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WO2010031312A1 - Régulation de la réponse à une privation d’azote - Google Patents

Régulation de la réponse à une privation d’azote Download PDF

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Publication number
WO2010031312A1
WO2010031312A1 PCT/CN2009/073780 CN2009073780W WO2010031312A1 WO 2010031312 A1 WO2010031312 A1 WO 2010031312A1 CN 2009073780 W CN2009073780 W CN 2009073780W WO 2010031312 A1 WO2010031312 A1 WO 2010031312A1
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Prior art keywords
lntl
plant
protein
plants
seq
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Inventor
Daowen Wang
Yurong Xie
Huanju Qin
Xin Liu
Yiping Tong
Zhensheng Li
Hongli Xie
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Institute of Genetics and Developmental Biology of CAS
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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
    • C07K14/415—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
    • C12N15/8271—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

Definitions

  • the present invention generally relates to methods and compositions for promoting plant tolerance to nitrogen deprivation. More particularly, the present invention relates to LNTl nucleic acids and proteins, binding partners and inhibitors thereof, LNTl knockout plants, and use of the same for improving plant growth in low nitrogen conditions.
  • Nitrogen (N) is the fourth most abundant element in plants, just after carbon, oxygen and hydrogen, and its assimilation is a vital process controlling plant growth and development. Plants utilize nitrogen substrates such as NO 3 " , NH4 + , urea, and amino acids, and most crop species grow optimally with a mixture of ammonium and nitrate, which is typically the most abundant nitrogen form in soil environments. See Crawford et al., Trends in Plant Science, 1998, 3: 389-395; Richard et al. , Journal ofExperimen tal Botany, 2001, 52: 435 -443. Although nitrogen is abundant in the biosphere, it has limited availability for plant use in the soil.
  • the Arabidopsis genome probably encodes more than 2,000 transcription factors or transcriptional regulators (Riechmann, In Somerville et al. (Eds.), The Arabidopsis Book. 2002, Rockville, Maryland, American Society of Plant Biologists:l-46; Davuluri et al., BMC Bioinformatics, 2003, 4: 25), and new classes of transcriptional regulators are still being discovered (Lin et al., Plant Cell, 2003, 15: 2241-2252).
  • the present invention provides LNTl (low nitrogen tolerance 1) knockout plants, which show increased nitrate content, increased nitrate uptake, increased lateral root growth, and increased biomass in low nitrogen conditions. Also provided are methods for making such plants, or alternatively, methods for mimicking the knockout phenotype using a LNTl inhibitor. Still further provided are isolated LNTl nucleic acids and proteins useful in the disclosed methods.
  • LNTl nucleic acids of the invention include nucleic acids comprising (a) an open reading frame encoding a LNTl protein comprising a DNA binding domain and an amino acid sequence at least 80% identical to SEQ K) NO:2, i.e., nucleic acids that differ from SEQ ID NO:1 only in use of a degenerate codon; (b) an open reading frame encoding a LNTl protein comprising an amino acid sequence of SEQ ID NO:2; (c) a nucleotide sequence at least 80% identical to SEQ ID NO:1 ; (d) a nucleotide sequence of SEQ ID NO:1; (e) a nucleic acid that specifically hybridizes to the complement of SEQ ID NO:1 under stringent hybridization conditions; and (f) a nucleotide sequence that is the complement of any one of
  • LNTl knockout plants including monocot and dicot plants.
  • the LNTl knockout plants are characterized by reduced or absent expression of LNTl.
  • Methods are also provided for producing a genetically modified plant by crossing a plant, which has been modified at the lntl locus, with a non-genetically modified plant.
  • the method can comprise (a) providing a LNTl protein; (b) contacting the LNTl protein with one or more test agents or a control agent under conditions sufficient for binding; (c) assaying binding of a test agent to the isolated LNTl protein; and (d) selecting a test agent that demonstrates specific binding to the LNTl protein.
  • the method can comprise (a) providing a cell expressing a LNTl protein; (b) contacting the cell with one or more test agents or a control agent; (c) assaying expression of a LNTl target gene; and (d) selecting a test agent that induces elevated expression of the target gene, which gene is normally subject to LNTl repression, when contacted with the test agent as compared to the control agent.
  • the method can comprise (a) providing a plant expressing a LNTl protein; (b) contacting the plant with one or more test agents or a control agent; (c) assaying nitrate content, nitrogen uptake, lateral root growth, or biomass of the plant; and (d) selecting a test agent that induces increased nitrate content, increased nitrate uptake, increased lateral root growth, and/or increased biomass when contacted with the test agent as compared to the control agent.
  • LNTl binding agents and LNTl inhibitors identified as disclosed herein are also provided.
  • methods of conferring to a plant tolerance to nitrogen deprivation by (a) disrupting a LNTl gene in the plant; or (b) inhibiting a LNTl nucleic acid or protein in the plant. These methods may be used, for example, when plants are subject to growth in soil or other medium containing less than about 2 mM nitrate content, to thereby result in increased plant biomass.
  • Figure 2 A shows the results of RT-PCR amplification of lntl transcripts in root (R), stem (S), leaf (L), flower (F), and silique (SI) following 24 cycles (24c) or 28 cycles (28c). Amplified tubulin transcripts in corresponding samples are also shown.
  • Figure 2B shows histochemical detection of lntl transcripts in Arabidopsis tissues. See Example 2.
  • Figure 3 shows transcriptional activation activity of LNTl protein using a GAL4 expression system. Yeast cells containing pBD-LNTl and the positive control plasmid pGAL4 all grew well on SD medium without histidine, while the negative control plasmid pBD could not grow. At the same time, all of these yeast cells grew well on YPAD medium. See Example 3.
  • Figures 4A-4B show the activity of a LNTl promoter-GUS fusion protein in transformed plants subjected to full nitrogen (FN), low nitrogen (LN), and recovery from low nitrogen (AN) conditions. Histochemical detection of GUS enzymatic activity shows reduced expression of the fusion protein in low nitrogen conditions ( Figure 4A). Quantitative GUS enzymatic assays showed 17% and 56% reduced activity in aerial and root tissues, respectively, when exposed to nitrogen limitation (Figure 4B). Upon return to normal nitrogen levels (AN), GUS activity in aerial and root tissues increased 205% and 293%, respectively, as compared to the low nitrogen (LN) expression levels ( Figure 3B). See Example 4.
  • Figure 5 A shows the genomic structure of lntl and the position of T-DNA insertions lntl-1 and lntl-2.
  • Figure 5B shows the results of PCR amplication experiments, in which lntl transcripts were not amplified in lnt-1 and lnt-2 homozygous mutant plants. See Example 5.
  • Figures 6A-6E shows the increased root growth response in lntl-1 and lntl-2 knockout plants as compared to wild type (WT) plants when the plants are subjected to low nitrogen conditions. No phenotypic differences were observed between lntl knockout plants and wild type plants under full nitrogen conditions (Figure 6A).
  • lntl knockout plants show approximately 60%- 70% increased lateral root number following growth for 14-16 days ( Figures 6B and 6E) or 30 days (Figure 6C) under low nitrogen conditions, lntl knockout plants and wild type plants showed similar primary root length either under full nitrogen or low nitrogen conditions ( Figure 6D). See Example 5.
  • Figures 7A- 7D show results of complementation assays as described in Example 5.
  • lntl-1 knockout plants expressing a lntl transgene (lntl-1 +LNT1) showed reduced lateral root number as compared to both wild type (WT) and lntl-1 plants subject to low nitrogen conditions ( Figures 7B and 7D).
  • lntl-1+LNTl, lntl-1, and wild type plants showed similar lateral root number under full nitrogen (FN) conditions, and similar primary root length under both full nitrogen (FN) and low nitrogen (LN) conditions ( Figures 7A and 7C).
  • Figure 8 shows the levels of N(V in wildtype (WT) and lntl knockout plants. N(V content was increased in lntl knockout plants subject to low nitrogen conditions (LN). Under full nitrogen (FN) conditions, N(V content was comparable in lntl knockout and wild type plants. See Example 5.
  • Figures 9A-9F show the results of real-time PCR of NRT2 family proteins.
  • most of the transporters NRT2.1 (Figure 9A), NRT2.2 (Figure 9B), NRT2.3 (Figure 9C), NRT2.4 (Figure 9D), NRT2.5 ( Figure 9E), and NRT2.6 ( Figure 9F)
  • NRT2.1 Figure 9A
  • NRT2.2 Figure 9B
  • NRT2.3 Figure 9C
  • NRT2.4 Figure 9D
  • NRT2.5 Figure 9E
  • NRT2.6 Figure 9F
  • Figure 10 shows levels of 15 NOs " influx in wildtype (WT) and lntl knockout plants.
  • N(V content was increased in lntl knockout plants subject to low nitrogen conditions (LN).
  • LN low nitrogen conditions
  • FN full nitrogen
  • AN nitrogen recovery
  • Figures 1 IA-I ID show aerial tissue and root biomass of wild type (WT) and lntl knockout plants. Biomass in both tissues was increased in lntl knockout plants subject to low nitrogen conditions (LN) before flower ( Figure 10B) or after flower ( Figure 10D). Under full nitrogen (FN) conditions before flower ( Figure 10A) or after flower (Figure 10C), aerial tissue and root biomass was comparable in lntl knockout and wild type plants. See Example 5.
  • LN low nitrogen conditions
  • FN full nitrogen
  • the present invention provides LNTl nucleic acids and proteins, variants thereof, and inhibitors thereof.
  • LNTl nucleic acids and proteins have not taught how to use such molecules for promoting nitrogen deprivation tolerance in plants, as presently disclosed.
  • a representative LNTl nucleic acid is set forth as SEQ ID NO:1, which encodes the representative LNTl protein of SEQ ID NO:2.
  • LNTl variants encompassed by the present invention include variant proteins having LNTl transcriptional activation activity.
  • LNTl is predicted to encode a MYB -like transcription factor. The Myb transcription factor family has more that two hundred members in plants (Yanhui et al., Plant Molecular Biology, 2006, 60:107- 124) and is the biggest superfamily in Arabidopsis .
  • the superfamily is further divided into three subfamilies, R2R3-Myb, R1R2R3-Myb, and MYB-related proteins (Rosinski et al., Journal of Molecular Evolution, 1998, 46: 74-83; Jin et al., Plant Molecular Biology, 1999, 41 : 577-585; Stracke et al., Current Opinion Plant Biology, 2001, 4:447-456).
  • LNTl is predicted to be within the MYB-related subfamily.
  • the LNTl protein includes a MYB/SANT-type DNA binding domain and an activation/repression domain, which operate together to regulate physiological responses to nitrogen deprivation.
  • Amino acids 35-83 of SEQ ID NO:2 constitute a MYB/SANT-type DNA binding domain consisting of three imperfect repeats, Rl, R2 and R3, each containing a helix-turn- helix motif variation. See Figure 1.
  • LNTl contains the motif SHAQK within the MYB repeats, a motif shared with other members of the CCAl -like subfamily of MYB-related proteins. See Yanhui, 2006. Tandem copies of the domain bind telomeric DNA tandem repeats as part of the capping complex.
  • LNTl variants encompassed by the present invention include variants in which residues 35-83 of SEQ ID NO:2 are unchanged, such residues are changed by conservative substitution, or such residues are changed in any other manner that does not compromise DNA binding activity of LNTl .
  • Nucleic acids are deoxyribonucleotides or ribonucleotides and polymers thereof in single- stranded, double-stranded, or triplexed form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural nucleic acid. The terms nucleic acid molecule or nucleic acid may also be used in place of gene, cDNA, mRNA, or cRNA. Nucleic acids may be synthesized, or may be derived from any biological source, including any organism. Representative methods for cloning nucleic acids that encode a LNTl protein are described in Example 1.
  • nucleic acids of the invention comprise the nucleotide sequence of SEQ ID NO:1 and substantially identical sequences encoding LNTl proteins with transcriptional activation activity, for example, sequences at least 50% identical to SEQ ID NO:1 , such as at least 55% identical; or at least 60% identical; or at least 65% identical; such as at least 70% identical; or at least 75% identical; or at least 80% identical; or at least 85% identical; or at least 90% identical, or as at least 91% identical; or at least 92% identical; or at least 93% identical; or at least 94% identical; or at least 95% identical; or at least 96% identical; or at least 97% identical; or at least 98% identical; or at least 99% identical. Sequences are compared for maximum correspondence using a sequence comparison algorithm using the full-length sequence of SEQ ID NO:1 as the query sequence, as described herein below, or by visual inspection.
  • Substantially identical sequences may be polymorphic sequences, Ie, 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 the full length of SEQ ID NO:1 under stringent conditions.
  • two nucleic acid sequences being compared may be designated a probe and a target.
  • a probe is a reference nucleic acid molecule
  • a target is a test nucleic acid molecule, often found within a heterogeneous population of nucleic acid molecules.
  • a target sequence is synonymous with a test sequence.
  • a preferred nucleotide sequence employed for hybridization studies or assays includes probe sequences that are complementary to at least an about 14 to 40 nucleotide sequence of a nucleic acid molecule of the present invention.
  • probes comprise 14 to 20 nucleotides, or even longer where desired, such as 30, 40, 50, 60, 100, 200, 300, or 500 nucleotides or up to the full length of SEQ ID NO:1.
  • Such fragments may be readily prepared, for example by chemical synthesis of the fragment, by application of nucleic acid amplification technology, or by introducing selected sequences into recombinant vectors for recombinant production.
  • Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex nucleic acid mixture (e.g., total cellular DNA or RNA). Specific hybridization may accommodate mismatches between the probe and the target sequence depending on the stringency of the hybridization conditions.
  • 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. Longer sequences hybridize specifically at higher temperatures. 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. 1993, part I chapter 2, Elsevier, New York, New York.
  • highly stringent hybridization and wash conditions are selected to be about 5 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • a probe will hybridize specifically to its target subsequence, but to no other sequences.
  • the Tm 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 Tm for a particular probe.
  • An example of stringent hybridization conditions for Southern or Northern Blot analysis of complementary nucleic acids having more than about 100 complementary residues is overnight hybridization in 50% formamide with 1 mg of heparin at 42 0 C.
  • An example of highly stringent wash conditions is 15 minutes in 0.1 X SSC at 65 0 C.
  • An example of stringent wash conditions is 15 minutes in 0.2X SSC buffer at 65 0 C.
  • a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example of medium stringency wash conditions for a duplex of more than about 100 nucleotides is 15 minutes in IX SSC at 45 0 C.
  • An example of low stringency wash for a duplex of more than about 100 nucleotides is 15 minutes in 4X to 6X SSC at 4O 0 C.
  • stringent conditions typically involve salt concentrations of less than about IM Na+ ion, typically about 0.01 to IM Na+ ion concentration (or other salts) at pH 7.0-8.3, and the temperature is typically at least about 3O 0 C.
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • a signal to noise ratio of 2-fold (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
  • a probe nucleotide sequence preferably hybridizes to a target nucleotide sequence in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO4, ImM EDTA at 50 0 C followed by washing in 2X SSC, 0.1% SDS at 50°C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO4, ImM EDTA at 50 0 C followed by washing in IX SSC, 0.1% SDS at 5O 0 C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO4, ImM EDTA at 50 0 C followed by washing in 0.5X SSC, 0.1% SDS at 50 0 C; more preferably, a probe and target sequence hybridize in 7% sodium dodecyl sulphate (SDS), 0.5M NaPO4, ImM EDTA at 50 0 C followed by washing in
  • nucleic acid sequences are substantially identical, share an overall three- dimensional structure, or are biologically functional equivalents. These terms are defined further herein below. 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.
  • 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 means nucleotide sequences which are substantially complementary, as may be assessed by the same nucleotide comparison methods set forth below, or is defined as being capable of hybridizing to the nucleic acid segment in question under relatively stringent conditions such as those described herein.
  • a particular example of a complementary nucleic acid segment is an antisense oligonucleotide.
  • subsequence refers to a sequence of nucleic acids that comprises a part of a longer nucleic acid sequence.
  • An exemplary subsequence is a probe, described herein above, or a primer.
  • primer refers to a contiguous sequence comprising about 8 or more deoxyribonucleotides or ribonucleotides, preferably 10-20 nucleotides, and more preferably 20-30 nucleotides of a selected nucleic acid molecule.
  • the primers of the invention encompass oligonucleotides of sufficient length and appropriate sequence so as to provide initiation of polymerization on a nucleic acid molecule of the present invention.
  • elongated sequence refers to an addition of nucleotides (or other analogous molecules) incorporated into the nucleic acid.
  • 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, intronic sequences, additional restriction enzyme sites, multiple cloning sites, and other coding segments.
  • the 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 When operatively linked to a nucleic acid, 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.
  • Nucleic acids 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.
  • a method for detecting a nucleic acid molecule that encodes a LNTl protein may be used to detect LNTl 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.
  • the nucleic acids of the present invention may be used to clone genes and genomic DNA comprising the disclosed sequences.
  • the nucleic acids of the present invention may be used to clone genes and genomic DNA of related sequences. Levels of a LNTl nucleic acid molecule may be measured, for example, using an RT-PCR assay. See Chiang, J. Chromatogr. A., 1998, 806:209-218, and references cited therein.
  • genetic assays using LNTl nucleic acids may be performed 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, 1983, 80(l):278-282), oligonucleotide ligation assays (OLAs) (Nickerson et al., Proc. Natl. Acad. Sd. USA, 1990, 87(22):8923-8927), single-strand conformation polymorphism (SSCP) analysis (Orita et al., Proc. Natl. Acad. Sd.
  • ASO allele-specific oligonucleotide
  • OVAs oligonucleotide ligation assays
  • SSCP single-strand conformation polymorphism
  • LNTl Proteins The present invention also provides isolated LNTl polypeptides. Polypeptides and proteins each refer to a compound made up of a single chain of amino acids joined by peptide bonds. A representative LNTl polypeptides is set forth as SEQ ID NO:2.
  • Additional polypeptides of the invention include lntl proteins with transcriptional activation activity, for example, sequences at least 50% identical to SEQ ID NO:2, such as at least 55% identical; or at least 60% identical; or at least 65% identical; such as at least 70% identical; or at least 75% identical; or at least 80% identical; or at least 85% identical; or at least 90% identical, or as at least 91% identical; or at least 92% identical; or at least 93% identical; or at least 94% identical; or at least 95% identical; or at least 96% identical; or at least 97% identical; or at least 98% identical; or at least 99% identical.
  • Sequences are compared for maximum correspondence using a sequence comparison algorithm using the full-length sequence of SEQ ID NO:2 as the query sequence, as described herein below, or by visual inspection.
  • the invention further encompasses polypeptides encoded by any one of the nucleic acids disclosed herein.
  • 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-methyl valine; norvaline; norleucine; and ornith
  • 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.
  • the present invention also provides functional fragments of a LNTl polypeptide, for example, fragments that have transcriptional activation activity similar to that of a full-length LNTl protein.
  • Functional polypeptide sequences that are longer than the disclosed sequences are also provided.
  • 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.
  • LNTl proteins of the invention include proteins comprising amino acids that are conservatively substituted variants of SEQ ID NO: 2.
  • a conservatively substituted variant refers to a polypeptide comprising an amino acid in which one or more residues have been conservatively substituted with a functionally similar residue.
  • conservative 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.
  • 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,
  • the present invention further provides methods for detecting a LNTl polypeptide.
  • the disclosed methods can be used, for example, to determine altered levels of LNTl protein, for example, induced levels of LNTl protein, or to detect LNTl in a myelin inhibitory complex.
  • the method may involve performing an immunochemical reaction with an antibody that specifically recognizes a LNTl protein.
  • Techniques for detecting such antibody- antigen conjugates or complexes are known in the art and include but are not limited to centrifugation, affinity chromatography and other immunochemical methods. See e.g., Ishikawa Ultrasensitive and Rapid Enzyme Immunoassay. 1999, Elsevier, Amsterdam/New York, United States of America; Law, Immunoassay: A Practical Guide. 1996, Taylor & Francis, London/Bristol, Pennsylvania, United States of America; Liddell et al., Antibody Technology.
  • nucleotide or protein sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms disclosed herein or by visual inspection.
  • substantially identical in regards to a nucleotide or protein sequence means that a particular sequence varies from the sequence of a naturally occurring sequence by one or more deletions, substitutions, or additions, the net effect of which is to retain biological function of a
  • LNTl nucleic acid or protein LNTl nucleic acid or protein
  • one sequence acts as a reference sequence to which one or more test sequences are compared.
  • test and reference sequences are entered into a computer program, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are selected.
  • sequence comparison algorithm calculates the percent sequence identity for the designated test sequence(s) relative to the reference sequence, based on the selected program parameters.
  • Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math, 1981, 2:482-489, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol., 1970, 48:443-453, by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444-
  • a preferred algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. MoI. Biol, 1990, 215:403-410.
  • Software for performing BLAST analyses is publicly available through the National Center for
  • the present invention further provides a system for expression of a recombinant LNTl protein. Such a system may be used for subsequent purification and/or characterization of a LNTl protein. A system for recombinant expression of a LNTl protein may also be used for identification of inhibitors of a LNTl protein, as described further herein below.
  • 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 LNTl nucleic acid encoding a LNTl protein operatively linked to a promoter, or a cell line produced by introduction of LNTl nucleic acids into a host cell genome.
  • the expression system may further comprise one or more additional heterologous nucleic acids relevant to LNTl function, such as targets of LNTl transcriptional activation or repression activity. These additional nucleic acids may be expressed as a single construct or multiple constructs.
  • Isolated proteins and recombinantly produced proteins 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. II. A. Expression Constructs
  • a construct for expression of a LNTl protein may include a vector sequence and a LNTl nucleotide sequence, wherein the LNTl nucleotide sequence is operatively linked to a promoter sequence.
  • a construct for recombinant LNTl 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 promoter from cauliflower mosaic virus (CaMV) (Odell et al., Nature, 1985, 313:810-812); 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. 5,378,619); the promoters from such genes as rice actin (McElroy et al.
  • 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. Sd. USA, 1993, 90:4567-4571); the promoter of the maize In2 gene which responds to benzenesulfonamide herbicide safeners (Hershey et al., MoI Gen. Genetics, 1991, 227:229-237 and Gatz et al., MoI Gen. Genetics, 1994, 243:32-38); and the promoter of the Tet repressor from TnIO (Gatz et al., MoI. Gen. Genet, 1991, 227:229-237).
  • 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. Sci. USA, 1991, 88:10421) 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., 2000, 24:265-273).
  • 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., 1995, 7:661-676 and PCT International Publication No. WO 95/14098 describing such promoters for use in plants.
  • 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., 1997, 6:143-156). 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 subcellular 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 also Guerineau et al., MoI. Gen. Genet, 1991, 262:141-144; Proudfoot, Cell, 1991, 64:671-674; Sanfacon et al., Genes Dev.
  • the vector and LNTl 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 improved 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, 1990, 92:1-11 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. 6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. Published Application Nos. 20040005600 and 20010003849, and Murray et al., Nucleic Acids Res., 1989, 17:477-498, herein incorporated by reference.
  • 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., 1991, 9:104-126; 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, herein incorporated by reference.
  • a plant expression cassette ⁇ i.e., a LNTl 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, 2000, 5:446-451).
  • 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). Also present on this plasmid vector 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. For example, 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).
  • virulence functions Vir genes
  • Several types of Agrobacterium strains e.g., LBA4404, GV3101, EHAlOl, EHAl 05, etc.
  • the second plasmid vector is not necessary for introduction of polynucleotides into plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc. ILB. Host Cells
  • 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 Bacillus subtilis.
  • Preferred host cells for functional assays substantially or completely lack endogenous expression of a LNTl 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 LNTl protein in a stable cell line.
  • 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 LNTl knockout plants comprising a disruption of a LNTl locus.
  • a disrupted gene may result in expression of an altered level of full-length LNTl protein or expression of a mutated variant LNTl protein.
  • Plants with complete or partial functional inactivation of the LNTl gene may be generated using standard techniques, such as T-
  • a knockout plant in accordance with the present invention may also be prepared using anti-sense, double-stranded RNA, or ribozyme LNTl constructs, which driven by a universal or tissue-specific promoter, to reduce levels of LNTl 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 LNTl .
  • the present invention also provides transgenic plants with specific "knocked-in” modifications in the disclosed LNTl gene, for example to create an over-expression mutant having a dominant negative phenotype.
  • "knocked-in” modifications include the expression of both wild type and mutated forms of a nucleic acid encoding a LNTl protein.
  • LNTl knockout plants may be prepared in mocot or dicot plants, for example, in corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Representative vegatables include tomatoes, lettuce, green beans, lima beans, peas, yams, onion, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.
  • a plant refers to a whole plant, a plant organ ⁇ e.g., leaves, stems, roots, etc), a seed, a plant cell, a propagule, an embryo, 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).
  • the LNTl knockout plants may be further modified at a locus other than LNTl to confer tolerance to nitrogen deprivation or other trait of interest.
  • Desired traits include improved crop yield; insect resistance; tolerance to broad-spectrum herbicides; resistance to diseases caused by viruses, bacteria, fungi, and worms; and enhancement of mechanisms for protection from environmental stresses such as heat, cold, drought, and high salt concentration.
  • Additional desired traits include output traits that benefit consumers, for example, nutritionally enhanced foods that contain more starch or protein, more vitamins, more anti-oxidants, and/or fewer trans-fatty acids; foods with improved taste, increased shelf-life, and better ripening characteristics; trees that make it possible to produce paper with less environmental damage; nicotine-free tobacco; ornamental flowers with new colors, fragrances, and increased longevity; etc.
  • desirable traits that may be used in accordance with the invention include gene products produced in plants as a means for manufacturing, for example, therapeutic proteins for disease treatment and vaccination; textile fibers; biodegradable plastics; oils for use in paints, detergents, and lubricants; etc.
  • the combination of a LNTl knockout plant and a second genetic modification can produce a synergistic effect, i.e., a change in gene expression, nitrate content, nitrate uptake, lateral root growth, or plant biomass that is greater than the change elicited by either genetic modification alone.
  • 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.
  • 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 ⁇ e.g., Hiei et al., Plant J., 1994, 6:271-282; Ishida et al., Nat. Biotechnol., 1996, 14:745-750).
  • a general description of the techniques and methods for generating transgenic plants are found in Ayres et al., CRC Crit. Rev. Plant Sci., 1994. 13:219-239, and Bommineni et al., Maydica, 1997, 42:107-120. 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.
  • 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-media ⁇ ed transformation), bombardment of plant cells with heterologous foreign DNA adhered to particles, and various other non-particle direct-mediated methods (e.g., Hiei et al.,
  • 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.
  • the efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens (Shahla et al., Plant Molec. Biol, 1987, 8:291-298). Alternatively, 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, etc. See e.g., Bidney et al., Plant Molec. Biol., 1992, 18:301- 313.
  • 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 McCabe, U.S. Patent No. 5,584,807, the entire contents of which are herein incorporated by reference. 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., 1994, 20:125).
  • 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., 1986, 5:81-84. 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.
  • 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.
  • LNTl antagonists/inhibitors are agents that alter chemical and biological activities or properties of a LNTl protein.
  • Methods of identifying inhibitors involve assaying a reduced level or quality of LNTl function in the presence of one or more test agents.
  • Representative LNTl inhibitors include small molecules as well as biological inhibitors, as described herein below.
  • a control level or quality of LNTl activity refers to a level or quality of wild type LNTl activity, for example, when using a recombinant expression system comprising expression of SEQ ID NO:2.
  • a control level or quality of LNTl activity comprises a level or quality of activity in the absence of the test agent.
  • Significantly changed activity of a LNTl protein refers to a quantifiable change in a measurable quality that is larger than the margin of error inherent in the measurement technique.
  • significant inhibition refers to LNTl activity that is reduced by about 2-fold or greater relative to a control measurement, or an about 5-fold or greater reduction, or an about 10- fold or greater reduction.
  • An assay of LNTl function may comprise determining a level of LNTl gene expression; determining DNA binding activity of a recombinantly expressed LNTl protein; determining an active conformation of a LNTl protein; or determining activation of signaling events in response to binding of a LNTl inhibitor ⁇ e.g., enhanced expression of nitrate transporters, increased nitrate content, increased nitrate uptake, increased lateral root sprouting, and/or increased biomass).
  • This screening method comprises separately contacting a LNTl protein with a plurality of test agents.
  • the plurality of test 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 LNTl protein, or a cell expressing a LNTl 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 LNTl protein to a substrate.
  • test agent refers to any agent that potentially interacts with a LNTl nucleic acid or protein, including any synthetic, recombinant, or natural product.
  • a test agent suspected to interact with a protein may be evaluated for such an interaction using the methods disclosed herein.
  • Representative test 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.
  • a test 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.
  • nucleic acids that may be used to disrupt LNTl function include antisense RNA and small interfering RNAs (siRNAs). See e.g., U.S. Published Application No. 20060095987. These inhibitory molecules may be prepared based upon the LNTl gene sequence and known features of inhibitory nucleic acids. See e.g., Van der Krol et al., Plant Cell, 1990, 2:291-299; Napoli et al., Plant Cell, 1990, 2:279-289; English et al., Plant Cell, 1996, 8:179-188; Waterhouse et al., Nature Rev. Genet, , 2003, 4:29-38.
  • Test 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 test agents in a library may be assayed simultaneously.
  • test 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.
  • 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.
  • IV.B Binding Assays
  • a method of identifying of a LNTl inhibitor comprises determining specific binding of a test agent to a LNTl protein.
  • a method of identifying a LNTl binding partner may comprise: (a) providing a LNTl protein of SEQ ID NO:2; (b) contacting the LNTl protein with one or more test agents under conditions sufficient for binding; (c) assaying binding of a test agent to the isolated LNTl protein; and (d) selecting a test agent that demonstrates specific binding to the LNTl protein.
  • Specific binding may also encompass a quality or state of mutual action such that binding of a test agent to a LNTl 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 a test agent to a LNTl protein may be considered specific if the binding affinity is about IxIO 4 IyT 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., 1989, 264:21613-21618.
  • Several techniques may be used to detect interactions between a LNTl protein and a test 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 LNTl 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.coR, 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 Ni2+ 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
  • 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 LNTl protein) immobilized on the layer.
  • a target protein e.g., a LNTl 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, 1999, 54:3-15 and references therein.
  • the present invention also provides methods of identifying LNTl binding partners and inhibitors that rely on a conformational change of a LNTl protein when bound by or otherwise interacting with a test agent. 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 LNTl protein.
  • a LNTl protein is purified, for example by ion exchange and size exclusion chromatography, and mixed with a test agent. The mixture is subjected to circular dichroism.
  • the conformation of a LNTl protein in the presence of a test agent is compared to a conformation of a LNTl protein in the absence of the test agent.
  • a change in conformational state of a LNTl protein in the presence of a test agent identifies a LNTl 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.
  • a method of identifying a LNTl inhibitor employs a functional LNTl protein, for example, as set forth in SEQ ID NO:2. Representative methods for determining LNTl function include assaying LNTl transcriptional activation activity (see Example 3) and assaying a physiological change elicited by LNTl activity, for example enhanced lateral root growth, increased nitrate uptake, increased nitrate content, and/or increased plant biomass ⁇ see Example 5).
  • a method of identifying a LNTl inhibitor may comprise (a) providing a cell expressing a LNTl protein; (b) contacting the cell with one or more test agents or a control agent; (c) assaying expression of a LNTl target gene; and (d) selecting a test agent that induces elevated expression of the LNTl target gene, which gene is normally repressed by LNTl ⁇ e.g., nitrate transporters), when contacted with the test agent as compared to the control agent.
  • a method of identifying a LNTl inhibitor may comprise (a) providing a cell expressing a LNTl protein; (b) contacting the cell with one or more test agents or a control agent; (c) assaying expression of LNTl target genes; and (d) selecting a test agent that results in reduced expression of LNTl target genes, which genes are normally subject to LNTl transcriptional activation, when contacted by the test agent as compared to the control agent.
  • cells expressing LNTl may be provided in the form of a kit useful for performing an assay of LNTl function.
  • a test kit for detecting a LNTl may include cells transfected with DNA encoding a full-length LNTl protein and a medium for growing the cells.
  • Assays of LNTl 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 LNTl expression, such that cells are simultaneously transfected with a nucleic acid molecule encoding LNTl 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.
  • a method of identifying a LNTl inhibitor may also comprise (a) providing a plant expressing a LNTl protein; (b) contacting the plant with one or more test agents or a control agent; (c) assaying (i) nitrate content; (ii) nitrate uptake, (iii) lateral root growth; or (iv) biomass; and (d) selecting a test agent that induces (i) increased nitrate content, (ii) increased nitrate uptake, (iii) increased lateral root growth, or (iv) increased biomass.
  • Assays employing cells expressing recombinant LNTl or plants expressing LNTl may additionally employ control cells or plants that are substantially devoid of native LNTl and, optionally, proteins substantially similar to a LNTl 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 LNTl protein, a control cell may comprise, for example, a parent cell line used to derive the LNTl -expressing cell line.
  • a control plant may include a LNTl knockout. In this instance, a LNTl inhibitor elicits a phenotype similar to a LNTl knockout, which is itself unresponsive to the inhibitor.
  • LNTl protein The knowledge of the structure of a native LNTl protein provides an approach for rational design of LNTl inhibitors.
  • the structure of a LNTl protein may be determined by X-ray crystallography and/or by computational algorithms that generate three-dimensional representations. See Saqi et al., Bioinformatics, 1999, 15:521-522; Huang et al., Pac. Symp. Biocomput, 2000, 230-241; and PCT International Publication No. WO 99/26966.
  • a working model of a LNTl protein structure may be derived by homology modeling (Maalouf et al., J. Biomol. Struct. Dyn.., 1998, 15(5):841 -851). Computer models may further predict binding of a protein structure to various substrate molecules that may be synthesized and tested using the assays described herein above. Additional compound design techniques are described in U.S. Patent Nos. 5,834,228 and 5,872,011.
  • a LNTl protein is a soluble protein, which may be purified and concentrated for crystallization.
  • the purified LNTl protein may be crystallized under varying conditions of at least one of the following: pH, buffer type, buffer concentration, salt type, polymer type, polymer concentration, other precipitating ligands, and concentration of purified LNTl.
  • Methods for generating a crystalline protein are known in the art and may be reasonably adapted for determination of a LNTl protein as disclosed herein. See e.g., Deisenhofer et al., J. MoI.
  • a crystallized LNTl protein may be tested for functional activity and differently sized and shaped crystals are further tested for suitability in X-ray diffraction. Generally, larger crystals provide better crystallography than smaller crystals, and thicker crystals provide better crystallography than thinner crystals. Preferably, LNTl crystals range in size from 0.1-1.5 mm.
  • crystals diffract X-rays to at least 10 A resolution, such as 1.5-10.0 A or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 A or less being preferred for the highest resolution.
  • a resolution such as 1.5-10.0 A or any range of value therein, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5 or 3, with 3.5 A or less being preferred for the highest resolution.
  • IV.F. LNTl Antibodies In another aspect of the invention, a method is provided for producing an antibody that specifically binds a LNTl protein.
  • a full-length recombinant LNTl 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, for example, an epitope within the LNTl DNA binding domain. Cell lines that produce anti-LNTl antibodies are also encompassed by the invention.
  • Specific binding of an antibody to a LNTl protein refers to preferential binding to a LNTl 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.
  • LNTl antibodies prepared as disclosed herein may be used in methods known in the art relating to the expression and activity of LNTl proteins, e.g., for cloning of nucleic acids encoding a LNTl protein, immunopurifi cation of a LNTl protein, and detecting a LNTl protein in a plant sample, and measuring levels of a LNTl 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.
  • LNTl binding partners and LNTl inhibitors are useful both in vitro and in vivo for applications generally related to assessing responses to nitrogen deprivation and for promoting nitrogen deprivation tolerance.
  • LNTl inhibitors may be used to repress or to disinhibit transcriptional targets of LNTl, to increase nitrate content in plants, to increase nitrate uptake into plant roots, and to induce lateral root sprouting.
  • the plants are typically subject to low nitrogen or nitrogen deprivation conditions, i.e., less than about 2 mM nitrate in the soil or growth medium, or other condition wherein a grower might consider application of a nitrogen fertilizer.
  • LNTl inhibitors of the invention may be applied to plants subject to nitrogen conditions of less than about 1.5 mM nitrate, less than about 1 mM nitrate, less than about 500 ⁇ M nitrate, less than about 400 ⁇ M nitrate, less than about 300 ⁇ M nitrate, less than about 200 ⁇ M nitrate, less than about 100 ⁇ M nitrate, or less than about 50 ⁇ M nitrate.
  • the present invention provides that an effective amount of a LNTl inhibitor is administered to a plant, i.e., an amount sufficient to elicit a desired biological response.
  • an effective amount of a LNTl inhibitor may comprise an amount sufficient to elicit elevated expression of genes normally subject to LNTl repression ⁇ e.g., genes encoding nitrate transporters), increased nitrate content in plant roots, increased nitrate uptake in plant roots, increased lateral root sprouting, and increased biomass.
  • Plants that may benefit from LNTl inhibition include, but are not limited to, corn (maize), sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers.
  • Representative vegetables include tomatoes, lettuce, green beans, lima beans, peas, yams, onions, and members of the genus Curcumis such as cucumber, cantaloupe, and musk melon.
  • Representative ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum. Any of the afore-mentioned plants may be wild type, inbred, or transgenic, e.g., plants strains and genetically modified plants as used in agricultural settings.
  • Plants treated with an LNTl inhibitor may be transgenic, i.e., genetically modified at LNTl, or at a locus other than LNTl to confer tolerance to nitrogen deprivation or other trait of interest.
  • desired traits include improved crop yield; insect resistance; tolerance to broad- spectrum herbicides; resistance to diseases caused by viruses, bacteria, fungi, and worms; and enhancement of mechanisms for protection from environmental stresses such as heat, cold, drought, and high salt concentration.
  • Additional desired traits include output traits that benefit consumers, for example, nutritionally enhanced foods that contain more starch or protein, more vitamins, more anti-oxidants, and/or fewer trans-fatty acids; foods with improved taste, increased shelf-life, and better ripening characteristics; trees that make it possible to produce paper with less environmental damage; nicotine-free tobacco; ornamental flowers with new colors, fragrances, and increased longevity; etc.
  • desirable traits that may be used in accordance with the invention include gene products produced in plants as a means for manufacturing, for example, therapeutic proteins for disease treatment and vaccination; textile fibers; biodegradable plastics; oils for use in paints, detergents, and lubricants; etc.
  • the combination of treatment with a LNTl inhibitor and a genetic modification can produce a synergistic effect, i.e., a change in gene expression, nitrate content, nitrate uptake, lateral root growth, or plant biomass that is greater than the change elicited by either genetic modification alone.
  • Example 1 Cloning and Characterization of LNTl A full-length LNTl cDNA was isolated from Arabidopsis thaliana using conventional RT-
  • LNTl contains the motif SHAQK within the MYB repeats, a motif shared with other members of the CCAl -like subfamily of MYB-related proteins. See Yanhui, 2006.
  • Tandem copies of the domain bind telomeric DNA tandem repeats as part of the capping complex. Binding is sequence dependent for repeats which contain the G/C rich motif [C2-3 A (CA)l-6].
  • a search of public sequence databases was conducted using the LNTl amino acid sequence as a query sequence. Three rice sequences and two grape sequences showing significant identities with the predicted full length amino acid sequence of LNTl were identified, as shown in Table 1.
  • Example 2 Expression of LNTl The organ-specific expression of LNTl in Arabidopsis plants was examined by RT-PCR
  • RNA isolation was performed essentially as described by Zhu et al., Plant MoI. Biol, 2005, 59(4):581-94.
  • the template for the probe was amplified from the 3' end of the LNTl cDNA with primers GlOl (SEQ K) NOs:3-4).
  • GlOl primers for RT-PCR analysis
  • cDNA was used as template for PCR amplification with specific primers Gl 02 (SEQ ID NOs:5-6).
  • Tubulin was used as an internal control using the primer Gl 03 (SEQ ID NO:7).
  • the results of RT-PCR showed that/JW7 is expressed in both vegetative and reproductive organs tested (root, stem, leaf, flower, and silique), with strong expression in leaf and root tissue, and moderate expression in other tissues tested. See Figure 2A.
  • the pLNTl-GXJS vector was constructed by cloning a promoter fragment (-1560 bp to -1 bp of the translational initiation site) of LNTl amplified by PCR using genomic DNA with primers Gl 09 (SEQ ID NOs: 18-19).
  • the 1560bp KpnVHindm PCR fragment was cloned into the pJIT166 vector (Guerineau et al., Plant Molecular Biology, 1992, 18:815-818) generating the p/JV77-GUS vector. Histochemical analysis of the GUS reporter enzyme activity was adapted from Jefferson, Plant Molecular Biology Report, 1987, 5:387-405.
  • Plantlets were incubated for 6 hours in reaction buffer without detergent, containing 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronid acid as the substrate.
  • samples were vacuum infiltrated for 30 minutes, incubated for 16 hours in reaction buffer containing 0.05% TRITON-X®100 and 2 mM 5-bromo- 4-chloro-3-indolyl-b-D-glucuronid acid, and plant pigments were cleared with 75% (v/v) ethanol. Samples were stored in 100% ethanol and whole plants were directly observed and photographed under a light microscope. Quantitative GUS enzymatic assays were performed essentially as described by Jefferson,
  • LNTl LNTl-GFP fusion gene in the plasmid pJIT163-hGFP was generated.
  • the fusion gene was expressed under the control of the double cauliflower mosaic virus (CaMV) 35S promoter.
  • CaMV cauliflower mosaic virus
  • LNTl encodes a MYB -like transcription factor.
  • LNTl was fused to the GAL4 DNA binding domain.
  • the yeast strain YRG-2 containing the HIS3 and lacZ reporter genes was used as an assay system (Stratagene of La Jolla, California, USA).
  • the coding sequence of LNTl was obtained by PCR using primers GI lO (SEQ ID NOs:20-21).
  • the PCR product was cloned into the vector containing the GAL-4 DNA binding domain to obtain pBO-LNTl. According to the manufacturer's protocol, pBO-LNTl, the positive control pGAL4, and the negative control pBD vector were all transformed into the yeast strain YRG2.
  • the transformed strains were confirmed by PCR and then were streaked on YPAD or SD/His " plates.
  • the transcription activation activities of each protein were evaluated according to their growth status.
  • Yeast cells containing pBD-LNTl and the positive control plasmid pGAL4 all grew well on SD medium without histidine, while the negative control plasmid pBD could not grow. At the same time, all of these yeast cells grew well on YPAD medium. See Figure 3. These results indicate that the full length LNTl protein has transcription activation activity.
  • Example 4 The Response of LNTl to Low Nitrogen Supply and Other Stresses LNTl has high expression at normal nitrogen levels (FN) both in aerial and root tissue, and repressed expression when subjected to low nitrogen treatment (LN). LNTl expression is rapidly up-regulated when plants are transferred from LN to FN (AN) ( Figure 4A). GUS enzymatic activity declined by 17% and 56% in aerial and root tissues, respectively, when exposed to nitrogen limitation (LN). Upon return to normal nitrogen levels (AN), GUS activity in aerial and root tissues increased 205% and 293%, respectively, as compared to the low nitrogen (LN) expression levels ( Figure 4B).
  • LNTl expression levels are also up-regulated or down-regulated in response to abiotic and biotic stresses, as shown in Table 2.
  • Two knockout mutants of LNTl, lntl-1 and lntl-2 were derived from the T-DNA insertion lines SALK 074896 and SALK 051843, respectively, which were supplied by the Arabidopsis Biological Resource Center, httBi//www J bipjcLpJii ⁇ iState 1 edu. Both of them have a T-DNA insertion in the fifth exon of LNTl, at 1164bp and 1618bp, respectively (Figure 5A).
  • PCR amplification techniques were used to identify homozygous mutant plants.
  • the PCR conditions were as follows: 35 cycles with each cycle consisting of 94 0 C for 30 seconds, 58 0 C for 45 seconds, and 72 0 C for 1 minute.
  • the T-DNA left border primer Gl 04 (SEQ ID NO:9), and gene-specific primers as follows were used in the PCR reactions: Gl 05 (SEQ ID NOs: 10-11) for lntl-1 and Gl 06 (SEQ ID NOs:12-13) for lntl-2.
  • LNTl in the lntl-1 and lntl-2 lines was examined using the probes synthesized from the LNTl cDNA.
  • the template was amplified with the primers Gl 07 (SEQ ID NOs: 14-15).
  • RNA gel blot analysis showed that the LNTl transcripts of the homozygous lntl-1 and lntl-2 were not detectable ( Figure 5B), which suggests that lntl-1 and lntl-2 are LNTl null mutant lines.
  • LNTl knockout plants grow better than wild type plants (i.e., show more aerial and root biomass) upon low nitrogen stress ( Figures HB and HD).
  • Figures HB and HD When exposed to low nitrogen deficiency, before flower lntl-1 and lntl-2 have accumulated nearly 27.2% and 31.9% more biomass than wild type in aerial tissue (0.0314, 0.0395 and 0.0414 g for WT, lntl-1 and lntl-2, respectively, Fig. 12b ), and with 54.9% and 46.7% in root biomass (0.0146, 0.0227 and 0.0215 g for WT, lntl-1 and lntl-2, respectively).
  • the biomass of lntl-1 and lntl-2 plants is 47.9% and 54.2% more than wild type in aerial tissue (0.0373, 0.0551 and 0.0575 g for WT, lntl-1 and lntl-2, respectively, Fig. 12d), and with 60.8% and 54.4% in root tissue (0.0242, 0.0390 and 0.0374 g for WT, lntl-1 and lntl-2, respectively, Fig. 12d).
  • No difference in biomass of LNTl knockout plants and wild type plants was observed under full nitrogen conditions ( Figures HA and HC).

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Abstract

Acides nucléiques et protéines de LNT1, partenaires de liaison et inhibiteurs de LNT1, souris inactivées pour LNT1, et leurs méthodes de fabrication et d’utilisation.
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WO2013067259A2 (fr) 2011-11-03 2013-05-10 Syngenta Participations Ag Acides nucléiques régulateurs et leurs procédés d'utilisation
CN103757020A (zh) * 2014-01-09 2014-04-30 浙江大学 用于调控烟草尼古丁合成和转运的基因及其应用
US8722072B2 (en) 2010-01-22 2014-05-13 Bayer Intellectual Property Gmbh Acaricidal and/or insecticidal active ingredient combinations
US9265252B2 (en) 2011-08-10 2016-02-23 Bayer Intellectual Property Gmbh Active compound combinations comprising specific tetramic acid derivatives
US9919507B2 (en) 2010-08-17 2018-03-20 The Boeing Company Process for inhibiting galvanic corrosion of an aluminum structure connected, without using a splice plate, to a composite structure having a fiber including graphite
CN110078804A (zh) * 2019-04-09 2019-08-02 中国农业科学院茶叶研究所 一种提高植物和微生物耐低氮胁迫能力的蛋白质及其基因

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
US8722072B2 (en) 2010-01-22 2014-05-13 Bayer Intellectual Property Gmbh Acaricidal and/or insecticidal active ingredient combinations
US9919507B2 (en) 2010-08-17 2018-03-20 The Boeing Company Process for inhibiting galvanic corrosion of an aluminum structure connected, without using a splice plate, to a composite structure having a fiber including graphite
US9265252B2 (en) 2011-08-10 2016-02-23 Bayer Intellectual Property Gmbh Active compound combinations comprising specific tetramic acid derivatives
WO2013067259A2 (fr) 2011-11-03 2013-05-10 Syngenta Participations Ag Acides nucléiques régulateurs et leurs procédés d'utilisation
CN103757020A (zh) * 2014-01-09 2014-04-30 浙江大学 用于调控烟草尼古丁合成和转运的基因及其应用
CN103757020B (zh) * 2014-01-09 2016-02-17 浙江大学 用于调控烟草尼古丁合成和转运的基因及其应用
CN110078804A (zh) * 2019-04-09 2019-08-02 中国农业科学院茶叶研究所 一种提高植物和微生物耐低氮胁迫能力的蛋白质及其基因

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