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WO2010076766A1 - Gènes associés au nombre de talles d'une plante et leurs utilisations - Google Patents

Gènes associés au nombre de talles d'une plante et leurs utilisations Download PDF

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Publication number
WO2010076766A1
WO2010076766A1 PCT/IB2009/055988 IB2009055988W WO2010076766A1 WO 2010076766 A1 WO2010076766 A1 WO 2010076766A1 IB 2009055988 W IB2009055988 W IB 2009055988W WO 2010076766 A1 WO2010076766 A1 WO 2010076766A1
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dlt
plant
nucleic acid
sequence
protein
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PCT/IB2009/055988
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English (en)
Inventor
Chu Chengcai
Tong Hongning
Yun Jin
Wenbo Liu
Feng Li
Jun Fang
Lihuang Zhu
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Institute Of Genetics And Developmental Biology
Syngenta Participations Ag
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Publication of WO2010076766A1 publication Critical patent/WO2010076766A1/fr

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    • 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
    • C12N15/8291—Hormone-influenced development
    • C12N15/8298—Brassinosteroids

Definitions

  • the invention relates generally to compositions and methods for altering plant tiller number using dwarf and low tillering (DLT) genes.
  • the aforementioned compositions include polynucleotides, polypeptides, vectors and host cells.
  • the present invention also relates to plants transformed by the aforementioned compositions and methods.
  • Brassinosteroids are an important class of phytohormones involved in various processes during plant growth and development. Brassinosteroids play a significant role in controlling the height and bending angle of the lamina joint of a plant. A plant's height directly correlates with its anti-lodging ability, and the bending angle of the lamina joint is associated with planting density and the efficiency with which plants capture sunlight. A compact morphology can greatly reduce the overshadowing between leaf blades, thus enhancing the capacity of each leaf to capture sunlight. This improves the collective photosynthetic capability of the population and leads to increased crop yield.
  • brassinosteroids may also improve tillering and the transport of assimilation products of photosynthesis from source to sink, subsequently improving the grain filling of rice seeds ⁇ see Wu, et al., Plant Cell, 20:2130-2145).
  • the OsD WARF4-dcF ⁇ cicnt mutant osdwarf4- 1 has a distinctly smaller lamina joint bending angle and is particularly suitable for close planting.
  • osdwarf4-l mutants can improve yield by 32% under dense planting conditions (44.4 plants/m 2 ) without any extra fertilizer ⁇ see Sakamoto et al., Nat. BiotechnoL, 24:105-109 (2006)).
  • OsBRIl loss-of-function mutants show a range of phenotypes, and the d61-l and d61-2 alleles produce agronomically useful traits such as semidwarf stature, erect leaves, and elongated neck internodes.
  • Two transgenic OsBRIl knock-down lines (BKDl 1 and BKD22) had grain yields that were calculated to be respectively 35% and 26% larger than the corresponding wild type plants planted at high density ⁇ see Morinaka et al., Plant Physiol., 141:924-931 (2006)).
  • the present invention relates to isolated dwarf and low-tillering (DLT) polynucleotides, polypeptides, vectors and host cells expressing isolated DLT polynucleotides capable of conferring desirable properties to plants, including altering tiller number.
  • DLT dwarf and low-tillering
  • the isolated DLT polynucleotides provided herein include nucleic acids comprising (a) a nucleotide sequence of SEQ ID NO: 1; (b) a nucleotide sequence of SEQ ID NO: 3; (c) a nucleotide sequence at least 70% identical to (a) or (b); (c) those that specifically hybridize to the complement of (a) or (b) under stringent hybridization conditions; (d) an open reading frame encoding a protein comprising a polypeptide sequence of SEQ ID NO: 2 or 4; (e) an open reading frame encoding a protein comprising a polypeptide sequence at least 70% identical to
  • SEQ ID NO: 2 or 4 SEQ ID NO: 2 or 4; and (f) a nucleotide sequence that is the complement of any one of (a)-(f).
  • the isolated DLT polypeptides provided herein include (a) the amino acid sequence of SEQ ID NO: 2 or 4, (b) an amino acid sequence derived from SEQ ID NO: 2 or 4 by substitution and/or deletion and/or addition of one or more amino acid residues wherein the amino acid sequence is capable of altering tiller number and (c) an amino acid sequence at least
  • the host cells provided herein include those comprising the isolated polynucleotides and vectors of the present invention.
  • the host cell can be from an animal, plant, or microorganism, such as E. coli. Plant cells are particularly contemplated.
  • the host cell can be isolated, excised, or cultivated.
  • the host cell may also be part of a plant.
  • the present invention further relates to a plant or a part of a plant that comprises a host cell of the present invention. Rice is particularly contemplated.
  • the present invention also relates to the transgenic seeds of the plants.
  • the present invention further relates to a method for producing a plant comprising regenerating a transgenic plant from a host cell of the present invention, or hybridizing a transgenic plant of the present invention to another non-transgenic plant. Plants produced by these methods are also encompassed by the present invention, and rice is particularly contemplated.
  • the present invention further relates to methods of altering a trait in a plant or part of a plant using the isolated polynucleotides, polypeptides, constructs and vectors of the present invention.
  • These traits include altering (i.e., increasing or decreasing) tiller number in comparison to a corresponding wild type plant and dwarf stature.
  • these traits are altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant.
  • the present invention further relates to the use of the isolated polynucleotides, polypeptides, constructs and vectors of the present invention to alter tiller number in a plant.
  • tiller number is altered by increasing the expression of DLT nucleic acids or proteins of the invention, such as SEQ ID NOs: 1-4, in a plant.
  • DLT nucleic acids or proteins of the invention such as SEQ ID NOs: 1-4
  • Figure 1 shows the phenotype of dlt mutant and wild type (WT) rice at (a) the vegetative phase and (b) the productive phase.
  • Figure 2(a) is a linkage map of DLT.
  • DLT is located between S240 and S1551 on chromosome 6, near the 5' telomere. Sequence-tagged site markers are named according to their chromosome physical positions, and relevant recombinant numbers are indicated above the linkage map.
  • PAC Pl -derived artificial chromosome
  • BAC BAC clones in the dlt mutation candidate region are shown under the linkage map.
  • Figure 2(b) shows the structure of the putative DL T full-length cDNA. The position of the mutation is shown. The underlined and italicized TGA is the putative new stop codon of an aberrant DLT in the dlt mutant.
  • Figure 2(c) is an RT-PCR of DL T. The band sizes of DLT are indicated. ACTINl was used as a control.
  • Figure 2(d) is a protein sequence alignment of DLT (OsGRAS32) and AtGRAS8 with AtGAI, AtRGAl, and AtSCR using Cluster W. The arrow indicates the mutation site. Five conserved motifs specific to GRAS proteins are indicated (leucine heptad I, VHIID, leucine heptad I, PFYRE, and SAW motifs). conserveed amino acids are highlighted in black and gray.
  • Figure 3 demonstrates the results of phenotypic complementation by introduction of the DLT gene into dlt mutants.
  • Figures 3 (a) and 3(b) show the gross morphology at (a) the vegetative phase and (b) the productive phase of WT, transgenic (dlt-c) and mutant plants.
  • Figure 3(c) is a PCR of WT, transgenic (dlt-c) and mutant plants. Arrows indicate band size.
  • Figure 4(a) shows the lamina joint bending response to various amounts of 24-epiBL by the micro-drop method.
  • Figure 4(b) shows the lamina joint bending response to 5 ng/ml 24- epiBL by the excised leaf segment method.
  • Figure 4(c) shows the coleoptile elongation response to 0.1 ⁇ M 24-epiBL.
  • Figure 5(a) shows the expression of DLT in various organs analyzed by quantitative RT-PCR analysis. Panicles and culms were collected when they had reached 1 cm length. The SAM, root, and the third leaf sheath and blade were harvested from 2-week-old plants.
  • Figure 5(b) shows the expression pattern of DLT in culm tissues. Intl to Int5 represent five internodes counted from top to bottom. The lowest zones of each internode were collected to extract RNA.
  • Figure 6 shows the GUS staining of PRO DLT :GUS transgenic line tissues. Pane 1 shows a longitudinal section after 3 days germination. The arrow indicates the SAM.
  • Pane 2 shows a longitudinal section of 7-day-old seedling after germination in dark. A primary root is also shown. The arrow indicates the SAM.
  • Pane 3 shows an unexpanded fourth leaf without greening from a 2-week old seedling.
  • Pane 4 shows the microscopic observation of a cross- section of pane 3.
  • Pane 5 shows a third leaf from a 2-week-old seedling. The arrow indicates the lamina joint.
  • Pane 6 shows a longitudinal section of a young culm. The arrow indicates the shoot apex.
  • Pane 7 shows a full-length elongating uppermost internode.
  • Pane 8 shows a full- length elongated third internode.
  • Pane 9 is a cross-section of the lowest part of pane 7.
  • Pane 10 is amagnified image of part of pane 9.
  • Pane 11 shows a young spikelet.
  • Pane 12 shows an older floret.
  • Pane 13 shows a young root with lateral root protruding.
  • Pane 14 shows a lateral root.
  • Scale bars 1 mm (panes 1-3, 5, 6, 9, and 11-14), 100 ⁇ m (panes 4 and 10) or 1 cm ( panes 7 and 8).
  • Figure 7(a) is a quantitative RT-PCR analysis of transcription levels for DLT at various times after exogenous 1 ⁇ M 24-epiBL treatment.
  • Figure 7(b) shows increased expression of DLT in d.2-1 and dll-2 mutants assayed by quantitative RT-PCR analysis. Shiokari plants are used as the WT control, and WT expression is set at 1.0.
  • Figure 8 shows a comparison of brassinosteroid (BR)-related gene expression between WT and dlt mutants with or without 24-epiBL. Gene expression was normalized to that of the rice ACTINl gene, and levels in WT or levels without 24-epiBL treatment are set as 1.0.
  • BR brassinosteroid
  • Figure 8(a) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT 'in WT and the dlt mutant.
  • Figure 8(b) shows the quantitative RT-PCR analysis of expression of BR biosynthetic genes and DLT in WT and the dlt mutant grown on half-strength MS with or without 1 ⁇ M 24-epiBL.
  • Figure 8(c) shows the quantitative RT-PCR analysis of expression of BR downstream genes and signaling genes.
  • Figure 9 shows an electrophoretic mobility shift assay.
  • a WT DNA probe derived from the DLT promoter was incubated with 200 ng recombinant OsBZRl . Competition reactions with either unlabeled WT probe or the mutant (Mt) form were performed to demonstrate the specific binding of OsBZRl to the BRRE in the DLT promoter.
  • Figure 10(a) shows the relative level of DLT expression in ten transgenic To plants.
  • Figure 10(b) shows the differing phenotypes of wild type and transgenic T 0 plants.
  • Figure 11 is a statistical analysis of the tiller number often T 2 transgenic plant lines performed during the heading stage.
  • Plant line number is shown on the x-axis, while tiller number is shown on the y-axis. The mean tiller number and standard deviation are provided. Statistically significant differences generated from a Student's t-test are indicated at the P ⁇ 0.05 (*) and P ⁇ 0.01 (**) levels.
  • nucleic acid As used herein, the terms “nucleic acid”, “polynucleotide”, “polynucleotide molecule”, “polynucleotide sequence” and plural variants are used interchangeably to refer to a wide variety of molecules, including single strand and double strand DNA and RNA molecules, cDNA sequences, genomic DNA sequences of exons and introns, chemically synthesized DNA and RNA sequences, and sense strands and corresponding antisense strands. Polynucleotides of the invention may also comprise known analogs of natural nucleotides that have similar properties as the reference natural nucleic acid.
  • polypeptide As used herein, the terms “polypeptide”, “protein” and plural variants are used interchangeably and refer to a compound made up of a single chain of amino acids joined by peptide bonds.
  • Polypeptides of the invention may comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Polypeptides may include both L-form and D-form amino acids.
  • non-genetically encoded amino acids include but are not limited to 2- aminoadipic acid; 3-aminoadipic acid; ⁇ -aminopropionic acid; 2-aminobutyric acid; 4- aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2- aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2'-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N- ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N- methylvaline; norvaline; norleucine;
  • Representative derivatized amino acids include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
  • Exemplary DLT polynucleotides of the invention are set forth as SEQ ID NOs: 1 and 2 and substantially identical sequences encoding proteins capable of altering the tiller number of a plant.
  • Exemplary DLT polypeptides of the invention are set forth as SEQ ID NOs: 2 and 4 and substantially identical proteins capable of altering the tiller number of a plant.
  • Substantially identical sequences are those that have at least 70%, preferably at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence using a sequence comparison algorithm or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • substantially identical nucleic acids or proteins perform substantially the same function.
  • substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population. An allelic difference may be as small as one base pair.
  • Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • a mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
  • Substantially identical nucleic acids are also identified as nucleic acids that hybridize specifically to or hybridize substantially to a reference sequence (e.g., SEQ ID NO: 1).
  • a reference sequence e.g., SEQ ID NO: 1
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol, 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI), or by visual inspection (see Ausubel et al., infra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • nucleic acid sequences are substantially identical.
  • Stringent conditions are those under which a nucleic acid probe will typically hybridize to its target sequence but to no other sequences when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA).
  • Stringent hybridization conditions and stringent hybridization wash conditions in the context of nucleic acid hybridization experiments are both sequence- and environment-dependent. 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, Elsevier, New York (1993).
  • highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Very stringent conditions are selected to be equal to the T m for a particular probe.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42 0 C, with the hybridization being carried out overnight.
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72 0 C for about 15 minutes.
  • stringent wash conditions is a 0.2X SSC wash at 65 0 C for 15 minutes ⁇ see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is IX SSC at 45 0 C for 15 minutes.
  • An example low stringency wash for a duplex of, e.g., more than 100 nucleotides is 4X - 6X SSC at 40 0 C for 15 minutes.
  • stringent conditions typically involve salt concentrations of less than about 1.0 M sodium ions, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 0 C.
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • a substantially identical nucleotide sequence preferably hybridizes to a reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 2X SSC, 0.1% SDS at 50 0 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in IX SSC, 0.1% SDS at 50 0 C, still more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 0.5X SSC, 0.1% SDS at 50 0 C, even more preferably in 7% sodium dode
  • nucleic acid sequences or proteins are substantially identical are that the that proteins encoded by the nucleic acids are substantially identical, share an overall three-dimensional structure, are biologically functional equivalents, or are immunologically cross-reactive with, or specifically bind to, each other. Nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially identical if the corresponding proteins are substantially identical. This may occur, for example, when two nucleotide sequences comprise conservatively substituted variants as permitted by the genetic code.
  • This also includes degenerate codon substitutions wherein the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acids Res., 19:5081(1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); and Rossolini et al. MoI. Cell Probes, 8:91-98 (1994)).
  • both the polynucleotides and the polypeptides of the present invention may be conservatively substituted at one or more residues.
  • conservative amino acid substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
  • Nucleic acids of the invention also comprise nucleic acids complementary to SEQ ID NOs: 1 and 3, and subsequences and elongated sequences of SEQ ID NOs: 1 and 3 and complementary sequences thereof.
  • Complementary sequences are two nucleotide sequences that comprise antiparallel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between base pairs.
  • complementary sequences maybe substantially similar to one another as described previously.
  • a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
  • a subsequence is a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence.
  • An exemplary subsequence is a probe or a primer.
  • An elongated sequence is one in which nucleotides (or other analogous molecules) are added to a nucleic acid sequence.
  • a polymerase e.g., a DNA polymerase
  • the nucleotide sequence may be combined with other DNA sequences, such as promoters, promoter regions, enhancers, polyadenylation signals, introns, additional restriction enzyme sites, multiple cloning sites, and other coding segments.
  • the present invention also provides vectors comprising the disclosed nucleic acids, including vectors for recombinant expression, wherein a nucleic acid of the invention is operatively linked to a functional promoter.
  • a promoter is in functional combination with the nucleic acid such that the transcription of the nucleic acid is controlled and regulated by the promoter region.
  • Vectors refer to nucleic acids capable of replication in a host cell, such as plasmids, cosmids, and viral vectors.
  • Polynucleotides of the present invention may be cloned, synthesized, altered, mutagenized, or combinations thereof. Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art. Site-specific mutagenesis to create base pair changes, deletions, or small insertions is also known in the art (see e.g., Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual.
  • Isolated polypeptides of the invention may be purified and characterized using a variety of standard techniques that are known to the skilled artisan (see e.g., Schroder et al., The Peptides, 1965, Academic Press, New York; Bodanszky, Principles of Peptide Synthesis, 2nd rev. ed. 1993, Springer- Verlag, Berlin/ New York; Ausubel (ed.), Short Protocols in Molecular Biology. 3rd ed., 1995, Wiley, New York).
  • the present invention also encompasses methods for detecting a nucleic acid molecule that encodes a DLT protein. Such methods may be used to detect gene variants or altered gene expression. Sequences detected by methods of the invention may detected, subcloned, sequenced, and further evaluated by any measure well known in the art using any method usually applied to the detection of a specific DNA sequence. Thus, the nucleic acids of the present invention may be used to clone genes and genomic DNA comprising the disclosed sequences. Alternatively, the nucleic acids of the present invention may be used to clone genes and genomic DNA of related sequences.
  • Levels of a DL T nucleic acid molecule may be measured, for example, using an RT-PCR assay (see e.g., Chiang, J Chromatogr. A., 806:209- 218 (1998) and references cited therein).
  • the present invention also encompasses genetic assays using DLT nucleic acids for quantitative trait loci (QTL) analysis and to screen for genetic variants, for example by allele-specific oligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad. Sd. USA, 80(l):278-282 (1983)), oligonucleotide ligation assays (OLAs) (Nickerson et al., Proc. Natl. Acad. ScL USA, 87(22):8923-8927 (1990)), single-strand conformation polymorphism (SSCP) analysis (Orita et al., Proc. Natl. Acad. Sci.
  • ASO allele-specific oligonucleotide
  • OVAs oligonucleotide ligation assays
  • SSCP single-strand conformation polymorphism
  • the present invention also encompasses functional fragments of a DLT polypeptide, for example, fragments that have the ability to alter tiller number similar to that of SEQ ID NOs: 2 and 4. Functional polypeptide sequences that are longer than the disclosed sequences are also encompassed.
  • one or more amino acids may be added to the N-terminus or C- terminus of an antibody polypeptide.
  • additional amino acids may be employed in a variety of applications, including but not limited to purification applications. Methods of preparing elongated proteins are known in the art.
  • the present invention also encompasses methods for detecting a polypeptide. Such methods can be used, for example, to determine levels of protein expression and correlate the level of expression with the presence or change in phenotype, trait, or level of expression in a different gene or gene product.
  • the method involves an immunochemical reaction with an antibody that specifically recognizes a protein.
  • An expression system refers to a host cell comprising a heterologous nucleic acid and the protein encoded by the heterologous nucleic acid.
  • a heterologous expression system may comprise a host cell transfected with a construct comprising a DLT nucleic acid encoding a protein operatively linked to a promoter, or a cell line produced by introduction of DLT nucleic acids into a host cell genome.
  • the expression system may further comprise one or more additional heterologous nucleic acids relevant to DLT function, such as targets of DLT transcriptional activation or repression activity. These additional nucleic acids may be expressed as a single construct or multiple constructs.
  • a construct for expressing a DLT protein may include a vector sequence and a DLT nucleotide sequence, wherein the DL T nucleotide sequence is operatively linked to a promoter sequence.
  • a construct for recombinant DL T expression may also comprise transcription termination signals and sequences required for proper translation of the nucleotide sequence. Preparation of an expression construct, including addition of translation and termination signal sequences, is known to one skilled in the art.
  • the promoter may be any polynucleotide sequence which shows transcriptional activity in the chosen plant cells, plant parts, or plants.
  • the promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the DNA sequence of the invention. Where the promoter is native or endogenous to the plant host, it is intended that the promoter is found in the native plant into which the promoter is introduced. Where the promoter is foreign or heterologous to the DNA sequence of the invention, the promoter is not the native or naturally occurring promoter for the operably linked DNA sequence of the invention.
  • the promoter may be inducible or constitutive. It may be naturally-occurring, may be composed of portions of various naturally-occurring promoters, or may be partially or totally synthetic.
  • promoters Guidance for the design of promoters is provided by studies of promoter structure, such as that of Harley et al., Nucleic Acids Res., 15:2343-61 (1987). Also, the location of the promoter relative to the transcription start may be optimized ⁇ see e.g., Roberts et al., Proc. Natl. Acad. Sd. USA, 76:760-4 (1979)). Many suitable promoters for use in plants are well known in the art. [0055] For example, suitable constitutive promoters for use in plants include the promoters from plant viruses, such as the peanut chlorotic streak caulimovirus (PClSV) promoter (U.S. Patent No.
  • PClSV peanut chlorotic streak caulimovirus
  • Suitable inducible promoters for use in plants include the promoter from the ACEl system which responds to copper (Mett et al., Proc. Natl. Acad. ScL USA, 90:4567-4571 (1993)); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., MoI. Gen. Genetics, 227:229-237 (1991); and Gatz et al., MoI. Gen. Genetics, 243:32-38 (1994)); and the promoter of the Tet repressor from TnIO (Gatz et al., MoI. Gen. Genet.
  • Another inducible promoter for use in plants is one that responds to an inducing agent to which plants do not normally respond.
  • An exemplary inducible promoter of this type is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone (Schena et al., Proc. Natl. Acad. ScL USA, 88:10421 (1991)) or the recent application of a chimeric transcription activator, XVE, for use in an estrogen receptor-based inducible plant expression system activated by estradiol (Zuo et al., Plant J., 24:265-273 (2000)).
  • inducible promoters for use in plants are described in EP 332104, PCT International Publication Nos. WO 93/21334 and WO 97/06269. Promoters composed of portions of other promoters and partially or totally synthetic promoters can also be used ⁇ see e.g., Ni et al., Plant J., 7:661-676 (1995) and PCT International Publication No. WO 95/14098 describing such promoters for use in plants).
  • Tissue-specific or tissue-preferential promoters useful for the expression of the novel DLT genes of the invention in plants including the cotton rubisco promoter disclosed in U.S. Patent No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent No. 5,604,121; and the cestrum yellow leaf curling virus promoter disclosed in PCT International Publication No. WO 01/73087.
  • Chemically inducible promoters useful for directing the expression of DLT genes in plants are disclosed in U.S. Patent No. 5,614,395.
  • the promoter may include, or be modified to include, one or more enhancer elements to thereby provide for higher levels of transcription.
  • Suitable enhancer elements for use in plants include the PClSV enhancer element (U.S. Patent No. 5,850,019), the CaMV 35S enhancer element (U.S. Patent Nos. 5,106,739 and 5,164,316) and the FMV enhancer element (Maiti et al., Transgenic Res., 6:143-156 (1997)). See also PCT International Publication No. WO 96/23898.
  • Such constructs can contain a 'signal sequence' or 'leader sequence' to facilitate co- translational or post-translational transport of the peptide of interest to certain intracellular structures such as the chloroplast (or other plastid), endoplasmic reticulum, or Golgi apparatus, or to be secreted.
  • the construct can be engineered to contain a signal peptide to facilitate transfer of the peptide to the endoplasmic reticulum.
  • a signal sequence is known or suspected to result in cotranslational or post-translational peptide transport across the cell membrane. In eukaryotes, this typically involves secretion into the Golgi apparatus, with some resulting glycosylation.
  • a leader sequence refers to any sequence that, when translated, results in an amino acid sequence sufficient to trigger co-translational transport of the peptide chain to a sub-cellular organelle. Thus, this includes leader sequences targeting transport and/or glycosylation by passage into the endoplasmic reticulum, passage to vacuoles, plastids including chloroplasts, mitochondria, and the like. Plant expression cassettes may also contain an intron, such that mRNA processing of the intron is required for expression.
  • Such constructs can also contain 5 ' and 3 ' untranslated regions.
  • a 3 ' untranslated region is a polynucleotide located downstream of a coding sequence.
  • Polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor are 3' untranslated regions.
  • a 5' untranslated region is a polynucleotide located upstream of a coding sequence.
  • the termination region may be native with the transcriptional initiation region, may be native with the sequence of the present invention, or may be derived from another source. Convenient termination regions are available from the Ti-plasmid of A.
  • tumefaciens such as the octopine synthase and nopaline synthase termination regions (see e.g., Guerineau et al., MoI. Gen. Genet., 262:141-144 (1991); Proudfoot, Cell, 64:671-674 (1991); Sanfacon et al., Genes Dev., 5:141-149 (1991); Mogen et al., Plant Cell, 2:1261-1272 (1990); Munroe et al., Gene, 91:151-158 (1990); Ballas et al., Nucleic Acids Res., 17:7891-7903 (1989); and Joshi et al., Nucleic Acid Res., 15:9627-9639 (1987)).
  • the vector and DLT sequences may be optimized for increased expression in the transformed host cell. That is, the sequences can be synthesized using host cell-preferred codons for improving expression, or may be synthesized using codons at a host- preferred codon usage frequency. Generally, the GC content of the polynucleotide will be increased ⁇ see e.g., Campbell et al., Plant Physiol, 92:1-11 (1990) for a discussion of host- preferred codon usage). Methods are known in the art for synthesizing host-preferred polynucleotides ⁇ see e.g., U.S. Patent Nos.
  • polynucleotides of interest are targeted to the chloroplast for expression.
  • the expression cassette may additionally contain a polynucleotide encoding a transit peptide to direct the nucleotide of interest to the chloroplasts.
  • transit peptides are known in the art (see e.g., Von Heijne et al., Plant MoI. Biol. Rep., 9:104-126 (1991); Clark et al., J. Biol.
  • the polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons (see e.g., U.S. Patent No.
  • a plant expression cassette i.e., a DLT open reading frame operatively linked to a promoter
  • a plant transformation vector which allows for the transformation of DNA into a cell.
  • Such a molecule may consist of one or more expression cassettes, and may be organized into more than one vector DNA molecule.
  • binary vectors are plant transformation vectors that utilize two non-contiguous DNA vectors to encode all requisite cis- and trans-acting functions for transformation of plant cells (Hellens et al., Trends in Plant Science, 5:446-451 (2000)).
  • a plant transformation vector comprises one or more DNA vectors for achieving plant transformation.
  • DNA vectors for achieving plant transformation.
  • These vectors are often referred to in the art as binary vectors.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mediated transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a polynucleotide of interest (i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired).
  • a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border)
  • a selectable marker that is engineered to be capable of expression in a plant cell
  • a polynucleotide of interest i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired.
  • different antibiotic or herbicide selectable markers may be preferred.
  • Selection markers used routinely in transformation include the nptllgene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra, Gene, 19:259-268 (1982); and Bevan et al., Nature, 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res., 18:1062 (1990), and Spencer et al., Theor. Appl. Genet., 79:625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, MoI. Cell.
  • sequences required for bacterial replication are sequences required for bacterial replication.
  • the cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein.
  • the selectable marker sequence and the sequence of interest are located between the left and right borders.
  • a second plasmid vector contains the transacting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as in understood in the art (Hellens et al., 2000).
  • a nucleotide sequence of the present invention is directly transformed into a plastid genome.
  • a major advantage of plastid transformation is that plastids are generally capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter.
  • Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817 and 5,545,818, in PCT International Application Publication WO 95/16783, and in McBride et al., Proc. Natl. Acad. Sci. USA, 91:7301-7305 (1994).
  • the basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation).
  • the 1 to 1.5 kb flanking regions facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome.
  • point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab et al., Proc. Natl. Acad. Sci. USA, 87:8526-8530 (1990); Staub et al., Plant Cell, 4:39-45 (1992)). This results in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves.
  • a nucleotide sequence of the present invention is inserted into a plastid-targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentially capable of high expression of the nucleotide sequence.
  • Host cells are cells into which a heterologous nucleic acid molecule of the invention may be introduced.
  • Representative eukaryotic host cells include yeast and plant cells, as well as prokaryotic hosts such as E.coli and B. subtilis.
  • Preferred host cells for functional assays substantially or completely lack endogenous expression of a DLT protein.
  • a host cell strain may be chosen which modulates the expression of the recombinant sequence, or modifies and processes the gene product in a specific manner. For example, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation of proteins).
  • Appropriate cell lines or host cells may be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system may be used to produce a non-glycosylated core protein product, and expression in yeast will produce a glycosylated product.
  • the present invention further encompasses recombinant expression of a DLT protein in a stable cell line.
  • Methods for generating a stable cell line following transformation of a heterologous construct into a host cell are known in the art ⁇ see e.g., Joyner, Gene Targeting: A Practical Approach. 1993, Oxford University Press, Oxford/New York).
  • transformed cells, tissues, and plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny or propagated forms thereof.
  • the present invention also provides DLT knockout plants comprising a disruption of a
  • DLT ⁇ ocMS DLT ⁇ ocMS.
  • a disrupted gene may result in expression of an altered level of full-length DLT protein or expression of a mutated variant DLT protein (e.g., SEQ ID NO: 4).
  • Plants with complete or partial functional inactivation of the DLT gene may be generated, e.g., by expressing a mutant DL T allele in the plant.
  • a knockout plant in accordance with the present invention may also be prepared using anti-sense, double-stranded RNA, or ribozyme DL T constructs, driven by a universal or tissue-specific promoter to reduce levels of DLT gene expression in somatic cells, thus achieving a "knock-down" phenotype.
  • the present invention also provides the generation of plants with conditional or inducible inactivation of DLT.
  • the present invention also encompasses transgenic plants with specific "knocked-in” modifications in the disclosed DLT gene, for example to create an over-expression mutant having a dominant negative phenotype.
  • "knocked-in” modifications include the expression of mutant alleles of the DLT gene.
  • DL T knockout plants may be prepared in monocot or dicot plants, such as maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees.
  • monocot or dicot plants such as maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach,
  • a plant refers to a whole plant, a plant organ (e.g., root, stem, leaf, flower bud, or embryo), a seed, a plant cell, a propagule, an embryo, other plant parts (e.g., protoplasts, pollen, pollen tubes, ovules, embryo sacs, zygotes) and progeny of the same.
  • Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, pollen).
  • introduction of a polynucleotide into plant cells is accomplished by one of several techniques known in the art, including but not limited to electroporation or chemical transformation ⁇ see e.g., Ausubel, ed. (1994) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Indiana). Markers conferring resistance to toxic substances are useful in identifying transformed cells (having taken up and expressed the test polynucleotide sequence) from non-transformed cells (those not containing or not expressing the test polynucleotide sequence). In one aspect of the invention, genes are useful as a marker to assess introduction of DNA into plant cells.
  • Stable transformation refers to introduction of a polynucleotide construct into a plant such that it integrates into the genome of the plant and is capable of being inherited by progeny thereof.
  • plant transformation methods involve transferring heterologous DNA into target plant cells (e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.), followed by applying a maximum threshold level of appropriate selection (depending on the selectable marker gene) to recover the transformed plant cells from a group of untransformed cell mass.
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely. Subsequently, the transformed cells are differentiated into shoots after placing on regeneration medium supplemented with a maximum threshold level of selecting agent (i.e., temperature and/or herbicide). The shoots are then transferred to a selective rooting medium for recovering rooted shoot or plantlet. The transgenic plantlet then grow into mature plant and produce fertile seeds (see e.g., Hiei et al., Plant J., 6:271-282 (1994); and Ishida et al., Nat. Biotechnol, 14:745-750 (1996)). A general description of the techniques and methods for generating transgenic plants are found in Ayres et al., CRC Crit. Rev.
  • Generation of transgenic plants may be performed by one of several methods, including but not limited to introduction of heterologous DNA by Agrobacterium into plant cells (Agrobacterium-mediated transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods to transfer DNA (see e.g., Hiei et al., Plant J., 6:271-282 (1994); Ishida et al., Nat. Biotechnol., 14:745-750 (1996); Ayres et al., CRC Crit. Rev. Plant ScL, 13:219-239 (1994); and Bommineni et al., Maydica, 1997, 42:107-120 (1997)).
  • the first method is co-cultivation of Agrobacterium with cultured isolated protoplasts. This method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts.
  • the second method is transformation of cells or tissues with Agrobacterium. This method requires (a) that the plant cells or tissues can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants.
  • the third method is transformation of seeds, apices or meristems with Agrobacterium. This method requires micropropagation. [0082] The efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art.
  • transformation efficiency may be enhanced by wounding the target tissue to be transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles ⁇ see e.g., Bidney et al., Plant Molec. Biol, 18:301-313 (1992).
  • the plant cells are transfected with vectors via particle bombardment (i.e., with a gene gun).
  • particle bombardment i.e., with a gene gun.
  • Particle mediated gene transfer methods are known in the art, are commercially available, and include, but are not limited to, the gas driven gene delivery instrument described in U.S. Patent No. 5,584,807. This method involves coating the polynucleotide sequence of interest onto heavy metal particles, and accelerating the coated particles under the pressure of compressed gas for delivery to the target tissue.
  • Other particle bombardment methods are also available for the introduction of heterologous polynucleotide sequences into plant cells.
  • these methods involve depositing the polynucleotide sequence of interest upon the surface of small, dense particles of a material such as gold, platinum, or tungsten.
  • the coated particles are themselves then coated onto either a rigid surface, such as a metal plate, or onto a carrier sheet made of a fragile material such as mylar.
  • the coated sheet is then accelerated toward the target biological tissue.
  • the use of the flat sheet generates a uniform spread of accelerated particles that maximizes the number of cells receiving particles under uniform conditions, resulting in the introduction of the polynucleotide sample into the target tissue.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the polypeptide of interest. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the polypeptide of interest, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers that are appropriate for the particular cell system that is used, such as those described in the literature (Scharf et al., Results Probl. Cell Differ., 20:125 (1994)).
  • the cells that have been transformed may be grown into plants in accordance with conventional ways ⁇ see e.g., McCormick et al., Plant Cell Rep., 5:81-84 (1986)). These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as transgenic seed) having a polynucleotide of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.
  • Transgenic plants of the invention can be homozygous for the added polynucleotides; i.e., a transgenic plant that contains two added sequences, one sequence at the same locus on each chromosome of a chromosome pair.
  • a homozygous transgenic plant can be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains the added sequences according to the invention, germinating some of the seed produced and analyzing the resulting plants produced for enhanced enzyme activity (i.e., herbicide resistance) and/or increased plant yield relative to a control (native, non-transgenic) or an independent segregant transgenic plant.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous polynucleotides. Selfing of appropriate progeny can produce plants that are homozygous for all added, exogenous polynucleotides that encode a polypeptide of the present invention. Back-crossing to a parental plant and outcrossing with a non-transgenic plant are also contemplated. [0089] Following introduction of DNA into plant cells, the transformation or integration of the polynucleotide into the plant genome is confirmed by various methods such as analysis of polynucleotides, polypeptides and metabolites associated with the integrated sequence. [0090] DLT Inhibitors
  • the present invention further discloses assays to identify DLT binding partners and DLT inhibitors.
  • DLT antagonists/inhibitors are agents that alter chemical and biological activities or properties of a DLT protein. Methods of identifying inhibitors involve assaying a reduced level or quality of DLT function in the presence of one or more agents. Exemplary DLT inhibitors include small molecules as well as biological inhibitors as described herein below.
  • the term "agent” refers to any substance that potentially interacts with a DLT nucleic acid or protein, including any of synthetic, recombinant, or natural origin. An agent suspected to interact with a protein may be evaluated for such an interaction using the methods disclosed herein.
  • Exemplary agents include but are not limited to peptides, proteins, nucleic acids, small molecules (e.g., chemical compounds), antibodies or fragments thereof, nucleic acid- protein fusions, any other affinity agent, and combinations thereof.
  • An agent to be tested may be a purified molecule, a homogenous sample, or a mixture of molecules or compounds.
  • a small molecule refers to a compound, for example an organic compound, with a molecular weight of less than about 1,000 daltons, more preferably less than about 750 daltons, still more preferably less than about 600 daltons, and still more preferably less than about 500 daltons.
  • a small molecule also preferably has a computed log octanol-water partition coefficient in the range of about -A to about +14, more preferably in the range of about -2 to about +7.5.
  • exemplary nucleic acids that may be used to disrupt DLT function include antisense RNA and small interfering RNAs (siRNAs) ⁇ see e.g., U.S. Application Publication No. 20060095987).
  • inhibitory molecules may be prepared based upon the DLT gene sequence and known features of inhibitory nucleic acids ⁇ see e.g., Van der Krol et al., Plant Cell, 2:291- 299 (1990); Napoli et al., Plant Cell, 2:279-289 (1990); English et al., Plant Cell, 8:179-188 (1996); and Waterhouse et al., Nature Rev. Genet, 2003, 4:29-38 (2003).
  • Agents may be obtained or prepared as a library or collection of molecules.
  • a library may contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more.
  • a molecule may comprise a naturally occurring molecule, a recombinant molecule, or a synthetic molecule.
  • a plurality of agents in a library may be assayed simultaneously.
  • agents derived from different libraries may be pooled for simultaneous evaluation.
  • Representative libraries include but are not limited to a peptide library (U.S. Patent Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Patent Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Patent Nos.
  • a library may comprise a random collection of molecules.
  • a library may comprise a collection of molecules having a bias for a particular sequence, structure, or conformation, for example, as for inhibitory nucleic acids (see e.g., U.S. Patent Nos. 5,264,563 and 5,824,483).
  • Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. patents cited herein above. Numerous libraries are also commercially available.
  • a control level or quality of DLT activity refers to a level or quality of wild type DLT activity, for example, when using a recombinant expression system comprising expression of SEQ ID NOs: 1 or 3.
  • a control level or quality of DLT activity comprises a level or quality of activity in the absence of the agent.
  • a control level may also be established by a phenotype or other measureable trait.
  • Assaying the inhibiting capacity of an agent may comprise determining a level of DLT gene expression; determining DNA binding activity of a recombinantly expressed DLT protein; determining an active conformation of a DLT protein; or determining a change in a trait in response to binding of a DLT inhibitor (e.g., increased or decreased tiller number).
  • a method of identifying a DLT inhibitor may comprise (a) providing a cell, plant, or plant part expressing a DLT protein; (b) contacting the cell, plant, or plant part with an agent; (c) examining the cell, plant, or plant part for a change in a trait as compared to a control; and (d) selecting an agent that induces a change in the trait as compared to a control. Any of the agents so identified in the disclosed inhibitory or binding assays (see hereinafter) may be subsequently applied to a cell, plant or plant part as desired to effectuate a change in that cell, plant or plant part.
  • the present invention also encompasses a rapid and high throughput screening method that relies on the methods described herein.
  • This screening method comprises separately contacting a DLT protein with a plurality of agents.
  • the plurality of agents may comprise more than about 10 4 samples, or more than about 10 5 samples, or more than about 10 6 samples.
  • the in vitro and cellular assays of the invention may comprise soluble assays, or may further comprise a solid phase substrate for immobilizing one or more components of the assay.
  • a DLT protein, or a cell expressing a DLT protein may be bound directly to a solid state component via a covalent or non-covalent linkage.
  • the binding may include a linker molecule or tag that mediates indirect binding of a DLT protein to a substrate.
  • the present invention also encompasses methods of identifying of a DLT inhibitor by determining specific binding of a substance (e.g., an agent described previously) to a DLT protein.
  • a method of identifying a DLT binding partner may comprise: (a) providing a DLT protein of SEQ ID NO: 2 or 4; (b) contacting the DLT protein with one or more agents under conditions sufficient for binding; (c) assaying binding of the agent to the isolated DLT protein; and (d) selecting an agent that demonstrates specific binding to the DLT protein.
  • Specific binding may also encompass a quality or state of mutual action such that binding of an agent to a DLT protein is inhibitory.
  • Specific binding refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biological materials.
  • the binding of an agent to a DLT protein may be considered specific if the binding affinity is about IxIO 4 M "1 to about 1x10 6 M "1 or greater.
  • Specific binding also refers to saturable binding.
  • Scatchard analysis may be carried out as described, for example, by Mak et al, J Biol. Chem., 264:21613-21618 (1989).
  • Several techniques may be used to detect interactions between a DLT protein and an agent without employing a known competitive inhibitor.
  • Representative methods include, but are not limited to, Fluorescence Correlation Spectroscopy, Surface-Enhanced Laser Desorption/Ionization Time-Of-Flight Spectroscopy, and BIACORE® technology, each technique described herein below. These methods are amenable to automated, high-throughput screening.
  • FCS Fluorescence Correlation Spectroscopy
  • the sample size may be as low as 10 3 fluorescent molecules and the sample volume as low as the cytoplasm of a single bacterium.
  • the diffusion rate is a function of the mass of the molecule and decreases as the mass increases.
  • FCS may therefore be applied to protein-ligand interaction analysis by measuring the change in mass and therefore in diffusion rate of a molecule upon binding.
  • the target to be analyzed e.g., a DLT protein
  • a sequence tag such as a poly-histidine sequence, inserted at the N-terminus or C-terminus.
  • the expression is mediated in a host cell, such as E.coli, yeast, Xenopus oocytes, or mammalian cells.
  • the protein is purified using chromatographic methods.
  • the poly-histidine tag may be used to bind the expressed protein to a metal chelate column such as Ni 2+ chelated on iminodiacetic acid agarose.
  • the protein is then labeled with a fluorescent tag such as carboxytetramethylrhodamine or BODIPYTM reagent (available from Molecular Probes of Eugene, Oregon).
  • the protein is then exposed in solution to the potential ligand, and its diffusion rate is determined by FCS using instrumentation available from Carl Zeiss, Inc. (Thornwood of New York, New York). Ligand binding is determined by changes in the diffusion rate of the protein.
  • SELDI Surface-Enhanced Laser Desorption/Ionization
  • a target protein e.g., a DLT protein
  • the target protein is bound to a SELDI chip either by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • a chip thus prepared is then exposed to the potential ligand via, for example, a delivery system able to pipet the ligands in a sequential manner (autosampler).
  • the chip is then washed in solutions of increasing stringency, for example a series of washes with buffer solutions containing an increasing ionic strength. After each wash, the bound material is analyzed by submitting the chip to SELDI-TOF.
  • Ligands that specifically bind a target protein are identified by the stringency of the wash needed to elute them.
  • BIACORE® relies on changes in the refractive index at the surface layer upon binding of a ligand to a target protein (e.g., a DLT protein) immobilized on the layer.
  • a target protein e.g., a DLT protein
  • a collection of small ligands is injected sequentially in a 2-5 microliter cell, wherein the target protein is immobilized within the cell. Binding is detected by surface plasmon resonance (SPR) by recording laser light refracting from the surface.
  • SPR surface plasmon resonance
  • the refractive index change for a given change of mass concentration at the surface layer is practically the same for all proteins and peptides, allowing a single method to be applicable for any protein.
  • a target protein is recombinantly expressed, purified, and bound to a BIACORE® chip. Binding may be facilitated by utilizing a poly-histidine tag or by other interaction such as ion exchange or hydrophobic interaction.
  • a chip thus prepared is then exposed to one or more potential ligands via the delivery system incorporated in the instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands in a sequential manner (autosampler).
  • the SPR signal on the chip is recorded and changes in the refractive index indicate an interaction between the immobilized target and the ligand. Analysis of the signal kinetics of on rate and off rate allows the discrimination between non-specific and specific interaction (see also Homola et al., Sensors and Actuators, 54:3-15 (1999) and references therein).
  • the present invention also encompasses methods of identifying DLT binding partners and inhibitors that rely on a conformational change of a DLT protein when bound by or otherwise interacting with a substance (e.g., an agent described previously). For example, application of circular dichroism to solutions of macromolecules reveals the conformational states of these macromolecules. The technique may distinguish random coil, alpha helix, and beta chain conformational states.
  • circular dichroism analysis may be performed using a recombinantly expressed DLT protein.
  • a DLT protein is purified, for example by ion exchange and size exclusion chromatography, and mixed with an agent. The mixture is subjected to circular dichroism.
  • the conformation of a DLT protein in the presence of an agent is compared to a conformation of a DLT protein in the absence of the agent.
  • a change in conformational state of a DLT protein in the presence of an agent identifies a DLT binding partner or inhibitor. Representative methods are described in U.S. Patent Nos. 5,776,859 and 5,780,242.
  • Antagonistic activity of the inhibitor may be assessed using functional assays, such assaying nitrate content, nitrate uptake, lateral root growth, or plant biomass, as described herein.
  • cells expressing DLT may be provided in the form of a kit useful for performing an assay of DLT function.
  • a kit for detecting a DLT may include cells transfected with DNA encoding a full-length DLT protein and a medium for growing the cells.
  • Assays of DLT activity that employ transiently transfected cells may include a marker that distinguishes transfected cells from non-transfected cells.
  • a marker may be encoded by or otherwise associated with a construct for DLT expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding DLT and the marker.
  • Representative detectable molecules that are useful as markers include but are not limited to a heterologous nucleic acid, a protein encoded by a transfected construct (e.g., an enzyme or a fluorescent protein), a binding protein, and an antigen.
  • Assays employing cells expressing recombinant DLT or plants expressing DLT may additionally employ control cells or plants that are substantially devoid of native DLT and, optionally, proteins substantially similar to a DLT protein.
  • a control cell When using transiently transfected cells, a control cell may comprise, for example, an untransfected host cell.
  • a control cell When using a stable cell line expressing a DLT protein, a control cell may comprise, for example, a parent cell line used to derive the -DZr-expressing cell line.
  • a method for producing an antibody that specifically binds a DLT protein.
  • a full-length recombinant DLT protein is formulated so that it may be used as an effective immunogen, and used to immunize an animal so as to generate an immune response in the animal.
  • the immune response is characterized by the production of antibodies that may be collected from the blood serum of the animal.
  • An antibody is an immunoglobulin protein, or antibody fragments that comprise an antigen binding site (e.g., Fab, modified Fab, Fab', F(ab') 2 or Fv fragments, or a protein having at least one immunoglobulin light chain variable region or at least one immunoglobulin heavy chain region).
  • Antibodies of the invention include diabodies, tetrameric antibodies, single chain antibodies, tretravalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and domain-specific antibodies that recognize a particular epitope. Cell lines that produce anti-DLT antibodies are also encompassed by the invention.
  • Specific binding of an antibody to a DLT protein refers to preferential binding to a DLT protein in a heterogeneous sample comprising multiple different antigens. Substantially lacking binding describes binding of an antibody to a control protein or sample, i.e., a level of binding characterized as non-specific or background binding.
  • the binding of an antibody to an antigen is specific if the binding affinity is at least about 10 ⁇ 7 M or higher, such as at least about 10 ⁇ 8 M or higher, including at least about 10 ⁇ 9 M or higher, at least about 10 "11 M or higher, or at least about 10 ⁇ 12 M or higher.
  • DLT antibodies prepared as disclosed herein may be used in methods known in the art relating to the expression and activity of DLT proteins, e.g., for cloning of nucleic acids encoding a DLT protein, immunopurification of a DLT protein, and detecting a DLT protein in a plant sample, and measuring levels of a DLT protein in plant samples.
  • an antibody of the present invention may further comprise a detectable label, including but not limited to a radioactive label, a fluorescent label, an epitope label, and a label that may be detected in vivo.
  • Methods for selection of a label suitable for a particular detection technique, and methods for conjugating to or otherwise associating a detectable label with an antibody are known to one skilled in the art.
  • dlt A dwarf and low tillering mutant (dlt) was identified from a library of rice T-DNA insertion mutants from the (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). As shown in Figure 1, the dlt mutant is 60% as tall and has only half the number of tillers of the corresponding Zhonghua 11 wild-type (WT) plants. In addition, the mutant plant has a compact morphology, erect leaves and panicles, reduced fertility, and a reduced ratio of the second internode to the total internode length. Microscopic observation also revealed that the dlt mutant had decreased cell length and less organized cellular morphology. These observations are consistent with plants that exhibit altered brassinosteroid signaling and/or synthesis.
  • DLT encodes a protein of 617 amino acids (DLT; SEQ ID NO: 2) that belongs to the plant-specific GRAS family.
  • the frameshift caused by the deletion generates an unrelated peptide (dlt; SEQ ID NO: 4) after amino acid 142 and a new stop codon (TGA) that is 2 bp downstream from the original stop codon (TAA) ⁇ see Figures 2(b) and (d)).
  • the mutation at amino acid 142 leads to loss of all conserved motifs, indicating that dlt is a knockout mutant ⁇ see Figure 2(d)).
  • RT-PCR analysis showed that aberrant DLT transcripts are expressed in the dlt plant ⁇ see Figure 2(c)).
  • a genomic fragment of 7577 bp containing the entire DLT coding sequence, 3253 bp of the 5' upstream region and 2471 bp of the 3' downstream region was digested from the BAC clone OSJNBa0038F22( Arizona Genomics Institute) using restriction enzymes BamHI and Kpnl (Promega). The fragment was recovered, ligated with a binary vector pCAMBIA1300 (Cambia, Australia), and transformed into Agrobacterium AGLl (ATCC). The transformed AGLl was used to infect the callus of a dit mutant as described, for example, in Yi et al., Journal of Genetics and Genomics, 28(4):352-358 (2001).
  • PCR amplification was performed on the genomic DNA of the regenerated plants using forward (5'- CATCAATCCATTGCAGGGACGAT-3' (SEQ ID NO: 5)) and reverse (5'- CGTTGAGCGTGAAGTGCAGGAA-S' (SEQ ID NO: 6) primers flanking the 62 bp deleted segment of the DLT gene. Forty positive transgenic plants were identified ⁇ see Figure 3(c)).
  • a third brassinosteroid response assay was performed to assess the effect of 24-epi- brassinolide on coleoptile elongation in dlt mutants (see e.g., Yamamuro et al., 2000). Seeds were germinated and grown on 0.7% agar medium supplemented with various concentrations of 24-epiBL. Comparison of coleoptile length showed that the dlt mutant has a much lower response to BL than WT does (see Figure 4(c)).
  • RNAs were extracted from various organs and tissues of the dlt mutant, using MMLV reverse transcriptase (Promega) to perform reverse transcription. Real-time fluorescence quantitative PCR was subsequently performed to detect the expression of DLT.
  • Rice ACTLNl was used as the internal control, and SYBR Green I was used as the dye.
  • Primers 5'- TGCGGATACTCAACGCCATC A-3' (forward; SEQ ID NO: 7) and 5'- ACTCGCCGACTCCGGTGATC-3' (reverse; SEQ ID NO: 8) were used to amplify DLT, and primers 5'-AGCAACTGGGATGATATGGA-S' (forward; SEQ ID NO: 9) and 5'-
  • CAGGGCGATGTAGGAAAGC-3' (reverse; SEQ ID NO: 10) were used to amplify ACTINl.
  • GUS staining showed that, in young seedlings, DLT is mainly expressed in the shoot apical meristem and elongating cells (Figure 6, panes 1 and 2). In leaves, DLT is expressed at significantly higher levels in unexpanded leaves than in green functional leaves, although with a slightly preferential expression in the leaf joint ( Figure 6, panes 3-5). DLT expression could not be detected in mature leaves by GUS staining.
  • DLT is expressed strongly in the internodes prior to rapid elongation
  • DLT also showed higher expression in the vascular cylinder and lateral root outgrowth locations, but much lower expression in cortex tissues ( Figure 6, panes 13 and 14). These expression patterns correlate well with DLTs putative function in cell elongation and division. Young tissues are those in which cells are actively dividing and elongating, and this is also where BR functions are most active.
  • d.2-1 is a dwarf mutant exhibiting a pleiotropic abnormal phenotype similar to that of the rice brassinosteroid-insensitive mutant, d61.
  • Hong et al. concluded that the D2 gene encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway ⁇ see Hong et al., Plant Cell, 15:2900-2910 (2003)).
  • dll-2 is a dwarf mutant that bears small round grains. Tanabe et al.
  • DIl gene also encodes a cytochrome P450 that plays a role in the late brassinosteroid synthesis pathway ⁇ see Tanabe et al., Plant Cell, 17:776-790 (2005)).
  • the experiment was performed in triplicate. As shown in Figure 7(b), DLT expression in d.2-1 and dll-2 plants was significantly greater than in wild type Shiokari plants. The observations from these two brassinosteroid-synthesizing mutants further reinforce the conclusion that brassinosteroids negatively regulate the expression of DLT
  • Fluorescence quantitative PCR was used to determine the expression levels of several known brassinosteroid synthesizing genes ⁇ D2, DIl, OsCPD and OsBR ⁇ ox) and two genes
  • BRs brassinosteroids
  • BRREs brassinosteroid response elements
  • the OsBZRl coding region was cloned into a maltose binding protein (MBP) fusion vector (pET-MALc-H vector) using primers Os-BES lNAsp718 (5'-CTCGGTACCGGAGCTGGTGGGTATGACGTC-S'; SEQ ID NO: 13) and OsBESlCHind3 (5'-CGCAAGCTTTCATTTCGCGCCGACGCCGAGC-S'; SEQ ID NO: 14).
  • MBP-OsBZRl was purified from E. coli using amylose resin (NEB).
  • Wild type oligos derived from the DLT promoter, and mutant forms in which each nucleotide in a brassinosteroid response element (CGTGCG; SEQ ID NO: 15) was replaced with an adenosine were synthesized and annealed.
  • the WT probe was labeled with 32 P-C-ATP, and approximately 0.5 ng of probe was used for each binding assay. For competition experiments, excess unlabeled probe was added to the reactions at indicated molar ratios compared to labeled probe.
  • OsBZRl can bind to the labeled WT probe and unlabeled WT probe competes for binding with OsBRZl. OsBZRl does not bind to the mutant probe in which the BRRE is altered as described above. These results demonstrate that OsBZRl can bind to the DLT promoter through the BRRE.
  • the recombinant expression vector was transformed into the callus of the rice cultivar Zhonghua 11 via an Agrobacterium AGl (ATCC) mediated method. Ten transgenic T 0 generation plants were obtained by resistance screening.
  • Agrobacterium AGl Agrobacterium AGl
  • RNAs were extracted from the ten To generation transgenic positive plants and expression levels of DLT were determined by quantitative fluorescence PCR as described previously.
  • the DLT expression level in wild type rice Zhonghua 11 was set as 1 and the ratio of the DLT expression level in each of the transgenic plants to that of the wild type was calculated.
  • the experiments were performed in triplicate.
  • T 0 generation transgenic plants had significantly upregulated DLT gene expression. Of these seven plants, five of them had more than 100-fold increase in DL T expression levels as compared to their Zhonghua 11 counterparts. These five plants exhibited a curly leaf blade, enlarged lamina joint angle, increased tiller number and slight dwarfism (see plant no. 5 in Figure 10(b)). The other two T 0 transgenic plants, which had less than 100-fold increases in expression of DLT, exhibited significantly increased tiller numbers and slightly increased height, (see plant no. 10 in Figure 10(b)). These observations indicate that increased DLT expression leads to increased tiller number in rice.
  • Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110
  • Asn Ala lie Thr Pro lie Pro Arg Phe Leu His Phe Thr Leu Asn Glu 305 310 315 320
  • Trp Glu Ala Arg Phe Ala Arg Ala Leu Arg Tyr Tyr Ala Ala Ala Phe 465 470 475 480
  • Tyr Gly Pro lie VaI Arg Ala Lys Arg Thr Arg Met Gly Gly Asp Gly 100 105 110

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Abstract

L'invention porte sur des compositions et des procédés destinés à modifier le nombre de talles d'une plante à l'aide de gènes de nanisme et de faible tallage (DLT).
PCT/IB2009/055988 2008-12-30 2009-12-29 Gènes associés au nombre de talles d'une plante et leurs utilisations WO2010076766A1 (fr)

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