EP0835311A2 - Facteurs transcriptionnels de la fibre de coton - Google Patents

Facteurs transcriptionnels de la fibre de coton

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Publication number
EP0835311A2
EP0835311A2 EP96921474A EP96921474A EP0835311A2 EP 0835311 A2 EP0835311 A2 EP 0835311A2 EP 96921474 A EP96921474 A EP 96921474A EP 96921474 A EP96921474 A EP 96921474A EP 0835311 A2 EP0835311 A2 EP 0835311A2
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EP
European Patent Office
Prior art keywords
cotton
cotton fiber
plant
sequence
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96921474A
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German (de)
English (en)
Inventor
Kevin Mcbride
David M. Stalker
Julie R. Pear
Luis Perez-Grau
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Monsanto Co
Original Assignee
Calgene LLC
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Publication date
Application filed by Calgene LLC filed Critical Calgene LLC
Publication of EP0835311A2 publication Critical patent/EP0835311A2/fr
Withdrawn legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8233—Female-specific, e.g. pistil, ovule
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    • 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
    • C12N15/825—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 pigment biosynthesis
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    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates to methods of using in vitro constructed DNA transcription or expression cassettes capable of directing fiber-tissue transcription of a DNA sequence of interest in plants to produce fiber cells having an altered phenotype, and to methods of providing for or modifying various characteristics of cotton fiber.
  • the invention is exemplified by methods of using cotton fiber promoters for altering the phenotype of cotton fiber, and cotton fibers produced by the method.
  • One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in fiber tissue, i.e. cotton fiber cells, so as to provide for altered or improved aspects of the mature cell type.
  • Cotton is a plant of great commercial significance. In addition to the use of cotton fiber in the production of textiles, other uses of cotton include food preparation with cotton seed oil and animal feed derived from cotton seed husks. Despite the importance of cotton as a crop, the breeding and genetic engineering of cotton fiber phenotypes has taken place at a relatively slow rate because of the absence of reliable promoters for use in selectively effecting changes in the phenotype of the fiber.
  • transcription initiation regions capable of initiating transcription in fiber cells during development are desired.
  • an important goal of cotton bioengineering research is the acquisition of a reliable promoter which would permit expression of a protein selectively in cotton fiber to affect such qualities as fiber strength-, length, color and dyability.
  • Cotton fiber-specific promoters are discussed in PCT publications WO 94/12014 and WO 95/08914, and John and Crow, Proc. Natl. Acad. Sci. USA, 89:5769-5773, 1992.
  • cDNA clones that are preferentially expressed in cotton fiber have been isolated.
  • One of the clones isolated corresponds to mRNA and protein that are highest during the late primary cell wall and early secondary cell wall synthesis stages. John and Crow, supra.
  • ras superfamily In animals, the ras superfamily is subdivided into the subfamilies ras which is involved in controlling cell growth and division, rah/YPT members which control secretory processes, and rho which is involved in control of cytoskeletal organization (Bourne et al. , (1991) Nature 349: 117-127), and number of homologous genes have now been identified in plants (for a review, see Terryn et al. , (1993) Plant Mol. Biol. 22: 143-152).
  • Rho In animals, two members of the rho subfamily, called Rac and Rho, have been shown to be involved in the regulation of actin organization (for a review, see Downward, (1992) Nature 359: 273- 274) .
  • RhoA regulates the formation of actin stress fibers associated with focal adhesions (Ridley and Hall, (1992) Cell 70: 389-399).
  • the CDC42 gene codes for a rho-type protein which also regulates actin organization involved in the establishment of cell polarity required for the localized deposition of chitin in the bud scar (Adams et al. , (1990) J Cell Biol 111: 131-143.
  • Cotton fiber represents an excellent system for studying cytoskeletal organization.
  • Cotton fibers are single cells in which cell elongation and secondary- wall deposition can be studied as distinct events. These fibers develop synchronously within the boll following anthesis, and each fiber cell elongates for about 3 weeks, depositing a thin primary wall (Meinert and Delmer, (1984) Plant Physiol. 59: 1088-1097;
  • Agrobacterium-mediated cotton transformation is described in Umbeck, United States Patents Nos. 5,004,863 and 5,159,135 and cotton transformation by particle bombardment is reported in WO 92/15675, published September 17, 1992. Transformation of Brassica has been described by Radke et al . (Theor. Appl. Genet. (1988) 75;685-694; Plant Cell Reports (1992) 11:499-505.
  • Novel DNA constructs and methods for their use are described which are capable of directing transcription of a gene of interest in cotton fiber, particularly early in fiber development and during secondary cell wall development.
  • the novel constructs include a vector comprising a transcriptional and translational initiation region obtainable from a gene expressed in cotton fiber and methods of using constructs including the vector for altering fiber phenotype. Both the endogenous 3 ' regions and 5' regions may be important in directing efficient transcription and translation.
  • Three promoters are provided from genes involved in the regulation of cotton fiber development.
  • Racl3 is from a protein in cotton which codes for an animal Rac protein homolog. Racl3, shows highly-enhanced expression during fiber development.
  • Rho3 is a gene that is moderately expressed during fiber development turning on at 9 dpa and shutting down approximately 24 dpa. It is maximally expressed between 17-21 dpa developing fiber.
  • Another promoter from a cotton protein is designated 4-4.
  • the 4-4 mRNA accumulates in fiber cells at day 17 post anthesis and continues towards fiber maturity, which occurs at 60 days or so post anthesis. Data demonstrates that the 4-4 promoter remains very active at day 35 post anthesis.
  • Ltp lipid transfer protein
  • the methods of the present invention include transfecting a host plant cell of interest with a transcription or expression cassette comprising a cotton fiber promoter and generating a plant which is grown to produce fiber having the desired phenotype.
  • Constructs and methods of the subject invention thus find use in modulation of endogenous fiber products, as well as production of exogenous products and in modifying the phenotype of fiber and fiber products.
  • the constructs also find use as molecular probes.
  • constructs and methods for use in gene expression in cotton embryo tissues are considered herein. By these methods, novel cotton plants and cotton plant parts, such as modified cotton fibers, may be obtained.
  • constructs and methods of use relating to modification of color phenotype in cotton fiber contain sequences for expression of genes involved in the production of colored compounds, such as anthocyanins, melanin or indigo, and also may contain sequences which provide for targeting of the gene products to particular locations in the plant cell, such as plastid organelles, or vacuoles.
  • Plastid targeting is of particular interest for expression of genes involved in aromatic amino acid biosynthesis pathways, while vacuolar targeting is of particular interest where the precursors required in synthesis of the pigment are present in vacuoles.
  • plants producing fibers which are color that is, with pigment produced in the fiber by the plant during fiber development, as opposed to fibers which are harvested and dyed or otherwise pigmented by separate processing.
  • Fibers from a plant producing such colored fiber may be used to produce colored yarns and/or fabric which have not been subjected to any dyeing process. While naturally colored cotton has been available from various domesticated and wild type cotton varieties, the instant application provides cotton fiber has a color produced by the expression of a genetically engineered protein.
  • the application provides constructs and methods of use relating to modification of color phenotype in cotton fiber.
  • Such constructs contain sequences for expression of genes involved in the production of colored compounds, such as melanin or indigo, and also contain sequences which provide for targeting of the gene products to particular locations in the plant cell, such as plastid organelles, or vacuoles.
  • Plastid targeting is of particular interest for expression of genes involved in the aromatic amino acid biosynthesis pathways, while vacuolar targeting is of particular interest where the precursors required in synthesis of the pigment are present in vacuoles.
  • Figure 1 shows the DNA sequence encoding the structural protein from cDNA 4-4.
  • Figure 2 shows the sequence to the promoter construct pCGN5606 made using genomic DNA from 4-4-6 genomic clone.
  • Figure 3 shows the sequence to the 4-4 promoter construct PCGN5610.
  • Figure 4 shows the cDNA sequence encoding the Racl3 gene expressed in cotton fiber.
  • Figure 5 shows the sequence the promoter region from the racl3 gene.
  • Figure 6 shows a restriction map for pCGN4735.
  • Figure 7 shows the sequence of the Ltp promoter region from a cotton fiber specific lipid transfer protein gene.
  • Figure 8 shows the arrangement of a binary vectors pCGN5148 and pCGN5616 for plant transformation to express genes for melanin synthesis and indigo synthesis, respectively.
  • Figure 9 provides the results of color measurements taken from fibers of the control Coker 130 cotton used in transformation using color constructs.
  • Figure 10 shows the results of measurements made of color of plants transformed by the pCGN5148 construct to express genes for melanin synthesis.
  • Figure 11 shows the results of measurements taken of the color of plants transformed by the pCGN5149 construct to express genes for melanin synthesis.
  • Figure 12 shows the results of measurements made of color of plants transformed to express genes for indigo synthesis, using construct pCGN5616.
  • Figure 13 shows control measurements made of naturally colored cotton plants which are produced by non-transgenic colored cotton plants.
  • novel constructs and methods are described, which may be used provide for transcription of a nucleotide sequence of interest in cells of a plant host, preferentially in cotton fiber cells to produce cotton fiber having an altered color phenotype.
  • Cotton fiber is a differentiated single epidermal cell of the outer integument of the ovule. It has four distinct growth phases; initiation, elongation (primary cell wall synthesis), secondary cell wall synthesis, and maturation. Initiation of fiber development appears to be triggered by hormones.
  • the primary cell wall is laid down during the elongation phase, lasting up to 25 days postanthesis (DPA) . Synthesis of the secondary wall commences prior to the cessation of the elongation phase and continues to approximately 40 DPA, forming a wall of almost pure cellulose.
  • DPA days postanthesis
  • the constructs for use in such cells may include several forms, depending upon the intended use of the construct.
  • the constructs include vectors, transcriptional cassettes, expression cassettes and plasmids.
  • the transcriptional and translational initiation region (also sometimes referred to as a "promoter,”), preferably comprises a transcriptional initiation regulatory region and a translational initiation regulatory region of untranslated 5' sequences, "ribosome binding sites,” responsible for binding mRNA to ribosomes and translational initiation. It is preferred that all of the transcriptional and translational functional elements of the initiation control region are derived from or obtainable from the same gene.
  • the promoter will be modified by the addition of sequences, such as enhancers, or deletions of nonessential and/or undesired sequences.
  • sequences such as enhancers, or deletions of nonessential and/or undesired sequences.
  • obtainable is intended a promoter having a DNA sequence sufficiently similar to that of a native promoter to provide for the desired specificity of transcription of a DNA sequence of interest. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
  • Cotton fiber transcriptional initiation regions chosen for cotton fiber modification may include the 4-4, racl3 and Ltp cotton fiber promoter regions provided herein.
  • a transcriptional cassette for transcription of a .nucleotide sequence of interest in cotton fiber will include in the direction of transcription, the cotton fiber transcriptional initiation region, a DNA sequence of interest, and a transcriptional termination region functional in the plant cell.
  • the cassette provides for the transcription and translation of a DNA sequence of interest it is considered an expression cassette.
  • One or more introns may be also be present.
  • Other sequences may also be present, including those encoding transit peptides and secretory leader sequences as desired.
  • Fiber-tissue transcription initiation regions of this invention are, preferably, not readily detectable in other plant tissues.
  • Transcription initiation regions capable of initiating transcription in other plant tissues and/or at other stages of fiber development, in addition to the foregoing, are acceptable insofar as such regions provide a significant expression level in cotton fiber at the defined periods of interest and do not negatively interfere with the plant as a whole, and, in particular, do not interfere with the development of fiber and/or fiber-related parts.
  • Downstream from, and under the regulatory control of, the cotton fiber transcriptional/translational initiation control region is a nucleotide sequence of interest which provides for modification of the phenotype of fiber.
  • the nucleotide sequence may be any open reading frame encoding a polypeptide of interest, for example, an enzyme, or a sequence complementary to a genomic sequence, where the genomic sequence may be an open reading frame, an intron, a noncoding leader sequence, or any other sequence where the complementary sequence inhibits transcription, messenger RNA processing, for example, splicing, or translation.
  • the nucleotide sequences of this invention may be synthetic, naturally derived, or combinations thereof. Depending upon the nature of the DNA sequence of interest, it may be desirable to synthesize the sequence with plant preferred codons.
  • the plant preferred codons may be determined from the codons of highest frequency in the proteins expressed in the largest amount in the particular plant species of interest.
  • Phenotypic modification can be achieved by modulating production either of an endogenous transcription or translation product, for example as to the amount, relative distribution, or the like, or an exogenous transcription or translation product, for example to provide for a novel function or products in a transgenic host cell or tissue.
  • an exogenous transcription or translation product for example to provide for a novel function or products in a transgenic host cell or tissue.
  • DNA sequences encoding expression products associated with the development of plant fiber including genes involved in metabolism of cytokinins, auxins, ethylene, abscissic acid, and the like. Methods and compositions for modulating cytokinin expression are described in United States Patent No. 5,177,307, which disclosure is hereby incorporated by reference.
  • genes from sources including other eukaryotic or prokaryotic cells, including bacteria, such as those from Agrobacterium tumefaciens T-DNA auxin and cytokinin biosynthetic gene products, for example, and mammals, for example interferons, may be used.
  • phenotypic modifications include modification of the color of cotton fibers.
  • genes involved in production of melanin and genes involved in the production of indigo are dark brown pigments found in animals, plants and microorganisms, any of which may serve as a source for sequences for insertion into the constructs of the present invention.
  • Specific examples include the tyrosinase gene which can be cloned from Streptomyces an iJ ioticus.
  • the ORF438 encoded protein in S. antibioticus also is necessary for melanin production, and may provide a copper donor function.
  • a tyrosinase gene can be isolated from any organism which makes melanin.
  • the gene can be isolated from human hair, melanocytes or melanomas, cuttle fish and red roosters, among others. See, for example, EP Application No. 89118346.9 which discloses a process for producing melanins, their precursors and derivatives in microorganisms. Also, See, Bernan et al . Gene (1985) 37:101-110; and della-Cioppa et aJ. Bio/Technology (1990) 8:634-638.
  • Indigo may be obtained by use of genes encoding a ono- oxygenase such as xylene oxygenase which oxidizes toluene and xylene to (methyl) benzyl alcohol and also transforms indole to indigo.
  • a ono- oxygenase such as xylene oxygenase which oxidizes toluene and xylene to (methyl) benzyl alcohol and also transforms indole to indigo.
  • nucleotide sequence in characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida See. Kurkela et al . Gene (1988) 73:355-362.
  • a tryptophanase gene sequence can be used in conjunction with an oxygenase to increase the amount of indole available for conversion to indigo.
  • Sources of tryptophanase gene sequences include E. coli (see, for example, Deeley et al . (1982) J. Bacteriol . 151 .942-951) .
  • Plastid targeting sequences are available from a number of plant nuclear-encoded plastid proteins, such as the small subunit (SSU) of ribulose bisphosphate carboxylase, plant fatty acid biosynthesis related genes including acyl carrier protein (ACP) , stearoyl-ACP desaturase, ⁇ -ketoacyl-ACP synthase and acyl-ACP thioesterase, or LHCPII genes.
  • the encoding sequence for a transit peptide which provides for transport to plastids may include all or a portion of the encoding sequence for a particular transit peptide, and may also contain portions of the mature protein encoding sequence associated with a particular transit peptide.
  • transit peptides which may be used to deliver a target protein into a plastid organelle.
  • the particular transit peptide encoding sequence used in the instant invention is not critical, as long as delivery to the plastid is obtained.
  • the desired constructs may be used to transform the plastid genome directly.
  • promoters capable of providing for transcription of genes in plant plastids are desired.
  • T7 promoter to provide for high levels of transcription.
  • T7 polymerase may be expressed from a nuclear construct and targeted to plastids using transit peptides as described above. (See McBride et al . (1994) Proc. Nat . Acad. Sci . 51:7301-7305; see also copending US patent application entitled “Controlled Expression of Transgenic Constructs in Plant Plastids", serial no.
  • Tissue specific or developmentally regulated promoters may be useful for expression of the T7 polymerase in order to limit expression to the appropriate tissue or stage of development.
  • vacuolar targeting constructs also encode a vacuolar localization signal (VLS) positioned at the carboxy terminus of the encoded protein.
  • VLS vacuolar localization signal
  • VLS regions may be obtained from various other plant genes and may be similarly used in the constructs of this invention.
  • Numerous vacuolar targetting peptides are known to the art, as are reviewed in Chrispeels et al . , Cell (1992) 68:613-616.
  • the Maize Al gene which encodes a dihydroflavonol reductase, an enzyme of the anthocyanin pigmentation pathway is one such gene.
  • dihydrokempferol is converted to 2-8 alkylleucopelargonidin, which may be further metabolized to pelargonidin pigment by endogenous plant enzymes.
  • Other anthocyanin or flavonoid type pigments may also be of interest for modification of cotton cell fibers, and have been suggested for use in plant flowers (for a review of plant flower color, see van Tunen et al . , Plant Biotechnology Series, Volume 2 (1990) Developmental Regulation of Plant Gene Expression, D. Grierson ed.) .
  • Anthocyanin is produced by a progression of steps from cellular phenylalanine pools.
  • the R anc Cl genes are maize regulatory proteins which are active by positively affecting upstream steps in the anthocyanin biosynthesis from these pools.
  • the maize R and Cl genes could be used in enhancing the levels of of anthocyanin produced in fiber cells.
  • the R and Cl proteins are proteins with a positive control at the regulatory level on anthocyanin pigment precursor biosynthesis, these proteins are expressed in the nucleus, and not targetted to plastids or vacuoles.
  • it is of interest to modify other aspects of the fiber For example, it is of interest to modify various aspects of cotton fibers, such as strength or texture of a fiber.
  • the appropriate gene may be inserted in the constructs of the invention, including genes for PHB biosynthesis (see, Peoples et al . J. Biol . Chem. (1989) 264: 15298-15303 and
  • Transcriptional cassettes may be used when the transcription of an anti-sense sequence is desired.
  • expression cassettes providing for transcription and translation of the DNA sequence of interest will be used.
  • Various changes are of interest; these changes may include modulation (increase or decrease) of formation of particular saccharides, hormones, enzymes, or other biological parameters. These also include modifying the composition of the final fiber that is changing the ratio and/or amounts of water, solids, fiber or sugars. Other phenotypic properties of interest for modification include response to stress, organisms, herbicides, brushing, growth regulators, and the like.
  • results can be achieved by providing for reduction of expression of one or more endogenous products, particularly an enzyme or cofactor, either by producing a transcription product which is complementary (anti-sense) to the transcription product of a native gene, so as to inhibit the maturation and/or expression of the transcription product, or by providing for expression of a gene, either endogenous or exogenous, to be associated with the development of a plant fiber.
  • endogenous products particularly an enzyme or cofactor
  • the termination region which is employed in the expression cassette will be primarily one of convenience, since the termination regions appear to be relatively interchangeable.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, may be derived from another source.
  • the termination region may be naturally occurring, or wholly or partially synthetic.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. In some embodiments, it may be desired to use the 3 ' termination region native to the cotton fiber transcription initiation region used in a particular construct.
  • nucleotide sequences will be present in the constructs to provide for targeting of a particular gene product to specific cellular locations.
  • coding sequences for synthesis of aromatic colored pigments are used in a construct, particularly coding sequences for enzymes which have as their substrates aromatic compounds such tyrosine and indole, it is preferable to include sequences which provide for delivery of the enzyme into plastids, such as an SSU transit peptide sequence.
  • targeting to the vacuole may provide for enhanced color modifications.
  • the tyrosinase and ORF438 genes from Streptomyces antibioticus are provided in cotton fiber cells for expression from a 4-4 and Racl3 promoter.
  • the ORF438 and tyrosinase proteins are expressed from the same promoter region.
  • the coding regions may be provided under the regulatory control of separate promoter regions. The promoter regions may be the same or different for the two genes. Alternatively, coordinate expression of the two genes from a single plant promoter may be desired.
  • Constructs for expression of the tyrosinase and ORF438 gene products from 4-4 and rac promoter regions are described in detail in the following examples. Additional promoters may also be desired, for example plant viral promoters, such as CaMV 35S, can be used for constitutive expression of one of the desired gene products, with the other gene product being expressed in cotton fiber tissues from the 4-4 and rac promoter.
  • plant viral promoters such as CaMV 35S
  • promoters may also be useful in certain applications, for example the mas, Mac or DoubleMac, promoters described in United States Patent No. 5,106,739 and by Comai et al . , Plant Mol . Biol . (1990) 15 : 373 -381 ) .
  • the plants may be obtained by co-transformation with both constructs, or by transformation with individual constructs followed by plant breeding methods to obtain plants expressing both of the desired genes.
  • plasmids can be prepared in E. coli which contain DNA homologous with the Ti-plasmid, particularly T-DNA.
  • the plasmid may or may not be capable of replication in Agrobacterium, that is, it may or may not have a broad spectrum prokaryotic replication system such as does, for example, pRK290, depending in part upon whether the transcription cassette is to be integrated into the Ti-plasmid or to be retained on an independent plasmid.
  • the Agrobacterium host will contain a plasmid having the vir genes necessary for transfer of the T-DNA to the plant cell and may or may not have the complete T-DNA. At least the right border and frequently both the right and left borders of the T-DNA of the Ti- or Ri-plasmids will be joined as flanking regions to the transcription construct.
  • T-DNA for transformation of plant cells has received extensive study and is amply described in EPA Serial No.
  • a disarmed Ti-plasmid lacking particularly the tumor genes found in the T-DNA region may be introduced into the plant cell.
  • the construct may be transferred to the A. tumefaciens and the resulting transfected organism used for transfecting a plant cell; explants may be cultivated with transformed A. tumefaciens or A. rhizogenes to allow for transfer of the transcription cassette to the plant cells.
  • terminal repeats of transposons may be used as borders in conjunction with a transposase.
  • transposase should be inducible, so that once the transcription construct is integrated into the genome, it should be relatively stably integrated.
  • Transgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells which are then grown to callus, shoots grown and plantlets generated from the shoot by growing in rooting medium.
  • transgenic plants may be grown to produce fiber having the desired phenotype.
  • the fibers may be harvested, and/or the seed collected.
  • the seed may serve as a source for growing additional plants having the desired characteristics.
  • transgenic plants and transgenic cells include plants and cells derived from either transgenic plants or transgenic cells.
  • sequences provided herein may be used as molecular probes for the isolation of other sequences which may be useful in the present invention, for example, to obtain related transcriptional initiation regions from the same or different plant sources.
  • Related transcriptional initiation regions obtainable from the sequences provided in this invention will show at least about 60% homology, and more preferred regions will demonstrate an even greater percentage of homology with the probes.
  • constructs can also be used in conjunction with plant regeneration systems to obtain plant cells and plants; thus, the constructs may be used to modify the phenotype of fiber cells, to provide cotton fibers which are colored as the result of genetic engineering to heretofor unavailable hues and/or intensities.
  • Cultivated cotton species include Gossypium hirsutum and G. babadense (extra-long stable, or Pi a cotton) , which evolved in the New World, and the Old World crops G. her-aceum and G. arbor eum.
  • Color phenotypes can be assessed by the use of a colorimeter, an instrument which is already used to provide objective measurements of the color of cotton samples.
  • a colorimeter uses a combination of light sources and filters to make various estimates of a samples colors, sometimes referred to as tristimulus values.
  • Munsell devised by the American artist A.. Munsell, uses a classification system of paper color chips assorted according to their hue (Munsell Hue) , lightness (Munsell Value) , and saturation (Munsell Chroma) for visual comparison with a specimen color.
  • the L*C*h color space uses the same diagram as the L*a*b* color space, but uses cylindrical coordinates instead of rectangular coordinates.
  • L* indicates lightness and is the same as the L* of the L*a*b* color space
  • C* is chroma
  • h is the hue angle.
  • the value of chroma C is 0 at the center and increases according to the distance from the center.
  • Hue angle is defined as starting at the +a axis of the L*a*b* space, and is expressed in degrees in a counterclockwise rotation.
  • Example 1 cDNA libraries Tissue preparation for cDNA synthesis
  • Leaf and root tissue were isolated from 8 inch tall greenhouse grown seedlings and immediately frozen in liquid nitrogen. Flowers were collected at the rapidly expanding 3 day preanthesis stage and also frozen. Seed was collected from 21 day postanthesis locules which had been removed from the boll and frozen entire in liquid nitrogen. Once frozen, the fiber was removed from the seed and the denuded seed used for RNA isolation. All fibers were removed from the seed under liquid nitrogen and the fiber was ground to a powder prior to RNA isolation. Fibers were from bolls which had been tagged at anthesis.
  • the lambda ZapIITM cDNA library system of Stratagene was used for screening, and was prepared from cDNA derived from poly-A + mRNA isolated from fibers of Gossypium hirsutum cultivar Acala SJ- 2. The fibers were isolated from bolls harvested at approximately 21 dpa using field-grown plants in Israel. Total RNA was isolated from 21 dpa seeds (G. hirsutum cv
  • RNAs were prepared according to Hall et al . ((1978), Proc Natl Acad Sci USA 75: 3196-3200), with the following modifications.
  • the pellet was dissolved in 1/10 original volume of 10 mM Tris pH7.5 and brought to 35mM potassium acetate pH6.5 and 1/2 volume EtOH was added slowly. The mixture was placed on ice for 15 minutes and then centrifuged at 20,000 x g for 15 minutes at 4°C. The potassium acetate concentration was brought to 0.2M, 2 1/2 volumes EtOH added and the RNA placed at -20 C for several hours.
  • Poly-A + RNA was prepared from total mRNA utilizing an oligo(dT) -cellulose kit (Becton Dickenson) and following the manufacturer's protocol. Cotton genomic DNA was prepared as follows.
  • the sample was centrifuged for 20 minutes at 21,000 x g and the supernatant was filtered through Miracloth into another tube and centrifuged as before. The supernatant was again filtered through Miracloth into 15 mis of room temperature isopropanol in an Oak Ridge tube. After gentle mixing, the sample was incubated at room temperature for 10-60 minutes until the DNA precipitated. The DNA was spooled and allowed to air dry before being resuspended in 4 mis of TE on ice for 1 hour. CsCl was added to 0.97g/ml final concentration and 300 ul lOmg/ml ethidium bromide was also added before filling VTi80 quick seal tubes.
  • the sample was centrifuged overnight at 225,000 x g overnight.
  • the DNA was extracted with water saturated butanol and enough water was added to bring the volume to 4 mis before adding 2 volumes EtOH.
  • the DNA was spooled, air dried and resuspended in 200 ul sterile water.
  • RNA was isolated from various tissues, separated by electrophoresis in 1.2% agarose-formaldehyde gels and transfered onto Nytran Plus membranes (Schleicher and Schuell) .
  • Hybridization conditions consisted of a solution containing 50% formamide(v/v) , 5xSSC, 0.1% SDS, 5mM EDTA, 10X Denhardts solution, 25mM sodium phosphate pH6.5 and 250 ug/ml carrier DNA. Washes were performed in 2xSSC, 0.1% SDS at 42°C 3 times for 30 minutes each time.
  • Cotton genomic DNA (12ug) was digested with various restriction endonucleases, electrophoresed in 0.9% agarose gels and blotted onto Nytran Plus membranes. Hybridization and filter washing conditions for both the 3 ' specific and full-length cDNA insert probes were as described for Northern analysis.
  • Probes derived from 3 ' -untranslated regions were synthesized via oligonucleotide primers from the Racl3 cDNA, corresponding to bases 600-619 and 843-864 ( Figure 4) .
  • Each set of primers was used in a polymerase chain reaction to synthesize copies of 3 ' - specific DNA sequences. These sequences were used as templates in the generation of single-stranded, 32P-labeled probes off the antisense strand in a polymerase chain reaction.
  • the full-length cDNA inserts for Racl3 were used as templates for double stranded, random primed probes using the Prime-It kit (Stratagene) .
  • Example 2 Isolation of cDNA Clones from Cotton cDNA to the 4-4 clone was isolated from the cotton fiber library described above, and shown to express in fiber but not other tissues. This sequence was not related to any known protein. Only 400 kb of encoding sequence was present in this clone, so the library was rescreened using the cDNA to obtain full-length clones. The full-length encoding sequence is provided in Figure 1. By comparing sequences of random cDNA clones against various sequence data banks via BLAST, a National Center for Biotechnology Information service, a clone, designated #105, was found to have an encoding sequence related to that of a reported lipid transfer protein. Another clone was sequenced which showed high homology to animal Rac proteins.
  • Figure 4 shows the cDNA sequence encoding the Racl3 gene expressed in cotton fiber.
  • Rho3 and 4-4 genes were assessed using mRNA prepared from various cotton tissues and from fibers at different stages of development. Blots were hybridized with probes derived from untranslated regions of Ltp, Racl3 and 4-4 genes.
  • the gene for Racl3 exhibits highly-enhanced expression in fibers; virtually no detectable mRNA is present in leaves, roots, or flower parts, even under conditions of extended development time.
  • Racl3 expression is detected in seeds at an age that corresponds to the highest expression levels observed in fiber tissue derived from seeds of this same age. The pattern of Racl3 expression in fibers is very dependent upon the developmental stage.
  • 4-4 mRNA is begins to accumulate in fiber cells only at day 17 post anthesis and continues through at least day 35 post anthesis. Levels peak at day 21 and remain high. 4-4 mRNA is not detected in other cotton tissues, and is not detected in fiber tissue before onset at 17 days post anthesis.
  • the #105 lipid transfer protein cDNA clone was used as a probe against cotton tissue and in a cotton fiber northern.
  • StratageneTM which was used to construct a genomic DNA library from cotton variety Coker 130 (Gossypium hirsutum cv. coker 130) , using DNA obtained from germinating seedlings.
  • the cotton genomic library was probed with a 3 ' -specific Ltp probe and 6 genomic phage candidates were identified and purified.
  • Figure 7 provides an approximately 2 kb sequence of the Ltp promoter region which is immediately 5 ' to the Ltp encoding region.
  • the pCGN5606 promoter construct comprises the 4-4 cotton fiber expression cassette in a first version, version I ( Figure
  • sequences from ntl to 65 and nt 5,494 to 5,547 correspond to fragments of the pBluescriptll polylinker where this cassette is cloned. Unique restriction enzyme sites present in these regions flanking the cassette allow the cloning of the fiber expression cassette into binary vectors including the pCGN 5138 and 1547 series.
  • sequences from nt57 to 5,494 are contained in a lambda phage clone of a cotton Coker 130 genomic library. This lambda genomic clone was given the designation 4-4(6).
  • nt 65 to nt 4,163 corresponds to the 5' flanking region of the 4-4(6) gene.
  • nt 4,163 there is a Ncol restriction site sequence that corresponds to the first codon of the 4-4 (6)ORF.
  • nucleotide 4,163 to 4,502 corresponds to part of the 4-4 (6)ORF.
  • sequence from nt 4,502 to 4,555 is a synthetic polylinker oligonucleotide that contains unique target sites for the restriction enzymes EcoRI, Smal, Sail, Nhel and
  • the genes to be expressed in cotton fiber cells using this cassette can be cloned between the Ncol restriction site and any of the polylinker sites. This operation will replace the stuffer fragment with the gene of interest.
  • the region from nt 4,555to 5,494 corresponds to the 940 nucleotides downstream of the stop codon and constitute the 3' flanking region of the 4-4 (6) gene.
  • PCGN5610 The pCGN5610 construct is a second version of a 4-4 cotton fiber expression cassette, version II, which is a modified version of pCGN5606.
  • the two versions of the 4-4 cotton fiber expression cassette are designed to allow the cloning of tandem arrays of two fiber cassettes in one binary plasmid.
  • the differences with respect to pCGN5606 are very minor and described below.
  • the Xbal restriction site in the region of nt 1 to 65 has been deleted by standard cloning manipulations.
  • the polylinker region is in the reverse orientation of pCGN5606.
  • the sequences from ntl to 57 and nt 5,494 to 5,518 of pCGN5610 correspond to fragments of the pBluescriptll polylinker where this cassette is cloned.
  • Unique restriction enzyme sites present in these regions allow the cloning of the fiber expression cassette into binary vectors of the pCGN 5138 and 1547 series.
  • the sequences from nt57 to 5,494 are contained a lambda phage clone of a Coker 130 genomic library. This clone is described in my notebook as lambda genomic clone 4-4(6).
  • the region from nt 57 to nt 4,155 corresponds to the 5' flanking region.
  • At nt 4,155 there is a Ncol restriction site sequence that corresponds to the first codon of the 4-4 ORF.
  • the region from nucleotide 4,156 to 4,500 corresponds to part of the 4-4 ORF.
  • This fragment from nt4,156 to 4,550 is a stuffer fragment and is left in place to facilitate the monitoring of cloning manipulations.
  • the sequence from nt 4,500 to 4,550 is a synthetic polylinker oligonucleotide containing unique target sites for the restriction enzymes Bglll, Nhel, Sail, Smal and EcoRI.
  • the genes to be expressed in cotton fiber cells using this cassette can be cloned between the Ncol restriction site and any of the polylinker sites. This operation replaces the stuffer fragment with the gene of interest.
  • the region from nt 4,550 to 5,494 corresponds to the 940 nucleotides downstream of .the stop codon and constitute the 3' flanking region of the 4-4 (6) gene.
  • mapping was done with restriction endonucleases. The largest fragment with the Racl3 coding region was identified. Theis was a Pst fragment, and when subcloned in the BluescriptTM KS+ vector (BSKS+; Stratagene) was named pCGN4722. The insert had a length of 9.2 kb.
  • the region of the Pst fragment with the Racl3 coding sequence was identified. DNA sequence was determined for approximately 1.7 kb 5 ' of the start codon and approximately 1.2 kb 3 ' of the stop codon. The entire Rac coding region (exons and introns) was conveniently flanked by Ndel sites. pCGN4722 was digested with Xbal, and a 2.7 kb fragment was removed. Religation gave pCGN4730, which was then digested with Ndel, dropping out a 1.7 kb fragment containing the entire Rac coding region. Religation yielded pCGN4731.
  • a polylinker region was created using overlapping synthetic oligonucleotides which were PCR'ed using primers homologous to the 5' and 3' ends of the resynthesized section.
  • the resulting product was digested with EcooRl and Hind III and ligated into BSKS+ at the EcoRI and Hind III sites.
  • the resulting plasmid was designated pCGN4733.
  • PCGN4731 and pCGN4633 were digested with Ndel and the Ndel fragment containing the synthesized polylinker region from pCGN4733 was dropped in the Ndel site of 4731, giving pCGN4734.
  • This last plasmid was digested with Sal and Xba, and so was
  • PCGN5133 was the 9.2 kb pst fragment in BSKS+ where the polylinker sites flanking the insert were altered to different sites for ease of manipulation. The fragment from pCGN4734 was then placed into the equivalent site of pCGN5143, giving pCGN4735.
  • a sequence for approximately 3 kb of the promoter construct pCGN4735 is provided in Figure 5. The resynthesized sequence falls between the Ndel sites located at bases 1706 and 1898 of the sequences. Thus, the sequence in Figure 5 includes approximately 1.7 kb 5' to the Ndel site 5' to the resynthesized polylinker region.
  • a binary construct for plant transformation to express genes for melanin synthesis is prepared as follows.
  • the melanin genes were originally isolated from the common soil bacterium
  • tyrA encodes the catalytic unit responsible for the polymerization of the amino acid tyrosine, the primary substrate, and is termed tyrosinase.
  • ORF438 is responsible for binding copper and delivering copper to the tyrosinase and activating the enzyme. Expression of both the ORF438 and tyrA genes ensures maximal tyrosinase activity.
  • ORF438 and tyrA were fully re-synthesized with respect to their DNA sequence. This was performed as the initial DNA sequence isolated from Streptomyces has a very high guanine and cytosine (G+C) DNA content. Thus, the ORF438 and tryA genes were re-synthesized to appear more "plant-like" (reduced G+C content) with respect to plant preferred codons encoding their corresponding amino acids.
  • Indigo production involves conversion of the amino acid tryptophan, the primary substrate, into indole which is then converted into indoxyl. Molecules of indoxyl spontaneously convert to indigo in the presence of oxygen.
  • a two gene system was used to affect indigo production in fiber cells.
  • the first gene ( tna) was obtained from the bacterium E. coli and encodes the enzyme tryptophanase.
  • the designation tna stands for the gene encoding tryptophanase from E. coli , an enzyme which converts tryptophan to indole (Stewart et al., (1986) J Bacteriol 166:217- 223).
  • the pig designation is used for the encoding sequence to the protein for indigo production from Rhodococcus, which produces indigo from indole (Hart et al . , (1990) J Gen Microbiol 136:1357- 1363) . Both tna and pig were obtained by PCR. Tryptophanase is responsible for the conversion of tryptophan to indole, while the second gene (pig) encodes an indole oxygenase enzyme responsible for the conversion of indole to indoxyl. Both these bacterial genes were utilized in their native form.
  • constructs for Targeting Pigment Synthesis contain a fragment of the tobacco ribulose bisphosphate carboxylase small subunit gene encoding the transit peptide and 12 amino acids of the mature protein (Tssu) positioned in reading frame with the appropriate encoding sequence.
  • constructs include a fragment of the metallocarboxypeptidase inhibitor gene, encoding the entire 32 amino acid N-terminus signal peptide of that protein plus 6 amino acids of the mature protein (CPI+6) (Martineau et al . , supra) , positioned in reading frame with the appropriate encoding sequences.
  • CPI+6 6 amino acids of the mature protein
  • VLS vacuolar localization signal
  • Constructs which contain encoding sequences for bacterial genes involved in biosynthesis of pigmented compounds and sequences for directing transport of the encoded proteins into plastids or vacuoles are prepared as follows.
  • the re-synthesized ORF438 and tyrA genes were treated in two distinct ways depending on which compartment in the fiber cell the final protein products would be localized.
  • One chimeric gene/plant binary construct (designated pCGN5148) contained the genes targeted to the fiber cell plastids. To do this, 12 amino acids of a gene for the small subunit of carboxylase (SSU) plus the original 54 amino acid SSU transit peptide were fused to the amino termini of both the ORF438 and tyrA gene products respectively. These peptide sequences allow the ORF438 and tyrA gene products (proteins) to be efficiently targeted to the plastid. This targeting was initiated as the plastid is the site of tyrosine production within the fiber cell.
  • SSU small subunit of carboxylase
  • the second chimeric gene/plant binary construct (designated pCGN5149) contained the ORF438 and tyrA genes targeted to the vacuole within the fiber cell. Based on information from other biological systems, it was postulated that the fiber cell vacuole may contain a high concentration of tyrosine for melanin polymerization.
  • Both the ORF438 and tryA genes contain the 29 amino acid signal peptide from a tomato carboxypeptidase inhibitor (CPI) protein as amino terminal gene fusions to direct these proteins to the endoplasmic reticulum (ER) secretory system of the fiber cell.
  • CPI carboxypeptidase inhibitor
  • the tyrA gene has an 8 amino acid vacuolar targeting peptide (VTP) from CPI fused at the carboxy terminus so that the mature copper-activated tyrosinase will eventually be targeted to the vacuole of the fiber cell.
  • VTP vacuolar targeting peptide
  • Both the ORF438 and tyrA proteins also had potential glycosylation sites removed via site-directed mutagenesis of the ORF438 and tyrA genes respectively. Potential plant cell glycosylation of these proteins upon their expression in fiber cells could result in tyrosinase inactivation, hence removal of potential glycosylation sites was deemed necessary.
  • the only modification to the indigo genes was the fusion of the tobacco SSU transit peptide encoding DNA sequences onto the amino terminal region of both the tna and pig genes to affect the localization of both the tryptophanase and indole oxygenase proteins to the fiber cell plastid. These are the same exact gene fusions that were made for the plastid-directed proteins for melanin production in construct 5148. The tna and pig gene products were targeted to the fiber cell plastid as that is the primary site of tryptophan synthesis.
  • the modified genes for both the plastid and vacuolar targeted ORF438 and tyrosinase proteins were placed into a fiber expression cassette to be "switched” on during development of the cotton fiber cell.
  • the "switch" (promoter) utilized for the melanin constructs was 4-4.
  • the modified ORF438 and tyrA genes were cloned into the 4-4 promoter cassette and these chimeric genes then inserted into a binary plasmid to create plasmids pCGN5148 and pCGN5149, containing the modified genes for plastid and vacuolar targeted ORF438 and tyrosinase proteins, respectively.
  • binary plasmids also contain genetic determinants for their stable maintenance in E. coli and Agrobacterium and also contain a chimeric gene for plant cell expression of the bacterial kanamycin resistance gene.
  • This kanamycin resistance marker allows for the selection of transformed versus non-transformed cotton cells when plant hypocotyl or leaf segments are infected with Agrobacterium containing the binary plasmids.
  • Plasmid pCGN5148 (not shown) is constructed the same as 5149, only pCGN5148 has plastid- targetting sequences.
  • plasmid pCGN5616 A block diagram of plasmid pCGN5616 is shown in Figure 8.
  • Anthocyanin A construct has been prepared for the expression of the maize R and CI genes in developing cotton fiber. These genes are known to be responsible for the production of Anthocyanin pigments by acting in a regulatory manner to turn on the chalcone pathway for production of anthocyanins (red spectrum colors) .
  • the R and CI genes were placed under the control of the Racl3 promoter cassette.
  • a binary plasmid designated pCGN4745 (not shown), contains both the R and CI genes each under control of the Racl3 promoter.
  • Coker 315 seeds are surface disinfected by placing in 50% Clorox (2.5% sodium hypochlorite solution) for 20 minutes and rinsing 3 times in sterile distilled water. Following surface sterilization, seeds are germinated in 25 x 150 sterile tubes containing 25 mis 1/2 x MS salts: 1/2 x B5 vitamins: 1.5% glucose: 0.3% gelrite. Seedlings are germinated in the dark at 28°C for 7 days. On the seventh day seedlings are placed in the light at 28 ⁇ 2°C.
  • Feeder plates are prepared one day before use by plating 1.0 ml tobacco suspension culture onto a petri plate containing Callus Initiation Medium CIM without antibiotics (MS salts: B5 vitamins: 3 % glucose: 0.1 mg/L 2,4-D: 0.1 mg/L kinetin: 0.3% gelrite, pH adjusted to 5.8 prior to autoclaving) .
  • a sterile filter paper disc (Whatman #1) was placed on top of the feeder cells prior to use. After all sections are prepared, each section was dipped into an A. tumefaciens culture, blotted on sterile paper towels and returned to the tobacco feeder plates.
  • Embryogenic callus was identified 2-6 months following initiation and was subcultured onto fresh regeneration medium. Embryos are selected for germination, placed in static liquid Embryo Pulsing Medium (Stewart and Hsu medium: 0.01 mg/1 NAA: 0.01 mg/L kinetin: 0.2 mg/L GA3) and incubated overnight at 30°C. The embryos are blotted on paper towels and placed into Magenta boxes containing 40 mis of Stewart and Hsu medium solidified with
  • Cotton growth conditions in growth chambers are as follows: 16 hour photoperiod, temperature of approximately 80-85°, light intensity of approximately 500 ⁇ Einsteins.
  • Cotton growth conditions in greenhouses are as follows: 14-16 hour photoperiod with light intensity of at least 400 ⁇ Einsteins, day temperature 90-95°F, night temperature 70-75°F, relative humidity to approximately 80%.
  • Transgenic fiber was collected from individual plant transformants at different stages of fiber development and analyze in two ways. One was to analyze fiber at a single developmental time point for each transgenic cotton plant to compare tyrosinase expression between transgenic events. The other was to screen developing fiber from selected plants to analyze the timing of tyrosinase expression under the control of the fiber-specific 4-4 promoter, by Western blots using antisera prepared against purified tyrosinase protein.
  • Fiber from pCGN5148 (plastid-directed) plants demonstrates a bluish-green color phenotype.
  • Coker 130 cotton fiber cells do not typically demonstrate a negative a* value.
  • These colored cotton cells also had a color located on the L*C*h color space with a relatively high hue angle value h, greater than 135'.
  • Normal Coker 130 fibers have a similar value which is not greater than about 90' as measured by this method.
  • Transgenic fiber was collected from individual plant transformants at different stages of fiber development.
  • the transgenic developing fiber is screened from selected plants to analyze the timing of tna and pig gene expression under the control of the fiber-specific 4-4 promoter and fiber is also analyzed at a single developmental time point for each transgenic cotton plant for comparison of both tryptophanase and indole oxygenase expression between transgenic events, by using Western blots with antisera prepared against the tryptophanase and indole oxygenase proteins.
  • the above results demonstrate that the color phenotype of a transgenic cotton fiber cell can be altered by expressing pigment synthesis genes .
  • the transgenic cotton fiber cells include both a pigment synthesizing protein, and pigment produced by the pigment synthesizing protein.
  • expression of a pigment gene of interest can result in cotton fiber cells in which the synthesis of pigments combined with appropriate targeting sequences results in modification of color phenotype in the selected plant tissue, yielding colored cotton fiber by expression from a genetically engineered construct.

Abstract

L'invention décrit des ADN de recombinaison de type nouveau pouvant être utilisés comme sondes moléculaires ou insérés à l'intérieur d'une plante hôte pour y effectuer la modification de la transcription d'une séquence d'ADN à étudier au cours de diverses étapes du développement de la fibre de coton. Lesdits ADN de recombinaison comportent une région de régulation d'initiation de la transcription de la fibre de coton associée à un gène exprimé dans la fibre de coton. L'invention décrit également un coton de type nouveau ayant une fibre de coton de couleur naturelle introduite par l'expression dans la cellule de fibre de coton, à l'aide d'une telle structure de recombinaison, de gènes de synthèse de pigment. L'invention inclut les cellules de fibre de coton ayant une couleur produite par génie génétique et les cellules de coton comprenant des pigments mélaniques et indigo.
EP96921474A 1995-06-07 1996-06-07 Facteurs transcriptionnels de la fibre de coton Withdrawn EP0835311A2 (fr)

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CA2221747A1 (fr) 1996-12-19
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WO1996040924A3 (fr) 1997-02-06
WO1996040924A2 (fr) 1996-12-19
MX9709724A (es) 1998-07-31

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