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WO1995008633A1 - Manipulation genetique de plante - Google Patents

Manipulation genetique de plante Download PDF

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Publication number
WO1995008633A1
WO1995008633A1 PCT/GB1994/002058 GB9402058W WO9508633A1 WO 1995008633 A1 WO1995008633 A1 WO 1995008633A1 GB 9402058 W GB9402058 W GB 9402058W WO 9508633 A1 WO9508633 A1 WO 9508633A1
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WIPO (PCT)
Prior art keywords
protein
dna
plant
sequence
fusion protein
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Application number
PCT/GB1994/002058
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English (en)
Inventor
Philip Mark Mullineaux
Gary Patrick Creissen
Original Assignee
John Innes Centre Innovations Limited
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by John Innes Centre Innovations Limited filed Critical John Innes Centre Innovations Limited
Priority to AU76620/94A priority Critical patent/AU7662094A/en
Publication of WO1995008633A1 publication Critical patent/WO1995008633A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • 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
    • C12N15/8221—Transit peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01007—Glutathione-disulfide reductase (1.8.1.7), i.e. glutathione reductase (NADPH)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • This invention relates to plant molecular biology.
  • it relates to the targeting of plant organelles by chimeric preproteins.
  • N-terminal pre-sequences of certain plant proteins are known to be able to target the protein either to the chloroplast or to the mitochondrion, with varying degrees of efficiency.
  • These pre-sequences which are known as 'transit peptides' or 'targeting sequences' , are necessary for nuclear-encoded chloroplastidic or mitochondrial proteins and are generally cleaved from the mature protein during or after translocation into the organelle.
  • Examples of transit peptides known to be chloroplast-specific include N-terminal pre-sequences derived from:
  • Transit peptides known to be mitochondrion- specific include N-terminal pre-sequences derived from: the j ⁇ -subunit of mitochondrial ATP synthase (Boutry et al . , Nature 328 340-342 (1987)) ; and
  • the present invention is based on the remarkable discovery that one of the transit peptides which was thought to be specific for the chloroplast does in fact meet this need.
  • the transit peptide in question is the glutathione reductase pre- sequence. Neither the Creissen et al . paper supra nor, it is believed, the rest of the literature actually discloses fusions between the glutathione reductase pre- sequence and a heterologous protein.
  • a fusion protein comprising a protein of interest and sufficient of the N-terminal pre-sequence of a glutathione reductase to cause targeting of the protein to both chloroplasts and mitochondria.
  • the glutathione reductase (GR) which supplies the pre- sequence may be derived from any suitable organism. All that is required is that the organism in question be such that its GR has the ability to co-target both chloroplasts and mitochondria in plants.
  • the GR will usually be derived from a plant, particularly a higher plant such as those of the class Gymnospermae or, preferably, Angiospermae . Angiosperms of the family Leguminosae are preferred, particularly species of the genus Pisum.
  • a highly suitable source of GR is the pea (Pisum sativum L.) .
  • the complete pre-GR sequence is set out in Creissen et ,al . supra and the transit peptide includes at least some of the following residues :
  • the natural transit peptide comprises about 60 to 70 residues, all of which may be present in embodiments of the present invention.
  • residues FAV may also be present to the C-terminal side of that sequence; and the further residues RAESQNGADPARQ may be further added to the C-terminal side.
  • transit peptides in the invention which are identical to natural GR transit peptides (particularly that of P. sativum)
  • a degree of divergence from the natural or consensus sequence can be tolerated in the invention provided only that the co- targeting ability of the transit peptide is not lost.
  • a mutant, variant or derivative transit peptide useful in the invention will be homologous with the natural sequence to the extent of about 60% or even 90% or 95%.
  • the invention is not limited by the protein of interest which may be targeted to both chloroplasts and mitochondria by means of the invention.
  • Some proteins of interest will be enzymes (even mature glutathione reductase) , but whatever their nature their presence will simply be dictated by the particular purpose of the embodiment of the invention in question.
  • enzymes even mature glutathione reductase
  • coli gamma glutanyl cysteine synthetase (EMBL accession code: ECGSHI) for increased scavenging of superoxide leading to enhanced tolerance to environmental stress (see, for example, Bowler et al . , The EMBO Journal 8(1) 31-38
  • Fusion proteins of the invention may in principle be made by any convenient process, including de novo chemical synthesis.
  • recombinant DNA technology provides the method of choice, and the fusion proteins will be expressed from a recombinant DNA molecule.
  • a recombinant DNA molecule encoding a fusion of a protein of interest: with sufficient of the N-terminal pre-sequence of a glutathione reductase to cause targeting of the protein to both chloroplasts and mitochondria.
  • Recombinant or isolated DNA molecules encoding the transit peptide alone, in the absence of the mature GR protein-coding sequence, are useful for ligation to DNA sequences encoding proteins of interest.
  • a recombinant or isolated DNA molecule encoding sufficient of the N-terminal pre-sequence of a ⁇ lutathione reductase to cause targeting of a protein to both chloroplasts and mitochondria provided that in the said isolated or recombinant DNA molecule the DNA encoding the pre-sequence is not precisely fused to DNA encoding mature glutathione reductase.
  • DNA molecules in accordance with the invention may, if encoding a natural GR transit peptide, correspond to a CDNA or genomic sequence; in other words the presence or absence of any natural introns is not critical to the functioning of the invention, although it may be expected that the presence of one or more natural introns can have implications for expression efficiency.
  • Recombinant DNA in accordance with the invention may be in the form of a vector.
  • the vector may for example be a plasmid, cosmid or phage.
  • Vectors will frequently include one or more selectable markers to enable selection of cells transfected (or transformed: the terms are used interchangeably in this specification) with them and, preferably, to enable selection of cells harbouring vectors incorporating heterologous DNA. Appropriate start and stop signals will generally be present. Additionally, if the vector is intended for expression, sufficient regulatory sequences to drive expression will be present; however, DNA in accordance with the invention will generally be expressed in cells containing both chloroplasts and mitochondria, e.g. plant cells and algae. Vectors not including microbial regulatory sequences are useful as cloning vectors.
  • a plant promoter will generally be present operably coupled to sequences to be expressed; any suitable promoter may be used, such as, for example, the 35S Cauliflower Mosaic Virus (CaMV) promoter, the rubisco small subunit (rbs c) , a ubiquitin, the plastocyanin or the Agrobacterium nopaline synthase (nos) promoter.
  • CaMV 35S Cauliflower Mosaic Virus
  • rbs c rubisco small subunit
  • a ubiquitin the plastocyanin
  • plastocyanin the Agrobacterium nopaline synthase
  • Cloning vectors can be introduced into E. coli or another suitable host which facilitate their manipulation. According to a fourth aspect of the invention, there is therefore provided a host cell transfected or transformed with DNA as described above.
  • DNA in accordance with the invention can be prepared by any convenient method involving coupling together successive nucleotides, and/or ligating oligo- and/or poly-nucleotides, including in vi tro processes, but again recombinant DNA technology forms the method of choice.
  • DNA in accordance with the invention will be introduced into plant cells, by any suitable means.
  • a plant cell including DNA in accordance with the invention as described above.
  • Plants transformed with the DNA segment containing the pre-sequence may be produced by standard techniques which are already known for the genetic manipulation of plants.
  • DNA can be transformed into plant cells using any suitable technology, such as a disarmed Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transfer ability (EP-A-270355, EP-A-0116718, Bevan, Nucleic Acids Research, 12(22) : 8711-8721 (1984)) , particle or microprojectile bombardment (US-A-5100792, EP-A-444882, EP-A-434616) , microinjection (WO 92/09696,
  • Agrobac terium transformation is widely used by those skilled in the art to transform dicotyledonous species. Although Agrobacterium has been reported to be able to transform foreign DNA into some monocotyledonous species (WO 92/
  • microprojectile bombardments are preferred where Agrobacterium is inefficient or ineffective.
  • electroporation is preferred where Agrobacterium is inefficient or ineffective.
  • a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-
  • transformation technology will be determined by its efficiency to transform certain plant species, as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce chimeric genes into the plant cells or algae is not essential to the invention.
  • the foreign DNA could be introduced directly into plant cells using a particle bombardment apparatus. This method is preferred where Agrobacterium is ineffective, for example where the recipient plant is monocotyledonous . Any other method that provides for the stable incorporation of the DNA within the nuclear DNA of any plant cell of any species would also be suitable.
  • DNA in accordance with some embodiments of the invention may also contain a second chimeric gene (a "marker" gene) that enables a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA.
  • a marker gene examples include antibiotic resistance (Herrera-Estrella et al , The E * MBO Journal , 2 987-995 (1983)) , herbicide resistance (EP-A-0242246) and glucuronidase (GUS) expression (EP-A-0344029) ; however, in some embodiments of the invention the protein of interest may serve as its own marker gene and so no second marker will necessarily be needed.
  • Expression of the marker gene if present, is preferably controlled by a second promoter (which may also be the 35S CaMV promoter) . However any other suitable second promoter could be used.
  • a whole plant can be regenerated from a single transformed plant cell, and the invention therefore provides in a sixth aspect transgenic plants (or parts of them, such as propagating material) including DNA in accordance with the invention as described above.
  • the regeneration can proceed by known methods .
  • a singular advantage, quite literally, of the invention is that, as discussed above, a plant does not have to be doubly transgenic if the same protein of interest is to be targeted to both mitochondria and chloroplasts.
  • a seventh aspect of the invention therefore, there is provided a plant having a transgene encoding a fusion of a protein and sufficient of the N- terminal leader sequence of a plant glutathione reductase to cause targeting of the said protein to both mitochondria and chloroplasts, wherein the plant does not have a further transgene which encodes a second mitochondrion- or chloroplast-targeting sequence fused to the said protein.
  • Transgenic plants in accordance with the invention are not limited by species. Much work on transgenic plants has been done in tobacco ⁇ Nicotiana tabacum) , which is consequently one of the better understood transgenic hosts (and which is represented in the examples of this invention, shown below) . However, the invention is in no sense limited in its usefulness to tobacco or any other individual species.
  • the invention also provides, in an eighth aspect, a method of targeting a protein to both mitochondria and chloroplasts, the method comprising expressing the said protein in a plant as a fusion with sufficient of the N- terminal leader sequence of a plant glutathione reductase to cause targeting of the protein to both organelles.
  • FIGURE 1 is a map of plasmid pGR46; 'LB' and 'RB' represent the left and right borders, respectively;
  • FIGURE 2 shows the complete cDNA sequence of pea glutathione reductase, with the deduced amino acid sequence
  • FIGURE 3 shows an amino acid alignment of glutathione reductases from pea (Peagr) , Pseudomonas aeruginosa (Psagr) , Escherichia coli (Ecgr) and man (Humgr) ; conserved regions are shaded;
  • FIGURE 4 is a map of plasmid pGR50; 'LB' and 'RB' represent the left and right borders, respectively; and
  • FIGURE 5 is a map of plasmid pGR42.
  • Glutathione reductase (GR) CDNAS were isolated from a bacteriophage ⁇ gtll cDNA expression library constructed from poly(A) * RNA isolated from 14 day old pea seedlings, ⁇ gtll is available from Amersham International pic, Amersham and the cDNA library was constructed essentially following the supplier's instructions and as indicated by Creissen et al . ( The Plant Journal 2(1) 129-131 (1992)) . The appropriate recombinant phage were identified by immunodetection using an anti-GR antiserum raised in rabbits against purified pea GR protein (Edwards et al . , Planta 180 278-284 (1990) ) .
  • Immunodetection was achieved by virtue of the recognition by anti-GR of antibodies of the phage-directed synthesis of a ⁇ -galactosidase-GR fusion protein. After purification of candidate phage and isolation of their DNA, cDNA inserts were subcloned into the plasmid vector PB UESCRIPT SKII+ (Stratagene Ltd. , Cambridge) as BamHI fragments. DNA sequence analysis was performed using the dideoxy-chain termination procedure
  • pGR27 The largest cDNA recovered at this stage was termed pGR27 which appeared to encode the mature GR peptide.
  • the cDNA pGR201 was subsequently recovered from the same cDNA library using the 5' -region (co-ordinate 254-393 of the published sequence) of pGR27 as a radiolabelled probe .
  • DNA sequence analysis of the subcloned cDNA in pGR201 revealed a coding sequence which was clearly identified as GR by amino-acid sequence homology to known GR sequences from other sources ⁇ Homo sapiens, Escherichia coli and Pseudomonas aeruginosa) .
  • the pGR201-encoded cDNA also encodes an N- terminai extension, which at the time of first analysis, was determined to be most likely a chloroplast targeting sequence (Creissen et al , supra) . Upstream of the first in-frame methionine initiator codon (AUG) was a translational stop codon. Therefore the cDNA in pGR201 was deemed to encode the full length GR preprotein.
  • GR cDNAs were manipulated in vi tro to produce the following chimeric genes .
  • pGR42 was constructed as follows: The -EcoRV-BamHI fragment (co-ordinates 18-2029) from pGR201 was recovered and inserted into the expression cassette pJIT163-BglII .
  • pJITl63-BglII contains 35S promoter and polyadenylation sequences from cauliflower mosaic virus (CaMV) separated by a restriction-site polylinker.
  • pJIT163-Bg.ilI was made from pJIT163 (Guerineau et al .
  • pBinLuc23 comprises the binary Ti vector pBinl9 (Bevan, Nucleic Acids Research, 12 8711-8721 (1984)) into which a 35S promoter-luciferase gene (Mullineaux et al . , Nucleic Acids Research, 18 7259-7265 (1990)) was inserted at the SacI site.
  • the chimeric GR gene was inserted between a Kanamycin resistance gene and the 35S- luciferase gene within the T-DNA borders of the vector.
  • the plasmid was designated pGR46 ( Figure 1) .
  • the plasmid pGR46 was introduced into Agrobacterium tumefaciens strain LBA4404 by a triparental mating technique (Ditta et al , Proc . Natl . Acad . Sci . USA, 77: 7374 (1990)) .
  • the Agrobacterium containing pGR46 was used to transform tobacco ⁇ Nicotiana tabacum L. cv. Samsun N ) .
  • Leaf discs of cv. Samsun NN were co-cultivated with A . tumefaciens containing the pGR46 essentially as described by Guerineau et al . , Plant Molecular Biology, 15 127-136 (1990) .
  • Potentially transformed shoots were identified as being resistant to 100 mg/1 kanamycin sulphate in the growth medium. Putative transformed shoots were rooted on 100 mg/1 kanamycin sulphate-containing rooting medium, and confirmed by screening the shoots for luciferase activity as described by Mullineaux et al . (1990) supra.
  • Kanamycin resistant, luciferase positive (Kan ⁇ luc 1" ) plantlets were potted in soil and grown to maturity in the glasshouse. Seed was collected from self-pollinated plants (Ti progeny) .
  • Two independently transformed lines were selected for further analysis. These were 46-23 and 46-27. Enhanced synthesis of GR was determined by immunodetection on Western blots. Luc' and luc " Ti plants from each of these lines were used as sources of chloroplast and mitochondria.
  • Chloroplast fractionation was carried out using the method of Boutry et al . , Nature, 328 340-342 (1987) with the following modifications:
  • the plant material was homogenised with a Polytron.
  • HEPES-sorbitol medium 50 mM HEPES, 330 mM sorbitol, 10 mM NaCl, 1 mM MgCl 2 , 2mM KH 2 P0 4 , pH7.6 and resuspended in the same medium.
  • Chloroplasts were lysed for enzyme assays by adding an equal volume of lysis buffer (50 mM Tris-HCl pH7.5, 5 mM MgCl 2 , 2 mM dithiothreitol, 1 mM EDTA, 0.2% Triton X-100) .
  • Mitochondria were layered onto a single concentration of Percoll (50% v/v) in grinding medium.
  • the marker enzymes used to establish the purity of the mitochondrion preparation were the same as those used for the chloroplast preparation .
  • the source of the glutathione reductase (GR) sequence was the plasmid pGR42 (see Example 2) . This contains the pea glutathione reductase cDNA clone (Creissen et al , 1992, supra) under the control of the CaMV 35S promoter and polyadenylation sequences.
  • phosphinothricin acetyl transferase (pat) coding sequence was obtained from the plasmid pIJ4102.
  • pIJ4102 is identical to plasmid pIJ4104 described by White et al , Nucl . Acids Res . , 18: 1063 (1990) .
  • the chimeric gene construct comprised the CaMV 35S promoter with duplicated enhancer region and CaMV polyadenylation signals (Guerineau et al , 1992, supra) , flanking a fusion between the 5' -region of the pea GR cDNA (pGR201; co-ordinates 18-392) and the pat coding sequence such that translation would be initiated at one of the GR AUG codons and continue to the translational stop codon at the 3 '-end of the pat coding sequence.
  • the pat coding sequence data is lodged with the EMBL database as entries SHBRPA and X17220.
  • the pat coding sequence was released from pIJ4102 by digestion with Xhol, followed by treatment with bacteriophage T4 DNA polymerase and subsequent digestion with Bglll.
  • the ca. 550bp pat coding sequence was eluted from an agarose gel.
  • the plasmid pGR42 was digested with Sad, followed by treatment with bacteriophage T4 DNA polymerase and subsequent digestion with BamHI .
  • the fragment comprising the vector plus CaMV promoter and polyadenylation sequences and 5' end of GR was eluted from an agarose gel.
  • the chimeric expression cassette pGR48 was generated by ligation of the two fragments from 1 and 2 above.
  • the chimeric gene was excised as a Bglll fragment and ligated into the unique BamHI site of the binary vector pBINLUC23 (see Example 3) to create pGR50.
  • the plasmid pGR50 was mobilised into Agrobacterium tumefaciens LBA4404 by the triparental mating procedure (Ditta et al , Proc . Na tl . Acad . Sci . USA, 77: 7374 (1990) ) and used to transform tobacco by leaf disc co- cultivation (Horsch et al , Science, 223: 496 (1984)) . Putative transgenic plants were identified by their ability to root on kanamycin-containing medium. Kanamycin-resistant shoots, which were found also to be expressing the firefly luciferase (T-DNA right border marker) , were transferred to the glasshouse and seeds were collected from self-pollinated plants.
  • T-DNA right border marker firefly luciferase
  • Seeds were sown on phosphinothricin-containing medium (10 ug/ml) and were found to exhibit the predicted 3:1 segregation of resistance, confirming that there was a single locus for the T-DNA and that the fusion protein expressed from the chimeric gene was biologically active.
  • the presence of the PAT protein in chloroplasts and mitochondria are confirmed using the methods detailed above (see Example 5) .

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Abstract

On connaît les 'peptides de transit' qui dirigent des protéines chloroplastidiques à codage nucléaire vers le chloroplaste. On connaît également les peptides de transit mitochondriaux correspondants. L'invention se rapporte à des peptides de transit qui orientent des protéines à codage nucléaire vers à la fois le chloroplaste et vers le mitochondrion. Le prototype est dérivé de la glutathione réductase du pois (Pisum sativum L.)). On peut utiliser une séquence d'ADN codant une protéine de fusion du peptide de transit fusionné à une protéine d'intérêt pour produire des plantes transgéniques.
PCT/GB1994/002058 1993-09-24 1994-09-22 Manipulation genetique de plante WO1995008633A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU76620/94A AU7662094A (en) 1993-09-24 1994-09-22 Plant genetic manipulation

Applications Claiming Priority (2)

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GB9319722.6 1993-09-24
GB939319722A GB9319722D0 (en) 1993-09-24 1993-09-24 Plant genetic manipulation

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WO1995008633A1 true WO1995008633A1 (fr) 1995-03-30

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6262340B1 (en) 1997-07-10 2001-07-17 Kosan Biosciences, Inc. Production of polyketides in plants
WO2001055169A1 (fr) * 2000-01-27 2001-08-02 Loma Linda University Vaccins a base de plantes transgeniques
US7422747B2 (en) 1997-10-07 2008-09-09 Loma Linda University Transgenic plant-based vaccines
WO2017198859A1 (fr) * 2016-05-20 2017-11-23 BASF Agro B.V. Peptides à double transit pour le ciblage de polypeptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, vol. 95, 1 May 1993, Philadelphia, PA, US; abstract no. 101542, AONO, M., ET AL.: "Enhanced tolerance to photooxidative stress of transgenic Nicotiana tabacum with high chloroplastic glutathione reductase activity" *
CREISSEN, G., ET AL.: "Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.)", THE PLANT JOURNAL, vol. 2, no. 1, January 1992 (1992-01-01), pages 129 - 131 *
HUANG, J.,: "A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria", THE PLANT CELL, vol. 2, December 1990 (1990-12-01), pages 1249 - 1260 *
PLANT CELL PHYSIOL., vol. 34, no. 1, 1993, pages 129 - 135 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6262340B1 (en) 1997-07-10 2001-07-17 Kosan Biosciences, Inc. Production of polyketides in plants
US7422747B2 (en) 1997-10-07 2008-09-09 Loma Linda University Transgenic plant-based vaccines
WO2001055169A1 (fr) * 2000-01-27 2001-08-02 Loma Linda University Vaccins a base de plantes transgeniques
WO2017198859A1 (fr) * 2016-05-20 2017-11-23 BASF Agro B.V. Peptides à double transit pour le ciblage de polypeptides
CN109154003A (zh) * 2016-05-20 2019-01-04 巴斯夫农业公司 用于靶向多肽的双转运肽
AU2017266411B2 (en) * 2016-05-20 2023-06-08 BASF Agro B.V. Dual transit peptides for targeting polypeptides
US11959086B2 (en) 2016-05-20 2024-04-16 BASF Agro B.V. Dual transit peptides for targeting polypeptides

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