MXPA97009724A - Fiber transcription factors of something - Google Patents

Abstract

Novel DNA constructs are provided which can be used as molecular probes, or can be inserted into a host plant, to provide a modification of the transcription of a DNA sequence of interest during different stages of the development of the cotton fiber. The DNA constructs comprise a transcriptional initiation region of cotton fiber. Also novel cotton is provided which has a cotton fiber which has a natural color introduced by the expression, in the cello of the cotton fiber, using this construction, of pigment synthesis genes. It includes cotton fiber cells that have the color produced by genetic engineering, and cotton cells that comprise the pigments of melanin eíndi

Description

TRANSCRIPTION FACTORS OF COTTON FIBER REFERENCE WITH RELATED APPLICATIONS This application is a partial continuation of the United States of America request with Serial No. 08 / 487,087 filed on June 7, 1995, and a partial continuation of the United States of America's Application Number Series 08 / 480,178, filed on June 7, 1995.

INTRODUCTION Technical Field This invention relates to the method for using transcription cassettes or DNA expression cassettes constructed in vitro, capable of directing the transcription of fiber tissue of a DNA sequence of interest, in plants, to produce fiber cells having a altered phenotype, and methods to provide or modify different characteristics of cotton fiber. The invention is exemplified by methods for using the cotton fiber promoters to alter the phenotype of the cotton fiber, and cotton fibers produced by the method.

Background In general, genetic engineering techniques have been directed at modifying the phenotype of individual prokaryotic and eukaryotic cells, especially in culture. Plant cells have proven to be more intransigent than other eukaryotic cells, due not only to a lack of adequate vector systems, but also as a result of the different goals involved. For many applications, it is desirable to be able to control the gene expression at a particular stage of the growth of a plant, or in a particular plant part. For this purpose, regulatory sequences are required that provide the desired transcription initiation in the appropriate cell types and / or at the appropriate time in the development of the plant, without having serious detrimental effects on the development and productivity of the plant. Accordingly, it is interesting to be able to isolate sequences that can be used to provide the desired regulation of transcription in a plant cell during the growth cycle of the host plant. One aspect of this interest is the ability to change the phenotype of particular cell types, such as differentiated epidermal cells that originate in the fiber tissue, i.e., the cells of the cotton fiber, to provide 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 cottonseed oil, and animal feed derived from cottonseed 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, due to the absence of reliable promoters to be used to selectively effect changes in the phenotype of cotton. the fiber. In order to effect the desired phenotypic changes, transcription initiation regions capable of initiating transcription in the fiber cells during development are desired. Therefore, an important goal of cotton bioengineering research is the acquisition of a reliable promoter that allows the expression of a protein selectively in cotton fiber, to affect qualities such as fiber strength, length, color , and the possibility of dyeing.

Relevant Literature Cotton fiber specific promoters are discussed in the publications of WO WO 94/12014 and WO 95/08914, and in John and Crow, Proc. Nati Acad. Sci. USA, 89: 5769-5773, 1992. cDNA clones which are preferably expressed on cotton fiber have been isolated. One of the isolated clones corresponds to mRNA and protein that are higher during the stages of synthesis of the late primary cell wall and the early secondary cell wall. John and Crow, supra. In animals, the ras superfamily is subdivided into the ras subfamilies, which are involved in the control of growth and cell division, the rab / YPT members that control the secretory processes, and rho that is involved in the control of the organization. cytoskeletal (Bourne et al. (1991) Nature 349: 117-127), and numbers of homologous genes have now been identified in plants (for a review, see Terryn et al. (1993) Plant Mol. Biol. 22: 143-152) . None have been found for the important ras subfamily, and all but one of the genes identified have been members of the rab / YPTl subfamily, and there is only one recent report of the cloning of a rho gene in peas (Yang and Watson ( 1993) Proc. Nati, Acad. Sci. USA 90: 8732-8736). Little work has been done to characterize the functions of these genes in plants, although a growing report has shown that a small G protein from Arabidopsis can functionally complement a mutant form in yeast involved in vesicle trafficking, suggesting a similar function for the plant gene (Bednarek et al. (1994) Plant Physiol 104: 591-596). In animals, it has been shown that two members of the rho subfamily, called Rae and Rho, are involved in the regulation of the actin organization (for a review, see Downward, (1992) Nature 359: 273-274). It has been shown that Racl mediates membrane puckering induced by the growth factor, by influencing the alignment of microfilament in the plasma membrane (Ridley et al., (1992) Cell 70: 401-410), while the RhoA regulates the formation of actin tension fibers associated with focal adhesions (Ridley and Hall, (1992) Cell 70: 389-399). In yeast, the CDC42 gene codes for a Rho-like protein, which also regulates the actin organization involved in establishing the cell polarity required for the localized deposition of chitin in the yolk crust (Adams et al., 1990 J. Cell Biol lll: 131-143). The alteration of the genetic function, either by changes in temperature with the temperature-sensitive mutant CDC42 in yeast (Adams et al., 1990), or by microinjection in fibroblasts of the Rae or Rho mutant proteins that exhibit a donation phenotype dominant (Ridley et al., 1992; Ridley and Hall, 1992), leads to the disorganization of the actin network. In plants, the control of the cytoskeletal organization is poorly understood, despite its importance for the regulation of cell division patterns, expansion, and the subsequent deposition of secondary cell wall polymers. The cotton fiber represents an excellent system to study the cytoskeletal organization. Cotton fibers are simple cells in which cell elongation and secondary wall deposition can be studied as separate events. These fibers develop synchronously within the sheath followed by the anthesis, and each fiber cell stretches for about 3 weeks, depositing a thin primary wall (Meinert and Delmer, (1984) Plant Physiol, 59: 1088-1097; Basra and Malik, (1984) Int Rev of Cytol 89: 65-113). At the time of transition to cellulose synthesis of the secondary wall, the fiber cells undergo a synchronous change in the cortical microtubule pattern and in the cell wall microfibril alignments, events that can be regulated upstream by The organization of actin (Seagull, (1990) Protoplasma 159: 44-59; and (1992) In: Proceedings of the Cotton Fiber Cellulose Conference, National Cotton Council of America, Memphis RN, pages 171-192. Agrobacterium mediated is described in Umbeck, US Pat. Nos. 5,004,863 and 5,159,135, and the transformation of cotton by bombardment of particles is reported in International Publication Number WO 92/15675, published on September 17, 1992. The Brassica transformation has been described by Radke et al. (Theor. Appl. Genet. (1988) 75: 685-694; Plant Cell Reports (1992) 11: 499-505.

SUMMARY OF THE INVENTION [0002] Novel DNA constructs and methods for their use are described, which are capable of directing the transcription of a gene of interest in cotton fiber, particularly early in the development of the fiber, and during the development of the wall. secondary cellular Novel constructs include a vector comprising a transcription and translational initiation region that can be obtained from a gene expressed in cotton fiber, and methods for using constructions that include the vector to alter the phenotype of the fiber. Both the 3 * regions and the endogenous 5 'regions can be important for directing efficient transcription and translation. These promoters are provided from genes involved in the generation of cotton fiber development. One, Racl3, is from a cotton protein, which codes for an animal Rae protein homologue. The Racl3, shows a highly improved expression during the development of the fiber. This expression pattern correlates well with the reorganization time of the cytoskeleton, suggesting that the Racl3 cotton gene, like its animal counterpart, may be involved in the signal transduction pathway for the cytoskeletal organization. Racl3 is a gene that is moderately expressed during fiber development, occurring 9 days after anthesis, and deactivating approximately 24 days after anthesis. It is expressed maximally between 17 and 21 days after the development anthesis of the fiber. Another promoter from a cotton protein, is designed as 4-4. The 4-4 mRNA accumulates in the fiber cells on day 17 after anthesis, and continues to the maturation of the fiber, which occurs at 60 days or something after anthesis. The data demonstrate that the 4-4 promoter remains very active at 35 after anthesis. A promoter is also provided from a lipid transfer protein (sometimes referred to herein after as "Ltp") which is preferably expressed in cotton fiber. 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 phenotype. wanted. Accordingly, the constructs and methods of the present invention find use in the modulation of endogenous fiber products, as well as in the production of exogenous products, and in the modification of the phenotype of fiber and fiber products. The constructions also find use as molecular probes. In particular, constructs and methods for use in gene expression in embryonic cotton fabrics are considered herein. By these methods, cotton plants and novel cotton plant parts, such as modified cotton fibers, can be obtained. Constructions and methods of use related to the modification of the color phenotype in cotton fiber are also provided. These constructs contain sequences for the expression of genes involved in the production of colored compounds, such as anthocyanins, melanin and indigo, and also contain sequences that provide the direction of genetic products to particular locations in the plant cell, such as organelles. of plastid, or vacuoles. The direction to the plastid is of particular interest for the expression of genes involved in the trajectories of aromatic amino acid biosynthesis, while the vacuolar direction is of particular interest where the precursors required in the synthesis of the pigment are present in vacuoles. Of particular interest are plants that produce fibers that are colored, that is, with pigment produced in the fiber by the plant during the development of the fiber, as opposed to the fibers that are harvested and stained or pigmented in another way. through separate processing. The fibers of a plant that produces this colored fiber, can be used to produce colored yarns and / or fabrics, which have not been subjected to any dyeing process. Although naturally colored cotton has been available from different domestic and wild type cotton varieties, the present application provides cotton fiber having a color produced by the expression of a genetically engineered protein. Accordingly, the application provides constructions and methods of use related to the modification of the color phenotype in cotton fiber. These constructs contain sequences for the expression of genes involved in the production of colored compounds, such as melanin or indigo, and also contain sequences that provide the direction of the genetic products towards particular places in the cell of the plant, such as plastid organelles, or vacuoles. The direction to the plastid is of particular interest for the expression of genes involved in the trajectories of aromatic amino acid biosynthesis, while the vacuolar direction is of particular interest where the precursors required in the synthesis of the pigment are present in the vacuoles.

DESCRIPTION OF THE DRAWINGS Figure 1 shows the DNA sequence encoding the structural protein from cDNA 4-4. Figure 2 shows the sequence for the construction of the pCGN5606 promoter, made using genomic DNA from the genomic clone 4-4-6. Figure 3 shows the sequence for the construction of promoter 4-4, pCGN5610. Figure 4 shows the cDNA sequence encoding the Racl3 gene expressed in cotton fiber. Figure 5 shows the sequence of the promoter region from the racl3 gene. Figure 6 shows a restriction map for pCGN4735. Figure 7 shows the sequence of the promoter region of the lipid transfer protein, from a lipid transfer protein gene specific for cotton fiber.

Figure 8 shows the configuration of the binary vectors pCGN5148 and pCGN5616 for the transformation of the plant, in order to express genes for the synthesis of melanin and the synthesis of indigo, respectively. Figure 9 provides the results of the color measurements taken from Coker 130 control cotton fibers, used in the transformation, using color constructions. Figure 10 shows the results of the measurements made of the color of the plants transformed by the construction pCGN5148, to express genes for the synthesis of melanin. Figure 11 shows the results of the measurements taken from the color of the plants transformed by the construction pCGN5149, to express genes for the synthesis of melanin. Figure 12 shows the results of the measurements made of the color of the transformed plants, to express genes for the synthesis of indigo, using the construction pCGN5616. Figure 13 shows the control measurements made of naturally colored cotton plants, which are produced by non-transgenic colored cotton plants.

DETAILED DESCRIPTION OF THE INVENTION In accordance with the present invention, novel constructs and methods, which can be used to provide transcription of a nucleotide sequence of interest, are described in cells of a host plant, preferably in cotton fiber cells. , to produce cotton fiber having an altered color phenotype. The cotton fiber is a simple epidermal cell differentiated from the outer integument of the ovule. It has four distinct growth phases: initiation, elongation (synthesis of the primary cell wall), synthesis of the secondary cell wall, and maturation. The initiation of fiber development seems to be triggered by lasts. The primary cell wall extends during the elongation phase, lasting up to 25 days after anthesis (DPA). The synthesis of the secondary wall begins before the elongation phase ceases, and continues until approximately 40 days after anthesis, forming an almost pure cellulose wall. The constructions for use in these cells can include various forms, depending on the intended use of the construction. Accordingly, the constructs include vectors, transcription cassettes, expression cassettes, and plasmid. The transcription and translation initiation region (also sometimes referred to as a "promoter", preferably comprises a transcription initiation regulatory region, and a translational initiation regulatory region of non-translated 5 'sequences. ribosomes ", responsible for the binding of mRNA to ribosomes, and initiation of translation It is preferred that all functional elements of transcription and translation of the initiation control region are derived from, or obtained from, , the same gene In some embodiments, the promoter will be modified by the addition of sequences, such as enhancers, or deletions of the essential and / or undesired na sequences. "What can be obtained" means a promoter having a sequence of DNA sufficiently similar to that of a native promoter, to provide the desired specificity of the transcription of a DNA sequence of interest. This includes natural and synthetic sequences, as well as sequences that can be a combination of synthetic and natural sequences. The cotton fiber transcription initiation regions selected for the modification of the cotton fiber may include the cotton fiber promoter regions 4-4, racl3, and Ltp provided herein. A transcription cassette, for the transcription of a nucleotide sequence of interest in cotton fiber, will include, in the direction of transcription, a transcription initiation region of cotton fiber, a DNA sequence of interest, and a functional transcription termination region in a plant cell. When the cassette provides for the transcription and translation of a DNA sequence of interest, it is considered an expression cassette. There may also be one or more introns present. There may also be other sequences present, including those encoding transit peptides and leader secretory sequences, as desired. The transcription initiation regions of the fiber tissue 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 in other stages of fiber development, in addition to the foregoing, are acceptable as far as those regions provide a significant expression level in the fiber of cotton in the defined periods of interest, and does not interfere negatively with the plant as a whole, and in particular, does not interfere with the development of the fiber and / or parts related to the fiber. Downstream from, and under the regulatory control of, the cotton fiber transcription / translation initiation control region, is a nucleotide sequence of interest, which provides for the modification of the fiber phenotype. The nucleotide sequence can be any open reading frame that encodes a polypeptide of interest, eg, an enzyme, or a sequence complementary to a genomic sequence, wherein the genomic sequence can be an open reading frame, an intron, a leader, non-coding sequence, or any other sequence in which the complementary sequence inhibits transcription, the processing of messenger RNA, for example, splicing or translation. The nucleotide sequences of this invention can be synthetic, naturally derived, or combinations thereof. Depending on the nature of the DNA sequence of interest, it may be desirable to synthesize the sequence with the preferred codons of the plant. The preferred codons of the plant can be determined from the highest frequency codons in the proteins expressed in the greatest amount in the particular plant species of interest. The phenotypic modification can be achieved by modulating the production, either of an endogenous transcription or translation product, for example, with respect to the amount, the relative distribution, or the like, or an exogenous transcription or translation product. , for example, to provide a novel function or products in a transgenic host cell or tissue. Of particular interest are DNA sequences that encode expression products associated with the development of plant fiber, including genes involved in the metabolism of cytokinins, auxins, ethylene, abscisic acid, and the like. Methods and compositions for modulating cytokinin expression are described in U.S. Patent No. 5,177,37, the disclosure of which is incorporated herein by reference. In an alternative way, different genes can be used, from sources, including other eukaryotic or prokaryotic cells, including bacteria, such as those of the biosynthetic gene products of auxin and cytokinin of T-DNA of Agrobacterium tumefaciens, for example, and of mammals, for example interferons. r phenotypic modifications include the color modification of cotton fibers. Of interest are the genes involved in the production of melanin, and the genes involved in the production of indigo. Melanins are brown pigments found in animals, plants, and microorganisms, any of which can serve as a source for the sequences to be inserted into the constructions of the present invention. Specific examples include the tyrosinase gene, which can be cloned from Streptomyces antibioticus. The protein encoded by ORF438 in S is necessary. antibioticus for the production of melanin, and can provide a copper donor function. In addition, a tyrosinase gene can be isolated from any organism that makes melanin. This gene can be isolated from human hair, melanocytes or melanomas, cuttlefish fish, and red roosters, among rs. See, for example, European Patent Application Number 89118346.9, which discloses a process for the production of melanins, their precursors and derivatives in microorganisms. Also, see Bernan et al., Gene (1985) 37: 101-110.; and della-Cioppa et al., Bio / Technology (1990) 8: 634-638. Indigo can be obtained by using genes that encode a mono-oxygenase, such as xylene oxygenase, which oxidizes toluene and xylene to obtain (methyl) benzyl alcohol, and also transforms indol into indigo. The cloning of the xylene oxygenase gene, and the nucleotide and amino acid sequences, are described in Japanese Unexamined Patent Application Kokai Number: 2-119777, published May 7, 1990. A dioxygenase, such as dioxygenase naphthalene, which also convert indol into indigo, also finds use; the naphthalene dioxygenase gene nahA is described in Science (1983) 222: 167. For cloning, we have the nucleotide sequence in the characterization of genes encoding naphthalene dioxygenase from Pseudomonas putida. See Kurkela et al., Gene (1988) 73: 355-362. A tryptophanase gene sequence in conjunction with an oxygenase can be used to increase the amount of indole available to become indigo.

The sources of tryptophanase gene sequences include E. coli (see, for example, Deeley et al. (1982) J. Bacteriol. 151: 942-951). Plastid targeting sequences (transit peptides) are available from a number of plastid proteins nuclearly coded by the plant, such as the small subunit (SSU) of ribulose bisphosphate carboxylase, the genes related to the biosynthesis of the plant fatty acid, including acyl carrier protein (ACP), stearoyl desaturase-acyl carrier protein, β-ketoacyl synthase-acyl carrier protein, and acyl thioesterase-acyl carrier protein, or the LHCPII genes. The coding sequence for a transit peptide that provides transport to plastids may include all or a portion of the coding sequence for a particular transit peptide, and may also contain portions of the sequence encoding the mature protein associated with a particular transit peptide. There are numerous examples in the art of transit peptides, which can be used to deliver an objective protein to a plastid organelle. The sequence encoding the particular plastid peptide used in the present invention is not critical, as long as delivery to the plastid is obtained. As an alternative to the use of transit peptides to direct the pigment synthesis proteins to the plastid organelles, the desired constructs can be used to transform the plastid genome directly. In this case, promoters capable of providing gene transcription in plant plastids are desired. Of particular interest is the use of a T7 promoter to provide high levels of transcription. Since the plastids do not contain a polymerase suitable for transcription from the T7 promoter, the T7 polymerase can be expressed from a nuclear construct, and can be targeted to the plastids using the transit peptides as described above. (See McBride et al. (1994) Proc. Nat. Acad. Sci. 91: 7301-7305; see also the pending United States Patent Application entitled "Controlled Expression of Transgenic Construets in Plant Plastids", with Series 08 / 472,719, filed June 6, 1995, and pending United States Patent Application Serial Number 08 / 167,638 filed December 14, 1993, and Patent Number PCT / US94 / 14574 filed on December 12, 1994). Tissue specific or developmentally regulated promoters may be useful for the expression of T7 polymerase, in order to limit expression to the tissue or to the appropriate stage of development. The direction of the genes for the synthesis of melanin to vacuoles is also of interest in the tissues of plants that accumulate the tyrosine substrate involved in the synthesis of melanin in vacuoles. The signal of the protein to be directed to the vacuoles can be provided from a plant gene that is normally transported through the crude endoplasmic reticulum, such as the 32 amino acid N-terminal region of the tomato metalocarboxypeptidase inhibitor gene. (Martineau et al. (1991) Mol. Gen. Genet, 228: 281-286). In addition to the signal sequence, vacuolar directed constructs also encode a vacuolar localization signal (VLS) placed at the carboxy terminus of the encoded protein. Appropriate signal sequences, and vacuolar localization signal regions, can be obtained from other different plant genes, and can be used similarly in the constructions of this invention. Numerous vacuolar direction peptides are known in the art and are reviewed in Chrispeels et al., Cell (1992) 68: 613-616. The Al Ma gene, which codes for a dihydroflavonol reductase, an enzyme from the path of anthocyanin pigmentation, is one of these genes. In cells expressing the Al gene, dihydroquempferol is converted to 2,8-alkyl-leucopelargonidine, which can be further metabolized into pelargonidin pigment by enzymes endogenous to the plant. Other pigments of the anthocyanin or flavonoid type may also be of interest for the modification of the fibers of the cotton cells, and have been suggested for use in the flowers plants (for a review of the color of the flower of the plant, see Tunen et al., Plant Biotechnology Series, Volume 2 (1990), Developmental Regulation of Plant Gene Expression, D. Grierson ed.). Anthocyanin is produced by progressing steps from cellular phenylalanine groups. The R and Cl genes are maize regulatory proteins that are active by positively affecting the upstream steps in anthocyanin biosynthesis from these groups. The R gene is described in Perot and Cone (1989) Nucí. Acids Res., 17: 8003, and the Cl gene is described in Paz-Ares et al. (1987) EMBO, 6: 3553-3558. Lloyd et al (1992) Science, 258: 1773-1775 discussed both genes. Although cotton fibers in commercially grown varieties are primarily white in color, other cotton varieties that occur naturally have chestnut or reddish-brown fibers. Additionally, a cotton line containing green fibers has been identified. The cotton lines provided by these fibers are available from different sources, including cottons of the BC variety (BC Cotton Inc., Box 8656, Bakersfield, CA 93389) and Fox Cottons (Natural Cotton Colors, Inc., PO Boz 791, Wasco, CA 93280).

The existence of these colored cotton lines suggests that the precursors required for the anthocyanin pigment trajectories are present in the cells of the cotton fibers, thus allowing other modifications of the color phenotype. Therefore, the corn R and Cl genes could be used to improve the levels of anthocyanin produced in the fiber cells. Since the R and Cl proteins are proteins with a positive control at the regulatory level on the biosynthesis of the anthocyanin pigment precursor, these proteins are expressed in the nucleus, and do not target plastids or vacuoles. For some applications, it is of interest to modify other aspects of the fiber. For example, it is of interest to modify different aspects of cotton fibers, such as the strength or texture of a fiber. Accordingly, the appropriate gene can be inserted into the constructs of the invention, including genes for PHB biosynthesis (see, Peoples et al., J. Biol. Chem. (1989) 264: 15298-15303, and Ibid. 15397; Saxema, Plant Molecular Biology (1990) 15: 673-683, which discloses the cloning and sequencing of the catalytic subunit gene of cellulose synthase, and Bowen et al., PNAS (1992) 89: 519-523, which discloses the chitin synthase genes of Sccharomyces cerevisiae and Candida albicans). Different constructs and methods for the use of hormones to effect changes in fiber quality are disclosed in the pending United States Patent Application entitled "Cotton Modification Using Ovary-Tissue Transcriptional Factors", serial number 08 / 397,652, filed on February 2, 1995, the teachings of which are incorporated herein by reference. Transcription cassettes can be used when transcription of an anti-sense sequence is desired. When the expression of a polypeptide is desired, expression cassettes that provide for the transcription and translation of the DNA sequence of interest will be used. Different changes are of interest; these changes may include modulation (increase or decrease) in the formation of particular saccharides, hormones, enzymes, or other biological parameters. These also include modifying the composition of the final fiber, that is, changing the proportion and / or the amounts of water, solids, fiber, or sugars. Other phenotypic properties of interest to be modified include stress response, organisms, herbicides, sow formation, growth regulators, and the like. These results can be achieved by providing reduction of the expression of one or more endogenous products, particularly an enzyme or a cofactor, either through the production of a transcription product that is complementary (anti-sense) to the transcription product of a native gene, to inhibit the maturation and / or expression of the transcription product, or by providing expression of a gene, either endogenous or exogenous, to be associated with the development of a plant fiber. The termination region that is used in the expression cassette will be primarily a convenience, since the termination regions appear to be relatively interchangeable. The termination region may be native to the transcription initiation region, may be native to the DNA sequence of interest, and may be derived from another source. The termination region may occur naturally, or may be wholly or partly synthetic. Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. In some embodiments, one may wish to use the native 3 'terminator region for the cotton fiber transcription initiation region used in a particular construct. As described in this, in some cases additional nucleotide sequences will be present in the constructs, to provide the direction of a particular gene product towards specific cellular locations. For example, where coding sequences are used for the synthesis of aromatic colored pigments in a construct, particularly coding sequences for enzymes having as their substrates, aromatic compounds such as tyrosine and indole, it is preferable to include sequences that provide the delivery of the enzyme to the plastids, such as a small subunit transit peptide sequence. Also, for the synthesis of pigments derived from tyrosine, such as melanin, the direction towards the vacuole can provide better color changes. For the production of melanin, the tyrosinase and ORF438 genes from Streptomyces antibioticus (Berman et al. (1985) 37: 101-110) are provided in cotton fiber cells for expression from a 4-promoter. 4 and Racl3. In Streptomyces, the ORF438 and tyrosinase proteins are expressed from the same promoter region. For expression from constructs in a transgenic plant genome, the coding regions can be provided under the regulatory control of separate promoter regions. The promoter regions may be the same or different for the two genes. Alternatively, a coordinated expression of the two genes can be desired from a single plant promoter. Constructs for expression of the tyrosinase and ORF438 gene products from promoter regions 4-4 and rae are described in detail in the following examples.

Additional promoters, for example, viral plant promoters, such as CaMV 35S, can also be desired, which can be used for the constitutive expression of one of the desired genetic products, the other genetic product being expressed in cotton fiber fabrics from promoter 4-4 and rae. In a similar manner, other constitutive promoters may also be useful in certain applications, for example the mas, Mac, or DoubleMac promoters, described in U.S. Patent No. 5,106,739, and by Comai et al., Plant Mol. Biol. (1990) 15: 373-381. When plants comprising multiple genetic constructions are desired, for example, plants that express the melanin, ORF438, and tyrosinase genes, plants can be obtained by cotransformation with both constructions, or by transformation with individual constructions, followed by breeding methods. plants, to obtain plants that express both desired genes. There are a variety of techniques available and known to those skilled in the art for the introduction of constructs into a plant cell host. These techniques include transfection with DNA using A. tumefaciens or A. rhizogenes as the transfection agent, protoplast fusion, injection, electroincorporation, acceleration of particles, etc. For transformation with AgroJacterium, the plasmids can be prepared in E. coli, which contains DNA homologous to the Ti plasmid, particularly T-DNA. The plasmid may or may not be able to replicate in Agrobacterium, that is, it may or may not have a broad-spectrum prokaryotic replication system, such as, for example, 90, depending in part on whether the cassette will be integrated. of transcription in the Ti plasmid, or if it is to be retained on an independent plasmid. The Agrojacterium host will contain a plasmid that has the vir genes necessary for the transfer of the T-DNA to the cells of the plant, and may or may not have the complete T-DNA. At least the right border, and often both the right border and the left border of the T-DNA of the Ti or Ri plasmids, will be joined as flanking regions to the transcription construct. The use of T-DNA for the transformation of plant cells, has received an extensive study, and is widely described in the European Patent Application EPA with Serial Number 120,516, of Hoekema, In: The Binary Plant Vector System Offset-drukkerij KantersB.V., Alblasserdam, 1985, Chapter V, Knauf et al., Genetic Analysis of Host Range Expression by Agrobacterium, En; Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed. , Springer-Verlag, NY, 1983, page 245, and An et al., EMBO J. (1985) 4: 277-284.

For infection, particle acceleration, and electroincorporation, a disarmed Ti plasmid may be introduced that lacks particularly the tumor genes that are found in the T-DNA region in the plant cell. By means of an auxiliary plasmid, the construction can be transferred to A. tumefaciens, and the resulting transfected organism can be used to transfect a plant cell; you can grow explants with A. tumefaciens or the A. transformed rhizogenes, to allow the transfer of the transcription cassette to the cells of the plants. Alternatively, to improve integration into the plant genome, terminal transposon repeats can be used as borders in conjunction with a transposase. In this situation the expression of the transposase must be inducible, in such a way that once the transcription construct is integrated into the genome, it must be integrated in a relatively stable manner. Then the cells of transgenic plants are placed in an appropriate selective medium for the selection of the transgenic cells, which are then grown to form calluses, grown shoots, and plants generated from the outbreaks, through the cultivation in a rooting medium. To confirm the presence of the transgenes in the cells of transgenic plants, a Southern blot analysis can be performed using methods known to those skilled in the art. The expression products of the transgenes can be detected in any of a variety of ways, depending on the nature of the product, and include immunoassay, enzyme assay, or visual inspection, for example, to detect the formation of the pigment in the part or in the cells of the appropriate plant. Once transgenic plants have been obtained, they can be grown to produce fiber having the desired phenotype. The fibers can be harvested and / or the seed can be harvested. The seeds can serve as a source to grow additional plants that have the desired characteristics. The terms "transgenic plants" and "transgenic cells" include plants and cells derived from transgenic plants or from transgenic cells. The different sequences provided herein can be used as molecular probes for the isolation of other sequences that may be useful in the present invention, for example, to obtain related transcription initiation regions from them or from different plant sources. . The related transcription initiation regions that can be obtained from the sequences provided in this invention will show at least a homology of about 60 percent, and the most preferred regions will show a still greater homology percentage with the probes. Of particular importance is the ability to obtain related transcription initiation control regions having the time and tissue parameters described herein. For example, using probe 4-4 and rae, at least seven additional clones have been identified, but have not been further characterized. Accordingly, by employing the techniques described in this application, and other techniques known in the art (such as Maniatis et al., Molecular Cloning, - A Laboratory Manual (Cold Spring Harbor, New York) 1982), can be determined other transcription initiation regions capable of directing transcription to the cotton fiber, as described in this invention. The constructions can also be used in conjunction with plant regeneration systems, to obtain plant cells, and plants; accordingly, the constructs can be used to modify the phenotype of fiber cells, to provide cotton fibers that are colored as the result of genetic engineering to provide nuances and / or intensities not yet available. Different varieties and lines of cotton can find use in the described methods. Cultured cotton species include Gossypium hirsutum and G. babadenee (extra-long stable, or Pima cotton), which evolved in the New World, and the Old World crops G. herbaceu and G. arboreum Color phenotypes can be evaluated by using a colorimeter, an instrument that is already used to provide objective color measurements of cotton samples. A colorimeter uses a combination of light sources and filters to make different estimates of sample colors, sometimes referred to as tristimulus values. In the past, these estimates have been used to calculate a value (Hunter + b, described below) that indicates the degree of yellowing of a cotton sample. The yellowing and reflectance (from Rd, the degree of illumination or darkness of the samples) have been used to provide color measurements of the cotton for graduation. The tests are typically conducted by exposing the face of a sample to a controlled light source. A typical color chart showing how official grade standards relate to the Rd and + b measurements is shown in Cotton, RJ Kohel and CF Lewis, Editors # 24 in AGRONOMY Series American Soc. Agronomy (see Figures 12-6). You can also use different colorimetric methods to quantify the color and express them numerically. The method of Munsell, devised by the American artist A. Munsell, uses a classification system of paper color chips classified according to their nuance (Matiz de Munsell), their illumination (Munsell value), and their saturation (Chroma) de Munsell) for visual comparison with the color of the sample. Other methods to express color numerically have been developed by an international organization concerned with light and color, the Commission Internationale de l'Eclairage (CIÉ), which has a Central Office located at Kegelgasse 27, A-1030 Vienna, AUSTRIA . The two most widely known methods of these methods are the Yxy color space, devised in 1931 based on the tristimulus value XYZ, as defined by the ICD, and the color space L * a * b *, devised in 1976 to provide more uniform color differences in relation to visual differences. Color spaces * such as these are now used throughout the world for color communication. The Hunter Lab color space was developed in 1948 by R.S. Hunter as a uniform color space that could be read directly from a photoelectric colorimeter (Tristimulus Method). The color space L * C * h uses the same diagram as the color space L * a * b *, but uses cylindrical coordinates instead of rectangular coordinates. In this color space, the L * indicates the illumination, and is equal to L * of the color space L * a * b *, C * is the chromaticity, and h is the hue angle. The value of chromaticity C is 0 at the center, and it increases according to the distance from the center. It is defined that the angle of the hue starts at the axis + a of the space L * a * b *, and is expressed in degrees in a rotation opposite to the hands of the clock. Therefore, in relation to the space L * a * b *, 0o and 360 ° would be on the line + a *, 90 ° would be on + b *, 180 ° would be on -a *, and 270 ° would be on -b *. All of the above methods can be used to obtain accurate measurements of a cotton fiber color phenotype.

EXPERIMENTAL The following examples are offered by way of illustration and not limitation.

Example 1 cDNA libraries Preparation of the tissue for cDNA synthesis Leaf and root tissue was isolated from nurseries grown in a 20.32 centimeter high greenhouse and frozen immediately in liquid nitrogen. The flowers were collected in the pre-anthesis phase rapidly expanding after 3 days, and they were also frozen. Seeds were collected from locules 21 days after anthesis, which had been removed from the pod, and frozen whole in liquid nitrogen. Once frozen, the fiber was removed from the seed, and the bare seed was used for RNA isolation. All fibers were removed from the seed under liquid nitrogen, and the fiber was milled to a powder prior to RNA isolation. The fibers were from pods that had been labeled in the anthesis.

DNA manipulations? RNA We used the Lambda ZapllMR cDNA library system from Stratagene, for screening, and was prepared from cDNA derived from poly-A + mRNA isolated from Gossypium hirsutum Acala SJ-2 cultivar fibers. The fibers were isolated from pods harvested about 21 days after anthesis using field-grown plants in Israel. Total RNA was isolated from seeds 21 days after anthesis (G. hirsutum cv Coker 130, from which the fiber had been removed), using the method of Hughes and Galau (1988) Plant Mol Biol Repórter, 6: 253- 257). All other RNAs were prepared according to Hall et al. (1978), Proc Nati Acad Sci USA 75: 3196-3200), with the following modifications. After the second wash with 2M LiCl, the pellet was dissolved in 1/10 of the original volume of 10 mM Tris, pH 7.5, and 35 mM potassium acetate, pH 6.5, and 1/2 volume of EtOH were slowly added. The mixture was placed on ice for 15 minutes, and then centrifuged at 20,000 x g for 15 minutes at 4 ° C. The concentration of potassium acetate was brought to 0.2M, 2-1 / 2 volumes of EtOH were added, and the RNA was placed at -20 ° C for several hours. The precipitate was centrifuged at 12,000 x g for 30 minutes at 4 ° C, and the pellet was resuspended in water treated with diethyl pyrocarbonate. Poly-A + RNA was prepared from total mRNA, using an oligo (dT) -cellulose kit (Becton Dickenson), and following the manufacturer's protocol. Cotton genomic DNA was prepared as follows. 4 grams of young cotton leaf tissue (coker 130 cv) were milled to a powder in N2, and placed in an Oak Ridge tube with 0.4 grams of polyvinyl pyrrolidone, and 20 milliliters of extraction buffer (200 Ches. mM / NaOH, pH 9.1, 200 mM NaCl, 100 M EDTA / NaOH, pH 9.0, 2 percent SDS, 0.5 percent Na deoxycholate, 2 percent Nonidet NP-40, 20 mM B-mercaptoethanol ) to the sample, mixed gently, and incubated at 65 ° C in a shaking water bath for 10 minutes. 7.0 milliliters of 5M potassium acetate, pH 6.5, were added and mixed thoroughly. Incubation was performed on ice for 30 minutes with gentle mixing every 5 minutes. The sample was centrifuged for 20 minutes at 21,000 x g, and the supernatant was filtered through Miracloth in another tube, centrifuged as before. The supernatant was again filtered through Miracloth in 15 milliliters of isopropanol at room temperature in an Oak Ridge tube. After gentle mixing, the sample was incubated at room temperature for 10 to 60 minutes, until the DNA was precipitated. The DNA was rolled up and allowed to air dry before being resuspended in 4 milliliters of TE on ice for 1 hour. CsCl was added to a final concentration of 0.97 grams / milliliter, and 300 microliters of a concentration of 10 milligrams / milliliter of ethidium bromide was also added before filling the VTÍ80 quick seal tubes. The sample was centrifuged overnight at 225,000 xg. The DNA was extracted with butanol saturated with water, and enough water was added to bring the volume up to 4 milliliters before adding two volumes of EtOH. The DNA was rolled up, dried in air, and resuspended in 200 microliters of sterile water.

Northern and Southern Analysis For the Northern, 10 micrograms of total RNA was isolated from different tissues, separated by electrophoresis on 1.2 percent agarose-formaldehyde gels, and transferred to Nytran Plus membranes (Schleicher and Schuell). Hybridization conditions consisted of a solution containing 50 percent (volume / volume) forraamide, 5xSSC, 0.1 percent SDS, 5 mM diethylenediaminetetraacetic acid, 10X Denhardt's solution, 25 mM sodium phosphate, pH 6.5, and 250 micrograms / milliliter of carrier DNA. The washes were performed in 2xSSC, SDS at 0.1 percent at 42 ° C three times for 30 minutes each time. Cotton genomic DNA (12 micrograms) was digested with different restriction endonucleases, electrophoresed on 0.1 percent agarose gels, and stained on Nytran Plus membranes. The hybridization and filter wash conditions for both full-length and 3 'specific cDNA insert probes were as described for Northern analysis. Probes derived from the 3 'non-translated regions were synthesized by means of 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 the 3 'specific DNA sequences. These sequences were used as templates in the generation of probes labeled with 32 P of a single chain, of the anti-sense chain 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 (Stratagene) kit.

EXAMPLE 2 Isolation of cDNA Clones from Cotton The cDNA for clone 4-4 was isolated from the cotton fiber library described above, and was shown to be expressed in the fiber, but not in other tissues. This sequence was not related to any known protein. Only 400 kb of the coding sequence in this clone was present, so that the library was screened again using the cDNA to obtain full-length clones. The full length coding sequence is provided in Figure 1. By comparing the sequences of random cDNA clones against different banks of sequence data by means of BLAST, a service from the National Center for Biotechnology Information was found to be a clone , designated # 105, has a coding sequence related to that of a reported lipid transfer protein. Another clone that showed a high homology with the Rae proteins of animals was sequenced. This clone, designated as Rae, was not fully full length, and the library was re-screened using this initial Rae DNA segment as a probe. Of approximately 130,000 traced primary plates, 56 were screened positive; of these, 14 clones were isolated and sequenced. Of these 14 clones, 12 showed identical sequence homology to the original Rae clone, and one of these cDNA clones encoded a full-length cDNA, and was named Racl3. Figure 4 shows the cDNA sequence encoding the Racl3 gene expressed in cotton fiber. Another clone of partial length cDNA, designated as Rac9, was clearly related, but different in DNA and in the amino acid sequence of Racl3. The re-tracking of 150,000 plates resulted in the isolation of 36 positive clones, of which only two clones corresponded to the Rac9 sequence (both full-length clones), with the remainder being Racl3. These results suggest that cotton contains genes for at least two different Rae proteins. Based on the frequency of isolation of the clone, Racl3 is expressed in a relatively high manner, and Rac9 does so less in cotton fibers at 21 days after anthesis (dpa), the time at which the Poly-A + mRNA for the construction of the library. Comparisons of the deduced amino acid sequence of Racl3 with other small G proteins showed that the cotton Rae proteins are very closely related to the deduced Rhol protein sequence from a freshly isolated cDNA clone from peas (Yang and Watson, supra). After the pea Rhol, the Rae proteins show the highest homology with the Rae proteins of cotton. Other proteins of the rho subfamily, such as yeast CDC42, and human RhoA, are also clearly related to cotton Rae genes. In contrast, the other small G proteins of the Rab / YPT subfamily isolated from plants, such as the example shown in the tobacco RAB5 protein, as well as the human Ras proteins, are less homologous with the cotton Rae proteins of all the proteins G small compared. The cotton and pea proteins, as well as the mammalian Raes, all have a pl greater than 9, while those of other rho and ras proteins are on the scale of 5.0 to 6.5.

EXAMPLE 3 Expression of Cotton Fiber Genes in Developing Fibers The expression of the Racl3 and 4-4 genes was evaluated using mRNA prepared from different cotton tissues, and from fibers at different stages of development. The spots were hybridized with probes derived from non-translated regions of the Ltp, Racl3, and 4-4 genes.

The gene for Racl3 exhibits a highly improved expression in fibers; there is virtually no detectable mRNA present in the leaves, roots, or parts of the flowers, even under conditions of a prolonged time of development. The expression of Racl3 is detected in seeds at an age corresponding to the highest expression levels observed in fiber tissue derived from seeds of this same age. The pattern of expression of Racl3 in fibers depends very much on the stage of development. The expression is very low during the stage of the synthesis of the primary wall (from 0 to 14 days after anthesis, see Meiner, 1977), and reaches a maximum during the transition to the synthesis of the secondary wall (approximately 15 to 18 days after anthesis) and declines during the stage of maximum cellulose synthesis of the secondary wall (from approximately 24 to 28 days after anthesis). The 4-4 mRNA begins to accumulate in the fiber cells only on day 17 after anthesis, and continues until at least the 35th day after anthesis. The levels reach the peak on day 21, and remain high. No 4-4 mRNA is detected in other cotton tissues, and it is not detected in the fiber tissue before establishment at 17 days after anthesis. The lipid transfer protein cDNA clone # 105 was used as a probe against cotton tissue and in a Northern cotton fiber. Northern showed that the cotton fiber lipid transfer protein is highly expressed in cotton fiber. The mRNA that codes for this protein is expressed throughout the development of the fiber at an extremely high level. Northern blots indicate that this mRNA is expressed at 5 days after anthesis, and is continuously expressed at a high level at 40 days after anthesis.

EXAMPLE 4 Genomic DNA The cDNA for both 4-4 and Racl3 was used to probe genomic clones. For both, full-length genomic DNA was obtained from a library made using the lambda dash 2 vector from StratageneMR, which was used to construct a genomic DNA library from cotton of the variety Coker 130 (Gossypium hirsutum cv. 130), using DNA obtained from germination seedbeds. The genomic cotton library was probed with a 3 'specific lipid transfer protein probe. and 6 genomic phage candidates were identified, and purified. Figure 7 provides a sequence of approximately 2 kb of the promoter region of the lipid transfer protein that is immediately to 51 of the coding region of the lipid transfer protein. Six clones of the genomic phage were identified from the cotton genomic library, using a 3 'specific probe for lipid transfer protein mRNA. This was done to select the promoter from the lipid transfer protein gene which is expressed maximally in the cotton fiber, from the gene family of lipid transfer protein in cotton. The lipid transfer protein promoter is active throughout the entire fiber development period.

Example 5 Preparation of Promoter Constructs 4-4 DCGN5606 The promoter construction pCGN5606 comprises the expression cassette of cotton fiber 4-4 in a first version, version I (Figure 2). The sequences from ntl to 65 and nt 5,494 to 5,547 correspond to the fragments of the polylinker pBluescriptII, where this cassette is cloned. The unique restriction enzyme sites present in these regions flanking the cassette allow the cloning of the fiber expression cassette into binary vectors, including the pCGN5138 and 1547 series. The sequences from nt57 to 5,494 are contained in a clone of lambda phage from a Coker 130 cotton genomic library. This lambda genomic clone was designated 4-4 (6). The region from nt 65 to nt 4,163 corresponds to the flanking region 5 'of the gene 4-4 (6). At nt 4,163, there is a Ncol restriction site sequence corresponding to the first codon of the 4-4 (6) ORF. The region from nucleotide 4,163 to 4,502 corresponds to part of the 4-4 (6) ORF. The sequence from nt 4,502 to 4,555 is a synthetic polylinker oligonucleotide containing unique targeting sites for the restriction enzymes EcoRI, SmaI, SalI, Nhel, and BglII. This fragment from nt 4,163 to 4,555 is an embossed fragment, and is left in place to facilitate the monitoring of cloning manipulations. 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 embossed fragment with the gene of interest. The region from nt 4,555 to 5,494 corresponds to the 940 nucleotides downstream from the stop codon, and constitute the flanking region 31 of the 4-4 (6) gene. There is a unique AscI restriction enzyme site in nt 5483.

PCGN5610 The construction of pCGN5610 is a second version of the 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 arrays in a row of two fiber cassettes in a binary plasmid. The differences with respect to pCGN5606 are very minor, and are described below. The Xbal restriction site in the region of nt 1 to 65 has been deleted by conventional cloning manipulations. The polylinker region is in the reverse orientation of pCGN5606. There is a unique Xbal restriction enzyme site at nt5484. The sequences from nt 1 to 57, and from nt 5,494 to 5,518 of pCGN5610, correspond to the fragments of the polylinker pBluescriptII, where this cassette is cloned. The unique restriction enzyme sites present in these regions allow the cloning of the fiber expression cassette in binary vectors of the series pCGN 5138 and 1547. The sequences from nt57 to 5,494 are contained in a lambda phage clone of a genomic library of Coker 130. This clone is described in my notebook as the lambda 4-4 genomic clone (6). The region from nt57 to nt 4.155 corresponds to the 5 'flanking region. At nt 4.155 there is a Ncol restriction site sequence corresponding 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 nt 4.156 to 4.550 is an embossed 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 targeting sites for the restriction enzymes BglII, Bhel, SalI, Smal, and EcoRI. The genes to be expressed in the cotton fiber cells using this cassette can be cloned between the Ncol restriction site and any of the polylinker sites. This operation replaces the embossed 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.

Example 6 Preparation of Constructions of the Racl3 Promoter Genomic Clone From a genomic clone designated 15-1, the mapping was done with restriction endonucleases. The largest fragment with the Racl3 coding region was identified.

This was a Pst fragment, and when subcloned into the Bluescript ™ KS + vector (BSKS +; stratagene) it was named pCGN4722. The insert had a length of 9.2 kb. The region of the Pst fragment was identified with the Racl3 coding sequence. The DNA sequence was determined for approximately 1.7 kb at 5 'of the initial codon, and approximately 1.2 kb at 3' of the stop codon. The entire coding region of Rae (exons and introns) was conveniently flanked by the Ndel sites. PCGN4722 was digested with Xbal, and a 2.7 kb fragment was removed. The religation gave pCGN4730, which was then digested with Ndel, leaving a 1.7 kb fragment containing the entire Rae coding region. The religation produced pCGN4731. A polylinker region was created using overlapping synthetic oligonucleotides, which were passed by polymerase chain reaction using homologous primers at the 5 'and 3' ends of the resynthesized section. The resulting product was digested with EcoRI and HindIII, and ligated into BSKS + at the EcoRI and HindIII sites. The resulting plasmid was designated pCGN4733. PCGN4731 and pCGN4633 were digested with Ndel, and the Ndel fragment containing the polylinker region synthesized from pCGN4733 was left at the Ndel site of 4731, giving pCGN4734. This last plasmid was digested with Sal and Xba, and thus was pCGN5133. PCGN5133 was the 9.2 kb pst fragment in BSKS +, where polylinker sites flanking the insert were altered to different sites for ease of manipulation. Then the fragment of pCGN4734 was placed in the equivalent site of pCGN5143, giving pCGN4735. In Figure 1 a sequence for approximately 3 kb of the pCGN4735 promoter construct is provided. The resynthesized sequence falls between the Ndel sites located at bases 1706 and 1898 of the sequences. Accordingly, the sequence of Figure 5 includes approximately 1.7 kb at 5 'of the Ndel site, at 5' for the resynchronized polylinker region, there is a sequence of almost 2.5 kb to 51 of this sequence which is not provided in Figure 5, in relation to the graft of 9.2 kb in total. The sequence of Figure 5 also includes approximately 1.1 kb to 3 'for the Ndel 3 * site. There are not provided in Figure 5 about 3 kb that are more 3 'in the Racl3 insert. A map for pCGN4735 is provided in Figure 6.

Example 7 Pigment Synthesis Genes Melanin A binary construct for the transformation of plants to express genes for the synthesis of melanin is prepared as follows. The melanin genes were originally isolated from the common ground bacteria Streptomycee antibioticus (Bernan et al. (1985) 34: 101-110). Melanin production is composed of a system of two genes. The first gene, tyrA, encodes the catalytic unit responsible for the polymerization of the amino acid tyrosine, the primary substrate, and is called tyrosinase. The second gene, ORF438, is responsible for fixing copper and delivering copper to tyrosinase, and for activating the enzyme. The expression of both ORF438 and tyrA ensures maximum tyrosinase activity. The genes for ORF438 and tyrA were completely resynthesized with respect to their DNA sequence. This was done, since the initial DNA sequence isolated from Streptomyces, has a very high content of guanine DNA and cytosine (G + C). Accordingly, the ORF438 and tyrA genes were resynthesized to appear more "plant-like" (reduced G + C content) with respect to the preferred plant codons that encode their corresponding amino acids.

Indigo Indigo production involves the conversion of the amino acid tryptophan, the primary substrate, in indole, which then becomes indoxyl. The indoxyl molecules spontaneously turn into indigo in the presence of oxygen. A two-gene system was used to affect the production of indigo in fiber cells. The first gene (tna) was obtained from the bacterium E. coli, and encodes the enzyme tryptophanase. The designation tna means the gene encoding tryptophanase from E. coli, an enzyme that converts tryptophan to indole (Stewart et al., (1986) J. Bacteriol 166: 217-223). The designation "pig" is used for the coding sequence for the protein for the production of indigo from Rhodococcus, which produces indigo from indole (Hart et al., (1990) J. Gen. Microbiol 136: 1357-1363). Both tna and pig were obtained by polymerase chain reaction. 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 bacterial genes were used in their native form.

Example 8 Constructs for Directing the Pigment Synthesis Genes For the address to the plastid, the constructs contain a fragment of the small subunit gene of tobacco ribulose bisphosphate carboxylase, which encodes the transit peptide and 12 amino acids of the mature protein ( Tssu) placed in the reading frame with the appropriate coding sequence. For the vacuolar direction of the melanin synthesis genes, the constructs include a fragment of the metallocarboxypeptidase inhibitor gene, which encodes all the N-terminus signal peptide of 32 amino acids of that protein plus 6 amino acids of the mature protein (CPI + 6 ) (Martineau et al., Supra), placed in the reading frame with the appropriate coding sequences. In addition to the signal peptide, a sequence encoding a vacuolar localization signal (VLS) is inserted 3 'of the protein coding sequence. Constructs containing coding sequences for bacterial genes, involved in the biosynthesis of pigmented compounds, and sequences for directing the transport of encoded proteins into plastids or vacuoles, are prepared as follows.

Melanin Resynthesized ORF438 and tyrA genes were treated in two different ways, depending on which compartment of the fiber cell the final protein products would be located. A chimeric gene / plant binary construct (designated pCGN5148) contained the genes directed towards the plastids of the fiber cell. To do this, 12 amino acids of a gene for the small carboxylase subunit (SSU) plus the original 54-amino acid small subunit transit peptide were fused with the amino termini of both ORF438 and tyrA gene products, respectively. These peptide sequences allow the ORF438 and tyrA (proteins) gene products to be efficiently directed towards the plastid. This direction was initiated when the plastid was the site of tyrosine production inside the fiber cell. The second chimeric gene / plant binary construct (designated pCGN5149) contained the ORF438 and tyrA genes directed towards the vacuole inside the fiber cell. Based on information from other biological systems, it was postulated that the vacuole of the fiber cell may contain a high concentration of tyrosine for the polymerization of melanin. Both ORF438 and tyrA genes contain the signal peptide of 29 amino acids from a tomato carboxypeptidase inhibitor protein (CPI) as amino-terminal gene fusions, to direct these proteins to the secretory system of the endoplasmic reticulum (ER) of the cell of fiber. In addition, the tyrA gene has a vacuolar direction peptide (VTP) of 8 amino acids from the carboxypeptidase inhibitor protein fused at the carboxy terminus, such that the tyrosinase activated by mature copper will eventually be directed towards the vacuole of the fiber cell. Both ORF438 and tyrA proteins also had potential glycosylation sites removed by site-directed mutagenesis of the ORF438 and tyrA genes, respectively. The glycosylation of potential plant cells of these proteins on their expression in fiber cells could result in the inactivation of tyrosinase, and therefore, the removal of potential glycosylation sites was considered necessary.

Indigo The only modification to the indigo genes was the fusion of the small tobacco subunit transit peptide that encodes DNA sequences on the amino-terminal region of both tna and tig genes, to affect the location of both tryptophanase and oxygenase proteins of indole, in the plastid of the fiber cell. These are the exact same gene fusions that were made for the plastid-targeted proteins for the production of melanin in the 5148 construct. The tria and pig genetic products were directed towards the plastid of the fiber cell, since that is the primary site of the synthesis of tryptophan.

Example 9 Expression Constructions Melanin The modified genes for both ORF438 and tyrosinase proteins directed to the plastid and the vacuole were placed in a fiber expression cassette to "switch" during the development of the cotton fiber cell. The "switch" (promoter) used for the melanin constructions was 4-4. The modified ORF438 and tyrA genes were cloned into the cassette of the 4-4 promoter, and these chimeric genes were then inserted into a binary plasmid to create the plasmids pCGN5148 and pCGN5149, which contained the modified genes for the ORF438 and tyrosinase proteins directed to the plastid and the vacuole, respectively. These binary plasmids also contain genetic determinants for their stable maintenance in E. coli and Agrobacterium, and also contain a chimeric gene for expression in the plant cell of the bacterial kanamycin resistance gene. This kanamycin resistance marker allows the selection of the transformed cotton cells against the untransformed ones, when the hypocotyl or the segments of the leaves of the plants are infected with Agrobacterium containing the binary plasmids. A block diagram of plasmid pCGN5149, which has vacuolar direction sequences, is shown in Figure 8. Plasmid pCGN5148 (not shown) is constructed in the same way as 5149, only that pCGN5148 has plastid targeting sequences.

Indigo As with the melanin genes, the tna and pig genes directed to the plastid were placed in the cassette of the fiber specific 4-4 promoter, and these chimeric genes were subsequently inserted into a binary plasmid to create the plasmid pCGN5616. A block diagram of plasmid pCGN5616 is shown in Figure 8.

Anthocyanin A construction has been prepared for the expression of maize R and Cl genes in the development of cotton fiber. It is known that these genes are responsible for the production of anthocyanin pigments by acting in a regulatory manner, to activate the chalcone path for the production of anthocyanins (red spectrum colors). The R and Cl genes were placed under the control of the Racl3 promoter cassette. A binary plasmid designated pCGN4745 (not shown) contains both R and Cl genes, each under the control of the Racl3 promoter.

Example 10 Cotton Transformation Extract Preparation 315 Coker seeds were surface disinfected by placing them in 50 percent Clorox (2.5 percent sodium hypochlorite solution) for 20 minutes, and rinsing 3 times in sterile distilled water. Following the surface sterilization, the seeds were germinated in sterile tubes of 25 x 150, containing 25 milliliters of salts 1/2 x mS: vitamins 1/2 x B5: 1.5% glucose: 0.3% gelrite. Seedlings were germinated in the dark at 28 ° C for 7 days. On the seventh day, the nurseries were placed in the light at 28 + 2 ° C.

Cocultivation and Regeneration of the Plant The simple colonies of A. tumefaciens strain 2760, containing the binary plasmids pCGN2917 and pCGN2926, are transferred to 5 milliliters of MG / L broth, and grown overnight at 30 ° C. Bacterial cultures are diluted to 1 x 108 cells / milliliter with MG / L, just before cocultivation. The hypocotyls of the 8-day-old seedlings are cut, cut into sections of 0.5 to 0.7 centimeters, and placed on tobacco feeder plates (Horsch et al., 1985). Feeder plates are prepared 1 day before use, coating with 1.0 milliliter of tobacco suspension culture on a petri dish containing Callus Initiation Medium, MIC, without antibiotics (MS salts: vitamins B5: 3 percent glucose: 0.1 milligrams / liter of 2,4-D: 0.1 milligrams / liter of kinetin: 0.3 percent gelrite, and the pH was adjusted to 5.8 before autoclaving). A sterile filter paper disk (Whatman # 1) was placed on top of the feeder cells before use. After all the sections are prepared, each section is submerged in a culture of A. tumefaciens, dried on sterile paper towels, and returned to the tobacco feeder plates. After two days of cocultivation in the feeder plates, the hypocotyl sections are placed in Callus Initiation Medium containing 75 milligrams / liter of kanamycin, and 500 milligrams / liter of carbenicillin. The tissue was incubated at 28 ± 2 ° C, 30uE 16: 8 light period: dark for 4 weeks. At four weeks, the whole explant was transferred to the fresh callus initiation medium containing antibiotics. After two weeks on the second pass, the callus was removed from the explants, and divided between Calcium Initiation Medium and Regeneration Medium (MS salts: 40mM KN03: NH4C110mM: vitamins B5: 3% glucose: gelrite at 0.3 percent: 00 milligrams / liter of carb: 75 milligrams / liter of kanamycin). The embryogenic callus was identified 2 to 6 months after initiation, and subcultured on fresh regeneration medium. Embryos are selected for generation, placed in static embryonic fluid medium (Stewart's medium and Hsu: 0.01 milligrams / liter of NAA: 0.01 milligrams / liter of kinetin: 0.2 milligrams / liter of GA3), and incubated during the night at 30 ° C. The embryos were dried on paper towels, and placed in magenta boxes containing 40 milliliters of Stewart medium and Hsu solidified with Gelrite. The germinating embryos are maintained at 28 + 2 ° C 50 uE m ~ 2s-1, photoperiod of 16: 8. The seedlings with roots are transferred to land, and settled in the greenhouse. The growth conditions of cotton in the culture chambers are as follows: photoperiod of 16 hours, temperature of approximately 80 to 85 ° C, light intensity of approximately 500μEinsteins. The growth conditions of cotton in the greenhouses are as follows: photoperiod of 14-16 hours with light intensity of at least 400μEinsteins, daytime temperature of 32 ° C-35 ° C, night temperature of 21 ° C to 24 ° C , and relative humidity of approximately 80 percent.

Analysis of the Plant The flowers of the IT plants grown in the greenhouse were labeled in the anthesis in the greenhouse. Cubes (cotton flower buds), flowers, pods, etc., were harvested from these plants at different stages of development, and were assayed for the activity of the enzyme. Fluorometric and GUS histochemical assays are performed on manually cut sections, as described in the pending application submitted to Martineau et al., Supra. For the color characteristics of the fiber, the plants are visually inspected, or Northern or Western analysis can be performed, if necessary.

Example 11 Expression of Transgenic Pigment Synthetic Genes Melanin Plants that exhibited resistance to the selectable marker of kanamycin by means of a leaf assay, and corresponding Western analysis, were considered transformed. The transgenic fiber was harvested from the individual transformants of the plant at different stages of fiber development, and analyzed in two ways. One was to analyze the fiber at a single point of development time for each transgenic cotton plant, to compare the expression of tyrosinase among the transgenic events. The other was to track the developing fiber of selected plants to analyze the time of tyrosinase expression under the control of the fiber specific 4-4 promoter, by Western blots, using antisera prepared against the purified tyrosinase protein. For the plastid-directed construct of pCGN5148, 9 of the 13 events tracked for tyrosinase expression were positive, while 13 of the 16 events of the pCGN5149 construct directed to the transformed vacuole, which were screened, were positive. The level of fiber expression in tyrosinase positive plants is about 0.1 to 0.5 percent fiber cell protein. Clearly, cotton fiber cells comprising DNA color constructs produce the necessary proteins required for the synthesis of a pigment. Visually, the fluff of the positive events for tyrosinase, exhibits the color to different degrees, while the plants that do not express the enzyme, do not exhibit any color. The colorimeter measurements of the cotton fiber taken from the Coker 130 control plants and from the plants of different events transformed with pCGN5148, are given in Figures 9 and 10, respectively. Plant fiber pCGN5148 (directed to the plastid) demonstrates a bluish green color phenotype. One event, 5148-50-2-1, included cotton fiber cells (lint) that were colored, and had a negative value less than -8.0, measured in the color space L * a * b *. Coker 130 cotton fiber cells typically do not show a negative value. These colored cotton cells also had a color localized on the color space L * C * h with a relatively high hue angle value h, greater than 135 °. Normal Coker 130 fibers have a similar value that is not greater than about 90 °, as measured by this method. The results of the measurements of the cotton fiber colorimeter, taken from plants transformed with pCGN5149, are given in Figure 11. Plant fibers expressing tyrosinase from the pCGN5149 construct (directed to the vacuole) tend to have a light chestnut phenotype.

Indigo The resistance to the selectable marker of kanamycin by means of the leaf test and Western analysis, was again the criterion to designate a plant as transformed by pCGN5616. The transgenic fiber was harvested from individual plant transformants at different stages of fiber development. The transgenic developing fiber is traced from the selected plants, to analyze the time of expression of the tna and pig gene under the control of the fiber-specific 4-4 promoter., and the fiber is also analyzed at a single point of development time for each transgenic cotton plant, for a comparison of the expression of both tryptophanase and indole oxygenase between the transgenic events, by using Western spots with prepared antisera. against indole tryptophanase and oxygenase proteins. For indigo events, 15 of the 24 plants traced were positive for the expression of both tryptophanase and indole oxygenase enzymes. The fiber expression levels of these proteins are between 0.05 and 0.5 percent fiber cell protein. Approximately half of these transformants are expressing both genes in the fiber, resulting in a very weak light blue phenotype. Visually, there is a weak blue color in most of these positive events, particularly in the fiber at 20-30 days after anthesis in the unopened sheath. The results of the measurements of the cotton fiber colorimeter taken from different events of plants transformed with pCGN5616 are given in Figure 12. Many of these events had relatively low a * values (less than 2), with high b * values (greater than 10), measured in the color space L * a * b *. In a similar way, several events of 5149 were also measured with a value a * less than 2, while maintaining a b * value greater than 10.

Cotton BC In Figure 13 colorimeter measurements taken on naturally colored fiber from four separate BC cotton lines are provided. The above results demonstrate that the color phenotype of a transgenic cotton fiber cell can be altered by the expression of pigment synthesis genes. The transgenic cotton fiber cells include both a protein that synthesizes the pigment, and a pigment produced by the protein that synthesizes the pigment. As shown from the results of Figures 9 to 13, the expression of a pigment gene of interest can result in cotton fiber cells where pigment synthesis, combined with appropriate targeting sequences, results in the modification of the color phenotype in the selected plant tissue, producing a colored cotton fiber, by means of the expression from a genetically designed construction. All publications and patent applications cited in this specification are hereby incorporated by reference as if each publication or individual patent application was specifically or individually indicated as being incorporated by reference. Although the invention has been described in some detail, by way of illustration and example for purposes of clarity and understanding, it will be readily apparent to ordinary experts in the field that certain changes and modifications may be made thereto, without departing from the spirit. or scope of the appended claims.

Claims (45)

1. A DNA construct comprising, as components operably linked in the direction of transcription, a cotton fiber transcription factor, and an open reading frame encoding a protein of interest, wherein the transcription factor is selected from of the group consisting of the promoter sequences Ltp, 4-4, and rae.

2. The DNA construct according to claim 1, further comprising a coding sequence for transport signal from a nuclear plant coding gene.

3. The DNA construct according to claim 2, wherein the transport signal coding sequence comprises a plastid transit peptide.

4. The DNA construct according to claim 1, wherein the transport signal coding sequence encodes a signal peptide that provides transport through the crude endoplasmic reticulum.

5. The DNA construct according to claim 4, wherein the sequence further comprises, at 3 'for the open reading frame, a vacuolar localization signal.

6. The DNA construct of claim 1, wherein the pigment is melanin or indigo.

The DNA construct of claim 6, wherein the open reading frame is from a bacterial gene.

The DNA construct of claim 7, wherein the bacterial gene is selected from the group consisting of ORF438, tyrA, the anthocyanin gene R, the anthocyanin 1, pig, and tna gene.

9. A plant cell comprising a DNA construct of claim 1.

10. A cotton plant cell according to claim 9.

11. A cotton fiber cell according to claim 10.

12. A plant comprising a cell of any of claims 9 to 11.

13. A method for modifying the fiber phenotype in a cotton plant, this method comprising: transforming a plant cell with DNA comprising a construct for the expression of a protein in a pigment biosynthesis pathway, wherein this construct comprises, as the operably linked components: a functional transcription initiation region in the cells of this cotton plant, an open reading frame encoding a protein of interest, and a functional transcription termination region in the cells of said cotton plant, wherein the plant cell comprises an treatment of this protein; and to grow this plant cell to produce a cotton plant, where the protein reacts with the substrate to produce the pigment.

The method of claim 13, wherein this construct further comprises a transport signal coding sequence, from a nuclear plant coding gene.

15. The method of claim 13, wherein the transport signal coding sequence encodes a signal peptide that provides transport through the crude endoplasmic reticulum.

The method of claim 13, wherein the DNA comprises constructs for the expression of two proteins in a pigment biosynthesis path, wherein each of the constructs comprises components i) to iv), and wherein these two Proteins are not encoded by the same gene.

17. The method of claim 16, wherein said pigment is melanin, and the proteins are encoded by tyrA and ORF438.

18. The method of claim 16, wherein the pigment is indigo, and the proteins are tna and pig.

The method of claim 16, wherein the pigment is anthocyanin, and the constructs comprise the anthocyanin regulatory genes R and Cl.

The method of claim 13, wherein the plant cell is a fiber cell of cotton, and wherein the transcription region is a region of transcription initiation of fiber tissue.

21. The method of claim 20, wherein the transcription region is selected from the group consisting of the promoter sequences Ltp, 4-4, and rae.

22. A recombinant DNA construct comprising the cotton tissue transcript sequence shown in Figure 2.

23. A recombinant DNA construct comprising the cotton tissue transcript sequence shown in Figure 5.

24. A DNA coding sequence isolated from Figure 1.

25. An isolated DNA coding sequence of Figure 4.

26. The method of claim 13, wherein the protein of interest is involved in the synthesis of a plant hormone.

27. An isolated DNA sequence comprising the cotton lipid transfer protein coding sequence of Figure 7.

28. A cotton fiber cell comprising a DNA sequence, wherein said DNA sequence comprises , as the components operably linked in the direction of transcription, a cotton fiber transcription factor, and an open reading frame that encodes a protein required for the synthesis of a pigment.

29. A cotton fiber cell according to claim 27, which comprises pigment produced by this pigment-synthesizing protein.

30. A cotton fiber cell according to claim 27, wherein said DNA sequence further comprises a transport signal encoding a sequence from a nuclear plant coding gene.

31. A cotton fiber cell according to claim 29, wherein this transport signal coding sequence comprises a plastid transit peptide.

32. A cotton fiber cell according to claim 29, wherein this transport signal coding sequence encodes a signal peptide, which provides transport through the crude endoplasmic reticulum.

33. A cotton fiber cell according to claim 31, wherein this sequence further comprises, at 3 'for the open reading frame, a vacuolar localization signal.

34. A cotton fiber cell according to claim 27, wherein the transcription factor is selected from the group consisting of the cotton fiber lipid transfer promoter sequence, the promoter sequence 4-4, and the promoter sequence rae.

35. A cotton fiber cell according to claim 27, wherein the pigment is melanin or indigo.

36. A cotton fiber cell according to claim 27, wherein the open reading frame is from a bacterial gene.

37. A cotton fiber cell according to claim 35, wherein the bacterial gene is selected from the group consisting of ORF438, tyrA, anthocyanin gene R, anthocyanin gene Cl, pig, and tna.

38. A cotton fiber cell comprising melanin.

39. A cotton fiber cell comprising indigo.

40. A cell of cotton fiber that is colored by genetic engineering, and that has a negative value of less than -1.0, measured in the color space L * a * b *.

41. The cotton fiber cell of claim 39, wherein the a * negative value is less than -5.0.

42. The cotton fiber cell of claim 40, wherein the a * negative value is less than -8.0.

43. A cell of cotton fiber that is colored by genetic engineering, and that has a value a * of less than 2, and the value b * greater than 10, measured in the color space L * a * b *.

44. A cotton fiber cell that is colored by genetic engineering, and that has a hue angle value greater than 100 °, measured in the color space L * C * h.

45. The cotton fiber cell of claim 43, wherein the value h is greater than 135 °.