METHODS OF INTRODUCTION OF A NUCLEIC ACID OF INTEREST IN A VEGETABLE CELL WITH CELL WALL, STABLE EXP

专利摘要:
quantum dot and vehicle peptide conjugates suitable for imaging and distribution applications in plants. methods for introducing a molecule of interest into a plant cell with a cell wall are presented using a qd-peptide conjugate with a quantum dot (qd) with one or more cell penetrating peptides (cpps). methods are presented for the genetic or other modification of plants and for the treatment or prevention of diseases in plant cells comprising a cell wall.
公开号:BR112013024220B1
申请号:R112013024220-5
申请日:2012-03-22
公开日:2020-12-15
发明作者:Jayakumar Pon Samuel；Narasimha Chary Samboju；Kerrm Y. Yau；Gaofeng Lin；Steven R. Webb；Frank G. Burroughs
申请人:Dow Agrosciences Llc；
IPC主号:

专利说明:

PRIORITY CLAIM
[0001] This application claims the benefit of Provisional US Patent Application Serial No. 61 / 466,804, filed on March 23, 2011. TECHNICAL FIELD
[0002] The present exhibition refers, generally, to methods for the introduction of a molecule of interest in a plant cell with a cell wall using a QD-peptide conjugate with a quantum dot (QD) with one or more peptides from cell penetration (CPPs). BACKGROUND
[0003] Nanoparticles have unique properties that have been explored for use in distributing DNA to cells. Metallic nanoparticles, such as (Au) nanoparticles, have been used for DNA distribution because of their low cytotoxicity and ease of functionalization with various ligands of biological significance. In addition to metallic nanoparticles, semiconductor nanoparticles (eg, quantum dots) ("QD") within the 3 - 5 nm size range have also been used as vehicles for delivering molecules to cells. DNA and proteins can be linked to the ligand attached to the surface of the QD (see, for example, F. Patolsky et al., J. Am. Chem. Soc. 125, 13918 (2003)).
[0004] Nanoparticles have been used to deliver plasmid DNA to a variety of animal cells. It has been found that when nanoparticles coated with DNA are incubated with cells without a cell wall, the cells capture the nanoparticles and begin to express any genes encoded in the DNA. However, the current distribution of plant genes is challenging due to the presence of walls in the plant cell, which leads to the common dependence on invasive means of distribution for the transformation of plants. When nanoparticle-mediated distribution is desired for cells normally with a cell wall, the cell wall is removed before adding the particles to the plant's protoplasts (see F. Torney et al., Nature Nanotechnol. 2 (2007)). In plant cells, the cell wall presents a barrier to the distribution of molecules applied exogenously. Many invasive methods, such as gene gun (biolistics), microinjection, electroporation and Agrobacterium, were used to achieve the distribution of genes and small molecules to these plant cells with walls, but the distribution of proteins was only achieved by microinjection. The distribution of small molecules and proteins in the presence of a plant cell wall remains unexplored and it would be advantageous to develop technologies that allow its implementation in plant cells / tissues or organs for manipulations in vitro and in vivo.
[0005] Cell penetrating peptides (CPPs) are a new, fast-growing class of short peptides that are known to play an important role in translocating a wide range of charge complexes, including proteins and DNA, through biomembranes in cell lines of mammals and humans. J. Schwartz and S. Zhang (2000), Peptide-Mediated Cellular Delivery, Curr. Opin. Mol. Ther. 2: 162-167; Ü. Langel (2002), Preface in Cell Penetrating Peptides; Processes and Applications, Ü. Langel, editor, CRC Press, Boca Raton; E. Vives and B. Lebleu (2002), The Tat-Derived Cell-Penetrating Peptide in Cell-Penetrating Peptides; Processes and Applications, Ü. Langel, editor, CRC Press, Boca Raton, pp. 3-22. Although CPPs have been shown to facilitate charge distribution in mammalian cells, the use of CPP in plant cells for transfection studies has been limited by a number of factors. A major obstacle to adapting this technology to plants is that, unlike animal cells, plant cells have a double barrier system (cell wall and plasma membrane) for the internalization of CPPs and their loads. Consequently, CPPs must overcome these two barriers for efficient translocation. CPPs have been used in plant cells, but are typically based on the use of agents and permeabilization techniques with the use of CPPs to effect charge distribution to plant cells. The HIV-1 TAT protein (PTD) transduction domain is one of the best studied translocation peptides. Recent reports have demonstrated the potential of TAT-PTD and its oligomers to distribute plasmids by forming a complex with negatively charged DNA in mammalian cells. I. Ignatovich, E. Dizhe, A. Pavlotskaya, B. Akifiev, S. Burov, S. Orlov and A. Perevozchikov (2003), Complexes of Plasmid DNA with Basic Domain 47-57 of the HIV-1 Tat Protein Are Transferred to Mammalian Cells by Endocytosis-mediated Pathways, J. Biol. Chem. 278: 42625-42636; C. Rudolph, C. Plank, J. Lausier, U. Schillinger, RH Müller and J. Rosenecker (2003), Oligomers of the Arginine-Rich Motif of the HIV-1 TAT Protein are Capable of Transferring Plasmid DNA into Cells, J Biol. Chem. 278: 11411-11418; Z. Siprashvili, F. Scholl, S. Oliver, A. Adams, C. Contag, P. Wender and P. Khavari (2003), Gene Transfer via Reversible Plasmid Condensation with Cysteine-Flanked, Internally Spaced Arginine-Rich Peptides, Hum Gene. The R. 14 (13): 1225-33; I. Hellgren, J. Gorman and C. Sylvén (2004), Factors Controlling the Efficiency of Tat-mediated Plasmid DNA Transfer, J. Drug. Target. 12 (1): 39-47. Other peptides that have been shown to have translocation properties include pVEC, transportan, penetratin, pep-1 peptides and fragments thereof.
[0006] The coating of QDs with peptides is an approach that has become popular in manipulating the surface of nanoparticles for various biotechnological processes. For example, the attachment of cell-penetrating peptides, such as polyarginine and TAT-derived peptides, to the surface of the QD allowed the translocation of QDs into animal cells. Recently, CPPs have become widely used as vehicles for the cellular distribution of molecules in basic and applied biomedical research. When aided, it is now possible to introduce impermeable substances into the membrane, such as peptic nucleic acids (PNA), proteins, oligonucleotides or nanoparticles in mammalian cells. There is a growing attraction of plant biologists for the use of CPPs for the distribution of biomolecules to cells and their transient expression. Thus, there is still a need for a method of stable incorporation of genes and other molecules of interest in plants through the use of nanoparticle-based distribution. DESCRIPTION
[0007] The following modalities are described together with systems, tools and methods that must be exemplary and illustrative and not limit the scope.
[0008] One embodiment of the invention includes a method of introducing a molecule of interest into a plant cell with cellular appearance to effect stable transformation of a plant and seeds. The method includes providing the plant cell with a cell wall and interacting a quantum dot (QD) with one or more cell penetrating peptides (CPPs) to form a QD-peptide conjugate, and fixing one or more molecules of interest to one or more CPPs to form an activated QD-peptide conjugate. The cell and the activated QD-peptide conjugate are brought into contact with each other, under conditions that allow their uptake by the cell with the cell wall.
[0009] Another embodiment of the invention includes a method of stable expression of a gene. The method includes providing a plant cell with a cell wall, interacting a quantum dot (QD) with one or more cell-penetrating peptides (CPPs) to form a QD-peptide conjugate, and attaching one or more genes to one or more CPPs to form an activated QD-peptide conjugate. The plant cell with cell wall and the activated QD-peptide conjugate are brought into contact with each other, and the QD-peptide conjugate and one or more genes are put under conditions that allow their uptake by the plant cell with cell wall. The gene in the offspring of a plant with the plant cell is then expressed.
[00010] Yet another embodiment of the invention includes a method for transferring a molecular substance to a plant cell. The method includes the interaction of a quantum dot (QD) with one or more cell-penetrating peptides (CPPs) to form a QD-peptide conjugate, and the interaction of the QD-peptide conjugate with a plasmid DNA to form a QD conjugate structure. -peptide activated. The structure of the activated QD-peptide conjugate is put in contact with an intact wall-bearing plant cell under conditions that allow the uptake of one or more CPPs and one or more plasmid DNA genes by the plant cell.
[00011] Another particular embodiment of the invention includes a method of screening and identifying transformation of the plant. The method includes providing a plant cell with a cell wall, interacting a quantum dot (QD) with one or more cell penetrating peptides (CPPs) to form a QD-peptide conjugate, and fixing one or more molecules of interest to one or more CPPs to form an activated QD-peptide conjugate. The cell with a cell wall and the activated QD-peptide conjugate are brought into contact with each other, and the QD-peptide conjugate and the molecule of interest are placed under conditions that allow its uptake by the plant cell with the cell wall. An image of the plant cell with the cell wall is then formed.
[00012] In addition to the exemplary aspects and modalities described above, additional aspects and modalities will be clear in view of the following descriptions. BRIEF DESCRIPTION OF THE DRAWINGS
[00013] FIG. 1 illustrates a modality of a quantum dot / peptide conjugate.
[00014] FIG. 2 illustrates a map of plasmid pDAB3831. MODE (S) FOR CARRYING OUT THE INVENTION
[00015] In the description and tables that follow, numerous terms are used. To provide a clear and consistent understanding of the report and claims, including the scope to be given to these terms, the following definitions are provided:
[00016] Backcross. Backcrossing can be a process in which a breeder repeatedly crosses the hybrid offspring with one of the ancestors, for example, a first generation F1 hybrid with one of the ancestor genotypes of the F1 hybrid.
[00017] Embryo. The embryo can be the small plant contained in the mature seed.
[00018] Resistant to a herbicide. Resistance to a herbicide dosage refers to the ability of a plant to survive (that is, the plant may not be killed) at that herbicide dosage. In some cases, tolerant plants may temporarily turn yellow or otherwise exhibit some herbicide-induced injury (for example, excessive tillering and / or growth inhibition), but they recover.
[00019] Stabilized. Stabilized refers to the characteristics of a plant that are passed reproducibly from one generation to the next generation of inbreeding plants of the same variety.
[00020] Funding. Uptake refers to the translocation of a particle, such as quantum dots, carrier peptides, cell penetrating peptides, and guided peptides, through a cell wall or a cell membrane, where translocation does not occur just as a result of the moment given to the particle by something different from the cell in which the particle is being captured. Non-limiting examples of devices or methods that cause translocation of a particle through a cell wall or a cell membrane only as a result of the moment given to the particles are biolistic, gene gun, microinjection and / or impalefection technologies.
[00021] In some embodiments of the invention, multiple attachment sites or the addition of an "additional" or "guest" molecule can be manipulated into one or more peptides at several and / or multiple sites. This property can be used, for example, in the specific selection and editing of molecular sites within cells for areas such as biomimetics, targeted distributions, for options of non-genetically modified organisms and options in various crops of trees or vegetables for options of traits and resistance to diseases. Modalities of the invention can also be employed to develop suitable biosensors. In addition, artificial chromosomes (ACES) can be used with the methods of the invention as an alternative to current eukaryotic vectors for options for precise targeting and homologous recombination.
[00022] Particular embodiments of the invention generally refer to the use of multifunctional fluorescent nanoparticles suitable for the distribution of negatively charged molecules, such as DNA / RNA. Vehicle and cell-penetrating peptides (CPPs) / guided peptides (HPs) (here collectively called "CPPs"), such as R9, TAT, MPG and Y-Zein, have been incorporated into the surface of luminescent quantum dots (QDs). QD-peptide conjugates were used for efficient DNA distribution in Arabidopsis in-plant pathways. QD-peptide bioconjugates did not exhibit any toxic effects with respect to the growth of Arabidopsis floral axes and seed set. Several stable T1 transformants were identified, and the seedlings were analyzed. Vehicle-based distribution of DNA and the establishment of stable transformation using QDs have been demonstrated in plants. Complex charges can be manipulated with smart options to distribute biomolecules that are targeted to cells and cellular compartments with precision. In particular modalities, the use of these autofluorescent QDs can be used for imaging options in plants.
[00023] According to certain embodiments of the invention, a method of introducing a molecule of interest into a plant cell with cellular appearance can be provided to effect stable transformation of a plant and seeds. The method includes providing the plant cell with a cell wall and interacting a quantum dot (QD) with one or more cell penetrating peptides (CPPs) to form a QD-peptide conjugate, and fixing one or more molecules of interest to one or more CPPs to form an activated QD-peptide conjugate. The cell and the activated QD-peptide conjugate are brought into contact with each other, under conditions that allow their uptake by the cell with the cell wall.
[00024] In some embodiments, several peptides were covalently coupled to QD nanoparticles and distributed to plant cells. Nanoparticles with R9, Y-Zein and MPG CPPs have been successfully distributed to plants to obtain stable plant transformation. In other modalities, labeled biomolecules were used to track the destination of the charge in the cytoplasm. Effective uptake of these QD-peptide-DNA conjugates was achieved, and the complex between DNA and the QD-peptide conjugate was stable, as demonstrated by the stable transformation that is transmitted by recovered resistant T1 seeds and seedlings.
[00025] In other respects, the invention relates to the application of QD-Peptide "vehicle" conjugates as a filler for multifunctionalization options for the intelligent distribution of biofunctionalized biomolecules (for example, DNA / RNA and enzyme distribution), formation of images and for various biotechnological diagnostics and reading functions. The present strategy can offer surface chemistry and encapsulation that is very adaptable, thus facilitating the synthesis of a wide range of molecules with different functionalities. The key properties in terms of the potential use of these materials in the distribution of biomolecules and genes are defined by the high density of terminal groups available in these systems. They contribute to the surface characteristics of molecules, offer multiple attachment sites (for example, for conjugation of signal or targeting fractions) and determine molecular volume, which is important for the ability to sequester other molecules to that complex. The conjugated vehicle peptides work simultaneously for the distribution of both the QD and the fixed charge. In addition, with the formation of a charge complex directly with the vehicle peptide, the compromises in the number of species fixed on the surface of the QD can be eliminated. Since negatively charged oligonucleotides are not able to translocate cell wall / membrane barriers and cell membrane by themselves, the present invention features, among others, effective delivery systems for integrating DNA, regulating genes and editing strategies.
[00026] In accordance with embodiments of the present invention, a plant cell with a cell wall can be any plant cell comprising an intact and entire cell wall. Examples of cells with a cell wall include, but are not limited to, algae, tobacco, carrots, corn, canola, rapeseed, cotton, palm, peanuts, soybeans, sugar cane, Oryza sp., Arabidopsis sp. and Ricinus sp., preferably tobacco, carrots, corn, cotton, canola, soybeans and sugar cane; more preferably tobacco and carrots. Modalities of the invention may include cells comprising a cell wall of any tissue or wherever found, including, but not limited to, in embryos, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds , pods, stems and tissue culture.
[00027] In embodiments of the invention, the molecule of interest can be any molecule that can be delivered to a plant cell according to the present invention. Molecules of interest, or components of molecules of interest, may comprise, but are not limited to, nucleic acids, DNA, RNA, RNAi molecules, genes, plasmids, cosmids, YACs, BACs, Artificial Plant Chromosomes, Plant Minicromosomes, Loci DNA of Traces Manipulated in Plants; polypeptides, enzymes, hormones, glyco-peptides, sugars, fats, signaling peptides, antibodies, vitamins, messengers, secondary messengers, amino acids, cAMP, drugs, herbicides, fungicides, antibiotics and / or their combinations.
[00028] Modalities of the invention include methods for preventing or treating disease. Exemplary non-limiting modalities include the delivery of fungicides, antibiotics and / or other drugs to cells in need using methods of the present invention.
[00029] In aspects of the invention, the QD-peptide conjugate can be captured in various parts of the cells. Examples of locations where the QD-peptide conjugate can be captured include, but are not limited to, cytosol, nucleus, tonoplasts, plastids, ethioplasts, chromoplasts, leukoplasts, elaioplasts, proteinoplasts, amyloplasts, chloroplasts and a double membrane lumen. In other embodiments of the invention, the QD-peptide conjugate can be captured by a cell comprising a cell wall via the symplastic or apoplastic pathway.
[00030] Additional embodiments of the invention include genetically modified plant cells and methods for their generation, wherein the plant cells have one or more nucleic acids introduced by methods of the present invention. In an example of an embodiment, a plasmid comprising a gene of interest and a selectable marker can be introduced into a plant cell with a cell wall by means of a QD-peptide conjugate according to the present invention. In additional modalities, it is possible to select stable transformants that have stably integrated the gene of interest and / or the selectable marker. In alternative embodiments, a plant cell now comprising the gene of interest can be propagated to produce other cells comprising a molecule of interest. In other embodiments, plant cells now comprising a molecule of interest can be a regenerable cell that can be used to regenerate an entire plant that includes the molecule of interest.
[00031] In another aspect, the present invention provides methods of creating regenerable plant cells comprising a molecule of interest for use in tissue culture. The tissue culture will preferably be able to regenerate plants with substantially the same genotype as the regenerable cells. The regenerable cells in these tissue cultures can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods or stems. In addition, one embodiment of the invention features plants regenerated from the tissue cultures of the invention.
[00032] Alternatively, the present invention presents a method of introducing a desired trait into a plant cell with a cell wall, in which the method comprises: placing a QD-peptide conjugate and a molecule of interest capable of conferring the desired trait to the plant cell in contact with the cell, allowing the uptake of the QD-peptide conjugate through the cell wall. Examples of desired traits include, but are not limited to, selected traits of male sterility, resistance to herbicides, resistance to insects and resistance to bacterial diseases, fungal diseases and / or viral diseases.
[00033] Additional aspects of the invention provide methods of generating stabilized plant lines comprising a desired trait or molecule of interest, wherein the desired trait or molecule of interest can first be introduced by capturing a QD-peptide conjugate through a wall plant cell. Methods of generating stabilized plant strains are well known to those skilled in the art and may include techniques such as, but not limited to, asexual reproduction, backcrossing, hybrid production, crossbreeding with populations and others. All plants and plant cells comprising a desired trait or molecule of interest first introduced into the plant cell (or its predecessors) by capturing a QD-peptide conjugate through a cell wall are within the scope of this invention. Advantageously, plant cells comprising a desired trait or molecule of interest first introduced into the plant or cell (or its predecessors) by capturing a QD-peptide conjugate through a cell wall can be used in crosses with other, or different, plant cells to produce first generation hybrid cells (F1), seeds and / or plants with superior characteristics.
[00034] In modalities in which the molecule of interest comprises one or more genes, the gene (s) can be a dominant or recessive allele. As an example, the gene (s) will confer traits such as herbicide resistance, insect resistance, bacterial resistance, fungal resistance, resistance to viral diseases, male fertility, male sterility, increased nutritional quality and industrial use.
[00035] With the advent of molecular biology techniques that allowed the isolation and characterization of genes encoding specific protein or RNA products (eg, RNAi), scientists in the field of plant biology have developed a strong interest in genome manipulation of cells to contain and express foreign genes or additional or modified versions of native or endogenous genes (perhaps triggered by different promoters) to alter a cell's traits in a specific way. These additional and / or modified foreign genes are here collectively referred to as "transgenes." In the last fifteen or twenty years, several methods for the production of transgenic cells have been developed and, in particular embodiments, the present invention relates to transformed versions of cells and methods for their production by introducing them into a cell with a cell wall of a transgene by capturing a QD-peptide conjugate through a cell wall. In embodiments of the invention, the transgene can be contained in an expression vector.
[00036] Cell transformation may involve building an expression vector that works in a particular cell. This vector can comprise DNA that includes a gene under the control of, or operationally linked to, a regulatory element (for example, a promoter). The expression vector can contain one or more of these operably linked gene / regulatory element combinations. The vector (s) can be in the form of a plasmid and can be used alone or in combination with other plasmids to provide transformed cells using transformation methods as described herein to incorporate a transgene (s) in the genetic material of a plant cell comprising a cell wall.
[00037] The use of QD-peptide conjugates according to methods of the present invention produced plants transformed in a stable manner and demonstrated the expression of the herbicidal gene transformed in a stable manner with the phenotype in which high tolerance to the herbicide was conferred to the transgenic T1 plant. This plant proved to be fertile, as it produced T2 seeds.
[00038] Expression Vectors For Uptake by QD-peptide Conjugate: Marker Genes
[00039] Expression vectors can include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be recovered by negative selection (that is, inhibiting the growth of cells that do not contain the selectable marker gene) or by positive selection (that is, screening of the product encoded by the genetic marker). Many selectable marker genes for transformation are well known in transformation techniques and include, for example, genes that encode enzymes that metabolically detoxify a selective chemical agent that may be an antibiotic or herbicide, or genes that encode an altered target that may be insensitive inhibitor. Some methods of positive selection are also known in the art.
[00040] A commonly used selectable marker gene suitable for plant transformation may include the neomycin phosphotransferase II (nptII) gene under the control of plant regulatory signals, which confers resistance to kanamycin. See, for example, Fraley et al., Proc. Natl. Acad. Sci. U.S.A. 80: 4803 (1983). Another selectable marker gene commonly used may be the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin. See, for example, Vanden Elzen and others, Plant Mol. Biol. 5: 299 (1985).
[00041] Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamicin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase and the bleomycin resistance determinant. See Hayford et al., Plant Physiol. 86: 1216 (1988); Jones et al., Mol. Gen. Genet. 210: 86 (1987); Svab et al., Plant Mol. Biol. 14: 197 (1990); Hille et al., Plant Mol. Biol. 7: 171 (1986). Other selectable marker genes confer resistance to herbicides, such as glyphosate, glufosinate or bromoxynil. See Comai et al., Nature 317: 741-744 (1985); Gordon-Kamm et al., Plant Cell 2: 603-618 (1990); and Stalker et al., Science 242: 419-423 (1988).
[00042] Other selectable marker genes suitable for plant transformation are not of bacterial origin. Such genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimato-3-phosphate synthase and plant acetolactate synthase. See Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987); Shah et al., Science 233: 478 (1986); Charest et al., Plant Cell Rep. 8: 643 (1990).
[00043] Another class of marker genes suitable for plant transformation requires screening presumably transformed plant cells, rather than direct genetic selection of transformed cells for resistance to a toxic substance, such as an antibiotic. These genes are particularly useful for quantifying or visualizing the spatial pattern of expression of a gene in specific tissues and are often called reporter genes, because they can be fused to a gene or gene regulatory sequence to investigate gene expression. Genes for screening commonly used transformed cells include β-glucuronidase (GUS), β-galactosidase, luciferase and chloramphenicol acetyltransferase. See R.A. Jefferson, Plant Mol. Biol. Rep. 5: 387 (1987); Teeri et al., EMBO J. 8: 343 (1989); Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84: 131 (1987), DeBlock et al., EMBO J. 3: 1681 (1984).
[00044] Recently, in vivo methods for visualizing GUS activity that do not require destruction of plant tissue have become available. Molecular Probes publication 2908, Imagene Green®, p. 1-4 (1993) and Naleway et al, J. Cell Biol. 115: 151a (1991). However, these in vivo methods for visualizing GUS activity have not been shown to be useful for the recovery of transformed cells because of low sensitivity, highly fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.
[00045] More recently, genes encoding Fluorescent Proteins (for example, GFP, EGFP, EBFP, ECFP, and YFP) have been used as markers for gene expression in prokaryotic and eukaryotic cells. See Chalfie et al., Science 263: 802 (1994). Fluorescent proteins and fluorescent protein mutations can be used as screening markers.
[00046] Expression Vectors For Uptake Through QD-Peptide Conjugates: Promoters
[00047] The genes included in expression vectors must be activated by a nucleotide sequence comprising a regulatory element, for example, a promoter. Various types of promoters are currently well known in transformation techniques, as well as other regulatory elements that can be used alone or in combination with promoters.
[00048] As used herein, "promoter" includes reference to a region of DNA that may be upstream of the start of transcription and that may be involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" can be a promoter capable of initiating transcription in plant cells. Examples of promoters under development control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids or sclerenchyma. These promoters are called "preferred by the fabric". Promoters that only initiate transcription in certain tissues are called "tissue-specific". A specific promoter for a "cell type" primarily triggers expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" prosecutor may be a prosecutor who may be under environmental control. Examples of environmental conditions that can affect transcription by inducible promoters include anaerobic conditions or the presence of light. Fabric-specific, tissue-preferred, cell-specific and inducible promoters constitute the "non-constitutive" class of promoters. A "constitutive" promoter can be a promoter that can be activated under most environmental conditions. A. Inducible promoters
[00049] An inducible promoter can be operationally linked to a gene for expression in a cell. Optionally, the inducible promoter can be operationally linked to a nucleotide sequence that encodes a signal sequence that can be operationally linked to a gene for expression in a cell. With an inducible promoter, the rate of transcription increases in response to an inducing agent.
[00050] Any inducible promoter can be used in the present invention. See Ward et al., Plant Mol. Biol. 22: 361-366 (1993). Exemplary inducible promoters include, but are not limited to: those in the ACEI system that responds to copper (Mett et al., PNAS 90: 4567-4571 (1993)); corn In2 gene that responds to benzenesulfonamide herbicide protective agents (Hershey et al., Mol. Gen. Genetics 227: 229-237 (1991); and Gatz et al., Mol. Gen. Genetics 243: 32-38 (1994) ); and Tn10 Tet repressor (Gatz et al., Mol. Gen. Genetics 227: 229-237 (1991)). A particularly useful inducible promoter may be a promoter that responds to an inducing agent to which plants normally do not respond. An exemplary inducible promoter may be the inducible promoter of a steroid hormone gene, whose transcriptional activity may be induced by a glucocorticoid steroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88: 0421 (1991). B. Constitutive promoters
[00051] A constitutive promoter can be operationally linked to a gene for expression in a cell or the constitutive promoter can be operationally linked to a nucleotide sequence that encodes a signal sequence that can be operationally linked to a gene for expression in a cell.
[00052] Different constitutive promoters can be used in the present invention. Exemplary constitutive promoters include, but are not limited to: plant virus promoters, such as the CaMV 35S promoter (Odell et al., Nature 313: 810-812 (1985)); promoters of the rice actin genes (McElroy et al., Plant Cell 2: 163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12: 619-632 (1989) and Christensen et al., Plant Mol. Biol. 18: 675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81: 581-588 (1991)); MAS (Velten et al., EMBO J. 3: 2723-2730 (1984)); and H3 corn histone (Lepetit et al., Mol. Gen. Genetics 231: 276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)). The ALS promoter, fragment Xba1 / NcoI 5 'in relation to the structural gene ALS3 of Brassica napus (or a nucleotide sequence similar to said fragment Xba1 / NcoI), represents a particularly useful constitutive promoter. See PCT application WO 96/30530. C. Fabric-specific or fabric-preferred promoters
[00053] A tissue-specific promoter can be operationally linked to a gene for expression in a cell. Optionally, the tissue-specific promoter can be operationally linked to a nucleotide sequence that encodes a signal sequence that can be operationally linked to a gene for expression in a cell. Plants transformed with a gene of interest operationally linked to a tissue-specific promoter can produce the transgene protein product exclusively, or preferably, in a specific tissue.
[00054] Any tissue-specific or tissue-preferred promoter can be used in the present invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as the phaseolin gene (Murai et al., Science 23: 476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82: 3320-3324 (1985)); a leaf-specific and light-induced promoter, such as that of cab or rubisco (Simpson et al., EMBO J. 4 (11): 2723-2729 (1985) and Timko et al., Nature 318: 579-582 (1985)) ; an anther-specific promoter, such as LAT52 (Twell et al., Mol. Gen. Genetics 217: 240245 (1989)); a pollen-specific promoter, such as that of Zm13 (Guerrero et al., Mol. Gen. Genetics 244: 161-168 (1993)) or a microspore-specific promoter, such as that of apg (Twell et al., Sex. Plant Reprod. 6: 217-224 (1993)).
[00055] The transport of the protein produced by transgenes to a subcellular compartment, such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondria, or for secretion in the apoplast, can be performed through the operational connection of the nucleotide sequence that encodes a signal sequence to the 5 'and / or 3' region of a gene encoding the protein of interest. Targeting sequences at the 5 'and / or 3' end of the structural gene can determine, during protein synthesis and processing, where the encoded protein can ultimately be compartmentalized. Alternatively, these proteins directed to the subcellular compartment can be linked directly to a QD-peptide conjugate to direct the QD-peptide conjugate coated with the molecule of interest to the desired subcellular compartment.
[00056] The presence of a signal sequence directs a polypeptide to an intracellular organelle or subcellular compartment, or to secretion in the apoplast. Many signal sequences are known in the art. See, for example, Becker et al., Plant Mol. Biol. 20:49 (1992); P.S. Close, Master’s Thesis, Iowa State University (1993); C. Knox et al., "Structure and Organization of Two Divergent Alpha-Amylase Genes from Barley," Plant Mol. Biol. 9: 3-17 (1987); Lerner et al., Plant Physiol. 91: 124-129 (1989); Fontes et al., Plant Cell 3: 483-496 (1991); Matsuoka et al., Proc. Natl. Acad. Sci. 88: 834 (1991); Gould et al., J. Cell. Biol. 108: 1657 (1989); Creissen et al., Plant J. 2: 129 (1991); Kalderon, et al., A short amino acid sequence able to specify nuclear location, Cell 39: 499-509 (1984); Steifel, et al., Expression of a maize cell wall hydroxyprolinerich glycoprotein gene in early leaf and root vascular differentiation, Plant Cell 2: 785-793 (1990). Genes of Strange Proteins and Agronomic Genes
[00057] With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, provide a plurality of transgenic plants that are harvested in a conventional manner, and a foreign protein can then be extracted from a tissue of interest or from biomass. total. The extraction of the protein from the plant's biomass can be carried out by known methods, which are discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6 (1981).
[00058] In aspects of the invention, the transgenic plant presented for commercial production of foreign protein can be a cell or a plant. In other respects, the biomass of interest may be a seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated mainly by conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton 269: 284 (1993). The map information regarding the chromosomal location can be useful for protecting rights to the transgenic plant in question. If an unauthorized propagation can be carried out, and crosses are made with another germplasm, the map of the integration region can be compared with similar maps for suspect plants to determine whether the latter have a common ancestry with the plant in question. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all conventional techniques.
[00059] Likewise, agronomic genes can be expressed in transformed cells or their progeny. More particularly, plants can be genetically manipulated using the methods of the invention to express various phenotypes of agronomic interest. Exemplary genes that can be used in this regard include, but are not limited to, those categorized below. 1. Genes that confer resistance to pests or diseases and that encode:
[00060] A) Genes of resistance to plant diseases. The plant's defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence gene (Avr) in the pathogen. A variety of plants can be transformed with cloned resistance genes to manipulate plants that are resistant to specific pathogen strains. See, for example, Jones et al., Science 266: 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262: 1432 (1993) (the tomato Pto gene for resistance to Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinos et al., Cell 78: 1089 (1994) (Arabidopsis may be the RSP2 gene for resistance to Pseudomonas syringae).
[00061] B) A gene that confers resistance to a pest, such as a soybean cyst nematode. See, for example, PCT Application WO 96/30517; PCT application WO 93/19181.
[00062] C) A protein from Bacillus thuringiensis, its derivative or a synthetic polypeptide modeled after it. See, for example, Geiser et al., Gene 48: 109 (1986), which presents the cloning and nucleotide sequence of a Bt δ-endotoxin gene. In addition, DNA molecules encoding δ-endotoxin genes can be purchased from the American Type Culture Collection, Manassas, Va., For example, under ATCC Accession No. 40098, 67136, 31995 and 31998.
[00063] D) A lectin. See, for example, the exhibition by Van Damme et al., Plant Molec. Biol. 24:25 (1994), which presents the nucleotide sequences of several mannose-binding genes from Clivia miniata.
[00064] E) A vitamin-binding protein, such as avidin. See PCT order US93 / 06487. The application teaches the use of avidin and avidin counterparts as larvicides against insect pests.
[00065] F) An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262: 16793 (1987) (nucleotide sequence of the rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21: 985 (1993) (nucleotide sequence of the cDNA encoding tobacco proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem. 57: 1243 (1993) (nucleotide sequence of the Streptomyces nitrosporeus alpha-amyl inhibitor) and U.S. Patent No. 5,494,813 (Hepher and Atkinson, issued February 27, 1996).
[00066] G) An insect-specific hormone or pheromone, such as an ecdiesteroid or juvenile hormone, its variant, a mimetic based on it or its antagonist or agonist. See, for example, the exposure of Hammock et al., Nature 344: 458 (1990), of the expression in cloned juvenile hormone esterase baculovirus, a juvenile hormone inactivator.
[00067] H) An insect-specific peptide or neuropeptide that, with the expression, disrupts the physiology of the affected pest. For example, see the exhibitions by Regan, J. Biol. Chem. 269: 9 (1994) (expression cloning provides DNA encoding the insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Comm. 163: 1243 (1989) (an allostatin can be identified in Diploptera puntata). See also US Patent No. 5,266,317, to Tomalski et al., Which contains genes that encode insect-specific paralytic neurotoxins.
[00068] I) A specific insect poison produced in nature by a snake, wasp or any other organism. For example, see Pang et al., Gene 116: 165 (1992), for exposure of heterologous plant expression of a gene that encodes an insect-toxic scorpion peptide.
[00069] J) An enzyme responsible for the hyperaccumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or other non-protein molecule with insecticidal activity.
[00070] K) An enzyme involved in the modification, including post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase , natural or synthetic. See PCT application WO 93/02197 on behalf of Scott et al., Which presents the nucleotide sequence of a calase gene. DNA molecules that contain chitinase coding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco caterpillar chitinase, and Kawalleck et al., Plant Molec. Biol. 21: 673 (1993), which present the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.
[00071] L) A molecule that stimulates signal transduction. For example, see the exhibition by Botella and others, Plant Molec. Biol. 24: 757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess et al., Plant Physiol. 104: 1467 (1994), which show the nucleotide sequence of a corn calmodulin cDNA clone.
[00072] M) A hydrophobic moment A peptide. See PCT application WO 95/16776 (exposure of peptide derivatives of Tachyplesin, which inhibits fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance).
[00073] N) A membrane permease, a channel former or a channel blocker. For example, see the exposition by Jaynes et al., Plant Sci. 89:43 (1993), of the heterologous expression of a cecropin-β peptide analog to make transgenic tobacco plants resistant to Pseudomonas solanacearum.
[00074] O) An invasive viral protein or complex toxin derived from it. For example, the accumulation of viral coat proteins in transformed plant cells confers resistance to viral infections and / or the development of disease caused by the virus from which the coat protein gene can be derived, as well as related viruses. See Beachy and others, Ann. Rev. Phytopathol. 28: 451 (1990). Coating protein-mediated resistance was conferred to plants transformed against the alfalfa mosaic virus, cucumber mosaic virus, white tobacco necrosis virus, potato X virus, potato Y virus, tobacco streak virus, virus tobacco rattle and tobacco mosaic virus. Id.
[00075] P) An insect specific antibody or an immunotoxin derived from it. Thus, an antibody targeting a critical metabolic function in the insect's intestine would inactivate an affected enzyme, killing the insect. See Taylor et al., Abstract No. 497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco by producing fragments of single chain antibodies).
[00076] Q) A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366: 469 (1993), which show that transgenic plants that express recombinant antibody genes are protected against virus attacks.
[00077] R) A protein that stops development produced in nature by a pathogen or parasite. For example, endo fungal α-1,4-D-polygalacturonases facilitate fungal colonization and release of plant nutrients by solubilizing the homo- α -1,4-D-galacturonase from the plant cell wall. See Lamb et al., Bio / Technology 10: 1436 (1992). The cloning and characterization of a gene encoding a bean endopolygalacturonase inhibiting protein can be described by Toubart et al., Plant J. 2: 367 (1992).
[00078] S) A protein that interrupts the development produced in nature by a plant. For example, Logemann et al., Bio / Technology 10: 305 (1992), demonstrated that transgenic plants that express the inactivating gene of the barley ribosome have an increased resistance to fungal diseases. 2. Genes that confer resistance to a herbicide:
[00079] A) An herbicide that inhibits the growth point or meristem, such as an imidazolinone, sulfonamide or sulfonylurea. Exemplary genes in this category encode the enzyme ALS and mutant AHAS, as described, for example, by Lee et al., EMBO J. 7: 1241 (1988), and Miki et al., Theor. Appl. Genet. 80: 449 (1990), respectively.
[00080] B) Glyphosate genes (resistance conferred, for example, by mutant 5-enolpyruvylshikimato-3-phosphate synthase (EPSPs)) (by introducing recombinant nucleic acids and / or various forms of in vivo EPSP gene mutagenesis native), aroA genes and glyphosate acetyl transferase (GAT) genes, respectively), other phosphono compounds such as glufosinate genes (phosphinothricin acetyl transferase (PAT) from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and pyridinoxy acids or propionic phenoxy and cyclohexones (genes encoding ACCase inhibitor). See, for example, U.S. Patent No. 4,940,835 to Shah, et al., And U.S. Patent No. 6,248,876 to Barry et al., Which have nucleotide sequences of forms of EPSPs that can resist glyphosate to a plant. A DNA molecule encoding a mutant aroA gene can be obtained under accession number ATCC 39256, and the nucleotide sequence of the mutant gene can be disclosed in U.S. Patent No. 4,769,061 to Comai. European patent application No. 0 333 033 to Kumada et al., And US patent No. 4,975,374 to Goodman et al., Show nucleotide sequences of glutamine synthetase genes that confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a PAT gene can be presented in European patent application No. 0 242 246 by Leemans and others DeGreef et al., Bio / Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes that encode PAT activity. Examples of genes that confer resistance to phenoxy propionic acids and cyclohexones, such as setoxidim and haloxifop, include the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theor. Appl. Genet. 83: 435 (1992). GAT genes capable of conferring resistance to glyphosate are described in WO 2005012515 by Castle and other Genes that confer resistance to the herbicides 2,4-D, phenoxypropionic acid and pyridyloxy auxin are described in WO 2005107437 assigned to Dow AgroSciences LLC.
[00081] C) A herbicide that inhibits photosynthesis, such as a triazine (psbA and gs + genes) or a benzonitrile (nitrilase gene). Przibila et al., Plant Cell 3: 169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are presented in U.S. Patent No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession No. 53435, 67441 and 67442. The cloning and expression of the DNA that encoding a glutathione S-transferase can be described by Hayes et al., Biochem. J. 285: 173 (1992). 3. Genes that confer or contribute to an added value trait, such as:
[00082] A) Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase the plant's stearic acid content. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89: 2624 (1992).
[00083] B) Decreased phytate content: 1) The introduction of a phytase encoding gene would increase phytate degradation, adding more free phosphate to the transformed plant. For example, see Van Hartingsveldt et al., Gene 127: 87 (1993), for an exposure of the nucleotide sequence of a phytase gene from Aspergillus niger. 2) A gene can be introduced that reduces the phytate content. In corn, for example, this could be accomplished by cloning and then reintroducing DNA associated with the single allele that may be responsible for corn mutants characterized by low levels of phytic acid. See Raboy et al., Maydica 35: 383 (1990).
[00084] C) Modified carbohydrate composition effected, for example, by transforming plants with a gene that encodes an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteol. 170: 810 (1988) (nucleotide sequence of the Streptococcus mutants fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20: 220 (1985) (Bacillus subtilis nucleotide sequence may be the levansucrase gene), Pen et al., Bio / Technology 10: 292 (1992) (the production of transgenic plants that express Bacillus lichenifonn may be α-amylase), Elliot et al., Plant Molec. Biol. 21: 515 (1993) (nucleotide sequences of tomato invertase genes), Sogaard et al., J. Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis may be from the barley α-amylase gene), and Fisher et al., Plant Physiol. 102: 1045 (1993) (branching enzyme II of corn endosperm starch). EXAMPLES
[00085] The present invention is further described in the following examples, which are offered by way of illustration and are not intended to limit the invention in any way. EXAMPLE 1 Peptide Synthesis
[00086] The following Cell Penetration Peptide (CPP) sequences; R9 (Futaki et al., 2001, Suzuki, et al., 2002), MPG (Morris, 1997 and Morris, 1999), and Y-ZEIN (Kogan et al., 2001 and 2002) are listed in Table 1. These peptides were synthesized by the American Peptide Company (Sunnyvale, CA) as C-terminal amides. The integrity of the samples was tested using a Mass Spectrophotometer with protocols recognized in the art. Table 1: Amino Acid Sequences and Molecular Masses of Synthetic Peptides
Preparation of Quantum Dot-Peptide Conjugates
[00087] Quantum dot (QD) and CPP conjugates were produced. Quantum dots functionalized with amine, purchased from EvidentTech (Troy, NY), were activated by adding approximately 1 mg of sulfo-SMCC to 200 μL of quantum points with amine (QD514; n = 2.3 nmoles) in 200 μL of phosphate 50 mM sodium, pH 7.4. CPPs were independently mixed with the maleimide-activated quantum dot in conjugation buffer (1 mM EDTA, 0.1 M phosphate, 0.15 M NaCl, pH 7.2) and incubated at 4 ° C overnight . After conjugation, the QD-CPP conjugates were centrifuged at 90,000 rpm for 3 hours, and the pellet was dissolved in a solution of PBS (Phosphate-Buffered Saline). QD-CPP conjugates were prepared with a molar ratio of QD to CPP from 1: 100 to 1: 300.
[00088] Cell Penetration Mediated DNA Distribution in QD Charge
[00089] A plasmid DNA complex, pDAB3831 (Figure 1), was formed with the QD-CPP conjugate. Before the formation of the DNA complex with the QD-CPP conjugate, the plasmid DNA was denatured and allowed to reseal. This was completed by diluting the DNA to a final volume of 10 μL in DNase-free water. The solution was denatured by heating the solution to 70 ° C for 5 minutes and then allowing the solution to cool slowly to room temperature. A DNA complex was then formed as the QD-CPP conjugate at final concentrations of 1: 100 of CPP-DNA to QD. The formation of the QD-CPP-DNA complex was carried out for 1 hour at 37 ° C. Finally, sterile 3% sucrose was added until a final volume of 10 ml was reached. The QD-CPP-DNA complex solution was used to transform Arabidopsis thaliana flower buds. Transformation of Arabidopsis thaliana with QD-CPP-DNA Complex Plant material for in-plant processing
[00090] Synchronized seed germination is important to ensure uniformity of floral development in T0 plants. The seeds of Arabidopsis thaliana cv. Columbia were suspended in 0.1% agar solution and incubated at 4 ° C for 48 hours to complete stratification. 60 mg of seeds were weighed and transferred to a 15 mL tube. 13 mL of 0.1% agar solution was added and swirled until the seeds were evenly dispersed. This creates a concentration of 4.6 mg of seeds / 1 ml of solution (or about 230 seeds / ml). 6 tubes (72 mL of solution) were prepared to seed 4 trays that contained 18 pots (8.89 cm (3-1 / 2 inches)) in each tray. The seeds were incubated at 4 ° C for 48 hours to complete stratification. Each pot was sown individually to 1.0 mL of stratified seeds per pot. When all the pots were sown, propagation domes were placed over the trays to keep the soil moist. The domes were removed 5 days after the sowing date. The seeds were germinated, and the plants were grown in a Conviron (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 hours of light / 8 hours of darkness) at light intensity 120 - 150 μmoles / m2s under constant temperature (22 ° C) and humidity (40 - 50%). The plants were watered 10 to 14 days after sowing the plants with Hoagland's solution and, subsequently, with DI water to keep the soil moist, but not wet. 4 weeks after the date of sowing, the flowers were cut to produce a more uniform growth of the secondary flowers. In the 5th week after sowing, the plants were prepared for the transformation process. In-plant transformation and screening of Ti resistant plants:
[00091] The transformation of Arabidopsis thaliana cv. Columbia was completed using a modified Clough and Bent protocol (S.J. Clough and A.F. Bent, 1998, Plant J. 16: 735-43). 10 mL of suspension was prepared with the QD-CPP-DNA solution, which was used to treat Arabidopsis plants (mainly clusters of immature flowers with some fertilized silica). Before dipping the plants, Silwet L-77 was added at a concentration of 0.05% (250 μL / 500 mL) - 0.005% to the QD-CPP-DNA solution, and mixed well. The above-ground parts of the plant were immersed in the QD-CPP-DNA solution for 2 to 30 seconds, with gentle agitation. The treated plants were kept under a plastic dome cover for 16 to 24 hours at 22 - 24 ° C. The plants were transferred to the Convirons and allowed to grow to maturity and to collect seeds. Selection trays (26.7 cm x 53.3 cm x 2.5 cm (10.5 "x 21" x 1 ") trays) were used for sorting raw harvest seeds from T0 plants, approximately 10,000 seeds Two controls were used to ensure that the selection spray was done correctly, Col-0 negative transformation control and Columbia homozygous seed for PAT selectable marker (phosphinothricin acetyl transferase) as a positive transformation control. , the seeds were stratified in a 0.1% agar solution for 48 hours before sowing.To provide 10,000 seeds per selection tray, 200 mg of seeds were added to a 0.1% agar solution and swirled until that the seeds were evenly distributed. The stratified seeds were then sown in selection trays filled with Sunshine LP5 mix and sub-irrigated with Hoagland's solution. z, it is important that the 40 mL of suspended seeds are sown evenly in the selection tray. After sowing, propagation domes were placed in each selection tray, and the plants were grown for selection. The propagation domes were removed approximately 5 days after sowing.
[00092] In addition, a control experiment was completed. In this experiment, a solution containing only DNA, not in complex with the QD-CPP conjugate, was used to transform Arabidopsis thaliana. The above protocol was used for DNA transformation only as a control. Selection of processed plants
[00093] Freshly harvested T1 seeds were allowed to dry for 7 days at room temperature. T1 seeds were grown in 26.5 x 51 cm germination trays, each receiving a 200 mg aliquot of stratified T1 seeds (~ 10,000 seeds) that had previously been suspended in 40 ml of 0% agarose solution , 1% and stored at 4 ° C for 2 days to complete the requirements for complete dormancy and ensure synchronous seed germination.
[00094] The Sunshine LP5 mixture was covered with fine vermiculite and soaked with Hoagland's solution until wet, then allowed to drain by gravity. Each 40 mL aliquot of stratified seeds was sown evenly over the vermiculite with a pipette and covered with moisture domes for 4 - 5 days. The domes were removed 1 day before the initial transformant selection using post-emergence spraying of glufosinate.
[00095] Seven days after planting (DAP), T1 plants (cotyledon and 2 - 4 leaf stage, respectively) were sprayed five times over five days with 0.2% Liberty herbicide solution (200 g ae / L of glufosinate, Bayer Crop Sciences, Kansas City, MO) at a spray volume of 10 mL / tray (703 L / ha) using a DeVilbiss compressed air spray nozzle to deliver an effective rate of 280 g ae / ha of glufosinate per application. The survivors (plants growing actively) were identified 4 - 7 days after the final spraying and transplanted individually in 7.62 cm (3 inch) pots prepared with potting medium (Metro Mix 360). The transplanted plants were covered with moisture domes for 3 - 4 days and placed in a growth chamber at 22 ° C as before or moved directly to the greenhouse. The domes were subsequently removed, and the plants grown in the greenhouse (22 ± 5 ° C, 50 ± 30% RH, 14 hours of light: 10 hours of darkness, minimum 500 μE / m2s1 of natural + supplemental light). Molecular Analysis
[00096] The genomic DNA of transgenic Arabidopsis plants was extracted from the total leaf material of 6-week-old plants using the Plant DNAZOL kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. PCR primers were designed to detect the yfp and pat. The yfp primers are presented as SEQ ID NO: 4 and SEQ ID NO: 5. The primers pat are presented as SEQ ID NO: 6 and SEQID NO: 7.
[00097] SEQ ID NO: 4: 5’-TGTTCCACGGCAAGATCCCCTACG-3 ‘
[00098] SEQ ID NO: 5: 5 '-TATTCATCTGGGTGTGATCGGCCA-3 ‘
[00099] SEQ ID NO: 6: 5’-GGAGAGGAGACCAGTTGAGATTAG-3 ‘
[000100] SEQ ID NO: 7: 5 '-AGATCTGGGTAACTGGCCTAACTG-3 ‘
[000101] PCR amplification reactions for pat and yfp were completed using the TaKaRa ExTaq kit (Takara, Otsu, Shiga, Japan). The gene products were amplified in a total reaction volume of 50 μL. The PCR reaction contained 100 ng of genomic DNA template, 1X ExTaq reaction buffer, 0.2 mM dNTP, 10 pMoles of each primer and 0.025 units / μL of ExTaq. The following PCR conditions were used: 1 cycle at 96 ° C for 5 min and 31 cycles of the following conditions: 94 ° C for 15 s, 65 ° C for 30 s, 72 ° C for 1 min and a final extension of 72 ° C for 7 min. The PCR amplification product was analyzed by agarose gel electrophoresis with 0.8% TAE and visualized by ethidium bromide staining. Table 2 shows the results of the amplification products that were obtained with these reactions. Table 2: PCR results for QD-based conjugates and vehicle used in this experiment, in which only representative samples are shown.
EXAMPLE 2: FORMATION OF LIVE IMAGE IN PLANT THROUGH QUANTUM POINT CONJUGATES AND CELLULAR PENETRATION PEPTIDE Live image formation using QD finished with DHLA and QD-CPP conjugate
[000102] Fusion proteins consisting of cell-penetrating peptides (CPP) and yellow fluorescent protein (YFP) were produced and isolated as previously described in Provisional U.S. Patent No. 61/319764 and in Chen et al., 2007. The various Cell penetrating peptides were subcloned upstream of the YFP coding sequence into the unique NcoI - SpeI restriction sites within a bacterial expression vector pET280. Protein expression was induced, and they were isolated and purified as described in Provisional U.S. Patent No. 61/319764 and in Chen et al., 2007. The sequences of CPP-YFP fusions are listed in Table 3. Table 3: Nucleotide sequences of cell-penetrating peptide fusions and yellow fluorescent protein


[000103] QDs finished off with DHLA with a maximum emission centered at 620 nm were synthesized using step reactions of organometallic precursors in hot coordination solvent mixtures according to the previously described procedures. See Aron et al., 2006; Lu et al., 2007; Doyon et al., 2006; Collins et al, 2003; and Lanio et al., 2000. The nanocrystals were made hydrophilic by replacing the native wrapper, composed mainly of trioctyl phosphine (TOP) and trioctyl phosphine oxide (TOPO), by bifunctional ligands as previously described. See Lie et al., 2002; Mani et al., 2006; Desjarlais and Berg, 1993. Two sets of hydrophilic QDs were used: (1) nanocrystals capped with dihydrolipoic acid, and (2) nanocrystals capped with a mixture of dihydrolipoic acid with a polyethylene glycol appendix (Mw ~ 600) ( DHLA-PEG) and DHLA polyethylene glycol (Mw ~ 400) terminated with biotin (DHLA-PEG-biotin) with a 9: 1 molar ratio of the binders. The resulting QDs were called DHLA-QDs and DHLA-PEG-biotin-QDs, respectively.
[000104] In addition to the CPP molecules described above (Y-ZEIN, MPG and R9), two additional molecules, PEP1 and TAT (Table 4), were assembled with the QDs topped with DHLA using the protocol previously described (Aron et al., 2006). QD-CPP conjugates of appropriate molar ratios as described above were added to 0.3 μM of QDs capped with DHLA emitting at 510 - 620 nm in 10 mM Tris-Cl buffer, pH 8.0, and incubated at room temperature for 30 minutes. The conjugates were characterized using gel electrophoresis, in which a change in the electrophoretic mobility of the QDs mounted with CPPs was observed. The samples were diluted in 1X TBE buffer (0.09 M Tris, 0.002 M Na2-EDTA, 0.09 M boric acid, pH 8.3) and run on 1% or 2% agarose gels. The effect of varying the number of CPP molecules per QD was monitored by observing the fluorescence of the complex. The images on the gel were produced by excitation of the QD and / or protein and observation of the images for separate fluorescent bands within the gels. In addition, conjugate formation was confirmed by monitoring changes in energy transfer between QDs and CPPs during self-assembly. Table 4: Amino acid sequences and molecular masses of the synthesized peptides

[000105] Capture and subcellular ocalization of the quantum dot-CPP conjugate within plant cells
[000106] Bioconjugates of QD were diluted with complete culture medium and added to agglomerated cell cultures of Arabidopsis, unique cell cultures of JTNT1 tobacco and carrots (Provisional US Patent No. 61/319764) with intact walls. The solution was incubated at 37 ° C for 1 - 4 hours at 40 - 150 μg / ml. Mixed QD conjugates consisting of QD / CPP ratios of 1: 5 or 1:10 to 50 CPP molecules per QD were incubated with the cell cultures. Excess unbound QD conjugates were removed by washing the culture at least three times with 1X PBS or cell culture medium. The cells were then incubated for 30 minutes at room temperature and washed twice with PBS.
[000107] Epifluorescence image collection was performed using a Leica confocal microscope. The fluorescence images divided side by side were collected and quantified using a dual visualization system equipped with a 565 nm dichroic filter. For 620 nm QDs, the QD-CPP complex was imaged. For cellular images, the samples were excited at 488 nm, and the emissions were collected / separated with the 565 nm dichroic filter and unconvoluted. The QD fluorescence was collected at À <620 nm, and the YFP fluorescent tail was collected at À> 537 nm if the CPP fusion tag was used alone, without the QDs. The leakage of YFP in the QD window is subtracted as part of the deconvolution. The 620 nm isolated QDs are excited at 488 nm, and their respective emissions are separated with the 565 nm dichroic filter and decoloured. The fluorescence of DAPI and Calcuofluor is excited using a xenon lamp (Xe), and the emission is collected using a DAPI cube (D350 / 50X for excitation, dichroic 400DCLP, D460 / 50m for detection). AF647-TF is excited using the Xe lamp, and fluorescence is detected using a Cy5 cube (HQ620 / 60X excitation, dichroic Q660LP, HQ700 / 75m emission). Both excitation / detection cubes are provided by Chroma Technology (Bellows Falls, VT). Differential interference contrast (DIC) images are collected using a bright light source.
[000108] Thus, functionalized QDs containing different cell-penetrating peptides to trace the location in cells with a single wall of Arabidopsis, carrot and JTNT1 tobacco were observed with a Zeiss LSM710 confocal microscope image. QDs have a higher resistance to metabolic degradation and a higher resistance to photo-targeting. QD complex amines with CPPs such as R9, MPG, Y-ZEIN, PEP1 and TAT were incubated with single cells of Arabidopsis, carrot and JTNT1 for 30 minutes, and the cells were washed with medium, taking the image with a confocal microscope Zeiss LSM710. A Zeiss LSM710 confocal scanner equipped with an Axio Observer Z1 inverted microscope was used with an excitation wavelength for a 3-hour capture experiment, and the 561 nm excitation wavelength was used for a 5-hour capture experiment. .
[000109] The results indicated that cells in suspension of Arabidopsis, JTNT1 tobacco and live carrots with intact walls did not show the fluorescence of À = 620 nm. However, when the QD620 were introduced into the cells, the internalization of the QD in the plant cells was observed. Channel images made in the blue and red emission for Calcofluor, the cell wall staining and the QD620 fluorescent band, respectively, showed blue fluorescence, indicating the presence of cell wall, or red fluorescence, indicating the presence of the nucleus where the QD620 is located due to targeting to the nucleus. An overlay of all the images illustrated that the internalized QDs were concentrated in the cytoplasm and nucleus.
[000110] The targeting of QD620 conjugated with CPP to the nucleus of cells in suspension of Arabidopsis, JTNT1 and carrot has been demonstrated. The targeting to the nucleus was confirmed by counterstaining the nucleus with the nuclear dye DAPI. DAPI is a vital nuclear dye, used very commonly in living plant cells. This dye is a fluorescent dye that is excited by ultraviolet light, showing blue fluorescence when bound to DNA in the nucleus. Although the Calcofluor emission range is in blue, it is only specific to the wall. The superposition of the images of the QD620 CPP conjugates and images stained with DAPI showed the colocalization of the QD-MPG conjugates in the nucleus.
[000111] QD620 conjugated with MPG, R9, Y-ZEIN, PEP1 and TAT internalized CPPs have characterized surface charge values, and their zeta potential values were measured in the range of 9.5466 - 10.1586 mv. The hydrodynamic values of the sizes of the conjugates were in the range of 122 - 342 nm. When these particulate conjugates were incubated with cells in suspension of Arabidopsis, JTNT1 tobacco and carrots, the conjugates were internalized in the intact cells and located in the nucleus, indicating the nuclear targeting of QD conjugates in living cells, as opposed to treatment with only QD620 amine conjugate, where the particles are seen mainly in the cytoplasm and occasionally in the nucleus. The translocation to the Arabidopsis cell nucleus, JTNT1 tobacco and carrot was observed with Quantum Dots in complex with the Cell Penetration Peptides (MPG, TAT, PEP1, R9 and Y-zein).
[000112] This example exemplifies the use of QDs labeled with Cell Penetration Peptides as vehicles of fluorescent particles for screening studies in living cells. QDs function as stable beacons. EXAMPLE 3: FORMATION OF LIVE IMAGES ON THE PLANT THROUGH POLYSTYRENE CONJUGATES AND CELLULAR PENETRATION PEPTIDE
[000113] Cell distribution and localization of polystyrene nanoparticles fused with CPPs has been completed. The complex polystyrene / CPP nanoparticles were translocated to live plant cells and directed to specific cell compartments.
[000114] The internalization of polystyrene nanoparticles was marked with the Cell Penetration Peptide, TAT, in single tobacco cells JTNT1.
[000115] Conjugation of Tat Peptide to Carboxylated FluoSpheres
[000116] The evaluation of the uptake of fluorescent polystyrene nanoparticles FluoSphere (20 nm in diameter) in single cells of JTNT1 tobacco with a wall was tested with the one without a partial TAT Cell Penetration Peptide.
[000117] FluoSpheres (Invitrogen, Carlsbad, CA) were obtained from storage at 4 ° C and prepared by adding 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDCL) and N-hydroxysuccinimide (NHS) . The resulting cocktail was incubated for 1 hour to allow the EDCL to react with the FluoSpheres. A TAT cell-penetrating peptide complex (with a tert-butanol-protected carboxy terminus) was formed with FluoSpheres. The TAT - FluoSphere complex was incubated overnight at room temperature to form a complex. The complex was purified using an Amicon Ultra-4 Centrifugal Filter Unit with a molecular weight cut-off limit of 50,000. The retained TAT - FluoSphere complex was transferred to a clean flask. The TAT - FluoSphere complex was stored at 4 ° C until needed for the experiments.
[000118] Cell uptake studies with TAT - Fluospheres and unconjugated Fluospheres in cells in JTNT1 tobacco suspension
[000119] JTNT1 single cell suspensions were prepared in NT1B medium, adjusted to contain 3% glycerol. 20 μL of freshly sonicated and swirled Tat- FluoSpheres and non-complex FluoSpheres were added to JTNT1 cells and incubated for 30, 60 or 120 minutes. Single JTNT1 cells were isolated by centrifuging the tubes for 5 minutes at 700 rpm. The supernatant was removed, and the cells rinsed twice using 2 ml portions of NT1B. The washed JTNT1 cells were resuspended in 1mL of the NT1B medium containing glycerol, and some of the cells were pipetted into separate glass slides and visualized using a vertical fluorescence microscope, in bright field mode and containing a filter cube set for record the fluorescence emission at 580 nm. The microscope was used to capture images of JTNT1 cells that had been treated with the TAT-FluoSphere complex. The ImageJ software was used to display and superimpose (stack) the bright field and fluorescence images to facilitate the determination of the location of the FluoSpheres within the JTNT1 tobacco cells.
[000120] The 120-minute treatment with the TAT-FluoSphere complex of the JTNT1 tobacco cells resulted in the internalization of the TAT-FluoSphere complex and targeting the cell nucleus. The 30-minute treatment did not result in significant cell uptake of the TAT-FluoSphere complex. It was noted that part of the TAT-FluoSphere complex was associated with the single cell walls with JTNT1 glycerol. The 60-minute treatment of the TAT-FluoSpheres complex resulted in the internalization of part of the complex within the unique JTNT1 cells. A large number of the TAT-FluoSphere complexes observed were observed close to the periphery of the cells; however, a few complexes appeared in close proximity to the nucleus of the JTNT1 tobacco cells. After 120 minutes of treatment, a significant number of the TAT-FluoSpheres complexes were captured within the JTNT1 tobacco cells and directed to the nucleus of these cells.
[000121] The internalization of polystyrene nanoparticles was marked with cell penetrating peptides (CPPS) TAT, MPG and Y-ZEIN in cells in suspension of Arabidopsis.
[000122] Conjugation of cell-penetrating peptides to carboxylated FluoSpheres and cell uptake in Arabidopsis suspension cells
[000123] Different types of CPPs have been marked to assess the uptake of nanoparticles through cell walls and cell membranes to the cytoplasm and nuclei of cells in suspension of live Arabidopsis using confocal microscopy. FluoSpheres complexes were formed with cell penetrating peptides (TAT, MPG, and À-zein) as described above. The resulting CPP-FluoSphere complex was mixed with 0.1 ml of Arabidopsis aggregate cell suspension and incubated at room temperature in the dark for 3 hours (first experiment) or for 5 hours (second experiment). The cell suspensions were centrifuged, and the supernatant was removed. The cells were resuspended in fresh culture medium. A drop of the resuspended cell suspensions was pipetted onto a glass cover slip, and vacuum grease was used to form a perimeter around the cell suspension droplet before placing a second glass cover slip over the cell suspension to form a cell sandwich. between the two coverslips. A confocal microscope (LSM710 Zeiss confocal scanner equipped with an inverted Axio Observer Z1 microscope with an excitation wavelength of 514 nm during the 3-hour capture experiment, and 561 nm for the 5-hour capture experiment) was used to form an image to the aggregated Arabidopsis cells 3 hours and 5 hours after exposure to the CPP-FluoSphere complex.
[000124] Arabidopsis aggregate cells did not exhibit any apparent autofluorescence when excited at 561 nm and 514 nm by the laser under the confocal microscope. However, Arabidopsis protoplasts exhibited some autofluorescence at this wavelength. The background autofluorescence of the cells did not interfere with the image formation of the CPP-FluoSphere complex uptake experiments.
[000125] Images of Arabidopsis protoplasts were formed 5 hours after exposure with CPP-FluoSpheres conjugates, which were then counter-dyed with a Calcofluor dye. The CPP - FluoSpheres conjugates were internalized by the protoplasts, even though the protoplasts were regenerating cell wall materials. The cells were determined to be alive, as evidenced by active protoplast filaments when viewed under confocal microscopy. In addition, a single cell was observed to capture the TAT and FluoSphere cell penetrating peptide complex in the cell nucleus. It was observed that the aggregated Arabidopsis cells 5 hours after exposure with the indicated cell penetrating peptide Y-zein and FluoSphere conjugates contained FluoSpheres within the cell nuclei. These images indicated that the cell-penetrating peptide Y-zein mediated the transport of FluoSpheres into cell nuclei.
[000126] Arabidopsis protoplasts observed 6 hours after exposure to unmodified FluoSpheres and counteracted with Calcofluor did not internalize in Arabidopsis protoplasts and were not transported to cell nuclei. Observations noted the association of collapsed and killed protoplasts with unmodified FluoSpheres. Thus, it was demonstrated that the unmodified FluoSpheres were not translocated into the cell of live Arabidopsis protoplasts. However, the collapsed cells or dead cells internalized the unmodified FluoSpheres. This observation indicates that when the cell membrane integrity is compromised, unmodified FluoSpheres are internalized, but unmodified FluoSpheres are not internalized in living cells that have an intact cell wall and cell membrane.
[000127] These examples show that fusion labels with CPP provide efficient distribution and targeting in the cell. In addition, depending on the type of CPP used, the cores can be specifically targeted. The use of these CPP-Fluosphere conjugates can facilitate the uptake of impermeable macromolecules in cells. Although the use of this approach allows cell labeling using less QDs or polystyrene NPs, it is still dependent on the cells' endocytic capacity, the labeling was abrogated in cells incubated at 4 ° C or incubated with an inhibitor, such as Wortmannin.
[000128] Dendrimer conjugates labeled with PAMAM-TRITC used as new probes for in vivo image formation of subcellular compartments in Arabidopsis plant cells.
[000129] A nanoparticle-based beacon that can be used to quantify endocytosis in living, walled and intact cells can allow the formation of images of the organelles involved in plant endocytosis in vivo. In addition, this image formation is carried out without fading or bleaching the dye. Finally, tracking the particle or charge distribution in living plant cells can be performed with this nanoparticle-based beacon.
[000130] This example depicts the formation of an endocytic compartment in living plant cells with marked particles of PAMAM-TRITC Dendrimer. The use of these particles for live imaging, instead of styrene dyes, such as FM4-64, which are known for use in studies of vacuolar traffic and endocytosis in plants. They are known to move from one compartment to another over time, thus providing an effective means of forming images of endocytic compartments within living plant cells. However, it is possible to use a fluorescently labeled PAMAM dendrimer particle to show endocytotic behavior, and biomolecules can be labeled or attached as charges in dendrimers to study the traffic in living plant cells (unlike styral dyes, which only dye the vesicles) . Treatment with wortmannin inhibited endocytosis in this study, demonstrating that the PAMAM dendrimer particle lines the endosome vesicles and could be used as a beacon in vesicular screening studies. The use of marked PAMAM-TRITC Dentrimer particles for the screening of endocytosis has been demonstrated, and has resulted in the inhibition of endocytosis with the use of wortmannin. Thus, an example of a new role for the particle as a beacon in the formation of images in living cells was presented for studies of vesicular tracking in plant cells. Marking with TRITC and PAMAM dentrimer
[000131] TRITC-labeled PAMAM dendrimers were labeled according to Pasupathy et al., 2008. TRITC-labeled PAMAM dendrimer was added to a 0.5 ml aliquot of cells suspended in Arabidopsis thaliana from 7-day-old cultures . In addition, the Calcofluor dye was added to the mixture 5 minutes after image formation. The plant cultures were incubated for 30 minutes with the TRITC-Dendrimer complexes and then immediately examined under a confocal microscope. For several control samples 25 μL of 10 μM wortmannin (MP Biomedicals, Solon, OH) were added to the culture 30 minutes before the addition of the TRITC-Dendrimer complexes. The samples were then incubated again for 30 minutes. Images of living cells and clusters were formed using the LSM710 confocal microscope, according to the protocol described above. Control cells that were not incubated with dendrimers did not produce a background when viewed using confocal microscopy. Confocal Laser Scanning Microscopy (CLSM)
[000132] To observe the cellular association of TRITC-PAMAM complexes in the treated cells and to visualize the untreated control with the respective duration of the incubation time, the cells were rinsed with culture medium or PBS buffer (pH 7.4) two times, Calcofluor was added to stain the cell wall for 5 minutes and then examined immediately under the confocal microscope. The cells were observed by CLSM (Carl Zeiss LSM-710, Germany) with a 600 - 620 nm argon laser to form images of the cells live. Here, the intracellular distribution of TRITC-PAMAM was observed in a single plane and also as z sections.
[000133] The results of the cells not treated with Wortmanin showed that the cell membranes and endosomes were stained with the TRITC-labeled PAMAM Dentrimer. TRITC-labeled PAMAM dentimers were transported into the endosomes. It was possible to track the PAMAM Dendrimer inside the translocated cell endosomes through the intact cell wall using confocal laser scanning microscopy. The PAMAM Dendrimer was translocated into the cell through the process of endocytosis. In the presence of Wortmannin, the TRAMC-labeled PAMAM dendrimer was located on the cell membrane. As the endocytosis process was inhibitory, the PAMAM - CPP Dentrimer complex did not translocate into the cytosol of Arabidopsis cells.
[000134] Although this invention has been described in certain embodiments, the present invention can be further modified within the spirit and scope of this exhibition. This application, therefore, is intended to cover any variations, uses or adaptations of the invention using its general principles. In addition, this application is intended to cover those departures from the present exhibition which are known or common practice in the art to which this invention refers and which are within the limits of the appended claims and their equivalents.

权利要求:
Claims (18)
[0001]
1. Method of introducing a nucleic acid of interest in a plant cell with a cellular appearance to effect a stable transformation of a plant and seeds, characterized by the fact that it comprises: the supply of the plant cell with a cell wall; the interaction of a quantum dot (QD) with one or more cell-penetrating peptides (CPPs) to form a QD-peptide conjugate; the attachment of one or more nucleic acids of interest to one or more CPPs to form an activated QD-peptide conjugate; placing the cell with the cell wall and the QD-activated peptide conjugate in contact with each other; and allowing the uptake of the QD-peptide conjugate and one or more nucleic acids of interest within the cell with the cell wall; and the selection of cells that have stably integrated the one or more nucleic acids of interest.
[0002]
2. Method, according to claim 1, characterized by the fact that the interaction of a QD with one or more CPPs comprises the assembly of the CPP on the surface of the QD.
[0003]
3. Method according to claim 1, characterized by the fact that the attachment of one or more nucleic acids of interest to the CPP comprises the interaction of negatively charged groups of the one or more nucleic acids of interest with amino groups charged at the C-terminus - CPP terminal.
[0004]
4. Method according to claim 1, characterized by the fact that it additionally comprises allowing uptake of the QD-peptide conjugate in a plant cell compartment comprising cell wall.
[0005]
5. Method, according to claim 4, characterized by the fact that the compartment is selected from the group consisting of cytosol, nucleus, tonoplasts, plastid, ethioplasts, chromoplasts, leukoplasts, elaioplasts, proteinoplasts, amyloplasts, chloroplasts and the lumen of double membrane.
[0006]
6. Method according to claim 1, characterized by the fact that the plant cell comprising cell wall is selected from the group consisting of tobacco cells, carrots, corn, canola, rapeseed, cotton, palm, peanuts, soybeans, Oryza sp., Arabidopsis sp., Ricinus sp. and sugar cane.
[0007]
7. Method, according to claim 1, characterized by the fact that the plant cell is a tissue selected from the group consisting of embryo, meristem, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds , pods and stems.
[0008]
8. Method, according to claim 1, characterized by the fact that the CPP is selected from the group consisting of peptides R9, MPG, TAT, Y-Zeína and MPG.
[0009]
9. Method, according to claim 1, characterized by the fact that the one or more nucleic acids of interest are selected from the group consisting of DNA, RNA, RNAi molecules, genes, plasmids, cosmids, YACs, BACs and their combinations.
[0010]
10. Method according to claim 9, characterized in that the nucleic acid of interest comprises a gene.
[0011]
11. Method according to claim 10, characterized by the fact that the gene is a foreign protein gene, an agronomic gene or a marker gene.
[0012]
12. Method according to claim 9, characterized by the fact that the selected cells are regenerable cells.
[0013]
13. Method according to claim 12, characterized by the fact that it additionally comprises the regeneration of a plant from regenerable cells.
[0014]
14. Method of stable expression of a gene, characterized by the fact that it comprises: the supply of a plant cell with a cell wall; the interaction of a quantum dot (QD) with a cell-penetrating peptide (CPP) to form a QD-peptide conjugate; the attachment of one or more genes to the CPP to form an activated QD-peptide conjugate; placing the cell with the cell wall and the QD-activated peptide conjugate in contact with each other; and allowing the uptake of the QD-peptide conjugate and the one or more genes within the cell with the cell wall; and selecting cells that stably express the gene to produce a progeny plant; gene expression in the offspring of a plant with the plant cell.
[0015]
15. Method according to claim 14, characterized by the fact that the gene is expressed in a chloroplast.
[0016]
16. Method for transferring a gene to a plant cell, characterized by the fact that it comprises: the interaction of a quantum dot (QD) with a cell penetrating peptide (CPP) to form a QD-peptide conjugate; the interaction of the QD-peptide conjugate with a plasmid DNA comprising one or more genes to form an activated QD-peptide conjugate structure; and the contact of the activated QD-peptide conjugate structure with a plant cell bearing an intact cell wall under conditions that allow the uptake of CPP and the one or more plasmid DNA genes by the plant cell.
[0017]
17. Method according to claim 16, characterized by the fact that it additionally comprises selecting cells that stablely express the gene to produce the progeny of a plant.
[0018]
18. Method of sorting and identifying transformed plants, characterized by the fact that it comprises: the supply of a plant cell with a cell wall; the interaction of a quantum dot (QD) with a cell-penetrating peptide (CPP) to form a QD-peptide conjugate; attaching one or more nucleic acids of interest to the CPP to form an activated QD-peptide conjugate; placing the cell with the cell wall and the activated QD-peptide conjugate in contact with each other; allowing the uptake of the QD-peptide conjugate and one or more nucleic acids of interest within the cell with cell wall; and the formation of an image of the plant cell with cell wall.

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同族专利:

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EP2697379A2|2014-02-19|

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US20120244569A1|2012-09-27|

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WO2012129443A3|2012-10-26|

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WO2012129443A2|2012-09-27|

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BR112013024220A2|2016-12-20|

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JP2014511678A|2014-05-19|

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CN103562397A|2014-02-05|

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AU2012230804A1|2013-10-17|

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KR20140020965A|2014-02-19|

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JP6067671B2|2017-01-25|

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RU2013147198A|2015-04-27|

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CA2830531C|2019-10-15|

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AR085555A1|2013-10-09|

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NZ616041A|2015-04-24|

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-06-25| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-09-29| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-15| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 22/03/2012, OBSERVADAS AS CONDICOES LEGAIS. |

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