WO1999005902A1 - Plantes transgeniques resistantes aux agressions dues a la salinite - Google Patents

Plantes transgeniques resistantes aux agressions dues a la salinite Download PDF

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WO1999005902A1
WO1999005902A1 PCT/US1998/015936 US9815936W WO9905902A1 WO 1999005902 A1 WO1999005902 A1 WO 1999005902A1 US 9815936 W US9815936 W US 9815936W WO 9905902 A1 WO9905902 A1 WO 9905902A1
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nucleotide sequence
cell
plant
seq
promoter
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Jose M. Pardo
Paul M. Hasegawa
Ray A. Bressan
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Purdue Research Foundation
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Purdue Research Foundation
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

  • the present invention relates to methods and materials in the field of molecular biology and to the utilization of isolated nucleotide sequences to genetically engineer cells. More particularly, the invention relates in certain preferred aspects to novel DNA constructs for transforming plants, and in other preferred aspects to methods for making transformed plants tolerant to salinity stress.
  • Salinity is a major environmental stress that is a substantial constraint to crop production both for dry-land and irrigated agriculture. The detrimental impact of this stress is perpetuated and exacerbated by management practices used to facilitate high-output crop production. Plants are typically damaged upon exposure to salt from ion toxicity and osmotic stress.
  • the present invention provides nucleotide sequences, originally isolated and cloned from Saccharomyces cerevisiae which encode functional calcineurin that may advantageously be used to transform a wide variety of plants. Inventive plants are tolerant of salt, which is normally toxic to plants absent an expressible calcineurin enzyme.
  • a recombinant DNA molecule having nucleotide sequences encoding a functional catalytic subunit is described.
  • the recombinant DNA molecule preferably also includes a nucleotide sequence encoding a functional calcineurin regulatory subunit.
  • the catalytic subunit preferably comprises a yeast CNA2 nucleotide sequence or a nucleotide sequence having substantial identity thereto.
  • the regulatory subunit preferably comprises from a yeast CNB1 nucleotide sequence or a nucleotide sequence having substantial identity thereto.
  • Inventive recombinant DNA molecules are advantageous in transforming cells, preferably plant cells, to form transgenic cells that are salt tolerant.
  • An inventive transgenic cell preferably includes a recombinant DNA molecule comprising a first nucleotide sequence encoding a functional calcineurin catalytic subunit.
  • the recombinant DNA molecule preferably also includes a functional calcineurin regulatory subunit.
  • inventive nucleotide sequence or sequences can be transcribed to produce mRNA encoding a catalytic and a regulatory subunit of calcineurin.
  • the respective mRNAs can then be translated to produce calcineurin, wherein calcineurin is functional in the plant.
  • the progeny of a plant transformed in accordance with the invention is also included within the scope of the present invention.
  • a transgenic cell having incorporated therein a foreign polynucleotide comprising a promoter that is operably linked to a nucleotide sequence encoding a catalytic subunit of calcineurin.
  • the cell preferably also has incorporated therein a foreign polynucleotide comprising a promoter that is operably linked to a nucleotide sequence encoding a regulatory subunit of calcineurin.
  • a method for producing a cell having increased tolerance to salinity is provided.
  • the method includes providing a vector including a nucleotide sequence encoding functional calcineurin and introducing the vector into a cell wherein the calcineurin is expressed in the cell. It is an object of the present invention to provide isolated nucleotide sequences, which may be introduced into the genome of a plant to increase the ability of the plant to withstand salt stress.
  • FIG. 1 sets forth the Agrobacterium tumefaciens binary vector used for co- expression of catalytic (CNA) and regulatory (CNB) subunits for reconstitution of constitutively-activated yeast calcineurin (CaN).
  • CNA catalytic
  • CB regulatory subunits for reconstitution of constitutively-activated yeast calcineurin
  • FIG. 2 sets forth an autoradiogram depicting yeast CNAtr and CNB transcripts from transgenic tobacco plants.
  • W38 represents untransformed plants, A2, A9, C8.
  • F3 and T7 represent 5 independent CNAtr and CNB expressing transgenic lines.
  • FIG. 3 sets forth a histogram and a photograph illustrating characteristics of salt-shocked tobacco plants from homozygous T 2 plants that either express npt-II (PKY) or both CNAtr and CNB (A9).
  • Left panel Percent surviving seedlings of 36 progeny from each line (significantly different at the 95% level) and growth parameters of all plants surviving the NaCl treatment ⁇ SE (6 and 28 plants from lines PKY and A9, respectively). Average fresh weights of PKY and A9 plants before the NaCl treatment were 1.63 ⁇ 0.1 lg and 1.76 ⁇ 0.10 g, respectively.
  • Right Panel Three representative plants from lines A9 (top) and PKY (bottom) that survived NaCl shock.
  • Functional PP2B phosphatase calcineurin is a heterodimer comprising a catalytic (CNA) subunit and a regulatory (CNB) subunit.
  • CaN is a component of a Ca 2+ -dependent signal transduction pathway that mediates Na + , Li + and Mn + tolerance of Saccharomyces cerevisiae (Mendoza et al., Activated Calcineurin Confers High Tolerance to Ion Stress and Alters the Budding Pattern and Cell Morphology of Yeast Cells, J. Biol. Chem., 271 :23061, (1996); Cunningham and Fink, Calcinuerin Inhibits VCX1 -Dependent I ⁇ /Ca + Exchange and Induces Ca 2+ ATPases in Saccharomyces cerevisiae, Mol. Cell.
  • the yeast genome contains two catalytic subunit genes designated CNA1 and CNA2, and one regulatory subunit gene, CNBl.
  • the present invention relates to the unexpected discovery that plants can be engineered to have improved salt tolerance by introducing therein a nucleotide sequence encoding all or a portion of a yeast CaN, thereby activating a Ca 2+ and calmodulin-dependent CaN salt stress adaptation cascade in plants.
  • the present invention therefore relates to methods and compositions for obtaining transformed plants, the plants expressing therein a form of functional calcineurin (CaN).
  • Inventive nucleotide sequences can be incorporated into vectors, which in turn can be used to transform plants. Expression of functional calcineurin in plants results in the plants acquiring altered susceptibility to salinity.
  • aspects of the present invention thus relate to nucleotide sequences encoding yeast CaN, which sequences may be introduced into target plant cells to provide a transformed plant more tolerant of salinity stress. Therefore, transformants harboring an expressible inventive nucleotide sequence demonstrate increased levels of CaN activity, and the transformants are more tolerant of salinity stress.
  • amino acid sequence is used herein to designate a plurality of amino acids linked in a serial array. Skilled artisans will recognize that through the process of mutation and/or evolution, polypeptides of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to a sequence set forth herein by virtue of amino acid sequence homology and advantageous functionality as described in detail herein.
  • CaN is used to refer generally to a heterodimeric protein having the features described herein and a preferred example comprises two subunits having the amino acid sequences of SEQ ID NOS: 1 and 2. Also included within the scope of the invention are variants of each which function in the salt stress adaptation cascade in plants, as described herein.
  • polypeptides which include such insertions, substitutions and/or deletions, and yet which effectively provide the same or a similar function.
  • an amino acid sequence isolated from another species may differ to a certain degree from the sequences set forth in SEQ ID NOS:l and 2, and yet have similar functionality with respect to catalytic and regulatory function. Amino acid sequences comprising such variations are included within the scope of the present invention and are considered substantially similar to a reference amino acid sequence.
  • a CaN variant is expected to be functionally similar to that set forth in SEQ ID NOS:l and 2, for example, if it includes amino acids which are conserved among a variety of species or if it includes non- conserved amino acids which exist at a given location in another species that expresses a functional CaN.
  • Nonpolar amino acids Alanine, valine, proline, leucine, phenylalanine, tryptophan, methionine, isoleucine, cysteine, glycine;
  • Group II Uncharged polar amino acids: Serine, threonine, asparagine, glutamine, tyrosine; Group III: Charged polar acidic amino acids: Aspartic, glutamic; and Group IV: Charged polar basic amino acids: Lysine, arginine, histidine.
  • inventive amino acid sequences similar to the amino acid sequences set forth herein that have at least about 50% identity thereto and that impart upon a plant tolerance to salinity stress.
  • inventive amino acid sequences have at least about 75% identity to these sequences, more preferably at least about 85% identity and most preferably at least about 95% identity.
  • Percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482,1981).
  • the GAP program defines identity as the number of aligned symbols (i.e., nucleotides or amino acids) which are the same, divided by the total number of symbols in the shorter of the two sequences.
  • the preferred default parameters for the GAP program include: (1) a uniary comparison matrix (containing a value of 1 for identities and 0 for non-identities), and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • SEQ ID NO:l sets forth an amino acid sequence comprising a truncated catalytic subunit of calcineurin, namely CNAtr.
  • CNAtr is truncated after Thr 459 .
  • SEQ ID NO:2 sets forth an amino acid sequence comprising a yeast regulatory subunit of calcineurin (CNBl). When expressed in a cell, CNAtr and CNBl impart to the cell salt tolerance.
  • nucleotide sequences encoding an inventive CaN enzyme, originally isolated from Saccharomyces cerevisiae, are set forth in SEQ ID NOS:l and 2.
  • a terminal portion of the yeast CNA2 including the nucleotide sequence comprising the calmodulin binding and autoinhibitory domains, were deleted such that the nucleotide sequence set forth in SEQ ID NO:l advantageously encodes a catalytic subunit CNAtr truncated after Thr 459 .
  • This truncated nucleotide sequence in the presence of the CNJ5 subunit encoded by the nucleotide sequence set forth in SEQ ID ⁇ O:2, allows the cell it is expressed in to better tolerate salt stress.
  • functional calcineurin encoded by nucleotide sequences set forth in SEQ ID NOS:l and 2 is expressed in a plant to form functional CaN, the plant is better able to tolerate salt stress than plants without such nucleotide sequences as explained more fully in the Examples below. It is of course not intended that the present invention be limited to these exemplary nucleotide sequences, but include sequences having substantial identity thereto and sequences which encode variant forms of functional calcineurin as described above.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof.
  • encoding and coding refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme.
  • the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence.
  • base changes i.e., insertions, deletions, substitutions
  • nucleic acid sequences encoding variant amino acid sequences are within the scope of the invention.
  • Modifications to a sequence, such as deletions, insertions, or substitutions in the sequence which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention.
  • alterations in a nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site are contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C- terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.
  • the present invention contemplates nucleotide sequences having substantial identity to the sequences set forth herein and variants thereof as described herein.
  • the term "substantial identity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence has a sequence sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions, this method of determining identity being well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al., Molecular Cloning: a Laboratory Manual, 2ed. Vol. 1, pp.
  • an inventive polynucleotide variant is that it must encode a polypeptide having similar functionality to the specific functional mutant calcineurin recited herein, i.e., functioning to increase salinity stress tolerance of a plant transformed therewith.
  • a suitable DNA sequence selected for use according to the invention may be obtained, for example, by cloning techniques using cDNA libraries corresponding to a wide variety of species, these techniques being well known in the relevant art.
  • Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of species by means of nucleic acid hybridization or PCR, using as hybridization probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NOS:l and 2; nucleotide sequences having substantial identity thereto, or portions thereof. Isolated sequences encoding CaN may then be positioned into an expression vector, which is subsequently used to transform a plant in accordance with the invention.
  • nucleic acid sequences encoding enzymes of the invention may be constructed using standard recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and DNA ligase.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate technology.
  • polymerase chain reaction PCR is used to accomplish splicing of nucleic acid sequences by overlap extension as is known in the art.
  • a transgenic cell comprising a foreign, recombinant DNA molecule or vector comprising nucleotide sequences encoding a catalytic subunit (e.g., CNAtr) and a regulatory subunit (e.g., CNBl) is provided as are methods for producing a cell having increased tolerance to salinity stress.
  • the inventive DNA sequences can be incorporated into the genome of a plant using conventional recombinant DNA technology, thereby making a transformed plant having the excellent features described herein.
  • the term "genome” as used herein is intended to refer to DNA which is present in a plant and which is heritable by progeny during propagation thereof.
  • an inventive transformed plant may alternatively be produced by producing FI or higher generation progeny of a directly transformed plant, wherein the progeny comprise the foreign nucleotide sequence.
  • Transformed plants and progeny thereof are all contemplated by the invention and are all intended to fall directly within the meaning of the term "transformed plant.”
  • the present invention contemplates the use of transformed plants which are selfed to produce an inbred plant.
  • the inbred plant produces seed containing the gene of interest. These seeds can be grown to produce plants that express the protein of interest.
  • the inbred lines can also be crossed with other inbred lines to produce hybrids.
  • Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are covered by the invention provided that these parts contain genes encoding and/or expressing the protein of interest. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention.
  • Polynucleotides encoding preferred CaN subunits can be used in conjunction with other plant regulatory elements to create plant cells expressing the polypeptides.
  • "Expressing,” as used herein, refers to the transcription and stable accumulation of mRNA inside a cell, the cell being of prokaryotic or eukaryotic origin. If it is desired for CaN to be targetted to a specific organelle in a given species, chimeric gene constructs encoding the mature CaN proteins covalently bound to transit peptides can be used. Generally, transformation of a cell involves inserting a DNA sequence into an expression vector in proper orientation and correct reading frame.
  • the vector may desirably contain the necessary elements for the transcription of the inserted polypeptide-encoding sequence.
  • vector systems known in the art can be advantageously used in accordance with the invention, such as plasmids, bacteriophage viruses or other modified viruses.
  • Suitable vectors include, but are not limited to the following viral vectors: lambda vector system gtl l, gtlO, Charon 4, and plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKClOl, pCDNAII, and other similar systems.
  • viral vectors lambda vector system gtl l, gtlO, Charon 4, and plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKClOl, p
  • the DNA sequences may be cloned into the vector using standard cloning procedures in the art, for example, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference in its entirety.
  • the plasmid pBI121 is available from Clontech Laboratories, Palo Alto, California. It is understood that known techniques may be advantageously used according to the invention to transform microorganisms such as, for example, Agrobacterium sp., yeast, E.coli and Pseudomonas sp.
  • a promoter be present in the expression vector and operably linked to the coding sequence (preferably two promoters are present where two coding sequences are present, one linked to each coding sequence).
  • the promoter is preferably a constitutive promoter, but may alternatively be a tissue-specific promoter or an inducible promoter.
  • the promoter is one isolated from a native gene which encodes a CaN subunit.
  • An expression vector according to the invention may be either naturally or artificially produced from parts derived from heterologous sources, which parts may be naturally occurring or chemically synthesized, and wherein the parts have been joined by ligation or other means known in the art.
  • the introduced coding sequence is preferably under control of the promoter and thus will be generally downstream from the promoter. Stated alternatively, the promoter sequence will be generally upstream (i.e., at the 5' end) of the coding sequence.
  • enhanced production of CaN may be achieved by inserting inventive nucleotide sequences in a vector downstream from and operably linked to a promoter sequence capable of driving expression in a host cell.
  • Two DNA sequences (such as a promoter region sequence and a CaN-encoding nucleotide sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter region sequence.
  • Constitutive promoter regulatory elements may be used thereby directing continuous gene expression in all cell types at all times (e.g., actin, ubiquitin,
  • Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin, and the like) and these may alternatively be used.
  • Promoter regulatory elements may also be active during a certain stage of the plants' development as well as active in plant tissues and organs. Examples of such include, but are not limited to, pollen-specific, embryo-specific, corn silk-specific, cotton fiber-specific, root-specific, seed endosperm-specific promoter regulatory elements, and the like. Under certain circumstances, it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as, for example, physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemicals and stress. Other desirable transcription and translation elements that function in plants may also be used. Numerous plant-specific gene transfer vectors are known in the art.
  • Plant promoter regulatory elements from a wide variety of sources can be used efficiently in plant cells to express foreign genes.
  • promoter regulatory elements of bacterial origin such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter, and promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, WO 97/13402 published April 17, 1997) and the like may be used.
  • Plant promoter regulatory elements include, but are not limited to, ribulose-l-5-bisphosphate (RUBP) carboxylase small subunit (ssu), beta- conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters, and tissue-specific promoters.
  • RUBP ribulose-l-5-bisphosphate
  • shu carboxylase small subunit
  • beta- conglycinin promoter beta- conglycinin promoter
  • beta-phaseolin promoter beta-phaseolin promoter
  • ADH promoter beta-phaseolin promoter
  • heat-shock promoters heat-shock promoters
  • tissue-specific promoters tissue-specific promoters.
  • RNA polymerase normally binds to the promoter and initiates transcription of a DNA sequence or a group of linked DNA sequences and regulatory elements (operon).
  • a transgene such as a nucleotide sequence selected in accordance with the present invention, is expressed in a transformed cell to produce in the cell a polypeptide encoded thereby.
  • transcription of the DNA sequence is initiated by the binding of RNA polymerase to the DNA sequence's promoter region.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • promoter there may or may not be other regulatory elements (e.g., enhancer sequences) which cooperate with the promoter and a transcriptional start site to achieve transcription of the introduced (i.e., foreign) coding sequence.
  • enhancer nucleotide sequence elements which can stimulate promoter activity in a cell such as those found in plants as exemplified by the leader sequence of maize streak virus (MSV), alcohol dehydrogenase intron 1 , and the like.
  • the recombinant DNA will preferably include a transcriptional termination sequence downstream from the introduced sequence. It may also be desirous to use a reporter gene. In some instances, a reporter gene may be used with or without a selectable marker.
  • Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al. (1988) Ann. Rev. Genetics, 22:421, which is incorporated herein by reference.
  • Preferred reporter genes include the beta- glucuronidase (GUS) of the uidA locus of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis.
  • GUS beta- glucuronidase
  • An assay for detecting reporter gene expression may then be performed at a suitable time after the gene has been introduced into recipient cells.
  • a preferred such assay entails the use of the gene encoding beta- glucuronidase (GUS) of the uidA locus of E. coli, as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) to identify transformed cells.
  • GUS beta- glucuronidase
  • a more preferred reporter gene or selectable marker is the gene neomycin phsophotransferase II which may be preferably assayed as described by Peng et al., (1991 , Plant Mol. Biol. Rep. 11, 38-47).
  • elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences, and the like, may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include, but are not limited to, Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
  • Plant tissue suitable for transformation of a plant in accordance with certain preferred aspects of the invention include, for example, whole plants, leaf tissues, flower buds, root tissues, callus tissue types I, II and III, embryogenic tissue, meristems, protoplasts, hypocotyls and cotyledons. It is understood, however, that this list is not intended to be limiting, but only to provide examples of plant tissues which may be advantageously transformed in accordance with the present invention. A wide variety of plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
  • Transformation of a plant or microorganism may be achieved using one of a wide variety of techniques known in the art.
  • the manner in which the transcriptional unit is introduced into a plant host is not critical to the invention.
  • a wide variety of methods which provide efficient transformation may be employed.
  • One technique of transforming plants with a DNA construct in accordance with the present invention is by contacting the tissue of such plants with an inoculum of bacteria transformed with a vector comprising the DNA construct.
  • This procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for about 48 to about 72 hours on regeneration medium without antibiotics at about 25-28°C.
  • Bacteria from the genus Agrobacterium may be advantageously utilized to transform plant cells.
  • Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • Agrobacterium tumefaciens e.g., strains LBA4404 or EHA105
  • Another technique which may advantageously be used is vacuum-infiltration of flower buds using Agrobacterium-based vectors.
  • Various methods for plant transformation include the use of Ti or Ri- plasmids and the like to perform Agrobacterium mediated transformation.
  • a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. Introduction of the vector may be performed via electroporation, tri-parental mating and other techniques for transforming gram- negative bacteria which are known to those skilled in the art.
  • the manner of vector transformation into the Agrobacterium host is not critical to the invention.
  • the expression construct being within the T-DNA borders will be inserted into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al. (PNAS USA (1980) 77:7347-7351 and EPO 0 120 515), which are incorporated herein by reference.
  • Explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof.
  • the Agrobacteria and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encouraged by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary.
  • the polynucleotide of interest is preferably incorporated into a transfer vector adapted to express the polynucleotide in a plant cell by including in the vector a plant promoter regulatory element, as well as 3' non-translated transcriptional termination regions such as an octopine synthase terminator (Ocs), Nos and the like.
  • a transfer vector adapted to express the polynucleotide in a plant cell by including in the vector a plant promoter regulatory element, as well as 3' non-translated transcriptional termination regions such as an octopine synthase terminator (Ocs), Nos and the like.
  • Plant RNA viral based systems can also be used to express genes for the purposes disclosed herein.
  • the chimeric genes of interest can be inserted into the coat promoter regions of a suitable plant virus under the control of a subgenomic promoter which will infect the host plant of interest.
  • Plant RNA viral based systems are described, for example, in U.S. Patent Nos. 5,500,360; 5,316,931 and 5,589,367, each of which is hereby incorporated herein by reference in its entirety.
  • Another approach to transforming plant cells with a DNA sequence selected in accordance with the present invention involves propelling inert or biologically active particles at plant tissues or cells. This technique is disclosed in U.S. Patent Nos.
  • An isolated DNA construct selected in accordance with the present invention may be utilized in an expression vector to transform a wide variety of cells and organisms, including monocotyledonous and dicotyledonous plants.
  • the invention finds advantageous use, for example, in transforming the following plants: rice, wheat, barley, rye, corn, potato, carrot, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
  • Crops grown on cultivated lands in arid and semi-arid areas in which irrigation with ground water is needed may advantageously benefit from the invention.
  • a particularly preferred plant for practice of the invention is a tomato plant. It is, of course, not intended that the invention be limited to the transformation of plants, but that it additionally includes the transformation of a wide variety of cells and microorganisms using transformation methods and techniques within the purview of a skilled artisan. Additional literature describing plant and/or microorganism transformation includes the following, each of which is incorporated herein by reference in its entirety: Zhijian Li et al.
  • PP2B protein phosphatase activity and in tobacco cells this mediates substantial salt tolerance of plants. While it is not intended that the invention be limited by any theory whereby it achieves its advantageous result, it appears that the essential components of a Ca - and calmodulin-dependent CaN salt stress signal pathway are present in plants and can function in conjunction with the yeast protein to facilitate salt adaptation. By analogy with yeast, this regulatory cascade is believed to exert control over numerous biochemical and physiological processes that function coordinately to regulate intracellular ion transport homeostasis. It is believed that salt tolerance of the CaN expressing plants may be the result of the activated phophatase either pre-adapting plants or potentiating the response of plants to the stress.
  • the gene encoding truncated yeast CNA2 which has been deleted of the calmodulin binding and autoinhibitory domains as described in Mendoza et al., J. Biol.Chem., 271 :23061, 1996, to form CNAtr, and a cDNA of the CNB open reading frame were constructed into separate cassettes in an Agrobacterium binary vector for co-expression of the subunits and reconstitution of activated CaN in situ ( Figure 1).
  • the truncated CNA2 gene was amplified by PCR with primers that annealed to the start codon and introduced a stop codon after Thr 459 as described in Mendoza et al., J.
  • This cDNA was inserted into the Xho I site between the CaMV 35S promoter and the Ocs terminator in the expression cassette pTEX which was contained in pUC19 and the entire cassette was cloned into the Hind III site of pBTEX containing CNAtr ( Figure 1).
  • CNA2 and CNB cDNA PCR products were confirmed by sequence analysis and functional complementation of the Na + /Li + sensitivity of yeast strains MCY100 (cnal cna2) and YP9 (CNB1)( Mendoza et al., J Biol.Chem., 271 :23061, 1996).
  • the binary vector contained the neomycin phophotransferase (nptll) gene as a selectable marker, which effects kanamycin resistance (Kn r ).
  • nptll neomycin phophotransferase
  • Leaf disks were co-cultivated with Agrobacterium tumefaciens strain EHA105 and tobacco plants
  • Ten ⁇ g of R ⁇ A were loaded into each lane, transferred to nitrocellulose filters and hybridized with (A) CNAtr or (B) CNB cD ⁇ A insert. Plants from different transformation events were obtained that expressed both CNAtr and CNB transcripts ( Figure 2).
  • the approximate transcript sizes of CNAtr and CNB are 1.4 kb and 0.5 kb, respectively.
  • Seeds were germinated on filter paper moistened with 1/4X Murashige and Skoog (MS) salt solution (Sigma Chemical Company, #M5524) and after 7 days transferred to tissue culture raft vessels containing the same nutrient solution.
  • Two-week-old Tj tobacco seedlings of the lines were transferred to 1/4X MS mineral nutrient solution containing 250 mM NaCl.
  • seedlings were transferred to nutrient solution without NaCl and left to recover for 10 days. Seedlings remaining viable after this period were transferred to agar solidified medium containing 1/4X MS salt mineral nutrients and 200 ⁇ g/ml kanamycin for evaluation of sensitivity (Kn s ) or resistance (Kn 1 ) to this antibiotic.
  • Kn s sensitivity
  • Kn 1 resistance
  • the T7 seedling population that survived NaCl stress was enriched with Kn r segregants (i.e., depleted of azygous seedlings) as indicated by the increase in the Kn r :Kn s from 3:1 for seedlings grown without salt to 9.4: 1 for seedlings after NaCl treatment.
  • Kn r :Kn s of 2.5:1 for PKY seedling progeny that survived NaCl treatment indicates that salt shock did not enrich this control population for Kn r seedlings.
  • the lack of enrichment for Kn r in the PKY population negates the possibility that salt survival is dependent on nptll expression.
  • Co-segregation of Kn r with NaCl survival in the T7 population indicates that the stress tolerance phenotype is linked to the CaN transgenes and is not the consequence of somaclonal variation in this line.
  • Ti progeny of 4 other independent CaN expressing lines exhibited comparable salt stress survival as those of the T7 line, salt tolerance did not result from insertional mutagenesis that would suppress a negative regulator.
  • Tj progeny of lines PKY, A9 and T7 were grown in hydroponic culture inside of a standard plant growth chamber rather than in tissue culture raft vessels.
  • the thirty-day-old hydroponically grown Tj progeny of lines PKY, A9 and T7 were transferred to fresh mineral nutrient (1/4X MS) solution containing 200 mM NaCl. After 7 days, the plants were transferred to solution without NaCl. Ten days subsequently, the number of surviving plants was determined. Kn r :Kn s of the T7 derived plants, after NaCl shock, was assessed using the NPT-II assay (Peng et al, Plant Mol. Biol. Rep., 11(1):38, 1993).

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Abstract

La présente invention concerne des procédés et des matières du domaine de la biologie moléculaire et la régulation de la synthèse de polypeptides à travers la production de cellules par génie génétique. Plus particulièrement, l'invention concerne l'introduction de séquences nucléotidiques étrangères dans le génome d'une plante pour obtenir une plante transformée, l'introduction des séquences nucléotidiques ayant pour effet d'augmenter la résistance de la plante aux agressions dues à la salinité. Les plantes transformées qui renferment une séquence nucléotidique comprenant un promoteur lié fonctionnellement à une séquence nucléotidique de l'invention, présentent des niveaux accrus d'activité de calcineurine fonctionnelle, ce qui permet d'obtenir une plante résistante à la salinité.
PCT/US1998/015936 1997-07-30 1998-07-30 Plantes transgeniques resistantes aux agressions dues a la salinite Ceased WO1999005902A1 (fr)

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WO2002081500A3 (fr) * 2001-04-06 2003-03-20 Univ California Procedes d'amelioration de la reaction des plantes au stress
WO2003087313A2 (fr) 2002-04-08 2003-10-23 Pioneer Hi-Bred International, Inc. Procedes d'amelioration de l'exsertion de la soie de mais lorsqu'elle est sujette a des agressions
EP1315410A4 (fr) * 2000-08-18 2004-06-23 Univ Connecticut Activite et competence meristematiques accrues par surexpression de pyrophosphatase de tonoplaste
EP1315795A4 (fr) * 2000-08-18 2004-06-23 Univ Connecticut Procedes destines a conferer des traits phenotypiques voulus, notamment la resistance a la secheresse, au gel et a des grandes quantites de sel, et procedes destines a augmenter la production de graines
EP1231831A4 (fr) * 1999-11-10 2004-06-30 Univ Connecticut Plantes transgeniques surdimensionnees resistant aux contraintes et capables de croitre dans un sol salinise
WO2003076597A3 (fr) * 2002-03-05 2004-10-14 Syngenta Participations Ag Sequences d'acides nucleiques isolees a partir d'arabidopsis thaliana et codant pour le gene bos1 et le promoteur de bos1, et utilisations correspondantes
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WO2006111512A1 (fr) 2005-04-19 2006-10-26 Basf Plant Science Gmbh Methodes ameliorees controlant une expression genique
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WO2006133983A1 (fr) 2005-04-19 2006-12-21 Basf Plant Science Gmbh Expression specifique de l'albumen farineux et/ou de l'embryon en germination dans des plantes monocotyledonees
WO2008099013A1 (fr) 2007-02-16 2008-08-21 Basf Plant Science Gmbh Séquences d'acides nucléiques pour la régulation de l'expression spécifique de l'embryon dans des plantes monocotyles
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EP2186903A2 (fr) 2005-02-09 2010-05-19 BASF Plant Science GmbH Cassettes d'expression pour la régulation de l'expression chez les plantes monocotylédones
WO2011001434A1 (fr) 2009-06-30 2011-01-06 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Introduction d'adn dans des cellules végétales
WO2011003901A1 (fr) 2009-07-10 2011-01-13 Basf Plant Science Company Gmbh Cassettes d'expression pour l'expression spécifiquement dans l'endosperme dans des plantes
WO2011067712A1 (fr) 2009-12-03 2011-06-09 Basf Plant Science Company Gmbh Cassette d'expression pour expression spécifique de l'embryon dans des plantes
US8022272B2 (en) 2001-07-13 2011-09-20 Sungene Gmbh & Co. Kgaa Expression cassettes for transgenic expression of nucleic acids
US8058515B2 (en) 1999-11-10 2011-11-15 University Of Connecticut Plant cells and plants overexpressing vacuolar proton pyrophosphatases
EP2436769A1 (fr) 2006-06-07 2012-04-04 Yissum Research Development Company of the Hebrew University of Jerusalem Ltd. Constructions d'expression végétale et leurs procédés d'utilisation
CN103172719A (zh) * 2013-03-06 2013-06-26 北京农业生物技术研究中心 抗逆相关sos3类似钙离子结合蛋白及其编码基因与应用
US8697950B2 (en) 1999-11-10 2014-04-15 University Of Connecticut Vacuolar pyrophosphatases and uses in plants
WO2015162608A1 (fr) 2013-04-25 2015-10-29 Morflora Israel Ltd. Procédés et compositions pour l'administration d'acides nucléiques dans des semences
JP2017506516A (ja) * 2014-02-28 2017-03-09 ダウ アグロサイエンシィズ エルエルシー キメラ遺伝子調節エレメントにより与えられる根特異的発現

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US8003852B2 (en) 1999-11-10 2011-08-23 University Of Connecticut Transgenic plants overexpressing a plant vacuolar pyrophosphatase
EP1231831A4 (fr) * 1999-11-10 2004-06-30 Univ Connecticut Plantes transgeniques surdimensionnees resistant aux contraintes et capables de croitre dans un sol salinise
US8058515B2 (en) 1999-11-10 2011-11-15 University Of Connecticut Plant cells and plants overexpressing vacuolar proton pyrophosphatases
EP1315410A4 (fr) * 2000-08-18 2004-06-23 Univ Connecticut Activite et competence meristematiques accrues par surexpression de pyrophosphatase de tonoplaste
EP1315795A4 (fr) * 2000-08-18 2004-06-23 Univ Connecticut Procedes destines a conferer des traits phenotypiques voulus, notamment la resistance a la secheresse, au gel et a des grandes quantites de sel, et procedes destines a augmenter la production de graines
US8168864B2 (en) 2000-08-18 2012-05-01 Beth Israel Deaconess Medical Center Transgenic pollen expressing exogenous plant vacuolar pyrophosphatase and methods for increasing seed production in plants
US7250555B2 (en) 2001-04-06 2007-07-31 The Regents Of The University Of California Methods for enhancing a plant stress response
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US8022272B2 (en) 2001-07-13 2011-09-20 Sungene Gmbh & Co. Kgaa Expression cassettes for transgenic expression of nucleic acids
US8604278B2 (en) 2001-07-13 2013-12-10 Sungene Gmbh & Co. Kgaa Expression cassettes for transgenic expression of nucleic acids
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