WO2002096198A2 - Procedes pour identifier des inhibiteurs de l'expression ou de l'activite de la 3-oxo-5-alpha-steroide 4-deshydrogenase dans des plantes - Google Patents

Procedes pour identifier des inhibiteurs de l'expression ou de l'activite de la 3-oxo-5-alpha-steroide 4-deshydrogenase dans des plantes Download PDF

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WO2002096198A2
WO2002096198A2 PCT/US2002/016786 US0216786W WO02096198A2 WO 2002096198 A2 WO2002096198 A2 WO 2002096198A2 US 0216786 W US0216786 W US 0216786W WO 02096198 A2 WO02096198 A2 WO 02096198A2
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det2
plant
compound
candidate
herbicide
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PCT/US2002/016786
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WO2002096198A3 (fr
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Robert Ascenzi
Douglas Boyes
Neil Hoffman
Keith Davis
Adel Zayed
Rao Mulpuri
Jeffrey Woessner
Jorn Gorlach
Carol Hamilton
Kenneth Phillips
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Paradigm Genetics, Inc.
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Priority to EP02739465A priority Critical patent/EP1407270A4/fr
Priority to AU2002312111A priority patent/AU2002312111A1/en
Publication of WO2002096198A2 publication Critical patent/WO2002096198A2/fr
Priority to US10/717,844 priority patent/US20040265789A1/en
Publication of WO2002096198A3 publication Critical patent/WO2002096198A3/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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to plant molecular biology.
  • the invention relates to methods for the identification of herbicides.
  • 3-oxo-5-alpha-steroid 4-dehydrogenase (EC 1.3.99.5) is a 5-alpha steroid which is implicated in the biosynthetic pathway of brassinosteroids, and which catalyzes the conversion of (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl- 5alpha-choIestan-3-one.
  • Other names for 3-oxo-5-alpha-steroid 4-dehydrogenase include steroid 5-alpha-reductase, DEETIOLATED2, and DET2.
  • the Arabidopsis 3- oxo-5-alpha-steroid 4-dehydrogenase has been cloned and shown to encode a protein that shares approximately 40% sequence identity with mammalian steroid 5-alpha- reductases.
  • the present inventors have discovered that antisense expression of a DET2 cDNA in Arabidopsis causes developmental abnormalities. Seedlings exhibited significant abnormalities, including being smaller and more chlorotic than controls. Thus, the present inventors have discovered that DET2 is essential for normal seed development and growth, and can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit DET2 expression or activity, comprising: contacting a candidate compound with a DET2 and detecting the presence or absence of binding between said compound and said DET2, or detecting a decrease in DET2 expression or activity. The methods of the invention are useful for the identification of herbicides.
  • Fig. 1 shows the 3-oxo-5-alpha-steroid 4-dehydrogenase (DET2) 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.
  • NADP refers to nicotinamide adenine dinucleotide phosphate, oxidized form.
  • NADPH refers to nicotinamide adenine dinucleotide phosphate, reduced form.
  • 3-oxo-5-alpha-steroid 4-dehydrogenase (EC 1.3.99.5) is synonymous with “DET2", “DEETIOLATED2”, and “steroid 5-alpha- reductase”, and refers to an enzyme that catalyses the conversion of (24R)-24- methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one during the biosynthesis ofbrassinosteroids, as shown in Fig. 1.
  • DNA means deoxyribonucleic acid
  • RNA means ribonucleic acid
  • mRNA messenger ribonucleic acid
  • cDNA complementary deoxyribonucleic acid
  • HPLC high pressure liquid chromatography
  • TLC thin layer chromatography
  • ELISA enzyme-linked immunosorbent assay
  • PCR means polymerase chain reaction
  • SDS sodium dodecyl sulfate
  • SDS-PAGE means sodium dodecyl sulfate - polyacrylimide gel electrophoresis.
  • GUS means ⁇ -glucouronidase
  • PKI plant growth inhibition
  • Ni refers to nickel
  • Ni-NTA refers to nickel sepharose.
  • LB means Luria-Bertani media.
  • TATA box refers to a sequence of nucleotides that serves as the main recognition site for the attachment of RNA polymerase in the promoter region of eukaryotic genes. Located at around 25 nucleotides before the start of transcription, it consists of the seven-base consensus sequence TATAAAA, and is analogous to the Pribnow box in prokaryotic promoters.
  • 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 DET2. 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 and Gish (1996) Meth Enzymol 255:460-480 and Altschul (1990) JMolBiol 275:403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 72:387), Genetics Computer Group (GCG), Madison, Wisconsin.
  • Plant refers to whole plants, plant organs and tissues (e.g., stems, roots, ovules, stamens, leaves, embryos, n eristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like) seeds, plant cells and the progeny thereof.
  • plant organs and tissues e.g., stems, roots, ovules, stamens, leaves, embryos, n eristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like
  • 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.
  • specific binding refers to an interaction between DET2 and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of DET2.
  • the present inventors have discovered that inhibition of DET2 gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that DET2 is a target for herbicides.
  • the invention provides methods for identifying compounds that inhibit DET2 gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for DET2 gene expression. Any compound that is a ligand for DET2, other than its substrate, (24R)-24-methylcholest- 4-en-3-one, 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 invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a DET2 with said compound; and b) detecting the presence and/or absence of binding between said compound and said DET2; wherein binding indicates that said compound is a candidate for a herbicide.
  • DET2 is meant any enzyme that catalyzes the interconversion of (24R)- 24-methylcholest-4-en-3-one with (24R)-24-methyl-5alpha-cholestan-3-one.
  • the DET2 may have the amino acid sequence of a naturally occurring DET2 found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence.
  • the DET2 is a plant DET2.
  • the cDNA (SEQ ID NO:l) encoding the DET2 protein or polypeptide (SEQ ID NO:2) can be found herein as well as in the TIGR database at locus T8P21.4.
  • plant DET2 is meant an enzyme that can be found in at least one plant, and which catalyzes the interconversion of (24R)-24-methylcholest-4-en-3-one with (24R)-24-methyl-5alpha-cholestan-3-one.
  • the DET2 may be from any plant, including both monocots and dicots.
  • the DET2 is an Arabidopsis DET2.
  • 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 DET2 is from Arabidopsis thaliana.
  • the DET2 can be from 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.
  • barnyard grass Echinochloa crus-galli
  • crabgrass Digitaria sanguinalis
  • green foxtail Setana viridis
  • perennial ryegrass
  • Fragments of a DET2 polypeptide may be used in the methods of the invention.
  • the fragments comprise at least 10 consecutive amino acids of a DET2.
  • 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 DET2.
  • the fragment is from an Arabidopsis DET2.
  • the fragment contains an amino acid sequence conserved among plant 3-oxo-5-alpha-steroid 4-dehydrogenases. Such conserved fragments are identified in Grima-Pettenuti et al. (1993) Plant MolBiol 27:1085-1095 and Taveres et al. (2000), supra. Those skilled in the art could identify additional conserved fragments using sequence comparison software.
  • Polypeptides having at least 80% sequence identity with a plant DET2 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 DET2. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant DET2. 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 thaliana DET2 protein.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with at least one polypeptide selected from the group consisting of: a plant DET2, a polypeptide comprising at least ten consecutive amino acids of a plant DET2, a polypeptide having at least 85% sequence identity with a plant DET2, and a polypeptide having at least 80% sequence identity with a plant DET2 and at least 50% of the activity thereof; and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.
  • at least one polypeptide selected from the group consisting of: a plant DET2, a polypeptide comprising at least ten consecutive amino acids of a plant DET2, a polypeptide having at least 85% sequence identity with a plant DET2, and a polypeptide having at least 80% sequence identity with a plant DET2 and at least 50% of the activity thereof; and
  • 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 DET2 protein or a fragment or variant thereof, the unbound protein is removed and the bound DET2 is detected.
  • bound DET2 is detected using a labeled binding partner, such as a labeled antibody.
  • DET2 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. See http://www.evotec.de/technology.
  • a compound Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit DET2 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 said herbicide candidate and detecting the presence or absence of a decrease in the growth or viability of said 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%>.
  • 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.
  • DET2 catalyzes the irreversible reaction of (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one.
  • Methods for detection of (24R)-24-methylcholest-4-en-3-one, and/or (24R)-24- methyl-5alpha-cholestan-3-one 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 (24R)-24-methylcholest-4-en-3-one with DET2; b) contacting said (24R)-24-methylcholest-4-en-3-one with DET2 and said candidate compound; and c) determining the concentration of (24R)-24-methyl-5alpha- cholestan-3-one after the contacting of steps (a) and (b).
  • a candidate compound inhibits DET2 activity, a higher concentration of the substrate ((24R)-24-methylcholest-4-en-3-one) and a lower level of the product ((24R)-24-methyl-5alpha-cholestan-3-one) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • the DET2 is a plant DET2.
  • Enzymatically active fragments of a plant DET2 are also useful in the methods of the invention.
  • a polypeptide comprising at least 100 consecutive amino acid residues of a plant DET 2 may be used in the methods of the invention.
  • a polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99% sequence identity with a plant DET2 may be used in the methods of the invention.
  • the polypeptide has at least 80% sequence identity with a plant DET2 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 (24R)-24-methylcholest-4-en-3-one with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant DET2, a polypeptide having at least 80% sequence identity with a plant DET2 and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant DET2; b) contacting said (24R)-24-methylcholest-4-en-3-one with said polypeptide and said compound; and c) determining the concentration of (24R)-24-methyl-5alpha- cholestan-3-one after the contacting of steps (a) and (b).
  • a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant DET2, a polypeptide having at least 80% sequence identity with a plant DET2 and at least
  • a candidate compound inhibits DET2 activity, a higher concentration of the substrate ((24R)-24-methylcholest-4-en-3-one) and a lower level of the product ((24R)-24-methyl-5alpha-cholestan-3-one) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • DET2 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. Preferably these proteins are produced using a baculovirus or E. coli expression system. Methods for the purification of 3- oxo-5-alpha-steroid 4-dehydrogenase may be described in Ordman et al. (1991) J Steroid Biochem MolBiol 39:487-92 and in Quemener et al. (1994) Steroids 59:712- 18. Other methods for the purification of DET2 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 ofDET2 in a plant or plant cell in the absence of said compound; b) contacting a plant or plant cell with said compound and measuring the expression of DET2 in said plant or plant cell; c) comparing the expression of DET2 in steps (a) and (b).
  • a reduction in DET2 expression indicates that the compound is a herbicide candidate.
  • the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
  • DET2 can be measured by detecting the DET2 primary transcript or mRNA, DET2 polypeptide or DET2 enzymatic activity.
  • Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention.
  • Methods for detecting DET2 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 DET2 promoter fused to a reporter gene, DNA assays and microarray assays.
  • amplification assays such as quantitative PCR
  • hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a DET2 promoter fused to a reporter gene, DNA assays and microarray assays.
  • Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, His Tag and ELISA assays, polyacrylamide gel elecfrophoresis, mass spectroscopy and enzymatic assays.
  • any reporter gene system may be used to detect DET2 protein expression.
  • a polynucleotide encoding a reporter protein is fused in frame with DET2, so as to produce a chimeric polypeptide.
  • Methods for using reporter systems are known to those skilled in the art. Examples of reporter genes include, but are not limited to, chloramphenicol acetylrransferase (Gorman et al.
  • Chemicals, compounds or compositions identified by the above methods as modulators of DET2 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.
  • the plates transferred into a growth chamber with a day and night temperature of 22 and 20°C, respectively, 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 1-137) fused to two tandem activation domains of herpes simplex virus protein VP16 (amino acid residues 413-490). Schwechheimer et al. (1998) Plant Mol Biol 35:195-204.
  • This chimeric driver is a rranscriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35S promoter.
  • the driver expression cassette was introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor were obtained.
  • SEQ ID TMO:l was ligated into the Pacl/Ascl sites of an E.coli/Agrobacterium binary vector in the antisense orientation. This placed 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 was transformed into E.coli. Kanamycin resistant clones were selected and purified. DNA was isolated from each clone and characterized by PCR and sequence analysis. The DNA was inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO: 1. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus T8P21.4. 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 was transformed into Agrobacterium tumefaciens by electroporation. Transformed Agrobacterium colonies were isolated using chemical selection. DNA was prepared from purified resistant colonies and the inserts were amplified by PCR and sequenced to confirm sequence and orientation.
  • the antisense expression cassette was 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 were clipped in order enhance the emergence of secondary bolts.
  • 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) was inoculated with a clonal glycerol stock of Agrobacterium carrying the desired DNA.
  • the cultures were incubated overnight at 28°C at 250 rpm until the cells reached stationary phase.
  • 200 ml LB in a 500 ml flask was inoculated with 500 ⁇ l of the overnight culture and the cells were grown to stationary phase by overnight incubation at 28°C at 250 rpm.
  • the cells were pelleted by centrifugation at 8000 rpm for 5 minutes.
  • the supernatant was removed and excess media was removed by setting the centrifuge bottles upside down on a paper towel for several minutes.
  • the cells were then resuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and 250 ⁇ l L Silwet L-77TM (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.
  • the previously clipped Arabidopsis plants were dipped into the Agrobacterium suspension so that all above ground parts were immersed and agitated gently for 10 seconds.
  • the dipped plants were then covered with a tall clear plastic dome in order to maintain the humidity, and returned to the growth room. The following day, the dome was removed and the plants were grown under normal light conditions until mature seeds were produced. Mature seeds were collected and stored desiccated at 4 °C.
  • Transgenic Arabidopsis Tl seedlings were selected. Approximately 70 mg seeds from an agrotransformed plant were mixed approximately 4:1 with sand and placed in a 2 ml screw cap cryo vial. One vial of seeds was then sown in a cell of an 8 cell flat. The flat was covered with a dome, stored at 4°C for 3 days, and then transferred to a growth room. The domes were removed when the seedlings first emerged. After the emergence of the first primary leaves, the flat was sprayed uniformly with a herbicide corresponding to the chemical resistance marker plus 0.005% Silwet (50 ⁇ l/L) until the leaves were completely wetted. The spraying was repeated for the following two days.
  • Tl antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above.
  • the resulting Fl 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. Four often seedlings were smaller than controls and two of those were chlorotic. Thus, DET2 is essential for normal plant growth and development.
  • DET2 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 DET2 may be determined in the presence and absence of candidate inhibitors in a suitable reaction mixture, such as described by the following fluorescence assay.
  • An assay mixture is prepared containing 5 ug of cell lysate protein, 0.30 - 1.0 uM (24R)-24-methylcholest-4-en-3-one, 2 mM NADPH, 0.1% bovine serum albumine, 0.1 % NOG (n-octyl-beta-D-glucopyranoside) and 0.1 M sodium phosphate buffer pH 6.8.
  • This reaction mixture is incubated at 37 degrees Celsius for thirty minutes, then the optical density is read at a wavelength of 340 nm.
  • the fluorescence of the reaction can be monitored by utilizing wavelengths of 340 nm (for excitation) and 460 nm (for emission). Quemener et al. (1994) Steroids 59:712-18.

Abstract

Les inventeurs du présent procédé ont découvert que la 3-oxo-5-alpha-stéroïde 4-déshydrogénase (DET2) est essentielle pour la croissance des plantes. Plus précisément, des semis de plantes deviennent chlorosés et d'une taille anormalement raccourcie lorsqu'ils sont inhibés de l'expression du gène DET2. La DET2 peut ainsi être utilisée comme cible pour l'identification d'herbicides. Par conséquent, le procédé ci-décrit a pour objet d'identifier les composants qui inhibent l'expression ou l'activité de la DET2, et il consiste à: mettre un composé en contact avec la DET2 et à détecter la présence et/ou l'absence de liaison entre ledit composé et ladite DET2, ou à détecter une baisse d'expression ou d'activité de DET2. Les procédés de l'invention ci-décrite sont utiles à l'identification d'herbicides.
PCT/US2002/016786 2001-05-30 2002-05-30 Procedes pour identifier des inhibiteurs de l'expression ou de l'activite de la 3-oxo-5-alpha-steroide 4-deshydrogenase dans des plantes WO2002096198A2 (fr)

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EP02739465A EP1407270A4 (fr) 2001-05-30 2002-05-30 Procedes pour identifier des inhibiteurs de l'expression ou de l'activite de la 3-oxo-5-alpha-steroide 4-deshydrogenase dans des plantes
AU2002312111A AU2002312111A1 (en) 2001-05-30 2002-05-30 Methods for the identification of inhibitors of 3-oxo-5-alpha-steroid 4-dehydrogenase expression or activity in plants
US10/717,844 US20040265789A1 (en) 2002-05-30 2003-11-20 Methods for the identification of inhibitors of 3-oxo-5-alpha-steroid 4-dehydrogenase expression or activity in plants

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EP1407270A4 (fr) 2005-05-25
EP1407270A2 (fr) 2004-04-14
WO2002096198A3 (fr) 2004-02-12
AU2002312111A1 (en) 2002-12-09

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