JP2002519035A - HMP-P kinase and TMP-PPase from Arabidopsis and their use in herbicide screening - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for screening chemicals for herbicidal activity using recombinantly produced enzymes having HMP-P kinase activity or TMP-PPase activity, and for inhibiting unwanted vegetation growth. Disclosed are their use for the identification of herbicide chemicals. Further, the present invention provides a method for developing herbicide tolerance in plants, plant tissues, plant seeds and plant cells using a gene encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, and Methods of using such transgenic plants to selectively control weeds growing in crop fields are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate kinase (HMP-P kinase) or thiamine monophosphate pyrophosphorylase (2-methyl-4-amino-5- (hydroxymethyl) Pyrimidine diphosphate: 4-methyl-5- (2-phosphoethyl) thiazole 2-methyl-4-aminopyrimidine-5-methyltransferase) or TMP-
The present invention relates to a method for screening a herbicide compound that inhibits the enzyme activity of PPase (all of these are enzyme activities involved in de novo thiamine biosynthesis). The present invention relates to the use of the herbicide compounds identified thereby for controlling unwanted vegetation growth. The invention can also be applied to the development of herbicide-tolerant plants, plant tissues, plant seeds and plant cells.

[0002] The use of herbicides to control undesirable vegetation, such as weeds in cereal fields, has become almost universally practiced. The herbicide market exceeds $ 15 billion annually. Despite this widespread use, weed control remains a significant and costly problem for farmers.

[0003] Effective use of herbicides requires sound management. For example, the timing and method of application and the stage of weed plant development are important for good weed control with herbicides. Due to the resistance of various weed species to herbicides, the production of effective new herbicides is of increasing importance. By the way, new herbicides can be discovered using high-throughput screens implementing recombinant DNA technology. Metabolic enzymes that have been found to be essential for plant growth and development can be produced recombinantly and utilized as herbicide targets in screens for novel inhibitors of enzyme activity. The novel inhibitors discovered by such screens can then be used as herbicides to control unwanted vegetation.

[0004] Herbicides that exhibit greater potency, a broader weed range and more rapid degradation in soil can, unfortunately, also have greater crop phytotoxicity. One solution applied to this problem has been to develop crops that are tolerant or tolerant to herbicides. Crop hybrids or varieties that are tolerant to herbicides allow the use of herbicides to kill weeds without the associated risk of damage to the crop. The development of tolerability allows the application of herbicides to crops whose use has previously been eliminated or restricted due to the sensitivity of the crop to herbicides (eg, for pre-emergence use).
For example, US Pat. No. 4,761,373 (Anderson et al.) Is directed to plants that are resistant to various imidazolinone or sulfonamide herbicides. Resistance is conferred by the modified acetohydroxy acid synthase (AHAS) enzyme. US Patent 4,975,374
No. (Goodman et al.) Encodes glutamine synthase (GS), for example a mutant glutamine synthase (GS) that is resistant to inhibition by herbicides known to inhibit phosphinothricin and methionine sulfoximine. It relates to plant cells and plants containing the gene. U.S. Patent No. 5,013,659 (Bedbrook et al.) Is directed to plants that express a mutant acetolactate synthase that renders the plant resistant to inhibition by sulfonylurea herbicides. US Patent 5,162,60
No. 2 (Somers et al.) Discloses plants that are tolerant to inhibition by chlorohexanedione and aryloxyphenoxypropanoic acid herbicides. Tolerability is conferred by altered acetyl coenzyme carboxylase (ACCase).

Definitions For clarity, certain terms used herein are defined and presented as follows: activatable DNA sequence : a gene in the genome, preferably in the genome of a plant DNA sequence that regulates the expression of The activatable DNA sequence is complementary to a target gene endogenous to the genome. When the activatable DNA sequence is introduced into the cell and expressed,
It suppresses the expression of the target gene. Activable DNA useful in connection with the present invention
Sequences include those that encode or act as inhibitors of major inhibitors, such as those in a stably transformed plant to unambiguously identify one or more genes that are essential for the normal growth and development of the plant. Translatable or non-translatable sense sequences capable of interrupting the gene function of the gene. Target genes are preferably proteins essential for plant growth or development, such as biosynthetic enzymes, receptors, signaling proteins,
Encodes a structural gene product or transport protein. In a particularly preferred embodiment, the target gene encodes an enzyme having HMP-P kinase activity or TMP-PPase activity. The interaction of the antisense sequence and the target gene causes a substantial inhibition of the expression of the target gene so as to kill the plant or at least suppress normal plant growth or development.

Activable DNA constructs : A recombinant DNA construct comprising a synthetic promoter operably linked to an activatable DNA sequence, when introduced into a cell, preferably a plant, binds to the synthetic promoter, It is not expressed, i.e., silent, unless a complete hybrid transcription factor capable of activating it is present. The activatable DNA construct is introduced into a cell, tissue or plant to form the activatable DNA.
Form stable transgenic lines capable of expressing the NA sequence.

Cofactor : A natural reactant required for an enzyme-catalyzed reaction, such as an organic molecule or metal ion. Cofactors include, for example, NAD (P), riboflavin (including FAD and FMN), folate, molybdopterin, thiamine, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone , Menaquinone. Optionally, cofactors can be adjusted and reused.

[0008] conjugate synthesis (Coupled Synthesis): the final product is a two or more enzymatic biosynthesis synthesized by sequential enzymatic steps, in this case, one or more partial biochemical pathways The substrate for the first enzymatic step in the branch pathway is captured in the reaction mixture and converted to an intermediate by the primary enzyme, which is finalized by one or more secondary enzymes without the addition of the intermediate. Converted to product.

A “chimera” is a DNA sequence, such as a vector or gene, that is a recombinant DNA
It is composed of more than one DNA sequence of different origin fused together by a technique and is used to indicate that it produces a DNA sequence that does not occur naturally and does not specifically occur in transformed plants. DNA shuffling : DNA shuffling is the exchange of DNA sequences, preferably at random, to introduce mutations or rearrangements into DNA molecules, or between two or more DNA molecules, preferably at random. Is a method for generating A DNA molecule resulting from DNA shuffling is a shuffled DNA molecule that is a non-natural DNA molecule derived from at least one template DNA molecule. Shuffled DNA encodes an enzyme that is modified with respect to the enzyme encoded by the template DNA, and has altered biological activity with respect to the enzyme encoded by the template DNA.

[0010] Enzyme activity : As used herein, means the ability of an enzyme to catalyze the conversion of a substrate to a product. Substrates for an enzyme include the natural substrate of the enzyme, but also include analogs of the natural substrate that can be converted into a product or an analog of the product by the enzyme. The activity of the enzyme is measured, for example, by determining the amount of product in the reaction after a period of time or by determining the amount of substrate remaining in the reaction mixture after a period of time. The activity of the enzyme can also be determined by determining the amount of unused cofactor in the reaction remaining in the reaction mixture after a period of time, or by determining the amount of spent cofactor in the reaction mixture after a period of time. Measured. The activity of the enzyme may be determined by determining the amount of free energy or donor of high energy molecules (eg, ATP, phosphoenolpyruvate, acetyl phosphate or phosphocreatine) remaining in the reaction mixture after a period of time, or It is also determined by determining the amount of free energy or spent donor of high energy molecules (eg, ADP, pyruvate, acetate or creatine) in the reaction mixture.

Expression refers to the transcription and / or translation of an endogenous or transgene in a plant. For example, in the case of an antisense construct, expression may indicate only transcription of the antisense DNA. A gene is defined as a coding sequence, as well as when the coding sequence is RNA, eg, mRNA, r
Refers to related regulatory sequences when transcribed into RNA, tRNA, snRNA, sense RNA or antisense RNA. Examples of regulatory sequences are promoter sequences, 5 'and 3' untranslated sequences, and termination sequences. Further elements that can be present are, for example, introns.

Herbicides : Chemicals used to kill or control plants, plant cells, plant seeds or plant tissues. Heterologous DNA sequence : A DNA sequence that is not naturally associated with the host cell into which it is introduced, eg, a non-naturally occurring multiple copy of a native DNA sequence. Homologous DNA sequence : A DNA sequence that is naturally associated with the host cell into which it is introduced.

Inhibitors : Chemicals that inactivate the enzymatic activity of proteins essential for plant growth or survival, such as biosynthetic enzymes, receptors, signaling gene structural gene products or transport proteins. In the context of the present invention, the inhibitor is HMP-P from a plant.
It is a chemical that inactivates kinase / TMP-PPase. The term "herbicide" is used herein to indicate an inhibitor when applied to a plant, plant cell, plant seed or plant tissue.

[0014] The same gene system: is a genetically identical, but may differ by the presence or absence of the transgene plant. Isolated : In the context of the present invention, an isolated DNA molecule or enzyme is a DNA molecule or enzyme that exists, by human hand, away from its natural environment and is therefore not a natural substance. An isolated DNA molecule or enzyme can exist in a purified form, or can exist in a non-natural environment, eg, in a transgenic host cell.

Mature protein : A protein that is normally targeted to an organelle, such as the chloroplast, from which the transit peptide has been removed. Minimal promoter : A promoter element, particularly a TATA element, that is inactive or has greatly reduced promoter activity in the absence of upstream activity. In the presence of an appropriate transcription factor, the minimal promoter functions to allow transcription.

Modified enzyme activity : One that occurs naturally in plants that is tolerable to inhibitors that inhibit the natural enzyme activity (ie, in the absence of direct or indirect manipulation of such activity by humans). Enzyme activity). Operatively linked means that a regulatory DNA sequence is " operably linked " to a DNA sequence encoding an RNA or protein when the two sequences are adapted to affect the expression of the coding DNA sequence. "Possibly linked" or "
Will be met. "

[0017] The plant refers to any plant, particularly a seed plant. Plant cells refer to the structural and physiological units of a plant, including protoplasts and cell walls. Plant cells can be in the form of isolated single cells or cultured cells, or can be present as part of a highly organized unit, such as a plant tissue or plant organ.

Plant materials include leaves, stems, roots, flowers or flower parts, fruits, pollen, pollen tubes, ovules, embryo sac, egg cells, zygotes, embryos, seeds, cuttings, cell or tissue cultures, or plant Refers to any other parts or products. Preprotein : A protein normally targeted to an organelle, such as a chloroplast, and still encompasses its transit peptide.

[0019] Recombinant DNA refers to a combination of DNA sequences that are brought together using recombinant DNA technology. Recombinant DNA technology includes, for example, Sambrook et al., 1989, Cold Spring Harbor,
NY: refers to the technique used to join DNA sequences together as described in the Cold Spring Harbor Laboratory Press.

Significant increase : An increase in enzyme activity that is greater than the error limits inherent in the assay technique, preferably about 2 times greater than the activity of the wild-type enzyme in the presence of the inhibitor, more preferably about 5 times or more. Increase, most preferably about 10-fold or more. Significantly lower : means that the amount of product of the enzymatic reaction is greater than the error limit inherent in the measurement technique, preferably by a factor of about 2 or more in the absence of the inhibitor from the activity of the wild-type enzyme. More preferably a reduction of about 5 times or more, most preferably a reduction of about 10 times or more.

In a broad sense, the term “substantially similar,” as used herein in reference to a nucleotide sequence, a polypeptide whose corresponding sequence has substantially the same structure as the polypeptide encoded by the reference nucleotide sequence Means, for example, only amino acid changes that do not affect polypeptide function. Desirably, a substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between substantially similar nucleotide sequences and the reference nucleotide sequence is desirably at least 85%, more desirably at least 90%, preferably at least 92%, more preferably at least 95%,
More preferably at least 97%, more preferably at least 99%. Sequence comparisons are performed using the Smith-Waterman sequence alignment algorithm (eg, Waterman, MS Introduction to Computational Biology; Maps, sequences).
and genomes. Chapman &Hall.London; 1995, ISBN 0-412-99391-0, or http
: //www-hto.usc.edu/software/seqaln/index.html). The local S program (version 1.16) is used with the following parameters: Match: 1
, Mismatch penalty: 0.33, open gap penalty: 2, extended gap penalty: 2. To a reference nucleotide sequence "substantially similar" nucleotide sequence in 7% sodium dodecyl sulfate 50 ℃ (SDS), 0.5 MNaPO 4, 1 mMED
Washing in 1 × SSC, 0.1% SDS at 65 ° C. in TA, more preferably
7% sodium dodecyl sulfate (SDS) at 50 ° C., 0.5 M NaPO ₄ , 1 mM EDTA
In 0.5 x SSC, 0.1% SDS at 65 ° C.
7% sodium dodecyl sulfate (SDS) at 50 ° C., 0.5 M NaPO ₄ , 1 mM EDTA
Washing at 65 ° C. in 0.2 × SSC, 0.1% SDS, preferably at 50 ° C.
% Sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA.
Hybridize with the reference nucleotide sequence while washing in 0.1 x SSC, 0.1% SDS at ° C.

The term “substantially similar” as used herein in reference to a protein means a protein corresponding to the reference protein, but in this case or having substantially the same structure as the reference protein. For example, only amino acid sequence changes that have a function and do not affect polypeptide function occur. When used with respect to a protein or amino acid sequence, the percentage of identity between substantially similar protein or amino acid sequence and the reference protein or amino acid sequence is desirably at least 65%, more desirably at least 75%, preferably at least 85%.
%, More preferably at least 90%, more preferably at least 95%, more preferably at least 99%.

Substrate : A substrate is a molecule that an enzyme naturally recognizes and whose function converts into a substance in a biochemical pathway that the enzyme naturally performs, or a substrate that is recognized by the enzyme and reacts naturally with the enzyme. A modified version of a molecule that is converted by an enzyme into a substance undergoing an enzymatic reaction similar to. Synthetic refers to nucleotide sequences that include structural features not present in the native sequence. For example, artificial sequences in which the G + C content and normal codon distribution of dicotyledonous and / or monocotyledonous genes are very similar are said to be synthetic sequences.

Tolerability : The ability to continue normal growth or function when exposed to inhibitors or herbicides. Transformation : A method for introducing heterologous DNA into cells, tissues or plants. A transformed cell, tissue or plant is understood to encompass not only the final product of the transformation process, but also its transgenic progeny.

Transgenic : Preferably, it has been stably transformed with a recombinant DNA molecule containing a suitable promoter operably linked to the DNA sequence. One object of the present invention is to provide a method for identifying new or improved herbicides. Another object of the present invention is to provide a method for using such new or improved herbicides to inhibit the growth of plants such as weeds. Yet another object of the present invention is to provide improved crop plants that are tolerant to such new or improved herbicides.

The present invention relates initially to Arabidopsis HMP-P kinase / T
Discloses the exact nucleotide sequence of the MP-PPase gene. The nucleotide sequence encoding the preprotein is set forth in SEQ ID NO: 1, and the nucleotide sequence encoding the putative mature protein is set forth in SEQ ID NO: 3. Arabidopsis HM
The exact amino acid sequences of PP kinase and TMP-PPase preprotein are SEQ ID NO: 2, and putative mature Arabidopsis HMP-P kinase and TM
The exact amino acid sequence of P-PPase is set forth in SEQ ID NO: 4. Therefore,
The present invention encompasses nucleotide sequences obtained from plants that encode enzymes having HMP-P kinase activity or TMP-PPase activity. In a preferred embodiment, such a nucleotide sequence is Arabidopsis thaliana
), And in a further preferred embodiment such a nucleotide sequence is
Is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, and its amino acid sequence is identical or substantially identical to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4 And an enzyme having HMP-P kinase activity or TMP-PP activity.

HMP-P kinase and TMP-PPase are present at enzymatic steps in the de novo thiamine biosynthetic pathway (see below). De novo thiamine biosynthesis finally results in the production of thiamine pyrophosphate, also known as thiamine diphosphate or vitamin B1, a form of this cofactor found in some enzymes (Schellenberger et al. (1997) Meth. Enz. 279, 131-146).
This de novo biosynthetic pathway occurs in bacteria, yeast and plants, but not in humans (Begley, TP (1996) Natl. Prod. Rep. 13, 177-186). HMP-P kinase enzyme activity catalyzes the pyrophosphorylation of 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) to produce 2-methyl-4-
Produces amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP). In E. coli, this step is performed by the protein encoded by the thiD gene. TMP-PPase enzyme activity is determined by thiamine pyrophosphate.
Corresponds to almost the last step in de novo biosynthesis, 4-methyl-5- (β-
(Hydroxyethyl) thiazole monophosphate (THZ-P) and HMP-PP
Catalyzes the formation of thiamine monophosphate (TMP).
In E. coli, this step is performed by the protein encoded by the thiE gene.

With knowledge of the exact nucleotide sequence of the HMP-P kinase / TMP-PPase gene, the inventors have determined that thiamin nutrition needs to be supplemented with exogenous thiamine to allow growth. Requirement strains are those HMP-P kinase / T
The coding sequence of the MP-PPase gene was shown to have a mutation. This certainly demonstrates the essentiality of this gene in plants at the molecular level. Such knowledge can be used to develop screens for chemicals that can inhibit HMP-P kinase activity or TMP-PPase activity. HMP-P kinase / T
Because of the essentiality of the enzymatic activity encoded by the MP-PPase gene, it is also anticipated that such chemicals will inhibit plant growth or viability and may therefore be good herbicides. Thus, purified HMP-P kinases or TMP-PPases are used to identify their inhibitors, which are then used as herbicides, e.g. for crops, especially for agriculturally important crops such as corn and other crops. Unwanted vegetation growth in cereal crops such as wheat, oats, rye, sorghum, rice, barley, millet, turf and forage grasses, and in areas where cotton, sugar cane, sugar beet, rapeseed and soybean are grown. It is an object of the present invention to provide a method that can be suppressed.

Included in the present invention is the HMP-P kinase activity or TMP-P of the present invention.
A chimeric gene comprising a promoter operably linked to a nucleotide sequence encoding an enzyme having Pase activity. Also encompassed is a recombinant vector comprising the chimeric gene of the present invention, in which case the vector can be stably transformed into a host cell. Also encompassed are host cells containing the vectors of the invention, wherein the nucleotide sequence is expressible in the cells. Preferred is a host cell of the invention that is a eukaryotic cell. Even more preferred are insect cells,
The host cell of the present invention is selected from the group consisting of yeast cells and plant cells.
Host cells that are prokaryotic are also preferred. Even more preferred are host cells of the present invention which are bacterial cells. Also included are isolated plant proteins involved in thiamine biosynthesis, wherein said proteins have HMP-P kinase activity or TMP-PPase activity. Preferred is an isolated protein, wherein the plant is Arabidopsis thaliana. More preferred is HM
It is a protein of the present invention having PP kinase activity. Particularly preferred is a protein of the invention comprising an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. Particularly preferred is a protein comprising the amino acid sequence set forth in SEQ ID NO: 2.

Preferred are those proteins of the invention that have TMP-PPase activity.
Even more preferred is a protein of the present invention that includes an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. Even more preferred is a protein of the invention comprising the amino acid sequence set forth in SEQ ID NO: 2. In a preferred embodiment, the present invention is a method of identifying a chemical to be tested for its ability to inhibit plant growth or viability, comprising: (a) conditions in which an enzyme can catalyze the synthesis of its product. Below, an enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture is combined with a substrate for HMP-P kinase or a substrate for TMP-PPase, and (b) the same as in the primary reaction mixture Under the same conditions, for the same time, combine the tested chemical and enzyme in the secondary reaction mixture with the substrate used in the primary reaction mixture, and (c) determine the activity of the enzyme in the primary and secondary reaction mixture And (d) the ability to inhibit plant growth or viability when the activity of the enzyme in the secondary reaction mixture is lower than the activity of the enzyme in the primary reaction mixture, and desirably significantly lower. It describes a method comprising the step of selecting the chemicals to be tested. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is a plant, preferably Arabidopsis thaliana.
Obtained from In another preferred embodiment, HMP-P kinase activity or TM
The enzyme having P-PPase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1, or is identical to the amino acid sequence set forth in SEQ ID NO: 2 Have or substantially similar amino acid sequences. In another preferred embodiment, the substrate for HMP-P kinase is 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (H
MP-P), and in another preferred embodiment, the substrate for TMP-PPase is
4-methyl-5- (β-hydroxyethyl) thiazole phosphate (THZ-
P). In yet another embodiment, the substrate for TMP-PPase is 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PPase).
PP). In yet another preferred embodiment, the activity of the enzyme is determined by measuring the TMP produced in the reaction mixture. In another preferred embodiment, the activity of the enzyme is determined by measuring the ADP obtained from ATP in the reaction mixture. The invention further provides the following: (a) HMP-P kinase activity or TMP-
Combining an enzyme having PPase activity with a substrate for HMP-P kinase or a substrate for TMP-PPase, and (b) under the same conditions and for the same time as in the primary reaction mixture,
The chemicals and enzymes in the secondary reaction mixture are combined with the substrate to determine (c) the activity of the enzymes in the primary and secondary reaction mixtures, where the activity of the conjugated enzyme in the secondary reaction mixture is An assay is described which comprises a step in which a chemical can inhibit the activity of an enzyme if it is significantly lower than the activity of the enzyme in the reaction mixture.

The present invention further provides a method of identifying a chemical to be tested for its ability to inhibit plant growth and viability, comprising: (a) under conditions where the enzyme may catalyze the binding synthesis of TMP. , An enzyme having HMP kinase activity in the primary reaction mixture and H
An enzyme having MP-P kinase activity or TMP-PPase activity is combined with a substrate for HMP kinase and a substrate for TMP-PPase, and (b) secondary reaction under the same conditions and for the same time as in the case of the primary reaction mixture. Combining the chemicals and enzymes in the mixture with the substrate; (b) determining the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary and secondary reaction mixtures; and (c) If the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the reaction mixture is significantly lower than the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture, A method is described that comprises selecting a chemical to be tested for its ability to inhibit growth or viability.
In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant, preferably Arabidopsis thaliana. In another preferred embodiment, the HMP-P kinase activity or TMP-P
The enzyme having Pase activity is identical to or substantially identical to the nucleotide sequence set forth in SEQ ID NO: 1, or is identical to the amino acid sequence set forth in SEQ ID NO: 2 Has an amino acid sequence that is or is substantially similar. In another preferred embodiment, the substrate for HMP kinase is HMP, and in yet another preferred embodiment, the substrate for TMP-PPase is THZ-P. In a further preferred embodiment, the activity of the enzyme is measured by determining the TMP produced in the reaction mixture. The present invention further provides:
Enzyme having MP kinase activity and HMP-P kinase activity or TMP-P
An enzyme having Pase activity is combined with a substrate for HMP kinase and a substrate for TMP-PPase, and (C) determine the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary and secondary reaction mixtures, wherein the HMP-P kinase in the secondary reaction mixture is determined. If the activity or activity of the enzyme having TMP-PPase activity is significantly lower than the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture, the chemical is HMP-P kinase activity or Also described is an assay comprising a step capable of inhibiting the activity of an enzyme having TMP-PPase activity.

The present invention is further directed to a method of identifying a chemical to be tested for its ability to inhibit plant growth and viability, comprising the steps of: (a) under conditions where the enzyme may catalyze the coupled synthesis of TMP. , An enzyme having THZ kinase activity in the primary reaction mixture and H
An enzyme having MP-P kinase activity or TMP-PPase activity is identified as HMP-P
(B) combining the chemicals and enzymes in the secondary reaction mixture with the substrate under the same conditions and for the same time as in the primary reaction mixture, and (c) combining the primary and Determining the activity of an enzyme having HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture, and (d) an enzyme having HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture If the activity of the enzyme is significantly lower than the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture, select a chemical to be tested for the ability to inhibit plant growth or viability. A method consisting of steps is described.
In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant, preferably Arabidopsis thaliana. In another preferred embodiment, the HMP-P kinase activity or TMP-P
The enzyme having Pase activity is identical to or substantially identical to the nucleotide sequence set forth in SEQ ID NO: 1, or is identical to the amino acid sequence set forth in SEQ ID NO: 2 Has an amino acid sequence that is or is substantially similar. In another preferred embodiment, the substrate for HMP-P kinase is HMP-P, and in yet another preferred embodiment, the substrate for THZ kinase is THZ. In another preferred embodiment, HMP-P kinase activity or T
The activity of the enzyme having MP-PPase activity is determined by the TMP produced in the reaction mixture.
Is determined by determining The present invention further provides the following:
An enzyme having THZ kinase activity and an enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture under conditions capable of catalyzing P bond synthesis. And (b)
C) combining the chemicals and enzymes in the secondary reaction mixture with the substrate under the same conditions and for the same time as in the primary reaction mixture, and (c) HMP-P in the primary and secondary reaction mixtures.
To determine the activity of an enzyme having kinase activity or TMP-PPase activity,
Here, the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture is determined by the HMP-P kinase activity or TMP activity in the primary reaction mixture.
If the activity of the enzyme with MP-PPase activity is significantly lower than the activity of
An assay comprising a step capable of inhibiting the activity of an enzyme having MP-P kinase activity or TMP-PPase activity is described.

[0033] In another preferred embodiment, the present invention relates to HMP-P kinase or T
A method for identifying a chemical having the ability to inhibit MP-PPase activity, preferably comprising the steps of: a) encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, HMP-P kinase or TMP-P
Obtaining a preferably stably transformed transgenic plant, plant tissue, plant seed or plant cell comprising a non-natural nucleotide sequence capable of overexpressing Pase, b) transgenic plant, plant cell, tissue Or applying the chemical to the part and to the isogenic non-transformed plant, plant cell, tissue or part,
c) determining the growth or viability of transgenic and non-transformed plants, plant cells, tissues after application of the chemical; d) transgenic and non-transformed plants, plant cells, after application of the chemical; A method comprising comparing the growth or viability of a tissue is described. Desirably, the chemical enhances the viability or growth of a non-transgenic plant, plant cell, tissue or part without significantly inhibiting the viability or growth of the isogenic transgenic plant, plant cell, tissue or part. Suppress. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is desirably identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.
Encoded by a nucleotide sequence obtained from a plant, preferably Arabidopsis thaliana. In another embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In yet another embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity has an amino acid sequence that is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. Having.

[0034] The present invention further provides for the use of a modified HMP-P kinase or TMP-PPase activity at a level that is normally inhibitory to native HMP-P kinase or TMP-PPase activity. Embodies plants, plant tissues, plant seeds and plant cells that are tolerable to suppression by herbicides. Herbicide-tolerant plants encompassed by the present invention include those that would otherwise be possible targets for normally controlling herbicides, especially the agriculturally important crops mentioned above. According to this embodiment, the plant, plant tissue, plant seed or plant cell is tolerant to suppression by a herbicide at a concentration that normally suppresses the activity of wild-type, unmodified HMP-P kinase or TMP-PPase. Modified HMP-P kinase or TMP-PP
It is transformed, preferably stably transformed, with a recombinant DNA molecule that includes a suitable promoter functional in plants operably linked to the nucleotide coding sequence encoding the enzyme. Modified HMP-P kinase activity or TMP-
PPase activity is increased by providing wild-type herbicide-sensitive HMP-P kinase or TMP-PPase expression by providing the plant with multiple copies of the wild-type HMP-P kinase or TMP-PPase gene; or Wild-type HMP-P kinase or TMP-PP under the control of a wild-reliable promoter
Plants can also be conferred by overexpression of the ase gene. The transgenic plant, plant tissue, plant seed or plant cell thus produced is then:
A herbicide-tolerant strain is selected, characterized and developed by conventional selection methods. Alternatively, using random or site-directed mutagenesis,
Herbicide-tolerant strains can be generated.

Thus, the present invention provides DNAs comprising nucleotides isolated from plants that encode enzymes having HMP-P kinase activity or TMP-PPase activity.
Providing a plant, plant cell, plant seed or plant tissue transformed with the molecule,
Here, the enzyme has HMP-P kinase activity or TMP-PPase activity,
The DNA molecule confers tolerance to a herbicide on a plant, plant cell, plant seed or plant tissue in an amount that normally suppresses natural HMP-P kinase activity or TMP-PPase activity. According to one example of this embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity has a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. It has an amino acid sequence that is identical to or substantially similar to the amino acid sequence encoded by the sequence or described in SEQ ID NO: 2 or SEQ ID NO: 4. The present invention also provides a method for inhibiting plant growth, comprising the step of applying to the plant a chemical that inhibits natural HMP-P kinase activity or TMP-PPase activity in the plant. In a related aspect, the present invention is a method for selectively inhibiting the growth of weeds in planted crop seeds or fields containing plant crops, comprising: (a) native HMP-P kinase activity or Planting a herbicide-tolerant crop or crop seed that is a plant or plant seed that is tolerant to a herbicide that inhibits TMP-PPase activity, and (b) inhibits native HMP-P kinase activity or TMP-PPase activity Applying herbicides to field crops or crop seeds and weeds
Here the herbicide is directed to a method comprising the step of inhibiting the growth of weeds without significantly inhibiting the growth of the crop.

[0036] Other objects and advantages of the present invention will become apparent to those skilled in the art from the following description of the invention and testing of non-limiting examples. Exogenous thiamine or thiamine precursor (ie, 4-methyl-5- (β-hydroxyethyl) thiazole (THZ) and / or 2-methyl-4-amino-5
-Hydroxymethylpyrimidine (HMP)) have been identified in Arabidopsis thaliana and have been mapped to four loci (Li and Redei (1969)). Biochemical
Genetics 3, 163-170). One locus th1 has been mapped to the first chromosome (Koornneef and Hanhart (1981) Arab. Info. Service 18, 52-58). Although the exact location of the block between the pyrimidine and thiazole precursors and the thiamine in the th1 mutant was not located by Koorneef and Hanhart, the th1 mutant was later identified using TMP- It has been shown to lack PPase enzyme activity (Komeda et al. (1988) Plant Physiol. 88, 248-250). Recently, a BAC sequence (BACF19G10) covering this region of the first chromosome has been submitted to GenBank. For this BAC, one gene had significant amino acid sequence similarity to both thiD and thiE from E. coli (nucleotide positions on the BAC sequence).
46133-48657). Based on this information, the present inventors have isolated an Arabidopsis thaliana cDNA encoding a novel protein having two functional domains. The amino acid sequence of the N-terminal domain (up to about 906 of SEQ ID NO: 1 or amino acid 302 of SEQ ID NO: 2) has homology to HMP-P kinase (thiD) and The amino acid sequence (from 925 nucleotides or amino acid 309 of SEQ ID NO: 2) has homology to TMP-PPase (thiE). The putative chloroplast transit peptide is the first 99 nucleotides of SEQ ID NO: 1 (
(The first 33 amino acids of SEQ ID NO: 2).

We have identified two different th1 mutants, CS79 and CS3530 (Arabidop
HMP-P kinase / TMP-PP from sis Stock Center, Nottingham, UK)
The sequence of the ase gene was also obtained. They found that in both mutants, the mutation was HMP
-P kinase / TMP-PPase gene was found to be present. CS79 has a single nucleotide change from C to T at position 188 of SEQ ID NO: 1 that changes amino acid 63 of SEQ ID NO: 2 from serine to phenylalanine. CS3530 converts position 87 of SEQ ID NO: 1 to amino acid 87 which changes isoleucine to asparagine
It has a seven nucleotide deletion from either 9-265 or positions 260-266. In this mutant, the codon for asparagine 87 is followed by a codon for 13 amino acids, followed by a TGA stop codon in the reading frame. Mutations need to include only a small portion of the domain homologous to thiD and result in the translation of a protein of only 100 amino acids lacking the domain homologous to thiE. These results indicate that the thiamine auxotroph phenotype is due to a mutation in the HMP-P kinase / TMP-PPase gene, and that the HMP-P kinase / TMP-PPase gene is essential in plants, It clearly demonstrates that it can represent a good target for herbicides.

Based on the cDNA sequence, we have further discovered that two incorrect splicing junctions were predicted in the BAC clone. The first predicted splice junction incorrectly resulted in the addition of 24 bases of the intron sequence at position 383 of SEQ ID NO: 1 (FIG. 2), and the second predicted splice junction was SEQ ID NO: 1. It incorrectly caused a deletion of 24 bases between positions 1066 and 1089 as well as the addition of 9 incorrect bases (FIG. 2). Based on this prediction,
The HMP-P kinase / TMP-PPase gene was considered ineffective for the purposes of the present invention and could not be used.

The HMP-P kinase / TMP-PPase gene is further referred to as encoding an enzyme having HMP-P kinase activity or TMP-PPase activity.
This means that, preferably, the HMP-P kinase / TMP-PPase gene encodes a single polypeptide having HMP-P kinase activity or TMP-PPase activity. In another preferred embodiment, the HM
A single polypeptide encoded by the PP kinase / TMP-PPase gene has HMP-P kinase activity and TMP-PPase activity.

The nucleotide sequence encoding the mature HMP-P kinase / TMP-PPase gene as described in SEQ ID NO: 3 was identified on July 8, 1998 by the clone name aththiDE
-ctp, deposit number NRRL B-30040, International Recognition of the Deposit o
f Agri as an International Depositary Authority, as established under the Budapest Treaty on Microorganisms for the Purposes of Patent Procedure
cultural Research Culture Collection (NRRL), 1815 N, University Street,
Deposited with Peoria, Illinois 61604, USA.

For recombinant production of HMP-P kinase / TMP-PPase in a host organism, the nucleotide sequence encoding the enzyme having HMP-P kinase activity or TMP-PPase activity is selected from the selected host. Inserted into an expression cassette intended for, and introduced into a recombinantly produced host. The selection of particular regulatory sequences suitable for the chosen host, such as promoters, signal sequences, 5 'and 3' untranslated sequences and enhancers, is within the level of ordinary skill in the art. The resulting molecule containing the individual elements linked in proper reading frame can be inserted into a vector that can be transformed in a host cell. Suitable expression vectors and methods for recombinant production of proteins are well known for host organisms such as E. coli, yeast and insect cells (see, eg, Luckow and Summers, Bio / Technol. 6: 4).
7 (1988)). Specific examples include plasmids such as pBluesctipt (Strata
gene, La Jolla, CA), pFLAG (International Biotechnologies, Inc., New Ha
ven, CT), pTrcHis (Invitrogen, La Jolla, CA) and baculovirus expression vectors such as Autographica californica nuclear polyhedrosis virus (AcMN
PV) genome. A preferred baculovirus / insect system is pVI1 1392 / Sf21 cells (Invitrogen, La Jolla, CA).

In a preferred embodiment, the nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a eukaryote such as yeast, but is preferably obtained from a plant. . In a further preferred embodiment, the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence is set forth in SEQ ID NO: 2 or SEQ ID NO: 4. Encodes an enzyme having HMP-P kinase activity or TMP-PPase activity identical or substantially similar to the amino acid sequence of The nucleotide sequence set forth in SEQ ID NO: 1 encodes an Arabidopsis thaliana HMP-P kinase / TMP-PPase preprotein whose amino acid sequence is set forth in SEQ ID NO: 2, and the nucleotide sequence set forth in SEQ ID NO: 3 is Arabidopsis deduced mature HMP whose amino acid sequence is described in SEQ ID NO: 4
Encoding P-kinase / TMP-PPase; In another preferred embodiment,
The nucleotide sequence is obtained from a prokaryote, preferably a bacterium, such as E. coli.
In this case, the enzyme having HMP-P kinase activity and the enzyme having TMP-PPase activity are encoded by the thiD and thiE genes, respectively.

[0043] Recombinantly produced HMP-P kinase / TMP-PPase is isolated and purified using various standard techniques. The practical techniques that can be used will depend on the host organism used, whether the enzyme is intended for secretion, and other such factors well known to those skilled in the art (eg, Ausubel, F. . et al.
, "Current Protocols in Molecular Biology", pub. By John Wiley & Sons, In
c. (See Chapter 16 of (1994)).

[0044] Recombinantly produced HMP-P kinase / TMP-PPase is useful for various purposes. For example, it indicates that they have HMP-P kinase activity or TM
It can be used in in vitro assays to screen for known herbicide chemicals whose targets have not been identified to determine if they inhibit P-PPase activity. Such in vitro assays can also be used as a more general screen to identify chemicals that suppress such enzymatic activity and are therefore novel candidate herbicides. Alternatively, recombinantly produced HMP-P kinase / TMP
-PPases elucidate the complex structure of these molecules and further characterize their association with known inhibitors in order to rationally design novel inhibitory herbicides and enzymes in herbicide-tolerant forms Can be used to transform

In vitro assays useful for identifying inhibitors of enzymes encoded by essential plant genes such as HMP-P kinase / TMP-PPase include: a) H
Reacting an enzyme having MP-P kinase activity or TMP-PPase activity and its substrate in the presence of a putative inhibitor of the function of the enzyme; b) determining the rate of enzyme activity in the presence of the putative inhibitor Comparing the rate of enzymatic activity under the same conditions in the absence of, and c) determining whether the putative inhibitor inhibits HMP-P kinase or TMP-PPase enzyme activity. I do. The inhibitory effect on HMP-P kinase activity or TMP-PPase activity is determined by a reduction or complete inhibition of TMP synthesis in the assay. In a preferred embodiment, such a determination is made by comparing the amount of TMP synthesized in an in vitro assay using a fluorescence assay in the presence and absence of the candidate inhibitor. In another preferred embodiment, such a determination is made by comparing the amount of ADP produced in an in vitro assay using absorbance detection in the presence and absence of a candidate inhibitor. A preferred substrate for HMP-P kinase is 2-methyl-4-amino-5
-Hydroxymethylpyrimidine phosphate (HMP-P). TMP-P
Preferred substrates for Pase are 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) and 4-methyl-5- (β
-Hydroxyethyl) thiazole phosphate (THZ-P).

The amount of HMP-P available for HMP-P kinase depends on the amount of bound HMP kinase / H
Augmented using the MP-P kinase / TMP-PPase assay, thereby increasing the detection limit of the assay and providing an improved screening procedure for chemicals that inhibit HMP-P kinase activity or TMP-PPase activity. It is. Such coupling assays include the following: (a) an enzyme having HMP kinase activity and HMP in the primary reaction mixture under conditions in which the enzyme can catalyze the conjugated synthesis of TMP.
Combining an enzyme having -P kinase activity or TMP-PPase activity with a substrate for HMP kinase and a substrate for TMP-PPase, and Combining the chemicals and enzymes in the substrate with (b) HMP-P kinase activity or TM in the primary and secondary reaction mixtures.
Determining the activity of the enzyme having P-PPase activity, and (c) determining the activity of the HMP-P kinase activity in the secondary reaction mixture or the activity of the enzyme having TMP-PPase activity in the primary reaction mixture. Selecting a chemical to be tested for its ability to inhibit plant growth or viability if the activity or activity of the enzyme having TMP-PPase activity is significantly lower. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant. In another preferred embodiment, HMP-P kinase activity or T
The enzyme having MP-PPase activity is encoded by a nucleotide sequence that is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or is encoded by SEQ ID NO: 2 or SEQ ID NO: 4. Has an amino acid sequence that is identical or substantially similar to the amino acid sequence described in In another preferred embodiment, the substrate for HMP kinase is HMP, and in yet another preferred embodiment, the substrate for TMP-PPase is THZ-P. In a further preferred embodiment, the activity of the enzyme is measured by determining the TMP produced in the reaction mixture.

Alternatively, the amount of THZ-P available for TMP-PPase is increased using a bound THZ kinase / HMP-P kinase / TMP-PPase assay, thereby increasing the detection limit of the assay. , HMP-P kinase activity or TMP-P
An improved screening approach is provided for chemicals that inhibit Pase activity. Such coupling assays include the following: (a) an enzyme with THZ kinase activity and HMP-P kinase activity or TMP-PPase in the primary reaction mixture under conditions where the enzyme can catalyze the coupled synthesis of TMP. HM enzyme
(B) combining the chemicals and enzymes in the secondary reaction mixture with the substrate under the same conditions and for the same time as for the primary reaction mixture, with the substrate for PP kinase and the substrate for THZ kinase; ) Determining the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the primary and secondary reaction mixture, and (d) HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture A chemical tested for its ability to inhibit plant growth or viability when the activity of an enzyme having HMP-P kinase activity or TMP-PPase activity in the primary reaction mixture is significantly lower than the activity of the enzyme having the activity. And selecting. In a preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant. In another preferred embodiment, the enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence that is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Or has an amino acid sequence that is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. In another preferred embodiment, the substrate for HMP-P kinase is HMP-P, and in yet another embodiment, the substrate for THZ kinase is THZ. In another preferred embodiment,
The activity of an enzyme having HMP-P kinase activity or TMP-PPase activity
It is determined by determining the TMP formed in the reaction mixture.

In one embodiment, putative herbicides identified, for example, by in vitro screening, are applied to plants at various concentrations. The putative herbicide is preferably sprayed on the plant. After application of the putative herbicide, its effect on the plant, such as death or growth inhibition, is recorded. In another embodiment, an in vivo screening assay for an inhibitor of HMP-P kinase activity or TMP-PPase activity comprises transgenic plants capable of overexpressing a nucleotide sequence having HMP-P kinase activity or TMP-PPase activity. Using plant tissue, plant seeds or plant cells, wherein HMP
-P kinase and TMP-PPase are enzymatically active in transgenic plants, plant tissues, plant seeds or plant cells. The nucleotide sequence is preferably obtained from a eukaryote, such as a yeast, but is preferably obtained from a plant. In a further preferred embodiment, the nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence is set forth in SEQ ID NO: 2 or SEQ ID NO: 4. HMP-P kinase or TMP-PP identical or substantially similar to the amino acid sequence
Encodes an enzyme having ase activity. In another preferred embodiment, the nucleotide sequence is obtained from a prokaryote, preferably a bacterium, such as E. coli. in this case,
The enzymes having HMP-P kinase and TMP-PPase activities are respectively
It is encoded by the iD and thiE genes.

Next, the chemical is applied to the transgenic plant, plant tissue, plant seed or plant cell, and to the same transgenic non-transformed plant, plant tissue, plant seed or plant cell, and the transgenic and non-transgenic Transformed plants, plant tissues,
The growth or viability of the plant seeds or plant cells is determined and compared after application of the chemical.

The present invention further relates to the activity of native HMP-P kinase or TMP in these plants.
-Directed to plants, plant tissues, plant seeds and plant cells that are tolerant to herbicides that inhibit PPase activity, where tolerance is imparted by modified HMP-P kinase activity or TMP-PPase activity You. Modified HMP-P kinase activity or TMP-PPase activity is indicated in plants by wild-type HMP-P kinase / TMP-
By providing a modified herbicide-tolerant HMP-P kinase / TMP-PPase in plants by providing a PPase gene, or by a combination of these techniques, the wild-type herbicide-sensitive HMP-P kinase / TMP-PPase expression can be added to the plant of the present invention by increasing the expression. Representative plants include any plant to which these herbicides are applied for their commonly intended purpose. Preferred are agriculturally important crops such as cotton, soybean, rapeseed, sugar beet, corn, rice, wheat, barley, oats,
Rye, sugar corn, millet, turf, lintel, turfgrass and the like.

Upon achieving altered HMP-P kinase activity or TMP-PPase activity by increasing expression, at least enough HMP-P kinase / TMP-PPase in the plant to overcome the growth inhibition caused by the herbicide Levels. The level of the expressing enzyme is generally at least two times, preferably at least five times, more preferably at least ten times the original expression level. Increased expression can be due to multiple copies of the wild-type HMP-P kinase / TMP-PPase gene, multiple occurrences of coding sequences within the gene (ie, gene amplification), or mutations in non-coding regulatory sequences of endogenous genes in plant cells. For. Plants having such alterations in gene activity can be obtained by direct selection in plants by methods known in the art (see, for example, US Pat. Nos. 5,162,602 and 4,761,373 and references cited therein). These plants can also be obtained by genetic engineering techniques known in the art. Herbicide-sensitive HMP-P kinase / TMP-PP
Increased expression of the ase gene is achieved by a set including a promoter capable of driving the expression of a related structural gene in a plant cell operably linked to a homologous or heterologous structural gene encoding HMP-P kinase / TMP-PPase. This is accomplished by transforming plant cells with the recombinant or chimeric DNA molecule. Preferably, the transformation is stable, thereby providing a hereditary transgenic property.

B. Expression of Modified Herbicide-Tolerant HMP-P Kinase / TMP-PPase According to this embodiment, the plant, plant tissue, plant seed or plant cell is a herbicide-tolerant form of HMP-P kinase / TMP-PPase. Is stably transformed with a recombinant DNA molecule that includes a suitable promoter that is functional in plants operably linked to the coding sequence that encodes. Herbicide-tolerant forms of the enzyme are:
At least one that confers tolerance to a herbicide that inhibits the enzyme in its unmodified natural form
It has one amino acid substitution, addition or deletion. The transgenic plant, plant tissue, plant seed or plant cell thus produced is then selected by conventional selection techniques, whereby a herbicide-tolerant strain is isolated, characterized and developed. You. A method for generating a gene encoding a herbicide-tolerant form of HMP-P kinase / TMP-PPase is described below: One general strategy involves direct or indirect mutagenesis techniques on microorganisms. For example, genetically engineered microorganisms, such as E. coli or brewer's yeast, may be subjected to random mutagenesis in vivo using ultraviolet light or a mutagen such as ethyl or methyl methanesulfonate. Mutagenesis techniques include, for example, Miller, Experiments in Molecular Genetics, Cold Spring Harbor Labora
tory, Cold Spring Harbor, NY (1972); Davis et al., Advanced Bacterial G
enetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980);
Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory
, Cold Spring Harbor, NY (1983); and U.S. Patent No. 4,975,374. The microorganism selected for mutagenesis is a normal inhibitor sensitive HMP-P
Contains the kinase / TMP-PPase gene and depends on the activity conferred by this gene. Mutant cells are grown in the presence of the inhibitor at a concentration that inhibits the unmodified gene. Colonies of mutagenized microorganisms that grow better in the presence of the inhibitor than non-mutated microorganisms (ie, exhibit resistance to the inhibitor) are selected for further analysis. The HMP-P kinase / TMP-PPase gene from these colonies is isolated by cloning or PCR amplification and their sequence elucidated. The sequences encoding the altered gene products are then cloned back into the microorganism, confirming their ability to confer inhibitor tolerance.

Methods for obtaining mutant herbicide-tolerant alleles of plant HMP-P kinase / TMP-PPase include direct selection in plants. For example, by plating sterilized seeds by methods known in the art on plates on simple minimal salt media containing increasing concentrations of inhibitors, the growth of plants such as Arabidopsis, soybean or corn can be inhibited. Effect of mutagenized HMP-P kinase / T
Determine the effect of the MP-PPase gene. Such concentrations are 0.001, 0.003,
0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 110, 300, 1000 and 3000 parts / million (pp
m). For subsequent experiments, the lowest dose at which significant growth inhibition can be reproducibly detected will be used. Determining the minimum dose is routine in the art.

[0054] The mutagenesis of plant material is used to increase the frequency of occurrence of resistance alleles in selected populations. Mutagenized seed material can be obtained from a variety of sources, for example, chemical or physical mutagenesis of seeds, or chemical or physical mutagenesis of pollen (Neuffer, In Maize for Biological Research Sheridan, ed. . Univ.
Press, Grand Forks, ND., Pp.61-64 (1982)), then pollinated plants and used to collect the resulting M ₁ mutant seeds. Typically, for Arabidopsis thaliana, the progeny seed of a plant grown from a seed mutated using a chemical such as ethyl methanesulfonate or using physical agents such as gamma rays or fast neutrons M ₂ seeds (Lehle seeds, Tucson, AZ) and 10,000 on minimal salts medium containing an inhibitor of appropriate concentration seeds / plate (10 cm in diameter
) And plate for tolerability. Seedlings that continue to grow after plating and retain the green color for 7-21 days are transplanted into soil and grown to maturity and seed development. The progeny of these seeds are tested for tolerance to herbicides.
If tolerated characteristic is dominant, the seeds 3: 1 / resistance: Plant for separating the sensitivity is estimated to have been heterozygous for resistance in M ₂ generation. Plants resulting total resistance seed is estimated to have been homozygous for resistance in M ₂ generation. Such mutagenesis and screening of their M ₂ progeny seeds in intact seeds, other species, which can be run on soybean example (e.g., U.S. Patent No. 5,084,
082). Alternatively, mutant seeds screened for herbicide tolerance are obtained as a result of pollination with pollen that has been mutagenized by chemical or physical means.

Confirmation that the genetic basis of herbicide tolerance is the modified HMP-P kinase / TMP-PPase gene is confirmed as demonstrated below. First, alleles of the HMP-P kinase / TMP-PPase gene from plants showing resistance to the inhibitor were identified by SEQ ID NO :? PCR based on the unaltered HMP-P kinase / TMP-PPase gene sequence from plants used to generate tolerable alleles, based on the Arabidopsis thaliana cDNA coding sequence shown in To isolate. After sequencing of the alleles to determine the presence of the mutation in the coding sequence, the putative tolerating alleles were evaluated for their ability to tolerate the inhibitor in the plant that had been transformed with the inhibitor. inspect. These plants can be Arabidopsis plants or any other plant whose growth is sensitive to HMP-P kinase / TMP-PPase inhibitors. Second, inserted HMP-P kinase / TMP
-Mapping the PPase gene relative to known restriction fragment length polymorphisms (RFLP) (eg Chang et al. Proc. Natl. Acad. Sci. USA 85: 6856-6860 (
1988); Nam et al., Plant Cell 1: 699-705 (1989)). HMP-P kinase / TMP-PPase inhibitor tolerability characteristics are separately mapped using the same marker. If tolerability is due to a mutation in the HMP-P kinase / TMP-PPase gene, the tolerability profile is found at a position indistinguishable from that of the HMP-P kinase / TMP-PPase gene.

Another method for obtaining the herbicide-tolerant allele of the HMP-P kinase / TMP-PPase gene is by selection in plant cell culture. Plant tissue,
For example, embryos, leaf discs, or the like, or actively growing callus or a piece of suspension culture of the plant, in the presence of increasing concentrations of inhibitory or similar herbicides suitable for use in a laboratory environment. , Grow on medium. Various degrees of growth are recorded in different cultures. Some cultures result in fast growing mutant colonies that usually continue to grow in the presence of inhibitory concentrations of the inhibitor. The frequency of such fast growing mutants is
It can be increased by treating the tissue or cell with a chemical or physical mutagen prior to exposure to the inhibitor. The putative tolerating allele of the HMP-P kinase / TMP-PPase gene is isolated and tested as described in the preceding paragraph. Alleles identified as conferring herbicide tolerance can then be engineered for optimal expression and transformed in plants. Alternatively, plants can be regenerated from tissue or cell cultures containing these alleles.

Yet another method is to use a wild type herbicide sensitive plant HMP in bacteria or yeast.
Mutagenesis of the -P kinase / TMP-PPase gene, followed by culturing the microorganism on a medium containing an inhibitory concentration of the inhibitor, followed by selection of colonies that grow in the presence of the inhibitor. . In particular, a plant cDNA, such as an Arabidopsis thaliana cDNA encoding HMP-P kinase / TMP-PPase, is cloned in a microorganism that would otherwise lack selected gene activity. Next, several chemical or enzymatic methods known in the art, such as sodium bisulfite (Shortle et al.,
Methods Enzymol. 100: 457-468 (1983)), methoxylamine (Kadonaga et al.)
., Nucleic Acids Res. 13: 1733-1745 (1985)), oligonucleotide-specific saturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83: 710-
714 (1986)) or various polymerase miscontamination strategies (eg, Shortle et al.).
Natl. Acad. Sci. USA, 79: 1588-1592 (1982); Shiraishi et al.,
Gene 64: 313-319 (1988); and see Leung et al., Technique 1: 11-15 (1989)) for in vivo mutagenesis or in vivo transformation of transformed microorganisms.
Perform in vitro mutagenesis. Colonies that grow in the presence of inhibitor, usually at inhibitory concentrations, are picked and purified by repeated re-streaking. The plasmids were purified and HMP-
Tested for their ability to confer tolerance to inhibitors by retransforming them in microorganisms lacking P-kinase / TMP-PPase gene activity. The DNA sequence of the cDNA insert that passes this test is then determined.

[0058] Herbicide-resistant HMP-P kinase / TMP-PPase enzymes can also be obtained using methods involving in vitro recombination, also called DNA shuffling. DNA shuffling introduces mutations, preferably random mutations, into the HMP-P kinase / TMP-PPase gene. DNA shuffling is HMP
-Recombination and rearrangement of sequences within the P-kinase / TMP-PPase gene or sequence recombination and exchange between two or more different HMP-P kinase / TMP-PPase genes. These methods allow for the production of countless mutated HMP-P kinase / TMP-PPase genes. The mutated or shuffled gene is screened for a desired property, such as improved tolerance to herbicides, and for mutations that provide broad tolerance to different types of inhibitor chemistry. Such screening is well within the skill of the artisan.

Included in the present invention is an HMP-P which exhibits enhanced tolerance to a herbicide that inhibits HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecule from which the shuffled DNA molecule is obtained. P kinase or TMP-PP
A shuffled DNA molecule that encodes an ase enzyme. Furthermore, a template DNA encoding an enzyme having HMP-P kinase activity or TMP-PPase activity
A mutagenized DNA molecule obtained by shuffling the molecule, wherein the HMP-P kinase activity or TMP encoded by the template DNA molecule is
Also included are mutagenized DNA molecules that encode HMP-P kinase or TMP-PPase enzymes that exhibit enhanced tolerance to herbicides that inhibit MP-PPase activity.

In a preferred embodiment, the mutagenized HMP-P kinase / TMP-PPase gene is generated from at least one template HMP-P kinase / TMP-PPase gene, wherein the template HMP-P kinase / TMP-PPase gene is used. The P-kinase / TMP-PPase gene has been cleaved into double-stranded random fragments of the desired size, and the resulting population of double-stranded random fragments has one or more single or double stranded fragments. Adding a oligonucleotide comprising the same region and a heterologous region to the double-stranded random fragment; combining the resulting mixture of double-stranded random fragment and oligonucleotide into one Denaturing into single-stranded fragments; annealing said single-stranded fragments with said regions of identity to produce pairs of annealed fragments, wherein said regions of identity are paired Incubating the resulting population of single-stranded fragments with a polymerase under conditions in which one member is sufficient to prime the other replication, thereby producing a mutagenized double-stranded polynucleotide; The second and third steps are then repeated at least two more times, wherein the mixture resulting from the second second step contains the mutagenized double stranded polynucleotide from the previous third step, and Performed once, yields an additional mutagenized double-stranded polynucleotide, wherein the mutagenized polynucleotide is tolerant to a herbicide that inhibits native HMP-P kinase activity or TMP-PPase activity. Being a mutated HMP-P kinase / TMP-PPase exhibiting enhancement.

In a further preferred embodiment, the mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity encodes an enzyme having HMP-P kinase activity or TMP-PPase activity. Adding at least one oligonucleotide generated from at least two non-identical template DNA molecules and including the same region for each of the template DNA molecules to the template DNA molecule; The resulting population of single-stranded molecules is incubated with the polymerase under conditions that cause the oligonucleotide to anneal to the template DNA molecule, in which case the conditions for polymerization by the polymerase are That a polymerization product corresponding to a part of the molecule is obtained. And repeating the second and third steps at least two more times, wherein the extension product obtained in the third step is switchable to a template DNA molecule for the next round of polymerization. Thereby producing a mutagenized double-stranded polynucleotide comprising sequences obtained from different template DNA molecules, wherein the mutagenized double-stranded polynucleotide comprises a HMP encoded by the template DNA molecule. Encoding HMP-P kinase activity or TMP-PPase enzyme that exhibits enhanced tolerance to herbicides that inhibit P-kinase activity or TMP-PPase activity.

In a preferred embodiment, the concentration of a single double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 polynucleotides. In another preferred embodiment, the size of the double-stranded random fragment is between about 5 bp and 5 kb. In a further preferred embodiment, the fourth step of the method comprises repeating the second and third steps at least 10 times. Such methods are described, for example, by Stemmer et.
al. (1994) Nature 370: 389-391, US Pat. No. 5,605,793 and Cramert et.
al. (1998) Nature 391: 288-291 and WO 97/20078, the contents of which are incorporated herein by reference.

In another preferred embodiment, for example, Zhao et al. (1998) Nature Biotechnolog
In vitro, any combination of two or more different HMP-P kinase / TMP-PPase genes is mutated by the adhesion extension method (StEP) as described in y 16: 258-261. Two or more HMP-P kinase / TMP-PPase genes are used as templates for PCR amplification,
The extension cycle of the reaction is preferably performed at a temperature below the optimal polymerization temperature of the polymerase. For example, when using a thermostable polymerase having an optimum temperature of about 72 ° C, the temperature for the extension reaction is desirably 72 ° C, more desirably 65 ° C.
Below, preferably 60 ° C. or lower, more preferably the temperature for the extension reaction is 55 ° C. Further, the duration of the extension reaction of the PCR cycle is desirably shorter than normally performed in the art, more desirably less than 30 seconds, preferably less than 15 seconds, more preferably the duration of the extension reaction. The time is 5 seconds. In each extension reaction, only short DNA fragments are polymerized, allowing template switching of extension products between starting DNA molecules after each cycle of denaturation and annealing, thereby resulting in structural dispersity between extension products. The optimal number of PCR reactions depends on the HMP to be mutated.
Depending on the length of the -P kinase / TMP-PPase coding region, it is preferably used 40 times or more, more preferably 60 times or more, preferably 80 times or more. HMP-
Optimal extension conditions and optimal PCR times for all combinations of P-kinase / TMP-PPase genes are determined as described using procedures well known in the art. Other parameters for the PCR reaction are essentially the same as those commonly used in the art. Primers for the amplification reaction are preferably a DNA sequence located outside the coding sequence of the HMP-P kinase / TMP-PPase gene and a DNA of a vector containing, for example, the HMP-P kinase / TMP-PPase gene. Designed to anneal with the sequence, thereby
The different HMP-P kinase / TMP-PPase genes used for the R reaction
Preferably they are contained in separate vectors. The primer is desirably HMP-
Less than 500 bp from the P-kinase / TMP-PPase coding sequence, preferably less than 200 bp from the HMP-P kinase / TMP-PPase coding sequence, more preferably the HMP-P kinase / TMP-PPase coding sequence. From 120 bp
Anneal with sequences located less than one distant apart. Preferably, the HMP-P kinase / TMP-PPase coding sequence is surrounded by restriction sites contained in the DNA sequence that is amplified during the PCR reaction, thereby facilitating cloning of the amplification product in a suitable vector. .

In another preferred embodiment, HMP-P kinase / TMP-
Fragments of the PPase gene are generated as described in WO 98/05765.
The cohesive end is used to replace the first oligonucleotide corresponding to a portion of the HMP-P kinase / TMP-PPase gene with a gene that is not present in the gene or that is not adjacent to the portion of the gene corresponding to the first oligonucleotide. Produced by ligating to a second oligonucleotide corresponding to the part, wherein the second oligonucleotide contains at least one ribonucleotide. Double-stranded DNA is generated using a first oligonucleotide as a template and a second oligonucleotide as a primer. The ribonucleotide is cleaved and removed. One or more nucleotides located 5 ′ to the ribonucleotide are also removed,
This produces a double-stranded fragment with cohesive ends. Such fragments are randomly reassembled by ligation to yield new combinations of gene sequences.

For in vitro recombination in the context of the present invention, any HMP-P kinase / T
Any combination of MP-PPase gene or HMP-P kinase / TMP-PPase gene may be used. For example, the HMP-P kinase / TMP-PPase gene is obtained from a plant, such as Arabidopsis thaliana,
For example, the HMP-P kinase / TMP-PPase gene is a gene as described in SEQ ID NO: 1 or SEQ ID NO: 3. Other genes suitable for in vitro recombination are, for example, those obtained from bacteria, such as the E. coli thiD or thiE gene (Vander H.
orn et al. (1993) J. Bacteriol. 175, 982-992; Backstrom et al. (1995) J.
Am. Chem. Soc. 117, 2351-2352), Salmonella typhimurium t
hiD gene (Petersen and Downs (1997) J. Bacteriol. 179, 4894-4900), Neurospora crassa thiD gene of Neurospora crassa (Akiyama and Nakashima (1996)
) Curr. Genet. 30, 62-67; Ouzonis and Kyrpides (1997) J. Mol. Evol. 45,
708-711) or brewer's yeast thiE gene (Nosaka et al. 19940) J. Biol. Chem.
269, 30510-30516 (the contents of which are incorporated herein by reference). The entire HMP-P kinase / TMP-PPase gene or a portion thereof is used in the context of the present invention. Mutated HMP obtained by the above method
The library of -P kinase / TMP-PPase genes is cloned in a suitable expression vector and the resulting vector is transformed in a suitable host, for example, algae such as Chlamydomonas, yeast or bacteria. A suitable host is preferably a host that otherwise lacks HMP-P kinase activity, such as E. coli NI500
Strains (Genetic Stock Center, New Haven, USA) or hosts lacking TMP-PPase activity, such as E. coli NI400 (Nakayama and Hayashi (1972) J. Bacterio
logy 112: 1118-1126). Host cells transformed with a vector comprising a library of mutated HMP-P kinase / TMP-PPase genes are cultured on media containing inhibitory concentrations of the inhibitor and grown in the presence of the inhibitor. Select a colony to clone. Collect colonies that grow in the presence of inhibitor, usually at inhibitory concentrations,
Purify by repeated re-streaking. The plasmids are purified and then the DNA sequence of the cDNA insert from the plasmid that passed this test is determined.

Assays to identify modified HMP-P kinase / TMP-PPase genes that are tolerant to inhibitors can be performed by assays to identify inhibitors of HMP-P kinase activity or TMP-PPase activity ( In the same manner as in the above inhibitor assay), the mutant HMP-P kinase / TMP-PPase can be used in one of the reaction mixtures, with the following modifications. Wild type HMP-P kinase /
Used in place of TMP-PPase. Second, inhibitors of the wild-type enzyme are present in both reaction mixtures. Third, comparing mutagenic activity (activity in the presence of inhibitors and mutating enzymes) and non-mutating activity (activity in the presence of inhibitors and wild-type enzymes), It is determined whether a significant increase in enzyme activity is observed in the mutating activity when compared to the mutating activity. Mutagenic activity is any measure of the activity of a mutated enzyme in the presence of a suitable substrate and inhibitor. Non-mutagenic activity is any measure of the activity of the wild-type enzyme in the presence of the appropriate substrate and inhibitor. A significant increase is defined as an increase in enzyme activity that is greater than the error limit inherent in the assay, preferably about a two-fold or more increase in the activity of the wild-type enzyme in the presence of the inhibitor, more preferably about 5 times. It is defined as more than a two-fold increase, most preferably about a ten-fold or more increase.

In addition to being used to produce herbicide-tolerant plants, herbicide-tolerant HMP-P
The gene encoding the kinase / TMP-PPase is also used as a selection marker in a plant cell transformation method. For example, a plant, plant tissue, plant seed or plant cell transformed with the transgene is a modified HM that can be expressed by the plant.
Genes encoding PP kinase / TMP-PPase can also be transformed. The transformed cells are transferred to a medium containing an inhibitor of the enzyme in an amount sufficient to inhibit the viability of plant cells that do not express the modified gene, where only the transformed cells survive. The method is applicable to any plant cell that can be transformed with a modified HMP-P kinase / TMP-PPase encoding gene and can be used with any such transgene. Expression of the transgene and the modified gene can be driven by the same promoter, which is functional in plant cells, or by separate promoters.

Preferred is a method for producing the mutagenized HMP-P kinase / TMP-PPase gene of the present invention. Even more preferred is the aforementioned mutagenized HMP-P kinase / TMP-P
This is a method for producing a Pase gene. Even more preferred is a method for producing a mutagenized DNA molecule of the present invention, wherein one template DNA molecule is obtained from a eukaryote. Particularly preferred is the method according to the invention, wherein said eukaryote is a plant.

Further preferred is a method according to the present invention, wherein the template DNA molecule of said species is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1. More preferred is a mutagenized DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of the present invention, wherein the HMP-P is encoded by the template DNA molecule. HM showing enhanced tolerance to herbicides that inhibit P-kinase activity or TMP-PPase activity
Mutagenized DN encoding PP kinase or TMP-PPase enzyme
A molecule.

[0070] The wild-type or herbicide-tolerant form of the HMP-P kinase / TMP-PPase gene can be incorporated into plant or bacterial cells using conventional recombinant DNA techniques. In general, this is accomplished using standard cloning techniques known in the art,
This involves inserting a DNA molecule encoding HMP-P kinase / TMP-PPase into an expression system in which the molecule is heterologous (ie, not normally present). Vectors contain the necessary elements for the transcription and translation of the inserted protein coding sequence in host cells containing the vector. Many vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Components of the expression system can also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be used. Almost any crop plant cell can be transformed under appropriate conditions using expression systems known in the art. The transgene, including the wild-type or herbicide-tolerant form of the HMP-P kinase / TMP-PPase gene, is preferably stably transformed and integrated into the genome of the host cell. In another preferred embodiment, the transgene, including the wild-type or herbicide-tolerant form of the HMP-P kinase / TMP-PPase gene, is located on a self-replicating vector. An example of a self-replicating vector is a virus, especially a geminivirus. The transformed cells contain selected forms of HMP-P kinase /
TMP-PPase can be regenerated in whole plants such that it confers herbicide tolerance in transgenic plants.

A. Requirements for Construction of Plant Expression Cassettes The gene sequences intended for expression in transgenic plants
It is assembled into an expression cassette after a suitable promoter that can be expressed in plants. The expression cassette also includes any additional sequences required or selected for expression of the transgene. Such sequences include transcription terminators,
These include, but are not limited to, intronic sequences for enhancing expression, life-threatening sequences, and sequences intended to target gene products to specific organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described below. The following is a description of the various components of a typical expression cassette.

1. Promoter The choice of promoter used for the expression cassette will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. The selection promoter expresses the transgene in a particular cell type (eg, leaf epithelial cells, mesophyll cells, root cortical cells) or in a particular tissue or organ (eg, root, leaf, flower), and The choice reflects the desired location of accumulation of the gene product. Alternatively, the selection promoter can drive expression of the gene under various inducing conditions. Promoters vary in their strength, ie, their ability to promote transcription. Depending on the host system used, any of a number of suitable promoters known in the art may be used. For example, for constitutive expression, the CaMV 35S promoter, rice actin promoter or ubiquitin promoter can be used. For regulated expression, a chemically inducible PR-1 promoter from tobacco or Arabidopsis thaliana may be used (see, eg, US Pat. No. 5,689,044).

2. Transcription Terminators A variety of transcription terminators are available for use in expression cassettes.
These are involved in the termination of transcription beyond the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants, examples of which include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledonous and dicotyledonous plants.

[0074] 3. Sequences for enhancing or regulating expression A number of sequences have been found to enhance gene expression from within a transcription unit,
These sequences can be used with the genes of the present invention to increase their expression in transgenic plants. For example, various intron sequences, such as those in the maize AdhI gene, have been shown to enhance expression, especially in monocot cells. In addition, a number of untranslated leader sequences from the virus are also known to enhance expression, and are particularly useful in dicot cells.

[0075] 4. Coding Sequence Optimization The coding sequence of the selected gene can be genetically engineered by altering the coding sequence for optimal expression in the crop species. Methods for modifying coding sequences to achieve optimal expression in particular crop species are well known (eg, Perlak et al.,
Natl. Acad. Sci. USA 88: 3324 (1991) and Koziel et al., Bio / tech.
nol. 11: 194 (1993)).

[0076] 5. Targeting gene products in cells Various mechanisms for targeting gene products are known to exist in plants, and the sequences that control the operation of these mechanisms have been characterized in considerable detail. I have. For example, targeting of gene products to chloroplasts is controlled by signal sequences found at the amino terminus of various proteins that are cleaved during chloroplast import to produce mature proteins (eg, Coma).
i et al. J. Biol. Chem. 263: 15104-15109 (1988)). Other gene products are localized to other organelles such as mitochondria and peroxisomes (eg, Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to perform targeting of heterologous gene products to these organelles. In addition, sequences that cause targeting of the gene product to multiple cell compartments have been characterized.
The amino-terminal sequence is involved in targeting ER, extracellular secretion from apoplasts and aleurone cells (Koehler & Ho, Plant Cell 2: 769-783).
(1990)). In addition, the amino terminal sequence, together with the carboxy terminal sequence, is involved in vacuolar targeting of the gene product (Shinshi et al. Plant Molec. Biol.
14: 357-368 (1990)). The fusion of the appropriate targeting sequence with the transgene sequence can direct the transgene product to any organelle or cell compartment.

B. Construction of Plant Transformation Vectors Numerous transformation vectors available for plant transformation are known to those skilled in the art of plant transformation, and the genes related to the present invention can be used with any such vector. The choice of vector will depend on the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be selected. Selectable markers routinely used for transformation include the nptII gene that confers resistance to kanamycin and related antibiotics (Messing & Vierra, Gene 19: 259-268 (1982); Bevan et al., Nature 304:
184-187 (1983)), bar gene that confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor.
Appl. Genet 79: 625-631 (1990)), hph gene that confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol Cell Biol 4: 2929-2931)
) And the dhfr gene (Bourouis et al.) Conferring resistance to metatrexate
., EMBO J. 2 (7): 1099-1104 (1983)), and the EPSPS gene that confers resistance to glyphosate (US Pat. Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Transformation of Agrobacterium Root Carcinoma A number of vectors are available from the Agrobacterium tumefaciens Agroba
It can be used for transformation using cterium tumefaciens. These typically carry at least one T-DNA border sequence and contain pBIN19 (Bevan, Nucl.
Acids Res. (1984)) and vectors such as pXYZ. Typical vectors suitable for apical carcinoma transformation include the binary vectors pCIB200 and pCIB200.
1, and the binary vector pCIB10 and its selective derivatives of hygromycin (see, eg, US Pat. No. 5,639,949).

2. Vectors Suitable for Non-Root Apex Carcinoma Transformation Transformation without the use of Agrobacterium tumefaciens blocks the requirement for a T-DNA sequence in the selected transformation vector, and therefore T-DN
In addition to vectors such as those containing the A sequence, vectors lacking these sequences may be used. Transformation techniques that do not rely on apical carcinoma include particle bombardment,
Protoplast uptake (eg, PEG and electroporation) and transformation by microinjection. The choice of vector is largely dependent on the preference for the species being transformed. Exemplary vectors suitable for non-apical carcinoma transformation include pCIB3064, pSOG19 and pSOG35 (see, eg, US Pat. No. 5,639,949).

C. Transformation techniques Once the coding sequence has been cloned in an expression system, it is transformed in plant cells. Methods for plant transformation and regeneration are well known in the art. For example,
Ti plasmid vectors have been used for delivery of foreign DNA, as well as for direct DNA uptake, liposomes, electroporation, microinjection and microprojectiles. In addition, bacteria from the genus Carcinoma can be utilized to transform plant cells.

[0081] Transformation techniques for dicots are well known in the art and include carcinoma based techniques and techniques that do not require carcinoma. Non-carcinoma techniques involve the direct uptake of exogenous genetic material by protoplasts or cells. This is PEG
Alternatively, it can be achieved by electroporation-mediated uptake, particle impact-mediated delivery, or microinjection. In each case, the transformed cells are regenerated into whole plants using standard techniques known in the art.

[0082] Transformation of most monocot species is also routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, particle bombardment into callus tissue, and carcinoma transformation. The wild-type or modified forms of the HMP-P kinase / TMP-PPase gene of the present invention can be used to confer herbicide tolerance on a wide variety of plant cells, such as gymnosperms, monocots and dicots. . The gene can be inserted into any plant cell within these wide variety, but it is not a plant cell such as rice, wheat, barley, rye, corn, potato, carrot, sweet potato, sugar beet, fava bean, pea, chicory, Lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, tounas, pumpkin, zucchini,
Cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sugar corn and sugar cane Particularly useful.

High level expression of wild-type HMP-P kinase / TMP-PPase gene and / or expression of herbicide-tolerant form of HMP-P kinase / TMP-PPase gene that confers herbicide tolerance in plants Can be incorporated into plant strains by breeding approaches and techniques known in the art, in combination with other properties important for production and quality.

If the allele of the herbicide-tolerant HMP-P kinase / TMP-PPase gene is obtained by direct selection in a crop plant or plant cell culture in which the crop plant can be regenerated, it is possible to engineer the allele. And transferred into commercial varieties using traditional breeding techniques to develop herbicide-tolerant crops without the need to transform them in plants.

Brief Description of Sequences in Sequence Listing SEQ ID NO: 1: DNA sequence of Arabidopsis HMP-P kinase / TMP-PPase preprotein. SEQ ID NO: 2: Amino acid sequence of Arabidopsis HMP-P kinase / TMP-PPase preprotein.

SEQ ID NO: 3: DNA sequence of putative mature Arabidopsis HMP-P kinase / TMP-PPase. SEQ ID NO: 4: Amino acid sequence of putative mature Arabidopsis HMP-P kinase / TMP-PPase. SEQ ID NO: 5: oligonucleotide aththiDEfor SEQ ID NO: 6: oligonucleotide aththiDErev SEQ ID NO: deposited oligonucleotide: aththiDEfor-ctp Deposited clone : aththiDE-ctp Deposit number : NRRL B-30040 Date of deposit : July 8, 1998 The invention will be further described with reference to examples. These examples are for illustrative purposes only and the invention is not limited thereto unless otherwise specified.

EXAMPLES Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook et al. ( Molecular Cloning , eds., Cold Spring Harbor Labora
tory Press, Cold Spring Harbor, NY (1989)), TJ Silhavy, ML
Berman and LW Enquist ( Experiments with Gene Fusions , Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY (1984)) and Ausubel,
FM etc. ( Current Protocols in Molecular Biology , pub. By Greene Publishin
g Assoc. and Wiley-Interscience (1987)).

Example 1 Expression of Recombinant Plant HMP-P Kinase and TMP-PPase in E. coli Landsbe of Arabidopsis thaliana in Plasmid Vector pFL61
Generate and amplify an rg ecotype cDNA library. Arabidopsis HMP-
PCR primers for amplifying the protein coding sequence for P-kinase and TMP-PPase are shown in SEQ ID NO: And uses it to amplify HMP-P kinase and TMP-PPase coding sequences from a plasmid library with Pfu DNA polymerase (Stratagene). For constructs containing coding regions for HMP-P kinase and TMP-PPase preprotein, the primer aththiDEfor (5 'ggt att gag ggt cgc atg aat agc tta gga gga at 3'
AththiDErev (5 ′ aga gga gag tta gag cct caa att ccc)
Using ctt ttg ctc tct tta a 3 ′, SEQ ID NO: 6), for constructs of putative mature HMP-P kinase and TMP-PPase, primers atiththiDErev and a
ththiDEfor-ctp (5 'ggt att gag ggt cgc tta aca gtg gcg gga tca gat 3'
SEQ ID NO: 7) is used. The coding regions for the pre-protein and the putative mature protein were subcloned in the expression vector pET35b (+) Xa / LIC (Novagen) and both were transformed into E. coli BL21 (DE3) by chemical transformation using the manufacturer's protocol.
) (Novagen).

Example 2 HMP-P Kinase and TMP-PPase Activity Assay HMP-P Kinase and TMP-PPase Activity Assay
From da et al. (1988) Plant Physiol. 88, 248-250. Reaction volume is preferred
Or as follows, but may vary depending on experimental requirements. 0.01-1.0
x 10^-3Having units of HMP-P kinase activity or TMP-PPase activity
Element (one unit of activity is defined as the amount of enzyme required to produce 1 mmol / min product)
And 10^-3~Ten^-Five M, but preferably 10^-Four 2-methyl-4-amino of M
-5-hydroxymethylpyrimidine phosphate (HMP-P) and 10^-3~Ten ^-Five M, but preferably 10^-Four M-methyl-5- (β-hydroxyethyl) thio
Azole phosphate (THZ-P) was added to a final volume of 100 mL of 50 mM Tris-HC.
1 (pH 7.0-8.0, preferably 7.4), 1-20 mM, preferably 10 mM MgCl_Two
And 0.1 to 10 mM, preferably 1 mM ATP. Preferably Kawasaki
 et al. (1990) J. Bacteriol. 172, 6145-6147, 50 mL of 10% (w /
v) Adding metaphosphoric acid or 20% (w / v) trichloroacetic acid (pH 1.4)
Thus, the production of thiamine monophosphate (TMP) is determined. Next, the mixture
Centrifuge to remove precipitated proteins. Remove 100 mL aliquot of supernatant
And add 50 mL of 0.3 M cyanogen bromide, mix and add 100 mL of 1 M NaOH.
Add. With an excitation wavelength of 360 ± 10 nm and an emission wavelength of 430 ± 10 nm,
Measure the light intensity.

Alternatively, ADP production is quantified by a binding reaction method. In this case, 3.5 units of pyruvate kinase, 4.7 units of lactate dehydrogenase, 1.0 mM of phosphenol pyruvate and 0.2 mM of NADH are added and the absorbance is measured at 340 nm. Example 3: Bound HMP kinase and HMP-P kinase and TMP-PP
Assay Activity Assay HMP Kinase Assay After conversion of HMP to HMP-P, the concomitant production of ADP is detected. ADP production was tracked using the enzymes pyruvate kinase and lactate dehydrogenase (reagent enzyme) and the NAD ⁺ of NADH in the presence of phosphenolpyruvate (PEP).
Detect conversion to This is monitored at 340 nm. Pyruvate kinase and PEP stimulate the regeneration of ATP from ADP. ATP is a required substrate for both HMP kinase and HMP-P kinase. Assay buffer was 10 mM Mg
Accompanied by the addition of Cl _2, 50 mM Tris-HCl, a pH 7.5 (buffer A).

HMP-P Kinase and TMP-PPase To assay HMP-P kinase and TMP-PPase, the substance HM
It is necessary to provide PP and THZ-P. HMP-P is provided by the conversion of HMP to HMP-P in the same reaction mixture. If NADH is added, conversion is followed using the HMP kinase assay. HMP to HMP-
Once the conversion to P has proceeded sufficiently (about 50 mM), then HMP-P kinase, TMP
-Add PPase and THZ-P. The addition of HMP-P kinase, TMP-PPase and THZ-P after the production of HMP-P ensures that the initial concentration of HMP-P is constant in all reaction wells. Kawasaki et al. (1990) J. Ba
The amount of TMP produced is assayed by the method of cteriol. 172, 6145-6147. T
After a sufficient time for MP generation (typically 15 minutes), metaphosphate or TCA
Stop the enzymatic reaction using. TMP is oxidized under alkaline conditions. 360 ± 10
At an excitation wavelength of nm and an emission wavelength of 430 ± 10 nm, the fluorescence of the resulting thiachrome derivative is measured.

Assay Protocol The assay is performed in the same manner, independent of the original source of the enzyme. Assay is 300 mL
Performed in 96 well microtiter plates. The total assay reaction volume is 100 mL. The substrates are mixed in ratios to give the following final concentrations (in microtiter plates): HMP (100 mM), ATP (1000 mM), PEP (1000 mM) and NADH.
(200 mM). The mixture of substrates at 10X can be pipetted at 10 mL / well. The reagent enzyme and HMP kinase may be mixed so that they are added simultaneously. The suggested amount of ADP detection / regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase / reaction. This should be used as a guideline, and the amount of enzyme adjusted empirically. After completion of the HMP kinase reaction at a rate of about 5 mM / min (which is within about 10-15 minutes), THZ-P was added to a final concentration of 50 mM and HMP-P kinase was added. And TMP-PPase. After a certain interval (determined by the activity of HMP-P kinase and TMP-PPase)
The reaction is stopped by adding 50 mL of 10% (w / v) metaphosphoric acid or 20% (w / v) trichloroacetic acid (pH 1.4). The plate is spun in a centrifuge to pellet the precipitated protein, and then the supernatant is transferred to a separate microtiter plate. Add 50 mL of 0.3 M cyanogen bromide and mix, then add 100 mL of 1 M NaOH. The plate is read on a fluorimetric microtiter plate reader at an excitation wavelength of 360 ± 10 nm and an emission wavelength of 430 ± 10 nm. Thiamine monophosphate is used as a standard.

[0093] Thiamine monophosphate, ATP, PEP, NADH, metaphosphoric acid, trichloroacetic acid, cyanogen bromide and NaOH are available from Sigma Chemicals. HMP is described in Schellenberger et al. (Hoppe-Seyler's Z. Physiol. Chem. (1967) 34
8, 501-505), and THZ-P was prepared according to Leder et al. (1970, Meth. Enz.
18A, 166-167).

Example 4: Conjugated THZ kinase and HMP-P kinase and TMP-PP
Assay activity assay THZ Kinase Assay Follow up the conversion of THZ to THZ-P by detecting co-production of ADP. ADP production is performed using enzymes pyruvate kinase and lactate dehydrogenase (reagent enzyme) in the presence of phosphenol pyruvate (PEP).
Follow up by detecting conversion of DH to NAD ⁺ . This is monitored at 340 nm. Pyruvate kinase and PEP promote the regeneration of ATP from ADP. ATP is a required substrate for both THZ kinase and HMP-P kinase. Assay buffer was buffer A, involves the addition of 10 mMMgCl _2.

B. HMP-P kinase and TMP-PPase To assay HMP-P kinase and TMP-PPase, the substance HM
It is necessary to provide PP and THZ-P. THZ-P is provided by the conversion of THZ to THZ-P in the same reaction mixture. If NADH is added, conversion is followed using the THZ kinase assay. THZ to THZ-
Once the conversion to P has proceeded sufficiently (about 20 mM), then HMP-P kinase, TMP
-Add PPase and HMP-P. TMP-PP after generation of THZ-P
The addition of ase and HMP-P ensures that the initial concentration of THZ-P is constant in all reaction wells. Kawasaki et al. (1990) J. Bacteriol. 172, 6145-6
The amount of TMP produced is assayed by the method of 147. After a sufficient time for TMP production (typically 15 minutes), the enzymatic reaction is stopped with metaphosphate or TCA. TMP is oxidized under alkaline conditions. 360 ± 10 nm excitation wavelength and 43
At an emission wavelength of 0 ± 10 nm, the fluorescence of the resulting thiachrome derivative is measured.

C. Assay Protocol The assay is performed in the same manner, independent of the original source of the enzyme. Assay is 300 mL
Performed in 96 well microtiter plates. The total assay reaction volume is 100 mL. The substrates are mixed in ratios to give the following final concentrations (in microtiter plates): THZ (50 mM), ATP (5 mM), PEP (1000 mM) and NADH (20 mM).
0 mM). The mixture of substrates at 10X can be pipetted at 10 mL / well. The reagent enzyme and HMP kinase may be mixed so that they are added simultaneously. Suggestive AD
The P detection / regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase / reaction. This should be used as a guideline, and the amount of enzyme adjusted empirically. After completion of the THZ kinase reaction at a rate of about 5 mM / min (which is within about 10-15 minutes), HMP-P was added to a final concentration of 10
Adjust to 0 mM and add HMP-P kinase and TMP-PPase. After a certain interval (determined by the activity of HMP-P kinase and TMP-PPase), 50 mL
The reaction is stopped by the addition of 10% (w / v) metaphosphoric acid or 20% (w / v) trichloroacetic acid (pH 1.4). The plate is spun in a centrifuge to pellet the precipitated protein, and then the supernatant is transferred to a separate microtiter plate. Add 50 mL of 0.3 M cyanogen bromide and mix, then add 100 mL of 1 M NaOH. The plate is read on a fluorimetric microtiter plate reader at an excitation wavelength of 360 ± 10 nm and an emission wavelength of 430 ± 10 nm. Thiamine monophosphate is used as a standard.

[0097] Thiamine monophosphate, ATP, PEP, NADH, metaphosphoric acid, trichloroacetic acid, cyanogen bromide and NaOH are available from Sigma Chemicals. HMP is described in Schellenberger et al. (Hoppe-Seyler's Z. Physiol. Chem. (1967) 34
8, 501-505), and THZ-P was prepared according to Leder et al. (1970, Meth. Enz.
18A, 166-167).

Example 5: Binding HMP Kinase, THZ Kinase and HMP-P Kinase and TMP-PPase Activity Assay HMP Kinase and THZ Kinase Assay HMP to HMP-P by detecting co-production of ADP, and T
Follow up conversion of HZ to THZ-P. ADP production is followed by detecting the conversion of NADH to NAD ⁺ in the presence of phosphenolpyruvate (PEP) using the enzymes pyruvate kinase and lactate dehydrogenase (reagent enzyme). This is monitored at 340 nm. Pyruvate kinase and PEP promote the regeneration of ATP from ADP. ATP is a required substrate for both HMP kinase and HMP-P kinase. The assay buffer is Buffer A,
Of 10 mMMgCl ₂ with additional.

HMP-P kinase and TMP-PPase To assay for HMP-P kinase and TMP-PPase, the substance HM
It is necessary to provide PP and THZ-P. HMP-P and THZ
-P to HMP-P of HMP and THZ of THZ in the same reaction mixture
Provided by conversion to -P. If NADH is added, conversion is followed using the HMP kinase and THZ kinase assays. Once the conversion of HMP to HMP-P and from THZ to THZ-P has progressed sufficiently (approximately 50 and 20 mM, respectively), HMP-P kinase and TMP-PPase are then added. H
The addition of HMP-P kinase and TMP-PPase after production of MP-P and THZ-P ensures that the initial concentrations of these substances are constant in all reaction wells. The amount of TMP produced is assayed by the method of Kawasaki et al. (1990) J. Bacteriol. 172, 6145-6147. After a sufficient time for TMP production (typically 15 minutes), the enzymatic reaction is stopped with metaphosphate or TCA. TMP is oxidized under alkaline conditions. At an excitation wavelength of 360 ± 10 nm and an emission wavelength of 430 ± 10 nm, the fluorescence of the resulting thiachrome derivative is measured.

Assay Protocol The assay is performed in the same manner, independent of the original source of the enzyme. Assay is 300 mL
Performed in 96 well microtiter plates. The total assay reaction volume is 100 mL. The substrates are mixed in a ratio to give the following final concentrations (in microtiter plates): HMP (100 mM), THZ (50 mM), ATP (5 mM), PEP (1000 mM)
And NADH (200 mM). The mixture of substrates at 10X can be pipetted at 10 mL / well. The reagent enzyme and HMP kinase may be mixed so that they are added simultaneously. The suggested amount of ADP detection / regeneration mix is 1.0 units pyruvate kinase and 1.0 units lactate dehydrogenase / reaction. This should be used as a guideline, and the amount of enzyme adjusted empirically. After the HMP kinase and THZ kinase reactions are allowed to proceed to completion at a rate of about 5 mM / min (which is within about 10-15 minutes), HMP-P kinase and TMP-PPase are added. At regular intervals (
After determination of the activity of HMP-P kinase and TMP-PPase), 50 mL of 1
The reaction is stopped by the addition of 0% (w / v) metaphosphoric acid or 20% (w / v) trichloroacetic acid (pH 1.4). The plate is spun in a centrifuge to pellet the precipitated protein, and then the supernatant is transferred to a separate microtiter plate. Add 50 mL of 0.3 M cyanogen bromide and mix, then add 100 mL of 1 M NaOH. The plate is read on a fluorimetric microtiter plate reader at an excitation wavelength of 360 ± 10 nm and an emission wavelength of 430 ± 10 nm. Thiamine monophosphate is used as a standard.

[0101] Thiamine monophosphate, ATP, PEP, NADH, metaphosphoric acid, trichloroacetic acid, cyanogen bromide and NaOH are available from Sigma Chemicals. HMP is described in Schellenberger et al. (Hoppe-Seyler's Z. Physiol. Chem. (1967) 34
8, 501-505), and THZ-P was prepared according to Leder et al. (1970, Meth. Enz.
18A, 166-167).

Example 6: In vitro recombination of the HMP-P kinase / TMP-PPase gene by DNA shuffling The Arabidopsis thaliana A. thaliana HMP-P kinase / TMP-PPase gene encoding the pre-protein is described in Example 6. Amplify by PCR as described above. The resulting DNA fragment is essentially synthesized as described above (Stemmer et al. (1994) PN
AS 91: 10747-10751), digested by DNase I treatment and the PCR primers are removed from the reaction mixture. A PCR reaction is performed without using primers, and then a PCR reaction using PCR is performed (both in Stemmer et al. (1994)).
PNAS 91: 10747-10751). The resulting DNA fragment was ligated with pTRC99a (Pharma
a, catalog number 27-5007-01), and electroporation using a Biorad Gene Pulser and manufacturer's conditions to E. coli NI500 (Genetic
Stock Center, New Haven, USA) or E. coli NI400 (Nakayama and Hayashi (
1972) in J. Bacteriology 112: 1118-1126). The transformed bacteria are grown on media containing an inhibitory concentration of the inhibitor to select for colonies that grow in the presence of the inhibitor. Colonies that grow in the presence of inhibitor, usually at inhibitory concentrations, are picked and purified by repeated re-streaking. The plasmids are purified and the DNA sequence of the cDNA insert from the plasmid that passed this test is then determined.

Example 7: In Vitro Recombination of HMP-P Kinase / TMP-PPase Gene by Adhesion Prolongation Method Arabidopsis thaliana A. thaliana HMP-P kinase / TMP-PPase gene encoding mature protein and E. coli thiD were each isolated Cloning in the polylinker of the pBluescript vector. Using "reverse primers" and "M1320 primers" (Stratagene catalog), essentially use the method described in (Zhao et al. (1998) N
The PCR reaction is carried out in the same manner as in the case of Nature Biotechnology 16: 258-261). Amplified PC
The R fragment is digested with the appropriate restriction enzymes, cloned in pTRC99a, and
The MP-P kinase / TMP-PPase gene is screened as in Example 6.

B. The Arabidopsis thaliana HMP-P kinase / TMP-PPase gene encoding the mature protein and E. coli thiE are each cloned in the polylinker of the pBluescript vector. Using “reverse primers” and “M1320 primers” (Stratagene catalog) essentially as described above (Zhao et al. (199
8) Perform a PCR reaction as in Nature Biotechnology 16: 258-261). The amplified PCR fragment is digested with appropriate restriction enzymes, cloned in pTRC99a, and the mutated HMP-P kinase / TMP-PPase gene is screened as in Example 6.

C. The Arabidopsis thaliana HMP-P kinase / TMP-PPase gene encoding the mature protein and E. coli thiD and thiE were isolated from pBlu
Clone in polylinker of escript vector. Using the “reverse primer” and the “M1320 primer” (Stratagene catalog), essentially as described above (Zhao
The PCR reaction is performed as in et al. (1998) Nature Biotechnology 16: 258-261). The amplified PCR fragment is digested with appropriate restriction enzymes, cloned in pTRC99a, and the mutated HMP-P kinase / TMP-PPase gene is screened as in Example 6.

Example 8: Initiation and Retention of Rice Embryogenic Cell Suspension Immature spikelets with milky endosperm of Japonica rice "Taipei 309" varieties were smashed to 70% (v / v) Surface sterilize with ethanol for 1 hour and calcium chloride for 20 minutes, then wash 3 times with sterile distilled water. Isolate immature embryos with 3% sucrose, 2 mg /
l of 2,4-dichlorophenoxyacetic acid (2,4-D), 0.35% containing pH 5.8
Culture at 28 ° C. on agarose-solidified MS medium (Murashige and Skoog, 1962).
One week later, the callus material generated from the scutellum is divided, transferred to a fresh medium every week and cultured. Four weeks after initiation, 3-4 calli were added to 20 ml of R2 medium (R2 salts and vitamins (Ohira et al., 1973), 1 mg / l 2,4-D, 500 mg / l 2-morpholinoethane. Transfer to a 50 ml culture vessel containing sulfonic acid (MES), 3% sucrose, pH 5.8). The culture is kept in dim light at 28 ° C. on a rotary shaker at 220 rpm, and the medium is replaced weekly with an equal volume of fresh medium. Quickly split, select fragile calli and subclon in a fresh container by transferring 2 ml of fine callus suspension into 20 ml of R2 medium.

Example 9: Microprojectile bombardment A 2-3 month old suspension medium, previously subcloned for 3-4 days, serves as target cells for bombardment. Four hours before particle bombardment, approximately 500 mg of cells were
On a 0.35% agarose solidified protoplast separation medium (R2 salts and vitamins, 1 mg / l 2,4-D, 3% sucrose, 0.5 M sucrose, pH 5.8) in a Petri dish, 2 c diameter
Spread as a single layer of m. DNA-coated gold particles (Aldrich Cat # 32,658-5, spherical gold powder 1.5-3.0) were used in embryogenic suspension cells using a particle inflow gun (Finer et al., 1992).
μm). Particle coating is performed essentially as in Vain et al. (1993): Dispense a 5 μl aliquot of the plasmid solution into a 0.5 ml reaction tube and place on ice. The particles are suspended at 100 mg / ml in 96% ethanol and stirred for 2 minutes. Replace the ethanol with an equal volume of sterile distilled water and stir the suspension for 1 minute. This washing step is repeated once. The particles are finally resuspended in sterile distilled water at 100 mg / ml. Add 25 μl of the particle suspension to each of the DNA aliquots and stir the tube for 1 minute, then immediately add 25 μl of sterile ice-cold CaCl ₂ (2.5 M in distilled water) and stir for an additional minute. 10 μl of sterile spermidine (0.1 M in distilled water) is added, the suspension is stirred again and placed on ice for 5 minutes, during which the particles precipitate. 50 μl of the particle-free supernatant was removed and the remaining suspension (15 μl) was used for 5 shocks. Prior to each impact, the particles need to be vigorously pipetted and resuspended.

The cells were covered with a 500 μm mesh baffle and the filter unit containing particles 14
cm below. The particles are released by a single 8 atm pulse of 50 msec in a partial vacuum (2 x 10 ⁴ Pa). Example 10: Transformation of wheat A preferred technique for wheat transformation involves particle bombardment of immature wheat embryos and involves a high sucrose or high maltose step prior to gene delivery. Before the shock,
Any number of embryos (0.75-1 mm in length) can be transferred to 3% sucrose (M
urashige and Skoog, 1962) and placed on MS medium containing 3 mg / l 2,4-D, which continues in the dark. On the day of impact selection, remove the embryos from the induction medium,
Place on infiltration medium (ie, induction medium containing sucrose or maltose added at the desired concentration, typically 15%). Embryos are subjected to protoplasmic separation for 2-3 hours and then subjected to bombardment. 24 embryos per target plate are typical, but not critical. The appropriate gene-bearing plasmid is precipitated on μm-sized gold particles using standard techniques. Each plate of embryos is shot on a DuPont Biolistics helium device using a huge pressure of ~ 1000 psi and using a standard 80 mesh screen. After bombardment, the embryos are returned to the dark and collected for about 24 hours (still on the permeation medium). After 24 hours, the embryos are removed from the infiltration medium and put back on the induction medium, where they remain for about one month before being regenerated. After about one month, the embryo explants with developing embryogenic callus are transferred to regeneration medium (MS + 1 mg / L NAA, 5 mg / L GA) further containing the appropriate selection agent. After about one month, the developed shoots are transferred to a larger sterile container known as GA7s containing half concentration of MS, 2% sucrose and the same concentration of selection agent. Stable transformation of wheat is described in detail in EP 0 674 715.

Example 11: Preparation of special callus of excellent inbred line Funk 2717 of maize Zea mays [0109] Maize Zea mays plants of inbred line Funk 2717 were grown, flowered, and self-pollinated in a greenhouse. Immature ears containing embryos of about 2-2.5 mm in length are removed from the plants and sterilized in a 10% Chlorox solution for 20 minutes. Embryos were aseptically removed from the kernels, with hypocotyls down and 0.1 mg / l 2,4-D, 6% sucrose and 25 mM L-solidified in 0.24% Gelrite (initial medium). O containing proline
Plate on MS medium. After culturing at 27 ° C. in the dark for 2 weeks, the callus that has developed on the scutellum is removed from the embryo and placed on B5 medium containing 0.5 mg / l of 2,4-D and solidified with 0.24% gellite. . The calli are subcultured every two weeks to fresh medium. A total of eight weeks after placing the embryos on the initial medium, a distinctive form of callus is identified by its characteristic morphology. This callus is further subcultured on the same medium. 2
After a month, the calli are transferred to N6 medium containing 2 mg / l of 2,4-D and solidified with gelrite, and subcultured sequentially thereon.

Example 12 Preparation of Suspension Culture of Maize Zea mays Inbred Line Funk 2717 The calli are subcultured for a total of at least 6 months. The type of callus selected for subculture is relatively non-mucous, granular and very fragile, so that when placed in liquid media, it separates into discrete small cell aggregates. Cultures containing aggregates with large expanded cells are not maintained. An approximately 500 mg aliquot of the special callus of the maize Zea mays inbred inbred line Funk 2717 is placed in a 125 ml Delong Delong flask in 30 ml N6 medium containing 2 mg / l 2,4-D. After culturing at 26 ° C. for 1 week in the dark on a rotary shaker (130 rpm, 2.5 cm stroke), the medium is replaced with fresh medium. The suspension is again subcultured in this way for another week. At that point, the culture is examined, leaving those that do not show large numbers of expanded cells. Discard the suspension culture containing the aggregate with large expanded cells. Preferred tissues consist of dense cytoplasmic split cell aggregates that have a characteristically smoother surface than conventional cell aggregates. The culture maintained has at least 50% of the cells resuspended in these subassemblies. This is the preferred form. This suspension also shows a rapid growth rate,
The doubling time is less than one week. Suspension culture is grown in 0.5 ml P
Weekly subcultures by transferring CVs. After 2-6 weeks of subculture in this manner, the culture grows 2-3 fold per week subculture. Cultures in which more than 75% of the cells are in the desired form are retained for further subculture. Lines are retained by usually selecting the flask whose contents show the best form for subculture. Periodically filtered every two weeks through a stainless steel sieve with a pore size of 630 μm,
In some cases, the dispersion of the culture is increased, but is not always necessary.

Example 13 Preparation of Protoplasts from Suspension Culture of Maize Zea mays 1-1.5 ml of PCV of suspension culture cells from the above consisted of 4% cellulase RS, KMC (8.65 g / l KCl , 16.47 g / l MgCl ₂ x6H ₂ O and 12.5 g / l
Incubate the MES of _{CaClx2H 2 O, 5 g / l} , pH5.6) salt 10 to 15 ml of filter-sterilized mixture containing 1% Rhozyme in solution. Perform digestion on a slowly rocking table at 30 ° C. for 3-4 hours. The preparation is monitored under an inverted microscope for protoplast release. The released protoplasts are collected as follows: the preparation is filtered through a 100 μm mesh screen and then through a 50 μm mesh screen. Wash the protoplasts through a sieve using a volume of KMC salt solution equal to the original volume of enzyme solution. Place 10 ml of the protoplast preparation in each of several disposable plastic centrifuge tubes and add 1.5-2 ml of 0.6 M sucrose solution (0.1% M
Buffer to pH 5.6 with ES and KOH) to layer underneath. 60 tubes
Centrifuge at 100100 × g for 10 minutes, collect protoplasts banding at the interface using a pipette and place in a new tube. Protoplast preparation is added to 10 ml of fresh KMC
Resuspend in salt solution and centrifuge at 60-100 xg for 5 minutes. The supernatant is removed and discarded, the protoplasts are gently resuspended in the dripping residue and 10 ml of a 13/14 KMC solution are added gradually. After centrifugation again for 5 minutes, the supernatant is removed again and the protoplasts are resuspended in a 6/7 KMC solution. Aliquots are taken for counting and the protoplasts are re-precipitated by centrifugation. 107 / m protoplast
Resuspend in KM-8p or in 0.5 M mannitol containing 6 mM MgCl ₂ or other suitable medium for use in transformation as described above in the Examples below. This protoplast suspension is used for transformation and cultured as described below.

Example 14 Transformation of Maize Zea mays by Electroporation All steps except the heat shock are performed at room temperature (22-28 ° C). The protoplasts were, in the last step, 0.5 M containing 0.1% MES and 6 mM MgCl _2.
Resuspend in mannitol. Resistance of this suspension is measured in chamber of Dialog Electroporator, adjusted to 1 to 1.2 k with 300 mMMgCl ₂ solution. The protoplasts are heat shocked by immersing the test tubes containing the specimens in a water bath at 45 ° C. for 5 minutes and then cooling to room temperature on ice. A plant-selective hygromycin resistance gene, as described by Rothstein et al. (1987), or a chimeric gene construct as described above, and 4 μg of the linearized plasmid containing 20 μg of calf thymus carrier DNA were added to this suspension. Add to a 0.25 ml aliquot of. 0.125 ml of a 24% PEG solution (molecular weight 8000) in 0.5 M mannitol containing 30 mM MgCl ₂ was
Add to protoplasts. Mix the mixture thoroughly but gently and incubate for 10 minutes. Transfer the sample to the electroporator chamber and transfer the sample to 1500,
Pulse three times at 10 second intervals with an initial voltage of 1800, 2300 or 2800 Vcm ^-1 and an exponential decay time of 10 msec.

Incubate the protoplasts as follows: Place the specimen in a 6 cm Petri dish at room temperature. After an additional 5-15 minutes, 1.2% SeaPlaque agarose and 1 mg / l 2,4-D
Add 3 ml of KM-8p medium containing Mix the agarose and protoplasts thoroughly and allow the medium to gel. B. This is repeated with one or more of the following modifications: (1) Adjust the resistance of the protoplast preparation to 0.5-0.7 k.

(2) The PEG used is a PEG having a molecular weight of 4000. (3) No PEG is added, or a half volume of 12% PEG is added. (4) Apply pulses at 3 second intervals. (5) Place the protoplasts into test tubes after electroporation in a dish placed on a plate cooled to room temperature of 16 ° C.

(6) After the electroporation step, put protoplasts into a test tube,
with 6/7 concentrated KMC solution or with W5 solution (380 mg / l KCl, 18.375 g / l C
aCl ₂ × 2H ₂ O, 9 g / l NaCl; 9 g / l glucose, pH 6.0)
And then collected by centrifugation at 60 xg for 10 minutes, resuspended in 0.3 ml of KM medium and plated as in A.

(7) No calf thymus carrier DNA is added. Example 15: Transformation of maize Zea mays protoplasts by treatment with PEG The protoplasts with the final step, 0 containing 12~30 mMMgCl _2.
Resuspend in 5 M mannitol. A heat shock at 45 ° C. for 5 minutes is performed as above. The protoplasts are dispensed in 0.3 ml suspension protoplasts / tube into aliquots for transformation in centrifuge tubes. In the next 10 minutes, add: DNA and PEG solution (molecular weight) to a final concentration of 20% PEG
40% containing 6000, 0.1 M Ca (NO ₃ ) ₂ and 0.4 M mannitol. PH is adjusted to 8-9 with KOH). Aliquots are incubated for 30 minutes with occasional gentle shaking, after which the protoplasts are placed in Petri dishes (0.3 ml of the original protoplast suspension / 6 cm diameter dish) and cultured as before.

B. This was repeated, and after incubating for 30 minutes in the PEG solution, 0.3 ml
Of W5 by adding 5 times at 2-3 minute intervals. The protoplast suspension is centrifuged, the supernatant is removed and the protoplasts are cultured as described above. C. The above is repeated with modifications to a final PEG concentration of 13-25%. Example 16: Callus regeneration from protoplasts Plates containing protoplasts in agarose are placed in the dark at 26 ° C. After 14 days, protoplasts give rise to colonies. Agarose containing colonies,
Transfer to the surface of a 9 cm diameter petri dish containing 2 mg / l 2,4-D and containing 30 ml N6 medium solidified with 0.24% gellite. This medium is called 2N6. Calli are further cultured in the dark at 26 ° C. and callus pieces are subcultured every two weeks on fresh solid 2N6 medium.

Example 17 Selection of Transformed Callus of Maize Zea mays To select for transformed cells, with the addition of adding 100 mg / l or 200 mg / l hygromycin B to 2N6 medium, The above embodiment is repeated. Example 18: Regeneration of corn plant A. The calli are placed on 2N6 medium for retention and O.
N6 (consisting of N6 medium lacking 2,4-D) and N61 medium (0.25 mg / l of 2
, 4-D and N6 medium containing 10 mg / l kinetin).
Calli growing on ON6 and N61 media were harvested in the light (840-8% from white fluorescent light).
Grow at 400 lx light for 16 hours / day). Callus grown on the N61 medium is transferred to the ON6 medium after two weeks because it is harmful if placed on the N61 medium for a long time. The callus is subcultured every two weeks, even if the callus is transferred again on the same medium formulation. When the seedlings are at least 2 cm high, transfer them to the ON6 medium in a GA7 container. Seedlings are transferred to soil in peat pots, protected from light for the first 4-7 days, when the roots form in 2-4 weeks and appear to have grown well enough to support growth. It is often useful to invert the transplanted plant with a clear plastic cap for 2-3 days to make it on top. Once the plants have settled, they are treated as ordinary corn plants, grown in a greenhouse and matured. Plants are self-pollinated or crossed with wild type to obtain progeny.

B. The above example is repeated with the modification that 100 mg / l or 200 mg / l hygromycin B is added to the 2N6 medium to retain callus. Example 19: DNA into protoplasts of Sorghum Sorghum bicolor
Introduction of the corn suspension FS 562 protoplasts with the corn Zea ma
Prepared essentially as for ys, resuspend after final wash at a density of 10 ⁷ / ml in the following solution: 0.2 M mannitol, 0.1% MES, 72 mM NaCl, 70 mM C
aCl ₂ , 2.5 mM KCl, 2.5 mM glucose. Adjust to pH 5.8 with KOH. Density 1.6
~ 2 x 10 ⁶ / ml. The protoplast suspension is dispensed as 1 ml aliquots into plastic disposable shallow bowls and 10 μg of DNA is added as before. The resistance of the solution at this point is in the range of 6, as measured between the electrodes of an electroporation device of the following 471 electrode set. For transformation, the DNA is added to 10 μl of sterile distilled water that has been sterilized as described by Paszkowski et al. (1984). After gently mixing the solution, a pulse of 400 Vcm ^-1 at room temperature with an exponential decay constant of 10 ms is applied from a BTX-Transfector 300 electroporation device using a 471 electrode assembly.

B. Repeat the above with one or more of the following modifications: (1) The voltage used is 200 Vcm ^-1 , or 100 Vcm ^{-1 to} 800 Vcm ^-1 . (2) The exponential decay constant is 5 ms, 15 ms or 20 ms. (3) Add 50 μg sheared calf thymus DNA in 25 μl sterile water along with plasmid DNA.

(4) By treating with an appropriate restriction enzyme (eg, BamHI),
The plasmid DNA is linearized. After transformation at a density of 2 × 10 ⁶ / ml in KM-8p medium without added solidifying agent,
Incubate protoplasts. Example 20: Introduction of DNA into protoplasts of soybean Glycine max Tricoil et al. (1986), Chowhury and Widholm (1985), or Klein et al. (1981)
The protoplasts of soybean Glycine max are prepared by a method similar to that described in (1). Essentially as before, DNA is introduced into these protoplasts. Without the addition of alginate to solidify the medium, Klein et al. (1981), C.
As described by howhury and Widholm (1985), or Tricoil et al. (1986),
Incubate protoplasts.

The above embodiments are illustrative. The disclosure of the present invention raises those skilled in the art that the present invention may have many variations. All such obvious and foreseeable modifications are intended to be covered by the appended claims.

[Sequence list]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/12 4B065 9/10 C12Q 1/48 Z 9/12 G01N 33/15 Z C12Q 1 / 48 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 AC (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG) , AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72 Inventor Bauer, Michael William United States, 27540 North Carolina, Holly Springs, St. Vincent Drive 617 F-term (reference) 2B030 AA02 AB03 AD05 CA06 CA17 CA19 CB02 CD03 CD07 CD09 CD13 CD17 2G045 AA31 AA40 CB20 DA12 DA13 DA36 FA11 FB AGC4 A08 AA11 BA10 CA04 DA01 EA04 GA12 GA14 GA21 4B050 CC03 DD13 LL03 LL10 4B063 QA01 QA18 QQ20 QR07 QR57 QS24 QS28 4B065 AA88X AA88Y AB01 AC14 BA03 BA04 BA10 CA29 CA46 CA53

Claims (58)

[Claims] 1. A nucleotide sequence encoding an enzyme involved in thiamine biosynthesis, wherein the enzyme has HMP-P kinase activity or TMP-PPase activity. 2. The nucleotide sequence of claim 1, wherein said enzyme comprises an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. 3. The nucleotide sequence of claim 1, wherein said enzyme comprises the amino acid sequence set forth in SEQ ID NO: 2. 4. The nucleotide sequence of claim 1, wherein said nucleotide sequence is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1. 5. The method of claim 1, wherein said nucleotide sequence is set forth in SEQ ID NO: 1.
The described nucleotide sequence. 6. A chimeric gene comprising a promoter operably linked to the nucleotide sequence of claim 1. 7. A recombinant vector comprising the chimeric gene according to claim 7, which can be stably transformed in a host cell. 8. A host cell comprising the vector of claim 14, wherein said nucleotide sequence is expressible in said cell. 9. A plant protein involved in thiamine biosynthesis, comprising HMP
The above-mentioned protein, which has -P kinase activity or TMP-PPase activity; 10. The protein of claim 9, wherein said enzyme comprises an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. 11. The method according to claim 9, which includes the amino acid sequence represented by SEQ ID NO: 2.
The described protein. 12. A method of identifying a chemical to be tested for its ability to inhibit the growth or viability of a plant, comprising the following steps: (a) Under conditions where the enzyme can catalyze the synthesis of its product, H in the reaction mixture
An enzyme having MP-P kinase activity or TMP-PPase activity is identified as HMP-P
(B) combining the chemical and enzyme to be tested in the secondary reaction mixture under the same conditions and for the same time as in the primary reaction mixture, in the primary reaction mixture (C) determine the activity of the enzyme in the primary and secondary reaction mixtures, and (d) the activity of the enzyme in the secondary reaction mixture is more significant than the activity of the enzyme in the primary reaction mixture Selecting a chemical to be tested for its ability to inhibit the growth or viability of the plant, if at all. 13. The method according to claim 13, wherein said enzyme is encoded by a nucleotide sequence that is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1.
Alternatively, the method according to claim 12, which has the same or substantially the same amino acid sequence as the amino acid sequence described in SEQ ID NO: 2. 14. The method of claim 12, wherein said substrate for HMP-P kinase is 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P). 15. The method according to claim 15, wherein the substrate of TMP-PPase is 4-methyl-5- (β-
13. Hydroxyethyl) thiazole phosphate (THZ-P).
The described method. 16. The TMP-PPase substrate is 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP).
The method according to claim 12. 17. The method of claim 12, wherein the activity of the enzyme is measured by measuring TMP produced in the reaction mixture. 18. A method for controlling unwanted vegetation growth, comprising the steps of:
Applying a chemical identified by the described method to the undesired vegetation. 19. A plant, plant cell, plant seed or plant tissue comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity, wherein the nucleotide sequence is HMP-P kinase activity in a wild-type plant. A plant, plant cell, plant seed or plant tissue that renders the plant, plant cell, plant seed or plant tissue tolerant to a chemical identified by the method of claim 12 in an amount that normally inhibits the following. 20. A method according to claim 12, comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity, and wherein the plant is tolerant to a chemical identified by the method of claim 12. A plant obtainable by a method comprising the step of transforming a plant or plant parent with an isolated DNA molecule capable of expressing a nucleotide sequence. 21. The method according to claim 19, wherein (a) H is present in the primary reaction mixture under conditions where the enzyme can catalyze the synthesis of the product
An enzyme having MP-P kinase activity or TMP-PPase activity is identified as HMP-P
(B) combining the chemicals and enzymes in the secondary reaction mixture with the substrate under the same conditions and for the same time as in the primary reaction mixture, with the substrate for the kinase or the substrate for TMP-PPase; The activity of the enzyme in the primary and secondary reaction mixture is determined, where the chemical is an enzyme if the activity of the bound enzyme in the secondary reaction mixture is significantly lower than the activity of the enzyme in the primary reaction mixture. Assay consisting of steps that can suppress activity. 22. A method of identifying a chemical to be tested for its ability to inhibit plant growth and viability, comprising: (a) a primary reaction under conditions in which an enzyme can catalyze the binding synthesis of TMP. H in the mixture
Enzyme having MP kinase activity and HMP-P kinase activity or TMP-P
Combining an enzyme with Pase activity with a substrate for HMP kinase and a substrate for TMP-PPase; (C) HMP-P kinase activity or TMP- in primary and secondary reaction mixtures
Determining the activity of the enzyme having PPase activity, and (d) determining the activity of the HMP-P kinase activity or the enzyme having TMP-PPase activity in the secondary reaction mixture as the HMP-P kinase activity or T
Selecting a chemical to be tested for its ability to inhibit plant growth or viability if the activity is significantly lower than the enzyme having MP-PPase activity. 23. The method according to claim 22, wherein the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant. 24. The enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1, or 23. The method of claim 22, having an amino acid sequence that is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. 25. The method of claim 22, wherein said substrate for HMP kinase is HMP. 26. The method of claim 22, wherein said substrate for TMP-PPase is THZ-P. 27. The method of claim 22, wherein the activity of an enzyme having HMP-P kinase activity or TMP-PPase activity is measured by measuring TMP produced in the reaction mixture. 28. An isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity,
23. A plant, plant cell, plant seed or plant tissue that is tolerant to a chemical identified by the method of claim 22 in an amount that normally inhibits HMP-P kinase activity or TMP-PPase activity in the plant. A plant, a plant cell, a plant seed or a plant tissue transformed with the DNA molecule imparted to the plant. 29. A plant comprising a nucleotide sequence which encodes an enzyme having HMP-P kinase activity and which renders the plant tolerant to a chemical identified by the method of claim 22. A plant obtained by a method comprising the step of transforming a plant or plant parent with an isolated DNA molecule capable of expressing a nucleotide sequence. 30. The following: (a) H in the primary reaction mixture under conditions where the enzyme can catalyze the conjugated synthesis of TMP.
Enzyme having MP kinase activity and HMP-P kinase activity or TMP-P
Combining an enzyme with Pase activity with a substrate for HMP kinase and a substrate for TMP-PPase; (b) removing the chemicals and enzymes in the secondary reaction mixture under the same conditions and for the same time as in the primary reaction mixture; (C) HMP-P kinase activity or TMP- in primary and secondary reaction mixtures
Determining the activity of an enzyme having PPase activity, wherein the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture is determined by determining the activity of HMP-P in the primary reaction mixture. An assay in which a chemical can inhibit the activity of an enzyme having HMP-P kinase activity or TMP-PPase activity when the activity is significantly lower than the activity of the enzyme having kinase activity or TMP-PPase activity. 31. A method for identifying a chemical substance to be tested for its ability to inhibit plant growth or viability, comprising: (a) a primary reaction under conditions in which an enzyme can catalyze the binding synthesis of TMP. T in the mixture
Enzyme having HZ kinase activity and HMP-P kinase activity or TMP-P
Combining an enzyme with Pase activity with a substrate for HMP-P kinase and a substrate for THZ kinase; (C) HMP-P kinase activity or TMP- in primary and secondary reaction mixtures
Determining the activity of the enzyme having PPase activity, and (d) determining the activity of the HMP-P kinase activity or the enzyme having TMP-PPase activity in the secondary reaction mixture as the HMP-P kinase activity or T
Selecting a chemical to be tested for its ability to inhibit plant growth or viability if the activity is significantly lower than the activity of an enzyme having MP-PPase activity. 32. The method according to claim 31, wherein the enzyme having HMP-P kinase activity or TMP-PPase activity is obtained from a plant. 33. The enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence that is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1, or 32. The method of claim 31, having an amino acid sequence that is identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. 34. The method of claim 3, wherein the substrate for HMP-P kinase is HMP-P.
The method of claim 1. 35. The method of claim 31, wherein the THZ kinase substrate is THZ. 36. The method of claim 31, wherein the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity is determined by determining the TMP produced in the reaction mixture. 37. A method of controlling unwanted vegetation growth, comprising:
Applying a chemical identified by the described method to the undesired vegetation. 38. An isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity and / or TMP-PPase activity, wherein the isolated DNA molecule comprises HMP-P kinase activity and / or TMP- DNA that confers to a plant, plant cell, plant seed or plant tissue tolerance to a chemical identified by the method of claim 31 in an amount that normally inhibits PPase activity.
Plant, plant cell, plant seed or plant tissue transformed with the molecule. 39. Includes a nucleotide sequence encoding an enzyme having HMP-P kinase activity and / or TMP-PPase activity, and
A method comprising transforming a plant or plant parent with an isolated DNA molecule capable of expressing a nucleotide sequence in a plant so as to render the plant tolerant to a chemical identified by the described method. The resulting plant. 40. The following: (a) T conditions in the primary reaction mixture under conditions where the enzyme can catalyze the binding synthesis of TMP.
Enzyme having HZ kinase activity and HMP-P kinase activity or TMP-P
Combining an enzyme having Pase activity with a THZ kinase substrate and a HMP-P kinase substrate, and (b) removing the chemicals and enzymes in the secondary reaction mixture under the same conditions and for the same time as in the primary reaction mixture. (C) HMP-P kinase activity or TMP- in primary and secondary reaction mixtures
Determining the activity of an enzyme having PPase activity, wherein the activity of the enzyme having HMP-P kinase activity or TMP-PPase activity in the secondary reaction mixture is determined by determining the activity of HMP-P in the primary reaction mixture. An assay in which a chemical can inhibit the activity of an enzyme having HMP-P kinase activity or TMP-PPase activity when the activity is significantly lower than the activity of the enzyme having kinase activity or TMP-PPase activity. 41. The method according to claim 41, wherein the chemical is HMP-P kinase activity or TMP
A method for identifying a chemical having herbicide activity that inhibits the ability to inhibit PPase activity, comprising: (a) encoding an enzyme having HMP-P kinase activity or TMP-PPase activity; Obtaining a transgenic plant, plant tissue, plant seed or plant cell comprising an isolated nucleotide sequence capable of over-expressing a highly active HMP-P kinase or TMP-PPase; (b) a transgenic plant, plant cell Applying the chemical to the transgenic and non-transformed plant, plant cell, plant cell, tissue or part, and to the isogenic transgenic and non-transformed plant, plant cell, Determining the growth or viability of the tissue; (d) transgenic and non-transformed plants, plant cells, groups after application of the chemical. Comparing the growth or viability of the tissue, wherein the chemical agent does not significantly inhibit the viability or growth of the isogenic non-transgenic plant, plant cell, tissue or part.
A method for inhibiting the viability or growth of a transgenic plant, plant cell, tissue or part. 42. The enzyme having HMP-P kinase activity or TMP-PPase activity is encoded by a nucleotide sequence that is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1. 42. The method of claim 41, wherein the method has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 2. 43. A method of controlling undesirable vegetation growth, comprising:
Or applying the chemical identified by the method of any of 41 to undesired vegetation. 44. An isolated DNA molecule comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity,
43. Tolerating the plant, plant cell, plant seed or plant tissue to a chemical identified by the method of claim 41 in an amount that normally inhibits HMP-P kinase activity or TMP-PPase activity in the plant. A plant, plant cell, plant seed or plant tissue transformed with the DNA molecule to be imparted. 45. A method comprising a nucleotide sequence encoding an enzyme having HMP-P kinase activity or TMP-PPase activity and rendering a plant tolerant to a chemical identified by the method of claim 41. Thus, a plant obtained by a method comprising the step of transforming a plant or plant parent with an isolated DNA molecule capable of expressing a nucleotide sequence in the plant. 46. A method for selectively inhibiting the growth of weeds in a field containing planted crop seeds or plant crops, comprising: (a) HMP-P kinase activity or TMP-PPase activity. Planting a herbicide-tolerant crop or crop seed which is a plant or plant seed transformed with an isolated DNA molecule comprising the nucleotide sequence having the nucleotide sequence having the nucleotide sequence capable of being expressed in the plant or plant seed. And (b) applying a herbicide to the field crop or crop seeds and weeds in an amount that inhibits native HMP-P kinase activity or TMP-PPase activity, wherein the herbicide is applied to the crop. A method for inhibiting weed growth without significantly inhibiting growth. 47. A shuffled DNA molecule, wherein said shuffled DN is
HM showing enhanced tolerance to herbicides that inhibit HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecule from which the A molecule is obtained
Shuffled DNA encoding PP kinase or TMP-PPase enzyme
. 48. A mutagenized DNA molecule obtained by shuffling a template DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity, wherein the mutagenized DNA molecule is encoded by the template DNA molecule. A mutagenized DNA molecule encoding an HMP-P kinase or TMP-PPase enzyme that exhibits enhanced tolerance to a herbicide that inhibits HMP-P kinase activity or TMP-PPase activity. 49. HMP-P kinase activity or T from a template DNA molecule encoding an enzyme having HMP-P kinase activity or TMP-PPase activity.
A method for producing a mutagenized DNA molecule encoding an enzyme having MP-PPase activity, comprising: (a) adding at least one single-stranded or double-stranded DNA to a resulting population of double-stranded random fragments; Adding a single-stranded oligonucleotide, wherein said oligonucleotide comprises a region identical to or heterologous to the template DNA molecule; (b) a single mixture of the resulting double-stranded random fragment and oligonucleotide (C) annealing the single-stranded fragment at the identity region to form a pair of annealed fragments, wherein the identity region is a member of one of the pairs that primes the other copy. The resulting population of single-stranded fragments is incubated with a polymerase under conditions sufficient to produce a mutagenized double-stranded polynucleotide. Is repeated at least two more times (d) is the second and third steps, but in this case, 2
The mixture resulting from the second round of the second step contains the mutagenized double-stranded polynucleotide from the previous third step and, when performed again, comprises a step that yields a further mutagenized double-stranded polynucleotide. The method, wherein the mutagenized double-stranded polynucleotide comprises a native H
A method encoding a mutated HMP-P kinase or TMP-PPase that exhibits enhanced tolerance to a herbicide that inhibits MP-P kinase activity or TMP-PPase activity. 50. The method of claim 49, wherein the one template DNA molecule is obtained from a eukaryote.
The described method. 51. The method of claim 49, wherein said eukaryote is a plant. 52. The method of claim 49, wherein said species of the template DNA molecule is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1. 53. The HMP-P kinase or TMP-protein, wherein the mutagenized DNA molecule exhibits enhanced tolerance to a herbicide that inhibits HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecule. 50. Mutagenized D encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of claim 49, which encodes a PPase enzyme.
NA molecule. 54. HMP-derived from at least two non-identical template DNA molecules encoding an enzyme having HMP-P kinase activity or TMP-PPase activity.
A method for producing a mutagenized DNA molecule encoding an enzyme having P-kinase or TMP-PPase activity, comprising: (a) at least one of the template DNA molecules comprising at least one region containing the same region. Adding two oligonucleotides to the template DNA molecule; (b) denaturing the resulting mixture into single-stranded molecules; and (c) combining the resulting population of single-stranded molecules with the oligonucleotide template DNA.
Incubate with the polymerase under conditions that result in annealing to the molecule, in which case the conditions for polymerization by the polymerase are that a polymerization product corresponding to a portion of the template DNA molecule is obtained, and ( d) repeating the second and third steps at least two more times, wherein the extension product obtained in the third step is switchable to a template DNA molecule for the next round of polymerization, Producing a mutagenized double-stranded polynucleotide comprising sequences derived from different template DNA molecules, wherein the mutagenized double-stranded polynucleotide comprises a template D
A method encoding an HMP-P kinase activity or a TMP-PPase enzyme that exhibits enhanced tolerance to a herbicide that inhibits the HMP-P kinase activity or TMP-PPase activity encoded by the NA molecule. 55. The method of claim 54, wherein the one template DNA molecule is obtained from a eukaryote. 56. The method of claim 54, wherein said eukaryote is a plant. 57. The method of claim 54, wherein said species of the template DNA molecule is identical or substantially similar to the nucleotide sequence set forth in SEQ ID NO: 1. 58. The HMP-P kinase or TMP-protein, wherein the mutagenized DNA molecule exhibits enhanced tolerance to a herbicide that inhibits HMP-P kinase activity or TMP-PPase activity encoded by the template DNA molecule. 55. A mutagenized DN encoding an enzyme having HMP-P kinase activity or TMP-PPase activity obtained by the method of claim 54, which encodes a PPase enzyme.
A molecule.