JP2002525061A - Uracil permease from Arabidopsis as a herbicidal target gene - Google Patents

Abstract

  (57) [Summary] The present invention relates to a gene isolated from Arabidopsis that encodes a protein essential for seedling growth. The invention also includes methods of using this protein to discover new herbicides based on the essentials of normal growth and development. The invention can also be used in screening assays to identify inhibitors that are potential herbicides. The invention also applies to the development of herbicide-tolerant plants, plant tissues, plant seeds and plant cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Arabidopsis (encoding proteins essential for seedling growth) Arabidopsis )). The present invention is
Use proteins as herbicidal targets based on the essence of genes for length and development
Including the method. The present invention provides a process for identifying inhibitors that are potential herbicides.
Also useful as a cleaning assay. The present invention relates to herbicide-tolerant plants, plant tissues,
It can also be applied to the development of plant seeds and plant cells.

[0002] The use of herbicides to control unwanted weeds in crop lands has become an almost common practice. The herbicide market exceeds $ 15 billion each year. Despite this extensive use, weed control continues to be a major and costly problem for farmers. Effective use of herbicides requires sound management. For example, the time and method of application as well as the stage of weed development are critical to obtaining good weed control with herbicides.
As various weed species are resistant to herbicides, the production of effective new herbicides is becoming increasingly important. New herbicides can now be discovered using high-throughput screens performing recombinant DNA technology. Metabolic enzymes, which have been found to be essential for plant growth and development, can be produced recombinantly via standard molecular biology techniques and are screened for novel inhibitors of enzyme activity with herbicides Can be used as a target. New inhibitors discovered through such screens can then be used as herbicides to control unwanted plants.

[0003] Herbicides that exhibit greater potency, wider weed coverage, and faster degradation in soil also have, unfortunately, greater crop toxicity. One solution applied to this problem is to develop crops that are resistant or tolerant to herbicides.
Crop hybrids or varieties that are resistant to herbicides allow the use of herbicides to kill weeds without the associated risk of damage to the crop. The development of tolerance may allow the application of the herbicide to the crop if its use has previously been hindered or limited (eg, to pre-emergence use) by the sensitivity of the crop to the herbicide. For example, Anderson
U.S. Patent No. 4,761,373 relates to plants that are resistant to various imidazolinone or sulfonamide herbicides. Its resistance is conferred by an altered acetohydroxy acid synthase (AHAS) enzyme. No. 4,975,374 to Goodman et al. Describes a mutant glutamine synthetase (GS) resistant to inhibition by herbicides known to inhibit GS, such as phosphinothricin and methionine sulfoximine. A) a plant cell and a plant comprising the gene encoding No. 5,013,659 to Bedbrook et al. Relates to plants expressing a mutant acetolactate synthase that renders the plants resistant to inhibition by sulfonylurea herbicides. No. 5,162,602 to Somers et al. Discloses plants that are resistant to inhibition by cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. Its resistance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).

[0004] Despite the advances described above, there is a continuing and intermittent problem of unwanted or harmful plant growth (eg, weeds). Furthermore, as the artificiality continues to increase,
Food shortages will increase. Therefore, there is a long felt need for an unmet need to find new, effective, and economical herbicides. Definitions For clarity, certain terms used herein are defined and indicated below: A chimera is a DNA sequence, such as a vector or gene, of more than one of different origins fused together by recombinant DNA technology. Used to refer to the creation of a DNA sequence composed of DNA sequences that does not occur in nature.

[0005] Cofactor : A natural reactant such as an organic molecule or metal ion required in an enzyme-catalyzed reaction. Cofactors include, for example, NAD (P), riboflavin (
FAD and FNN), folic acid, molybdopterin, thiamine, biotin, lipoic acid, pantothenic acid and coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone, menaquinone. Optionally, the cofactor can be regenerated and reused.

[0006] DNA shuffling : DNA shuffling is a method of introducing mutations or rearrangements, preferably randomly, into DNA molecules or exchanging DNA sequences between two or more DNA molecules, preferably randomly. It is. The DNA molecule resulting from DNA shuffling is shuffled DNA, which is a non-natural DNA derived from at least one template DNA molecule. The shuffled DNA encodes an enzyme that is modified relative to the enzyme encoded by the template DNA, and preferably has an altered biological activity with respect to the enzyme encoded by the template DNA.

[0007] Enzymatic activity refers to the ability of an enzyme to catalyze the conversion of a substrate to a product. Substrates for an enzyme include the natural substrate for the enzyme, but also include analogs of the natural substrate that can also be converted into products or analogs of the product by the enzyme. The activity of the enzyme is measured, for example, by measuring the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time. The activity of the enzyme is determined by measuring the amount of unused cofactor of the reaction remaining in the reaction mixture after a certain period of time, or after a certain period of time, measuring the amount of used cofactor in the reaction mixture. It is also measured by The activity of the enzyme is determined by the free energy donor or energy-rich molecule (eg, A
By measuring the amount of TP, phosphoenol pyruvate, acetyl phosphate or phosphocreatine) or after a certain period of time the free energy donor or energy-rich molecule used in the reaction mixture (eg ADP, pyruvate, acetate) Or creatine).

[0008] Expression refers to the transcription and / or translation of an endogenous gene or transgene in a plant. In the case of an antisense construct, for example, expression may refer to transcription of only the antisense DNA. A gene refers to a coding sequence and related regulatory sequences, the coding sequence of which may be RNA, eg, mRNA, rRNA, tRNA, snRNA, sense R
Transcribed to NA or antisense RNA. Examples of regulatory sequences are promoter sequences,
5 'and 3' untranslated sequences. Further elements that may be present are, for example, introns.

The gene in question, when transferred to a plant, provides the plant with the required characteristics, such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide resistance, improvement. Any gene that confers an improved nutritional value, an improved ability in an industrial process, or an altered regeneration ability. A “gene of interest” may be one that is transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

Herbicides : Chemicals used to kill or inhibit the growth of plants, plant cells, plant seeds, or plant tissues. Heterologous DNA sequence : A DNA sequence that is not naturally associated with the introduced host cell, including unnatural multiple copies of the native DNA sequence. Homologous DNA sequence : A DNA sequence that is naturally associated with the introduced host cell.

Identity : Sequence identity (%) is determined using a computer program based on a kinetic programming algorithm. Preferred computer programs within the scope of the present invention include BLAST (Basi) designed to check all available sequence databases, whether protein or DNA.
c There is a Local Alignment Search Tool search program. This search tool version BLAST 2.0 (Gap
ped BLAST) is the Internet (currently http: //www.ncbi.nlm.nih.go)
v / BLAST /) for public use. It is the opposite of global alignment,
Use a heuristic algorithm that looks for locals, and therefore
Relationships in sequences sharing only isolated regions can be detected. BLA
The score assigned in the ST search is based on well-defined statistics.

Inhibitors : Chemicals that inactivate the enzymatic activity of proteins, such as biosynthetic enzymes, receptors, signaling proteins, structural gene products, or transport proteins that are essential for plant growth or survival. In context, an inhibitor is a chemical that inactivates 4788 enzyme activity from a plant. Isogenic : A plant that is genetically identical except that it may differ due to the presence or absence of the transgene .

Isolation : In the context of the present invention, an isolated DNA molecule or an isolated enzyme is
A DNA molecule or enzyme that exists apart from its native environment by the human hand and is therefore not a natural product. An isolated DNA molecule or enzyme may be present in purified form or may be in a non-native environment such as a transgenic host cell.

Marker gene : A gene encoding a selectable or screenable trait. Mature protein : A protein , such as chloroplast, that is usually targeted to cellular organelles from which temporary peptides are removed. Minimal promoter : A promoter element, particularly a TATA element, that is inactive or has significantly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.

Modified enzyme activity : those that are natural in plants that are resistant to inhibitors that inhibit the natural enzyme activity (ie, the natural enzyme activity without direct or indirect manipulation of such activity by humans). Different enzyme activities. Operably linked / ligated : A regulatory DNA sequence is "operably linked" to a DNA sequence that encodes an RNA or protein if the two sequences are located such that the regulatory DNA sequence affects the expression of the coding DNA sequence. "Or" connected ".

Plant refers to any plant, especially a seed plant. Plant cell : The structural and physiological unit of a plant, including protoplasts and cell walls. Plant cells may be in the form of isolated single cells or cultured cells, or as part of a highly organized unit such as a plant tissue or plant organ.

The plant material may be a leaf, stem, root, flower or flower part, fruit, pollen, pollen tube, ovule, embryo sac, egg cell, zygote, embryo, seed, cutting, cell or tissue culture, or plant Any other part or product. Preprotein: A protein that is targeted to cellular organelles such as chloroplasts and still contains its plasma peptide.

Recombinant DNA molecule : A combination of DNA sequences linked together using recombinant DNA technology. Recombinant DNA technology : see, for example, Sambrook et al. (1989, Cold Spring Harbor, NY: Col.
d The procedure used to ligate DNA together, as described in Spring Harbor Laboratory Press).

Selectable marker : A gene whose expression in a plant cell gives the cell a selective advantage. The selective advantage of cells transformed with a selectable marker gene may be due to the ability to grow in the presence of a negative selection agent, such as an antibiotic or herbicide, over the growth of non-transformed cells. The selective advantage of transformed cells over untransformed cells is that
It may also be due to an enhanced or novel ability to utilize compounds added as nutrients, growth factors or energy sources. Selectable marker genes also refer to genes and combinations of genes whose expression in a plant cell confers on the cell both negative and positive selective advantages.

Significant increase : an increase in enzyme activity that is greater than the error limits inherent in the assay technique, preferably about a two-fold or more increase, more preferably about a five-fold increase, over the activity of the wild-type enzyme in the presence of the inhibitor. Or more, and most preferably about a 10-fold or more increase. A significant decrease is that the amount of the product of the enzymatic reaction is greater than the error limit inherent in the assay technique, preferably about a two-fold or more decrease in the activity of the wild-type enzyme in the absence of the inhibitor, more preferably about a 5 fold or more reduction, and most preferably about 10
Means a fold or greater reduction.

In its broadest sense, the term “ substantially similar ” as used herein in reference to a nucleotide sequence refers to a polypeptide in which the corresponding sequence has substantially the same structure and function as the polypeptide encoded by the referenced nucleotide sequence. It means that only the nucleotide sequence corresponding to the quoted nucleotide sequence encoding the peptide, for example, an amino acid change that does not affect polypeptide function, occurs. Optionally, a substantially similar nucleotide sequence encodes a polypeptide encoded by the referenced nucleotide sequence. The term "substantially similar" is specifically intended to include nucleotide sequences whose sequences have been modified to optimize expression in a particular cell.
The percentage of identity between the substantially similar nucleotide sequence and the referenced nucleotide sequence may be at least 65%, more preferably at least 75%, preferably at least 85%, more preferably at least 85%. 90%, even more preferably at least 95%, even more preferably at least 99%. For sequence comparisons, use Smi
Performed using the th-Waterman sequence alignment algorithm (eg,
Waterman, MS, Introduction to Computational Biology: Maps, Sequence an
d 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 uses the following parameters: match: 1, mismatch penalty
: 0.33, open-gap penalty: 2, extended-gap penalty: 2. Nucleotide sequences that are "substantially similar" to the referenced nucleotide sequence are those that have 5
Wash with 2 × SSC at 0 ° C., 0.1% SDS and at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, more preferably 5 mM
Wash with 1 × SSC, 0.1% SDS at 0 ° C. and at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, more preferably 0.5 × at 50 ° C. Wash with SSC, 0.1% SDS and at 50 ° C. in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, preferably at 50 ° C. with 0.1 × SSC, 0.1% SDS. Wash and wash at 50 ° C. with 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA, more preferably at 65 ° C. with 0.1 × SSC, 0.1% SDS; Hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO ₄ , 1 mM EDTA.

The term “ substantially similar ” as used herein with respect to a protein, refers to a protein corresponding to a reference protein, eg, a polypeptide function, wherein the protein has substantially the same structure and function as the reference protein. Means that only the change in the amino acid sequence desired to be given occurs. When used for protein or amino acid sequences, the percentage of identity between substantially similar and referenced protein or amino acid sequences is preferably at least 65%, more preferably at least 75%, and preferably at least 85%. %, More preferably at least 90%, even more preferably at least 95%, even more preferably at least 99%.

Substrate : A substrate is a molecule that an enzyme recognizes and converts into a product in a biochemical pathway in which the enzyme naturally performs its function, or an enzymatic reaction that is recognized by an enzyme and mimics the natural reaction. Is a modified version of a molecule that is converted to a product by an enzyme. Tolerance : The ability to continue normal growth or function when exposed to an inhibitor or herbicide in an amount sufficient to suppress normal growth and function of native unmodified plants.

Transformation : A method for introducing heterologous DNA into cells, tissues, or plants. A transformed cell, tissue or plant is understood to include not only the end product of the transformation method, but also its transgenic progeny. Transgenic : Stably transformed with a recombinant DNA molecule preferably containing a suitable promoter operably linked to the DNA sequence in question.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING SEQ ID NO: 1 Genomic DNA sequence of the Arabidopsis 4788 gene. SEQ ID NO: 2 cDNA sequence of Arabidopsis 4788 gene. SEQ ID NO: 3 amino acid sequence for Arabidopsis 4788 protein. SEQ ID NO: 4 oligonucleotide SLP346for.

It is an object of the present invention to provide an effective and valuable method for identifying new herbicides. A feature of the present invention is the identification of a putative permease gene in Arabidopsis . Another feature of the present invention is the discovery that putative permease genes are essential for seedling growth and development.
An advantage of the present invention is that the newly discovered essential genes, including aspects of the function of the new herbicide, allow those skilled in the art to easily and quickly identify the new herbicide.

One object of the present invention is to provide essential genes in plants for the development of assays for inhibitory compounds with herbicidal activity. Genetic results indicate that if the putative permease gene is mutated in Arabidopsis, the resulting phenotype is homozygous and lethal seedlings. This suggests a critical role for the gene product encoded by the mutated gene.

Using T-DNA insertion mutagenesis, the inventors of the present invention have demonstrated that its activity is essential in Arabidopsis seedlings. This means that chemicals that inhibit protein function in plants appear to have detrimental effects on plants and are potentially excellent herbicidal candidates. Therefore, the present invention provides a method using the purified protein encoded by the gene sequence described below to identify the inhibitor. It is then used as a herbicide to control unwanted plant growth, e.g. on crop-growing lands, especially agriculturally important crops, e.g. corn and other cereals, e.g. wheat, oats, rye, sorghum, rice , Barley, millet, turf and forage grass, as well as land where cotton, sugar cane, sugar beet, rape and soybean grow.

The present invention discloses a novel nucleotide sequence from Arabidopsis called the 4788 gene. The nucleotide sequence of the genomic clone is shown in SEQ ID NO: 1, the nucleotide sequence of the corresponding cDNA clone is shown in SEQ ID NO: 2, and the amino acid sequence of Arabidopsis 4788 protein is shown in SEQ ID NO: 3. The invention also includes nucleotide sequences that are substantially similar to the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2. The invention also includes nucleotide sequences substantially similar to the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, which are plant nucleotide sequences. SEQ ID NO: 1 and SEQ ID NO: a nucleotide sequence substantially similar to the sequence set forth in 2 Arabidopsis thaliana (Arabidopsis thali ana) what is the nucleotide sequence are preferred.

Furthermore, nucleotide sequences substantially similar to the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the encoded protein has permease activity, are also included. More preferably, the nucleotide sequence is substantially similar to the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the encoded protein has purine or pyrimidine permease activity. Particularly preferred are nucleotide sequences substantially similar to the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, wherein the encoded protein has uracil permease activity. Also encompassed are amino acid sequences that include an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2. Amino acid sequences that include the amino acid sequence encoded by SEQ ID NO: 1 or SEQ ID NO: 2 are also included.

The present invention also includes a protein whose amino acid sequence is substantially similar to the amino acid sequence set forth in SEQ ID NO: 3. Also encompassed are amino acid sequences comprising SEQ ID NO: 3 and amino acid sequences substantially similar thereto. The amino acid sequence comprising SEQ ID NO: 3 and the amino acid sequence SEQ ID NO: 3 are preferred.

An amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the protein has permease activity is included. More preferred is an amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the protein has purine or pyrimidine permease activity. Particularly preferred is an amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the protein has uracil permease activity.

At least 2 of the amino acid sequence encoded by SEQ ID NO: 1 or SEQ ID NO: 2
It further includes amino acid sequences containing 0 consecutive amino acid residues. It further includes an amino acid sequence comprising at least 20 contiguous amino acid residues of the amino acid sequence of SEQ ID NO: 3. A further embodiment is a chimeric gene comprising a promoter operably linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2.

Furthermore, a recombinant vector comprising a chimeric gene comprising a promoter operably linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, which can be stably transformed into a host cell Things. Further, a host cell comprising a vector comprising a chimeric gene comprising a promoter operably linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the vector is stably transformed into the host cell. And those whose nucleotide sequence can be expressed in the cell.

[0035] Preferred host cells according to the invention are eukaryotic cells, more preferably insect cells,
A host cell selected from the group consisting of yeast cells and plant cells. Further preferred host cells are prokaryotic cells, with bacterial cells being more preferred. A plant comprising a vector comprising a chimeric gene comprising a promoter operably linked to a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the vector stably transforms plant cells. Also include plants that are capable of growing; also include the progeny and seeds of the plants, the seeds being optionally treated (eg, priced or coated) and / or packaged, eg, in a bag or other form with instructions for use. Put in a container. More preferred are plants according to the invention that are resistant to inhibitors of permease activity; also include progeny and seeds for the plants, the seeds optionally being treated (eg, priced or coated) and / or packaged And placed, for example, in a bag or other container with instructions for use.

A further embodiment of the present invention is a method for producing a nucleotide sequence encoding a gene product having altered permease activity, comprising: a) substantially as SEQ ID NO: 1 or SEQ ID NO: 2. Shuffling (mixing) similar nucleotide sequences; b) expressing the resulting shuffled nucleotide sequence; and c) selecting for altered permease activity relative to the permease activity of the gene product of the unmodified nucleotide sequence. It is a method including.

Preferred is a method according to the invention wherein the nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2. More preferred is a method according to the present invention wherein said permease activity is purine or pyrimidine permease activity. Particularly preferred is
The method according to the invention, wherein said permease activity is uracil permease activity.

Also included in the invention is a method for producing a nucleotide sequence encoding a gene product having altered permease activity, comprising: a) substantially as SEQ ID NO: 1 or SEQ ID NO: 2. Shuffling similar nucleotide sequences; b) expressing the resulting shuffled nucleotide sequences; and c) selecting for altered permease activity relative to the permease activity of the gene product of the unmodified nucleotide sequence. A shuffled DNA molecule obtainable by the method.

A further embodiment of the present invention is a shuffled DNA molecule produced by a method according to the present invention. Also included in the present invention are shuffled DNA molecules obtained by the method according to the present invention, which encode a gene product having increased resistance to inhibitors of permease activity.

A further embodiment of the present invention is a chimeric gene comprising a promoter operably linked to a shuffled DNA molecule produced by a method according to the present invention. A further embodiment of the present invention is a recombinant vector comprising a chimeric gene comprising a promoter operably linked to a shuffled DNA molecule produced by a method according to the present invention, wherein said vector is stably maintained in a host cell. It can be transformed.

[0041] Also included is a host cell comprising a vector according to the claims comprising a chimeric gene comprising a promoter operably linked to the shuffled DNA molecule produced by the method, wherein the host cell comprises A vector that can stably transform a host cell and whose nucleotide sequence can be expressed in said cell. Preferred are host cells according to the invention that are eukaryotic cells, more preferably said cells are selected from the group consisting of insect cells, yeast cells, and plant cells.

Also preferred are host cells according to the invention which are prokaryotic cells, more preferably host cells according to the invention which are bacterial cells. A further substantial form is a plant cell comprising a vector according to the invention comprising a chimeric gene comprising a promoter operably linked to a shuffled DNA molecule produced by a method according to the invention, wherein said vector is stable in plant cells. A plant whose nucleotide sequence can be expressed in said cell;
Also includes progeny and seeds for the plant, the seeds are optionally treated (e.g., priced or coated and / or packaged), e.g., placed in a bag or other container with instructions for use.

A further substantial form is a plant according to the invention that is resistant to inhibitors of permease activity; also includes progeny and seeds for the plant, the seed optionally being treated (eg, priced or Coated) and / or packaged and placed in a bag or other container, for example, with instructions for use.

A further embodiment is a method of identifying a compound having herbicidal activity, comprising: a) a protein comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2 And the compound to be tested for its ability to bind to the protein are combined under conditions conducive to binding; b) selecting the compound identified in step (a) that is capable of binding to said protein; c) selecting applying the compound identified in b) to the plant to be tested for herbicidal activity, and d) selecting a compound having herbicidal activity.

Also included are compounds having herbicidal activity that can be identified by the method according to the present invention. Also included are methods for identifying inhibitors of permease activity having herbicidal activity, comprising: a) combining permease and the compound to be tested for its ability to inhibit the activity of permease under conditions conducive to the inhibition; b. A) selecting the compound identified in step (a) capable of inhibiting said permease activity; c) applying the compound identified in step (b) to the plant to be tested for herbicidal activity; Selecting a compound having activity.

Included by the present invention is a method according to the present invention, wherein said permease is a purine or pyrimidine permease, more preferably said permease is uracil permease. A further embodiment of the present invention is a compound having herbicidal activity that can be identified by the method according to the present invention.

A further embodiment is a method of identifying an inhibitor of permease activity, comprising: a) replacing a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or substantially similar thereto with a permease-deficient host cell, such as E. coli uraA is introduced such that the sequence is functionally expressible; b) host cells containing the nucleotide sequence should be tested with a minimal inhibitory concentration of 5-fluorouracil and the ability to inhibit the activity of the permease C) measuring host cell growth under the conditions of step (b); and d) selecting said compound that inhibits host cell growth in step (c). It is a method that includes.

Also included are compounds having herbicidal activity identifiable by the method according to the invention. Further included is a method for inhibiting plant growth, wherein the plant comprises:
A compound that inhibits the activity of an amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2 is applied in an amount sufficient to inhibit the growth of said plant. It is a method including that.

Also included is a method of identifying a compound having herbicidal activity, comprising: a) combining the protein of claim 10 and the compound to be tested for its ability to bind to the protein under conditions conducive to binding. B) selecting the compound identified in step (a) that can bind to the protein; c) applying the compound identified in step (b) to the plant to be tested for herbicidal activity; and d) A method comprising selecting a compound having herbicidal activity; and a compound having herbicidal activity that can be identified by the method according to the present invention.

Also included is a herbicide-tolerant plant or seed which is resistant to a permease inhibitor selected from the group consisting of a plant or seed according to the present invention, A method comprising applying the compound in an amount that inhibits the growth of an unnecessary plant without significantly suppressing the growth of a drug-resistant plant or seed. The invention also includes methods of using the 4788 gene product as a herbicide target, based on the nature of the gene for normal growth and development. Further, the present invention can be used in screening assays to identify inhibitors that are potential herbicides.

In another preferred embodiment, the present invention provides a method for identifying a chemical capable of inhibiting 4788 activity in a plant, comprising: a) a non-native nucleotide sequence encoding an enzyme having 4788 activity. Obtaining a preferably stably transformed transgenic plant, plant tissue, plant seed or plant cell capable of overexpressing the enzymatically active 4788 gene product; b) the transgenic plant, plant Applying the chemical to the cells, tissues or parts or to the isogenic non-transformed plants, plant cells, tissues or parts; c) the growth of the transgenic and non-transformed plants, plant cells or tissues after application of the chemicals Or determining viability; d) transgenic and non-transformed plants, plants after application of the chemical Comparing cells, tissues; and e) increasing the viability or growth of non-transgenic plants, plant cells, tissues without significantly inhibiting the viability or growth of the isogenic transgenic plants, plant cells, tissues or parts. A method is described that preferably includes the step of selecting a chemical to suppress. In a preferred embodiment, the enzyme having 4788 activity is encoded by a nucleotide sequence from a plant, preferably Arabidopsis thaliana, optionally identical or substantially identical to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Similar to In another embodiment, the enzyme having 4788 activity is encoded by a nucleotide sequence that can encode the amino acid sequence of SEQ ID NO: 3. In yet another embodiment, 47
The enzyme having 88 activities has an amino acid sequence identical or substantially similar to the amino acid sequence set forth in SEQ ID NO: 3.

The present invention provides, in a further embodiment, a plant that has altered 4788 activity, and is therefore resistant to inhibition by herbicides at levels that normally inhibit native 4788 activity, Plant tissue, and plant cells; also includes progeny and seeds of the plant, wherein the seeds are optionally treated (eg, priced or coated) and / or packaged, eg, in a bag or bag with instructions for use. Put in another container. Herbicide-tolerant plants encompassed by the present invention include those that would otherwise be potential targets for the inhibition of herbicides, especially those crops of agricultural significance. According to this embodiment, a plant, plant tissue, plant seed,
Alternatively, the plant cell is capable of acting on a nucleotide coding sequence encoding a modified 4788 gene that is resistant to inhibition by a herbicide at a concentration that would normally inhibit the activity of the wild-type unmodified 4788 gene product. It is transformed, preferably stably transformed, with a recombinant DNA molecule containing a suitable promoter that functions in the ligated plant. The altered 4788 activity may be due to expression of the wild-type herbicide-sensitive 4788 protein by providing multiple copies of the wild-type 4788 gene to the plant or by over-expression of the wild-type 4788 gene under the control of a stronger promoter than the wild-type. Can also be given to plants by increasing the amount. The transgenic plant, plant tissue, plant seed, or plant cell thus formed is then selected by conventional selection techniques, whereby the herbicide-tolerant system is isolated, characterized and developed. You. Alternatively, random or site-directed mutagenesis can be used to create a herbicide-tolerant system.

Thus, the present invention provides a plant, plant cell or plant tissue transformed with a DNA molecule comprising a nucleotide sequence isolated from a plant encoding an enzyme having 4788 activity; Seeds, optionally treated (e.g., priced or coated) and / or packaged, e.g., placed in a bag or other container with instructions for use. Here, the enzyme has 4788 activity and the DNA molecule
In a quantity that normally inhibits 88 activity, it confers resistance to a herbicide on a plant, plant cell, plant seed, or plant tissue. According to an example of this embodiment, the enzyme having 4788 activity is encoded by a nucleotide sequence identical or substantially similar to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 3 Has the same or substantially similar amino acid sequence as the amino acid sequence described in (1).

The present invention also provides a method for inhibiting plant growth comprising applying to the plant a chemical that inhibits natural 4788 activity in the plant. In a related aspect, the invention provides a method for selectively inhibiting the growth of unwanted plants in planted crop seed crops or land containing plants, comprising: (a) herbicidal activity that inhibits natural 4788 activity. Optionally planting herbicide-tolerant crops or crop seeds that are plants or plant seeds that are resistant to the herbicide; Including applying in the amount
Here, the present invention relates to a method in which the herbicide suppresses the growth of weeds without significantly suppressing the growth of crops.

[0055] 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 the study of non-limiting examples. I. 478 in Arabidopsis demonstrated by T-DNA insertion mutagenesis
8 Gene Indispensability As shown in the examples below, the identification of novel gene structures and the essentiality of the 4788 gene for normal plant growth and development was first determined in Arabidopsis using T-DNA insertion mutagenesis. Proven. By establishing the essentiality of 4788 function in plants and identifying the gene that encodes this essential activity, we provide an important and in-demand tool for the development of new herbicides.

The Arabidopsis insertion mutant system isolated for the seedling lethal mutation is identified as the first step in identifying essential proteins. Starting with T2 seeds collected from a single T1 plant containing a T-DNA insertion in the genome, we identify these lines isolated from homozygous seedlings. These systems are found by placing seeds in minimal plant growth medium containing the fungicides benomyl and maxim and screening for non-viable seedlings after 7-14 days in the light at room temperature. Non-viable phenotypes include altered colors or altered forms. These phenotypes are observed directly on the plate or in soil after seedling transplantation.

If the system is identified as segregating seedling lethality, determine if the resistance marker in the T-DNA segregates the same with its lethality (Errampalti et al. (1991).
) The Plant Cell, 3: 149-157). Co-segregation analysis is performed by placing the seeds in a medium containing a selective agent and scoring the seedlings for resistance or sensitivity to the agent. Examples of selective agents used are hygromycin or phosphinothricin. Approximately 35 resistant seedlings are transplanted into soil and their progeny are tested for segregation of seedlings. If the T-DNA insertion disrupts an essential gene, there is a simultaneous segregation of the resistant phenotype and the seedling lethal phenotype in each plant. Therefore, in such cases, all resistant plants segregate seedlings and lethals in the next generation; this result indicates that each of the resistant plants is heterozygous for DNA causing both phenotypes.

For those systems that show a co-segregation of the T-DNA resistance marker and the seedling lethal phenotype, a Southern analysis is performed as the first step in characterizing the molecular properties of each insert. Southern analysis was performed on genome D isolated from heterozygotes.
This is performed using a probe capable of hybridizing with the T-DNA vector DNA together with NA. Often, the T-DNA insertion in a given plant is shown to contain multiple copies of the T-DNA inserted at a single genomic locus. The results of the Southern analysis are used to select suitable restriction enzymes for performing plasmid rescue in order to molecularly clone Arabidopsis genomic DNA flanking one or both sides of the T-DNA insert. Plasmids obtained in this way are analyzed by restriction enzyme digestion to classify the plasmids into classes based on their digestion pattern. The DNA sequence is determined for each class of plasmid clone. The resulting sequence is analyzed for the presence of a non-T-DNA vector sequence. If such sequences are found, they are called BLAST and BLAST2.
(Altschul et al. (1990), J. Mol. Biol. 215: 403-410; Altschul et al. (19)
97) Nucleic Acid Res 25: 3389-3402). Additional genomic and cDNA sequences for each gene are identified by standard molecular biology procedures.

II. Sequence of the Arabidopsis 4788 Gene The Arabidopsis 4788 gene was obtained from the tagged seedling lethal system # 4788 by T-DN.
It is sequenced by isolating the DNA adjacent to the A border. T-DNA
The Arabidopsis DNA adjacent to the border is identical to the internal region of the sequenced BAC of Arabidopsis (BAC T9 J22, chromosome 2). This BA
The C clone contains 115,851 bp of sequence, a very small portion of which is 478
It corresponds to a genomic region containing eight genes. Despite the BAC information, we first clearly established the entire gene sequence and first demonstrated that the 4788 gene product was essential for normal growth and development, Define the function of the gene product. The present invention discloses the nucleotide sequence of the Arabidopsis 4788 gene and the amino acid sequence of the Arabidopsis 4788 protein. The nucleotide sequence corresponding to the genomic clone is shown in SEQ ID NO: 1, the corresponding clone is shown in SEQ ID NO: 2, and the amino acid sequence encoding the mature protein is shown in SEQ ID NO: 3. The present invention relates to an isolated amino acid sequence derived from a plant, wherein the amino acid sequence is identical or substantially similar to the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and The amino acid sequence also includes an amino acid sequence having 4788 activity. B by default setting
Using the LAST and BLAST2 programs, the sequence of the 4788 gene shows similarity to uracil permease.

[0060] The 4788 gene is also a member of the gene family in Arabidopsis. This gene family consists of at least six members (Genbank Acc.
#s: AC002505 (2739376), BAC T9J22; AC004481 (3337350), BAC F13P17; AC001229
(2190545), BAC F5I14; AB009053 (n / a), clone MQB2 (13 ^th ORF); U83501 (1791
307); and AA712474 (EST clone 194H6T7) and AA605567 (EST clone 205J16XP
)).

III. Recombinant Production of 4788 and Use Thereof For the recombinant production of 4788 in a host organism, the nucleotide sequence encoding the 4788 protein is inserted into an expression cassette designed for the selected host, which is produced recombinantly. Introduce into host. Specific regulatory sequences suitable for the chosen host, such as promoters, signal sequences, 5 'and 3' untranslated sequences, and enhancers are within the skill of the art. The resulting molecule, containing the individual factors operably linked in the correct reading frame, can be inserted into a vector that can be transformed into a host cell. Suitable expression vectors and methods for recombinant production of proteins include host cells, such as E. coli, yeast, and insect cells (eg, Luckow and Summers, Bio / Technol. 6:47 (1988)), and baculovirus expression. Vectors are known, such as those derived from the genome of Autographa californica nuclear polynucleosis virus (AcMNPV). A preferred baculovirus / insect system is the linear autographa californica baculovirus DNA (Pharmigen, San Die
go, p used Sodoputera in the presence of a CA) · frugiperda a (Spodoptera fru giperda) Sf9 cells (ATCC) to transfect
AcHLT (Pharmingen, San Diego, CA). The resulting virus is Hi
gh Five Tricoplusia ni cells (Invitrogen, La Jolla,
CA).

In a preferred embodiment, the nucleotide sequence encoding the protein having 4788 activity is obtained from a eukaryote, such as a mammal, a fly or 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 shown in SEQ ID NO: 1 or SEQ ID NO: 2, or its amino acid is the same or substantially the same as the amino acid sequence shown in SEQ ID NO: 3. Encodes a protein having 4788 activity that is similar to The nucleotide sequence shown in SEQ ID NO: 2 has the amino acid sequence represented by SEQ ID NO:
In another preferred embodiment, encoding the Arabidopsis 4788 protein shown in Figure 3, the nucleotide sequence is obtained from a prokaryote, preferably a bacterium, such as the uraA gene in E. coli (Andersen et al. (1995) J. Bacteriol. 177:
2008-2013). Recombinantly produced 4788 is isolated and purified using various standard techniques. The actual techniques that can be used will vary depending on the host cell used, whether the protein is designed for secretion, and other similar factors known to those of skill in the art (eg, Ausubel, F. et al., “Current Protocols i
n Molecular Biology ”, pub by John Wiley & Sons, Inc. (1994) chapter 1
See 6).

Assays for Characterizing 4788 Protein Recombinantly produced 4788 protein serves a variety of purposes. For example, they can be used in in vitro assays to screen for well-known herbicide chemicals that have not been identified to determine if their target inhibits 4788. Such in vitro assays inhibit such enzyme activity,
Therefore, it can be used as a more general screen to identify chemicals that are new herbicide candidates. Alternatively, the recombinantly produced 4788 proteins may be used to elucidate the complex structure of these molecules and their association with well-known inhibitors to rationally design novel inhibitory herbicides and herbicide-resistant forms of enzymes. Can be used to further characterize gender. Nucleotide sequences substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2 and proteins substantially similar to SEQ ID NO: 3 from any source, including microbial sources, can be used in the assays exemplified herein. Can be used. Optionally, such nucleotide sequences and proteins are obtained from plants. More preferably, they are obtained from dicotyledonous plants. Alternatively, these nucleotide sequences and proteins are obtained from a non-corn source or a non-monocot source.

Assay for Permease Activity The 4788 gene product is a permease, more specifically a uracil permease similar to the maize Lpel protein (Schultes et al. (1996) The Plant Cell
, 8: 463-475). The maize leaf permease 1 gene product is known to have similarities to purine and pyrimidine permease from bacteria and fungi, and mutations in lpel are known to adversely affect chloroplast development. Therefore, lpel-encoded proteins are potential herbicide targets, which are further supported by the evidence herein of the essentiality of the Arabidopsis 4788 gene product. New assays can be developed based on the expression of plant proteins in bacterial hosts lacking the corresponding activity (Anders
en et al. (1995) J. Bacteriol. 177: 2008-2013).

Simple assays can be developed to screen for compounds that affect the normal function of the activity encoded by the plant. Such compounds promise an in vitro tade compound that can be tested for herbicidal activity in vivo. One assay consists of growing E. coli uraA , which has the 4788 gene and functionally expresses it, in minimal medium in the presence of a minimal inhibitory concentration of 5-fluorouracil. This is done in a 96-well format for automated high-throughput screening. Compounds that are effective at blocking the function of the 4788 protein inhibit the ability of cells to take up 5-FU, resulting in bacterial growth. This growth is measured by a simple turbidimetric method.

Other assays based on plant genes in corresponding bacterial variants have been described. However, in addition to being a novel herbicide target, a particular advantage of this assay is that, since uracil permease is expressed on the cell surface, compounds that are effective at inhibiting its function may be useful for their activity. It does not need to penetrate cells. This can be a major problem using the bacterial system as a model to find compounds that will be effective in plants. This is because many potential herbicides are known to lack activity on bacteria due to poor uptake of the compound by the bacteria.

In Vitro Inhibitor Assays: Finding Small Molecule Ligands That Interact with Proteins of Unknown Function Once a protein has been identified as a potential herbicidal target, the next step is to determine if it interacts with that protein To develop assays that allow screening of a large number of chemicals to do so. Developing assays for proteins of known function is straightforward, but developing assays for proteins of unknown function is more difficult.

To address this problem, we will examine new techniques that can detect the interaction between a protein and a ligand without knowing the biological function of the protein. A short description of three methods is provided, including fluorescence corrected spectroscopy, surface enhanced laser desorption / ionization, and biacore technology. More of these methods have been discovered and some may be acceptable for automated large-scale screening within the scope of this disclosure.

[0069] The theory of fluorescence-corrected spectroscopy (FCS) was developed in 1972, but the technique of performing FCS has only recently become available (Madge et al. (1972).
Phys. Rev. Lett., 29: 705-708; Maiti et al. (1997) Proc. Natl. Acad. Sci.
USA, 94: 11753-11757). FCS measures the average diffusivity of fluorescent molecules in a small sample volume. The sample size can be as low as 10 ³ fluorescent molecules and the sample volume is as small as the cytoplasm of a single bacterium. The diffusivity is a function of the mass of the molecule, which decreases with increasing mass. Therefore, FCS can be applied to protein-ligand interaction analysis by measuring the change in mass and therefore the diffusivity of the molecule upon binding.

Surface enhanced laser desorption / ionization (SELDI) was invented by Hutchens and Yib in the late 1980's (Hutchens and Yib (1993) Rapid Commun. Mass
Spectrom. 7: 576-580). When combined with time of flight mass spectrometry (TOF),
SELDI provides a means to rapidly analyze molecules retained on a chip. It can be applied to ligand-protein interaction analysis by covalently binding to a target protein on the chip, and small molecules retained by this protein can be analyzed by MS (Worrall et al. (1998) Anal. . Biochem. 70
: 750-756).

Biacore relies on changes in the refractive index at the surface layer due to binding of ligands to proteins immobilized on the layer. In this system, a collection of small ligands is continuously injected into a 2-5 μL cell with the immobilized protein. Binding is achieved by recording laser light refracting from the surface, resulting in surface plastin resonance (
SPR). In general, the refractive index change for a given change in mass concentration at the surface layer is, in fact, the same for all proteins and peptides and is applicable for any protein in a single way (Liedberg et al.). (1983
) Sensors Actuators. 4: 299-304; Malmquist (1993) Nature, 361: 186-18
7).

IV. In Vivo Inhibitor Assays In one embodiment, putative herbicides, such as those identified by in vitro screening, are applied to plants at various concentrations. The putative herbicide is preferably sprayed on the plant. After the application of the putative herbicide, the effect on the plant, for example the inhibition of death or growth, is recorded.

In another embodiment, an in vivo screening assay for an inhibitor of 4788 activity employs a transgenic plant, plant tissue, plant seed or plant cell capable of overexpressing a nucleotide sequence having 4788 activity.
Here, the 4788 gene product is enzymatically active in transgenic plants, plant tissues, plant seeds or plant cells. The nucleotide sequence is preferably
Obtained from eukaryotes, such as yeast, but preferably from plants. In a further preferred embodiment, the nucleotide sequence is identical or substantially similar to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or 4788.
Encodes an enzyme that has activity. Its amino acid sequence is identical or substantially similar to the amino acid sequence shown in SEQ ID NO: 3. In another preferred embodiment, the nucleotide sequence is obtained from a prokaryotic, preferably bacterial, eg, the uraA gene of E. coli.

Next, the chemical is applied to the transgenic plant, plant tissue, plant seed or plant cell, and to the isogenic plant, plant tissue, plant seed or plant cell, and the transgenic and non-transformed plant, The growth or viability of the plant tissue, plant seed or plant cell is determined and compared after application of the chemical. Compounds that can inhibit the growth of non-transgenic plants but do not affect the growth of the transgenic plants are selected as specific inhibitors of 4788 activity.

V. The present invention also relates to plants, plant tissues, plant seeds, and plant cells that are resistant to herbicides that inhibit native 4788 activity in these plants, wherein the resistance has been altered. Regarding what is given by the activity. 4788 changed
The activity can be determined by providing the plant with an additional wild-type 4788 gene, expressing a modified herbicide-tolerant 4788 gene in the plant, or by combining these techniques, to increase the expression of wild-type herbicide sensitivity 4788. It can be given to the plant according to the invention by increasing it. Representative plants include any plants to which these herbicides are applied for their usually intended purpose. Preferred are agriculturally important crops, such as cotton, soybean, rape, sugar beet, corn, rice, wheat, barley, oats, rye, sorghum, cereals (millet), turf, porridge, 4-fubras and the like. is there.

A. Increased Expression of Wild-Type 4788 Changing the 4788 activity through increased expression results in at least enough levels of 4788 in the plant cells to overcome the growth inhibition caused by the herbicide. The level of expressed enzyme is generally at least two times, preferably at least five times, and more preferably at least ten times the amount expressed natively. Increased expression may be due to multiple copies of the wild-type 4788 gene; multiple occurrences of coding sequences within the gene or mutations in non-coding regulatory sequences of endogenous genes in plant cells. Plants having such altered gene activity can be prepared by methods known in the art (eg, US Pat. Nos. 5,162,602 and 4,763).
No. 1,373 and their references) by direct selection in plants. These plants can also be obtained by genetic engineering techniques known in the art. A set of cells comprising a promoter capable of driving expression of a relevant structural gene in a plant cell operably linked to a homologous or heterologous structural gene encoding a plant 4788 protein with increased expression of a herbicide-sensitive 4788 gene. It can also be achieved by transforming with a recombinant or chimeric DNA molecule. Preferably, the transformation is stable, thereby providing a hereditary transgenic trait.

B. Expression of Modified Herbicide-Resistant 4788 Protein According to this embodiment, the plant, plant tissue, plant seed, or plant cell is 478
Transformants are stably transformed with a recombinant DNA molecule containing a suitable promoter that is functional in plants operably linked to a coding sequence encoding herbicide resistance.
The herbicide-resistant form of the enzyme has at least one amino acid substitution, addition or deletion that confers resistance to a herbicide that inhibits the unmodified native form of the enzyme. The transgenic plant, plant tissue, plant seed, or plant cell thus formed is then selected by conventional selection techniques, whereby a herbicide-tolerant system is isolated, characterized and developed. . The following describes a method for obtaining genes encoding 4788 herbicide-resistant forms: One general strategy involves direct or indirect mutagenesis in microorganisms. For example, a microorganism that can be genetically manipulated, such as E. coli or S. aureus. cerevisi
ae can be subjected to in vivo random mutagenesis with a mutagen such as UV light or ethyl or methyl methanesulfonate. Mutagenesis procedures are described, for example, in Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laborato
ry, Cold Spring Harbor, NY (1972); Davis et al., Advanced Bacterial Gen
etics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1980); Sh
erman 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 normally inhibitor sensitive 478
8 genes, depending on the activity provided by this gene. The mutagenized cells are grown in the presence of the inhibitor at a concentration that inhibits the unmodified gene.
Colonies of mutagenized microorganisms that grow better than non-mutagenized microorganisms in the presence of the inhibitor (ie, exhibit resistance to the inhibitor) are selected for further analysis. The 4788 genes from these colonies are isolated by cloning or by PCR amplification and their sequence elucidated. The sequences encoding the altered gene products are then cloned back into the microorganism to confirm their ability to confer inhibitor resistance.

A method for obtaining a mutant herbicide resistance allele of the plant 4788 gene involves direct selection by the plant. For example, the effect of the mutagenized 4788 gene on growth inhibitors of plants such as Arabidopsis, soybean, or maize can be determined by art-recognized methods on plates on simple minimal salt media containing increasing concentrations of the inhibitor. Determined by plating sterilized seeds.
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 per million (ppm). The lowest dose at which significant growth inhibition can be reproducibly detected is used for later experiments. Determination of the lowest dosage is routine in the art.

[0079] 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 including chemical or physical mutagenesis or seed, or chemical or physical mutagenesis or pollen (Neuffer In Maize for Biological Research Sheri).
dan, ed. Univ. Press. Grand Forks, ND., pp. 61-64 (1982))
Is used to fertilize plants, M ₁ mutant seeds obtained is collected. Taking Arabidopsis example, chemicals such as ethyl methanesulfonate, or physical agents, for example, gamma rays or fast neutrons are descendants seeds of plants grown from mutagenized seeds M ₂ seeds (Lehle Seeds, Tucson, AZ) are plated at a density of 10,000 seeds / plate (10 cm diameter up to 1) on minimal salt medium containing the appropriate concentration of inhibitor to select for resistance. Continue growing, 7-21 after plating Seedlings that remain green for days are transplanted into the soil and allowed to grow to maturity and set seeds.The progeny of these seeds are tested for herbicide resistance. There 3: 1 / resistance: plant for separating the sensitivity is estimated to be resistant heterozygous for at M ₂ generation.
All which resistance seed plant is estimated constant as resistant homozygous for at M ₂ generation. Such complete mutagenesis and screening of their M ₂ progeny seeds in seed, other species, (see for example U.S. Pat. No. 5,084,082) for example can also be carried out in soybean. Alternatively, mutant seeds to be screened for herbicide resistance are obtained as a result of fertilization with pollen mutated by chemical or physical means.

The confirmation of the herbicide tolerance gene base or the 4788 gene is confirmed as follows. Initially, 478 from plants showing resistance to the inhibitor
The alleles of the eight genes were based on the Arabidopsis cDNA coding sequence shown in SEQ ID NO: 2, or more preferably, PC with primers based on the unchanged 4788 gene sequence from the plant used to generate the resistance allele.
Isolated using R. After screening the allele to determine the presence of a mutation in the coding sequence, the allele is tested for the ability to confer resistance to the inhibitor on the plant in which the allele conferring putative resistance has been transformed. . These plants are Arabidopsis plants or their growth is 478
It can be any other plant that is susceptible to 8 inhibitors. Second,
The inserted 4788 gene has a known restriction fragment length polymorphism (RFLP).
s) (eg, Chang et al. Proc. Natl. Acad, Sci.
, USA 85: 6856-6860 (1988); Nam et al., Plant Cell 1: 699-705 (1989),
cleaved amplified polymorphic sequences (CAPS) (Konieczny and Ausubel (
1993) The Plant Journal, 4 (2): 403-410), or SSLPs (Bell and Ecker (1
994) Genomics, 19: 1377-144). 4788 inhibitor resistance properties are independently mapped using the same marker. If the resistance is due to a mutation in the 4788 gene, the resistance trait maps to a position that is indistinguishable from the position of the 4788 gene.

Another way to obtain the herbicide resistance allele of the 4788 gene is by selection in plant cell culture. Explants of plant tissue, such as embryos, leaf disks, etc., or callus or suspension cultures that grow to the activity of the plant in question, have increased concentrations of inhibitory herbicides or analog inhibitors suitable for use in a laboratory environment. Grow in medium in the presence.
Different degrees of growth are recorded in different cultures. In certain cultures, rapidly growing mutant colonies that continue to grow even in the presence of normal inhibitory concentrations of inhibitors are produced. The frequency of occurrence of such rapidly growing mutants can be increased by treatment with a chemical or physical mutagen prior to exposing the tissue or cell to the inhibitor. Alleles that confer the putative resistance of the 4788 gene are isolated and tested as described in the preceding paragraph. These alleles identified as conferring herbicide tolerance can then be manipulated for optimal expression and transformed into plants. Alternatively, plants can be regenerated from tissue or cell culture containing these alleles.

Still another method involves mutagenizing the 4788 gene of a wild-type herbicide-sensitive plant in bacteria or yeast, culturing the microorganism on a medium containing the next inhibitory concentration of the inhibitor, and growing in the presence of the next inhibitor. To select these colonies. More specifically, a plant cDNA, for example, an Arabidopsis cDNA encoding 4788, is cloned into a microorganism that otherwise lacks the activity of the selected gene.
The transformed microorganism is then subjected to any of several chemical or enzymatic methods known in the art, such as sodium bisulfite (Shortle et al., Meth.
ods Enzymol. 100: 457-468 (1983); methoxylamine (Kadonaga et al., N
ucleic Acids Res. 13: 1733-1745 (1985); Oligonucleotide-induced saturation mutagenesis (Hutchinson et al., Proc. Natl. Acad. Sci. USA, 83: 710-714 (1986))
Or various polymerase miss incorporation strategies (eg, Shor
tle et al., Proc. Natl. Acad. Sci. USA, 79: 1588-1592 (1982); Shiraish
i et al., Gene 64: 313-319 (1988); and Leung et al., Technique 1: 11-1.
5 (1989)) in vivo or in vitro mutagenesis. Colonies that grow in the presence of the usual inhibitory concentrations of the inhibitor are harvested and purified by subsequent retreaking. Those plasmids make them 478
Purified and tested for the ability to confer resistance to an inhibitor by re-transforming a microorganism lacking 8 gene activity. The DNA sequence of the cDNA insert from the plasmid passing this test is then determined.

The herbicide-tolerant 4788 protein can also be obtained using a method for in vitro recombination, also called DNA shuffling. By DNA shuffling, a mutation, preferably a random mutation, is introduced into the 4788 gene. DNA shuffling leads to the recombination and rearrangement of sequences within the 4788 gene, or the recombination and exchange of sequences between two or more different 4788 genes. These methods allow the production of millions of mutated 4788 genes. The mutated or shuffled genes are screened for the required properties, such as improved resistance to herbicides and for mutations that provide a wide range of resistance to different classes of inhibitor chemicals. Such screening is within the skill of the art.

In a preferred embodiment, the mutagenized 4788 gene is formed from at least one template 4788 gene, wherein the template 4788 gene has been cleaved into a double-stranded random fragment of the required size. And adding one or more single or double-stranded oligonucleotides to the resulting population of double-stranded random fragments, wherein said oligonucleotides are heterologous to the identified region and to the double-stranded random fragment. Denaturing the resulting mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; transforming the resulting population of single-stranded fragments in the same region as the polymerase with the single-stranded fragments To form a pair of the single-stranded fragments. Wherein the same region is sufficient for one member of the pair to prime the other's replication, thereby forming a mutagenized double-stranded polynucleotide; and Additional cycles, second
And repeating the third step, wherein the resulting mixture in the second step of the further cycle comprises the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and But also forms a mutagenized double-stranded polynucleotide, wherein the mutagenized polynucleotide has enhanced activity against a herbicide that inhibits native 4788 activity. In a preferred embodiment, the concentration of a single species of double-stranded random fragment in the population of double-stranded random fragments is less than 1% by weight of the 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 for at least 10 cycles. Such a method is described, for example, in Stemmer et al. (1994
) Nature 370: 389-391; U.S. Patent 5,605,793 and Crameri et al. (19)
98) Nature 391: 288-291, Cherry et al. (1999) Nature Biotechnology, 17
: 379-384, as well as WO 97/20078. These references are incorporated herein by reference.

In another preferred embodiment, any combination of two or more different 4788 genes can be used, for example, in Zhao et al. (1988) Nature Biotechnology 16: 258-261.
Staggered extension process (StE) as described in
P) is mutagenized in vitro. Two or more 4788 genes are PCR
It is used as a template for PCR amplification in the extension cycle of the reaction and is preferably performed at a temperature lower than the optimal polymerization temperature of the polymerase. For example, when using a thermostable polymerase at an optimum temperature of about 72 ° C, the temperature for the extension reaction is less than 72 ° C, more preferably less than 65 ° C, preferably 60 ° C.
C., more preferably the temperature for the extension reaction is 55.degree. In addition, the duration of the extension reaction of the PCR cycle may be shorter, if necessary, than is normally done in the art, more preferably less than 30 seconds, preferably less than 15 seconds, more preferably 5 minutes. Seconds. Short DNA
Only the fragments polymerize in each extension reaction, allowing template switching of the extension product between the starting DNA molecules after each cycle of denaturation and annealing, thereby creating diversity in the extension reaction. The optimal number of PCR reactions will depend on the length of the 4788 coding region to be mutagenized, but will be greater than 40 cycles, more preferably greater than 60 cycles, as needed.
Preferably more than 80 cycles are used. Optimal extension conditions and optimal number of PCR cycles for each combination of 4788 genes are determined as described using procedures known in the art. Other parameters for the PCR reaction are essentially the same as commonly used in the art. Primers for the amplification reaction are preferably designed to anneal to a DNA sequence located outside the coding sequence of the 4788 gene, for example, to the DNA sequence of a vector containing the 4788 gene, such that different 4788 genes used in the PCR reaction are used. However, a separate vector is preferably included. Primers were optionally 4788
Located less than 500 bp from the coding sequence, preferably less than 200 bp from the 4788 coding sequence, more preferably 72 bp from the 4788 coding sequence.
Less than 0 bp away. Preferably, the 4788 coding sequence is surrounded by restriction sites and included within the DNA sequence that is amplified during the PCR reaction, thereby facilitating cloning of the amplified product into a suitable vector.

[0086] In another preferred embodiment, a fragment of the 4788 gene with cohesive ends is produced as described in WO 98/05765. The sticky end is 47
A first oligonucleotide corresponding to a portion of the 88 gene to a second oligonucleotide not present in the gene or a second oligonucleotide corresponding to a portion of the gene not adjacent to the portion of the gene corresponding to the first oligonucleotide. Produced by linking to an oligonucleotide. Wherein the second oligonucleotide is at least 1
Of ribonucleotides. Double-stranded DNA is produced using a first oligonucleotide as a template and a second oligonucleotide as a primer. The ribonucleotide is cleaved and removed. The nucleotide located 5 'of the ribonucleotide is also removed, yielding a double-stranded fragment with sticky ends. Such fragments are reassembled randomly by ligation to yield new combinations of gene sequences.

[0087] Herbicide-resistant proteins can also be obtained using methods involving in-situ modification of target genes. Techniques for targeting and mutating genes in vivo can be used based on self-complementary chimeric oligonucleotides. This approach has been developed for the modification of endogenous genes in a site-specific and hereditary manner (Beet
Ham et al. (1999) Proc. Natl. Acad. Sci. 96: 8774-8778: U.S. Pat.
No. 6,325, US Pat. No. 5,871,984, US Pat. No. 5,731,18
No. 1). These references are incorporated herein by reference. Furthermore, a method is described for producing plants exhibiting agronomically required properties, including mutating or modifying genes in situ in plant cells (WO 98/54330).
). This reference is incorporated herein by reference. Such modifications are
It can be made through directed mutagenesis such as homologous recombination and is selected based on the resulting herbicide-tolerant phenotype (eg, Pazkowski et al., EMBO J. 7:40).
21-4026 (1988) and U.S. Pat. No. 5,487,992, especially columns 18-19.
And Example 18). These references are incorporated herein by reference.

[0088] Any 4788 gene or combination of 4788 genes can be used for in vitro recombination in the context of the present invention, for example, a plant such as Arabidopsis.
A 4788 gene derived from Thaliana, such as the 4788 gene shown in SEQ ID NO: 1 or SEQ ID NO: 2, a bacterium such as Bacillus caldolyticus (Ghim and Neuhard (1994) J. Bacteriol. 176:
3698-3707) or E. coli (Andersen et al. (1995) J. Bacteriol. 177: 2008-2)
013), 4788 gene from Zea mays (Schultes
(1996) The Plant Cell, 8: 463-475). All of which are incorporated herein by reference. The entire 4788 gene or a part thereof is used in the present invention. Library of 4788 genes mutations obtained by the method described above are cloned have suitable expression vector, resulting vector is transformed into a suitable host, for example, algae such as Chl Amydomonas, yeast or bacteria . Suitable hosts are preferably those that would otherwise lack 4788 gene activity, such as the E. coli uraA mutant (Andersen et al. (1995) J. Bacteriol. 177: 2008-2013).
It is. Vector-transformed host cells containing a library of mutated 4788 genes are cultured on a medium containing an inhibitory concentration of the inhibitor, and colonies that grow in the presence of the inhibitor are selected. Colonies that grow in the presence of the usual inhibitory concentrations of the inhibitor are picked and purified by repeated restreaking. The plasmids are purified and the DNA sequence of the cDNA insert from the plasmid that passes this test is then selected.

Assays for identifying modified 4788 genes that are resistant to inhibitors can be performed similarly to assays for identifying inhibitors of 4788 activity with the following modifications (inhibitor assays, supra). : First, mutant 4788 was substituted in one of the reaction mixtures for wild-type 4788 of the inhibitor assay. Second, inhibitors of host-type enzymes are present in both reaction mixtures. Third, a mutated activity (activity in the presence of an inhibitor and a mutated enzyme) and a non-mutated activity (activity in the presence of an inhibitor and a wild-type enzyme), Observed in mutant activity when compared. Mutation activity is any measure of the activity of the mutant enzyme in the presence of a suitable substrate and inhibitor. Non-mutant activity is any measure of the activity of the wild-type enzyme in the presence of a suitable substrate and inhibitor. A significant increase is defined as an increase in enzyme activity that is greater than a margin of error inherent in the measurement technique, preferably at least a 2-fold or more increase in the activity of the wild-type enzyme in the presence of the inhibitor, more preferably The increase is at least 5-fold or more, most preferably at least 10-fold or more.

In addition to being used to create herbicide-tolerant plants, the gene encoding herbicide-tolerant 4788 can be used as a selectable marker in plant cell transformation methods. For example, a transgene-transformed plant, plant tissue, plant seed,
Alternatively, plant cells can be transformed with a gene encoding an altered 4788 that the plant can express. 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. Here, only the transformed cells will survive. The method can be applied to any plant cell that can be transformed with the modified 4788 coding gene and can use any transgene of interest.
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.

VI. Plant Transformation Techniques The wild-type or herbicide-resistant form of the 4788 gene can be incorporated into plant or bacterial cells using conventional recombinant DNA techniques. Generally, this involves using standard cloning procedures well known in the art to insert the DNA molecule encoding 4788 into an expression system in which the DNA molecule is heterologous (ie, usually not present).
The vector contains the necessary elements for transcription and translation of the inserted protein coding sequence in a host cell containing the vector. Many vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. The components of the expression system can also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications can be used. Essentially any crop cell can be transformed under suitable conditions using expression systems known in the art. A transgene containing a wild-type or herbicide-resistant form of the 4788 gene is preferably stably transformed and integrated into the genome of the host cell. In another preferred embodiment,
The transgene containing the wild-type or herbicide-resistant form of the 4788 gene is located on a self-replicating vector. An example of a self-replicating vector is a virus, especially a geminivirus. The transformed cells can be regenerated into whole plants so that the selected form of the 4788 gene confers herbicide resistance on the transgenic plants.

A. Requirements for the Production of a Plant Expression Cassette Gene sequences intended for expression in transgenic plants are first assembled in an expression cassette behind a suitable promoter that can be expressed in the plant. The expression cassette may also include any additional sequences required or selected for expression of the transgene. Such sequences include, but are not limited to, transcription terminators, external sequences to enhance expression, such as introns, essential sequences, and sequences intended for targeting gene products to particular organelles and cellular components. . These expression cassettes can then be easily transferred to the plant transformation vectors described above. The following describes 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 selected promoter will express the transgene in a particular cell type (eg, leaf epidermal cells, mesophyll cells, root cortex cells) or a particular tissue or organ (eg, root, leaf or flower), and the selection may be Will reflect the required location of gene product accumulation. Alternatively, the selected promoter can drive the expression of the gene under various inducing conditions. Promoters vary in their strength, ie, in their ability to promote transcription. Depending on the host cell system utilized, any one of several suitable promoters known in the art can be used. For example, for constitutive expression, the CaMV35S promoter, rice actin promoter, or ubiquitin promoter can be used. For regulatable expression, chemically inducible PR- from tobacco or arabitopsis
One promoter can be used (see, eg, US Patent No. 5,689,044).

[0094] 2. Transcription terminators Various transcription terminators are available for use in expression cassettes. These are responsible for the termination of transcription over the transgene and its correct polyadenylation. Suitable transcription terminators are those known to function in plants, and include the CaMV35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator.
These can be used in both monocots and dicots.

[0095] 3. Sequences for enhancing or regulating expression Numerous sequences have been found to increase gene expression within transcription units, and these sequences may be used to increase their expression in transgenic plants. Can be used in conjunction with For example, various intron sequences, such as those in the maize Adhl gene, have been shown to enhance expression, especially in monocots. In addition, some untranslated leader sequences from the virus are also known to enhance expression, which are particularly effective in dicotyledonous cells.

[0096] 4. Coding sequence optimization The coding sequence of a selected gene can be engineered by changing the coding sequence for optimal expression in the crop species in question. Methods for modifying coding sequences to achieve optimal expression in particular crop species are known (eg, Perlak et al., Proc. Natl Acad. Sci. USA 88: 3324).
(1991); and Koziel et al., Bio / technol. 11: 194 (1993)).

[0097] 5. Targeting gene products in cells Various mechanisms for targeting gene products are known to exist in plants, and the sequences that control the functionality of these mechanisms have been characterized in some detail. 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 create the mature protein (see, for example, Comai et al., J. Am. 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 target heterologous gene products to these organelles. Furthermore,
Sequences that cause targeting of the gene product to other cellular components have also been characterized. Amino-terminal sequences are responsible for targeting to extracellular secretions from ER, apoplast and aleurone cells (Koehler & Ho, Plan
t Cell 2: 769-783 (1990). Furthermore, amino terminal sequences combined with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al., Plant
Molec. Biol. 14: 357-368 (1990)). By fusing the suitable targeting sequence described above to the transgene sequence in question, it is possible to direct the transgene product to any organelle or cellular component.

B. Construction of Plant Transformation Vectors A number of transformation vectors available for plant transformation are available from plant transformation technology.
Genes known to those of ordinary skill in the
It can also be used in combination with a tar. Selection of the vector depends on the preferred transformation technique.
It will depend on the target species for surgery and transformation. For a particular target species,
A different antibiotic or herbicide selection marker may be provided. Conventionally used for transformation
Selectable markers confer resistance to kanamycin and related antibiotics mptII Gene (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al.
., Nature304 : 184-187 (1983)), conferring tolerance to the herbicide phosphinothricin
barGene (White et al., Nucl. Acids Res18 : 1062 (1990), Spencer et
 al., Theor. Appl. Genet79 : 625-631 (1990)), the antibiotic hygromycin
Gives resistance tohphGene (Blochinger & Diggelmann, Mol Cell BiolFour : 2929-2931) and resistance to methotrexatedhfrgene(
Bourouis et al., EMBO J.2 (7) : 1099-1104 (1983)) and glyphosate
EPSPS gene that confers resistance to infection (U.S. Pat.
8, 642).

1. Many vectors for transformation using Agrobacterium (Agrobacterium) vector suitable for transformation Agrobacterium tumefaciens (Agrobacterium tu mefaciens) are available.
These typically have at least one T-DNA border sequence and have a pBIN
19 (Bevan, Nucl. Acids Res (1984)) and vectors such as pXYZ.
Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, and the binary vector pCIB2001.
There are IB10 and its selective derivatives of hygromycin (eg, US Pat. No. 5,633).
9, 949).

[0100] 2. Vectors Suitable for Non-Agrobacterium Transformations Transformation without Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the selected transformation vectors and results in the absence of these sequences. Such vectors can be used in addition to vectors such as those described above containing a T-DNA sequence. Agrobacterium-free transformation techniques include particle bombardment, protoplast uptake (eg, PE
G and electroporation) and transformation via microinjection. The choice of vector depends largely on the preferred choice for the species to be transformed. Exemplary vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG19, and pSOG35 (see, eg, US Pat. No. 5,639,949).

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

[0102] Transformation techniques for dicots are known in the art and include Agrobacterium-based techniques and techniques that do not require Agrobacterium. Non-Agrobacterium technology involves the direct uptake of exogenous genetic material by protoplasts or cells. This can be done by PEG or electroporation mediated uptake, particle bombardment mediated delivery, or microinjection. In each case, the transformed cells are regenerated into whole plants using standard techniques well known in the art. Transformation of most monocot species is now routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation, particle bombardment into callus tissue, and Agrobacterium-mediated transformation.

VII. Breeding The wild type or mutated forms of the present invention can be utilized to confer herbicide tolerance on a wide variety of plant cells, including those of gymnosperms, monocots, and dicots. Genes can be inserted into any of these broad classes of plant cells, but can be from crop cells, especially rice, wheat, barley, rye, corn, potatoes, carrots, sweet potatoes, sugar beets, beans, peas, and lilies. , Lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, carrot, pumpkin (Squash, pumpkin), zucchini, cucumber, apple, pear, quince, melon, Plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado,
Particularly useful in papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane. The high level expression of the wild-type 4788 gene and the expression of the herbicide-resistant form of the 4788 gene that confers herbicide resistance in plants, combined with other features important for production and quality, are well known in breeding approaches and in the art. And can be incorporated into plant systems via techniques.

If the herbicide-resistant 4788 gene allele is obtained by direct selection in the crop or by plant cell culture capable of regenerating the crop, it is not necessary to engineer the allele and transform the plant with it Become a commercial variety using traditional breeding techniques to develop herbicide-tolerant crops. The present invention will be further described by reference to the following detailed examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated.

EXAMPLES Standard recombinant DNA and molecular cloning techniques used herein are known in the art and are described in Sambrook, et al., Molecular Cloning , eds., Cold Spring Harbor Labo.
ratory Press, Cold Spring Harbor, Ny (1989), TJSilhavy, MLBerman, an
d LWEnquist, Experiments with Gene Fusions , Cold Spring Harbor Laborat
ory, Cold Spring Harbor NY (1984), Ausubel, FM et al., Current Protocols in Molecular Biology , pub. by Greene Publishing Assoc. and Wiley- Interscience (1987), Reiter et al., Methods in Arabidopsis Research , Wor
ld Scientific Press (1992), and Schultz et al., Plant Molecular Biology Manual , Kluwer Academic Publishers (1998). These references describe all steps in tagging and cloning genes from the T-DNA mutagenized population of Arabidopsis: plant infection and transformation; screening for identification of seedling variants; co-segregation analysis; and plasmids Describes the standard techniques used for rescue.

Example 1 Sequence Analysis of Lethal Line # 4788 from a Tagged Seedling-Arabidopsis T-DNA Mutagenized Population The Arabidopsis Genome Flanked on One or Both Sides of a T-DNA Insert Resulting from T-DNA Mutagenesis Plasmid rescue technology is used to molecularly clone DNA. Plasmids obtained in this manner are analyzed by restriction digestion to separate plasmids into classes based on their digestion pattern. For each class of plasmid clones, the resulting sequences that determine the DNA sequence are analyzed for the presence of non-T-DNA vector sequences. Plasmids recovered from the plasmid rescue protocol are sequenced using the slp346 primer. Primer slp346 informs the flanking sequence immediately adjacent to the left T-DNA border. Plasmid rescue is demonstrated by PCR of genomic DNA from heterozygotes for the 4788 mutation. This PCR experiment uses the primers immobilized within the predicted flanking sequence and the slp346 primer (immobilized in the T-DNA insert). Reasonable rescue is confirmed by finding a PCR product of the expected size based on the sequence of the plasmid rescue clone.

The sequences obtained from the above clones are used for NCBIWWW blastn searches against nucleotide sequence databases (Altschul et al. (1990) J. Mol. Bi
ol. 215: 403-410: Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402)
. The search results indicate that the recovered sequence is Arabidopsis chromosome II BACT9J
22 (Genbank Acc. AC002505 (2739376)). The region of the genomic DNA where the insertion has taken place is annotated as encoding a putative permease (ACCESSION # 2 739 376). Next, primers are designed for the 5 'and 3' ends of the expected mRNA, and PCR is performed using DNA from an Arabidopsis cDNA library as a template. The obtained PCR product was TA-linked and cloned (Original TA Cloning Kit,
Invitrogen) and sequence. The cDNA sequence is the same as the sequence predicted by Genbank annotation, thereby first confirming the putative open reading frame annotation.

Example 2 Expression of Recombinant 4788 Protein Consisting of Escherichia coli The coding region of the putative mature protein corresponding to the cDNA clone is subcloned into the above expression vector and transformed into E. coli using the manufacturer's conditions. . Specific examples include plasmids such as pBluescript (Stratagene
, La Jolla, CA), pFLAG (International Biotechndogies, Inc., New Hav
en, CT), and pTrcHis (Invitrogen, La Jolla, CA).

Example 3: In vitro recombination of the 4788 gene by DNA shuffling A. encoding the 4788 protein The Thaliana 4788 gene is amplified by PCR. The resulting DNA fragment is essentially described (Stemmer et al. (19
94) Digest by DNase I treatment as in PNAS 91: 10747-10751) and remove the PCR primers from the reaction mixture. Perform a PCR reaction without primers,
Next, a PCR reaction is performed with the primers. Both listed (Stemmer et al. 1994) PNAS
91: 10747-10751). The obtained DNA fragment was ligated with pTRC99a
(Pharmacia, Cat no: 27-5067-01) and E. coli uraA strain (Anders) by electroporation using Biorad Gene Pulser and manufacturer conditions.
(1995) Journal of Bacteriol. 177 (8): 2008-2013).
The transformed bacteria are grown on a medium containing an inhibitory concentration of the inhibitor, and colonies that grow in the presence of the inhibitor are selected. Colonies that grow in the presence of the usual inhibitory concentrations of the inhibitor are picked and purified by repeated restreaking, the plasmids are purified, and then the DNA sequence of the cDNA insert from the plasmid passing this test is determined. I do. In a similar reaction, A. The PCR-amplified DNA fragment containing the Thaliana 4788 gene and the PCR-amplified DNA fragment containing the E. coli uraA gene are recombined in vitro and the resulting mutants with improved resistance to the inhibitor are recovered as described above.

Example 4: 47 by the Staggered extension method
In vitro recombination of 88 genes Thaliana 4788 gene and E. coli ur
The aA gene is each cloned into the polylinker of the pBluescript vector. “Reverse primer” and “M13-20 primer” (Stratage
(Nata Catalog) (Zhao et al. (1998) Nature
Biotechnology 16: 258-261). The amplified PCR fragment is digested with appropriate restriction enzymes, cloned into pTRC99a, and the mutated 4788 gene is screened as described in Example 3.

Example 5 In Vitro Binding Assay Recombinant 4788 protein is obtained, for example, according to Example 2. The protein is immobilized on a chip suitable for a ligand binding assay. The proteins immobilized on the chip are exposed to sample compounds in solution according to methods known in the art. The sample compound is contacted with the immobilized protein and a measurement is taken that can detect protein-ligand interactions. Examples of such measurements are SELDI, biacore and FLC described above. Compounds found to bind to proteins are immediately discovered in this manner and are subjected to further characterization, for example, according to Example 6 below.

Example 6: Assay for uracil permease activity A simple assay is developed to screen for compounds that affect the normal functionality of 4788 activity. Such compounds promise an in vitro lead compound that can be tested for herbicidal activity in vivo. The assay is 4
E. coli uraA carrying the 788 gene and functionally expressing it is grown in minimal medium in the presence of a minimal inhibitory concentration of 5-fluorouracil. This is done in a 96-well format for automated high-throughput screening. Compounds that are effective to block the function of the 4788 protein are 5
-Inhibits the ability to ingest FU and causes bacterial growth. This growth is measured by simple turbidimetric means. More particularly, the invention relates to a method of identifying an inhibitor of permease activity, comprising the steps of: a) replacing SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence substantially similar thereto, with a permease-deficient host cell, such as E. coli; introducing the sequence into uraA such that the sequence is functionally expressible; b) testing the host cell containing the nucleotide sequence for a minimal inhibitory concentration of 5-fluorouracil and the ability to inhibit the activity of the permease. C) measuring host cell growth under the conditions of step (b); and d) selecting a compound that inhibits host cell growth in step (c). And, optionally, e) applying the compound identified in step (d) to the plant to be tested for herbicidal activity, and It said method comprising selecting a compound which has a herbicidal activity at f) Step (e).

The above embodiments are illustrative. The disclosure of the present invention will give those skilled in the art many variations of the invention. All such obvious and predictable variations are intended to be within the scope of the appended claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12Q 1/02 4H011 9/00 1/25 C12Q 1/02 C12R 1:91) 1/25 C12N 15/00 ZNAA // (C12N 5/10 5/00 B C12R 1:91) C (81) Designated country 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, A T, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, 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 Badtsitsszewski, Gregory Joseph Joseph United States, North Carolina 27705, Durham, Englewood Avenue 2016 (72) Inventor Ward, Eric Russell United States of America, North Carolina 27707, Durham, Bentley Dora Eve 3761 (72) Inventor Burnasconi, Paul United States, North Carolina 27514, Chapel Hill, Windhover Drive 102 F-term (reference) 2B030 AA02 AB03 AD05 CA06 CA17 CA19 CB03 CD14 4B024 AA07 AA08 BA07 CA04 DA01 DA02 DA05 DA12 EA01 EA02 EA04 FA02 GA11 HA11 4B050 CC01 CC03 DD13 LL10 4B063 QA01 QA08 QQ05 QQ21 QR01 QR57 QR59 QR74 QR80 QS05 QS36 QX01 4B065 AA01X AA72X AA88X AA88Y AA90X AB01 BA01 CA27 CA47 CA53 4H011 AB04 BB19 DH11

Claims (42)

[Claims] 1. A nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2. 2. The nucleotide sequence according to claim 1, which encodes an amino acid sequence substantially similar to SEQ ID NO: 3. 3. The nucleotide sequence according to claim 1, wherein said nucleotide sequence is a plant nucleotide sequence. 4. The nucleotide sequence according to claim 1, wherein said protein has permease activity. 5. The nucleotide sequence according to claim 4, wherein said protein has purine or pyrimidine permease activity. 6. An amino acid sequence comprising an amino acid sequence encoded by a nucleotide sequence substantially similar to SEQ ID NO: 1 or SEQ ID NO: 2. 7. An amino acid comprising an amino acid sequence substantially similar to SEQ ID NO: 3. 8. The amino acid sequence according to claim 6, wherein the protein has permease activity. 9. The amino acid sequence according to claim 8, wherein the protein has purine or pyrimidine permease activity. 10. An amino acid sequence comprising at least 20 contiguous amino acid residues of the amino acid sequence encoded by SEQ ID NO: 1 or SEQ ID NO: 2. 11. An amino acid sequence comprising at least 20 consecutive amino acid residues of the amino acid sequence of SEQ ID NO: 3. 12. A chimeric gene comprising a promoter operably linked to the nucleotide sequence according to claim 1. 13. A recombinant vector comprising the chimeric gene according to claim 12, wherein the vector is capable of stably transforming a host cell. 14. A host cell comprising the vector of claim 13, wherein said host cell is capable of expressing said nucleotide sequence in said cell. 15. The host cell according to claim 14, wherein said host cell is a eukaryotic cell. 16. The host cell according to claim 15, wherein said host cell is selected from the group consisting of an insect cell, a yeast cell, and a plant cell. 17. The host cell according to claim 15, wherein said host cell is a prokaryotic cell. 18. A plant or seed comprising the plant cell according to claim 16. 19. The plant according to claim 18, wherein said plant is resistant to an inhibitor of permease activity. 20. A method for making a nucleotide sequence encoding a gene product having altered permease activity, comprising: a) shuffling the nucleotide sequence of claim 1; b) the resulting shuffled nucleotide sequence. And c) selecting for altered permease activity relative to the permease activity of the gene product of the unmodified nucleotide sequence. 21. The method of claim 20, wherein said nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 2. 22. The method of claim 20, wherein said permease activity is purine or pyrimidine permease activity. 23. A shuffled DNA molecule obtainable by the method of claim 20. 24. A shuffled DNA molecule obtainable by the method of claim 20, wherein the shuffled DNA molecule encodes a gene product having increased resistance to inhibitors of permease activity. 25. A chimeric gene comprising a promoter operably linked to the nucleotide sequence according to any one of claims 23 to 24. 26. A recombinant vector comprising the chimeric gene according to claim 25, wherein the vector is capable of stably transforming a host cell. 27. A host cell comprising the vector of claim 26, wherein said nucleotide sequence is capable of being expressed in said cell. 28. The host cell according to claim 27, wherein said host cell is a eukaryotic cell. 29. The host cell according to claim 27, wherein said host cell is selected from the group consisting of an insect cell, a yeast cell, and a plant cell. 30. The host cell according to claim 27, wherein said host cell is a prokaryotic cell. A plant or seed comprising the plant cell according to claim 29. 32. The plant according to claim 31, wherein said plant is resistant to an inhibitor of permease activity. 33. A method for identifying a compound having herbicidal activity, comprising: a) combining the protein of claim 6 and a compound to be tested for its ability to bind to said protein under conditions conducive to binding; b. A) selecting the compound identified in step (a) that is capable of binding said protein; c) applying the compound identified in step (b) to the plant to be tested for herbicidal activity; Selecting a compound having activity. 34. A compound having herbicidal activity that can be identified by the method according to claim 33. 35. A method for identifying an inhibitor of permease activity having herbicidal activity, comprising: a) combining permease and a compound to be tested for its ability to inhibit the activity of permease under conditions conducive to the inhibition; b. A) selecting the compound identified in step (a) capable of inhibiting said permease activity; c) applying the compound identified in step (b) to the plant to be tested for herbicidal activity; and d) Selecting a compound having herbicidal activity. 36. The method of claim 35, wherein said permease is a purine or pyrimidine permease. 37. A compound having herbicidal activity that can be identified by the method of claim 35. 38. A method for identifying an inhibitor of permease activity, comprising: a) transferring SEQ ID NO: 1 or SEQ ID NO: 2, or a nucleotide sequence substantially similar thereto, to a permease deficient host cell, such as E. coli uraA . B) introducing a host cell containing said nucleotide sequence into a host cell containing said nucleotide sequence with a minimal inhibitory concentration of 5-fluorouracil and the ability to inhibit the activity of said permease. C) measuring host cell growth under the conditions of step (b); and d) selecting a compound that inhibits host cell growth in step (c). . 39. A compound having a herbicidal activity that can be identified by the method according to claim 38. 40. A method for inhibiting the growth of a plant, which comprises adding the compound that inhibits the activity of the amino acid sequence according to claim 6 to the plant in an amount sufficient to inhibit the growth of the plant. A method that includes applying at. 41. The method of claim 40, wherein said compound is selected from the group consisting of the compound of claim 37 and the compound of claim 39. 42. A method for improving a crop, comprising the steps of providing a plant or seed according to claim 19 and a herbicide-tolerant plant or seed selected from the group consisting of the plant or seed according to claim 32. 34, a compound having herbicidal activity selected from the group consisting of the compound according to claim 37 and the compound according to claim 39, without significantly suppressing the growth of herbicide-tolerant plants or seeds. A method comprising applying in an amount that inhibits unwanted plant growth.