WO2002090596A1 - Methodes permettant d'identifier les inhibiteurs de l'expression ou de l'activite de la biotine synthase chez les plantes - Google Patents

Methodes permettant d'identifier les inhibiteurs de l'expression ou de l'activite de la biotine synthase chez les plantes Download PDF

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WO2002090596A1
WO2002090596A1 PCT/US2002/014473 US0214473W WO02090596A1 WO 2002090596 A1 WO2002090596 A1 WO 2002090596A1 US 0214473 W US0214473 W US 0214473W WO 02090596 A1 WO02090596 A1 WO 02090596A1
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plant
compound
candidate
herbicide
contacting
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PCT/US2002/014473
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Neil Hoffman
Jorn Gorlach
Douglas Boyes
Adel Zayed
Jeffrey Woessner
Carol Hamilton
Keith Davis
Robert Ascenzi
Kenneth Phillips
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Paradigm Genetics, Inc.
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Priority to EP02769381A priority Critical patent/EP1386008A4/fr
Publication of WO2002090596A1 publication Critical patent/WO2002090596A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates generally to plant molecular biology.
  • the invention relates to methods for the identification of herbicides.
  • Biotin synthase from plants has been fairly well characterized.
  • Biotin synthase (bioB, BIO2, BS) (EC 2.8.1.6) is involved in the conversion of dethiobiotin to biotin in bacteria, yeast, and higher plants. Bui et al., 440 FEBS LETT. 226-30 (1998) (PMID: 9862460); Baldet et al., 217 EUR. J. BiOCHEM 479-85 (1993) (PMID: 8223585); and Baldet et al., 319 CR ACAD. Sci. in 99-106 (1996) (PMID: 8680961).
  • the threonine- 173 residue which is highly conserved in biotin synthases, was farther shown to be important for catalytic competence of the enzyme through site-specific mutagenesis.
  • Weaver et al. 110 PLANT PHYSIOL. 1021-28 (1996) (PMID: 8819873).
  • the primary sequence of the Arabidopsis biotin synthase is most similar to biotin synthases from E. coli, Serratia marcescens, and Saccharomyces cerevisiae (about 50% sequence identity) and more distantly related to the Bacillus sphaericus enzyme (33% sequence identity).
  • BIO2 is a single-copy nuclear gene in Arabidopsis that is expressed at high levels in the tissues of immature plants. Expression of BIO2 was higher in the light relative to dark and was induced 5 -fold during biotin-limited conditions. These results demonstrate that expression of at least one gene in this pathway is regulated in response to developmental, environmental, and bio-chemical stimuli. Patton et al., 112 PLANT PHYSIOL. 371-78 (1996) (PMID: 8819333).
  • the purified A. thaliana bioB gene product is a homodimer (100 kDa) with a reddish color and has an absorbance spectrum characteristic of protein with [2Fe-2S] clusters.
  • the present inventors have discovered that antisense expression of a biotin synthase complementary DNA (BS cDNA) in Arabidopsis causes developmental abnormalities, and short and extremely stunted plant seedlings.
  • BS is essential for normal seed development and growth, and can be used as a target for the identification of herbicides.
  • the present invention provides methods for the identification of compounds that inhibit BS expression or activity, comprising: contacting a candidate compound with a BS and detecting the presence or absence of binding between the compound and the BS, or detecting a decrease in BS expression or activity. The methods of the invention are useful for the identification of herbicides.
  • FIG. 1 shows the biotin synthase reaction.
  • binding refers to a noncovalent interaction that holds two molecules together.
  • two such molecules could be an enzyme and an inhibitor of that enzyme.
  • Noncovalent interactions include hydrogen bonding, ionic interactions among charged groups, van der Waals interactions and hydrophobic interactions among nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other.
  • Biotin synthase (EC 2.8.1.6) is synonymous with “BS” and refers to an enzyme that catalyses the conversion of dethiobiotin and a sulfur donor to biotin, as shown in Fig. 1.
  • the cDNA (SEQ ID No. 1) encoding the BS polypeptide or protein (SEQ ID No. 2) is found herein as well as in the TIGR database at locus T 1024.10.
  • herbicide refers to a compound that may be used to kill or suppress the growth of at least one plant, plant cell, plant tissue or seed.
  • inhibitor refers to a chemical substance that inactivates the enzymatic activity of AS. The inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof.
  • a polynucleotide may be "introduced" into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection and the like.
  • the introduced polynucleotide may be maintained in the cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome.
  • the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.
  • the "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to the either the BLAST program (Basic Local Alignment Search Tool: Altschul & Gish, 266 METH. ENZYMOL. 460- 480 (1996); Altschul, 215 J. MOL. BIOL. 403-10 (1990)) in the Wisconsin Genetics Software Package (Devererreux et al., 12 NUCL. ACID RES. 387 (1984)), Genetics Computer Group, Madison, Wisconsin (NCBI, Version 2.0.11, default settings) or using Smith Waterman Alignment (Smith & Waterman, 2 ADV. APPL.
  • Plant refers to whole plants, plant organs and tissues (e.g., stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like) seeds, plant cells, and the progeny thereof.
  • polypeptide is meant a chain of at least four amino acids joined by peptide bonds.
  • the chain may be linear, branched, circular, or combinations thereof.
  • the polypeptides may contain amino acid analogs and other modifications, including, but not limited to glycosylated or phosphorylated residues.
  • binding refers to an interaction between BS and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of BS.
  • the present inventors have discovered that inhibition of BS gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that BS is a target for herbicides.
  • the invention provides methods for identifying compounds that inhibit BS gene expression or activity. Such methods include ligand-binding assays, assays for enzyme activity and assays for BS gene expression. Any compound that is a ligand for BS, other than its substrates, dethiobiotin and a sulfur donor, may have herbicidal activity.
  • ligand refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as herbicides.
  • the sulfur donor may be, but is not limited to, cysteine, sulfur, glutathione, N- acetyl cysteine, methionine, cystine, 4,4'-dithio-bis-morpholine, dithiodicaprolactam, alkylphenol disulfide, or amylphenol disulfide.
  • the sulfur donor is cysteine.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a BS with the compound; and b) detecting the presence and/or absence of binding between the compound and the BS, wherein binding indicates that the compound is a candidate for a herbicide.
  • BS is meant any enzyme that catalyzes the interconversion of dethiobiotin and a sulfur donor with biotin.
  • the BS may have the amino acid sequence of a naturally occurring BS found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence.
  • the BS is a plant BS.
  • plant BS an enzyme that can be found in at least one plant, and which catalyzes the interconversion of dethiobiotin and a sulfur donor with biotin.
  • the BS may be from any plant, including both monocots and dicots.
  • the BS is an Arabidopsis BS.
  • Arabidopsis species include, but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis griffithiana, Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii.
  • the Arabidopsis BS is from Arabidopsis thaliana.
  • the BS can be from barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solarium nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like.
  • barnyard grass Echinochloa crus-galli
  • crabgrass Digitaria sanguinalis
  • green foxtail Setana viridis
  • perennial ryegrass Lo
  • Fragments of a BS polypeptide may be used in the methods of the invention.
  • the fragments comprise at least 10 consecutive amino acids of a BS.
  • the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or at least 100 consecutive amino acids residues of a BS.
  • the fragment is from an Arabidopsis BS.
  • the fragment contains an amino acid sequence conserved among plant biotin synthases. Such conserved fragments have been previously reported. (Grima-Pettenuti et al., 21 PLANT MOL. BlOL. 1085-1095 (1993)). Those skilled in the art could identify additional conserved fragments using sequence comparison software.
  • Polypeptides having at least 80% sequence identity with a plant BS are also useful in the methods of the invention.
  • the sequence identity is at least 85%o, more preferably the identity is at least 90%, most preferably the sequence identity is at least 95% or 99%.
  • the polypeptide has at least 50%> of the activity of a plant BS. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant BS. Most preferably, the polypeptide has at least 50%, at least 60%, at least 70%, at least 80%> or at least 90% of the activity of the A. thaliana BS protein.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting the compound with at least one polypeptide selected from the group consisting of: a plant BS, a polypeptide comprising at least ten consecutive amino acids of a plant BS, a polypeptide having at least 85% sequence identity with a plant BS, and a polypeptide having at least 80% sequence identity with a plant BS and at least 50% of the activity thereof; and b) detecting the presence and/or absence of binding between the compound and the polypeptide, wherein binding indicates that the compound is a candidate for a herbicide.
  • any technique for detecting the binding of a ligand to its target may be used in the methods of the invention.
  • the ligand and target are combined in a buffer.
  • Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand- target complex or the detection of a change in the properties of a target when it is bound to a ligand.
  • an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a BS protein or a fragment or variant thereof, the unbound protein is removed and the bound BS is detected.
  • bound BS is detected using a labeled binding partner, such as a labeled antibody.
  • BS is labeled prior to contacting the immobilized candidate ligands.
  • Preferred labels include fluorescent or radioactive moieties.
  • Preferred detection methods include fluorescence correlation spectroscopy ("FCS") and FCS-related confocal nanofluorimetric methods.
  • a compound Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit BS enzyme activity.
  • the compounds can be tested using either in vitro or cell based enzyme assays.
  • a compound can be tested by applying it directly to a plant or plant cell, or expressing it therein, and monitoring the plant or plant cell for changes or decreases in growth, development, viability or alterations in gene expression.
  • the invention provides a method for determining whether a compound identified as a herbicide candidate by an above method has herbicidal activity, comprising: contacting a plant or plant cells with the herbicide candidate and detecting the presence or absence of a decrease in the growth or viability of the plant or plant cells.
  • decrease in growth is meant that the herbicide candidate causes at least a 10%) decrease in the growth of the plant or plant cells, as compared to the growth of the plants or plant cells in the absence of the herbicide candidate.
  • a decrease in viability is meant that at least 20% of the plants cells, or portion of the plant contacted with the herbicide candidate are nonviable.
  • the growth or viability will be at decreased by at least 40%. More preferably, the growth or viability will be decreased by at least 50%>, 75% or at least 90% or more. Methods for measuring plant growth and cell viability are known to those skilled in the art. It is possible that a candidate compound may have herbicidal activity only for certain plants or certain plant species.
  • BS catalyzes the irreversible or reversible reaction of dethiobiotin and a sulfur donor to biotin.
  • Methods for detection of dethiobiotin, a sulfur donor, and/or biotin include spectrophotometry, mass spectroscopy, thin layer chromatography ("TLC”) and reverse phase HPLC.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a dethiobiotin and a sulfur donor with BS; b) contacting the dethiobiotin and a sulfur donor with BS and the candidate compound; and c) determining the concentration of biotin after the contacting of steps (a) and (b).
  • a candidate compound inhibits BS activity, a higher concentration of the substrates (dethiobiotin and a sulfur donor) and a lower level of the product (biotin) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • the BS is a plant BS.
  • Enzymatically active fragments of a plant BS are also useful in the methods of the invention.
  • a polypeptide comprising at least 100 consecutive amino acid residues of a plant BS may be used in the methods of the invention.
  • a polypeptide having at least 80%, 85%>, 90%), 95%o, 98%o or at least 99%> sequence identity with a plant BS may be used in the methods of the invention.
  • the polypeptide has at least 80% sequence identity with a plant BS and at least 50%, 75%, 90% or at least 95% of the activity thereof.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting dethiobiotin and a sulfur donor with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant BS, a polypeptide having at least 80% sequence identity with a plant BS and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant BS; b) contacting the dethiobiotin and a sulfur donor with the polypeptide and the compound; and c) determining the concentration of biotin after the contacting of steps (a) and (b).
  • a candidate compound inhibits BS activity, a higher concentration of the substrate (dethiobiotin and a sulfur donor) and a lower level of the product (biotin) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • BS protein and derivatives thereof may be purified from a plant or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture.
  • these proteins are produced using a baculovirus or E. coli expression system.
  • Methods for the purification of biotin synthase have been described. Baldet et al., 319 CR ACAD. Sci. Ill 99-106 (1996) (PMID: 8680961).
  • Other methods for the purification of BS proteins and polypeptides are known to those skilled in the art.
  • the invention also provides plant and plant cell based assays.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the expression of BS in a plant or plant cell in the absence of the compound; b) contacting a plant or plant cell with the compound and measuring the expression of BS in the plant or plant cell; and c) comparing the expression of BS in steps (a) and (b).
  • a reduction in BS expression indicates that the compound is a herbicide candidate.
  • the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
  • BS expression of BS can be measured by detecting BS primary transcript or mRNA, BS polypeptide or BS enzymatic activity.
  • Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See e.g., Current Protocols in Molecular Biology, (Ausubel et al., eds., Greene Publishing and Wiley-Interscience) (1995). The method of detection is not critical to the invention.
  • Methods for detecting BS RNA include, but are not limited to amplification assays such as quantitative PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a BS promoter fused to a reporter gene, bDNA assays and microarray assays.
  • Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, His Tag and ELIS A assays, polyacrylamide gel electrophoresis, mass spectroscopy and enzymatic assays. Also, any reporter gene system may be used to detect BS protein expression.
  • reporter genes include, but are not limited to: chloramphenicol acetyltransferase (Gorman et al., 2 MOL. CELL BlOL. 1104 (1982); Prost et al., 45 GENE 107-111 (1986)); ⁇ -galactosidase (Nolan et al., 85 PROC. NAT. ACAD. SCI.
  • Chemicals, compounds, or compositions identified by the above methods as modulators of BS expression or activity can then be used to control plant growth.
  • compounds that inhibit plant growth can be applied to a plant or expressed in a plant, in order to prevent plant growth.
  • the invention provides a method for inhibiting plant growth, comprising contacting a plant with a compound identified by the methods of the invention as having herbicidal activity.
  • Herbicides and herbicide candidates identified by the methods of the invention can be used to control the growth of undesired plants, including both monocots and dicots.
  • undesired plants include, but are not limited to barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like.
  • Scotts Company or a similar soil mixture in an environmental growth room at 22°C, 65%> humidity, 65%) humidity and a light intensity of -100 ⁇ -E m "2 s "1 supplied over 16 hour day period.
  • the "Driver” is an artificial transcription factor comprising a chimera of the
  • DNA-binding domain of the yeast GAL4 protein (amino acid residues 147) fused to two tandem activation domains of herpes simplex virus protein VP16 (amino acid residues 413-490).
  • This chimeric driver is a transcriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35 S promoter.
  • the driver expression cassette is introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor are obtained.
  • a fragment, fragment or variant of an Arabidopsis thaliana cDNA corresponding to SEQ ID NO: 1 is ligated into the Pacl/Ascl sites of an
  • E.coli/Agrobacterium binary vector in the antisense orientation places transcription of the antisense RNA under the control of an artificial promoter that is active only in the presence of the driver transcription factor described above.
  • the artificial promoter contains four contiguous binding sites for the GAL4 transcriptional activator upstream of a minimal promoter comprising a TATA box.
  • the ligated DNA is transformed into E.coli. Kanamycin resistant clones are selected and purified. DNA is isolated from each clone and characterized by PCR and sequence analysis. The DNA is inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO:l. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus T 1024.10. The coding sequence for this locus is shown as SEQ ID NO: 1. The protein encoded by these mRNAs is shown as SEQ ID NO:2. The antisense expression cassette and a constitutive chemical resistance expression cassette are located between right and left T-DNA borders. Thus, the antisense expression cassettes can be transferred into a recipient plant cell by agroinfection.
  • the vector is transformed into Agrobacterium tumefaciens by electroporation.
  • Transformed Agrobacterium colonies are isolated using chemical selection.
  • DNA is prepared from purified resistant colonies and the inserts are amplified by PCR and sequenced to confirm sequence and orientation.
  • the antisense expression cassette is introduced into Arabidopsis thaliana wild-type plants by the following method. Five days prior to agroinfection, the primary inflorescence of Arabidopsis thaliana plants grown in 2.5 inch pots are clipped in order enhance the emergence of secondary bolts. At two days prior to agroinfection, 5 ml LB broth (10 g/L Peptone, 5 g/L
  • Yeast extract 5 g/L NaCl, pH 7.0 plus 25 mg/L kanamycin added prior to use
  • a clonal glycerol stock of Agrobacterium carrying the desired DNA The cultures are incubated overnight at 28°C at 250 rpm until the cells reached stationary phase.
  • 200 ml LB in a 500 ml flask is inoculated with 500 ⁇ l of the overnight culture and the cells are grown to stationary phase by overnight incubation at 28°C at 250 rpm.
  • the cells are pelleted by centrifugation at 8000 rpm for 5 minutes.
  • the supernatant is removed and excess media is removed by setting the centrifuge bottles upside down on a paper towel for several minutes.
  • the cells are then resuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and 250 ⁇ l/L SILWET L-77 (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16%) allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.
  • the previously clipped Arabidopsis plants are dipped into the Agrobacterium suspension so that all above ground parts are immersed and agitated gently for 10 seconds.
  • the dipped plants are then cover with a tall clear plastic dome in order to maintain the humidity, and returned to the growth room. The following day, the dome is removed and the plants are grown under normal light conditions until mature seeds are produced.
  • Mature seeds are collected and stored desiccated at 4 °C.
  • Transgenic Arabidopsis TI seedlings are selected. Approximately 70 mg seeds from an agrotransformed plant are mixed approximately 4:1 with sand and placed in a 2 ml screw cap cryo vial.
  • One vial of seeds is then sown in a cell of an 8 cell flat.
  • the flat is covered with a dome, stored at 4°C for 3 days, and then transferred to a growth room.
  • the domes are removed when the seedlings first emerged.
  • the flat is sprayed uniformly with a herbicide corresponding to the chemical resistance marker plus 0.005%> SILWET (50 ⁇ l/L) until the leaves are completely wetted. The spraying is repeated for the following two days.
  • the TI antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above.
  • the resulting FI seeds were then subjected to a PGI plate assay to observe seedling growth over a 2-week period. Seedlings were inspected for growth and development.
  • the transgenic plant line containing the antisense construct exhibited significant developmental abnormalities during early development. A clear 1:1 segregation ratio was observed in 2 antisense lines demonstrating that the antisense expression of the gene results in significantly impaired growth and represents an essential gene for normal plant growth and development.
  • Two transgenic lines containing the antisense construct for argininosuccinate synthase exhibited significant seedling abnormalities. Seedlings had chlorotic cotyledons, and red patches on the abaxial sides of the cotyledons.
  • BS gene can be cloned into E. coli (pET vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags. Evaluate the expression of recombinant protein by SDS-PAGE and Western blot analysis.
  • the enzymatic activity of BS may be determined in the presence and absence of candidate inhibitors in a suitable reaction mixture, such as described by the following known assay protocols and others known in the art:
  • This assay is based on the conversion of [ 14 C] dethiobiotin and a sulfur donor to [ 14 C] biotin. Birch et al., 275 J. Bio. CHEM. 32277-80 (2000).
  • This assay is based on the separation and quantitation of dethiobiotin and biotin. Azoulay et al., 26 J. CHROMATOGRAPHY 272-76 (1984).

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Abstract

Selon l'invention, la biotine synthase (BS) est essentielle à la croissance des plantes. D'une manière plus spécifique, l'inhibition de l'expression du gène BS dans les plantules produit une chlorose grave et une croissance réduite. De cette façon, la BS peut servir de cible pour l'identification des herbicides. En conséquence, la présente invention concerne des méthodes permettant d'identifier des composés qui inhibent l'expression ou l'activité de BS, et à ce titre, les méthodes selon l'invention conviennent pour l'identification des herbicides.
PCT/US2002/014473 2001-05-07 2002-05-07 Methodes permettant d'identifier les inhibiteurs de l'expression ou de l'activite de la biotine synthase chez les plantes WO2002090596A1 (fr)

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EP02769381A EP1386008A4 (fr) 2001-05-07 2002-05-07 Methodes permettant d'identifier les inhibiteurs de l'expression ou de l'activite de la biotine synthase chez les plantes

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014018775A1 (fr) * 2012-07-25 2014-01-30 Amvac Chemical Corporation Compositions herbicides dispersibles et procédés d'utilisation
US10512262B2 (en) 2012-07-25 2019-12-24 Amvac Chemical Corporation Dispersible herbicidal compositions and methods of use
US10531659B2 (en) 2012-07-25 2020-01-14 Amvac Chemical Corporation Dispersible herbicidal compositions and methods of use
US10575518B2 (en) 2012-07-25 2020-03-03 Amvac Chemical Corporation Dispersible herbicidal compositions and methods of use
US11576375B2 (en) 2012-07-25 2023-02-14 Amvac Chemical Corporation Dispersible herbicidal compositions and methods of use

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