transformed plant cells, plant parts or plant tissues, or progeny derived from the regenerated transformed plants, may be used directly as feed or food, or further processing may occur. All publications cited in this application are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
• plant tissue samples were pulverized in liquid nitrogen before the addition of the TRIzol Reagent, and then were further homogenized with a mortar and pestle. Addition of chloroform followed by centrifugation was conducted for separation of an aqueous phase and an organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.
• cDNA Library Construction cDNA synthesis was performed and unidirectional cDNA libraries were constructed using the Superscript Plasmid System (Life Technology Inc. Gaithersburg, MD). The first stand of cDNA was synthesized by priming an oligo(dT) primer containing a Not I site. The reaction was catalyzed by Superscript Reverse Transcriptase II at 45°C. The second strand of cDNA was labeled with alpha- 32 P-dCTP and a portion of the reaction was analyzed by agarose gel electrophoresis to determine cDNA sizes. cDNA molecules smaller than 500 base pairs and unligated adapters were removed by Sephacryl-S400 chromatography. The selected cDNA molecules were ligated into pSPORTI vector in between of Not I and Sal I sites.
• Colony hybridization was conducted as described by Sambrook, J., Fritsch, E.F. and Maniatis, T., (in Molecular Cloning: A laboratory Manual, 2 nd Edition). The following probes were used in colony hybridization: 1. First strand cDNA from the same tissue from which the library was made to remove the most redundant clones.
• a Sal-A20 oligo nucleotide TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.
• the image of the autoradiography was scanned into computer and the signal intensity and cold colony addresses of each colony was analyzed. Re-arraying of cold-colonies from 384 well plates to 96 well plates was conducted using Q-bot.
• Gene identities were determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et ai., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches under default parameters for similarity to sequences contained in the BLAST "nr" database (comprising all non- redundant GenBank CDS translations, sequences derived from the 3-dimensionai structure Brookhaven Protein Data Bank, the last major release of the SWISS- PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences were analyzed for similarity to all publicly available DNA sequences contained in the "nr" database using the BLASTN algorithm.
• cDNA libraries representing mRNAs from various corn, poppy, soybean and Vernonia tissues were prepared (see Table 1 ). The characteristics of the libraries are described below.
• Soybean embryo 6 to 10 days after flowering
• cDNA libraries were prepared in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA). Conversion of the Uni-ZAPTM XR libraries into plasmid libraries was accomplished according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript. cDNA inserts from randomly picked bacterial colonies containing recombinant pBluescript plasmids were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences or plasmid DNA was prepared from cultured bacterial cells.
• Amplified insert DNAs or plasmid DNAs were sequenced in dye- primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or "ESTs"; see Adams, M. D. et ai., (1991) Science 252:1651). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
• ESTs encoding plant transcription factors were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST "nr" database (comprising all non- redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS- PROT protein sequence database, EMBL, and DDBJ databases).
• BLAST Basic Local Alignment Search Tool
• Example 1 The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the "nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the "nr” database using the BLASTX algorithm (Gish, W. and States, D. J. (1993) Nature Genetics 3:266-272 and Altschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402) provided by the NCBI.
• NCBI National Center for Biotechnology Information
• HAP3 homologs were identified in our EST database and aligned. By analyzing sequence homology amongst the plant HAP3 family of transcriptional activators these sequences were observed to fall into a least two distinctive groups. All of the HAP3 sequences derived from seed or embryo specific libraries form a distinctive LEC1 group that suggests a common evolutionary origin (confirmed by phylodentrograms).
• LEC1 genes are highly divergent outside of the region spanning the DNA binding and subunit interaction motifs. The low levels of homology between these genes make it difficult to identify these based solely on a hybridization strategy.
• LEC1 polynucleotide of the invention
• the LEC1 polynucleotide was operably linked to a constitutive promoter such as nos, or an inducible promoter, such as In2, plus a plasmid containing the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25- 37) that confers resistance to the herbicide Bialaphos fused to the Green Fluorescence protein. Transformation was performed as follows.
• the ears were surface sterilized in 50% Chlorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water.
• the immature embryos were excised and placed embryo axis side down (scutellum side up), 25 embryos per plate. These were cultured on 560 L medium 4 days prior to bombardment in the dark.
• Medium 560 L is an N6-based medium containing Eriksson's vitamins, thiamine, sucrose, 2,4-D, and silver nitrate.
• the day of bombardment the embryos were transferred to 560 Y medium for 4 hours and were arranged within the 2.5-cm target zone.
• Medium 560Y is a high osmoticum medium (560L with high sucrose concentration).
• a plasmid vector comprising a polynucleotide of the invention operably linked to the selected promoter was constructed.
• This plasmid DNA plus plasmid DNA containing a PAT selectable marker was precipitated onto 1.1 ⁇ m (average diameter) tungsten pellets using a CaCI 2 precipitation procedure as follows: 100 ⁇ l prepared tungsten particles (0.6 mg) in water, 20 ⁇ l (2 ⁇ g) DNA in TrisEDTA buffer (1 ⁇ g total), 100 ⁇ l 2.5 M CaC1 2 , 40 ⁇ l 0.1 M spermidine. Each reagent was added sequentially to the tungsten particle suspension.
• the final mixture was sonicated briefly. After the precipitation period, the tubes were centrifuged briefly, liquid removed, washed with 500 ml 100% ethanol, and centrifuged again for 30 seconds. Again the liquid was removed, and 60 ⁇ l 100% ethanol was added to the final tungsten particle pellet.
• the tungsten/DNA particles were briefly sonicated and 5 ⁇ l spotted onto the center of each macrocarrier and allowed to dry about 2 minutes before bombardment.
• sample plates were bombarded at a distance of 8 cm from the stopping screen to the tissue, using a Dupont biolistics helium particle gun. All samples received a single shot at 650 PSI, with a total of ten aliquots taken from each tube of prepared particles/DNA.
• the embryos were moved to 560P (a low osmoticum callus initiation medium similar to 560L but with lower silver nitrate), for 3-7 days, then transferred to 560R selection medium, an N6 based medium similar to 560P containing 3 mg/liter Bialaphos, and subcultured every 2 weeks. Multicellular GFP cell clusters became visible after two weeks and their numbers were periodically recorded. After approximately 10 weeks of selection, selection-resistant GFP positive callus clones were sampled for PCR and activity of the polynucleotide of interest. Positive lines were transferred to 288J medium, an MS-based medium with lower sucrose and hormone levels, to initiate plant regeneration.
• 560P a low osmoticum callus initiation medium similar to 560L but with lower silver nitrate
• 560R selection medium an N6 based medium similar to 560P containing 3 mg/liter Bialaphos
• somatic embryo maturation 2-4 weeks
• somatic embryos were transferred to medium for germination and transferred to the lighted culture room.
• developing plantlets were transferred to medium in tubes for 7-10 days until plantlets were well established.
• Plants were then transferred to inserts in flats (equivalent to 2.5" pot) containing potting soil and grown for 1 week in a growth chamber, subsequently grown an additional 1 -2 weeks in the greenhouse, then transferred to ClassicTM 600 pots (1.6 gallon) and grown to maturity. Plants are monitored for expression of the polynucleotide of interest.
• embryos were isolated and cultured on 560L medium for 3-5 days. Four to twelve hours before bombardment these embryos were transferred to high osmotic 560Y medium. Expression cassettes containing the LEC1 cDNA were then co-introduced into the scutella of these embryos along with an expression cassette containing the Pat gene fused to the Green Fluorescent protein using methods described in Example 7. Embryos from a single ear were divided evenly between treatments. Four to 12 hours following bombardment embryos were then transferred back to a low osmoticum callus initiation medium (560P) and incubated in the dark at 26°C. After 3-7 days of culture these embryos were moved to 560R selection medium.
• 560P low osmoticum callus initiation medium
• Ectopic expression of the maize LEC1 polynucleotide in tobacco is sufficient to induce somatic embryogenesis in tobacco leaves
• a maize LEC1 polynucleotide was placed into an agrobacterium expression cassette driven by the maize safener-induced In2 promoter (this promoter is leaky and expresses at low levels without induction). Also between the left and right T-DNA borders was the bar gene driven by 35S promoter and the Green Fluorescence Protein driven by the ubiquitin promoter.
• a similar construct was made without the LEC1 polynucleotide to be used as a control. Tobacco leaf discs from variety SR1 were co-cultured with Agrobacterium as described by Horsch et ai.
• Transformation frequency was improved by LEC1 introduced using particle-mediated DNA delivery.
• a series of expression cassettes were made to evaluate the effects of LEC1 expression on maize transformation.
• the maize LEC1 polynucleotide was placed under the control of the In2 promoter (weakly induced with the auxin levels used under normal culture conditions and strongly-induced with safener), the barley NUC1 promoter (expressed strongly in the nucellus), the Ubiquitin promoter (strongly expressed constitutively), and the nos promoter (weakly expressed constitutively).
• a frame-shift version of the ln2:LEC1 cassette was made along with an ln2:ZM-NF-YB (designated as ln2:HAP3 henceforth) construct
• the maize ZM NF-YB is non-LEC1 type of HAP3 transcriptional activator (Li et al Nucleic Acids Res. 20:1087-1091) for use as negative controls. All of these constructs were co-bombarded with the Pat ⁇ GFP fusion construct (designated as PAT-GFP) into high type II embryos as described in Example 7.
• Example 7 immature embryos were harvested from separate ears, and the embryos from each ear were divided equally between treatments to account for ear-to-ear variability (for example, in an experiment comparing a control plasmid with this same plasmid + LEC1, one-half the total embryos from each ear would be used for each treatment.
• the control treatment contained the Pat ⁇ GFP construct co-bombarded with GUS. Transformation frequency was determined by counting the numbers of embryos with large multicellular GFP- positive cells clusters using a GFP microscope, and representing these as a percentage of the original number of embryos bombarded. No distinction was made between embryos with single or multiple events.
• the functional LEC1 expression cassettes increased transformation frequencies over the control treatment (the LEC1 expression cassette also increased the incidence of multiple, i.e. 2-3, multicellular transgenic clones growing from the same immature embryo, but as stated above we only scored these as a single event, and are providing a conservative representation of LECI's ability to improve transformation).
• transformation frequencies in control treatments for three consecutive experiments were 5.1 , 7.4 and 0.8%.
• transformation frequencies with the LEC1 polynucleotide (ln2::LEC1::pinll) were 28.8, 25.7 and 12.4%, respectively.
• LEC1 transformants appeared earlier than the control transformants (suggesting that the LEC1 polynucleotide also stimulated growth rates).
• Increasing the promoter strength increased transformation frequencies. For example, an experiment was performed to compare the In2, nos and UBI promoters. Based on our experience with these two promoters driving other genes, the In2 promoter (in the absence of an inducer other than auxin from the medium) would drive expression at very low levels.
• the nos promoter has been shown to drive moderately-low levels of transgene expression (approximately 10- to 30-fold lower than the maize ubiquitin promoter, but still stronger than In2 under the culture conditions used in this experiment).
• Transformation frequency was improved by LEC1 introduced using agrobacterium.
• the Agrobacterium strains containing the superbinary plasmids described in Example 8A were used to transformed High type II embryos. Briefly, colonies containing the engineered Agrobacterium were grown to log phase in minimal A medium. Log phase cells were collected by centrifugation and resuspended in 561 Q medium (N6 salts, Eriksson's vitamins, 1.5mg/l 2,4-D , 68.5g/l sucrose, 36g/l glucose, plus 20mg/l acetosyringone). Immature embryos, 1.5-2mm in length, were excised and immersed in this solution at a concentration of 5 X 10 8 bacterial cells/ml. Embryos were vortexed in this medium and allowed to sit for 5 minutes.
• 562P medium 560P medium with 10OmM acetosyringone and incubated at 20°C for 3 days. Embryos were moved again to 563N medium (an agar solidified medium similar to 560P with 100mg/l carbenicillin, 0.5g/l MES and reduced 2,4-D) and cultured at 28°C for 3 days. Embryos were then moved to 5630 medium (563N medium with 3mg/l bialaphos) and transferred thereafter every 14 days to fresh 5630 medium. Bialaphos resistant GFP+ colonies were counted using a GFP microscope and transformation frequencies were determined as described in example 8B. Similar to particle gun experiments, transformation frequencies were greatly increased in the LEC1 treatment.
• transformation frequencies for the control treatment across embryos taken from 7 separate ears were 7.1, 40.9, 11.1 , 7.4, 11.5, 12, 30.8, and 16.6%.
• the side-by-side comparison for the LEC treatment showed that transformation frequencies were 13.5, 47, 55.8, 37.1, 40.6, 30, 57.1 and 40.8%.
• Averaged across all 7 ears, the average transformation frequency for the control was 16.6% while that of the LEC1 treatment was 40.8%. This represents a substantial increase for an already high baseline produced by Agrobacterium-mediated transformation. Comparing across ears, it was observed that the beneficial effects on transformation frequency were the greatest when the control frequencies were low.
• normal bialaphos-containing selection medium with normal auxin levels of 2 mg/l 2,4-D
• medium with no bialaphos and reduced 2,4-D levels 0.5 mg/l
• the LEC1 polynucleotide in combination with GFP+ expression can be used to recover transformants without chemical selection.
• the recovery of transformants was relatively efficient (16% compared to 18% for bialaphos selection), but this required more diligence than the low- or no-auxin treatments above to separate the GFP-expressing colonies from the growing callus population.
• E. LEC1 improves the embryogenic phenotype and regeneration capacity of inbreds.
• Immature embryos from the inbred PHP38 were isolated, cultured and transformed as described in example 4 with the following changes.
• Embryos were initially cultured on 601 H medium (a MS based medium with 0.1 mg/l zeatin, 2mg/l 2,4-D, MS and SH vitamins, proline, silver nitrate, extra potassium nitrate, casein hydrolysate, gelrite, 10g/l glucose and 20g/l sucrose).
• Prior to bombardment embryos were moved to a high osmoticum medium (modified Duncan's with 2mg/l 2,4-D and 12% sucrose).
• Post bombardment embryos were moved to 601 H medium with 3mg/l bialaphos for two weeks.
• Embryos were then moved to 601 H medium without proline and casein hydrolysate with 3mg/l bialaphos and transferred every two weeks. Transformation frequency was determined by counting the numbers of bialaphos resistant GFP-positive colonies. Colonies were also scored on whether they had an embryogenic (regenerable) or non- embryogenic phenotype.
• the LEC1 polynucleotide increased transformation frequency and improved the regenerative potential of the callus.
• a balanced experiment (the embryos from each harvested ear were divided equally between treatments) was conducted in which PHP38 immature embryos were bombarded with the control plasmid (UBI::PAT ⁇ GFP::pinll) in one treatment, with the UBI::PAT ⁇ GFP::pinll plasmid + ln2::LEC1, or with the UBI::PAT ⁇ GFP::pinll plasmid + nuc1 ::LEC1 (a maize nucellus-specific promoter driving LEC1 expression).
• the frequency of GFP+ calli growing on bialaphos- containing media was determined 6 weeks after bombardment.
• the transformation frequency was 1.2%, while for the ln2:LEC1 and nuc1 ::LEC1 treatments the transformation frequencies were 3.2 and 2.0% respectively.
• the presence of the LEC1 polynucleotide appeared to greatly improve the regeneration capacity of the recovered transformants. None of the control transformants (UBI::PAT ⁇ GFP::pinll alone) had an embryogenic, regenerable phenotype, while the transformants recovered from the ln2:LEC1 and nuc1 ::LEC1 treatments all exhibited a more vigorous, embryogenic growth pattern. This has been born out in the ability to recover plants. Callus from the ln2:LEC1 and nuc1::LEC1 treatments has produced many healthy plants.
• LEC1 5'capped polyadenylated RNA expression cassettes containing LEC-1 DNA, or LEC-1 protein. All of these molecules can be delivered using a biolistics particle gun.
• 5'capped polyadenylated LEC1 RNA can easily be made in vitro using Ambion's mMessage mMachine kit. Following the procedure outline above RNA is co-delivered along with DNA containing an agronomically useful expression cassette. The cells receiving the RNA will immediately form somatic embryos and a large portion of these will have integrated the agronomic gene. Plants regenerated from these embryos can then be screened for the presence of the agronomic gene.
• Maize expression cassettes directing LEC1 expression to the inner integument or nucellus can easily be constructed.
• An expression cassette directing expression of the LEC1 polynucleotide to the nucellus was made using the barley Nucl promoter. Embryos were co-bombarded with the selectable marker PAT fused to the GFP gene along with the nucellus specific LEC1 expression cassette described above. Both inbred (PHP38) and GS3 transformants were obtained and regenerated as described in examples 4 and 5. Transformation frequencies were also increased over the control using the nuc1:LEC1 polynucleotide (see Example 8 above).
• nud :LEC1 transformations could be done using a FIE-null genetic background which would promote both de novo embryo development and endosperm development without fertilization (see Ohad et ai. 1999 The Plant Cell 11:407-415; also pending US application Serial No. 60/151575 filed August 31 , 1999). Upon microscopic examination of the developing embryos it will be apparent that apomixis has occurred by the presence of embryos budding off the nucellus.
• LEC1 polynucleotide could be delivered as described above into a homozygous zygotic-embryo-lethal genotype. Only the adventive embryos produced from somatic nucellus tissue would develop in the seed. EXAMPLE 11 Expression of Chimeric Genes in Microbial Cells
• the cDNAs encoding the instant transcription factors can be inserted into the T7 E. coli expression vector pBT430.
• This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
• Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
• Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
• Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1 % NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
• the fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above.
• the vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above.
• the prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL).
• Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the polynucleotide encoding the transcription factor are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
• a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. Biol. 789: 113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 hours at 25°C.
• IPTG isopropylthio- ⁇ -galactoside, the inducer
• Cells are then harvested by centrifugation and re- suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride.
• a small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator.
• the mixture is centrifuged and the protein concentration of the supernatant determined.
• One ⁇ g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
• the transcription factors described herein may be produced using any number of methods known to those skilled in the art. Such methods include, but are not limited to, expression in bacteria as described in Example 6, or expression in eukaryotic cell culture, in planta, and using viral expression systems in suitably infected organisms or cell lines.
• the instant transcription factors may be expressed either as mature forms of the proteins as observed in vivo or as fusion proteins by covalent attachment to a variety of enzymes, proteins or affinity tags.
• Common fusion protein partners include glutathione S-transferase ("GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/or N-terminal hexahistidine polypeptide ("(His) 6 ").
• the fusion proteins may be engineered with a protease recognition site at the fusion point so that fusion partners can be separated by protease digestion to yield intact mature enzyme.
• proteases include thrombin, enterokinase and factor Xa.
• any protease can be used which specifically cleaves the peptide connecting the fusion protein and the enzyme.
• Purification of the instant transcription factors may utilize any number of separation technologies familiar to those skilled in the art of protein purification. Examples of such methods include, but are not limited to, homogenization, filtration, centrifugation, heat denaturation, ammonium sulfate precipitation, desalting, pH precipitation, ion exchange chromatography, hydrophobic interaction chromatography and affinity chromatography, wherein the affinity ligand represents a substrate, substrate analog or inhibitor.
• the purification protocol may include the use of an affinity resin which is specific for the fusion protein tag attached to the expressed enzyme or an affinity resin containing ligands which are specific for the enzyme.
• a transcription factor may be expressed as a fusion protein coupled to the C-terminus of thioredoxin.
• a (His) 6 peptide may be engineered into the N-terminus of the fused thioredoxin moiety to afford additional opportunities for affinity purification.
• Other suitable affinity resins could be synthesized by linking the appropriate ligands to any suitable resin such as Sepharose-4B.
• a thioredoxin fusion protein may be eluted using dithiothreitol; however, elution may be accomplished using other reagents which interact to displace the thioredoxin from the resin. These reagents include ⁇ -mercaptoethanol or other reduced thiol.
• the eluted fusion protein may be subjected to further purification by traditional means as stated above, if desired.
• Proteolytic cleavage of the thioredoxin fusion protein and the enzyme may be accomplished after the fusion protein is purified or while the protein is still bound to the ThioBondTM affinity resin or other resin.
• Crude, partially purified or purified enzyme may be utilized in assays for the evaluation of compounds for their ability to inhibit enzymatic activition of the transcription factors disclosed herein. Assays may be conducted under well-known experimental conditions that permit optimal enzymatic activity.
• Example 13 LEC1 expression resulted in increased growth rates, which could be used as a screening criterion for positive selection of transformants.
• Example 14 The use of LEC1 polynucleotide as a positive selection system for wheat transformation and for improving the regeneration capacity of wheat tissues
• the growth conditions used were; 1) soil composition: 75% L&P fine-grade peat, 12% screened sterilized loam, 10% 6 mm screened, lime-free grit, 3% medium grade vermiculite, 3.5 kg Osmocote per m 3 soil (slow-release fertiliser, 15-11-13 NPK plus micronutrients), 0.5 kg PG mix per m 3 (14-16-18 NPK granular fertiliser plus micronutrients, 2) 16 h photoperiod (400W sodium lamps providing irradiance of ca.
• scutellar and inflorescence tissues Two sources of primary explants were used; scutellar and inflorescence tissues.
• scutella early-medium milk stage grains containing immature translucent embryos were harvested and surface-sterilized in 70% ethanol for 5 min and 0.5% hypochlorite solution for 15-30 min.
• tillers containing 0.5-1.0 cm inflorescences were harvested by cutting below the inflorescence-bearing node (the second node of a tiller). The tillers were trimmed to approximately 8-10 cm length and surface-sterilized as above with the upper end sealed with Nescofilm (Bando Chemical Ind. Ltd, Japan).
• the standard callus induction medium for scutellar tissues consisted of solidified (0.5% Agargel, Sigma A3301) modified MS medium supplemented with 9% sucrose, 10 mg I "1 AgN0 3 and 0.5 mg I "1 2,4-D (Rasco- Gaunt et al., 1999).
• Inflorescence tissues were cultured on L7D2 which consisted of solidified (0.5% Agargel) L3 medium supplemented with 9% maltose and 2 mg I "1 2,4-D (Rasco-Gaunt and Barcelo, 1999).
• the basal shoot induction medium, RZ contained L salts, vitamins and inositol, 3% w/v maltose, 0.1 mg P 2,4-D and 5 mg I "1 zeatin (Rasco-Gaunt and Barcelo, 1999). Regenerated plantlets were maintained in RO medium with the same composition as RZ, but without 2,4-D and zeatin.
• Submicron gold particles (0.6 ⁇ m Micron Gold, Bio-Rad) were coated with a plasmid containing the maize ln-2:LEC1 construct following the protocol modified from the original Bio-Rad procedure (Barcelo and Lazzeri, 1995).
• the standard precipitation mixture consisted of 1 mg of gold particles in 50 ⁇ l SDW, 50 ⁇ l of 2.5 M calcium chloride, 20 ⁇ l of 100 mM spermidine free base and 5 ⁇ l DNA (concentration 1 ⁇ g ⁇ l "1 ). After combining the components, the mixture was vortexed and the supernatant discarded. The particles were then washed with 150 ⁇ l absolute ethanol and finally resuspended in 85 ⁇ l absolute ethanol.
• DNA/gold ethanol solution was kept on ice to minimise ethanol evaporation.
• 5 ⁇ l of DNA/gold ethanol solution (ca. 60 ⁇ g gold) was loaded onto the macrocarrier.
• Particle bombardments were carried out using DuPont PDS 1000/He gun with a target distance of 5.5 cm from the stopping plate at 650 psi acceleration pressure and 28 in. Hg chamber vacuum pressure.
• Genomic DNA was extracted from calluses or leaves using a modification of the CTAB (cetyltriethylammonium bromide, Sigma H5882) method described by Stacey and Isaac (1994). Approximately 100-200 mg of frozen tissues was ground into powder in liquid nitrogen and homogenised in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA, 0.1 M Tris-CI pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65°C. Homogenised samples were allowed to cool at room temperature for 15 min before a single protein extraction with approximately 1 ml 24:1 v/v chloroform:octanol was done.
• CTAB cetyltriethylammonium bromide
• RNAse A was added to the samples and incubated at 37°C for 1 h.
• gel electrophoresis was performed using an 0.8% agarose gel in 1x TBE buffer. One microlitre of the samples were fractionated alongside 200, 400, 600 and 800 ng ⁇ l "1 ⁇ uncut DNA markers.
• the presence of the maize LEC1 polynucleotide was analyzed by PCR using 100-200 ng template DNA in a 30 ml PCR reaction mixture containing 1X concentration enzyme buffer (10 mM Tris-HCl pH 8.8, 1.5 mM magnesium chloride, 50 mM potassium chloride, 0.1 % Triton X-100), 200 ⁇ M dNTPs, 0.3 ⁇ M primers and 0.022 U TaqDNA polymerase (Boehringer Mannheim).
• Thermocycling conditions were as follows (30 cycles): denaturation at 95°C for 30 s, annealing at 55°C for 1 min and extension at 72°C for 1 min.
• the polynucleotide was then introduced into wheat scutellar and inflorescence explants, driven by the maize In2 promoter. Both tissues are used for wheat transformation.
• calluses were assessed prior to transfer onto shoot regeneration medium.
• inflorescence tissues as explants for the tissue culture and transformation of wheat offer several advantages over seed explants such as scutella (Rasco-Gaunt and Barcelo, 1999).
• responses of these tissues to culture are highly genotype-dependent and calluses are often non-regenerative despite having a 'highly-embryogenic' appearance.
• LEC was introduced into inflorescence tissues to see whether regeneration could be enhanced on a poorly regenerating line such BO 014.
• the LEC1 polynucleotide can also be used to improve the transformation of soybean.
• the construct consisting of the In2 promoter and LEC1 coding region were introduced into embryogenic suspension cultures of soybean by particle bombardment using essentially the methods described in Parrott, W.A., L.M. Hoffman, D.F. Hildebrand, E.G. Williams, and G.B. Collins, (1989) Recovery of primary transformants of soybean, Plant Cell Rep. 7:615-617. This method with modifications is described below.
• Seed was removed from pods when the cotyledons were between 3 and 5 mm in length. The seeds were sterilized in a Chlorox solution (0.5%) for 15 minutes after which time the seeds were rinsed with sterile distilled water. The immature cotyledons were excised by first cutting away the portion of the seed that contains the embryo axis. The cotyledons were then removed from the seed coat by gently pushing the distal end of the seed with the blunt end of the scalpel blade. The cotyledons were then placed (flat side up) SB1 initiation medium (MS salts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH 5.8).
• SB1 initiation medium MS salts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH 5.8.
• soybean embryogenic suspension cultures were maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights (20 ⁇ E) on a 16:8 hour day/night schedule. Cultures were sub-cultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
• Soybean embryogenic suspension cultures were then be transformed using particle gun bombardment (Klein et ai. (1987) Nature (London) 327:70, U.S. Patent No. 4,945,050).
• a BioRad BiolisticTM PDS1000/HE instrument was used for these transformations.
• a selectable marker gene which was used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et ai. (1985) Nature 373:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
• Approximately 300-400 mg of a two-week-old suspension culture was placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette.
• Membrane rupture pressure was set at 1100 psi and the chamber was evacuated to a vacuum of 28 inches mercury.
• the tissue was placed approximately 8 cm away from the retaining screen, and was bombarded three times. Following bombardment, the tissue was divided in half and placed back into 35 ml of FN Lite medium.
• 92B91 and 93B82 Two different genotypes were used in these experiments: 92B91 and 93B82. Samples of tissue were either bombarded with the hygromycin resistance gene alone or with a 1:1 mixture of the hygromycin resistance gene and the LEC1 construct. Embryogenic cultures generated from 92B91 generally produce transformation events while cultures from 93B82 are much more difficult to transform. For transformation experiments with 92B91 , approximately equal numbers of transformants were recovered from bombardments conducted with the LEC1 polynucleotide as without it. Twenty-nine transformants were recovered from the LEC1 -treated 92B91 tissue while 27 transformants were recovered from tissue receiving only the hygromycin resistance gene.
• transformants were only recovered from 93B82 tissue receiving the LEC1 polynucleotide (none were recovered from the treatment using only the hygromycin resistance gene). Five transformants were recovered from 93B82 tissue bombarded with the LEC1 polynucleotide while no transformants were recovered from tissue treated with only the hygromycin resistance gene.

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