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WO2001021781A2 - Procede de modification associee a un site de l'activite genique et de la structure d'un acide nucleique - Google Patents

Procede de modification associee a un site de l'activite genique et de la structure d'un acide nucleique Download PDF

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Publication number
WO2001021781A2
WO2001021781A2 PCT/US2000/025778 US0025778W WO0121781A2 WO 2001021781 A2 WO2001021781 A2 WO 2001021781A2 US 0025778 W US0025778 W US 0025778W WO 0121781 A2 WO0121781 A2 WO 0121781A2
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nucleic acid
plant
site
molecule
effector molecule
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PCT/US2000/025778
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WO2001021781A3 (fr
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Richard A. Jefferson
Andrzej Kilian
Carol Nottenburg
Paul Konrad Keese
Jorge Mayer
Scott E. Stachel
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Center For The Application Of Molecular Biology To International Agriculture
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Priority to EP00966763A priority Critical patent/EP1220937A2/fr
Priority to AU77055/00A priority patent/AU7705500A/en
Publication of WO2001021781A2 publication Critical patent/WO2001021781A2/fr
Publication of WO2001021781A3 publication Critical patent/WO2001021781A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • This invention relates generally to methods for making transgenic plants with modified gene activity or nucleic acid structure.
  • a strategy has been devised to identify and gain functional insight into such genes. This strategy seeks to identify regions of the genome that are expressed under specific developmental conditions, with the goal of finding genes that mediate specific processes, such as root development or flower formation. It entails an intensive effort to find genes that are differentially expressed. As expression differences that lead to dramatic phenotypic changes may involve subtle pattern differences or as there may be multiple genes necessary for mediating processes, this strategy may fail to identify the key genes.
  • a second strategy seeks to generate gain-of-function mutations, in which a developmental pathway is expressed in the wrong place and/or time, to result in a novel phenotype that provides insight into the function of the mis-expressed gene.
  • a desired gene is placed under control of a promoter that is active only in a particular tissue or developmental time.
  • This method requires first that a promoter and desired gene be identified and cloned.
  • a reporter target gene is separated from its transcriptional activator in two distinct transgenic lines (Brand and Perrimon, Development 118: 401, 1993; Guyer et al, Genetics 149: 633, 1998).
  • the activator When the gene encoding the transcriptional activator is introduced under control of a minimal promoter, the activator is only expressed when the element integrates near genomic regulatory sequences.
  • the target gene In the second transgenic line, the target gene is linked to a sequence recognized by the transcriptional activator.
  • lines By screening for expression of the reporter in different tissues and times, lines can be identified in which the transcriptional activator is nearby to a desired regulatory sequence. Thus, lines are produced that can be used to express any gene of interest with the same expression pattern as that exhibited by the transgenic organism containing the transcriptional activator gene.
  • the present invention discloses methods for generating transgenic plants that exhibit novel phenotypes, transgenic plants, and further provides other related advantages.
  • methods comprising, cross fertilizing two transgenomic plant lines to produce seed, wherein the first transgenomic plant line contains an introduced first nucleic acid molecule that expresses a non-native site-specific nucleic acid effector molecule under control of a minimal promoter, and the second transgenomic plant line contains a second introduced nucleic acid molecule containing a binding site for the heterologous site-specific nucleic acid effector molecule; and growing the seed to produce a plant; wherein the introduced first nucleic acid molecule is operably linked to an endogenous enhancer sequence; and wherein the binding of the effector molecule to the binding site modifies gene activity in a binding site-associated manner.
  • the non-native nucleic acid effector molecule is a fusion protein of a plant-derived effector molecule with a heterologous DNA binding domain. In another embodiment, the non-native effector molecule is a plant-derived molecule with an altered DNA binding domain that recognizes a different recognition sequence.
  • the method is used to affect nucleic acid structure.
  • transgenic plants and seeds produced by the cross of the two transgenic plants are provided.
  • methods for modifying gene activity in a plant comprising the steps of: generating a transformed plant line with an introduced nucleic acid molecule that expresses a non-native site-specific nucleic acid effector molecule under control of a minimal promoter and a transposable element containing a minimal promoter operably linked to at least one binding site for the nucleic acid effector molecule; cross-fertilizing the transformed plant line with a second plant line that expresses a transposase to produce seed; and growing the seed into a plant.
  • plants that display a desired phenotype are selected.
  • the transposable element is Ds and the transposase is Ac and the introduced nucleic acid molecule further comprises a reporter gene operably linked to at least one binding site for the nucleic acid effector molecule.
  • the site-specific nucleic acid effector molecule is a transactivator.
  • Figure 1 is a schematic of an exemplary transactivating vector.
  • Figure 2 is a schematic of an exemplary transactivating vector, pSMR- J18R, which contains a GFP reporter gene.
  • FIG. 3 is a schematic of an exemplary transactivating vector, DsMutagenvector, which contains a GUS reporter gene.
  • site-specific nucleic acid effector molecule refers to a molecule that binds a specific recognition sequence in a nucleic acid molecule and has a function that affects nucleic acid.
  • the effector molecule may be protein, peptide, DNA, RNA, a chimera of these molecules, or the like.
  • functions include, but are not limited to, activating transcription, unwinding DNA, methylating DNA. Generally, the nucleic acid binding and function will reside in separate domains.
  • a “binding site” or “recognition sequence” for a site-specific nucleic acid effector molecule” refers to the specific nucleic acid sequence that the nucleic acid effector molecule binds to.
  • “enhancer” refers to any one of a class of cis-acting DNA sequences that function in a non-directional manner and that increase transcriptional activity of an operably linked promoter.
  • a “promoter” means a nucleotide sequence comprising one or more sequences that proteins and other molecules that are involved in the transcription process bind to and initiate transcription from.
  • a “minimal promoter” refers to a promoter sequence that cannot initiate detectable transcription in the absence of additional transcriptional elements.
  • transcriptional elements refers to nucleotide sequences involved, directly or indirectly, in regulating transcription of cis-linked genes. Such elements include, but are not limited to, enhancers, promoters, transcriptional termination sequences, and the like.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence.
  • a "plant line” means a group of individual plants derived from a common ancestry.
  • phenotype or “trait” refers to a set of observable physical characteristics of an individual organism, which is the observed manifestation of a genotype.
  • a phenotype may be expressed physically, biochemically, or physiologically.
  • the parental transgenic plant lines comprise a line containing an introduced nucleic acid molecule that expresses a site- specific nucleic acid effector molecule and a second line containing a second introduced nucleic acid molecule having a sequence that the effector molecule specifically binds.
  • the parental lines are crossed to produce progeny that have modified endogenous gene activity or modified nucleic acid structure.
  • the parental line contains an introduced nucleic acid molecule that has a site-specific nucleic acid effector molecule (e.g., a transcriptional activator) operably linked to a minimal promoter and at least one binding site operably linked to a minimal promoter.
  • a site-specific nucleic acid effector molecule e.g., a transcriptional activator
  • gene activity may be enhanced, suppressed, or the like and the nucleic acid structure may be modified by unwinding, overwinding, nicked, cut, or the like.
  • one parental transgenic plant line contains a nucleic acid molecule that expresses a site-specific nucleic acid effector molecule.
  • the nucleic acid effector molecule may be proteinaceous, such as a peptide or protein, a nucleic acid, such as single or double-stranded DNA or RNA or DNA:RNA hybrids, protein-nucleic acid chimeras, such as PNA or conjugates, and the like. Whatever its form, the nucleic acid effector molecule must bind to a nucleotide recognition sequence in a site-specific manner.
  • site-specificity means that that the nucleic acid effector molecule binds in a manner dependent upon the sequence of the recognition site.
  • a restriction enzyme such as EcoRI binds to the sequence 5'-GAATTC-3' when present in a double stranded form.
  • the nucleic acid effector molecule may be any molecule that affects either the structure or function of nucleic acids.
  • the nucleic acid effector molecule should affect nucleic acids that are cis-linked to the recognition sequence. It is preferred that the site of action occurs only or preferentially occurs in a localized area to the recognition sequence.
  • the nucleic acid effector molecule comprises at least an active fragment of a transcription factor, a methylase, a repressor, a gyrase, a kinkase, a histone deactylase, a histone acetylase, a topoisomerase, an enhancer, any one of a suite of transcriptional factors, a restriction enzyme, or the like.
  • Methylases, gyrases, histone deactylases and acetylases, and the like are well-known in the art. Sequences that encode these proteins may be found in GenBank for example.
  • the nucleic acid effector molecule has a null function, such that binding of the molecule to the recognition sequence blocks other molecules from binding, but otherwise has no direct effect on the nucleic acid.
  • One means to constructing a null function effector molecule is to use only the nucleic acid binding domain.
  • a gene encoding it may be synthesized, cloned from host genomic or cDNA libraries, or amplified from cDNA or genomic DNA.
  • transcription factors useful in the context of this invention include gus repressor (U.S. Patent No: 5,879,906); Gal4 (U.S. Patent No: 5,968,793); HIV rev; cro, lac repressor, glucocorticoid receptor, trp repressor, TFIIIA, Sp-1, GCN4, AP-2, and the like (Pabo and Sauer, Ann. Rev. Biochem. 61: 1052-1095, 1992).
  • the nucleic acid effector molecule is a fusion protein comprising a peptide or polypeptide that confers binding to a recognition sequence and a protein that affects the function or structure of nucleic acids.
  • nucleic acid binding transcription factors may be generated as a fusion protein of a nucleic acid-binding domain and a transcriptional effector domain.
  • transcriptional effector domain means a polypeptide that effects transcription, such as activating and inhibiting.
  • the domain may be a portion or fragment of a larger molecule (e.g., the activation domain of Gal4) or a full-length molecule (e.g., VP16 or Gal4).
  • fusion proteins are preferably accomplished by amplification of the desired domain regions and ligation of the amplified products.
  • primers flanking a DNA binding domain selected from a DNA-binding protein and a effector domain, including activators and repressors, are useful within the context of this invention.
  • Compatible restriction sites are preferably incorporated into the primers, such that the products when joined are in the same reading frame.
  • Amplified products of the two domains are restricted and ligated together and inserted into an appropriate vector. Verification of the resulting clone is readily done by restriction mapping and DNA sequence analysis. DNA sequence analysis is preferable so that an in- frame reading frame can be verified.
  • DNA sequence analysis is preferable so that an in- frame reading frame can be verified.
  • the nucleic acid effector molecule is a conjugate of a protein that affects the function or structure of nucleic acids and a oligonucleotide or polynucleotide that binds to the recognition sequence.
  • an oligonucleotide is a short polynucleotide. Typically, an oligonucleotide is from a few bases to a hundred or so, and more typically from 10 to 100 bases.
  • the oligo- or polynucleotide preferably comprises a sequence complementary to the recognition sequence.
  • the nucleic acid effector molecule is selected to bind a defined recognition sequence.
  • desirable recognition sequences include sequences found in natural transposable elements, such as Ds from maize. Techniques for selecting such characteristics are well-known in the art and include phage display (see for example, U.S. Patent No: 5,223,409, gene shuffling, site-directed mutagenesis, and the like. In another embodiment, variants are generated by "exon shuffling” (see U.S. Patent No. 5,605,793). Variant sequences may also be generated by "molecular evolution” techniques (see U. S. Patent No. 5,723,323). In other embodiments, the nucleic acid effector molecule may be a nucleic acid, such as single-stranded or double-stranded DNA or RNA.
  • the nucleic acid effector molecule may be complementary to the recognition sequence and effect binding as well as affecting the function or structure of cis-linked nucleic acids. In other cases, the nucleic acid effector molecule may be a conjugate of a nucleic acid and an effector protein.
  • a nucleic acid effector molecule will affect either the function or structure of nucleic acids that are cis-linked to the recognition sequence. Function may be affected either directly or indirectly.
  • direct action includes the enhancement of gene expression through activation of a promoter and the diminishing of gene expression through action of a repressor.
  • Indirect action includes, but is not limited to, alterations of nucleic acid structure, such as by deactylation of histones, association of the nucleic acid effector molecule with a factor necessary for transcription thus inhibiting the action of the factor, and the like. Structure can be affected in a large variety of means, such as by unwinding or increasing the winding of nucleic acids, increasing or decreasing the number or type of chromatin-associated proteins, causing nucleic acid kinking, and the like.
  • the recognition sequence for the nucleic acid effector molecule is a specific sequence such that the nucleic acid effector molecule binds in a sequence-specific manner.
  • the recognition sequence will be at least three nucleotides and preferably at least four nucleotides, at least five, six, seven or eight nucleotides. While there is no theoretical upper limit to the length of the recognition sequence, pragmatically the sequence will not be more than 50 nucleotides and in certain embodiments, not more than 40, 30, 25 or 20 nucleotides.
  • the recognition sequence for many DNA-binding protein is well-known.
  • a widely-used recognition sequence for Gal4 is a consensus sequence of the four Gal4 sites found in the UAS controlling expression of the yeast GALl and GAL 10 genes and the one Gal4 site found in the GAL7 promoter (Giniger et al., Cell 40:767-774, 1985). These different sequences demonstrate some of the variation that can be made in the Gal4 binding site while maintaining the ability of Gal4 to bind.
  • Other suitable binding sequences include sequences found in natural transposons.
  • one of the parental lines carries one or more, and preferably many copies of the transposon and is crossed to a line that expresses a site-specific nucleic acid effector molecule under control of a minimal promoter.
  • parental plant lines containing nucleic acid effector molecules and recognition sequences are generated.
  • multiple plant lines of each parental type are desirable. As each transgenic line will likely carry the introduced nucleic acid molecule in a unique location, a larger number of lines will increase the number of new phenotypes observed.
  • the first method is based on T-DNA integration into a plant genome during Agrobacterium transformation. Examples of suitable vectors for this method are described below.
  • 10,000 independent primary transgenic lines of rice are generated, resulting in insertions in approximately 10-20% of rice genes (with an average number of T-DNA copies of 1.5-2 per transgenic).
  • 251 lines analyzed about 48% of lines have a single T-DNA insertion.
  • each T-DNA insertion can be mapped by standard techniques, such as fluorescent-labeled hybridization to chromosomes, similarity searching against DNA sequence data, restriction mapping, and each corresponding line scored for enhancer trap activity and homozygous phenotype.
  • standard techniques such as fluorescent-labeled hybridization to chromosomes, similarity searching against DNA sequence data, restriction mapping, and each corresponding line scored for enhancer trap activity and homozygous phenotype.
  • a subset of these lines that carry single copy insertions evenly distributed throughout the genome and exhibit interesting patterns of enhancer trap expression may be chosen for saturation mutagenesis.
  • Saturation mutagenesis or coverage of a complete genome with insertional mutagens may be achieved in one of a variety of methods. Briefly, in one such method, a Ds transposable element, which is part of the Agrobacterium vector is activated to transpose itself from the T-DNA insertion site. Other transposition systems in which transposition can be controlled, such as by expression of a transposase are also suitable within the context of the present invention. Transposition is achieved by crossing transgenic plants carrying a T-DNA insert with plants expressing Ac transposase. Further control of transposition is attained by using different promoters to regulate Ac transposase. Promoters that are active in the germline can be used.
  • promoters driving transcription during inflorescence meristem development are likely to provide a high frequency of germinal excisions of the Ds element while exhibiting low levels of somatic excisions.
  • Somatic excisions may contribute unwanted "background” variation, potentially masking/modifying mutant phenotypes resulting from germinal excisions.
  • a reporter gene such as ⁇ -glucuronidase or green fluorescent protein, in the construct under control of the same promoter, the activity of the promoter can be readily monitored.
  • promoter fusions with Ac transposase are introduced into Nipponbare rice plants that are already transformed with a construct comprising the Ds element and the marker gene ⁇ -glucuronidase (GUS). These plants do not express GUS unless the Ds element is excised. These tester plants allow quantification of germinal and somatic excision events by simple GUS staining of respective tissues.
  • a selected set of transcriptional activator facilitated enhancer trap/Ds transposon (TA ET/Ds) lines are crossed with an Ac transposase-expressing line. The number of TA ET/Ds lines and the number of crosses per line is determined by the frequency of germinal excisions.
  • the integrated T-DNA of the TA-ET/Ds comprises a 'Launching Pad' and contains a non-autonomous Ds transposable element.
  • This element ( Figure 1) contains not only a dominant herbicide resistance marker that can be used under field conditions, but an 'outward-facing' UAS-minimal promoter at one side.
  • UAS is but one example of a recognition sequence.
  • This Ds element is positioned between a promoter active in seedlings, and an antibiotic resistance (e.g., hygromycin resistance; kanamycin resistance) gene.
  • antibiotic resistance e.g., hygromycin resistance; kanamycin resistance
  • Other selectable markers for plants are well known and may alternatively be used.
  • the Ds-element when the Ds-element is induced to transpose by provision of the appropriate transposase enzyme through a genetic cross to a line engineered to express the transposase, the promoter and the resistance gene are brought together, and result in a seedling resistant to the antibiotic. This is referred to as an 'excision marker'.
  • the biology of the Ac/Ds system ensures that most of the excision events are correlated with an integration event somewhere in the genome, typically at a genetically linked site.
  • the progeny of TA ET/Ds lines and Ac-expressing lines are selected for Ds excision and reinsertion using Selectable Marker I and Selectable Marker II ( Figure 1). Selection against the presence of Ac transposase gene is performed at the same time (germination to seedling stage) using a negative selection marker present on the same T-DNA as Ac-construct. Although not required, elimination of Ac -positive plants will stabilize Ds in the new location.
  • transposon e.g., Ac/Ds
  • a gemini virus is used as a vehicle for generating high copy number Ds element substrates for Ac transposase.
  • the Gemini virus vector contains the Ds element with recognition sequences operably linked to a minimal promoter.
  • the virus may also contain a transposase to initiate transposition.
  • the plant cell infected with the Gemini virus may be super-infected with a vector expressing transposase or the plant cell may express transposase.
  • a second strategy involves a transposon-independent approach to recognition-sequence mutagenesis based on co- transformation of DNA molecules that provide selection function in callus and a molar excess of DNA molecules that contain recognition sequence-minimal promoter mutagen.
  • Each insertion has a "unique identifier" - DNA sequence tag, which allows not only easy identification of specific mutagens elements in segregating populations, but also quantification of the transcripts originating from a specific recognition sequence-minimal promoter.
  • the nucleic acid effector molecule is under control of a minimal promoter and requires the presence of a cis-acting enhancer sequence for expression.
  • the introduced nucleic acid comprising the nucleic acid effector molecule construct integrates near an endogenous enhancer the nucleic acid effector molecule will be expressed.
  • the nucleic acid effector molecule will be expressed in different cell types and at various developmental stages depending on when and where the enhancer is activated.
  • a reporter gene under control of a minimal promoter in the construct, transcription can be readily monitored. The reporter gene is not expressed by the minimal promoter per se.
  • An exemplary vector ( Figure 1) functions as a transactivator enhancer trap, as it carries the Gal4 VP16 gene under the control of a minimal promoter adjacent to the right T-DNA border.
  • the construct also carries marker genes, either GUSPlusTM (see, WO 99/13085) or both the GUSPlusTM and GFP (green fluorescent protein) reporter genes, under the control of UAS elements to provide sensitive readouts of patterns of expression of the transactivator protein.
  • GUSPlusTM see, WO 99/13085
  • GFP green fluorescent protein reporter genes
  • the reporter can be any protein that allows convenient and sensitive measurement or facilitates isolation of the gene product and does not interfere with the function of the telomerase.
  • ⁇ -glucuronidase U.S. Patent No: 5,268,463 and 5,599,670
  • green fluorescent protein GFP
  • GUSPlusTM WO 99/13085
  • ⁇ -galactosidase are readily available as DNA sequences.
  • a peptide tag may be additionally be used.
  • a tag is a short sequence, usually derived from a native protein, which is recognized by an antibody or other molecule.
  • Peptide tags include FLAG®, Glu-Glu tag (Chiron Corp., Emeryville, CA) KT3 tag (Chiron Corp.), T7 gene 10 tag (Invitrogen, La Jolla, CA), T7 major capsid protein tag (Novagen, Madison, WI), His 6 (hexa-His), and HSV tag (Novagen).
  • tags other types of proteins or peptides, such as glutathione-S-transferase may be used.
  • selectable markers include, hygromycin resistance (U.S.
  • the present invention also provides an opportunity to use the information and genetic resources developed through many years of plant genetic research. For example, a large number of "classical" mutants are mapped genetically and genes responsible for mutant phenotypes may be targeted for Ds mutagenesis by launching TA ET Ds element mapping in the vicinity. This is done through crossing respective TA ET/Ds lines with Ac transposase expressing lines. Alternatively, DNA isolated from the primary and secondary mutant populations may be used in a reverse genetics strategy to identify lines that carry insertional mutations within specified genes.
  • the information and genetic resources available are not limited to a single species. For example, one can also exploit the enormous information resources generated for other cereals, due to grass genome synteny. Mutant phenotypes identified and mapped in other cereal species, are likely to be in syntenic location in the rice genome, and their "tagging" may be done in rice. Searching for corresponding genes in rice will not only offer a shortcut, but, once a target gene is isolated through this synteny-with-rice approach, sequence similarity between homologues will provide many clues about regions important for gene regulation and function as a bonus. There is little doubt that this evolutionary approach will be beneficial both for rice and other cereal and grass research and breeding.
  • the transgenic parental lines are constructed by introduction of a nucleic acid molecule, typically a DNA-based vector, containing one or more of the genes noted above.
  • the vectors should be functional in plant cells. Vectors and procedures for cloning and expression in E. coli and animal cells are discussed herein and, for example, in Sambrook et al (supra) and in Ausubel et al. (supra).
  • Vectors that are functional in plants are preferably binary plasmids derived from Agrobacterium plasmids. Such vectors are capable of transforming plant cells. These vectors contain left and right border sequences that are required for integration into the host (plant) chromosome. At minimum, between these border sequences is the gene to be expressed under control of a promoter. In preferred embodiments, a selectable marker and a reporter gene are also included. The vector also preferably contains a bacterial origin of replication. Available vectors include co- integrative vectors and variants (U.S. Patent No: 5,731,179, 5,635,381 ; 4,693,976;), binary vectors (U.S. Patent Nos: 4,940,838; 5,464,763) and variants (U.S. Patent No: 5, 149,645).
  • the promoter should be functional in a plant cell.
  • the promoter may be derived from a host plant gene, but promoters from other plant species and other organisms, such as insects, fungi, viruses, mammals, and the like, may also be suitable, and at times preferred.
  • the promoter region is designed narrowly to comprise a transcription initiation site, but lack sequences and elements necessary for activity.
  • One such promoter is the minimal promoter derived from CaMV 35S promoter, which comprises a TATA box, needed for assembly of active RNA transcription complex, and supports only basal, non-detectable reporter activity.
  • the minimal promoter region typically used encompasses nucleotides -46 to +8 of CaMV 35S promoter (see U.S. Patent 5,097,025).
  • the vector contains a selectable marker for identifying transformants.
  • a selectable marker confers a growth advantage under appropriate conditions.
  • selectable markers are drug resistance genes, such as neomycin phosphotransferase.
  • Other drug resistance genes are known to those in the art and may be readily substituted.
  • hygromycin resistance U.S. Patent Nos: 4,727,028; 4,960,704; and 5,668,298
  • G418 resistance ampicillin resistance
  • kanamycin resistance genes are commonly employed.
  • positive selection systems such as taught in U.S. Patent Nos. 5994629; 5767378; and 5,599,670 may be used.
  • the selectable marker also preferably has a linked constitutive or inducible promoter and a termination sequence, including a polyadenylation signal sequence.
  • a bacterial origin of replication and a selectable marker for bacteria are preferably included in the vector.
  • a colEI origin of replication is preferred.
  • Most preferred is the origin from the pUC plasmids, which allow high copy number.
  • Selectable markers for bacteria include, ampicillin resistance, tetracycline resistance, kanamycin resistance, chloramphenicol resistance, and the like.
  • General vectors suitable for use in the present invention are based on the vectors described above and in U.S. Patent No. 4,940,838 and 5,464,763 and pBI121 (U.S. Patent No. 5,432,081) a derivative of pBIN19.
  • the plasmid pSMR-J18R contains a left and right border sequence for integration into a plant host chromosome and also contains bacterial origins of replication and ampicillin selectable marker for bacterial growth.
  • the border sequences flank several genes.
  • One is a hygromycin resistance gene driven by a CaMV 35S promoter and using a nopaline synthase polyadenylation site.
  • a second gene is the Streptomyces GUS gene (GUSPlusTM or BoGUS) operably linked to six copies of Gal4 UAS.
  • a gene encoding a fusion protein of a nucleic acid binding domain (Gal4) and a transcriptional activator (VP16) is located near the right border.
  • Other elements may also be included.
  • Plants may be transformed by any of several methods.
  • plasmid DNA may be introduced by Agrobacterium co-cultivation (U.S. Patents 4,940,838 and 5,591,616, or bombardment.
  • Other transformation methods include electroporation (U.S. Patent No. 5,859,327), CaPO 4 -mediated transfection, gene transfer to protoplasts, microinjection, and the like (see, Gene Transfer to Plants, Ed. Potrykus and Spangenberg, Springer, 1995, for procedures).
  • vector DNA is first transfected into Agrobacterium and subsequently introduced into plant cells. Most preferably, the infection is achieved by co-cultivation.
  • the choice of transformation methods depends upon the plant to be transformed.
  • monocots can be transformed by Agrobacterium using the methods claimed in U.S. Patent 5,591,616 and U.S. 6,037,522.
  • U.S. Patent No 6,074,877 for transformation of cerials See also, U.S. Patent No. 6,177, 010 and 5, 981,840 for transformation of maize; U.S. Patent No. 5, 187,073 and 6,020,539 for transformation of Gramineae
  • Agrobacterium transformation by co-cultivation is also appropriate for dicots and for mitotically active tissue.
  • Non-mi totic dicot tissues can be efficiently infected by Agrobacterium when a projectile or bombardment method is utilized (U.S. Patent No: 5,932,782). Projectile methods are also generally used for transforming plants. (See U.S.
  • co-cultivation is performed by first transforming Agrobacterium by freeze-thawing (Holsters et al., Mol Gen. Genet. 163: 181-187, 1978) or by other suitable methods (see, Ausubel, et al. supra; Sambrook et al., supra).
  • a culture of Agrobacterium containing the plasmid is incubated with leaf disks, protoplasts or meristematic tissue to generate transformed plants (Bevan, Nucl. Acids. Res. 72:8711, 1984) or for monocots, incubated with callus derived from explants.
  • Transgenic plant lines generated according to the teachings of the present invention will allow gene identification and generation of new phenotypes via a combination of approaches:
  • the Ds element harbored on the primary T-DNA insertion in each of these lines is launched to generate a large second set of insertional mutations.
  • These mutations are then mapped and screened in a variety of ways, including for gain- of-function phenotypes in a heterozygous state and for loss-of-function phenotypes in a homozygous state.
  • Insertional mutagenesis has proved effective for cloning genes in a variety of organisms and causing a wide range of novel phenotypes.
  • T-DNA or transposable element insertion within the coding region of the gene will usually completely abolish gene function leading to a null mutation.
  • insertion occurs in the promoter region, 5' or 3' untranslated region, or intron region it may lead to substantial reduction of the amount of gene product being expressed and a hypomorph mutation as a result.
  • LoF mutations While there is almost no limit to the type of phenotypic alterations one can search for in the mutagenized population, several classes of LoF mutations are of especial interest.
  • One of the most interesting and abundant types of mutations to be identified in the T-DNA and Ds-tagged population include those leading to male and/or female sterility.
  • Male sterility mutations may prove very useful in developing genetic systems for hybrid seed production.
  • Female sterility will be an indication of an insertion of the mutagenic element into a gene essential for female reproductive functions, which may result in discovery of a gene important for development of apomictic character.
  • Another class of desirable phenotypes are related to floral structure or other development alterations that can facilitate cross-pollination, such as precocious lodicule expansion, stigma exersion or prolonged flower-opening times.
  • the present invention will also allow access to the genes leading to lethal phenotypes, due to the ability to maintain recessive lethals using the dominant herbicide locus associated with the insertional lesion. Amplifying sequences proximal to the sterility-causing insertion site, together with molecular characterization (high- resolution mapping and sequencing) will allow rapid access to candidate genes.
  • Gain-of-function mutations can result in organisms having novel phenotypes. Many of the most striking examples of such mutations involve the mis- expression of genes that control developmental pathways. Two striking gain-of- function mutations in plants are Knl in maize (Veit B, et al. Genetics 725:623-31, 1990) and LEC1 in Arabidopsis, (Lotan T, et al. Cell 25:1 195-205. 1998), which respectively display grossly modified leaf mo ⁇ hology, and the ectopic formation of embryo-like structures in adult tissues. Many new and useful traits that have arisen during evolution are also likely the result of the changed expression of specific genes.
  • certain gain-of-function mutations in plants might be predicted to have agriculturally useful phenotypes.
  • the inappropriate expression of a rice gene that controls panicle initiation in the vegetative meristem of the node could lead to plants having increased panicle number.
  • the Ds element carries a Gal4-responsive UAS recognition sequence and a minimal promoter adjacent to its rightward inverted repeat and oriented to read outward into the genomic region following transposition ( Figure 1).
  • the UAS recognition sequence that is operably linked to a minimal promoter is in a separate parental transgenic plant line.
  • the strategy is amplification-based and relies on the availability of a mutagenized population for which any given gene has a high probability of carrying at least one transposon insertion.
  • a DNA sequence is first selected for which the identification of the loss-of- function phenotype is desired, and corresponding PCR primers are generated.
  • DNA from all the transposon-tagged lines is pooled and used as a target for PCR reactions employing a combination of primers to the ends of the insertion mutagen (in our invention either T-DNA or Ds terminal sequences) and the gene of interest.
  • Amplification of a product indicates that the population contains a line with an insertion in the selected sequence.
  • two independent plant transformation vectors are described: one is an enhancer trap vector and the other one is UAS mutagenic vector.
  • a number of enhancer trapping vectors are available using various reporter genes.
  • the main component of the vector is transcriptional activator (transactivator) under the control of minimal promoter (supporting only basal transcription level).
  • transactivator is a GAL4 VP16 fusion protein with the DNA binding domain derived from the yeast GAL4 protein and the activation domain derived from the VP16 protein of Herpes Simplex Virus.
  • the transactivator activates the reporter gene by binding to the UAS element containing the recognition sequence for the DNA binding domain of the transactivator.
  • GUS, GFP and BoGUS BoGUS is the preferred reporter molecule.
  • Enhancer trapping vectors that are available are: pSK66.1 (GUS reporter); pFX (EGFP reporter); pFX (GUS/EGFP fusion reporter); pFX (BoGUS/EGFP fusion reporter); and pSR (BoGUS reporter).
  • the second vector comprises the DNA binding domain recognition sequence (UAS) of the transactivator.
  • UAS sequences are included on the transposable element (e.g. Ds element of maize) or in the vicinity of the Right Border of T-DNA when UAS mutagenesis is performed using Agrobacterium transformation.
  • transposon mutagenesis plants with UAS elements on the transposable element are crossed with line expressing transposase, to allow transposition of UAS element into new location in the genome.
  • an excision marker is often used to identify (usually in the F2 generation of the cross) the plants with transposon excision from its original location. Chloramphenicol acetyl transferase can be used as an excision marker in plants.
  • An alternative method uses a single vector for enhancer trapping and UAS mutagenesis.
  • This vector has a composition similar to the one exhibited in the Figure 1.
  • This vector is used for Agrobacterium transformation and upon insertion of T-DNA into plant genomic DNA minimal promoter of transactivator is up-regulated through the activity of genomic enhancers that are in the vicinity of insertion site. After selecting enhancer trap lines with interesting pattern of transactivator activity, this line is crossed with transposase expressing line and the plants with UAS element transposed into new location identified through the use of excision marker selection and/or screening.
  • Plant material Surface sterilized rice seeds are grown on 2N6 medium containing auxin (2,4-D) in darkness at 26°C for three weeks (21 d) to form calluses. Scutellum-derived calli obtained from these seeds are used for transformation.
  • Agrobacterium strains Agrobacterium vir helper strains LBA4404, EHA105, and AGL-1 that harbor pCAMBIA vectors are used for transformation.
  • LBA4404 carries a vir helper Ti plasmid, pAL4404, derived from the octopine Ti plasmid pTiAch5 (Ooms et al. Plasmid 1, 15-19, 1982).
  • the vir helper Ti plasmids in strains EHA105 (Hood et al, Transgenic Res. 2, 208-218, 1993) and AGL-1 (Lazo et al, Bio/Technology 9, 963-967, 1991) are derived from leucinopine type supervirulent Ti plasmid pTiBo542.
  • Protocol Day 1: After three weeks of callusing, scutellum-derived calli are subdivided into 4 to 8 mm diameter pieces and placed on plates containing 2N6 medium and incubated at 26°C in the dark for four to seven days. Day 3: Agrobacterium strains are streaked on AB medium with appropriate antibiotics and incubated at 28-29°C for two days. At this time, Agrobacterium forms a lawn on the plates.
  • Agrobacterium strains are resuspended in AAM medium containing 100 ⁇ M acetosyringone by scraping the bacteria from plates with an inoculation loop, shaking vigorously for a minute, and incubated for 3 h.
  • the OD of the bacterial suspension is measured at 600 nm, and approximately 1.0 OD of bacteria are used for transformation.
  • 20 mL of the bacterial suspension is transferred into a petri dish or other suitable sterile container.
  • Four to seven-day incubated calli are added to the bacterial suspension, swirled and left for 30 min. The calli are then blotted dry on sterile Whatman No. 1 filter papers and transferred to 2N6-AS plates. These calli and are then co-cultivated for two days in the dark 26°C.
  • the calli are transferred to fresh selection medium once every two weeks.
  • Small, transgenic hygromycin resistant calli start proliferating after four weeks of selection on hygromycin.
  • Such proliferated calli are sub-cultured and independent proliferating lines are made. These sub-cultured calli further proliferate within two weeks and are transferred onto regeneration medium and cultured in the dark for one week.
  • the calli After a week, the calli are transferred to light. 5-10 days later calli start turning green and in 2-3 weeks time shoots start differentiating. These shoots are then transferred onto rooting medium, and once roots are formed, plants are hardened and transferred to the glass house.
  • T-DNA and Ds transposon Three methods are used to isolate DNA fragments adjacent to DNA tag (T-DNA and Ds transposon) insertions into the plant (e.g., rice) genome: TAIL-PCR, ST-PCR and plasmid rescue.
  • TAIL-PCR This strategy, called thermal asymmetric interlaced (TAIL)- PCR, utilizes nested sequence-specific primers together with a shorter arbitrary degenerate primer so that the relative amplification efficiencies of specific and nonspecific products can be thermally controlled.
  • One low-stringency amplification cycle is carried out to create annealing site(s) adapted for the arbitrary primer within the unknown target sequence bordering the known segment.
  • This sequence is then preferentially and geometrically amplified over non-target ones by interspersion of high-stringency amplification cycles with reduced-stringency cycles.
  • the procedure is described in Liu et al. (Plant J. 8: 457-463, 1995).
  • the three degenerate TAIL-PCR oligos that are described in Liu et al (1995) are used in combination with DNA-tag specific primers.
  • Degenerate primers are as follows:
  • T-DNA primers and Ds primers are selected from T-DNA right and left border sequences or Ds terminal repeat sequences. Three rounds of amplification are performed essentially as described.
  • a line with transactivator expression in the panicle during male meiosis stage is identified in the pattern line population. After crossing with transposase expressing line, progeny are selected (usually in the F2 generation of the cross) that has UAS element transposed into rice genome. These lines are identified through the activity of the excision marker (resistance to chloramphenicol treatment, for example). Chloramphenicol resistant lines are grown in the glasshouse and, during male meiosis stage, are subjected to cold stress (14-15 C). Plants with seed set significantly better than control (untransformed) plants are selected and further tested under the field conditions for increased resistance to cold treatment.
  • a line with expression of the transactivator in the root is identified in the pattern line population. After crossing with a transposase expressing line, progeny are selected (usually in the F2 generation of the cross) that have the UAS element transposed into rice genome. These lines are identified through the activity or the excision marker (resistance to chloramphenicol treatment, for example). Chloramphenicol resistant lines are grown in agar medium and development of the root system is compared by visual inspection with control plants. Plants with seed set significantly larger root system than control (untransformed) plants are selected and further tested under the field conditions for increased yielding potential.
  • a line with expression of the transactivator in the node tissue (especially nodes III and IV) is identified in the pattern line population. After crossing with transposase expressing line, a progeny is selected (usually in the F2 generation of the cross) that has UAS element transposed into rice genome. These lines are identified through activity ot the excision marker (resistance to chloramphenicol treatment, for example). Chloramphenicol resistant lines are grown in the glasshouse, and plants with panicles initiating from nodes III and IV are identified. These plants are further analyzed under field conditions for increased yield as compared to control (untransformed) plants.
  • Transactivation in rice is tested using 3 different GAL4/VP16 constructs in an enhancer trap vector: (1) a full length GAL4/VP16 transactivator; (2) a full length GAL4/VP16 transactivator with an intron of castor bean catalase (CAT1) inserted into the 5' untranslated region; and (3) a GAL4 VP16 transactivator with a deletion of 36 amino acids from the DNA binding domain (DBDdel).
  • CAT1 castor bean catalase
  • An enhancer trap vector with a UAS-GUS reporter is used in Agrobacterium transformation of rice calli. After two weeks of co-cultivation, expression of the reporter is analyzed through histochemical staining with X-Gluc (5- Bromo-4-chloro-3-indolyl ⁇ -D glucuronide). Foci with GUS expression are counted and results compared among three constructs. In this test method, reporter gene expression in UAS-reporter gene transgenomic plants is demonstrated as dependent upon expression of the transactivator. When deletion variants of the transactivator DNA binding domain (DBDdel) are used, reporter gene expression is absent, whereas use of a full-length transactivator was very effective in inducing expression of the reporter.
  • DBDdel deletion variants of the transactivator DNA binding domain
  • an enhancer trap vector with UAS-EGFP reporter is used in Agrobacterium transformation of rice calli. After two weeks of co-cultivation, expression of the reporter is analyzed through fluorescent microscopy. Calli with EGFP expression are counted and results compared among three constructs. UAS-EGFP expression in enhancer trap constructs
  • reporter gene expression results from co-transformation of a transactivator construct and a UAS-reporter gene construct.
  • re-transformation of a rice line without expression of a reporter gene from UAS-promoter with 35S CaMV transactivator construct resulted in high level reporter gene expression.
  • T-DNA insertions The number of T-DNA insertions is analyzed by Southern blot hybridization using nucleic acid encoding GAL4/VP16 as a probe. DNA is extracted from young leaves (1-2 weeks after transferred to the glass house). Some 251 Transactivator Facilitated Enhancer Trap (TAFET) lines with GUS and GUSPlusTM reporter genes are analyzed for T-DNA insertion. The number of T-DNA insertions varies from 1 to 7 but about 48.6% of lines (122) have a single insertion.
  • TFET Transactivator Facilitated Enhancer Trap
  • the data on the population with GUS as a reporter gene are summarized to provide a sense of the high efficiency of the system to trap specific patterns of transactivator/reporter gene expression.
  • In shoot/leaf tissues we identified 19 patterns with expression in shoot base, node, coleoptile, leaf blade, collar, ligule or auricle, or a combination of the above.

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Abstract

L'invention porte sur des procédés de génération de plantes transgéniques dont l'activité génique ou la structure d'un acide nucléique est modifiée. Selon certains procédés, les plantes sont issues de graines provenant d'un croisement entre deux plantes transgéniques. Un parent possède un gène codant une molécule effectrice d'acide nucléique sous la maîtrise d'un promoteur minimal et l'autre parent possède une séquence de reconnaissance de la molécule effectrice d'acide nucléique. Lorsque le gène codant la molécule effectrice se trouve à proximité d'un gène endogène, la molécule effectrice est exprimée et active le ou les gènes endogènes situés près de la séquence de reconnaissance.
PCT/US2000/025778 1999-09-20 2000-09-20 Procede de modification associee a un site de l'activite genique et de la structure d'un acide nucleique WO2001021781A2 (fr)

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