WO2010097746A1 - Gène de métallothionéine conférant une tolérance au stress abiotique dans des plantes et ses utilisations - Google Patents

Gène de métallothionéine conférant une tolérance au stress abiotique dans des plantes et ses utilisations Download PDF

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WO2010097746A1
WO2010097746A1 PCT/IB2010/050755 IB2010050755W WO2010097746A1 WO 2010097746 A1 WO2010097746 A1 WO 2010097746A1 IB 2010050755 W IB2010050755 W IB 2010050755W WO 2010097746 A1 WO2010097746 A1 WO 2010097746A1
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plant
osmtia
protein
sequence
expression
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Chengcai Chu
Zhao Yang
Yaorong Wu
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Syngenta Participations AG
Institute of Genetics and Developmental Biology of CAS
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Syngenta Participations AG
Institute of Genetics and Developmental Biology of CAS
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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
    • C07K14/825—Metallothioneins
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the invention relates generally to compositions and methods for conferring abiotic stress tolerance to plants, including polynucleotides, polypeptides, vectors and host cells.
  • the present invention also relates generally to plants transformed by the aforementioned compositions and methods.
  • MTs Metallothioneins
  • cysteine-rich proteins that can bind metals via mercaptide bonds. Since the first MT was characterized from horse kidneys as cadmium-binding proteins, numerous MT genes have been identified in both eukaryotes and prokaryotes. Based on the number and arrangement of cysteine residues, all the plant MTs belong to class II (in contrast to the vertebrate class I) and can be further subdivided into four types based on their amino acid sequences.
  • typel MTs are expressed preferentially in roots, whereas type 2 MTs are found mainly in the leaves, type 3MTs are expressed at high levels in the ripe fruits and Arabidopsis leaves, and expression of type 4 MTs, also known as the Ec type, is only found in developing seeds.
  • ROS reactive oxygen species
  • antioxidants including low molecular mass antioxidants (glutathione, ascorbate, carotenoids) and ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX).
  • SOD superoxide dismutase
  • CAT catalase
  • APX ascorbate peroxidase
  • the present invention relates to isolated OsMTIa polynucleotides, polypeptides, vectors and host cells expressing isolated OsMTIa polynucleotides capable of imparting a variety of properties to plants, such as improved drought tolerance.
  • the isolated OsMTIa polynucleotides provided herein include nucleic acids comprising (a) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1 ; (b) a nucleic acid comprising a nucleotide sequence at least 90% identical to (a); (c) a nucleic acid comprising a nucleotide sequence that specifically hybridizes to the complement of (a) under stringent hybridization conditions and which encodes a metallothionein; (d) a nucleic acid comprising an open reading frame encoding a OsMTIa protein comprising a polypeptide sequence of SEQ ID NO: 2; (e) a nucleic acid comprising an open reading frame encoding a OsMTIa protein comprising a polypeptide sequence derived from (d) by substitution, deletion or addition of one or several amino acids in the amino acid sequence in (d) and having metallothionein activity; and (f) a nucleic acids comprising
  • the isolated OsMTIa polypeptides provided herein include (a) a polypeptide sequence of SEQ ID NO: 2; and (b) a polypeptide sequence derived from (a) by substitution, deletion or addition of one or several amino acids in the amino acid sequence in (a) and having metallothionein activity.
  • 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. Plants such as rice, maize and soybean are 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 plants having improved tolerance to drought and improved zinc uptake are particularly contemplated, as are crop plants, such as rice, maize and soybean.
  • 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 improved tolerance to drought and improved zinc uptake.
  • the aforementioned traits are altered so that they are increased or otherwise improved.
  • one or more traits of a plant are altered by expressing in a plant an isolated nucleic acid such as (a) a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1; (b) a nucleic acid comprising a nucleotide sequence at least 90% identical to (a); (c) a nucleic acid comprising a nucleotide sequence that specifically hybridizes to the complement of (a) under stringent hybridization conditions and which encodes a metallothionein; (d) a nucleic acid comprising an open reading frame encoding a OsMTIa protein comprising a polypeptide sequence of SEQ ID NO: 2; (e) a nucleic acid comprising an open reading frame encoding a OsMTIa protein comprising a polypeptide sequence derived from (d) by substitution, deletion or addition of one or several amino acids in the amino acid sequence in (d) and having metallothionein activity; and (f) a nucleic acid such as
  • one or more traits of a plant are altered by expressing in a plant an isolated hypermorphic OsMTIa allele. In another embodiment, one or more traits of a plant are altered by increasing the expression of a OsMTIa nucleic acid or polypeptide in the plant. In yet another embodiment, one or more traits of a plant are altered by altering the function of a OsMTIa polypeptide in the plant.
  • the present invention further relates to plants, plant parts and transgenic seeds created through the aforementioned methods of altering a trait in a plant.
  • Such contemplated plants, plant parts and transgenic seeds may be created directly from the aforementioned methods.
  • the contemplated plants, plant parts and transgenic seeds may be derived from a host cell (e.g., regenerated from a host cell) or produced by crossing a transgenic plant with one or more altered traits with a non-transgenic plant.
  • the present invention further relates to methods of increasing enzymatic activity in a plant comprising expressing an isolated OsMTIa polynucleotide in accordance with the invention in the plant, wherein the enzymatic activity is that of an enzyme selected from the group consisting of catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX).
  • CAT catalase
  • POD peroxidase
  • APX ascorbate peroxidase
  • the activity of one or more of these antioxidative enzymes is increased by increasing the expression one or more OsMTIa polypeptides in a host cell, plant or plant part.
  • the activity of one or more of these antioxidative enzymes is decreased by decreasing the expression of one or more OsMTIa polypeptides in a host cell, plant or plant part.
  • the present invention further relates to methods of increasing the expression of a zinc finger transcription factor in a plant comprising expressing an isolated OsMTIa polynucleotide in accordance with the invention in the plant, wherein the zinc finger transcription factor is encoded by a gene selected from the group consisting of Ossiz, ZFl and WRKY71.
  • the expression of one or more of these zinc finger transcription factor genes is increased by increasing the expression one or more OsMTIa polypeptides in a host cell, plant or plant part.
  • the activity of one or more of these zinc finger transcription factor genes is decreased by decreasing the expression of one or more OsMTIa polypeptides in a host cell, plant or plant part.
  • the present invention further relates to methods of identifying OsMTIa binding agents and inhibitors.
  • the method comprises (a) providing an isolated OsMTIa protein; (b) contacting the isolated OsMTIa protein with an agent under conditions sufficient for binding; (c) assaying binding of the agent to the isolated OsMTIa protein; and (d) selecting an agent that demonstrates specific binding to the isolated OsMTIa protein.
  • the method comprises (a) providing a host cell expressing a OsMTIa protein; (b) contacting the host cell with an agent; (c) assaying expression of OsMTIa protein; and (d) selecting an agent that induces altered expression of OsMTIa protein.
  • the method comprises (a) providing a plant or part of a plant expressing a OsMTIa protein; (b) contacting the plant or the part of the plant with an agent; (c) assaying for alteration of a trait of the plant or the part of the plant; and (d) selecting an agent that alters the trait.
  • the traits to be assayed are those known to be affected by OsMTIa expression (e.g., drought tolerance, zinc uptake).
  • agents that increase or otherwise improve these traits are selected.
  • agents that negatively impact a trait are contemplated as well.
  • the present invention also relates to methods of inhibiting OsMTIa in a plant using the binding agents and inhibitors identified by the methods herein.
  • Figure 1 shows OsMTIa expression in Nipponbare by Northern blotting. Lane 1 is from flower. Lane 2 is from leaf. Lane 3 is from shoots. Lane 4 is from root.
  • Figure 2 shows OsMTIa expression in metal-treated Brazil upland rice.
  • Figure 3 shows OsMTIa expression in non-biologically stressed Brazil upland rice.
  • Figure 4 shows the phenotypical change and dehydration rate in wild type
  • Figure 5 shows CAT, APX and POD activity assay results.
  • Figure 6A shows the relative expression of Ossiz, ZFl and WRKY71 in OsMTIa- transformed plants.
  • Figure 6B shows the synergistic expression of exogenous OsMTIa and
  • FIG. 6C shows the time course of expression of Ossiz in zinc treated plants.
  • OsMT 1 a Nucleic Acids and Proteins
  • 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; norleu
  • 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.
  • isolated refers to polynucleotides and polypeptides that, but for at least one act of man, do not exist in whatever form or amount they are found.
  • Exemplary embodiments include polynucleotides and polypeptides that are partially, substantially or wholly purified from other molecular species; polynucleotides and polypeptides that are heterologous to a particular cell, organism, or part of an organism; polynucleotides and polypeptides that are not heterologous to a particular cell, organism, or part of an organism, but are expressed at an altered level as a result of the at least one act of man; and polynucleotides and polypeptides that are expressed in the progeny or other downstream products (e.g., fruit) of a cell, organism, or part of an organism subject to the at least one act of man.
  • progeny or other downstream products e.g., fruit
  • Exemplary OsMTIa polynucleotides of the invention are set forth as SEQ ID NO: 1 and substantially identical sequences encoding OsMTIa proteins capable of altering a trait of a plant, for example, drought tolerance.
  • Exemplary OsMTIa polypeptides of the invention are set forth as SEQ ID NO: 2 and substantially identical proteins capable of altering a trait of a plant, for example, drought tolerance.
  • Substantially identical sequences are those that have at least 60%, 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).
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • 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.
  • HSPs high scoring sequence pairs
  • 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).
  • M forward score for a pair of matching residues
  • N penalty score for mismatching residues; always ⁇ 0.
  • 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.
  • W wordlength
  • E expectation
  • BLOSUM62 scoring matrix see Henikoff & Henikoff, Proc. Natl. Acad. ScL 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. ScL 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.
  • 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.
  • 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. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical.
  • 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 dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50 0 C with washing in 0.
  • SDS 7% sodium do
  • nucleic acid sequences or proteins are substantially identical is 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.
  • 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 NO: 1 and subsequences and elongated sequences of SEQ ID NO: 1 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 operative Iy 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, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Silhavy et al., Experiments with Gene Fusions. 1984, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover & Hames, DNA Cloning: A Practical Approach. 2nd ed., 1995, IRL Press at Oxford University Press, Oxford/New York; Ausubel (ed.) Short Protocols in Molecular Biology. 3rd ed., 1995, Wiley, New York).
  • 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 OsMTIa protein. Such methods may be used to detect OsMTIa 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.
  • OsMTIa nucleic acid molecule levels 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 OsMTIa 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. Sci.
  • QTL quantitative trait loci
  • ASO allele-specific oligonucleotide
  • OLAs oligonucleotide ligation assays
  • SSCP single-strand conformation polymorphism
  • enzyme mismatch cleavage direct sequence analysis of amplified exons (Kestila et al., MoI. Cell, l(4):575-582 (1998); Yuan et al., Hum.
  • Preferred detection methods are non- electrophoretic, including, for example, the TAQMANTM allelic discrimination assay, PCR- OLA, molecular beacons, padlock probes, and well fluorescence (see Landegren et al., Genome Res., 8:769-776 (1998) and references cited therein).
  • the present invention also encompasses functional fragments of a OsMTIa polypeptide, for example, fragments that have the ability to alter a plant trait similar to that of SEQ ID NO: 2.
  • 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. Such 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 OsMTIa polypeptide. Such methods can be used, for example, to determine levels of OsMTIa 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. In certain embodiments, the method involves an immunochemical reaction with an antibody that specifically recognizes a OsMTIa 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 OsMTIa nucleic acid encoding a OsMTIa protein operatively linked to a promoter, or a cell line produced by introduction of OsMTIa nucleic acids into a host cell genome.
  • the expression system may further comprise one or more additional heterologous nucleic acids relevant to OsMTIa function, such as targets of OsMTIa transcriptional activation or repression activity. These additional nucleic acids may be expressed as a single construct or multiple constructs.
  • a construct for expressing a OsMTIa protein may include a vector sequence and a OsMTIa nucleotide sequence, wherein the OsMTIa nucleotide sequence is operatively linked to a promoter sequence.
  • a construct for recombinant OsMTIa 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.
  • 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. 5,850,019); the 35S and 19S promoters from cauliflower mosaic virus (CaMV) (Odell et al., Nature, 313:810-812 (1985) and U.S. Patent No. 5,352,605); the promoters of Chlorella virus methyltransferase genes (U.S. Patent No. 5,563,328) and the full-length transcript promoter from figwort mosaic virus (FMV) (U.S. Patent No.
  • PClSV peanut chlorotic streak caulimovirus
  • CaMV cauliflower mosaic virus
  • FMV figwort mosaic virus
  • Suitable inducible promoters for use in plants include the promoter from the ACEl system which responds to copper (Mett et al., Proc. Natl. Acad. Sci. 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., 227:229-237 (1991)).
  • 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 genes of the invention in plants are disclosed in WO 93/07278.
  • tissue specific promoters useful in the present invention include 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 the novel dense and erect panicle gene 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., MoL 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 OsMTIa 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. 5,380,831).
  • a plant expression cassette i.e., a OsMTIa 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.
  • selection markers used routinely in transformation include the nptll gene, 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.
  • the hph gene which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, MoI. Cell. Biol, 4:2929-2931 (1984)), the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J., 2(7): 1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642), and the mannose-6-phosphate isomerase gene, which provides the ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629).
  • 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 trans-acting 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).
  • Agrobacterium strains e.g., LBA4404, GV3101, EHAlOl, EHA105, etc.
  • the second plasmid vector is not necessary for introduction of polynucleotides into plants by other methods such as, e.g., microprojection, microinjection, electroporation, and polyethylene glycol.
  • 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. ScL 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 termed targeting sequences, 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.
  • Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab et al., Proc. Natl. Acad. ScL USA, 90:913-917 (1993)).
  • this marker had been used successfully for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, Nucl. Acids Res., 19:4083-4089 (1991)).
  • telomere 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 OsMTIa 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.
  • 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 OsMTIa 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.,
  • 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 OsMTIa knockout plants comprising a disruption of a OsMTIa locus.
  • a disrupted gene may result in expression of an altered level of full-length OsMT 1 a protein or expression of a mutated variant OsMT 1 a protein.
  • Plants with complete or partial functional inactivation of the OsMTIa gene may be generated, e.g., by expressing an amorphic (i.e., null mutation) or hypomorphic OsMTIa 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 OsMTIa constructs, driven by a universal or tissue-specific promoter to reduce levels of OsMTIa 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 OsMTIa.
  • the present invention also encompasses transgenic plants with specific "knocked- in” modifications in the disclosed OsMTIa gene.
  • a "knocked-in" transgenic plant expresses an antimorphic (i.e., dominant negative) allele.
  • a "knocked-in” transgenic plant expresses a hypermorphic (i.e., a gain of function) allele.
  • OsMTIa 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,
  • 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.
  • Transgenic plants, transformed plants, or stably transformed plants, or cells, tissues or seed of any of the foregoing, refer to plants that have incorporated or integrated exogenous polynucleotides into the plant cell.
  • 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.
  • target plant cells e.g., immature or mature embryos, suspension cultures, undifferentiated callus, protoplasts, etc.
  • a maximum threshold level of appropriate selection depending on the selectable marker gene
  • Explants are typically transferred to a fresh supply of the same medium and cultured routinely.
  • 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. Plant ScL, 13:219-239 (1994); and Bommineni et al., Maydica, 42:107-120 (1997). Since the transformed material contains many cells, both transformed and non-transformed cells are present in any piece of subjected target callus or tissue or group of cells.
  • 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.
  • acetosyringone AS
  • 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 Pr obi. 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.
  • OsMTIa antagonists/inhibitors are agents that alter the function of a OsMTIa protein e.g., by altering chemical and biological activities or properties. Methods of identifying inhibitors involve assaying a reduced level or quality of OsMTIa function in the presence of one or more agents. Exemplary OsMTIa inhibitors include small molecules as well as biological inhibitors as described herein below.
  • agent refers to any substance that potentially interacts with a OsMTIa 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 -4 to about +14, more preferably in the range of about -2 to about +7.5.
  • Exemplary nucleic acids that may be used to disrupt OsMTIa function include antisense RNA and small interfering RNAs (siRNAs) (see e.g., U.S. Application Publication No. 20060095987. These inhibitory molecules may be prepared based upon the OsMTIa 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).
  • siRNAs small interfering RNAs
  • 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. 7,338,762; 7,329,742; 6,949,379; 6,180,348; and 5,756,291), a small molecule library (U.S. Patent Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (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 OsMTIa activity refers to a level or quality of wild type OsMTIa activity, for example, when using a recombinant expression system comprising expression of SEQ ID NO: 2.
  • a control level or quality of OsMTIa 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.
  • Methods of identifying OsMTIa inhibitors also require that the inhibiting capacity of an agent be assayed.
  • Assaying the inhibiting capacity of an agent may comprise determining a level of OsMTl a gene expression; determining DNA binding activity of a recombinantly expressed OsMTIa protein; determining an active conformation of a OsMTIa protein; or determining a change in a trait in response to binding of a OsMTIa inhibitor (e.g., drought tolerance, zinc uptake).
  • a method of identifying a OsMT Ia inhibitor may comprise (a) providing a cell, plant, or plant part expressing a OsMTIa 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 OsMTIa 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 OsMTIa protein, or a cell expressing a OsMTIa 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 OsMTIa protein to a substrate.
  • the present invention also encompasses methods of identifying of a OsMTIa inhibitor by determining specific binding of a substance (e.g., an agent described previously) to a OsMTIa protein.
  • a method of identifying a OsMTIa binding partner may comprise: (a) providing a OsMTIa protein of SEQ ID NO: 2; (b) contacting the OsMTIa protein with one or more agents under conditions sufficient for binding; (c) assaying binding of the agent to the isolated OsMTIa protein; and (d) selecting an agent that demonstrates specific binding to the OsMTIa protein.
  • Specific binding may also encompass a quality or state of mutual action such that binding of an agent to a OsMTIa 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 OsMTIa protein may be considered specific if the binding affinity is about IxIO 4 M "1 to about IxIO 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 OsMTIa 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 OsMTIa 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.col ⁇ , 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).
  • SELDI Surface-Enhanced Laser Desorption/Ionization
  • TOF time-of- flight mass spectrometer
  • SELDI provides a technique to rapidly analyze molecules retained on a chip. It may be applied to ligand-protein interaction analysis by covalently binding the target protein, or portion thereof, on the chip and analyzing by mass spectrometry the small molecules that bind to this protein (Worrall et al., Anal Chem., 1998, 70(4):750-756 (1998)).
  • a target protein e.g., a OsMTIa 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 OsMTIa protein) immobilized on the layer.
  • a target protein e.g., a OsMTIa 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 nonspecific 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 OsMTIa binding partners and inhibitors that rely on a conformational change of a OsMTIa 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.
  • a OsMTIa 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 OsMTIa protein in the presence of an agent is compared to a conformation of a OsMTIa protein in the absence of the agent. A change in conformational state of a OsMTIa protein in the presence of an agent identifies a OsMTIa 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 as assaying for altered stress tolereance as described herein.
  • cells expressing OsMTIa may be provided in the form of a kit useful for performing an assay of OsMTIa function.
  • a kit for detecting a OsMTIa may include cells transfected with DNA encoding a full-length OsMTIa protein and a medium for growing the cells.
  • Assays of OsMTIa 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 OsMTIa expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding OsMTIa 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 OsMTIa or plants expressing OsMTIa may additionally employ control cells or plants that are substantially devoid of native OsMTIa and, optionally, proteins substantially similar to a OsMTIa 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 OsMTIa protein, a control cell may comprise, for example, a parent cell line used to derive the 0sM77 ⁇ -expressing cell line.
  • a method for producing an antibody that specifically binds a OsMTIa protein.
  • a full-length recombinant OsMTIa 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-OsMTla antibodies are also encompassed by the invention.
  • Specific binding of an antibody to a OsMTIa protein refers to preferential binding to a OsMTIa 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 ⁇ n M or higher, or at least about 10 ⁇ 12 M or higher.
  • OsMTIa antibodies prepared as disclosed herein may be used in methods known in the art relating to the expression and activity of OsMTIa proteins, e.g., for cloning of nucleic acids encoding a OsMTIa protein, immunopurification of a OsMTIa protein, and detecting a OsMTIa protein in a plant sample, and measuring levels of a OsMTIa 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.
  • Example 1 The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teachings provided herein. [00123] Example 1
  • OsMTIa was directly obtained from Brazil upland rice using suppression subtractive hybridization.
  • the 222 bp cDNA sequence of OsMTIa is composed of an open reading frame of 74 amino acids (SEQ ID NO: 2).
  • the predicted protein contains two cysteine -rich domains in the N- and C-terminal regions, which is characteristic of metallothioneins.
  • OsMTIa contains six Cys-Xaa-Cys motifs (where Xaa represents another amino acid) that are distributed equally among the N- and C-terminal domains, and an approximately 40-amino acid spacer containing aromatic amino acids, which is typical for most type 1 MTs in plants.
  • OsMTIa showed high homology with many other type 1 plant MT proteins, especially with monocotyledonous Zea mays (73.7%) and Hordeum vulgare (67.6%).
  • OsMTIa expression in Iapar 9 significantly increased after 0.5 hours as a result of 7% PEG 6000 treatment, though OsMTIa expression did not change in response to 150 mM NaCl and ABA treatment (see Figure T).
  • the standard thermal profile was as follows: 95 0 C for 4 min; 30 cycles of 95 0 C for 30 seconds; 56 0 C (OsMTIa, AK108249, JRC0332)/61 0 C (JRCO 189) for 30 seconds; and 72 0 C for 30 seconds; then 72 0 C for 10 minutes.
  • the fluorescence signal was captured at the end of each cycle and melting curve analysis was performed from 65 to 95 0 C with data capture every 0.3 0 C during a 1 second hold. Actinl was used as a quantification control. Reactions with the cDNA template replaced by nuclease-free water were run with each primer pair as a blank control. Quantification consisted of at least three independent replicates.
  • OsMTIa levels in Iapar 9 increased dramatically (5.9-fold at 6 h and 15.7-fold at 24 h; see Figure 3). A similar pattern was observed in Nipponbare (data not shown).
  • OsMTIa To examine whether OsMTIa is involved in accumulating certain heavy metals, the full-length ORF of OsMTIa was introduced into the binary vector under the control of the rice Actinl promoter, generating plasmi ⁇ pActini:OsMTla.
  • the pActini: OsMTl ⁇ plasmid was introduced into Agrob ⁇ cterium tumef ⁇ ciens AGLl and transformed into rice as described by Liu et al. (2007).
  • the independent transgenic plants were further confirmed by PCR assay, and were then transferred to a greenhouse.
  • Wild type and 0sM77 ⁇ -overexpressing plants were further cultivated, and the aerial parts of mature plants and grains (s ⁇ ns hull) were collected and washed thoroughly in distilled water, dewatered and then ovendried at 80 0 C for 24 hours.
  • the dry samples were milled and 200 mg aliquots were digested in a microwave oven (Ethos touch control advanced microwave lab station; Milestone, Inc, Italy) with 9 mL concentrated HNO 3 and 2 mL H 2 O 2 at 180 0 C for 20 minutes.
  • the digests were diluted to 25 mL with distilled water, and analyzed for metal content using an Optima-2000TM DV Inductively Coupled Plasma-Optic Emission spectrometer (ICP-OES, Perkin Elmer, USA).
  • ICP-OES Inductively Coupled Plasma-Optic Emission spectrometer
  • the level of different metals was measured in transgenic plants harboring the pACTINl::OsMTla using inductive coupled plasma spectroscopy (ICP-OES). The data represent the average from six independent measurements with standard deviation. The asterisks (**) indicate significant differences from the control at P ⁇ 0.01 by Student's t-test.
  • OsMTIa was ectopically expressed in yeast.
  • the yeast Sacharomyces cerevisiae
  • BY4741 wild type MATa his 3 Ieu2 met 15 ura3 was employed for the heavy metal accumulation assay.
  • the expression vector pi 8 IAINE carries the yeast alcohol dehydrogenase (ADH2) promoter and terminator and b-isopropylmalate dehydrogenase gene (LEU2) as the selective marker.
  • ADH2 yeast alcohol dehydrogenase
  • LEU2 b-isopropylmalate dehydrogenase gene
  • yeast cells were collected for metal uptake analysis.
  • yeast cells expressing OsMTIa accumulated 2.4-fold more zinc than controls, while no difference in manganese, copper or iron levels were observed between transgenic and wild type yeast (see Table 2).
  • Protein content was determined as described by Bradford (1976).
  • CAT activity was determined as described by Aebi (1983), by monitoring the disappearance of H2O2 by measuring the decrease in absorbance at 240 nm of a reaction mixture containing 0.3 mL of
  • Ossiz expression was also induced by Zn 2+ treatment. The expression reached plateau at 6 hours and then declined gradually, and the final expression pattern was similar to

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Abstract

L'invention porte sur des compositions et des procédés pour conférer des propriétés améliorées à des plantes à l'aide du gène de métallothionéine OsMT1a et ses produits, comprenant des polypeptides et des plantes et graines transgéniques. Des propriétés améliorées comprennent une tolérance améliorée à la sécheresse et une absorption du zinc accrue.
PCT/IB2010/050755 2009-02-26 2010-02-19 Gène de métallothionéine conférant une tolérance au stress abiotique dans des plantes et ses utilisations Ceased WO2010097746A1 (fr)

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