EP1109910A2 - Homologues de thioredoxine h - Google Patents
Homologues de thioredoxine hInfo
- Publication number
- EP1109910A2 EP1109910A2 EP99968680A EP99968680A EP1109910A2 EP 1109910 A2 EP1109910 A2 EP 1109910A2 EP 99968680 A EP99968680 A EP 99968680A EP 99968680 A EP99968680 A EP 99968680A EP 1109910 A2 EP1109910 A2 EP 1109910A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- isolated polynucleotide
- thioredoxin
- seq
- isolated
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
-
- 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
-
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
Definitions
- This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding thioredoxin proteins in plants and seeds.
- Thioredoxin H is a cytosolic member of the thioredoxin family of proteins. These small proteins (typical mass of 12 kD) have been shown to play a central role in the activation of proteins by influencing the redox status of sulfhydryl groups on target proteins (Holmgren et al. (1985) Annu. Rev. Biochem 54:131-111; and Holmgren et al. (1995) Structure 3(3) :239-243). Two other thioredoxin classes, F and M, are located in plastids and have been shown to be involved in redox mediated activation/inactivation of various photosynthetic enzymes during light/dark transitions.
- cytosolic (H) form of thioredoxin has been shown to be involved in disassembly of seed storage proteins during germination and in the bread making process.
- storage proteins are held together in clusters by S-S bonds.
- thioredoxin H reduces the S-S bonds and the subunits dissociate, facilitating attack by proteases.
- Reduction of the S-S bonds causes the protein complexes to disassemble allowing them to be distributed through out the dough during mixing.
- Thioredoxin H has also been shown to inactivate snake and bee venom toxins and has been shown to reduce the allergenicity of cereal proteins. In the later, the process is presumably the same as described above; by reducing the S-S bonds holding the storage protein clusters together they are more susceptible to denaturation and proteolysis in the gut. Thioredoxin H may also be overexpressed in transformed corn kernels and other cereal crops.
- the wet milling industry which is primarily focused on starch extraction, steeps corn in liquors of sodium metabisulphite or SO2. Although this has many secondary effects (e.g., suppression of microbial activity), the primary function is to cause a dissociation of the storage proteins which leads to more efficient starch extraction.
- thioredoxin H Small increases in extractable starch translate into significant increases in the profit margins for the wet millers.
- Overexpressing thioredoxin H in maize kernels and other cereals it may possible to improve starch recoveries, reduce steep times, and reduce or eliminate the use of sulfur compounds in the steeping process.
- Overexpression of thioredoxin H in maize kernels and other cereals may have the added advantage of reducing the allergenicity of any transgenic protein engineered into cereal crops with high sulfhydryl content.
- the present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 100 amino acids that has at least 80% identity based on the Clustal Method of alignment when compared to a polypeptide selected from the group consisting of a Momordica charantia thioredoxin polypeptide of SEQ ID NO:2, a Catakoa speciosa thioredoxin polypeptide of SEQ ID NO:4, a soybean thioredoxin polypeptide of SEQ ID NO:6, a soybean thioredoxin polypeptide of SEQ ID NO:8, and a Vernonia thioredoxin polypeptide of SEQ ID NO: 10.
- the present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above. It is preferred that the isolated polynucleotides of the claimed invention consists of regions of the isolated polynucleotide selected from the group SEQ ID NO:l, 3, 5, 7 and 9 that codes for the polypeptide selected from the group consisting of SEQ ID NO:2, 4, 6, 8 and 10.
- the present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NO:l, 3, 5, 7, 9 and the complement of such nucleotide sequences.
- the present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
- the present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention.
- the host cell may be eucaryotic, such as a yeast or a plant cell, or procaryotic, such as a bacterial cell.
- the present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
- the present invention relates to a process for producing an isolated host cell comprising a chimeric gene or isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
- the present invention relates to a thioredoxin polypeptide of at least 100 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NO:2, 4, 6, 8 and 10.
- the present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a thioredoxin polypeptide in a plant cell, the method comprising the steps of: constructing an isolated polynucleotide or chimeric gene of the present invention; introducing the isolated polynucleotide into a plant cell; ⁇ measuring the level of thioredoxin polypeptide in the plant cell containing the polynucleotide; and comparing the level of thioredoxin polypeptide in the plant cell containing the isolated polynucleotide with the level of thioredoxin polypeptide in a plant cell that does not contain the isolated polynucleotide.
- the present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a thioredoxin gene, preferably a plant thioredoxin gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer.
- the amplified nucleic acid fragment preferably will encode a portion of a thioredoxin polypeptide amino acid sequence.
- the present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a subsantial portion of the amino acid sequence encoding a thioredoxin protein comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
- Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing.
- the sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.821-1.825.
- a "polynucleotide” is a nucleotide sequence such as a nucleic acid fragment.
- a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
- a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.
- substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co- suppression technology.
- Substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% ⁇ sequence identity with the gene to be suppressed.
- alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide are well known in the art.
- a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
- changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine can also be expected to produce a functionally equivalent product.
- a polynucleotide comprising a nucleotide sequence of at least one of 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NO:l, 3, 5, 7, 9 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a thioredoxin polypeptide in a plant cell.
- substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds.
- Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
- Post-hybridization washes determine stringency conditions.
- One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
- a more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60°C.
- Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.
- Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (polynucleotides) encode amino acid sequences that are 80% identical to the amino acid sequences reported herein.
- PENALTY 10).
- a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises.
- Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer- based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J Mol. Biol. 275:403-410; see also www.ncbi.nlm.nih.gov/BLAST/).
- a sequence often or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
- gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques).
- oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
- a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
- the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.
- Codon degeneracy refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein.
- the skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
- Synthetic nucleic acid fragments can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. "Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines.
- nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell.
- the skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
- Gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.
- “Native gene” refers to a gene as found in nature with its own regulatory sequences.
- Chimeric gene refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
- Endogenous gene refers to a native gene in its natural location in the genome of an organism.
- a “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
- a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
- Coding sequence refers to a nucleotide sequence that codes for a specific amino acid sequence.
- Regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
- Promoter refers to a nucleotide sequence capable of controlling the expression ⁇ fa coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence.
- the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
- an "enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions.
- Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 75:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
- the "translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence.
- the translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence.
- the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 5:225-236).
- the "3' non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
- the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
- the use of different 3' non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1 :671 -680.
- RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
- Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into polypeptide by the cell.
- cDNA refers to a double-stranded DNA that is complementary to and derived from mRNA.
- Sense RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell.
- Antisense RNA refers to an RNA transcript that is — complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Patent No. 5,107,065, incorporated herein by reference).
- the complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
- “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
- operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
- a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
- Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
- expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
- Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
- Overexpression refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms.
- Co-suppression refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Patent No. 5,231,020, incorporated herein by reference).
- altered levels refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
- Measure protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed.
- Precursor protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.
- chloroplast transit peptide is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made.
- Chloroplast transit sequence refers to a nucleotide sequence that encodes a chloroplast transit peptide.
- a “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53).
- a vacuolar targeting signal can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added.
- an endoplasmic reticulum retention signal may be added.
- any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1611-1632).
- Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic” organisms.
- methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:111) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:10-13; U.S. Patent No. 4,945,050, incorporated herein by reference). Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Maniatis”).
- nucleic acid fragments encoding at least a portion of several thioredoxin proteins have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art.
- the nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art.
- sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).
- genes encoding other thioredoxin H proteins could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art.
- Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis).
- the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems.
- primers can be designed and used to amplify a part or all of the instant sequences.
- the resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.
- two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA.
- the polymerase chain reaction may also be — performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding plant genes.
- the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc.
- a polynucleotide comprising a nucleotide sequence of at least one of 40 (preferably 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NO:l, 3, 5, 7, 9 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a thioredoxin polypeptide. Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries.
- Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36: -34; Maniatis).
- nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of thioredoxin activity in those cells.
- Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development.
- the chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3' Non-coding sequences encoding transcription termination signals may also be provided.
- the instant chimeric gene may also comprise one or more introns in order to facilitate gene expression. Plasmid vectors comprising the instant chimeric gene can then be constructed. The- choice of plasmid vector is dependent upon the method that will be used to transform host plants.
- the chimeric gene described above may be further supplemented by altering the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:141-153), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:11-53), or nuclear localization signals (Raikhel (1992) Plant Phys.100:1611 -1631) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.
- a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences.
- a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.
- tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
- tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.
- special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes.
- the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
- the instant polypeptides may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art.
- the antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts.
- Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides.
- This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded thioredoxin protein.
- An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).
- nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes.
- the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers.
- RFLP restriction fragment length polymorphism
- Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 7:174-181) in order to construct a genetic map.
- nucleic- acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 52:314-331).
- nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154).
- FISH direct fluorescence in situ hybridization
- nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 77:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:315-331), allele- specific ligation (Landegren et al. (1988) Science 247:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 75:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet.
- Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinge and Benzer (1989) Proc. Natl. Acad. Sci USA 5(5:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways.
- short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra).
- the amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptides.
- the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor.
- an arbitrary genomic site primer such as that for a restriction enzyme site-anchored synthetic adaptor.
- composition of cDNA Libraries Isolation and Sequencing of cDNA Clones cDNA libraries representing niRNAs from various barley, Catalpa, pear, soybean and Vernonia tissues were prepared. The characteristics of the libraries are described below.
- cDNA libraries may be prepared by any one of many methods available.
- the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAPTM XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA).
- the Uni-ZAPTM XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript.
- the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).
- plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or
- ESTs see Adams et al., (1991) Science 252:1651-1656).
- the resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.
- the data in Table 4 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8 and 10 and the Arabidopsis thaliana, Nicotiana tabacum and Ricinus communis sequences (SEQ ID NOs:l 1, 12 and 13 respectively).
- the percent identity between the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 8 and 10 ranged from 49% to 80%.
- a chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5' to the cDNA fragment, and the 10 kD zein 3' end that is located 3' to the cDNA fragment, can be constructed.
- the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (Ncol or Smal) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below.
- Plasmid pML 103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Boulevard., Manassas, VA 20110-2209), and bears accession number ATCC 97366.
- the DNA segment from pML103 contains a 1.05 kb Sall-Ncol promoter fragment of the maize 27 kD zein gene and a 0.96 kb Smal-Sall fragment from the 3' end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega).
- Vector and insert DNA can be ligated at 15°C overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL 1 -Blue (Epicurian Coli XL-1 BlueTM; Stratagene).
- Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (SequenaseTM DNA Sequencing Kit; U.S. Biochemical).
- the resulting plasmid construct would comprise a chimeric gene encoding, in the 5' to 3' direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3' region.
- the chimeric gene described above can then be introduced into com cells by the following procedure.
- Immature com embryos can be dissected from developing caryopses derived from crosses of the inbred com lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27°C.
- Friable embryogenic callus consisting of undifferentiated masses of cells with somatic — proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos.
- the embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
- the plasmid, p35S/Ac obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt,
- This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transf erase (PAT).
- PAT phosphinothricin acetyl transf erase
- the enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin.
- the pat gene in p35S/Ac is under the control of the 35 S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.
- the particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells.
- gold particles (1 ⁇ m in diameter) are coated with DNA using the following technique.
- Ten ⁇ g of plasmid DNAs are added to 50 ⁇ L of a suspension of gold particles (60 mg per mL).
- Calcium chloride 50 ⁇ L of a 2.5 M solution
- spermidine free base (20 ⁇ L of a 1.0 M solution) are added to the particles.
- the suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed.
- the particles are resuspended in 200 ⁇ L of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 ⁇ L of ethanol.
- An aliquot (5 ⁇ L) of the DNA-coated gold particles can be placed in the center of a KaptonTM flying disc (Bio-Rad Labs). The particles are then accelerated into the com tissue with a BiolisticTM PDS-1000/He (Bio-Rad Instruments, Hercules CA), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
- the embryogenic tissue is placed on filter paper over agarose- solidified N6 medium.
- the tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter.
- the petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen.
- the air in the chamber is then evacuated to a vacuum of 28 inches of Hg.
- the macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
- tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate- supplemented medium. These calli may continue to grow when sub-cultured on the " ⁇ selective medium.
- Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 5:833-839).
- a seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the ⁇ subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261 :9228-9238) can be used for expression of the instant polypeptides in transformed soybean.
- the phaseolin cassette includes about 500 nucleotides upstream (5') from the translation initiation codon and about 1650 nucleotides downstream (3') from the translation stop codon of phaseolin. Between the 5' and 3' regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Smal, Kpnl and Xbal. The entire cassette is flanked by Hind III sites.
- the cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.
- PCR polymerase chain reaction
- Soybean embroys may then be transformed with the expression vector comprising sequences encoding the instant polypeptides.
- Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26°C with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.
- Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 527:70-73, U.S. Patent No. 4,945,050).
- a DuPont BiolisticTM PDS1000/HE instrument helium retrofit
- a selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Vims (Odell et al. (1985) Nature 575:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacte ⁇ um tumefaciens.
- the seed expression cassette comprising the phaseolin 5' region, the fragment encoding the instant polypeptides and the phaseolin 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
- Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60x15 mm petri dish and the residual liquid removed from the tissue with a pipette.
- approximately 5-10 plates of tissue are normally bombarded.
- Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury.
- the tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
- Five to seven days post bombardment the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly.
- green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
- the cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430.
- This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 5(5:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system.
- Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector.
- Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis.
- Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTGTM low melting agarose gel (FMC). Buffer and agarose contain 10 ⁇ g/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELaseTM (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 ⁇ L of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, MA).
- the fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above.
- the vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above.
- the prepared vector pBT430 and fragment can then be ligated at 16°C for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL).
- Transformants can be selected on agar plates containing LB media and 100 ⁇ g/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.
- a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25°C. At an optical density at 600 run of approximately 1, IPTG (isopropylthio- ⁇ -galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°.
- IPTG isopropylthio- ⁇ -galactoside, the inducer
- Cells are then harvested by centrifugation and re-suspended in 50 ⁇ L of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride.
- a small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator.
- the mixture is centrifuged and the protein concentration of the supernatant determined.
- One ⁇ g of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US9950198P | 1998-09-08 | 1998-09-08 | |
US99501P | 1998-09-08 | ||
PCT/US1999/020420 WO2000014239A2 (fr) | 1998-09-08 | 1999-09-07 | Homologues de thioredoxine h |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1109910A2 true EP1109910A2 (fr) | 2001-06-27 |
Family
ID=22275311
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99968680A Withdrawn EP1109910A2 (fr) | 1998-09-08 | 1999-09-07 | Homologues de thioredoxine h |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1109910A2 (fr) |
AU (1) | AU6028099A (fr) |
WO (1) | WO2000014239A2 (fr) |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20010108021A (ko) * | 1998-12-17 | 2001-12-07 | 릴리 엠 씨즐러 스피허, 아네뜨 워너 | 티오레독신 및 곡물 처리방법 |
US6531648B1 (en) | 1998-12-17 | 2003-03-11 | Syngenta Participations Ag | Grain processing method and transgenic plants useful therein |
US7009087B1 (en) | 2000-12-01 | 2006-03-07 | Pioneer Hi-Bred International, Inc. | Compositions and methods for altering the disulfide status of proteins |
US20060064784A1 (en) | 2002-08-07 | 2006-03-23 | Basf Plant Science Gmbh | Nucleic acid sequences encoding proteins associated with abiotic stress response |
ES2354537B1 (es) * | 2008-06-27 | 2012-01-23 | Universidad Pública de Navarra | Tiorredoxinas plastidiales: sobreexpresión y aplicaciones biotecnológicas. |
ES2384777B1 (es) * | 2008-06-27 | 2013-06-19 | Universidad Pública de Navarra | Tiorredoxinas plastidiales: sobreexpresión y aplicaciones biotecnológicas. |
CN107232059B (zh) * | 2017-06-28 | 2019-09-13 | 山东博华高效生态农业科技有限公司 | 一种山苦瓜的组培快繁方法 |
CN117264971B (zh) * | 2023-11-09 | 2024-05-03 | 海南大学 | 大豆硫氧还原蛋白基因GmTrx1及其应用 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5614395A (en) * | 1988-03-08 | 1997-03-25 | Ciba-Geigy Corporation | Chemically regulatable and anti-pathogenic DNA sequences and uses thereof |
EP0672127B1 (fr) * | 1991-10-12 | 1999-01-07 | The Regents Of The University Of California | Utilisation de proteines d'oxydoreduction a base de thiol pour reduire des liaisons bisulfures |
US5792506A (en) * | 1991-10-12 | 1998-08-11 | The Regents Of The University Of California | Neutralization of food allergens by thioredoxin |
FR2723097B1 (fr) * | 1994-07-26 | 1996-10-18 | Agronomique Inst Nat Rech | Thioredoxines h de ble tendre et de ble dur et proteines presentant des similitudes, fragments d'adncodant pour ces proteines et procedes d'obtention |
-
1999
- 1999-09-07 EP EP99968680A patent/EP1109910A2/fr not_active Withdrawn
- 1999-09-07 WO PCT/US1999/020420 patent/WO2000014239A2/fr active Application Filing
- 1999-09-07 AU AU60280/99A patent/AU6028099A/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO0014239A3 * |
Also Published As
Publication number | Publication date |
---|---|
AU6028099A (en) | 2000-03-27 |
WO2000014239A2 (fr) | 2000-03-16 |
WO2000014239A3 (fr) | 2000-09-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2002057439A2 (fr) | Facteurs de transcription pour vegetaux | |
WO2002044390A2 (fr) | Genes de developpement floral | |
EP1097221A2 (fr) | Genes codant pour des proteines d'assimilation des sulfates | |
WO2000014207A2 (fr) | Farnesyltransferases vegetales | |
US7129088B2 (en) | Plant caffeic acid 3-O-methyltransferase homologs | |
WO2000014239A2 (fr) | Homologues de thioredoxine h | |
EP1062355A1 (fr) | Inhibiteurs de proteines d'apoptose dans les plantes | |
US7572951B2 (en) | Plant viral movement protein genes | |
US7029889B2 (en) | Plant sorbitol biosynthetic enzymes | |
WO2000075340A2 (fr) | Chelatase de magnesium | |
WO2000037652A2 (fr) | Enzymes biosynthetiques flavonoides | |
US20040048382A1 (en) | Plant UDP-glucose dehydrogenase | |
US7087813B2 (en) | Plant lipoxygenases | |
US6897356B1 (en) | Thioredoxin H homologs | |
US6893853B1 (en) | Chromatin associated proteins | |
US7202398B2 (en) | Chalcone isomerase | |
US20050074891A1 (en) | Starch branching enzyme IIb | |
EP1056863A1 (fr) | FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX | |
WO2000036121A2 (fr) | Proteines phosphatases de plantes | |
WO1999049047A2 (fr) | 1,3-beta-d-glucane synthases vegetales et sequences codant brittle-1 | |
WO2000032782A2 (fr) | Facteurs de tanscription de vegetaux | |
US6939710B2 (en) | Genes encoding sulfate assimilation proteins | |
WO2001014540A2 (fr) | Isoforme v de la synthase de l'amidon | |
WO2004096995A2 (fr) | Ribonuclease dicer | |
WO2001019995A1 (fr) | Genes de regulation de la floraison des plantes |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20010219 |
|
AK | Designated contracting states |
Kind code of ref document: A2 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
RBV | Designated contracting states (corrected) |
Designated state(s): DE FR GB |
|
17Q | First examination report despatched |
Effective date: 20040819 |
|
17Q | First examination report despatched |
Effective date: 20040819 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20071228 |