CA2185339C - Enhanced expression in a plant plastid - Google Patents
Enhanced expression in a plant plastid Download PDFInfo
- Publication number
- CA2185339C CA2185339C CA002185339A CA2185339A CA2185339C CA 2185339 C CA2185339 C CA 2185339C CA 002185339 A CA002185339 A CA 002185339A CA 2185339 A CA2185339 A CA 2185339A CA 2185339 C CA2185339 C CA 2185339C
- Authority
- CA
- Canada
- Prior art keywords
- plastid
- plant
- construct
- dna
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Classifications
-
- 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
- C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—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 biotic stress resistance, pathogen resistance, disease resistance for insect resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal protein (delta-endotoxin)
-
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8214—Plastid transformation
-
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
-
- 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
Abstract
Novel compositions and methods useful for genetic engineering of plant cells to provide increased expression in the plastids of a plant or plant cell of a protein which produces a phenotype which is present when the plant or plant cell is grown in the absence of means for selecting transformed cells. Expression of the Bacillus thuringiensis bacterial protoxin in a plant chloroplast is exemplified.
Description
0 WO 95/24493 2185339 pC1yCTS95J02901 ENHANCED ESPRESSION IN A PLANT PLASTID
INTRODUCTION
Field of the ?nvention This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to compositions and methods for enhancing expression of a peptide of interest in the plastid of a plant cell.
Backaround Plastids of higher plants, i.e. chloroplasts, amyloplasts and chromoplasts, have the same genetic content, and thus are believed to be derived from a common precursor, known as a proplastid. The plastid genome is circular and varies in size among plant species from about 120 to about 217 kilobase pairs (kb). The genome typically includes a large inverted repeat, which can contain up to about 76 kilobase pairs, but which is more typically in the range of about 20 to about 30 kilobase pairs. The inverted repeat present in the plastid genome of various organisms has been described (Palmer, J. D. (1990) Trends Genet. 6:115-120).
One advantage of plant plastid transformation over nuclear transformation is that the plastids of most plants are maternally inherited, and consequently heterologous plastid genes are not pollen disseminated. This feature is particularly attractive for transgenic plants having altered agronomic traits, as introduced resistance or tolerance to natural or chemical conditions will not be transmitted to wild-type relatives.
WO 95124493 z 185339 Plant plastids are also major biosynthetic cerXters. In addition to photosynthesis in chloroplasts, plastids are responsible for production of importa~t compounds such as amino acids, complex carbohydrates'-; 'fatty acids, and ';
pigments.
Plastids can also express two or more genes from a single plastid promoter region. A DNA sequence expressed in a plastid may thus include a number of individual structural gene encoding regions under control of one set of regulatory components. Thus, it is possible to introduce and express multiple genes in a plant cell, either from an engineered synthetic sequence or from a pre-existing prokaryotic gene cluster. -- -Such an expression method makes possible-large scale and inexpensive production of certain proteins and fine chemicals that are not practically produced through standard nuclear transformation methods. In nuclear expression from introduced genes, each encoding sequence must be engineered under the control of a separate regulatory region, i.e., a monocistron. As a consequence, gene expression levels vary widely among introduced sequences, and generation of a number of transgenic plant lines is-required, with crosses necessary, to introduce all of the cistrons into one plant and to get proper coordinated expression in the target biochemical pathway.
Plastids can be present in a plant cell at a very high copy number, with up to 50,000 copies per cell present for the chloroplast genome (Bendich, A. J. (1987) BioEssays 6:279-282). Thus, through plastid transformation plant cells can be engineered to maintain an introduced gene of interest at a very high copy number.
Forall of the above reasons, the plastids of higher plants present an attractive target for genetic engineering.
INTRODUCTION
Field of the ?nvention This invention relates to the application of genetic engineering techniques to plants. More specifically, the invention relates to compositions and methods for enhancing expression of a peptide of interest in the plastid of a plant cell.
Backaround Plastids of higher plants, i.e. chloroplasts, amyloplasts and chromoplasts, have the same genetic content, and thus are believed to be derived from a common precursor, known as a proplastid. The plastid genome is circular and varies in size among plant species from about 120 to about 217 kilobase pairs (kb). The genome typically includes a large inverted repeat, which can contain up to about 76 kilobase pairs, but which is more typically in the range of about 20 to about 30 kilobase pairs. The inverted repeat present in the plastid genome of various organisms has been described (Palmer, J. D. (1990) Trends Genet. 6:115-120).
One advantage of plant plastid transformation over nuclear transformation is that the plastids of most plants are maternally inherited, and consequently heterologous plastid genes are not pollen disseminated. This feature is particularly attractive for transgenic plants having altered agronomic traits, as introduced resistance or tolerance to natural or chemical conditions will not be transmitted to wild-type relatives.
WO 95124493 z 185339 Plant plastids are also major biosynthetic cerXters. In addition to photosynthesis in chloroplasts, plastids are responsible for production of importa~t compounds such as amino acids, complex carbohydrates'-; 'fatty acids, and ';
pigments.
Plastids can also express two or more genes from a single plastid promoter region. A DNA sequence expressed in a plastid may thus include a number of individual structural gene encoding regions under control of one set of regulatory components. Thus, it is possible to introduce and express multiple genes in a plant cell, either from an engineered synthetic sequence or from a pre-existing prokaryotic gene cluster. -- -Such an expression method makes possible-large scale and inexpensive production of certain proteins and fine chemicals that are not practically produced through standard nuclear transformation methods. In nuclear expression from introduced genes, each encoding sequence must be engineered under the control of a separate regulatory region, i.e., a monocistron. As a consequence, gene expression levels vary widely among introduced sequences, and generation of a number of transgenic plant lines is-required, with crosses necessary, to introduce all of the cistrons into one plant and to get proper coordinated expression in the target biochemical pathway.
Plastids can be present in a plant cell at a very high copy number, with up to 50,000 copies per cell present for the chloroplast genome (Bendich, A. J. (1987) BioEssays 6:279-282). Thus, through plastid transformation plant cells can be engineered to maintain an introduced gene of interest at a very high copy number.
Forall of the above reasons, the plastids of higher plants present an attractive target for genetic engineering.
WO 95124493 21 V J S' u J PGT/US95/02901 Stabla transformation of plastids has been reported in the green algae Chlamydomonas (Boynton et al. (1988) Science 240:1534-1538) and more recently in higher plants (Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530: Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917); (Staub, J. M. and Maliga, P. (1993), EMBO J. 12:601-606). The method disclosed for plastid transformation in higher plants relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination.
Many examples exist where expression levels greater than what is possible from nuclear expression would be desirable.
One example can be found in those instances where it is desired to produce a novel substance in a mature plant for subsequent extraction and purification. Other examples of proteins which may need to be expressed at very high levels are those producing resistance or tolerance phenotypes in the plant. One example of such a phenotype is a toxin active against plant pests.
in particular, there is a continuing need to introduce newly discovered or alternative Bacillus thuringiensis genes into crop plants. Cry proteins (d-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against a number of Lepidopteran, Dipteran, and Coleopteran insects.
These proteins are classified Cryl to CryV, based on amino acid sequence homology and insecticidal activity. Most Cryl proteins are synthesized as protoxins (ca. 130-140 kDa) then solubilized and proteolytically processed into active toxin fragments (ca. 60-70 kDa).
The poor expression of the protoxin genes from the nucleus of plants has heretofore required the use of 'truncated' versions ofthese genes. The truncated versions code only for the active toxin fragments. Other attempts to increase the expression efficiency have included i resynthesizing the Bacillus thuringiensis toxin genes to utilize plant preferred codons. Many problems can arise in such extensive reconstruction of these large cry genes (approximately 3.5 Kb), and the process is both laborious and expensive.
Problems can also arise as new insect pests become endemic, or as existing populations develop resistance to a particular level or type of Bacillus thuringiensis toxin.
Thus, there is a particular need for producing higher and thereby more effective levels of the Bacillus thuringiensis toxin in plants, a need which will only increase with time.
SUNWARF OF THE INVENTION
Various embodiments of this invention provide a construct comprising the following as operably joined components in the 5' to 3' direction of transcription:
(a) a promoter functional in a plant plastid;
(b) a DNA sequence encoding a peptide of interest; and (c) a transcription termination region capable of terminating transcription in a plant plastid wherein a native DNA sequence encoding said peptide of interest has a given adenine and thymine content and wherein said DNA encoding sequence in (b) encodes the same amino acid sequence as said native DNA sequence and has an enriched adenine and thymine content. Also provided are plant cells containing such a construct.
Various embodiments of this invention provide a method for producing a plant cell expressing a peptide of interest, said method comprising introducing the construct of this invention into a plant cell and expressing said peptide in a plastid of said plant cell from the construct. Also provided is a plant cell produced by this method or a progeny plant cell thereof.
Various embodiments of this invention provide a method for producing a cell having enhanced production of a peptide of interest, said method comprising introducing a DNA
construct functional in a chioroplast for expression of a peptide of interest into a plant cell and expressing said peptide in chloroplasts of said plant cell, wherein said peptide is expressed as a component of about 5% or greater by weight of the total protein of said plant cell. The construct may be a DNA construct as described above which may have enriched adenine and thymine content as referred to above. Also provided is a plant cell produced by this method or a progeny plant cell thereof.
By this invention, plastid expression constructs are provided which are useful for genetic engineering of plant cells and which provide for enhanced expression of a foreign peptide in plant cell plastids. The transformed plastid is preferably a metabolically active plastid, such as the chloroplasts found in green plant tissues including leaves or cotyledons. The plastid is preferably one which is maintained at a high copy number in the plant tissue of interest.
The plastid expression constructs for use in this invention generally include a plastid promoter region and a DNA sequence of interest to be expressed in transformed plastids. The DNA sequence of interest may be a single encoding region, or may contain a number of consecutive encoding regions, to be expressed as an operon, for example where introduction of a foreign biochemical pathway into plastids is desired.
In one embodiment, the DNA encoding sequence of the construct encodes the same amino acid sequence as the native DNA sequence, while having a codon usage enriched for adenine and thymine content. As an example, a native DNA sequence 4a ~ WO 95124493 2185339 PCT/US95/02901 may be resynthesized to include an adenine and thymine content preferred by the plant plastid. While the adenine and thymine percentage content of the nuclear genome varies from organism to organism, in plants the codon utilization generally comprises-moreguanine and cytosine pairings than adenine and thymine, thus the content is considered enriched ~ for guanine plus cytosine.
Plastid expression constructs of this invention may be linked to a construct having a DNA sequence encoding a selectable marker which can be expressed in a plant plastid.
Expression of the selectable marker allows the identification of plant cells comprising a plastid expressing the marker.
In a preferred embodiment, transformation vectors for transfer of the construct into a plant cell include means for inserting the expression and selection constructs into the plastid genome. This preferably comprises regions of homology to the target plastid genome which flank the constructs.
Also by this invention a method is provided whereby a plastid expression construct is used to produce a peptide of interest in a plant cell. The peptide may be expressed in a plastid of the plant cell from-the native DNA encoding sequence to the peptide.= Alternatively, the DNA encoding sequence of the construct can be one enriched for adenine and thymine.
By this invention the insecticidal Bacillus thuringiensis toxin is produced in plastids of a plant cell from the native DNA encoding sequence, with enhanced levels of expression of an insect resistant phenotype, as measured . by insect feeding assays. The native Bacillus thuringiensis DNA encoding sequence may be the truncated version specific to the active fragment. This invention also provides the expression of the Bacillus thuringiensis toxin from the non-truncated sequence which encodes the protoxin.
;. .
Plant cells and plants produded by a method of the invention and comprising a plastid expression construct are also considered in this invention.
DESCRIPTION OF THE FIGURES
Figure 1 shows integration of cry genes from vectors pZS223 and pZS224 into the wild-type plastid genome (Nt-ptDNA) to yield transplastomes Nt-pZS223 ptDNA and Nt-pZS224 ptDNA, respectively.
DETAILED DESCRIPTION OF THE INVENTION
A plastid expression construct of this invention generally comprises a promoter functional in a plant plastid, a DNA sequence encoding a peptide of interest and a transcription termination region-capable of terminating transcription in a plant plastid. These elements are provided as operably joined components in the 5' to 3' direction.of transcription. - -- -Any DNA encoding sequence which is enriched for adenine plus thymine content, and which can be inserted into the plastid genome of a plant cell to provide enhanced expression of a peptide of interest from the DNA encoding sequence, can be utilized.
In developing the constructs the various fragments comprising the regulatory regions and open reading frame may be subjected to different processing conditions, such as ligation, restriction enzyme digestion, PCR, in vitro mutagenesis, linkers and adapters addition, and the like.
Thus, nucleotide transitions, transversions, insertions, deletions, or the like, may be performed on the DNA which is WO 95l24493 2185339 PCT/US95/02901 employed in the regulatory regions or the DNA sequences of interest for expression in the plastids. Methods for restriction digests, Klenow blunt end treatments, ligations, and the like are well known to those in the art and are described, for example, by Maniatis et al. (in Molecular cloning: a laboratory manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
During the preparation of the constructs, the various fragments of DNA will often be cloned in an appropriate cloning vector, which allows for amplification of the DNA, modification of the DNA or manipulation of the DNA by joining or removing sequences, linkers, or the like. Preferably, the vectors will be capable of replication to at least a relatively high copy number in E. coli. A number of vectors are readily available for cloning, including such vectors as pBR322, vectors of the pUC series, the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla, CA).
In order to provide a means of selecting the desired plant cells, vectors for-plastid transformation typically contain a construct which provides for expression of a selectable marker gene. Marker genes are plant-expressible DNA sequences which express a polypeptide which resists a natural inhibition by, attenuates, or inactivates a selective substance, i.e., antibiotic, herbicide etc..
Alternatively, a marker gene may provide some other visibly reactive response, i.e., may cause a distinctive appearance or growth pattern relative to plants or plant cells not expressing the selectable marker gene in the presence of some substance, either as applied directly to the plant or plant cells or as present in the plant or plant cell growth media.
In either case, the plants or plant cells containing such selectable marker genes will have a distinctive WO 95/24493 '21O 5939 PCT/US95/02901 phenotype for purposes of identification, i.e., they will-be distinguishable from non-transformed cells. The characteristic phenotype allows the identification of cells, cell groups, tissues, organs, plant parts or whole plants containing the construct.
Detection of the marker phenotype makes possible the selection of cells having a second gene to which the marker gene'has been linked. This second gene typically comprises a desirable phenotype which is not readily identifiable in transformed cells, but which-is present when the plant cell or derivative thereof is grown to maturity, even under -conditions wherein the selectable marker phenotype itself is not apparent. _ - - - -The use of such a marker for identification of plant cells containing a plastid construct has been described.
Svab et al. (1993 supra). In the examples provided below, a bacterial aadA gene is expressed as the marker under the regulatory control of chloroplast 5' promoter and 3' transcription termination regions, specifically the tobacco 16S rRNA promoter rsn region and rpsl6 3' termination region.
Numerous additional promoter regions may also be used to drive expression of the selectable marker gene, including various plastid promoters and bacterial promoters which have been shown to function in plant plastids. -Expression of the aadA gene confers resistance to spectinomycin and streptomycin, and thus allows for the identification of plant cells expressing this marker. The aadA gene product allows for continued growth and greening of cells whose chloroplasts comprise the selectable marker gene product. Cells which do not contain the selectable marker gene product are bleached. Selection for the aadA gene marker is thus based on identification of-plant cells which are not bleached by the presence of streptomycin, or more preferably spectinomycin, in the plant growth medium.
Many examples exist where expression levels greater than what is possible from nuclear expression would be desirable.
One example can be found in those instances where it is desired to produce a novel substance in a mature plant for subsequent extraction and purification. Other examples of proteins which may need to be expressed at very high levels are those producing resistance or tolerance phenotypes in the plant. One example of such a phenotype is a toxin active against plant pests.
in particular, there is a continuing need to introduce newly discovered or alternative Bacillus thuringiensis genes into crop plants. Cry proteins (d-endotoxins) from Bacillus thuringiensis have potent insecticidal activity against a number of Lepidopteran, Dipteran, and Coleopteran insects.
These proteins are classified Cryl to CryV, based on amino acid sequence homology and insecticidal activity. Most Cryl proteins are synthesized as protoxins (ca. 130-140 kDa) then solubilized and proteolytically processed into active toxin fragments (ca. 60-70 kDa).
The poor expression of the protoxin genes from the nucleus of plants has heretofore required the use of 'truncated' versions ofthese genes. The truncated versions code only for the active toxin fragments. Other attempts to increase the expression efficiency have included i resynthesizing the Bacillus thuringiensis toxin genes to utilize plant preferred codons. Many problems can arise in such extensive reconstruction of these large cry genes (approximately 3.5 Kb), and the process is both laborious and expensive.
Problems can also arise as new insect pests become endemic, or as existing populations develop resistance to a particular level or type of Bacillus thuringiensis toxin.
Thus, there is a particular need for producing higher and thereby more effective levels of the Bacillus thuringiensis toxin in plants, a need which will only increase with time.
SUNWARF OF THE INVENTION
Various embodiments of this invention provide a construct comprising the following as operably joined components in the 5' to 3' direction of transcription:
(a) a promoter functional in a plant plastid;
(b) a DNA sequence encoding a peptide of interest; and (c) a transcription termination region capable of terminating transcription in a plant plastid wherein a native DNA sequence encoding said peptide of interest has a given adenine and thymine content and wherein said DNA encoding sequence in (b) encodes the same amino acid sequence as said native DNA sequence and has an enriched adenine and thymine content. Also provided are plant cells containing such a construct.
Various embodiments of this invention provide a method for producing a plant cell expressing a peptide of interest, said method comprising introducing the construct of this invention into a plant cell and expressing said peptide in a plastid of said plant cell from the construct. Also provided is a plant cell produced by this method or a progeny plant cell thereof.
Various embodiments of this invention provide a method for producing a cell having enhanced production of a peptide of interest, said method comprising introducing a DNA
construct functional in a chioroplast for expression of a peptide of interest into a plant cell and expressing said peptide in chloroplasts of said plant cell, wherein said peptide is expressed as a component of about 5% or greater by weight of the total protein of said plant cell. The construct may be a DNA construct as described above which may have enriched adenine and thymine content as referred to above. Also provided is a plant cell produced by this method or a progeny plant cell thereof.
By this invention, plastid expression constructs are provided which are useful for genetic engineering of plant cells and which provide for enhanced expression of a foreign peptide in plant cell plastids. The transformed plastid is preferably a metabolically active plastid, such as the chloroplasts found in green plant tissues including leaves or cotyledons. The plastid is preferably one which is maintained at a high copy number in the plant tissue of interest.
The plastid expression constructs for use in this invention generally include a plastid promoter region and a DNA sequence of interest to be expressed in transformed plastids. The DNA sequence of interest may be a single encoding region, or may contain a number of consecutive encoding regions, to be expressed as an operon, for example where introduction of a foreign biochemical pathway into plastids is desired.
In one embodiment, the DNA encoding sequence of the construct encodes the same amino acid sequence as the native DNA sequence, while having a codon usage enriched for adenine and thymine content. As an example, a native DNA sequence 4a ~ WO 95124493 2185339 PCT/US95/02901 may be resynthesized to include an adenine and thymine content preferred by the plant plastid. While the adenine and thymine percentage content of the nuclear genome varies from organism to organism, in plants the codon utilization generally comprises-moreguanine and cytosine pairings than adenine and thymine, thus the content is considered enriched ~ for guanine plus cytosine.
Plastid expression constructs of this invention may be linked to a construct having a DNA sequence encoding a selectable marker which can be expressed in a plant plastid.
Expression of the selectable marker allows the identification of plant cells comprising a plastid expressing the marker.
In a preferred embodiment, transformation vectors for transfer of the construct into a plant cell include means for inserting the expression and selection constructs into the plastid genome. This preferably comprises regions of homology to the target plastid genome which flank the constructs.
Also by this invention a method is provided whereby a plastid expression construct is used to produce a peptide of interest in a plant cell. The peptide may be expressed in a plastid of the plant cell from-the native DNA encoding sequence to the peptide.= Alternatively, the DNA encoding sequence of the construct can be one enriched for adenine and thymine.
By this invention the insecticidal Bacillus thuringiensis toxin is produced in plastids of a plant cell from the native DNA encoding sequence, with enhanced levels of expression of an insect resistant phenotype, as measured . by insect feeding assays. The native Bacillus thuringiensis DNA encoding sequence may be the truncated version specific to the active fragment. This invention also provides the expression of the Bacillus thuringiensis toxin from the non-truncated sequence which encodes the protoxin.
;. .
Plant cells and plants produded by a method of the invention and comprising a plastid expression construct are also considered in this invention.
DESCRIPTION OF THE FIGURES
Figure 1 shows integration of cry genes from vectors pZS223 and pZS224 into the wild-type plastid genome (Nt-ptDNA) to yield transplastomes Nt-pZS223 ptDNA and Nt-pZS224 ptDNA, respectively.
DETAILED DESCRIPTION OF THE INVENTION
A plastid expression construct of this invention generally comprises a promoter functional in a plant plastid, a DNA sequence encoding a peptide of interest and a transcription termination region-capable of terminating transcription in a plant plastid. These elements are provided as operably joined components in the 5' to 3' direction.of transcription. - -- -Any DNA encoding sequence which is enriched for adenine plus thymine content, and which can be inserted into the plastid genome of a plant cell to provide enhanced expression of a peptide of interest from the DNA encoding sequence, can be utilized.
In developing the constructs the various fragments comprising the regulatory regions and open reading frame may be subjected to different processing conditions, such as ligation, restriction enzyme digestion, PCR, in vitro mutagenesis, linkers and adapters addition, and the like.
Thus, nucleotide transitions, transversions, insertions, deletions, or the like, may be performed on the DNA which is WO 95l24493 2185339 PCT/US95/02901 employed in the regulatory regions or the DNA sequences of interest for expression in the plastids. Methods for restriction digests, Klenow blunt end treatments, ligations, and the like are well known to those in the art and are described, for example, by Maniatis et al. (in Molecular cloning: a laboratory manual (1982) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
During the preparation of the constructs, the various fragments of DNA will often be cloned in an appropriate cloning vector, which allows for amplification of the DNA, modification of the DNA or manipulation of the DNA by joining or removing sequences, linkers, or the like. Preferably, the vectors will be capable of replication to at least a relatively high copy number in E. coli. A number of vectors are readily available for cloning, including such vectors as pBR322, vectors of the pUC series, the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla, CA).
In order to provide a means of selecting the desired plant cells, vectors for-plastid transformation typically contain a construct which provides for expression of a selectable marker gene. Marker genes are plant-expressible DNA sequences which express a polypeptide which resists a natural inhibition by, attenuates, or inactivates a selective substance, i.e., antibiotic, herbicide etc..
Alternatively, a marker gene may provide some other visibly reactive response, i.e., may cause a distinctive appearance or growth pattern relative to plants or plant cells not expressing the selectable marker gene in the presence of some substance, either as applied directly to the plant or plant cells or as present in the plant or plant cell growth media.
In either case, the plants or plant cells containing such selectable marker genes will have a distinctive WO 95/24493 '21O 5939 PCT/US95/02901 phenotype for purposes of identification, i.e., they will-be distinguishable from non-transformed cells. The characteristic phenotype allows the identification of cells, cell groups, tissues, organs, plant parts or whole plants containing the construct.
Detection of the marker phenotype makes possible the selection of cells having a second gene to which the marker gene'has been linked. This second gene typically comprises a desirable phenotype which is not readily identifiable in transformed cells, but which-is present when the plant cell or derivative thereof is grown to maturity, even under -conditions wherein the selectable marker phenotype itself is not apparent. _ - - - -The use of such a marker for identification of plant cells containing a plastid construct has been described.
Svab et al. (1993 supra). In the examples provided below, a bacterial aadA gene is expressed as the marker under the regulatory control of chloroplast 5' promoter and 3' transcription termination regions, specifically the tobacco 16S rRNA promoter rsn region and rpsl6 3' termination region.
Numerous additional promoter regions may also be used to drive expression of the selectable marker gene, including various plastid promoters and bacterial promoters which have been shown to function in plant plastids. -Expression of the aadA gene confers resistance to spectinomycin and streptomycin, and thus allows for the identification of plant cells expressing this marker. The aadA gene product allows for continued growth and greening of cells whose chloroplasts comprise the selectable marker gene product. Cells which do not contain the selectable marker gene product are bleached. Selection for the aadA gene marker is thus based on identification of-plant cells which are not bleached by the presence of streptomycin, or more preferably spectinomycin, in the plant growth medium.
A number of markers have been developed for use with plant cells, such as resistance to chloramphenicol, the aminoglycoside G418, hygromycin, or the like. Other genes which encode a product involved in chloroplast metabolism may also be used as selectable markers. For example, genes which provide resistance to plant herbicides such as glyphosate, bromoxynil or imidazolinone may find particular use. Such genes have been reported (Stalker et al., J. Biol. Chem.
(1985) 260:4724-4728 (glyphosate resistant EPSP); Stalker et al., J. Biol.Chem. (1985) 263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan et al., Nuc1. Acids Res. (1990) 18:2188 (AHAS imidazolinone resistance gene)).
Stable transformation of tobacco plastid genomes by particle bombardment is-reported (Svab et.al. (1990 supra) and Svab et al. (1993 supra)). The methods described therein may be employed to obtain plants homoplasmic for plastid expression constructs.
Generally, bombarded tissue is cultured for approximately 2 days on a cell division-promoting media, after which the plant tissue is transferred to a selective media containing an inhibitory amount of the particular selective agent, as well as the particular hormones and other ---substances necessary to obtain regeneration for that particular plant species. Shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots.
Homoplasmy is verified by southern analysis. In the examples provided below, BamHI-digested total cellular DNA is tested with various probes, specifically, a part of the = plastid targeting fragment, an aadA fragment, a 1.8 kb czylA
fragment and a 3.5 kb fragment of the c.ry73 coding region.
Southern-blot analysis with these probes confirms the integration of the chimeric cry genes in the tobacco plastid genome to yield transplastome linh4s.
As an alternative to a second round of shoot formation, the initial selected shoots may be grown to mature plants and segregation relied-upon to provide transformed plants homoplastic for the inserted gene construct.
Where transformation and regeneration methods have been adapted for a given plant species, either by Agrobacterium-mediated transformation, bombardment or some other method, the established techniques may be modified for use in selection and regeneration methods to produce plastid- -transformed plants. For example, the methods described herein for tobacco are readily adaptable to othersolanaceous species, such as tomato, petunia and potato.
In Brassica, Agrobacterium-mediated transformation and regeneration protocols generally involve the use of hypocotyl tissue, a non-green tissue which might contain a low plastid content. Thus, for Brassica, preferred target tissues would include microspore-derived hypocotyl or cotyledonary tissues (which are green and thus contain numerous plastids) or leaf tissue explants. W'hile the regeneration rates from such tissues may be low, positional effects, such as seen with Agrobacterium-mediated transformation, are not expected, thus it would not be necessary to screen numerous successfully transformed plants in order to obtain a desired phenotype.
The vectors for use in plastid transformation preferably include means for providing a stable transfer of the plastid expression construct and selectable marker construct into the plastid genome. This is most conveniently provided by regions of homology to the target plastid genome. The regions of homology flank the construct to be transferred and provide for transfer to the plastid genome by homologous recombination, via a double crossover into the genome. The SUBSTITUTE SHEET (RULE 26) W O 95/24493 218 5 3 3 9 pCT/pS95/OZ901 complete DNA sequence of the plastid genome of tobacco has been reported (Shinozaki et al.,(1986) EMBO J. 5:2043-2049).
Complete DNA sequencesot the plastid genomes from liverwort (Ohyama et al. (1986) Nature 322:572-574) and rice (Hiratsuka et al. (1989) Mol. Gen. Genet. 217:185-194), have also been reported.
Where the regions of homology are present in the inverted repeat regions of the plastid genome (known as IRA
and IRB), two copies of the transgene are expected per transformed plastid. The regions of homology within the plastid genome are approximately ikb in size. Smaller regions of homology may also be used, and as little as 100 bp can provide for homologous recombination into the plastid genome. However, the frequency of recombination and thus the frequency of obtaining plants having transformed plastids decreases with decreasing size of the homology regions.
Examples of constructs having regions of homology the plastid genome are described in Svab et.al. (1990 supra) and Svab et al. (1993 supra). Regions useful for recombination into tobacco and Brassica plastid genomes are also identified in the following examples, but homologous recombination and selection constructs may be prepared using many plastid DNA
sequences, and to any target plant species. In the examples provided herein, the flanking tobacco plastid homology regions of the plastid expression construct direct the insertion of a Bacillus thuringiensis transgene into the tobacco genome between trnV and the rps12 operon. Since integration into the plastid genome occurs by homologous recombination and the target site is in an inverted repeat region of the plastid genome, two copies of the transgene per plastid genome are expected. Selection is made for the spectinomycin resistance marker phenotype expressed by the aadA gene.
SUBSTITUTE SHEET (RULE 26) In the examples the native cry gene, i.e., having an unmodified coding region to the protoxin, is placed into a plastid expression construct for expression of Bacillus 4 ~
thuringiensis toxin from the plant pl?astid.
A synthetic Bacillus thuringiensis gene is placed in the same expression construct as the protoxin gene. The synthetic gene is designed to have tobacco RuBPCO small subunit codon usage, with an overall intrease in the guanine plus cytosine content to 55% (with respect to the native gene content of 39%), and has been truncated to leave only those sequences which enode the active fragment of the toxin. Such a gene is lrnown to provide optimal expression from the plant nuclear genome. Both the bacterial gene which has been resynthesized for increased expression from plant nuclear transformation and the non-resynthesized, non-truncated wild-type gene to the protoxin are introduced via a chloroplast transformation vector (Fig. 1).
Unexpectedly, it is found that expression of the toxin is greatly enhanced from the native encoding sequence for the gene, as opposed to a version of the gene resynthesized to approximate the preferred codons of the plant genome.
Tobacco lines containing the native encoding sequence demonstrate strong insecticidal bioactivity, as measured by insect feeding assays. Tobacco lines having a synthetic cryIA(c) gene demonstrate no observable bioactivity. As in both cases the constructs are introduced in a controlled manner:by homologous recombination from the same plastid vector, the differences cannot be accounted for by positional effects.
In transformed plants containing the native encoding sequence, the Bacillus thuringiensis toxin is present as a component of up to about 5% or greater of the total leaf protein, a level which is much higher than is present in-the leaf of plants resulting from nuclear transformation. In WO95124493 PCT1US95l02901 plants containing the gene resynthesized to approximate the preferred codons of the plant genome, the mRNA to the toxin appears degraded, and little br no toxin protein appears present in the leaf.
That a native Bacillus thuringiensis toxin gene is expressed to such a high level in the plastid, while an otherwise identical construct containing a Bacillus thuringiensis gene resynthesized for efficient nuclear expression is very poorly expressed in the plastid, despite having the same copy number in the plastid, suggests that the adenine plus thymine content of the plastid transgene heavily influences expression. As previously noted, the guanine plus cytosine content of the synthetic gene was increased to 55%, which is high relative to that of the plastid genome content of less than 40% guanine plus cytosine. This difference may cause inefficient processing of the mRNA, or lead to an increase in its rate of degradation. The native Bacillus thuringiensis gene has a guanine plus cytosine percentage which more closely matches that of the plastid genome, and thus more closely favors the codon usage of a plastid gene.
The adenine plus thymine content of the respective genes may not entirely explain the dramatic differences in expression of the native and synthetic Bacillus thuringiensis toxin proteins. One additional factor which could be postulated is that unwanted or highly inefficient plastid RNA
processing signals are introduced into the synthetic crylA(c) gene. Such signals, if present, could greatly reduce or even eliminate expression of the toxin.
In any case, it is now shown that the codon usage of the native Bacillus thuringiensis gene achieves an expression level which is much higher in plastid expression than is possible with resynthesized sequence to the same gene, thus demonstrating that a gene having bacterial codon usage can achieve high levels of expression in a plant plastid. The WO 95/24493 PCT/US95102901 ~
above results eliminate the need to resynthesize a certain class of genes for high level expression in plants.
The DNA sequence of interest may have a natural codon usage high in adenine and thymine, as is the case for the Bacillus thuringiensis gene, or may alternatively be resynthesized to enrich the adenine plus thymine content. In fact, while the constructs and methods described herein may be employed with a wide variety of native bacterial DNA
encoding sequences, a wider range of potential gene targets for high level plastid expression can be obtained by resynthesizing genes, for instance plant nuclear genes, to increase the adenine and thymine content of the encoding sequence.
The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.
EXAMPLES
In the experimental disclosure which follows, all temperatures are given in degrees centigrade ( ), weights are given in grams (g), milligram (mg) or micrograms ( .g), concentrations are given as molar (M), millimolar (mM) or micromolar ( M) and all volumes are given in liters (1), milliliters (ml) or microliters ( l), unless otherwise indicated.
EXAMPLE 1. PLASTID TRANSFORMATION VECTORS
Constructs and methods for use in transforming the plastids of higher plants are described in Svab et al. (1990 supra), Svab et al. (1993 supra) and Staub et al. (1993 .
supra). The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (1986 supra). All WO95124493 2185339 _ plastid DNA references in the following description are to the nucleotide number from tobacco.
The cryIA(c) gene is obtained from plasmid pBtkHD73 (Toagosei Chemical Co., Japan). This gene is further processed by digestion with SmaI/Nsil and a synthetic adapter is inserted (top strand:-5'-CCCGGATCCATGGATAACAATCCGA-ACATCAATGAATGCA-3'; bottom strand: 5'-TTCATTGATGTTCGGATT-GTTATCCATGGATCCGGG-3'). The entire 5' untranslated region from the cryIA(c) gene is then removed, and an NcoI site is introduced at the natural start codon (position 163 of the nucleotide sequence (Adang et aI. (1985) Gene 36;289-300). A
BamHI site is introduced just upstream of the Ncol site.
Oligonucleotide mutagenesis is performed to introduce BglII
and SaII sites directly adjacent to the stop codon of the crylA(c) gene, to facilitate removal of unwanted DNA 3' of the coding region. The remaining sequence includes the entire encoding region to the protoxin.
A synthetic cryIA(c) gene encoding the active toxin fragment is constructed by annealing and ligating 70 and 90 base oligonucleotides, in a method as described (Wosnick et al. (1987) Gene 60;115-127). The synthetic gene is designed to have tobacco RuBISCO small subunit codon usage, including a guanine and cytosine content of 55%, with an NcoI site at the start codon and a Sa2I site at the stop codon, while still encoding the amino acid sequence of the toxin. This synthetic gene is also truncated, however, so that the encoding region only provides the amino acid sequence to the active fragment of the protoxin.
A plastid transformation vector is used which carries a passenger gene in a Prrn(L)rbcL(S)/Trps16 expression cassette, with polylinker restriction sites. The Prrn(L)rbcL(S) fragments are described in Svab et al. (1993 supra). To further secure the stability of the mRNAs, the Trps16 fragment is cloned downstream of the passenger gene W O 95/24493 21 V51"9 PCTIUS95/02901 encodingregion. The Trps16 fragment comprises the rpsl6 gene 3'-regulatory region from,nucleotides 5,087 to 4,939 in the tobacco plasmid DNA. -Chimeric genes are preferably inserted into the vector to direct their transcription towards the rrn operon. Thus, in the plastid genome, chimeric genes are transcribed from the Prsn(L)rbcL(S) 5'-regulatory region comprising the long rrn operon promoter fragment from nucleotides 102,561 to 102,677 of the tobacco plastid genome, which is fused with a syntheticleader sequence designed after the rbcL gene leader between nucleotides 57,569 to 57,584 in the plastid DNA.
The plastid transformation vector also carries a selectable spectinomycin resistance gene (aadA) under control of psbA gene expression signals. The regulatory and encoding sequences are also flanked by plastid DNA homology regions whose limits are bp 138,447 (EcoRI) to 140,219 (Hincll) and 140,219 (HincII) to 141,382 (Bg1II) of the tobacco plastid genome (Shinozaki et al. (1986 supra)). This directs insertion of foreign genes located between the flanking regions into the plastid between the traV gene and the rpsl2/7 operon.
This plastid transformation vector is digested with the NcoI/SalI restriction endonucleases to remove the encoding region of the passenger gene, which is then replaced with a Ncol/Saii fragment containing the synthetic crylA(c) coding region, yielding a vector whichis designated pZS223 (Fig.
1). The wild type crylA(c) protoxin gene is similarly cloned as an NcoI/Sa1I fragment, yielding a plasmid designated pZS224. By this approach Bacillus thuringiensis DNA 3' of the protein coding region is omitted for both plasmids, pZS223 and pZS224.
The insertion of the respective cry genes from vectors pZS223 and pZS224 into the wild-type plastid genome (Nt-ptDNA) to yield transplastomes Nt-pZS223 and Nt-pZS224, respectively, is shown in Fig. 1. The abbreviations used in Fig. 1 are as follows: 16S, 16S rRNA gene; trnV, tznV gene;
aadA, spectinomycin resistance gene; crylA and cry73 are synthetic and iiative Bacillus thuringiensis d-endotoxin genes, respectively. The restriction endonuclease cleavage sites are designated as follows: B, BamHI; Bg, BgIII; H, HindII2; N, Ncol; RI, EcoRI, RV, EcoRV; S, Sa1I.
EXAMPLE 2. PLANT PLASTID TRANSFORMATION
Stable transformation of tobacco plastid genomes by particle bombardment is reported in Svab et.al. (1990 supra) and Svab et al. (1993 supra). The methods described therein may be employed to obtain plants transformed with the plastid expression constructs described herein. Such methods generally involve DNA bombardment of a target host explant, preferably an explant made from a tissue which is rich in metabolically active plastids, such as green plant tissues including leaves or cotyledons.
Tobacco seeds (N. tabacum v. Xanthi N/C) are surface sterilized in a 50% chlorol solution (2.5% sodium , hypochlorite) for 20 minutes and rinsed 4 times in sterile H20. These are plated asceptically on a 0.2x MS salts media and allowed to germinate. The seedlings are grown on agar solidified MS media with 30g/l sucrose (Murashige et al.
(1962) Physiol. Plant 15:493-497).
Tungsten microprojectiles (1.O M) are coated with plasmid DNA according to Maliga (Maliga, P. (1993) Methods in Plant Molecular Biology - A Laboratory Manual, eds. Pal Maliga, Daniel Klessig, Anthony Cashmore, Wilhelm Gruissem and Joseph Varner; Cold Spring Harbor Press) and used to bombard mature leaves, placed abaxial side up on RMOP media;
MS salts, 1 mg/1 BAP, 0.1 mg/1 NAA, 30 g/l sucrose and 0.7%
phytagar # Svab et al. (1990) Proc. Natl. Acad. Sci. USA
*Trademark WO 95/24493 PCTI[JS95102901 87:8526-8530 (using the Bio-Rad PDS 1000 He system (Sanford et al., An improved, helium-driven Biolistic device, Technique 3:3-16)). Plasmids pZS223 and pZS224 are used as the coating plasmid DNA.
The bombarded tissue is then cultured for approximately 2 days on a cell division-promoting media, after which the plant tissue is transferred to a selective media containing an inhibitory amount of the particular selective agent.
Transformed explants form green shoots in approximately 3-8 weeks. Leaves from these shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots.
EXAMPLE 3_ DNA GEL BLOT ANALYSIS OF TRANSPLASTOMIC LINES
Transformed plants selected for marker aadA marker gene expression are analyzed to determine whether the entire plastid content of the plant has been transformed (homoplastic transformants). Typically, following two rounds of shoot formation and spectinomycin selection, approximately 50% of the transgenic plantlets which are analyzed are homoplastic, as determined by Southern blot analysis of plastid DNA. Homoplasmic plantlets are selected for further cultivation.
Following a second round of shoot formation and spectinomycin selection, two transplastomic lines for each construct are obtained, Nt-pZS223 and Nt-pZS224. These lines are checked for homoplasmy. Southern blot analysis is used to confirm the integration of the chimeric cry genes in the tobacco plastid genome. Preparation, electrophoresis, and transfer of DNA to filters is as described (Svab et al., (1993 supra)).
The complete disappearance of the 3.3 Kb native tobacco BamHI fragment in the Nt-pZS223 and Nt-pZS224 transformants ~ W0951]A493 PCT/US95102901 with a probe covering the region of integration, and the appearance of expected sized bands for the inserted DNA
fragments in those transformants, 5.5 kb and 7.3 kb, respectively (see Fig. 1), establishes that the transformed plants are homoplasmic for the intended constructs. Probing identical filters with the aadA, crylA(c) protoxin, and synthetic czylA(c) genes demonstrated a linkage of the aadA
and cryIA(c) genes to the expected 5.5 and 7.3 Kb BamHI
fragments as well as the lack of these genes in the negative control.
EXAMPLE 4. INSECT BIOASSAYS
As described, the development of transformed plant lines Nt-pZS223 and Nt-pZS224 is accomplished on RMOP media supplemented with 500 mg/1 Spectinomycin dihydrochloride.
Plants are subcloned on the same selective medium, by the method according Svab et al.(1990 supra). Selected plants are then rooted in MS media containing 1 mg/1 IBA, 500 mg/1 Spectinomycin dihydrochloride and 0.6% phytagar.
Helicoverpa zea and Heliothis virescens eggs are obtained from the USDA-ARS in Stoneville, MS. and allowed to hatch. Neonate larva are placed on Tobacco Budworm Diet from Bioserve (Frenchtown, NJ), and incubated in a 16:8 photoperiod at 28'C_for=5 days. The larva develop during this time to late second or early third instar.
At 5 days, fully expanded leaves are excised from the tobacco plants and placed on 3 ml of 2% agar in a 32 well rearing tray from CD International (Pitman, NJ). The larva are placed 1 per well, sealed and incubated for 5 days at the same conditions. At day 10, the leaf material consumed by the insect is estimated and insects checked for mortality.
The larva are considered dead if they showed no movement = after prodding with forceps.
WO 95/24493 ~y1Q5'~ '~ 9 PCTIIIS95/02901 EXAMPLE S. INSEC~rTI VCIDAL FEEDING ACTIVITY
To determine the presence-and relative amount of active Bacillus thuringiensis d-toxin in the tobacco lines homoplasmic for native protoxin and synthetic 'truncated' cryIA(c) gene expression construcfts,'efficacy of these plants to third instar Heliothis virescens (tobacco budworm) and He.iicoverpa zea (corn earworm/cotton bollworm) larvae is =
tested (See table). Both test insects are sensitive to the crylA(c) toxin with H. zea being 10-fold more resistant than H. virescens (Maclntosh et al. (1990) J. invertebr. Pathol.
56:258-266.).
Third instar larvae are chosen for the bioassay since the insects are more resistant to the toxin at this stage than are first instar larvae thus allowing a more stringent comparison between the control and test plants. Tobacco-lines designated 4083 and 4084, derived by nuclear -transformation with the same synthetic cryIA(c) gene as used in pZS223 and shown to be highly toxic to third instar H.
virescens larvae, are used as positive controls in the bioassay. Nicotiana tabacum var. 'Petite Havana' serves as the negative control since this-is the genetic background used to generate the transplastomic lines.
Table 1 is a summary of Bacillus thuringiensis tobacco insect feeding assays. The data demonstrates that _ transplastomic line Nt-pZS224 is very toxic to both H.
virescens and H. zea as it causes 100% mortality to these insects while sustaining less than 2% total leafdamage.
This result compares favorably to the results for positive control 4083 and 4084 tobacco plants. The 4083-2-4 plant when assayed with H. zea causes 100% mortality but sustains a much greater level of leaf feeding damage than the Nt-pZS224 tobacco line indicating less toxin production. -Tobacco line 4084-4-1 performed comparably to Nt-pZS224 tobacco in _ feeding, although it does not compare to the levels of toxin produced in Nt-pZS224 when measured as a component of total leaf protein.
Tobacco line Nt-pZS223 shows no detectable bioactivity.
SIMNAARY OF ST TOBACCO INSECT FEEDING ASSAYS
piants Heiiothis %i.ea1 Hel'iocoverpa % Leai ChlorOplast Vector tested virescena~" Eaten Zea"~% Eaten synthetic loxin gane pZS223 223-3 NO mortaNty 100% NO mortatity 100%
223-5 NO morlaUty 75% NO nwrlalily 100%
223-12 NT' NO mortality 100 /.
223-13 NO mortaqly 75% NT' wiid lype protoxin gene pZS224 224-5 100% mortality 2% 100% mortality 2%
224-9 100% mortality 2% 100% mortality 2%
Nuclear Controis synthetic toxin gene pCGN4083 4083-1-2 100% mortality 2% NT' 4083-2-4 W. 100 .4 mortality 40%
pCGN4084 4084-8-5 100% mortality 2% NT=
4084-1-1 NT= 100% mortality 29'.
Untransiormed Controls control 1 25% mortality 75% NO mortality 100%
control 2 NO monatlty 100% NT=
control 3 50% mortality 75%. NT=
M 10 third instar larva were individuaNy tested per plant 'NT: Plant not tesled All publications and patent applications meutioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.
35 F1lthougti the foregoing invention has been described in sonie detail by way of illustration and example for purposes of-c'sarity of understanding, it will be obvious ttiat certain changes and modifications may be practiced within ltie scope of the appended claim.
(1985) 260:4724-4728 (glyphosate resistant EPSP); Stalker et al., J. Biol.Chem. (1985) 263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan et al., Nuc1. Acids Res. (1990) 18:2188 (AHAS imidazolinone resistance gene)).
Stable transformation of tobacco plastid genomes by particle bombardment is-reported (Svab et.al. (1990 supra) and Svab et al. (1993 supra)). The methods described therein may be employed to obtain plants homoplasmic for plastid expression constructs.
Generally, bombarded tissue is cultured for approximately 2 days on a cell division-promoting media, after which the plant tissue is transferred to a selective media containing an inhibitory amount of the particular selective agent, as well as the particular hormones and other ---substances necessary to obtain regeneration for that particular plant species. Shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots.
Homoplasmy is verified by southern analysis. In the examples provided below, BamHI-digested total cellular DNA is tested with various probes, specifically, a part of the = plastid targeting fragment, an aadA fragment, a 1.8 kb czylA
fragment and a 3.5 kb fragment of the c.ry73 coding region.
Southern-blot analysis with these probes confirms the integration of the chimeric cry genes in the tobacco plastid genome to yield transplastome linh4s.
As an alternative to a second round of shoot formation, the initial selected shoots may be grown to mature plants and segregation relied-upon to provide transformed plants homoplastic for the inserted gene construct.
Where transformation and regeneration methods have been adapted for a given plant species, either by Agrobacterium-mediated transformation, bombardment or some other method, the established techniques may be modified for use in selection and regeneration methods to produce plastid- -transformed plants. For example, the methods described herein for tobacco are readily adaptable to othersolanaceous species, such as tomato, petunia and potato.
In Brassica, Agrobacterium-mediated transformation and regeneration protocols generally involve the use of hypocotyl tissue, a non-green tissue which might contain a low plastid content. Thus, for Brassica, preferred target tissues would include microspore-derived hypocotyl or cotyledonary tissues (which are green and thus contain numerous plastids) or leaf tissue explants. W'hile the regeneration rates from such tissues may be low, positional effects, such as seen with Agrobacterium-mediated transformation, are not expected, thus it would not be necessary to screen numerous successfully transformed plants in order to obtain a desired phenotype.
The vectors for use in plastid transformation preferably include means for providing a stable transfer of the plastid expression construct and selectable marker construct into the plastid genome. This is most conveniently provided by regions of homology to the target plastid genome. The regions of homology flank the construct to be transferred and provide for transfer to the plastid genome by homologous recombination, via a double crossover into the genome. The SUBSTITUTE SHEET (RULE 26) W O 95/24493 218 5 3 3 9 pCT/pS95/OZ901 complete DNA sequence of the plastid genome of tobacco has been reported (Shinozaki et al.,(1986) EMBO J. 5:2043-2049).
Complete DNA sequencesot the plastid genomes from liverwort (Ohyama et al. (1986) Nature 322:572-574) and rice (Hiratsuka et al. (1989) Mol. Gen. Genet. 217:185-194), have also been reported.
Where the regions of homology are present in the inverted repeat regions of the plastid genome (known as IRA
and IRB), two copies of the transgene are expected per transformed plastid. The regions of homology within the plastid genome are approximately ikb in size. Smaller regions of homology may also be used, and as little as 100 bp can provide for homologous recombination into the plastid genome. However, the frequency of recombination and thus the frequency of obtaining plants having transformed plastids decreases with decreasing size of the homology regions.
Examples of constructs having regions of homology the plastid genome are described in Svab et.al. (1990 supra) and Svab et al. (1993 supra). Regions useful for recombination into tobacco and Brassica plastid genomes are also identified in the following examples, but homologous recombination and selection constructs may be prepared using many plastid DNA
sequences, and to any target plant species. In the examples provided herein, the flanking tobacco plastid homology regions of the plastid expression construct direct the insertion of a Bacillus thuringiensis transgene into the tobacco genome between trnV and the rps12 operon. Since integration into the plastid genome occurs by homologous recombination and the target site is in an inverted repeat region of the plastid genome, two copies of the transgene per plastid genome are expected. Selection is made for the spectinomycin resistance marker phenotype expressed by the aadA gene.
SUBSTITUTE SHEET (RULE 26) In the examples the native cry gene, i.e., having an unmodified coding region to the protoxin, is placed into a plastid expression construct for expression of Bacillus 4 ~
thuringiensis toxin from the plant pl?astid.
A synthetic Bacillus thuringiensis gene is placed in the same expression construct as the protoxin gene. The synthetic gene is designed to have tobacco RuBPCO small subunit codon usage, with an overall intrease in the guanine plus cytosine content to 55% (with respect to the native gene content of 39%), and has been truncated to leave only those sequences which enode the active fragment of the toxin. Such a gene is lrnown to provide optimal expression from the plant nuclear genome. Both the bacterial gene which has been resynthesized for increased expression from plant nuclear transformation and the non-resynthesized, non-truncated wild-type gene to the protoxin are introduced via a chloroplast transformation vector (Fig. 1).
Unexpectedly, it is found that expression of the toxin is greatly enhanced from the native encoding sequence for the gene, as opposed to a version of the gene resynthesized to approximate the preferred codons of the plant genome.
Tobacco lines containing the native encoding sequence demonstrate strong insecticidal bioactivity, as measured by insect feeding assays. Tobacco lines having a synthetic cryIA(c) gene demonstrate no observable bioactivity. As in both cases the constructs are introduced in a controlled manner:by homologous recombination from the same plastid vector, the differences cannot be accounted for by positional effects.
In transformed plants containing the native encoding sequence, the Bacillus thuringiensis toxin is present as a component of up to about 5% or greater of the total leaf protein, a level which is much higher than is present in-the leaf of plants resulting from nuclear transformation. In WO95124493 PCT1US95l02901 plants containing the gene resynthesized to approximate the preferred codons of the plant genome, the mRNA to the toxin appears degraded, and little br no toxin protein appears present in the leaf.
That a native Bacillus thuringiensis toxin gene is expressed to such a high level in the plastid, while an otherwise identical construct containing a Bacillus thuringiensis gene resynthesized for efficient nuclear expression is very poorly expressed in the plastid, despite having the same copy number in the plastid, suggests that the adenine plus thymine content of the plastid transgene heavily influences expression. As previously noted, the guanine plus cytosine content of the synthetic gene was increased to 55%, which is high relative to that of the plastid genome content of less than 40% guanine plus cytosine. This difference may cause inefficient processing of the mRNA, or lead to an increase in its rate of degradation. The native Bacillus thuringiensis gene has a guanine plus cytosine percentage which more closely matches that of the plastid genome, and thus more closely favors the codon usage of a plastid gene.
The adenine plus thymine content of the respective genes may not entirely explain the dramatic differences in expression of the native and synthetic Bacillus thuringiensis toxin proteins. One additional factor which could be postulated is that unwanted or highly inefficient plastid RNA
processing signals are introduced into the synthetic crylA(c) gene. Such signals, if present, could greatly reduce or even eliminate expression of the toxin.
In any case, it is now shown that the codon usage of the native Bacillus thuringiensis gene achieves an expression level which is much higher in plastid expression than is possible with resynthesized sequence to the same gene, thus demonstrating that a gene having bacterial codon usage can achieve high levels of expression in a plant plastid. The WO 95/24493 PCT/US95102901 ~
above results eliminate the need to resynthesize a certain class of genes for high level expression in plants.
The DNA sequence of interest may have a natural codon usage high in adenine and thymine, as is the case for the Bacillus thuringiensis gene, or may alternatively be resynthesized to enrich the adenine plus thymine content. In fact, while the constructs and methods described herein may be employed with a wide variety of native bacterial DNA
encoding sequences, a wider range of potential gene targets for high level plastid expression can be obtained by resynthesizing genes, for instance plant nuclear genes, to increase the adenine and thymine content of the encoding sequence.
The invention now being generally described, it will be more readily understood by reference to the following examples which are included for purposes of illustration only and are not intended to limit the present invention.
EXAMPLES
In the experimental disclosure which follows, all temperatures are given in degrees centigrade ( ), weights are given in grams (g), milligram (mg) or micrograms ( .g), concentrations are given as molar (M), millimolar (mM) or micromolar ( M) and all volumes are given in liters (1), milliliters (ml) or microliters ( l), unless otherwise indicated.
EXAMPLE 1. PLASTID TRANSFORMATION VECTORS
Constructs and methods for use in transforming the plastids of higher plants are described in Svab et al. (1990 supra), Svab et al. (1993 supra) and Staub et al. (1993 .
supra). The complete DNA sequences of the plastid genome of tobacco are reported by Shinozaki et al. (1986 supra). All WO95124493 2185339 _ plastid DNA references in the following description are to the nucleotide number from tobacco.
The cryIA(c) gene is obtained from plasmid pBtkHD73 (Toagosei Chemical Co., Japan). This gene is further processed by digestion with SmaI/Nsil and a synthetic adapter is inserted (top strand:-5'-CCCGGATCCATGGATAACAATCCGA-ACATCAATGAATGCA-3'; bottom strand: 5'-TTCATTGATGTTCGGATT-GTTATCCATGGATCCGGG-3'). The entire 5' untranslated region from the cryIA(c) gene is then removed, and an NcoI site is introduced at the natural start codon (position 163 of the nucleotide sequence (Adang et aI. (1985) Gene 36;289-300). A
BamHI site is introduced just upstream of the Ncol site.
Oligonucleotide mutagenesis is performed to introduce BglII
and SaII sites directly adjacent to the stop codon of the crylA(c) gene, to facilitate removal of unwanted DNA 3' of the coding region. The remaining sequence includes the entire encoding region to the protoxin.
A synthetic cryIA(c) gene encoding the active toxin fragment is constructed by annealing and ligating 70 and 90 base oligonucleotides, in a method as described (Wosnick et al. (1987) Gene 60;115-127). The synthetic gene is designed to have tobacco RuBISCO small subunit codon usage, including a guanine and cytosine content of 55%, with an NcoI site at the start codon and a Sa2I site at the stop codon, while still encoding the amino acid sequence of the toxin. This synthetic gene is also truncated, however, so that the encoding region only provides the amino acid sequence to the active fragment of the protoxin.
A plastid transformation vector is used which carries a passenger gene in a Prrn(L)rbcL(S)/Trps16 expression cassette, with polylinker restriction sites. The Prrn(L)rbcL(S) fragments are described in Svab et al. (1993 supra). To further secure the stability of the mRNAs, the Trps16 fragment is cloned downstream of the passenger gene W O 95/24493 21 V51"9 PCTIUS95/02901 encodingregion. The Trps16 fragment comprises the rpsl6 gene 3'-regulatory region from,nucleotides 5,087 to 4,939 in the tobacco plasmid DNA. -Chimeric genes are preferably inserted into the vector to direct their transcription towards the rrn operon. Thus, in the plastid genome, chimeric genes are transcribed from the Prsn(L)rbcL(S) 5'-regulatory region comprising the long rrn operon promoter fragment from nucleotides 102,561 to 102,677 of the tobacco plastid genome, which is fused with a syntheticleader sequence designed after the rbcL gene leader between nucleotides 57,569 to 57,584 in the plastid DNA.
The plastid transformation vector also carries a selectable spectinomycin resistance gene (aadA) under control of psbA gene expression signals. The regulatory and encoding sequences are also flanked by plastid DNA homology regions whose limits are bp 138,447 (EcoRI) to 140,219 (Hincll) and 140,219 (HincII) to 141,382 (Bg1II) of the tobacco plastid genome (Shinozaki et al. (1986 supra)). This directs insertion of foreign genes located between the flanking regions into the plastid between the traV gene and the rpsl2/7 operon.
This plastid transformation vector is digested with the NcoI/SalI restriction endonucleases to remove the encoding region of the passenger gene, which is then replaced with a Ncol/Saii fragment containing the synthetic crylA(c) coding region, yielding a vector whichis designated pZS223 (Fig.
1). The wild type crylA(c) protoxin gene is similarly cloned as an NcoI/Sa1I fragment, yielding a plasmid designated pZS224. By this approach Bacillus thuringiensis DNA 3' of the protein coding region is omitted for both plasmids, pZS223 and pZS224.
The insertion of the respective cry genes from vectors pZS223 and pZS224 into the wild-type plastid genome (Nt-ptDNA) to yield transplastomes Nt-pZS223 and Nt-pZS224, respectively, is shown in Fig. 1. The abbreviations used in Fig. 1 are as follows: 16S, 16S rRNA gene; trnV, tznV gene;
aadA, spectinomycin resistance gene; crylA and cry73 are synthetic and iiative Bacillus thuringiensis d-endotoxin genes, respectively. The restriction endonuclease cleavage sites are designated as follows: B, BamHI; Bg, BgIII; H, HindII2; N, Ncol; RI, EcoRI, RV, EcoRV; S, Sa1I.
EXAMPLE 2. PLANT PLASTID TRANSFORMATION
Stable transformation of tobacco plastid genomes by particle bombardment is reported in Svab et.al. (1990 supra) and Svab et al. (1993 supra). The methods described therein may be employed to obtain plants transformed with the plastid expression constructs described herein. Such methods generally involve DNA bombardment of a target host explant, preferably an explant made from a tissue which is rich in metabolically active plastids, such as green plant tissues including leaves or cotyledons.
Tobacco seeds (N. tabacum v. Xanthi N/C) are surface sterilized in a 50% chlorol solution (2.5% sodium , hypochlorite) for 20 minutes and rinsed 4 times in sterile H20. These are plated asceptically on a 0.2x MS salts media and allowed to germinate. The seedlings are grown on agar solidified MS media with 30g/l sucrose (Murashige et al.
(1962) Physiol. Plant 15:493-497).
Tungsten microprojectiles (1.O M) are coated with plasmid DNA according to Maliga (Maliga, P. (1993) Methods in Plant Molecular Biology - A Laboratory Manual, eds. Pal Maliga, Daniel Klessig, Anthony Cashmore, Wilhelm Gruissem and Joseph Varner; Cold Spring Harbor Press) and used to bombard mature leaves, placed abaxial side up on RMOP media;
MS salts, 1 mg/1 BAP, 0.1 mg/1 NAA, 30 g/l sucrose and 0.7%
phytagar # Svab et al. (1990) Proc. Natl. Acad. Sci. USA
*Trademark WO 95/24493 PCTI[JS95102901 87:8526-8530 (using the Bio-Rad PDS 1000 He system (Sanford et al., An improved, helium-driven Biolistic device, Technique 3:3-16)). Plasmids pZS223 and pZS224 are used as the coating plasmid DNA.
The bombarded tissue is then cultured for approximately 2 days on a cell division-promoting media, after which the plant tissue is transferred to a selective media containing an inhibitory amount of the particular selective agent.
Transformed explants form green shoots in approximately 3-8 weeks. Leaves from these shoots are then subcultured on the same selective media to ensure production and selection of homoplasmic shoots.
EXAMPLE 3_ DNA GEL BLOT ANALYSIS OF TRANSPLASTOMIC LINES
Transformed plants selected for marker aadA marker gene expression are analyzed to determine whether the entire plastid content of the plant has been transformed (homoplastic transformants). Typically, following two rounds of shoot formation and spectinomycin selection, approximately 50% of the transgenic plantlets which are analyzed are homoplastic, as determined by Southern blot analysis of plastid DNA. Homoplasmic plantlets are selected for further cultivation.
Following a second round of shoot formation and spectinomycin selection, two transplastomic lines for each construct are obtained, Nt-pZS223 and Nt-pZS224. These lines are checked for homoplasmy. Southern blot analysis is used to confirm the integration of the chimeric cry genes in the tobacco plastid genome. Preparation, electrophoresis, and transfer of DNA to filters is as described (Svab et al., (1993 supra)).
The complete disappearance of the 3.3 Kb native tobacco BamHI fragment in the Nt-pZS223 and Nt-pZS224 transformants ~ W0951]A493 PCT/US95102901 with a probe covering the region of integration, and the appearance of expected sized bands for the inserted DNA
fragments in those transformants, 5.5 kb and 7.3 kb, respectively (see Fig. 1), establishes that the transformed plants are homoplasmic for the intended constructs. Probing identical filters with the aadA, crylA(c) protoxin, and synthetic czylA(c) genes demonstrated a linkage of the aadA
and cryIA(c) genes to the expected 5.5 and 7.3 Kb BamHI
fragments as well as the lack of these genes in the negative control.
EXAMPLE 4. INSECT BIOASSAYS
As described, the development of transformed plant lines Nt-pZS223 and Nt-pZS224 is accomplished on RMOP media supplemented with 500 mg/1 Spectinomycin dihydrochloride.
Plants are subcloned on the same selective medium, by the method according Svab et al.(1990 supra). Selected plants are then rooted in MS media containing 1 mg/1 IBA, 500 mg/1 Spectinomycin dihydrochloride and 0.6% phytagar.
Helicoverpa zea and Heliothis virescens eggs are obtained from the USDA-ARS in Stoneville, MS. and allowed to hatch. Neonate larva are placed on Tobacco Budworm Diet from Bioserve (Frenchtown, NJ), and incubated in a 16:8 photoperiod at 28'C_for=5 days. The larva develop during this time to late second or early third instar.
At 5 days, fully expanded leaves are excised from the tobacco plants and placed on 3 ml of 2% agar in a 32 well rearing tray from CD International (Pitman, NJ). The larva are placed 1 per well, sealed and incubated for 5 days at the same conditions. At day 10, the leaf material consumed by the insect is estimated and insects checked for mortality.
The larva are considered dead if they showed no movement = after prodding with forceps.
WO 95/24493 ~y1Q5'~ '~ 9 PCTIIIS95/02901 EXAMPLE S. INSEC~rTI VCIDAL FEEDING ACTIVITY
To determine the presence-and relative amount of active Bacillus thuringiensis d-toxin in the tobacco lines homoplasmic for native protoxin and synthetic 'truncated' cryIA(c) gene expression construcfts,'efficacy of these plants to third instar Heliothis virescens (tobacco budworm) and He.iicoverpa zea (corn earworm/cotton bollworm) larvae is =
tested (See table). Both test insects are sensitive to the crylA(c) toxin with H. zea being 10-fold more resistant than H. virescens (Maclntosh et al. (1990) J. invertebr. Pathol.
56:258-266.).
Third instar larvae are chosen for the bioassay since the insects are more resistant to the toxin at this stage than are first instar larvae thus allowing a more stringent comparison between the control and test plants. Tobacco-lines designated 4083 and 4084, derived by nuclear -transformation with the same synthetic cryIA(c) gene as used in pZS223 and shown to be highly toxic to third instar H.
virescens larvae, are used as positive controls in the bioassay. Nicotiana tabacum var. 'Petite Havana' serves as the negative control since this-is the genetic background used to generate the transplastomic lines.
Table 1 is a summary of Bacillus thuringiensis tobacco insect feeding assays. The data demonstrates that _ transplastomic line Nt-pZS224 is very toxic to both H.
virescens and H. zea as it causes 100% mortality to these insects while sustaining less than 2% total leafdamage.
This result compares favorably to the results for positive control 4083 and 4084 tobacco plants. The 4083-2-4 plant when assayed with H. zea causes 100% mortality but sustains a much greater level of leaf feeding damage than the Nt-pZS224 tobacco line indicating less toxin production. -Tobacco line 4084-4-1 performed comparably to Nt-pZS224 tobacco in _ feeding, although it does not compare to the levels of toxin produced in Nt-pZS224 when measured as a component of total leaf protein.
Tobacco line Nt-pZS223 shows no detectable bioactivity.
SIMNAARY OF ST TOBACCO INSECT FEEDING ASSAYS
piants Heiiothis %i.ea1 Hel'iocoverpa % Leai ChlorOplast Vector tested virescena~" Eaten Zea"~% Eaten synthetic loxin gane pZS223 223-3 NO mortaNty 100% NO mortatity 100%
223-5 NO morlaUty 75% NO nwrlalily 100%
223-12 NT' NO mortality 100 /.
223-13 NO mortaqly 75% NT' wiid lype protoxin gene pZS224 224-5 100% mortality 2% 100% mortality 2%
224-9 100% mortality 2% 100% mortality 2%
Nuclear Controis synthetic toxin gene pCGN4083 4083-1-2 100% mortality 2% NT' 4083-2-4 W. 100 .4 mortality 40%
pCGN4084 4084-8-5 100% mortality 2% NT=
4084-1-1 NT= 100% mortality 29'.
Untransiormed Controls control 1 25% mortality 75% NO mortality 100%
control 2 NO monatlty 100% NT=
control 3 50% mortality 75%. NT=
M 10 third instar larva were individuaNy tested per plant 'NT: Plant not tesled All publications and patent applications meutioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.
35 F1lthougti the foregoing invention has been described in sonie detail by way of illustration and example for purposes of-c'sarity of understanding, it will be obvious ttiat certain changes and modifications may be practiced within ltie scope of the appended claim.
Claims (12)
1. A construct comprising the following as operably joined components in the 5' to 3' direction of transcription:
(a) a promoter functional in a plant plastid;
(b) a DNA sequence encoding a peptide of interest; and (c) a transcription termination region capable of terminating transcription in a plant plastid, wherein a native DNA sequence encoding said peptide of interest has a given adenine and thymine content and wherein said DNA encoding sequence in (b) encodes the same amino acid sequence as said native DNA sequence and has an enriched adenine and thymine content.
(a) a promoter functional in a plant plastid;
(b) a DNA sequence encoding a peptide of interest; and (c) a transcription termination region capable of terminating transcription in a plant plastid, wherein a native DNA sequence encoding said peptide of interest has a given adenine and thymine content and wherein said DNA encoding sequence in (b) encodes the same amino acid sequence as said native DNA sequence and has an enriched adenine and thymine content.
2. The construct of claim 1, wherein said construct further comprises (d) a gene encoding a marker for selection of plant cells expressing said marker, and (e) DNA regions of homology to the genome of said plant plastid, wherein said regions of homology in (e) flank at least one of components (a), (b), (c) and (d) of said construct.
3. The construct of claim 1 or 2, wherein said plant plastid is a chloroplast.
4. The construct of claim 1, 2, or 3, wherein said DNA encoding sequence comprises an adenine and thymine content of greater than 50%.
5. The construct of any one of claims 1 to 4, wherein said DNA encoding sequence encodes two or more peptides of interest.
6. A plant cell containing the construct of any one of claims 1 to 5.
7. A method for producing a plant cell expressing a peptide of interest, said method comprising introducing the construct of claim 1 into a plant cell and expressing said peptide in a plastid of said plant cell from the construct.
8. The method of claim 7, wherein said construct further comprises (d) a gene encoding a marker for selection of plant cells expressing said marker and (e) DNA regions of homology to the genome of said plastid, wherein said regions of homology in (e) flank at least one of components (a), (b), (c) and (d) of said construct.
9. The method of claim 7 or 8, wherein said plant plastid is a chloroplast.
10. The method of claim 7, 8, or 9, wherein said DNA
encoding sequence comprises an adenine and thymine content of greater than 50%.
encoding sequence comprises an adenine and thymine content of greater than 50%.
11. The method of any one of claims 7 to 10, wherein said DNA encoding sequence encodes two or more peptides of interest.
12. A plant cell produced by the method of any one of claims 7 to 11 or a progeny plant cell thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002565191A CA2565191A1 (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/209,649 | 1994-03-11 | ||
| US08/209,649 US5545817A (en) | 1994-03-11 | 1994-03-11 | Enhanced expression in a plant plastid |
| PCT/US1995/002901 WO1995024493A1 (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002565191A Division CA2565191A1 (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2185339A1 CA2185339A1 (en) | 1995-09-14 |
| CA2185339C true CA2185339C (en) | 2008-06-03 |
Family
ID=22779658
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002565191A Abandoned CA2565191A1 (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
| CA002185339A Expired - Lifetime CA2185339C (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002565191A Abandoned CA2565191A1 (en) | 1994-03-11 | 1995-03-10 | Enhanced expression in a plant plastid |
Country Status (9)
| Country | Link |
|---|---|
| US (2) | US5545817A (en) |
| EP (2) | EP0749491B1 (en) |
| JP (1) | JPH09510100A (en) |
| AT (2) | ATE310094T1 (en) |
| CA (2) | CA2565191A1 (en) |
| DE (2) | DE69534621T2 (en) |
| DK (1) | DK0749491T3 (en) |
| ES (1) | ES2252740T3 (en) |
| WO (1) | WO1995024493A1 (en) |
Families Citing this family (154)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5545818A (en) * | 1994-03-11 | 1996-08-13 | Calgene Inc. | Expression of Bacillus thuringiensis cry proteins in plant plastids |
| US5545817A (en) * | 1994-03-11 | 1996-08-13 | Calgene, Inc. | Enhanced expression in a plant plastid |
| US5767373A (en) | 1994-06-16 | 1998-06-16 | Novartis Finance Corporation | Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms |
| US6808904B2 (en) * | 1994-06-16 | 2004-10-26 | Syngenta Participations Ag | Herbicide-tolerant protox genes produced by DNA shuffling |
| US6084155A (en) * | 1995-06-06 | 2000-07-04 | Novartis Ag | Herbicide-tolerant protoporphyrinogen oxidase ("protox") genes |
| GB9517674D0 (en) * | 1995-08-30 | 1995-11-01 | Lynxvale Ltd | Plastid inner envelope membrane targeting polypeptides;manufacture and use thereof |
| US7285700B2 (en) * | 1996-03-06 | 2007-10-23 | Rutgers, The State University Of New Jersey | Plastid directed DNA constructs comprising chimeric plastid targeting regions, vectors containing same and methods of use thereof |
| AU732479B2 (en) * | 1996-03-06 | 2001-04-26 | Rutgers University | Plastid transformation in arabidopsis thaliana |
| US5994526A (en) | 1996-06-21 | 1999-11-30 | Plant Genetic Systems | Gene expression in plants |
| NZ502742A (en) * | 1997-08-07 | 2004-04-30 | Univ Auburn | Universal chloroplast integration and expression vectors, transformed plants and products thereof |
| US7741536B2 (en) * | 1997-08-07 | 2010-06-22 | University Of Central Florida Research Foundation, Inc. | Expression of human serum albumin in plastids |
| US6362398B1 (en) | 1998-03-11 | 2002-03-26 | Syngenta Participations Ag | ClpP plastid promoter sequence |
| AR020078A1 (en) | 1998-05-26 | 2002-04-10 | Syngenta Participations Ag | METHOD FOR CHANGING THE EXPRESSION OF AN OBJECTIVE GENE IN A PLANT CELL |
| US6271444B1 (en) * | 1998-07-10 | 2001-08-07 | Calgene Llc | Enhancer elements for increased translation in plant plastids |
| MXPA01000372A (en) * | 1998-07-10 | 2002-10-17 | Calgene Llc | Expression of herbicide tolerance genes in plant plastids. |
| US6492578B1 (en) | 1998-07-10 | 2002-12-10 | Calgene Llc | Expression of herbicide tolerance genes in plant plastids |
| US6498286B1 (en) | 1998-08-20 | 2002-12-24 | Ffr Cooperative | Germplasm containing an identifier nucleotide sequence and method for identifying germplasm |
| AR022383A1 (en) | 1998-09-18 | 2002-09-04 | Univ Kentucky Res Found | SYNTHESES |
| US6489542B1 (en) | 1998-11-04 | 2002-12-03 | Monsanto Technology Llc | Methods for transforming plants to express Cry2Ab δ-endotoxins targeted to the plastids |
| US6515206B1 (en) * | 1998-12-23 | 2003-02-04 | Calgene Llc | Plastid transformation of Brassica |
| IL128111A (en) | 1999-01-18 | 2007-03-08 | Yissum Res Dev Co | A method of silencing expression of a target sequence in a plant genome |
| US7439018B2 (en) * | 1999-01-29 | 2008-10-21 | Evolutionary Genomics, Inc. | EG1117 Polynucleotides and uses thereof |
| US7252966B2 (en) * | 1999-01-29 | 2007-08-07 | Evolutionary Genomics Llc | EG307 polynucleotides and uses thereof |
| US20080047032A1 (en) * | 1999-01-29 | 2008-02-21 | Evolutionary Genomics Llc | Eg307 nucleic acids and uses thereof |
| ATE446370T1 (en) | 1999-04-12 | 2009-11-15 | Monsanto Technology Llc | OIL CONTAINING BRASSICASTANOL |
| EP1194578B1 (en) * | 1999-07-10 | 2008-05-28 | Calgene LLC | Enhanced expression of proteins using gfp |
| AU3008601A (en) * | 1999-12-08 | 2001-06-18 | International Centre For Genetic Engineering And Biotechnology | Plastid transformation |
| US20030041353A1 (en) * | 2001-04-18 | 2003-02-27 | Henry Daniell | Mutiple gene expression for engineering novel pathways and hyperexpression of foreign proteins in plants |
| WO2004005521A1 (en) * | 2002-07-03 | 2004-01-15 | University Of Central Florida | Expression of the human igf-1 in transgenic plastids |
| AU2001281464A1 (en) | 2000-03-13 | 2001-09-24 | Monsanto Technology Llc | Recombinant proteins containing repeating units |
| US7226787B2 (en) | 2000-03-22 | 2007-06-05 | Icon Genetics, Inc. | Methods for transforming plant plastids and making transplastomic plants |
| GB0018876D0 (en) * | 2000-08-01 | 2000-09-20 | Applied Research Systems | Method of producing polypeptides |
| JP2004506427A (en) | 2000-08-11 | 2004-03-04 | シンジェンタ パーティシペーションズ アクチェンゲゼルシャフト | Method for stable transformation of plants |
| US20030167524A1 (en) * | 2000-12-19 | 2003-09-04 | Rooijen Gijs Van | Methods for the production of multimeric protein complexes, and related compositions |
| AR035799A1 (en) | 2001-03-30 | 2004-07-14 | Syngenta Participations Ag | INSECTICIDE TOXINS ISOLATED FROM BACILLUS THURINGIENSIS AND ITS USES. |
| EP1925672A1 (en) | 2001-06-22 | 2008-05-28 | Syngeta Participations AG | Abiotic stress responsive polynucleotides and polypeptides |
| EP1947201A3 (en) | 2002-01-16 | 2009-05-06 | Evolutionary Genomics, LLC | Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals |
| AU2008202053B2 (en) * | 2002-04-23 | 2012-11-01 | The Scripps Research Institute | Expression of polypeptides in chloroplasts, and compositions and methods for expressing same |
| CA2483337C (en) * | 2002-04-23 | 2015-10-27 | The Scripps Research Institute | Expression of polypeptides in chloroplasts, and compositions and methods for expressing same |
| EP1521770B1 (en) * | 2002-06-14 | 2008-11-26 | Monier Tadros | Method for the production of protamine |
| US7667099B2 (en) * | 2002-06-20 | 2010-02-23 | Board Of Trustees Of Michigan State University | Plastid division and related genes and proteins, and methods of use |
| AU2003298545A1 (en) * | 2002-08-08 | 2004-04-23 | Evolutionary Genomics, Llc | Detection of evolutionary bottlenecking by dna sequencing as a method to discover genes of value |
| WO2004052085A1 (en) | 2002-12-06 | 2004-06-24 | Del Monte Fresh Produce Company | Transgenic pineapple plants with modified carotenoid levels and methods of their production |
| EP1576178A4 (en) | 2002-12-26 | 2008-03-05 | Syngenta Participations Ag | Cell proliferation-related polypeptides and uses therefor |
| US7655833B2 (en) * | 2003-05-29 | 2010-02-02 | Brookhaven Science Associates, Llc | ADS genes for reducing saturated fatty acid levels in seed oils |
| US20050044593A1 (en) * | 2003-07-01 | 2005-02-24 | Biolex, Inc. | Chloroplast transformation of duckweed |
| ATE495264T1 (en) | 2003-10-06 | 2011-01-15 | Syngenta Participations Ag | PROMOTOR FUNCTIONAL IN PLANT PLASTIDS |
| US20070122864A1 (en) * | 2003-11-05 | 2007-05-31 | The Regents Of The University Of California | Methods for the determination of protein three-dimensional structure employing hydrogen exchange analysis to refine computational structure prediction |
| US7363171B2 (en) | 2003-11-26 | 2008-04-22 | The Regents Of The University Of California | Enhanced methods for crystallographic structure determination employing hydrogen exchange analysis |
| US20070169227A1 (en) | 2003-12-16 | 2007-07-19 | Pioneer Hi-Bred International Inc. | Dominant Gene Suppression Transgenes and Methods of Using Same |
| EP1696721B1 (en) | 2003-12-16 | 2010-02-17 | Pioneer Hi-Bred International, Inc. | Dominant gene suppression transgenes and methods of using same |
| EP2166097B1 (en) | 2004-10-05 | 2014-08-06 | SunGene GmbH | Constitutive expression cassettes for regulation of plant expression |
| EP1655364A3 (en) | 2004-11-05 | 2006-08-02 | BASF Plant Science GmbH | Expression cassettes for seed-preferential expression in plants |
| EP2163631B1 (en) | 2004-11-25 | 2013-05-22 | SunGene GmbH | Expression cassettes for guard cell-preferential expression in plants |
| EP1666599A3 (en) | 2004-12-04 | 2006-07-12 | SunGene GmbH | Expression cassettes for mesophyll- and/or epidermis-preferential expression in plants |
| EP1669455B1 (en) | 2004-12-08 | 2009-10-28 | SunGene GmbH | Expression cassettes for vascular tissue-preferential expression in plants |
| EP1669456A3 (en) | 2004-12-11 | 2006-07-12 | SunGene GmbH | Expression cassettes for meristem-preferential expression in plants |
| CN100362104C (en) * | 2004-12-21 | 2008-01-16 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
| EP1856264B1 (en) | 2005-02-26 | 2014-07-23 | BASF Plant Science GmbH | Expression cassettes for seed-preferential expression in plants |
| AU2006230352A1 (en) * | 2005-03-29 | 2006-10-05 | Evolutionary Genomics Llc | EG1117 and EG307 polynucleotides and uses thereof |
| EP2444496A1 (en) | 2005-04-15 | 2012-04-25 | Del Monte Fresh Produce Company | Plant promoters, terminators, genes, vectors and related transformed plants |
| CA2604807C (en) | 2005-04-19 | 2018-06-12 | Basf Plant Science Gmbh | Improved methods controlling gene expression |
| US20070037230A1 (en) * | 2005-04-29 | 2007-02-15 | Petrenko Valery A | Peptides for selectively and specifically binding Bacillus anthracis spores and use thereof in landscape phages |
| WO2006120197A2 (en) | 2005-05-10 | 2006-11-16 | Basf Plant Science Gmbh | Expression cassettes for seed-preferential expression in plants |
| CA2608717A1 (en) * | 2005-05-18 | 2006-11-23 | The Board Of Trustees Operating Michigan State University | Resistance to soybean aphid in early maturing soybean germplasm |
| AP3827A (en) | 2005-07-05 | 2016-09-30 | Univ California | Polynucleotides encoding isoprenoid modifying enzymes and methods of use thereof |
| US7671254B2 (en) * | 2005-08-25 | 2010-03-02 | The Board Of Trustees Of The University Of Illinois | Herbicide resistance gene, compositions and methods |
| US7842856B2 (en) | 2005-08-25 | 2010-11-30 | The Board Of Trustees Of The University Of Illinois | Herbicide resistance gene, compositions and methods |
| US20080256659A1 (en) * | 2005-09-02 | 2008-10-16 | Evolutionary Genomics, Inc. | Eg8798 and Eg9703 Polynucleotides and Uses Thereof |
| EP1931789B1 (en) | 2005-09-20 | 2016-05-04 | BASF Plant Science GmbH | Methods for controlling gene expression using ta-siran |
| US20090012029A1 (en) * | 2006-01-06 | 2009-01-08 | Hussey Richard S | Cyst Nematode Resistant Transgenic Plants |
| WO2007133558A2 (en) * | 2006-05-09 | 2007-11-22 | The Scripps Research Institute | Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast |
| EP2021476B1 (en) | 2006-05-26 | 2014-07-09 | Monsanto Technology, LLC | Corn plant and seed corresponding to transgenic event mon89034 and methods for detection and use thereof |
| US7847152B2 (en) * | 2006-06-30 | 2010-12-07 | The Board Of Trustees Of The University Of Illinois | Use of tryptophan indole and anthranilate analogs as plant transformation selection agents |
| US7977535B2 (en) | 2006-07-12 | 2011-07-12 | Board Of Trustees Of Michigan State University | DNA encoding ring zinc-finger protein and the use of the DNA in vectors and bacteria and in plants |
| US20090061492A1 (en) * | 2006-11-15 | 2009-03-05 | The Board Of Trustees For Michigan State University System | Method for producing biodiesel |
| US7745698B2 (en) * | 2006-11-16 | 2010-06-29 | Wisconsin Alumni Research Foundation | Peptide extension for enhancement of transgene expression in chloroplasts |
| PT2102349T (en) | 2006-12-07 | 2017-10-26 | Univ Kansas State | Acetolactate synthase herbicide resistant sorghum |
| US20100115663A1 (en) | 2007-01-12 | 2010-05-06 | Tuinstra Mitchell R | Acetyl-CoA Carboxylase Herbicide Resistant Sorghum |
| ATE554174T1 (en) | 2007-02-06 | 2012-05-15 | Basf Plant Science Gmbh | USE OF ALANINE RACEMASE GENES TO PROVIDE NEMATODE RESISTANCE TO PLANTS |
| BRPI0808389A2 (en) | 2007-03-15 | 2014-07-08 | Basf Plant Science Gmbh | TRANSGENIC PLANT, SEED, EXPRESSION VECTOR, AND METHOD FOR INCREASING NEMATODE RESISTANCE ON A PLANT. |
| NZ581473A (en) | 2007-06-01 | 2012-02-24 | Scripps Research Inst | Use of genetically modified organisms to generate biomass degrading enzymes |
| CN101889068A (en) * | 2007-09-11 | 2010-11-17 | 蓝宝石能源公司 | Methods of producing organic products with photosynthetic organisms and products and compositions thereof |
| NZ598302A (en) | 2007-09-11 | 2013-08-30 | Sapphire Energy Inc | Molecule production by photosynthetic organisms |
| US20100050301A1 (en) * | 2007-10-05 | 2010-02-25 | Sapphire Energy, Inc. | System for capturing and modifying large pieces of genomic dna and constructing vascular plants with synthetic chloroplast genomes |
| US20090269816A1 (en) * | 2007-10-05 | 2009-10-29 | Sapphire Energy, Inc. | System for capturing and modifying large pieces of genomic DNA and constructing organisms with synthetic chloroplasts |
| US8314222B2 (en) * | 2007-10-05 | 2012-11-20 | Sapphire Energy, Inc. | System for capturing and modifying large pieces of genomic DNA and constructing organisms with chloroplasts |
| CA2705542A1 (en) * | 2007-11-13 | 2009-05-22 | Sapphire Energy, Inc. | Production of fc-fusion polypeptides in eukaryotic algae |
| US20090172832A1 (en) * | 2007-12-28 | 2009-07-02 | Timothy Caspar | Expression of rubisco enzyme from a non-rubisco locus |
| US8487159B2 (en) * | 2008-04-28 | 2013-07-16 | Metabolix, Inc. | Production of polyhydroxybutyrate in switchgrass |
| WO2009158658A2 (en) | 2008-06-27 | 2009-12-30 | Sapphire Energy, Inc. | Induction of flocculation in photosynthetic organisms |
| US8344209B2 (en) | 2008-07-14 | 2013-01-01 | Syngenta Participations Ag | Plant regulatory sequences |
| DE112009002061T5 (en) | 2008-08-27 | 2011-07-14 | BASF Plant Science GmbH, 67063 | Nematode resistant transgenic plants |
| US9133475B2 (en) | 2008-11-26 | 2015-09-15 | Board Of Trustees Of Michigan State University | Aphid resistant soybean plants |
| WO2010066600A1 (en) | 2008-12-11 | 2010-06-17 | Basf Plant Science Gmbh | Plant root-specific nematode resistance |
| US8362318B2 (en) | 2008-12-18 | 2013-01-29 | Board Of Trustees Of Michigan State University | Enzyme directed oil biosynthesis in microalgae |
| CN101768213B (en) | 2008-12-30 | 2012-05-30 | 中国科学院遗传与发育生物学研究所 | Protein related to plant tillering number and coding gene and application thereof |
| AR074941A1 (en) | 2009-01-07 | 2011-02-23 | Bayer Cropscience Sa | TRANSPLASTOMIC PLANTS EXEMPTED FROM SELECTOR MARKER |
| US8373025B2 (en) | 2009-02-09 | 2013-02-12 | Chromatin Germplasm, Llc | Herbicide resistant sorghum |
| CN101817879A (en) | 2009-02-26 | 2010-09-01 | 中国科学院遗传与发育生物学研究所 | Metallothionein and encoding gene and application thereof |
| CA2754261A1 (en) * | 2009-03-05 | 2010-09-10 | Metabolix, Inc. | Stable, fertile, high polyhydroxyalkanoate producing plants and methods of producing them |
| AU2010221135A1 (en) | 2009-03-05 | 2011-09-29 | Metabolix, Inc. | Propagation of transgenic plants |
| WO2010102293A1 (en) | 2009-03-06 | 2010-09-10 | Metabolix, Inc. | Method of positive plant selection using sorbitol dehydrogenase |
| AU2010270309A1 (en) | 2009-07-10 | 2012-02-02 | Basf Plant Science Company Gmbh | Expression cassettes for endosperm-specific expression in plants |
| US20120151629A1 (en) | 2009-08-25 | 2012-06-14 | Basf Plant Science Company Gmbh | Nematode-Resistant Transgenic Plants |
| UA110925C2 (en) | 2009-10-02 | 2016-03-10 | Сінгента Партісіпейшнс Аг | CRY1BA STRUCTURED PROTEIN ACTIVE TO FISH INSULTS |
| US20110154541A1 (en) * | 2009-11-24 | 2011-06-23 | Technion Research And Development Foundation Ltd. | Bio-engineered photosystems |
| CA2782290A1 (en) | 2009-12-03 | 2011-06-09 | Basf Plant Science Company Gmbh | Expression cassettes for embryo-specific expression in plants |
| US20130055471A1 (en) | 2009-12-15 | 2013-02-28 | Edwin Henricus Antonius HOLMAN | Transgenic Ozone-Resistant Plants |
| EP2336310A1 (en) | 2009-12-16 | 2011-06-22 | Isobionics B.V. | Valencene synthase |
| WO2011082253A2 (en) | 2009-12-30 | 2011-07-07 | Board Of Trustees Of Michigan State University | A method to produce acetyldiacylglycerols (ac-tags) by expression ofan acetyltransferase gene isolated from euonymus alatus (burning bush) |
| BR112013005973A2 (en) | 2010-09-15 | 2019-09-24 | Metabolix Inc | increased carbon flow for polyhydroxybutyrate production in biomass crops |
| DE112011104462T5 (en) | 2010-12-20 | 2013-09-12 | Basf Plant Science Company Gmbh | Nematode resistant transgenic plants |
| US8846370B2 (en) | 2010-12-23 | 2014-09-30 | Exxonmobil Research And Engineering Company | Genetically engineered microorganisms comprising 4-hydroxybenzoyl-coa thioesterases and methods of using the same for producing free fatty acids and fatty acid derivatives |
| EP2655615A4 (en) | 2010-12-23 | 2014-04-23 | Exxonmobil Res & Eng Co | Prokaryotic acyl-acp thioesterases for producing fatty acids in genetically engineered microorganisms |
| EP2537926A1 (en) | 2011-06-21 | 2012-12-26 | Isobionics B.V. | Valencene synthase |
| WO2013005211A2 (en) | 2011-07-05 | 2013-01-10 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Boron complexing plant materials and uses thereof cross-reference to related applications |
| WO2013006861A1 (en) | 2011-07-07 | 2013-01-10 | University Of Georgia Research Foundation, Inc. | Sorghum grain shattering gene and uses thereof in altering seed dispersal |
| WO2013015993A1 (en) | 2011-07-28 | 2013-01-31 | Syngenta Participations Ag | Methods and compositions for controlling nematode pests |
| WO2013050318A1 (en) | 2011-10-07 | 2013-04-11 | Basf Plant Science Company Gmbh | Method of producing plants having increased resistance to pathogens |
| WO2013050611A1 (en) | 2011-10-07 | 2013-04-11 | Basf Plant Science Company Gmbh | Method of producing plants having increased resistance to pathogens |
| WO2013050593A1 (en) | 2011-10-07 | 2013-04-11 | Basf Plant Science Company Gmbh | Method of producing plants having increased resistance to pathogens |
| WO2013053686A1 (en) | 2011-10-10 | 2013-04-18 | Basf Plant Science Company Gmbh | Method of producing plants having increased resistance to pathogens |
| WO2013053711A1 (en) | 2011-10-10 | 2013-04-18 | Basf Plant Science Company Gmbh | Method of producing plants having increased resistance to pathogens |
| MX356581B (en) | 2012-02-16 | 2018-06-05 | Syngenta Participations Ag | Engineered pesticidal proteins. |
| SI3241902T1 (en) | 2012-05-25 | 2018-07-31 | The Regents Of The University Of California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
| WO2013184768A1 (en) | 2012-06-05 | 2013-12-12 | University Of Georgia Research Foundation, Inc. | Compositions and methods of gene silencing in plants |
| MX349486B (en) | 2012-06-22 | 2017-08-01 | Syngenta Participations Ag | Biological control of coleopteran pests. |
| CA2881787A1 (en) | 2012-08-13 | 2014-02-20 | University Of Georgia Research Foundation, Inc. | Compositions and methods for increasing pest resistance in plants |
| US20140123339A1 (en) | 2012-10-31 | 2014-05-01 | Pioneer Hi Bred International Inc | Transformed Plants Having Increased Beta-Carotene Levels, Increased Half-Life and Bioavailability and Methods of Producing Such |
| CA2836403A1 (en) | 2013-01-04 | 2014-07-04 | Board Of Trustees Of Michigan State University | New sources of aphid resistance in soybean plants |
| EP3071695A2 (en) | 2013-11-18 | 2016-09-28 | Crispr Therapeutics AG | Crispr-cas system materials and methods |
| US10392629B2 (en) | 2014-01-17 | 2019-08-27 | Board Of Trustees Of Michigan State University | Increased caloric and nutritional content of plant biomass |
| EP3191594A4 (en) | 2014-09-11 | 2018-06-13 | Marrone Bio Innovations, Inc. | Chromobacterium subtsugae genome |
| CN106998697B (en) | 2014-12-12 | 2021-09-03 | 先正达参股股份有限公司 | Compositions and methods for controlling plant pests |
| BR112017013528A2 (en) | 2014-12-23 | 2018-03-06 | Syngenta Participations Ag | biological control of beetle pests |
| US10392607B2 (en) | 2015-06-03 | 2019-08-27 | The Regents Of The University Of California | Cas9 variants and methods of use thereof |
| AU2016339053A1 (en) | 2015-09-24 | 2018-04-12 | Crispr Therapeutics Ag | Novel family of RNA-programmable endonucleases and their uses in genome editing and other applications |
| AR109205A1 (en) | 2016-08-05 | 2018-11-07 | Syngenta Participations Ag | PATHOPE CONTROL OF COLEOPTERS USING RNA MOLECULES |
| AR109206A1 (en) | 2016-08-05 | 2018-11-07 | Syngenta Participations Ag | PATHOPE CONTROL OF COLEOPTERS USING RNA MOLECULES |
| NL2018457B1 (en) | 2017-03-02 | 2018-09-21 | Isobionics B V | Santalene Synthase |
| CA3061718A1 (en) | 2017-04-27 | 2018-11-01 | Regents Of The University Of California | Microorganisms and methods for producing cannabinoids and cannabinoid derivatives |
| NL2019473B1 (en) | 2017-09-01 | 2019-03-11 | Isobionics B V | Terpene Synthase producing patchoulol and elemol, and preferably also pogostol |
| AR113761A1 (en) | 2017-10-18 | 2020-06-10 | Syngenta Participations Ag | PEST CONTROL OF HEMIPTERS USING RNA MOLECULES |
| CA3084825A1 (en) | 2017-12-14 | 2019-06-20 | Crispr Therapeutics Ag | Novel rna-programmable endonuclease systems and their use in genome editing and other applications |
| US20200407719A1 (en) | 2018-02-26 | 2020-12-31 | Devgen Nv | Control of insect pests using rna molecules |
| EP3768834A1 (en) | 2018-03-19 | 2021-01-27 | CRISPR Therapeutics AG | Novel rna-programmable endonuclease systems and uses thereof |
| WO2019206780A1 (en) | 2018-04-27 | 2019-10-31 | Devgen Nv | Control of insect pests using rna molecules |
| WO2020181264A1 (en) | 2019-03-07 | 2020-09-10 | The Trustees Of Columbia University In The City Of New York | Rna-guided dna integration using tn7-like transposons |
| GB2595606B (en) | 2019-03-07 | 2022-09-21 | Univ California | CRISPR-Cas effector polypeptides and methods of use thereof |
| CA3129553A1 (en) | 2019-03-21 | 2020-09-24 | Lien DE SCHRIJVER | Control of insect pests using rna molecules |
| WO2020249811A1 (en) | 2019-06-14 | 2020-12-17 | Bioinsectis S.L. | Bacillus thuringiensis strain |
| CA3212047A1 (en) | 2021-04-15 | 2022-10-20 | Jeroen Stuurman | Co-regeneration recalcitrant plants |
| WO2024023578A1 (en) | 2022-07-28 | 2024-02-01 | Institut Pasteur | Hsc70-4 in host-induced and spray-induced gene silencing |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU638438B2 (en) * | 1989-02-24 | 1993-07-01 | Monsanto Technology Llc | Synthetic plant genes and method for preparation |
| US5409823A (en) * | 1992-09-24 | 1995-04-25 | Ciba-Geigy Corporation | Methods for the production of hybrid seed |
| US5545817A (en) * | 1994-03-11 | 1996-08-13 | Calgene, Inc. | Enhanced expression in a plant plastid |
-
1994
- 1994-03-11 US US08/209,649 patent/US5545817A/en not_active Expired - Lifetime
-
1995
- 1995-03-10 EP EP95913608A patent/EP0749491B1/en not_active Revoked
- 1995-03-10 EP EP05110350A patent/EP1657308B1/en not_active Expired - Lifetime
- 1995-03-10 AT AT95913608T patent/ATE310094T1/en not_active IP Right Cessation
- 1995-03-10 WO PCT/US1995/002901 patent/WO1995024493A1/en active IP Right Grant
- 1995-03-10 JP JP7523624A patent/JPH09510100A/en active Pending
- 1995-03-10 AT AT05110350T patent/ATE471987T1/en not_active IP Right Cessation
- 1995-03-10 CA CA002565191A patent/CA2565191A1/en not_active Abandoned
- 1995-03-10 DE DE69534621T patent/DE69534621T2/en not_active Revoked
- 1995-03-10 DK DK95913608T patent/DK0749491T3/en active
- 1995-03-10 CA CA002185339A patent/CA2185339C/en not_active Expired - Lifetime
- 1995-03-10 ES ES95913608T patent/ES2252740T3/en not_active Expired - Lifetime
- 1995-03-10 DE DE69536087T patent/DE69536087D1/en not_active Expired - Lifetime
-
1996
- 1996-01-29 US US08/593,205 patent/US5866421A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP1657308A1 (en) | 2006-05-17 |
| DE69534621D1 (en) | 2005-12-22 |
| DE69536087D1 (en) | 2010-08-05 |
| JPH09510100A (en) | 1997-10-14 |
| ATE310094T1 (en) | 2005-12-15 |
| DK0749491T3 (en) | 2005-12-12 |
| US5545817A (en) | 1996-08-13 |
| EP1657308B1 (en) | 2010-06-23 |
| DE69534621T2 (en) | 2006-08-10 |
| EP0749491B1 (en) | 2005-11-16 |
| CA2185339A1 (en) | 1995-09-14 |
| ES2252740T3 (en) | 2006-05-16 |
| EP0749491A1 (en) | 1996-12-27 |
| CA2565191A1 (en) | 1995-09-14 |
| US5866421A (en) | 1999-02-02 |
| WO1995024493A1 (en) | 1995-09-14 |
| ATE471987T1 (en) | 2010-07-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2185339C (en) | Enhanced expression in a plant plastid | |
| US5545818A (en) | Expression of Bacillus thuringiensis cry proteins in plant plastids | |
| CA2299609C (en) | Universal chloroplast integration and expression vectors, transformed plants and products thereof | |
| US7129391B1 (en) | Universal chloroplast integration and expression vectors, transformed plants and products thereof | |
| EP0142924A2 (en) | Insect resistant plants | |
| US20190390209A1 (en) | Fertile transplastomic leguminous plants | |
| US6891086B2 (en) | Plastid transformation of Brassica | |
| US6541682B1 (en) | Plastid transformation of solanaceous plants | |
| JP5414136B2 (en) | Translational regulatory elements for the expression of high levels of proteins in plastids of higher plants and uses thereof | |
| CA2185340C (en) | Expression of bacillus thuringiensis cry proteins in plant plastids | |
| EP1571220B1 (en) | Universal chloroplast integration and expression vectors, transformed plants and products thereof | |
| Jing-Yu et al. | Chloroplast genetic engineering in higher plants |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20150310 |