WO1992004456A1 - Synthase recombinee de l'enzyme acc (acide 1-aminocyclopropane-1-carboxylique) - Google Patents

Synthase recombinee de l'enzyme acc (acide 1-aminocyclopropane-1-carboxylique) Download PDF

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WO1992004456A1
WO1992004456A1 PCT/US1991/006453 US9106453W WO9204456A1 WO 1992004456 A1 WO1992004456 A1 WO 1992004456A1 US 9106453 W US9106453 W US 9106453W WO 9204456 A1 WO9204456 A1 WO 9204456A1
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acc synthase
acc
sequence
plant
dna
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PCT/US1991/006453
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Athanasios Theologis
Takahide Sato
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Priority to JP3515173A priority Critical patent/JPH06502759A/ja
Priority to AU85114/91A priority patent/AU657276B2/en
Priority to CA002091243A priority patent/CA2091243C/fr
Publication of WO1992004456A1 publication Critical patent/WO1992004456A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8249—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving ethylene biosynthesis, senescence or fruit development, e.g. modified tomato ripening, cut flower shelf-life
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)

Definitions

  • the invention relates to the plant enzyme ACC syn ⁇ thase which is essential for the production of ethylene in higher plants. More particularly, the invention concerns recombinant methods and materials for the production of this enzyme and their use in controlling plant develop ⁇ ment, and in particular, plant genescence.
  • the enzyme ACC synthase is essential to the produc ⁇ tion of ethylene in higher plants. It is well known that ethylene is related to various events in plant growth and development including fruit ripening, seed germination, abscission, and leaf and flower senescence.
  • Ethylene production is strictly regulated by the plant and is induced by a variety of external factors, including the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought, and ions such as cadmium and lithium ions.
  • auxins including the application of auxins, wounding, anaerobic conditions, viral infection, elicitor treatment, chilling, drought, and ions such as cadmium and lithium ions.
  • control of the level of this enzyme permits control of ethylene levels and thus regulation of the plant growth and development aspects that are controlled by ethylene.
  • the availability of the ACC synthase gene, as provided by the invention herein, permits the construc ⁇ tion of recombinant materials which permit such regula ⁇ tion.
  • the availability of ACC synthase makes possible large-scale production of an ethylene precursor useful in industrial production of this chemical and its products, such as ethanol.
  • Van Der Straeten, D., et al. reported the cloning and sequences of cDNAs encoding ACC synthase from tomato (Proc Natl Acad Sci USA (1990) 82:4859-4863). Although the cDNA, which corresponded to an open reading frame of approximately 55 kd, produced a 55 kd peptide in E. coli, it is not clear from the data provided that this protein represents ACC synthase. Disclosure of the Invention The invention provides recombinant materials and techniques which permit control of the level of ACC synthase in plants and portions thereof and also provides methods for large scale nonpetroleum-dependent production of ethylene.
  • the recovery of a cDNA encoding the ACC synthase of zucchini provides access to recombinant materials corresponding to alternate ACC synthases from zucchini as well as the range of ACC synthases in higher plants. This permits the control of plant development and activity in a wide variety of plant materials of commer- cial interest.
  • the invention is directed to DNA in purified and isolated form consisting essen ⁇ tially of a DNA sequence encoding the enzyme ACC synthase of a higher plant.
  • the invention is directed to expression systems effective in expressing the DNA encoding said ACC synthase, to recombinant hosts transformed with this expression system, and to methods of producing ACC synthase using these transformed hosts.
  • the invention is directed to methods to control ACC synthase production using the coding sequences for ACC synthase in an antisense construct or by replacing the ACC synthase gene by a mutated form thereof.
  • the invention is directed to a novel method to isolate an inducible cDNA without necessity for the purified protein which it encodes.
  • Figure 1A shows a restriction map of two clones encoding the zucchini ACC synthase enzyme
  • Figure IB shows the nucleotide and deduced amino acid sequence of one of these clones.
  • Figure 2A-2C show elution patterns in chromatographic steps in the purification of ACC synthase from zucchini.
  • Figure 3 shows the elution pattern of the final step in the purification of ACC synthase from zucchini.
  • Figure 4 shows an SDS-polyacrylamide gel of the fractions of Figure 3.
  • Figure 5 shows a restriction map of genomic clones obtained by hybridization to the cDNA encoding zucchini ACC synthase.
  • Figure 5A shows the alignment of the retrieved clones with the position of the coding sequences on the genome;
  • Figure 5B shows a restriction map of the sequences on the genome;
  • Figure 5C shows the functional portions of the two zucchini ACC synthase genes CP-ACC 1A and CP-ACC IB.
  • Figure 6 shows the complete nucleotide sequence and deduced amino acid sequence of the genomic clone repre- senting CP-ACC 1A. Both control regions and coding regions are shown.
  • Figure 7 shows the complete nucleotide sequence and deduced amino acid sequence of the genomic clone repre ⁇ senting CP-ACC IB. Both control regions and coding regions are shown.
  • Figure 8 shows the nucleotide and deduced amino acid sequence of a cDNA encoding the tomato ACC synthase.
  • Figure 9 shows a diagram and restriction map of several clones of the cDNA encoding tomato ACC synthase.
  • Figure 10 shows a comparison of the amino acid sequence of the ACC synthase encoded by the zucchini genomic sequence CP-ACC 1A and the tomato genomic sequence ACC 2.
  • Figure 11 shows the pattern of genomic clones and functional diagrams thereof for the tomato genome containing coding and control sequences for LE-ACC 1A, LE-ACC IB, and LE-ACC 3.
  • Figure 12 shows the pattern of genomic clones and the organization of the gene for LE-ACC 2.
  • Figure 13 shows the complete genomic and deduced amino acid sequence of LE-ACC 2, including the control sequences.
  • Figure 14 shows a comparison of the deduced amino acid sequence from the two genomic zucchini clones and the four genomic tomato clones for ACC synthase.
  • Figure 15 shows the junction region and a restriction map of a bacterial expression vector for the production of tomato ACC synthase in bacteria.
  • Figure 16 shows the production of ACC by bacterial cultures transformed with the vector of Figure 15 in the presence and absence of the inducer IPTG.
  • Figure 17 is a half tone photograph of a two- dimensional chromatographic gel of bacterial extracts wherein the bacterial culture is transformed with an expression vector for tomato ACC synthase having the coding sequence in the correct and incorrect orientations.
  • Figure 18 shows the construction of an expression vector for the tomato ACC-synthase gene oriented in the antisense direction.
  • Figures 19 and 20 show the ethylene production by tomato plants regenerated from tomato cotyledons trans ⁇ formed with the vector of Figure 18 as a function of days from pollination.
  • recombinant refers to a nucleic acid sequence which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar recombinant techniques as described by, for example, Maniatis et al. "Recombinant”, as used in the present application, does not refer to naturally-occurring genetic recombinations.
  • ACC synthase includes all enzymes which are capable of catalyzing the conversion of AdoMet to ACC and methyl thioadenosine (MTA) .
  • the amino acid sequence of the synthase may or may not be identical with the amino acid sequence which occurs natively in higher plants.
  • An example of such native sequence is shown in Figure 1 which occurs in the zucchini fruit (Cucurbita pepo) .
  • Naturally occurring allelic variants undoubtedly occur as well. Similar proteins are present in a wide variety of higher plants.
  • artifi ⁇ cially induced mutations are also included so long as they do not destroy activity. In general, conservative amino acid substitutions can be made for most of the amino acids in the primary structure as shown without affecting destruction of activity.
  • the definition of ACC synthase used herein includes these variants which are derived by direct or indirect manipulation of the dis ⁇ closed sequences.
  • the primary structure may be altered by post-translational processing or by subse- quent chemical manipulation to result in a derivatized protein which contains, for example, glycosylation substi ⁇ tuents, oxidized forms of, for example, cysteine or pro- line, conjugation to additional moieties, such as carriers, solid supports, and the like.
  • alterations do not remove the protein from the definition of ACC synthase so long as its capacity to convert AdoMet to ACC and MTA is maintained.
  • an enzyme as "ACC synthase” can be confirmed by its ability to effect the production of ethylene in an assay performed as follows: the enzyme to be tested is incubated with 200 ⁇ M AdoMet, 10 ⁇ M pyridoxal phosphate, 40 ⁇ g BSA in 200 mH Hepes buffer, pH 8.5 in a total volume of 600 ⁇ l at 30°C for 30 minutes, and the amount of ACC formed is assayed by conversion to ethylene using hypochlorite as described, for example, by Lisada, C.C., et al., Anal Biochem (1979) 100:140-145.
  • ACC synthases of the invention include those thus illustrated herein, and those derivable therefrom by systematic mutation of the genes. Such systematic mutation may be desirable to enhance the ACC synthase properties of the enzyme, to enhance the characteristics of the enzyme which are ancillary to its activity, such as stability, or shelf life, or may be desirable to provide inactive forms useful in the control of ACC activity in vivo.
  • ACC synthase refers to a protein having the activity assessed by the assay set forth above; a “mutated ACC synthase” refers to a protein which does not necessarily have this activity, but which is derived by mutation of a DNA encoding an ACC synthase.
  • derived from mutation is meant both direct physical derivation from a DNA encoding the starting material ACC synthase using, for example, site specific mutagenesis or indirect derivation by syntheses of DNA having a sequence related to, but deliberately different from, that of the ACC synthase.
  • oligonucleotide of the required length are available, such DNAs can be constructed wholly or partially from their individual constituent nucleotides.
  • higher plant refers to those plants whose development and activity are controlled by ethylene. These includes all common agricultural plants and various flowering species.
  • the ACC synthase cDNA was isolated initially from Cucurbita pepo (zucchini) using a novel method which is applicable to inducible proteins.
  • the method does not require pure protein in order to design probes or to prepare monoclonal antibodies; the method relies on the production of a cDNA expression library from induced tissue and identification of positive clones using an antibody preparation which has been purified by taking advantage of the inducible nature of the protein.
  • the method comprises the steps of preparing partially purified inducible protein of interest from the cells or tissue which have been induced for this production and preparing a composition of antibodies to these purified proteins.
  • the composition of antibodies is prepared in a conventional manner by immunization of a suitable mammal with a protocol designed to enhance the production of antiserum.
  • the first composition of antibodies which will also contain antibodies to the protein contaminants in the preparation, is then purified to obtain a second antibody composition enriched in the antibodies immunoreactive for the desired protein.
  • This enrichment is effected by reacting the first prepared composition with a protein extract from uninduced tissue. This will selectively remove those antibodies immunoreactive with background contaminants.
  • This purified preparation of antibodies is then used to screen a cDNA expression library which has been prepared from tissue expressing the gene encoding the inducible protein. As the purified antibody preparation is immunospecific for this protein, identification of the positive clones is simplified.
  • the application of this method to the recovery of cDNA encoding ACC synthase is described in detail in Example l herein. However, a similar method can be used to obtain the cDNA for any inducible protein, even without isolation and purification of the desired target protein.
  • the availability of the cDNA encoding zucchini ACC synthase makes accessible both the multigene family which provides the variety of ACC synthases found in the same plant host-i.e., zucchini, as well as other cDNAs encoding ACC synthase from other species of higher plants and their corresponding multiple gene families.
  • the cDNAs or portions thereof are used as probes to hybridize to the additional genomic or cDNA sequences by hybridization under standard conditions. Typical standard conditions of stringency include those set forth, for example, in Example 4.
  • recovered sequences can also be engineered to effect the expression of ACC synthase, to make modification which result in ACC synthase mutants, or "mutated ACC synthase" and to construct antisense vectors to control the production of indigenous ACC synthase. Recombinant Production of ACC Synthase
  • the availability of the ACC synthase gene permits its production in a variety of recombinant systems.
  • Recombi ⁇ nant production of this enzyme in single cellular systems including procaryotic and eucaryotic systems, provides the tools for the recombinant production of ethylene nd ethanol, the products of the ACC synthesized.
  • Large scale production of these chemicals can be effected by utilizing suitable large scale recombinant production of the ACC synthase enzyme to effect the endogenous production of ACC followed by chemical conversion of the ACC to ethylene and/or ethanol.
  • large scale production such as in large algae cultures, is preferred.
  • the ACC synthase can also be produced in transgenic plants both in enhanced amounts and in an antisense mode, as further set forth below, to control the aspects of plant development which are ethylene sensitive, and in particular, to delay plant genescence. Accordingly, a variety of expression systems and hosts can be used for the production of this enzyme.
  • a variety of procaryotic hosts and appropriate vectors is known in the art; most commonly used are E___ coli or other bacterial hosts such as _______ subtilis or Pseudomonas and typical bacterial promoters include the trp, lac, tac, and jS-lactamase promoters.
  • a readily controllable, inducible promoter, the ⁇ -phage promoter can also be used.
  • a large number of control systems suitable for procaryote expres ⁇ sion is known in the art.
  • a large number of recombinant systems have been developed for expression in eucaryotic hosts, including yeast, insect cells, mammalian cells, and plant cells. These systems are well characterized, and require the ligation of the coding sequence under the control of a suitable transcription initiating system (promoter) and, if desired, termination sequences and enhancers.
  • promoter transcription initiating system
  • ACC synthase genes of the present invention are expression systems which are operable in plants. These include systems which are under control of a tissue-specific promoter, as well as those which involve promoters that are operable in all plant tissues.
  • Transcription initiation regions include the various opine initiation regions, such as octopine, mannopine, nopaline and the like.
  • Plant viral promoters can also be used, such as the cauliflower mosaic virus 35S promoter.
  • plant promoters such as ribulose-l,3-diphosphate carboxylase, fruit-specific pro ⁇ moters, heat shock promoters, seed-specific promoters, etc. can also be used.
  • the cauliflower mosaic virus (CaMV) 35S promoter has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants including tobacco and petunia, and has been shown to confer expression in protoplasts of both dicots and monocots.
  • the CaMV 35S promoter has been demonstrated to be active in at least the following monocot and dicot plants with edible parts: blackberry, Rubus; blackberry/rasp ⁇ berry hybrid, Rubus, and red raspberry; carrot, Daucus carota; maize; potato, Solanum tuberosum; rice, Oryza sativa; strawberry, Fragaria x ananassa; and tomato, Lycopersicon esculentum.
  • Nos nopaline synthase promoter
  • apple Malus pumila
  • cauli ⁇ flower Brassica oleracea
  • celery Apiu graveolens: cucumber, Cucumis sativus
  • eggplant Solanum melongena: lettuce, Lactuca sativa
  • potato Solanum tuberosum: rye, Secale cereale strawberry, Fragaria x ananassa
  • tomato Lycopersicon esculentum
  • walnut Juglans regia.
  • Organ-specific promoters are also well known.
  • the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO J (1988) 2:3315; Giovannoni et al., The Plant Cell (1989) 1:53).
  • the activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes.
  • Other organ-specific promoters appropriate for a desired target organ can be isolated using known proce ⁇ dures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Phil, Trans R Soc London (1986) B314:343) .
  • RNA isolated from ripening avocado fruit but that did not hybridize with labeled RNAs isolated from unripe avocado fruit. Many of these clones represent mRNAs encoded by genes that are transcriptionally activated at the onset of avocado fruit ripening.
  • E8 cDNA clone The gene that encodes the organ-specific mRNA is then isolated by constructing a library of DNA genomic sequences from the plant. The genome library is screened with the organ-specific DNA clone, and the sequence is determined. The promoter is then isolated.
  • Either a constitutive promoter or a desired organ- specific promoter is then ligated to the gene encoding ACC synthase or a mutated form thereof using standard tech ⁇ niques now common in the art.
  • the expression system may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer ele ⁇ ments.
  • the recombinant expression cassette will contain in addition to the ACC synthase-encoding sequence, a plant promoter region, a transcription initiation site (if the coding sequence to be transcribed lacks one) , and a transcription termination sequence.
  • Unique restriction enzyme sites at the 5' and 3' ends of the cassette are typically included to allow for easy insertion into a pre-existing vector.
  • Promoter sequence elements include the TATA box consensus sequence (TATAAT) , which is usually 20 to 30 base pairs (bp) upstream of the transcription start site. In most instances the TATA box is required for accurate transcription initiation. By convention, the start site is called +1. Sequences extending in the 5' (upstream) direction are given negative numbers and sequences extending in the 3' (down- stream) direction are given positive numbers.
  • TATAAT TATA box consensus sequence
  • a promoter element In plants, further upstream from the TATA box, at positions -80 to -100, there is typically a promoter element with a series of adenines surrounding the trinu- cleotide G(or T)NG (Messing, J. et al., in Genetic Engi- neering in Plants. Kosage, Meredith and Hollaender, eds. (1983) pp. 221-227) .
  • Other sequences conferring tissue specificity, response to environmental signals, or maximum efficiency of transcription may also be found in the promoter region. Such sequences are often found within 400 bp of transcription initiation site, but may extend as far as 2000 bp or more.
  • the promoter is preferably posi ⁇ tioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • any of a number of promoters which direct transcription in plant cells is suitable.
  • the promoter can be either constitutive or inducible.
  • Promoters of bacterial origin include the octopine syn- thase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids (Herrera- Estrella et al., Nature (1983) 3_03_:209-213) .
  • Viral promoters include the 35S and 19S RNA promoters of cauli ⁇ flower mosaic virus (O'Dell et al., Nature (1985) 313:810- 812).
  • Plant promoters include the ribulose-1,3- diphosphate carboxylase small subunit promoter and the phaseolin promoter.
  • the promoter sequence from the E8 gene and other genes in which expression in induced by ethylene may also be used.
  • the isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer, EMBO J (1988) 2:3315-3320 which is incorporated herein by reference.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • DNA sequences which direct polyadenylation of the RNA are also commonly added to the vector construct (Albert and Kawasaki, Mol and Appl Genet. (1982) 1:419-434). Polyadenylation is of importance for expression of the ACC synthase-encoding RNA in plant cells. Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J . (1984) 2 :83 5:-846) or the nopaline synthase signal (Depicker et al., Mol and Appl Genet (1982) 1:561-573) .
  • the resulting expression system or cassette is ligated into or otherwise constructed to be included in a recombinant vector which is appropriate for higher plant transformation.
  • the vector will also typically contain a selectable marker gene by which transformed plant cells can be identified in culture. Usually, the marker gene will encode antibiotic resistance. These markers include resistance to G418, hygromycin, bleomycin, kanamycin, and gentamicin. After transforming the plant cells, those cells having the vector will be identified by their ability to grow on a medium containing the particular antibiotic.
  • Replication sequences, of bacterial or viral origin are generally also included to allow the vector to be cloned in a bacterial or phage host, preferably a broad host range procaryotic origin of replication is included.
  • a selectable marker for bacteria should also be included to allow selection of bacterial cells bearing the desired construct. Suitable procaryotic selectable markers also include resistance to antibiotics such as kanamycin or tetracycline.
  • DNA sequences encoding additional functions may also be present in the vector, as is known in the art. For instance, in the case of Agrobacterium transformation, T-DNA sequences will also be included for subsequent transfer to plant chromosomes.
  • vectors can also be constructed that contain in-frame ligations between the sequence encoding the ACC synthase protein and sequences encoding other molecules of interest resulting in fusion proteins, by techniques well known in the art.
  • transgenic plants are prepared which contain the desired expression system.
  • a number of techniques are available for transformation of plants or plant cells. All types of plants are appropriate subjects for "direct” transformation; in general, only dicots can be transformed using Agrobacterium-mediated infection.
  • the vector is microinjected directly into plant cells by use of micro- pippettes to mechanically transfer the recombinant DNA (Crossway, Mol Gen Genetics (1985) 202:179-185) .
  • the genetic material is transferred into the plant cell using polyethylene glycol (Krens, et al., Nature (1982) 296:72-74) , or high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, is used (Klein, et al.. Nature (187) 327:70-73) .
  • protoplasts are fused with other entities which contain the DNA whose introduction is desired. These entities are minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley, et al., Proc Natl Acad Sci USA (1982) 2£:1859- 1863.
  • DNA may also be introduced into the plant cells by electroporation (Fromm et al., Proc Natl Acad Sci USA
  • plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide and regenerate. For transformation mediated by bacterial infection, a plant cell is infected with Agrobacterium tumefaciens or
  • A. rhizogenes previously transformed with the DNA to be introduced.
  • Agrobacterium is a representative genus of the gram-negative family Rhizobiaceae. Its species are responsible for crown gall (A. tumefciens) and hairy root disease (A. rhizogenes) .
  • the plant cells in crown gall tumors and hairy roots are induced to produce amino acid derivatives known as opines, which are catabolized only by the bacteria.
  • the bacterial genes responsible for expres ⁇ sion of opines are a convenient source of control elements for chimeric expression cassettes.
  • assaying for the presence of opines can be used to identify transformed tissue.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes.
  • the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome (Schell, J. , Science (1987) 13_7:1176-1183) .
  • Ti and Ri plasmids contain two regions essential for the production of transformed cells. One of these, named transferred DNA (T-DNA) , is transferred to plant nuclei and induces tumor or root formation. The other, termed the virulence (vir) region, is essential for the transfer of the T-DNA but is not itself transferred.
  • T-DNA transferred DNA
  • vir virulence
  • the T-DNA will be transferred into a plant cell even if the vir region is on a different plasmid (Hoekema, et al., Nature (1983) 303:179-189) .
  • the transferred DNA region can be increased in size by the insertion of heterologous DNA without its ability to be transferred being affected.
  • a modified Ti or Ri plasmid in which the disease-causing genes have been deleted, can be used as a vector for the transfer of the gene constructs of this invention into an appropriate plant cell.
  • cointegrate the shuttle vector containing the gene of interest is inserted by genetic recombination into a non- oncogenic Ti plasmid that contains both the cis-acting and tans-acting elements required for plant transformation as, for example, in the pMLJl shuttle vector of DeBlock et al., EMBO J (1984) 2:1681-1689 and the non-oncogenic Ti plasmid pGV2850 described by Za bryski et al., EMBO J (1983) 2:2143-2150.
  • the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation.
  • the other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Research (1984) 12:8711-8721 and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al., Nature (1983) 303:179-180.
  • Agrobacterium There are two common ways to transform plant cells with Agrobacterium: co-cultivation of Agrobacterium with cultured isolated protoplasts, or transformation of intact cells or tissues with Agrobacterium.
  • the first requires an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cul- tured protoplasts.
  • the second method requires (a) that the intact plant tissues, such as cotyledons, can be transformed by Agrobacterium and (b) that the transformed cells or tissues can be induced to regenerate into whole plants. Most dicot species can be transformed by
  • Agrobacterium as well as species which are a natural plant host for Agrobacterium are transformable in vitro. Monocotyledonous plants, and in particular, cereals, are not natural costs to Agrobacterium. Attempts to transform them using Agrobacterium have been unsuccessful until recently (Hooykas-Van Slogteren et al., Nature (1984) 311:763-764) . However, there is growing evidence now that certain monocots can be transformed by Agrobacterium.
  • Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obtaining evidence of successful bacterial infection. Plant cells which have been transformed can also be regenerated using known techniques.
  • Plant regeneration from cultured protoplasts is described in Evans et al. , Handbook of Plant Cell Cultures, Vol. l: (MacMillan Publishing Co. New York, 1983); and Vasil I.R. (ed.), Cell Culture and Somatic Cell Genetics of Plants. Acad. Press, Orlando, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugar- cane, sugar beet, cotton, fruit trees, and legumes.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regene ⁇ ration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • a large number of plants have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants.
  • regeneration has been shown for dicots as follows: apple, Malus pumila; blackberry, Rubus. Blackberry/raspberry hybrid, Rubus , red raspberry, Rubus; carrot, Daucus carota; cauliflower, Brassica oleracea; celery, Apium graveolens; cucumber, Cucumis sativus: eggplant, solanum elongena; lettuce, Lactuca sativa: potato, Solanum tuberosum; rape, Brassica napus: soybean (wild) , Glycine canescens; strawberry, Fragaria x ananassa: tomato, Lycopersicon esculentum; walnut, Juglans regia; melon, Cucumis melo; grape, Vitis vinifera; mango, Mangifera indica; and for the following monocots: rice, Oryza sativa; rye, Secale cereale;
  • the expression cassette After the expression cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • Antisense Expression When the ACC synthase coding sequence is placed in correct orientation in the expression systems described above, the ACC synthase protein is produced. However, when placed in the opposite orientation, the expression vector has an antisense effect which can interfere with the indigenous production of this enzyme.
  • Antisense tech ⁇ nology can work at a variety of levels including hybridi ⁇ zation to a messenger RNA encoding the ACC synthase, hybridization to single-stranded intermediates in the production of this mRNA, or triplex formation with the DNA duplex which contains the ACC synthase genes. All of these modalities can be employed in effecting antisense control of ACC synthase production.
  • ripening of tomato fruit can be controlled and inhibited by suitable antisense expression of the ACC synthase coding sequence supplied in a vector under the control of the cauliflower 35S promoter.
  • Other properties which are controlled by ethylene can also be influenced by appropriate choice of control systems and/or the particular AC synthase encoded.
  • ACC synthase in higher plants is a dimer.
  • a decoy can be pro- prised to obtain inactive monomer and thereby regulate the levels of ACC synthase in the plant.
  • An additional embodiment of the invention involves the mutated ACC synthase and expression systems therefor.
  • Example 1 Recovery of Zucchini ACC Synthase cDNA
  • a cDNA encoding ACC synthase in zucchini fruit was recovered as follows: Slices 1 mm thick were prepared from zucchini fruits of the species Cucurbita pepo. To induce production of ACC synthase, slices were incubated for 18-24 hours in induction medium (50 ⁇ M potassium phosphate buffer , pH 6.8; 0.5 mM indole acetic acid (IAA) ; 0.1 mM benzyl adenine (BA) ; 50 mM LiCl; 0.6 mM aminooxyacetic acid (AOA) ; and 50 ⁇ g/ml chloramphenicol.
  • IAA indole acetic acid
  • BA 0.1 mM benzyl adenine
  • AOA aminooxyacetic acid
  • RNA was isolated from 18-hr tissue prepared as described above, and in vitro translated in a wheat germ lysate as described by Theologis, A., et al., J Mol Biol (1985) 183:53-68, in the presence of labeled methionine (greater than 1,000 Ci/ ⁇ mol) to verify the presence of ACC synthase encoding mRNA.
  • a cDNA library was prepared in ⁇ gtll as described by Huynh, T.V. , et al., in "DNA Cloning Techniques," Glover, E. , ed. (1985) IRL Press, Oxford, 1:49-88. The insert sizes were 200-500 bp.
  • the library was screened with purified ACC synthase anti- serum prepared as follows:
  • the antisera were prepared to 1500-fold purified ACC synthase preparations.
  • Purified ACC synthase can be prepared from tissue homogenates sequentially bound to and eluted from Butyl Toyopearl (Toyo Soda Tokyo) , SP- Sephadex, and QAE-Sephadex. (Higher purification can be obtained by subsequent chromatography sequentially through columns containing Butyl Toyopearl, Sephacryl S-300, Bio Gel-Ht, and finally FPLC mono-Q.
  • the application of all of the following steps results in approximately a 6000- fold purification.
  • the antibodies are prepared in New Zealand white rabbits by immunization protocols involving four immunizations at three-week intervals with 5000 nmol of ACC synthase activity/hr (specific activity 1500 nmol of ACC/hr/mg protein obtained from the Bio Gel-HT column) .
  • Crude antiserum (2 ml) was purified by incubation with 10 ml Sepharose 4B coupled with soluble proteins from intact noninduced Cucurbita fruit.
  • pACC7 is identical to a portion of the sequence of pACCl.
  • the open reading frame encodes a protein of 493 amino acids, corresponding to a 55.779 kd polypeptide.
  • the positive clones from the ⁇ gtll library could also be used to prepare further purified antiserum for immuno- blotting as follows:
  • the positive cones from the expression library was plated on E. coli strain Y1090 to obtain 10 5 plaque-form ⁇ ing units per 90-mm plate.
  • Dry nitrocellulose filters presoaked in 10 mM isopropyl -D-thiogalacto-pyranoside (IPTG) were laid on the lawn after incubation for two hours at 42°C and then incubated for an additional four hours at 37°C.
  • bound antibody was eluted by shaking for three minutes at room temperature with 0.2M glycine hydro- chloride buffer, pH 2.3, containing 1% milk protein. The antibody solution was neutralized and used for immuno- blotting.
  • Example 2 Purification of Native ACC Synthase From Cucurbita ACC synthase was purified 6000-fold from induced Cucurbita homogenates according to a multistep protocol as shown below.
  • Various buffers used in the purification are as follows:
  • Buffer A Tris-HCl 100 mM, pH 8.0, EDTA 20 mM, pyridoxal phosphate 10 ⁇ M, PMSF 0.5 mM, ⁇ -mercaptoethanol
  • Buffer B Tris-HCl 20 mM, pH 8.0, EDTA 10 mM, pyridoxal phosphate 10 ⁇ M, DTT 0.5 mM
  • Buffer C Na- acetate 20 mM, pH 6.0, pyridoxal phosphate 10 ⁇ M, EDTA 10 mM, DTT 0.5 mM
  • Buffer D K-phosphate 10 mM, pH 8.0, pyridoxal phosphate 10 ⁇ M, EDTA ImM, DTT 0.5 mM
  • Buffer E Tris-HCl 20mM, pH 8.0, pyridoxal phosphate 5 ⁇ M, EDTA 1 mM, DTT 0.5 mM
  • Buffer F Hepes-KOH 500 mM, pH 8.5, pyridoxal phosphate 40 ⁇ M, BSA 400 ⁇ g/ml.
  • Chroma- tographic elutions were assayed for ACC synthase activity and by absorption at 280 nm.
  • the adsorbed proteins were eluted from the matrix by washing (twice) with 750 ml of buffer B, batch- wise.
  • the combined eluates were dialyzed three times against 10 liters of buffer B for 36 hr.
  • SP-Sephadex Fractionation The dialyzed fraction above was clarified by centri ⁇ fugation at 17,000 x g for 30 min. The volume was adjusted to 4 liters with buffer B and the pH was brought to pH 6.0 with 5% acetic acid.
  • Two liters of packed SP-Sephadex C-50 equilibrated with buffer C were added and the suspension was stirred for 60 min.
  • the matrix was recovered by filtration through two sheets of Whatman filter paper #1 and washed with 2 liters of buffer C.
  • the adsorbed proteins were eluted twice with 1 liter of buffer B containing 1M KC1, batchwise.
  • the eluant was recovered by suction through #1 Whatman filter paper and solid ammonium sulfate was added to achieve 40% saturation.
  • 100 ml of Butyl Toyopearl-packed matrix equilibrated with buffer B/40% ammonium sulfate was added to the eluate.
  • the suspension was stirred for 30 min and the matrix was collected by filtration through a layer of Nitex nylon cloth (30 ⁇ m mesh) .
  • the matrix was resus ⁇ pended in a small volume of buffer B/40% ammonium sulfate and poured in a column (2.5 x 20 cm).
  • Figure 2A shows the elution pattern. Solid ammonium sulfate was added to enzymatically active fractions to achieve 80% saturation and the solution was incubated at 4°C for at least 4 hr. The precipitate was collected by centrifugation at 30,000 x g for 30 min at 4°C and dis ⁇ solved in 3 ml of buffer D.
  • the concentrated active fractions (-0.5 ml) from the Bio Gel-HT column were applied to a monc-Q H5/5 column.
  • the column then was eluted with a 15 ml linear gradient: 0.1 to 0.4M KCl in buffer E.
  • the flow rate of the gradient was 0.5 ml/min.
  • Figure 3 shows the elution pattern.
  • Table 1 shows the overall purification sequence and the increase in specific activity with each successive step. The overall process results in a 6000-fold purifi ⁇ cation with a recovery of 7.5%. The enzyme has a specific activity of 35,590 nmol ACC produced /hr/mg of protein. Table 1
  • the purified ACC synthase was also subjected to size exclusion chromatography o Sephacryl S-300. In this protocol, the ACC synthase eluted as an 86 kd species. This suggests that the Cucurbita ACC synthase consists of two identical 46 kd subunits. Further characterization showed that the pH optimum for ACC synthase activity is 9.5, and the isoelectric point is estimated at 5 using mono-P H 5/20 FPLC column chromatography. The Km for AdoMet is 16.7 mM, and pyridoxal phosphate is a cofactor. The enzyme is stable at -20°C or -80°C for over a year.
  • DNA corresponding to 20 kb in size was ligated into EcoRI/BamHI cut EMBL 3 ⁇ (Frischholz, A.M., et al., J Mol Biol (1983) 170:827-842: Raleigh, E.A. , et al., Proc Natl Acad Sci USA (1986) £:9070-9074; Maniatis, T. , et al.. Molecular Cloning (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • the ligation mixture was pack ⁇ aged and the library was screened without amplification by plating on E. coli strain K802 and screening with nick translated ACCl cDNA (the full length zucchini cDNA clone) as described by Benton, D.
  • FIG. 5A-C The results after restriction analysis of the various genomic clones recovered is shown in Figure 5A-C. As shown in the figure, two genomic clones reside on the same DNA strand, but are oriented in opposite directions. CP-ACC 1A and CP-ACC IB each contain four introns. The complete genomic sequences of these clones are shown in Figures 6 and 7 respectively. As shown in Figures 6 and 7, the entire upstream regulatory sequences are encoded in the clones.
  • cDNA libraries were constructed into ⁇ gtlO as described by Huynh, T.V., et al. cDNA Cloning Techniques: A Practical Approach (1985) (Glover, D.M. ed.), IRL Press, London, 49-78.
  • cDNAs greater than 500 bp were inserted into the EcoRI site of the Cl repressor gene.
  • the packaged DNA was plated on C600 HFL, a derivative of C600, to select for phage- containing inserts.
  • 5X SSPE is 0.1 8M NaCl, 10 mM sodium phosphage, pH 7.0, 1 mM sodium EDTA
  • the gel purified 1.8 kb EcoRI fragment of the zucchini pACCl prepared in Example 1 was labeled to a specific activity of 2 x 10 8 cpm/mg using random hexamer printing and ⁇ -32P dCTP as described by Feinberg, A.P., et al.. Anal Biochem (1983) 132:6-13.
  • the labeled probe was separated from starting material and used to probe the ⁇ gtlO library.
  • the probe was denatured with 0.25 volumes 1M NaOH for 10 minutes at room temperature and neutralized with 0.25 volume 2M Tris HCl, pH 7.2 and then added to the hybridi ⁇ zation mixture at 1 x 10 6 cpm/ml.
  • the filters were washed once in 30% formamide, 5X SSPE, 0.1% SDS at 37°C for 20 min and then four times in 2X SSPE, 0.1% SDS at 37°C for 20 min. The final wash was in 2X SSPE at 50°C for 10 min.
  • the filters were air dried, covered with Saran wrap and exposed at -70°C to X-ray film.
  • Hybridization conditions were employed using 2 x 10 5 pfu of the ⁇ gtlO library on C600 wherein nitrocellulose platelets were prehybridized at 42°C and 50% formamide, 5X
  • Genomic DNA was isolated from etiolated Rutgers seed- lings using a modification of the method described by Davis, R.W., et al. Meth Enzvmol (1980) 65:404-411. Briefly, seedlings were grown on moist filter paper in the dark for 5 days at 22°C. Fifty g frozen hypocotyl and cotelydon tissue, seed coat removed, was ground in a coffee grinder.
  • the powdered tissue was added to 200 ml of ice cold extraction buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaEDTA, 0.25 M NaCl, 15% sucrose (w/v)) and homogenized on ice using a hand held glass-glass homogenizer.
  • the nuclei were pelleted at 2000 x g for 10 min at 4°C.
  • the crude nuclei we resuspended in 100 ml of cold extraction buffer without the salt.
  • 10 ml of 10% Nasarcosine was added, the suspension was gently inverted and incubated on ice for 10 min, then 120 g of CsCl was added and dissolved by gently shaking. To remove debris the solution was centrifuged at 26,000xg for 20 min at 4°C and the supernatant was decanted through Miracloth.
  • Ethidium bromide (10 mg/ml) was added to a final concentration of 0.4 mg/ml and the density of the solution was adjusted to 1.55 g/ml.
  • Equilibrium centrifugation was carried out in a Beckman Ri70 rotor at 40,000 rpm for 48 hr at 20°C.
  • the DNA was further purified by a second round of equilibrium centrifugation in a VTi65 rotor at 50,000 rpm for 16 hr at 20°C.
  • Ethidium bromide was extracted from the DNA with isopropanol saturated with water containing 5 M NaCl and the DNA was dialyzed twice against 5000 volumes of TE (pH 7.5) to remove the CsCl.
  • the typical yield was 15 ⁇ g/g fresh weight tissue.
  • Two genomic libraries were constructed, one with 15- 23 kb Sau3A partially digested DNA in ⁇ EMBL3 and another with 6-8 kb DNa after complete Hindlll digestion into ⁇ 2001.
  • 100 ⁇ g of genomic DNA was digested with 1.5 units of Sau3A at 37°C in 300 ⁇ l of medium salt buffer (MSB) plus 2 mM dithiothreitol (DTT) (IX MSB is 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgS0 4 ) .
  • MSB medium salt buffer
  • DTT dithiothreitol
  • One third of the reaction was removed at 7.5 min, at 10 min and at 12.5 min.
  • the DNA was size fractionated in a 0.5% low melting temperature agarose gel by electrophoresis at 0.8 Volts/cm for 24 h.
  • the agarose gel electrophoresis buffer was IX TAE, 40 mM Tris-HOAc, pH 8.0, 2 mM NaWDTA.
  • the gel was soaked at room temperature for 3 hr in IX TAE buffer containing 0.3 M NaCl. DNA was visualized with 365 nm ultraviolet light and the 15-23 kb side range was excised.
  • the agarose was melted at 65°C for 15 min and extracted twice the TE (pH 7.5)-saturated phenol, prewarmed to 37°C. The aqueous phase was extracted three times with ether and two volumes of EtOH were added. After overnight at -20°C the DNA was collected by centrifugation and dissolved in TE, pH 7.5 Two ⁇ g of EMBL3 arms and 2 ⁇ g size selected DNA were com ⁇ bined in a final volume of 6 ⁇ l, 1 ⁇ l of 10X ligase buffer (IX ligase buffer is 66 mM Tris-HCl, pH 7.5 5 mM MgCl 2 ) was added and the cohesive ends annealed at 42°C for 15 min.
  • IX ligase buffer is 66 mM Tris-HCl, pH 7.5 5 mM MgCl 2
  • Clones corre ⁇ sponding to other genes were obtained by probing the Sau3A partial digest library in EMBL3 with the cDNA at low stringency. In two separate screenings, phage were plated on the hosts C600 or TC410, lifted and fixed to nitro ⁇ cellulose filters as described above. Low stringency prehybridization and hybridization were done in 30% formamide, 5X SSPE, 5X BFP, 100 ⁇ g/ml denatured salmon sperm DNA, 0.2% SDS at 37°C for 18 h each. Probe was used at a concentration of 10 6 cpm/ml.
  • HSB high salt buffer
  • IX HSB is 100 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM MgS0 4
  • genomic DNA gel blots 7.15 ⁇ g of genomic DNA was digested in 100 ⁇ l of IX HSB with 80 units of EcoRI and Hindlll or 40 units of Bglll, at 37°C for 6 h.
  • Digested DNAs were loaded on 1 cm thick, 0.8% agarose gels and electrophoresed at 3 V/cm in IX TAE buffer containing 0.5 ⁇ g/ml ethidium bromide. After electrophoresis the gel was photographed, the DNA was nicked with two 15 min treatments of 0.25 M HCl, denatured with two 20 min treatments of 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl and neutralized with two 20 min treat ⁇ ments of 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl. The nucleic acids were transferred in 2OX SSPE to a Nytran nylon membrane.
  • nucleic acids were fixed with 1.2 joules of 254 nm ultraviolet radiation.
  • the membranes were prehybridized in 50 ml of 50% formamide, 5X SSPE, 5X BFP, 1.0% SDS and 100 ⁇ g/ml heat denatured salmon sperm DNA at 42°C for 12 h.
  • Figure 11A shows a series of three genomic clones which were identified to three separate genes LE-ACC 1A; LE-ACC IB; and LE-ACC 3;
  • Figure 12 shows genomic clones which were identified with LE-ACC 2.
  • Figure 13 shows the complete nucleotide sequence of LE-ACC 2.
  • Figure 14 compares the amino acid sequences encoded by the six genomic clones recovered—two from zucchini and four from tomato. Again, conserved sequences are found and there is considerable homology; however, there are numerous differences in sequence.
  • pETC3C Rosenberg, A.H., et al. Gene (1987) .56:125-155) was modified by cutting with EcoRI and ECoRV and filling in with Klenow to remove a 375 bp fragment downstream of the T7 promoter.
  • the resulting religatad plasmid was named pP07.
  • pP07 was cut with BamHI and Ndel and the large DNA segment was purified and ligated to a BaMHI/Ndel polylinker containing an EcoRI site to obtain the intermediate plasmid pP09.
  • the 1.4 kb EcoRI fragment from ptACCl was then ligated into the EcoRI site of pP09 to obtain the junction shown in Figure 15 and designated pP046.
  • This plasmid was then used to transform 7______ coli BL21 (DE3) (Rosenberg et al. (supra)).
  • the cultures were induced by diluting fresh overnight cultures into 2x TY (Maniatis et al. (supra)) and grown at 37° to an absorp ⁇ tion at 600 nm of 0.7-0.8.
  • IPTG was added to a final concentration of 2 mM and growth was continued for another two hours.
  • the cells were harvested and the recombinant polypeptide was purified as described by Nagai, K. and Thogersen, A.C., Meth Enzvmol (1987) 153.:461-481.
  • Figure 16 shows the synthesis of ACC synthase in nmol/15 ⁇ l of culture transformed with the tACC-containing vector in the presence and absence of IPTG.
  • the cDNA when the cDNA is ligated in the antisense direction, no ACC synthase is produced either in the presence or absence of IPTG (solid squares) .
  • the cDNA is oriented in the correct orientation, after 180 min, over 2 nmol ACC synthase are produced after 15 ⁇ l in the presence of IPTG (solid circles), and between 0.5 and 1.0 nmol. in the absence of IPTG (open circles).
  • FIG. 17 The production of ACC using labeled C 14 -carboxyl- labeled S-adenosyl-methionine is shown in Figure 17.
  • #1 is methionine
  • #2 is methionyl sulfite
  • #3 is methionyl sulfoxide
  • #4 is unidentified.
  • ACC is clearly labeled.
  • Figure 17A shows the results in the absence of IPTG; a little ACC is formed.
  • Figure 17C shows the results when the cDNA is ligated in the wrong orientation; no ACC is formed.
  • Figure 17B shows the production of labeled ACC when the correct orientation of the cDNA is used. A large quantity of ACC is produced.
  • the EcoRI fragment representing cDNA clone ACCl was subcloned into the EcoRI site of the yeast expression vector pBM258 (Johnston, M. , et al. Mol Cell Biol (1984) 4.:1440-1448) and introduced into yeast strain YM2061.
  • the yeast cells were grown on YP medium (Sherman, F. , et al.. Methods in Yeast Genetics (1979) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) at 37°C for 24 hr.
  • the medium either contained 2% galactose or 2% glucose. After this culture, the cells were harvested and the supernatant was assayed for ACC released into the medium.
  • the pelleted cells were resuspended in buffer containing 5 gm glass beads and vortex-mixed 10 times for 30 sec and centrifuged at 2000 x g for 3 min. This supernatant was also collected. Solid ammonium sulfate was added to achieve 80% saturation an the precipitate was collected and dissolved in 2 ml of 20 mM Tris-HCl, pH 8.0, 10 ⁇ M pyridoxal phosphate, 10 mM EDTA, 0.5 mM dithiothreitol; and dialyzed against the same buffer. No ACC was produced in medium containing 2% glucose regardless of the construction of the vector.
  • Control host vector and control vector with the ACCl cDNA inserted in the anti ⁇ sense direction also gave no production of ACC in the cellular extract.
  • the medium contained 2% galactose the pBM-ACCl vectors containing the cDNA in the correct orientation did show the production of ACC in the crude extract as well as ACC activity in the extracted protein.
  • ACC synthase activity was 2.6 nmol/hr/mg protein in the crude extract; 354 nmol of ACC were formed per 100 ml of culture.
  • Example 8 Antisense Inhibition of Ethylene Production in Tomato Plants
  • the ripening of tomatoes was shown to be preventable by the transformation of tomato plants with an antisense construction of the tomato ACC synthase gene which, therefore, putatively inhibited the synthesis of indige ⁇ nous ACC synthase.
  • the cDNA clone was inserted in the opposite sense direction under the control of the cauli ⁇ flower CAMV 35S promoter and used to transform tomato plantlets using the A ⁇ tumefaciens mediated method.
  • the regenerated plants produced tomatoes which failed to ripen, and which produced no ethylene at the times after pollination wherein ethylene was produced in control plants.
  • the antisense vector was constructed as follows: the 35S promoter was obtained as a 302 bp fragment from pJ024D (Ow, D.W., Proc Natl Acad Sci USA (1987) 84:4870-4874) .
  • the plasmid pJ024D was digested with Hindlll, treated with Klenow, and then cut with BamHI to isolate the 302 bp fragment using gel electrophoresis. This was ligated to 3.5 kb of tomato ACC synthase cDNA by excising the coding sequence from ptACCl by digestion with Xbal, filling with Klenow, and then cutting with BamHI. The two BamHI frag ⁇ ments were ligated and the resulting ligation transformed into E. coli stain DH5A for cloning.
  • the recovered plasmid was named pP032.
  • the plasmid pP032 was partially digested with Sad and Sail and the digest was ligated into Sall/SacI digested pBHOl binary Ti vector (Clonetech) .
  • pBHOl further contains the NOS 3' terminating sequences, as shown in Figure 17.
  • the resultant vector, designated pP035 was transformed into E. coli DH5A for cloning. The sequences at the junctions were verified by sequence analysis.
  • pP035 or a control vector without the ACC-synthase gene was purified and introduced into Agrobacterium strain LBA4404 by a standard procedure described briefly as follows: A.
  • tumefaciens LBA-4404 (2 ml) was grown over ⁇ night at 28°C in LB broth, and this used to inoculate 50 ml of LB broth to obtain the desired culture.
  • the inocu- lated medium was grown at 28°C until the OD 600 was 0.5 - 1.0.
  • the cells were collected by centrifugation and the pellet was resuspended in 1 ml, 20 mM ice cold CaCl 2 .
  • To 100 ⁇ l of the cell suspension 1 ⁇ m of pP035 DNA was added, and the mixture was incubated on ice for 30 min before snap-freezing in liquid nitrogen.
  • the cells were then thawed at 37°C for 5 min and used to inoculate 1 ml LB. After 2 h growth at 28° C with agitation , 100 ⁇ l of the culture were plated on LB+Kan 50 medium; colonies appeared in 2-3 days at 28°C. The cells were recultured by picking several colonies and streaking on LB+Kan 50 medium; again, 3-4 colonies were picked from independent streaks and 5 ml cultures in LB+Kan 50 medium were grown. Stationary phase of these cultures were used for transfection of tomato plants. The cells can be frozen using 15% glycerol at -80°C to store for later use. Preparation of Host Plants
  • Tomato seeds were sterilized using a protocol which consisted of treatment with 70% ethanol for 2 min with mixing; followed by treatment with 10% sodium hypochlorite and 0.1% SDS for 10 min with mixing, followed by treatment with 1% sodium hypochlorite, 0.1% SDS for 30 min with mixing, and washing with sterile water 3X for 2 min each wash.
  • 0.8 g of the sterilized seeds were placed in a Seed Germination Medium in a filled magenta box and grown for 2 weeks at low light in a growth room.
  • the magenta box contained 30 ml of the medium 1 .
  • the harvested cotyledons were placed abaxial side up in tobacco feeder plates and grown for 48 h.
  • the feeder plates were prepared from a tobacco cell suspension in liquid medium 3 at 25°C prepared with shaking at 130-150 rpm. The suspension was transferred to fresh medium at 1:10 dilution per every 3-5 days. 1 ml of rapidly dividing culture was placed on the feeder plate, overlaid with filter paper and placed in low light in a growth room. The feeder plates were supplemented with 10
  • Seed Germination Medium contains, per liter
  • 2 MSO contains per liter 4.3 g of Murashige-Skoog salts, 2 ml of 500 X B5 vitamins; 30 g of sucrose and 980 ml of water made 1 N in KOH to a final pH of 5.8.
  • Tobacco Suspension Medium contains in 1 liter 4.3 g Murashige-Skoog salts, 2 ml of 50OX B5 vitamins, 30 g 3% sucrose, 10 ⁇ l of a 0.5 mg/ml solution of kinetin stored at -20°C, 2 ml of a 2 mg/ml solution of pCPA, and 980 ml of water made 1 N in KOH for a pH of 5.8 and autoclaved in 50 ml portions per 250 ml flask.
  • ml Feeder Medium 4 The Agrobacterium containing the pP035 vector was inoculated into 50 ml LB containing kanamycin with a single colony of the strain.
  • the culture was grown shaking vigorously at 30°C to saturation (OD>2.0 at 600 nm) .
  • the strain was chosen to come to full growth in less than 24 h.
  • the culture was then diluted to 5 x 10 8 cell/ml with MSO nd split into 50 ml portions in plastic tubes.
  • Cotyledons from two of the feeder plates were scraped into each tube and rocked gently for 10-30 min.
  • the cotyledons were then removed from the bacterial culture into sterile filter paper abaxial side up on a tobacco feeder plate and incubated for 48 h in low light in a growth room.
  • the cotyledons were then transferred axial side up to
  • the Callus Inducing Medium approximately four plates will be used per magenta box, and the explant are crowded.
  • the box is place in a growth room for three weeks, and small masses of callus formed at the surface of the cotyledons.
  • the explants are transferred to fresh plates containing the callus inducing medium every three weeks.
  • Feeder Medium contains 0.43 g Murashige-Skoog salts, 2 ml 50OX B5 vitamins, 30 g of sucrose and 980 ml water made 1 N in KOH to a pH of 5.8, including 0.8% agar.
  • the foregoing components are autoclaved in two 500 ml portions and hormones are added when pouring plates to obtain 1 ⁇ /ml benzyl adenine (BA) and 0.2 ⁇ g/ml of indole acetic acid (IAA) .
  • BA benzyl adenine
  • IAA indole acetic acid
  • Callus Inducing Medium contains per liter 4.3 g of Murashige-Skoog salts, 2 ml of 50OX B5 vitamins, 30 g of sucrose and 980 ml of water brought to 1 N KOH at a pH of 5.8.
  • the medium contains 0.8% agar and is autoclaved in two 500 ml portions.
  • the following hormones are added to the following concentra ⁇ tions: l ⁇ m/ml BA, 0.2 ⁇ g/ml IAA, 100 ⁇ g/ml kanamycin, 500 ⁇ g/ml carbenicillin (Geopen) . to plates containing shoot inducing medium. 6
  • the shoots were dissected from the calli and the shoots were transferred to root inducing medium-containing plates. 7 After a vigorous root system was formed on the plants, the plantlets were transferred to soil. To do this, they were taken from the plates, removing as much agar as possible and placed in a high peat content soil in a small peat pot which fits into a magenta box with cover. When the seedling leaves reached the top of the box, the lid was loosened and continued to be uncovered slowly over a period of 4-5 days. The plants were then transferred to a light cart and larger pots, and kept moist. The regenerated tomato plants were allowed to flower and pollinated. Seeds from the regenerated plants were replanted and grown to maturity.
  • Figures 19 and 20 show the results for two sets of individual plants VI.1-4 (which contains the control vector) and All.2-24 (which contains the antisense vector) in Figure 19 and VI.1-6 (which contains the control vector) and All.2-7 (which contains the antisense vector) in Figure 20. As shown in these figures, in both cases, the control plants which had
  • 6 Shoot Inducing Medium contains, per liter, 4.3 g of Murashige-Skoog salts, 2 ml of 500X B5 vitamins, 0.6 g of MES and 900 ml of water made 1 N in KOH for a pH of 5.8, and 0.8% agar.
  • the medium is autoclaved in two 450 ml portions and then is added 100 ml of a 30% filtered, sterilized glucose solution. When the plates are poured, additional components are added as follows: 0.1 mg/ml zeatin, 100 ⁇ g/ml kanamycin, 500 ⁇ g/ml carbenicillin.
  • 7 Root Inducing Medium contains, per liter, 4.3 g
  • Murashige-Skoog salts 2ml 50OX B5 vitamins, 30 g of sucrose and 980 ml of water, 1 N in KOH to a pH of 5.8 in 0.8% agar.
  • the medium is autoclaved in two 500 ml por ⁇ tions and when pouring plates, hormones are added to a concentration of 100 ⁇ g/ml kanamycin and 500 ⁇ g/ml or carbenicillin.
  • control vector produced high levels of ethylene up to 8 ng/g fruit/h after approximately 50 days after pollination either in the presence of propylene or in the presence of air.
  • the antisense pP035 vector there was no production of ethylene in those plants which had been transformed with the antisense pP035 vector.
  • the tomatoes which failed to produce ethylene also failed to ripen, whereas the control plants did ripen at this time.

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Abstract

L'isolation de l'ADNc codant la synthase de l'enzyme ACC de zucchini au moyen d'un nouveau procédé d'isolation, permet de récupérer des gènes de synthase de l'ACC à partir d'une grande variété de sources de plantes supérieures. Les synthases de l'ACC sont codées par des familles à gènes multiples dont les membres peuvent être responsables de caractéristiques diverses du développement des plantes induites par l'éthylène. L'utilisation de ce gène permet également la production recombinée de la synthase de ACC et, de ce fait, la production in vitro de l'ACC, d'éthylène et d'éthanol. De plus, on peut maîtriser les processus induits dans les plantes par la synthase de l'ACC, en appliquant une technique de recombinaison non codante ou en utilisant des gènes mutés de synthase de l'ACC.
PCT/US1991/006453 1990-09-10 1991-09-10 Synthase recombinee de l'enzyme acc (acide 1-aminocyclopropane-1-carboxylique) WO1992004456A1 (fr)

Priority Applications (3)

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JP3515173A JPH06502759A (ja) 1990-09-10 1991-09-10 組換えａｃｃシンターゼ
AU85114/91A AU657276B2 (en) 1990-09-10 1991-09-10 Recombinant ACC synthase
CA002091243A CA2091243C (fr) 1990-09-10 1991-09-10 Lyase recombinante acc

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US57989690A 1990-09-10 1990-09-10
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Cited By (28)

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EP0690919A4 (fr) * 1992-10-15 1995-11-16 Gen Hospital Corp Synthase acc de crucifere et utilisations de ladite synthase
WO1996007742A1 (fr) * 1994-09-02 1996-03-14 Asgrow Seed Company Plantes transgeniques exprimant les genes de l'oxydase de l'acide 1-aminocyclopropane-1-carboxylique
EP0716808A2 (fr) * 1994-12-15 1996-06-19 Basf Aktiengesellschaft Utilisation des herbicides du type Auxin pour le traitement des récoltes
WO1996021027A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques exprimant le gene de l'acc synthase
WO1997011166A1 (fr) * 1995-09-20 1997-03-27 The University Of Queensland Nouveaux genes de l'acc synthase
WO1998006852A1 (fr) * 1996-08-12 1998-02-19 University Of Hawaii Proteines purifiees, sequences d'adn de recombinaison et procedes de regulation du mûrissement des cafeiers
WO1998045445A1 (fr) * 1997-04-09 1998-10-15 The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Nothern Ireland Promoteurs de plantes activables
US5952546A (en) * 1996-06-27 1999-09-14 Dna Plant Technology Corporation Delayed ripening tomato plants with T-DNA bearing a truncated ACC2 synthase gene
US6043409A (en) * 1995-06-07 2000-03-28 Seminis Vegetable Seeds, Inc. Transgenic plants expressing ACC oxidase genes
US6075184A (en) * 1996-03-26 2000-06-13 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for producing caffeine free beverages
US6194639B1 (en) 1996-05-01 2001-02-27 The University Of Queensland ACC synthase genes from pineapple
WO2001034812A1 (fr) * 1999-11-09 2001-05-17 Zhejiang Academy Of Agricultural Sciences Procede de regulation de la teneur en proteines/graisses de graines de plantes
US6448474B1 (en) 1995-06-07 2002-09-10 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plants
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
EP1663466A2 (fr) * 2003-06-23 2006-06-07 Pioneer Hi-Bred International Inc. Creation technique d'une aptitude a rester vert dans des plantes
EP2267139A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés ét moyens d'obtention de phénotypes modifies
WO2011001434A1 (fr) 2009-06-30 2011-01-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Introduction d'adn dans des cellules végétales
EP2436769A1 (fr) 2006-06-07 2012-04-04 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. Constructions d'expression végétale et leurs procédés d'utilisation
WO2013078520A2 (fr) * 2011-11-30 2013-06-06 Universidade Estadual De Campinas - Unicamp Procédé pour la production de plantes génétiquement modifiées avec capacité de contrôle de la production de l'hormone éthylène, plantes génétiquement modifiées ainsi obtenues, vecteur à adn recombinant et utilisations correspondantes
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
WO2014079896A1 (fr) 2012-11-21 2014-05-30 Nunhems B.V. Plantes de solanum lycopersicum présentant des altérations non-transgéniques dans le gène acs2
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
WO2015162608A1 (fr) 2013-04-25 2015-10-29 Morflora Israel Ltd. Procédés et compositions pour l'administration d'acides nucléiques dans des semences
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression

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Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0690919A1 (fr) * 1992-10-15 1996-01-10 The General Hospital Corporation Synthase acc de crucifere et utilisations de ladite synthase
EP0690919A4 (fr) * 1992-10-15 1995-11-16 Gen Hospital Corp Synthase acc de crucifere et utilisations de ladite synthase
WO1996007742A1 (fr) * 1994-09-02 1996-03-14 Asgrow Seed Company Plantes transgeniques exprimant les genes de l'oxydase de l'acide 1-aminocyclopropane-1-carboxylique
EP0716808A3 (fr) * 1994-12-15 1997-04-16 Basf Ag Utilisation des herbicides du type Auxin pour le traitement des récoltes
EP0716808A2 (fr) * 1994-12-15 1996-06-19 Basf Aktiengesellschaft Utilisation des herbicides du type Auxin pour le traitement des récoltes
US5670454A (en) * 1994-12-15 1997-09-23 Basf Aktiengesellschaft Herbicides of the auxin type for treating transgenic crop plants
WO1996021027A1 (fr) * 1994-12-30 1996-07-11 Asgrow Seed Company Plantes transgeniques exprimant le gene de l'acc synthase
US5998702A (en) * 1994-12-30 1999-12-07 Seminis Vegetable Seeds, Inc. Transgenic plants expressing ACC synthase gene
US6448474B1 (en) 1995-06-07 2002-09-10 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plants
US5874269A (en) * 1995-06-07 1999-02-23 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plant
US6043409A (en) * 1995-06-07 2000-03-28 Seminis Vegetable Seeds, Inc. Transgenic plants expressing ACC oxidase genes
US6727406B2 (en) 1995-06-07 2004-04-27 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plants
WO1997011166A1 (fr) * 1995-09-20 1997-03-27 The University Of Queensland Nouveaux genes de l'acc synthase
US6864406B1 (en) 1995-09-20 2005-03-08 University Of Queensland ACC synthase gene
US6124525A (en) * 1995-09-20 2000-09-26 The University Of Queensland ACC synthase genes from pineapple, papaya and mango
US6075184A (en) * 1996-03-26 2000-06-13 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for producing caffeine free beverages
US6194639B1 (en) 1996-05-01 2001-02-27 The University Of Queensland ACC synthase genes from pineapple
US5952546A (en) * 1996-06-27 1999-09-14 Dna Plant Technology Corporation Delayed ripening tomato plants with T-DNA bearing a truncated ACC2 synthase gene
WO1998006852A1 (fr) * 1996-08-12 1998-02-19 University Of Hawaii Proteines purifiees, sequences d'adn de recombinaison et procedes de regulation du mûrissement des cafeiers
AU735202B2 (en) * 1996-08-12 2001-07-05 University Of Hawaii Purified proteins, recombinant DNA sequences and processes for controlling the ripening of coffee plants
WO1998045445A1 (fr) * 1997-04-09 1998-10-15 The Minister Of Agriculture Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Nothern Ireland Promoteurs de plantes activables
US9029527B2 (en) 1998-03-20 2015-05-12 Commonwealth Scientific And Industrial Research Organisation Synthetic genes and genetic constructs
US9963698B2 (en) 1998-03-20 2018-05-08 Commonwealth Scientific And Industrial Research Organisation Control of gene expression
EP2267139A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés ét moyens d'obtention de phénotypes modifies
US9441239B2 (en) 1998-04-08 2016-09-13 Commonwealth Scientific & Industrial Research Organisation Methods and means for obtaining modified phenotypes
US8598332B1 (en) 1998-04-08 2013-12-03 Bayer Cropscience N.V. Methods and means for obtaining modified phenotypes
EP2267138A2 (fr) 1998-04-08 2010-12-29 Commonwealth Scientific and Industrial Research Organization Procédés et moyens d'obtention de phénotypes modifiés
EP3214177A2 (fr) 1998-04-08 2017-09-06 Commonwealth Scientific and Industrial Research Organisation Procédés et moyens pour obtenir des phénotypes modifiés
US10190127B2 (en) 1999-08-13 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US9708621B2 (en) 1999-08-13 2017-07-18 Commonwealth Scientific And Industrial Research Organisation Methods and means for obtaining modified phenotypes
US7176350B2 (en) 1999-11-09 2007-02-13 Zhejiang Academy Of Agricultural Science Method for controlling ratio of proteins/lipids in crop seeds
WO2001034812A1 (fr) * 1999-11-09 2001-05-17 Zhejiang Academy Of Agricultural Sciences Procede de regulation de la teneur en proteines/graisses de graines de plantes
WO2003078629A1 (fr) 2002-03-20 2003-09-25 Basf Plant Science Gmbh Produit de synthese et procede de regulation de l'expression genique
EP1663466A2 (fr) * 2003-06-23 2006-06-07 Pioneer Hi-Bred International Inc. Creation technique d'une aptitude a rester vert dans des plantes
US8124860B2 (en) 2003-06-23 2012-02-28 Pioneer Hi-Bred International Inc. Zea mays seeds and plants with reduced expression of the ACS6 gene
AU2011202041B2 (en) * 2003-06-23 2012-09-13 Pioneer Hi-Bred International, Inc. Engineering single-gene-controlled staygreen potential into plants
US7230161B2 (en) 2003-06-23 2007-06-12 Pioneer Hi-Bred International, Inc. Engineering single-gene-controlled staygreen potential into plants utilizing ACC synthase from maize
EP1663466A4 (fr) * 2003-06-23 2007-07-04 Pioneer Hi Bred Int Creation technique d'une aptitude a rester vert dans des plantes
US8129587B2 (en) 2003-06-23 2012-03-06 Pioneer Hi-Bred International, Inc. Zea mays seeds and plants with reduced expression of the ACS2 gene
US7763773B2 (en) 2003-06-23 2010-07-27 Pioneer Hi-Bred International Inc. Engineering single-gene-controlled staygreen potential into plants
US7838730B2 (en) 2003-06-23 2010-11-23 Pioneer Hi-Bred International Inc. Engineering single-gene-controlled staygreen potential into plants
US8779235B2 (en) 2003-06-23 2014-07-15 Pioneer Hi-Bred International, Inc. Engineering single-gene-controlled staygreen potential into plants
EP2980220A1 (fr) 2005-09-20 2016-02-03 BASF Plant Science GmbH Procédés améliorés de contrôle de l'expression de gènes
EP2436769A1 (fr) 2006-06-07 2012-04-04 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. Constructions d'expression végétale et leurs procédés d'utilisation
WO2011001434A1 (fr) 2009-06-30 2011-01-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Introduction d'adn dans des cellules végétales
WO2013078520A3 (fr) * 2011-11-30 2013-09-19 Universidade Estadual De Campinas - Unicamp Procédé pour la production de plantes génétiquement modifiées avec capacité de contrôle de la production de l'hormone éthylène, plantes génétiquement modifiées ainsi obtenues, vecteur à adn recombinant et utilisations correspondantes
WO2013078520A2 (fr) * 2011-11-30 2013-06-06 Universidade Estadual De Campinas - Unicamp Procédé pour la production de plantes génétiquement modifiées avec capacité de contrôle de la production de l'hormone éthylène, plantes génétiquement modifiées ainsi obtenues, vecteur à adn recombinant et utilisations correspondantes
WO2013184768A1 (fr) 2012-06-05 2013-12-12 University Of Georgia Research Foundation, Inc. Compositions et méthodes d'inactivation génique dans les plantes
WO2014079896A1 (fr) 2012-11-21 2014-05-30 Nunhems B.V. Plantes de solanum lycopersicum présentant des altérations non-transgéniques dans le gène acs2
US9832943B2 (en) 2012-11-21 2017-12-05 Nunhems B.V. Solanum lycopersicum plants having non-transgenic alterations in the Acs2 gene
WO2015162608A1 (fr) 2013-04-25 2015-10-29 Morflora Israel Ltd. Procédés et compositions pour l'administration d'acides nucléiques dans des semences

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AU8511491A (en) 1992-03-30
MX9100993A (es) 1992-05-04
CA2091243A1 (fr) 1992-03-11

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