WO2000011196A1 - Polynucleotide sequences - Google Patents
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- WO2000011196A1 WO2000011196A1 PCT/GB1999/002720 GB9902720W WO0011196A1 WO 2000011196 A1 WO2000011196 A1 WO 2000011196A1 GB 9902720 W GB9902720 W GB 9902720W WO 0011196 A1 WO0011196 A1 WO 0011196A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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
- C12N15/8282—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 fungal resistance
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to recombinant DNA technology, and in particular to nucleotide sequences (and expression products thereof) which are used in the production of transgenic plants.
- the present invention provides, inter alia, nucleotide sequences useful in the production of plants which show improved resistance to infection by microorganisms such as bacteria and fungi.
- polynucleotide comprising a sequence selected from those depicted in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.
- the invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 53 to 385 in SEQ ID No. 1, nucleotides 11 to 334 in SEQ ID No. 2, nucleotides 24 to 317 in SEQ ID No. 3, nucleotides 20 to 343 in SEQ ID No. 4 or nucleotides 1 to 446 in SEQ ID No. 5. Also included within the invention is the translation product of the region comprised by nucleotides 53 to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3 or by nucleotides 20 to 343 in SEQ ID No.
- the invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 137 to 286 in SEQ ID No. 1, nucleotides 95 to 244 in SEQ ID No. 2, nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No. 5.
- polynucleotide sequences are especially preferred. Also included within the invention and especially preferred is the translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3 or by nucleotides 104 to 253 in SEQ ID No. 4 and protein having an amino acid sequence which is at least 95% similar to said product.
- the translation product is an antimicrobial protein.
- the related antimicrobial proteins DmAMPl and Dm-AMP2 have been described in Published International Patent Application No. WO 93/05153 and in Osborn et al (1995) FEBS Lett. 368 257-262.
- the invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5.
- These nucleotides are particularly preferred according to the invention and encode protein sequences which may be used as cleavable linkers in the co-expression of multiple proteins as is described further herein.
- the invention further extends to the translation product of nucleotides 287 to 385 in SEQ ID NO.
- the invention further provides a polynucleotide sequence comprising a sequence selected from the group consisting of nucleotides 53 to 136 in SEQ ID No. 1, nucleotides 11 to 94 in SEQ ID No.2, nucleotides 24 to 107 in SEQ ID No. 3, nucleotides 20 to 103 in SEQ ID No.
- nucleotide sequences are signal sequences which may be linked to homologous and heterologous protein encoding regions to transport proteins extracellular ly.
- the invention further extends to the use of said sequences as signal sequences.
- the invention further extends to the translation product of nucleotides 53 to 136 in SEQ ID No. 1, nucleotides 11 to 94 in SEQ ID No.2, nucleotides 24 to 107 in SEQ ID No. 3, nucleotides 20 to 103 in SEQ ID No. 4 or nucleotides 1 to 176 in SEQ ID No. 5 and protein having an amino acid sequence which is at least 85% similar to said product.
- the degree of similarity is at least 90%, more preferred that the degree of similarity is at least 95% and still more preferred that the degree of similarity is at least 97%.
- two amino acid sequences with at least 85% similarity to each other have at least 85% similar (identical or conservatively replaced) amino acid residues in a like position when aligned optimally allowing for up to 3 gaps, with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected.
- two amino acid sequences with at least 90% similarity to each other have at least 90% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 3 gaps with the proviso that in respect of the gaps a total of not more than 15 amino acid residues is affected.
- a conservative amino acid is defined as one which does not alter the activity/function of the protein when compared with the unmodified protein.
- conservative replacements may be made between amino acids within the following groups:
- Sequence alignments to measure sequence similarity may be produced using the Lasergene program MegAlign (supplied by DNASTAR Inc. 1228 S. Park St. Madison WI 53715, USA).
- the Clustal Method and the PAM250 residue weight table with the following parameters may be used: Multiple Alignment Parameters gap penalty 10 gap length penalty 10 Pairwise alignment parameters Ktuple 1 gap penalty 3 window 5 Diagonals saved 5
- the invention also includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from those encoded by SEQ ID No. 1 , SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No.
- polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted in SEQ ID No 1, SEQ ID No 2, SEQ ID No3, SEQ ID No.4 or SEQ ID No.5 with the proviso that the sequence is not that described in SEQ ID No.6 or 7.
- 5 X SSC saline sodium citrate buffer
- polynucleotide sequence provided in SEQ ID Nos 6 and 7 is the predicted DNA sequence for Dm- AMP 1 and Dm-AMP2 as described in Figure 31A of Published International Patent Application No. WO 93/05153.
- the invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 53 to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3, by nucleotides 20 to 343 in SEQ ID No.
- nucleotides 1 to 446 in SEQ ID No.5 which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 53to 385 in SEQ ID No. 1, by nucleotides 11 to 334 in SEQ ID No. 2, by nucleotides 24 to 317 in SEQ ID No. 3, by nucleotides 20 to 343 in SEQ ID No. 4 or by nucleotides 1 to 446 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No. 6 or SEQ ID No. 7.
- the invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3, by nucleotides 104 to 253 in SEQ ID No.
- nucleotides 177 to 326 in SEQ ID No.5 which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65°C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 137 to 286 in SEQ ID No. 1, by nucleotides 95 to 244 in SEQ ID No. 2, by nucleotides 108 to 257 in SEQ ID No. 3, by nucleotides 104 to 253 in SEQ ID No. 4 or by nucleotides 177 to 326 in SEQ ID No.5. with the proviso that said sequence is not that described in SEQ ID No. 6 or SEQ ID No. 7.
- the invention still further includes a polynucleotide encoding a protein having a substantially similar activity to any one of the group selected from that encoded by nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5.
- polynucleotide which polynucleotide is complementary to one which when incubated at a temperature of between 55 and 65 °C in a solution containing 5 X SSC (saline sodium citrate buffer) containing 0.1% SDS and 0.25% powdered skimmed milk followed by washing at the same temperature with 0.1, 0.5 or 2x SSC containing 0.1% SDS still hybridises with a sequence depicted by nucleotides 287 to 385 in SEQ ID NO. 1, nucleotides 245 to 334 in SEQ ID No. 2, nucleotides 258 to 317 in SEQ ID No. 3, nucleotides 254 to 343 in SEQ ID No. 4 or nucleotides 327 to 446 in SEQ ID No.5.
- 5 X SSC saline sodium citrate buffer
- SDS powdered skimmed milk
- polynucleotide comprises sequences encoding chloroplast transit peptides, cell wall targeting sequences etc. immediately 5' of the regions encoding the said translation products.
- Translational expression of the protein encoding sequences contained within the polynucleotide may be relatively enhanced by including known non translatable translational enhancing sequences 5' of the said protein encoding sequences.
- the skilled man is very familiar with such enhancing sequences, which include the TMV-derived sequences known as omega, and omega prime, as well as other sequences derivable, inter alia, from the regions 5' of other viral coat protein encoding sequences.
- the polynucleotide is modified in that mRNA instability motifs and/or fortuitous splice regions are removed, or plant preferred codons are used so that expression of the thus modified polynucleotide in a plant yields substantially similar protein having a substantially similar activity/function to that obtained by expression of the unmodified polynucleotide in the organism in which the protein encoding regions of the unmodified polynucleotide are endogenous, with the proviso that if the thus modified polynucleotide comprises plant preferred codons, the degree of identity between the modified polynucleotide and a polynucleotide endogenously contained within the said plant and encoding substantially the same protein is less than about 60%.
- the invention also includes a plant transformation vector comprising a plant operable promoter, a polynucleotide sequence comprising all or part of the sequence selected from those depicted in SEQ ID'No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.5 under the transcriptional control thereof and encoding an antimicrobial protein, and a plant operable transcription terminator.
- the promoter may be constitutive or inducible. In particular, the promoter may be such that it induces transcription in response to application to the plant material containing it of a chemical.
- the invention further provides a plant transformation vector comprising a polynucleotide sequence selected from the group consisting of nucleotides 137 to 286 in SEQ ID No.
- nucleotides 95 to 244 in SEQ ID No. 2 nucleotides 108 to 257 in SEQ ID No. 3, nucleotides 104 to 253 in SEQ ID No. 4 or nucleotides 177 to 326 in SEQ ID No.5 under the transcriptional control of a plant operable promoter, and a plant operable transcriptional terminator.
- the polynucleotide sequences provided in SEQ ID No 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.5 are related sequences with the translated products thereof showing a high degree of sequence similarity and it is believed that they may belong to a multi gene family.
- the invention still further includes plant tissue transformed with the said polynucleotide or vector, and material derived from the said transformed plant tissue, as well as morphologically normal fertile whole plants comprising the tissue or material.
- Such transformed plants include but are not limited to, field crops, fruits and vegetables such as canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion, etc.
- Particularly preferred genetically modified plants are bananas.
- the invention still further includes the progeny of the plants of the preceding paragraph, which progeny comprises a polynucleotide of the invention stably incorporated into its genome and heritable in a mendelian manner and the seeds of such plants and such progeny.
- the invention also provides a method of producing plants which are substantially tolerant or substantially resistant to antimicrobial infection, comprising the steps of:
- Plant transformation, selection and regeneration techniques which may require routine modification in respect of a particular plant species, are well known to the skilled man.
- the invention also provides the use of a polynucleotide as described herein or a vector described herein in the production of plant tissues and/or morphologically normal fertile whole plants which are substantially tolerant or substantially resistant to microbial infection.
- the invention provides a method of selectively controlling microorganisms at a locus comprising the plants, progeny and/or seeds described herein comprising applying to the locus a microorganism controlling amount of the translation product of the region comprised by nucleotides 137 to 286 in SEQ ID No. 1.; nucleotides 95 to 244 in SEQ ID No.2, nucleotides 108 to 257 in SEQ ID No. 3, or nucleotides 104 to 253 in SEQ ID No. 4.
- the invention provides the use of a polynucleotide according to the invention described herein , or a vector as described herein in the production of an antimicrobial protein.
- Figure 1 shows the polynucleotide and corresponding amino acid sequences for A) Dml (SEQ ID No5) and B) Dm2.18 (SEQ ID Nol),
- Figure 2 shows the polynucleotide and corresponding amino acid sequences for A) Dm2.1 (SEQ ID No2) and B) Dm2.3 (SEQ ID No3)
- Figure 3 shows the polynucleotide and corresponding amino acid sequence for Dm2.5 (SEQ ID No3)
- Figure 4 shows a diagrammatic map of plasmids pMJBl, pDmAMPD and pDmAMPE;
- Figure 5 shows a diagrammatic map of plasmid pFAJ3106
- Figure 6 shows a diagrammatic map of plasmid pFAJ3109
- Figure 7 shows the nucleotide sequence between the Xhol and Sad sites of pFAJ3106
- Figure 8 shows the nucleotide sequence between the Xhol and Sad sites of pFAJ3109
- Figure 9 shows a diagrammatic map of plasmid pZPS38
- Figure 10 shows a diagrammatic map of plasmid pZPS34
- Figure 11 shows a diagrammatic map of plasmid pZPS35
- Figure 12 shows a diagrammatic map of plasmid pZPS37.
- Figure 13 shows a plan of the construction of the Dm- AMP gene
- Figure 14 shows one predicted polynucleotide sequence for DmAMPl (SEQ ID No. 6) and
- FIG. 15 shows a diagrammatic map of plasmid pAID-MR7
- RNA was collected by spinning at 13,000 rpm in an Eppendorf centrifuge and the RNA pellet re-suspended in 5 mM Tris-HCI, pH 7.5. A second overnight lithium chloride precipitation was carried out and the RNA collected and re-suspended in 5 mM Tris-HCI, pH 7.5.
- RNA 0.6 mg was obtained from 2 g of D. merkii seed.
- PolyATract magnetic beads (Promega) were used to isolate approximately 2 ⁇ g poly-A + RNA from 0.2 mg of total RNA.
- the poly-A + RNA was used to construct a cDNA library using a ZAP-cDNA synthesis kit (Stratagene). Following first and second strand synthesis double stranded cDNA was size fractionated on a Sephacryl S-400 column. The three largest cDNA size fractions were pooled and ligated with vector DNA. After phage assembly using Gigapack Gold (Stratagene) packaging extracts, approximately 1 x 10 5 pfu were obtained. Probing
- Dahlia genomic DNA was prepared from 100 mg of developing Dahlia seeds and flower tissue. Tissue was homogenised in a 1.5 ml Eppendorf tube with a conical plastic pestle. 400 ⁇ l of a solution containing 0.2M Tris-HCI pH 8.5, 0.25M NaCl, 0.025 M EDTA and 0.5% SDS was added and the tube vortexed for 5 seconds. Cell debris was pelleted by spinning at 13,000 rpm for 1 minute in a MSE bench top micro centrifuge. 300 ⁇ l of aqueous extract was transferred to a fresh Eppendorf tube. Genomic DNA was precipitated by the addition of 300 ⁇ l isopropyl alcohol and incubation at room temperature for 2 minutes.
- Genomic DNA was pelleted by spinning at 13,000 rpm for 5 minutes. The ethanol/aqueous supernatant was removed from the tube by pipette and the genomic DNA pellet allowed to air dry. Genomic DNA was then resuspended in 30 ⁇ l H 2 O.
- PCR was carried out with Dahlia genomic DNA and oligonucleotides AFP-5 (based on Dm- AMP1 N-terminal amino acid sequence CEKASKTW) and AFP-3EX (based on Dm- AMP 1 C-terminal amino acid sequence MCFCYFNC). Using the following conditions 94°C, 60 seconds, 48°C, 12 seconds and 72°C, 60 seconds for 35 cycles.
- a PCR product of approximately 150 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation.
- the PCR product was cloned into pBluescript by ligating blunt Bluescript vector and gel isolated PCR product together using T4DNA ligase and transforming into competent E. coli MCI 022 cells. Transformation mixes were plated onto L-agar plates containing 100 ⁇ g/ml a picillin and incubated at 37°C for 16 hours. Colonies were picked and cells shaken for 16 hours in 3 ml L-broth containing 100 ⁇ g/ml ampicillin at 37"C. Plasmid DNA was prepared from colonies using a Promega Wizard mini-prep kit. The inserts of 10 transformants were sequenced using a Sequenase kit (United States Biochemical). The cloned PCR product sequences represented 3 Dm-AMPl related genes. PCR clone 4 contained the DNA sequence
- the 144 bp PCR product mixture labelled with ⁇ 32 -P d-CTP was used to probe Hybond N (Amersham) filter lifts made from plates containing a total of 6 x 10 4 pfu of the cDNA library.
- the filters were hybridised at 46°C for 18 hrs in 5 x SSC, 0.1% SDS, 0.25% skimmed milk powder. Filters were washed in 2 x SSC, 0.1% SDS at 60°C. Autoradiography was carried out at -70°C with intensifying screens. Thirty potentially positive signals were observed. 22 plaques were picked and taken through two further rounds of screening. After in vivo excision 13 clones were characterised by DNA sequencing.
- Dm- AMP related peptide Four classes of Dm- AMP related peptide were encoded by the 13 cDNA clones and the sequences of these peptides are provided in SEQ ID Nos 1- 4 in the accompanying figures. Three versions of the Dm- AMP core region were represented in the four classes. One of the classes (Dm2.5 type) contained a core region which may correspond to Dm-AMP2. None of the cDNAs encoded a core region equivalent to the observed mature Dm-AMPl peptide sequence.
- a PCR was carried out with Dahlia genomic DNA and oligonucleotides MATAFP-5P (based on the codons present in Dm2.1, Dm2.3, Dm2.18 and Dm2.5 encoding the N-terminal amino acid sequence M(AV)(KN)(NR)SVAF) and MATAFP-5 (based on the mature Dm- AMP 1 amino acid sequence NGKHMCF) using the following conditions; 94°C, 60 seconds, 53°C, 12 seconds and 72°C, 60 seconds for 40 cycles.
- a PCR product of approximately 220 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation. The PCR product was cloned into pBluescript and clones sequenced as described above. A clone containing the
- a PCR was carried out with Dahlia genomic DNA and oligonucleotides MATAFP-3 (based on the mature Dm-AMPl amino acid sequence GACHVRN) and DM25MAT-3 (based on the last two amino acids and the 3' untranslated region of Dm2.5) using the following conditions; 94°C, 60 seconds, 53°C, 12 seconds and 72°C, 60 seconds for 40 cycles.
- a PCR product of approximately 170 bp was isolated from a 2% agarose gel by electroelution and ethanol precipitation. The PCR product was cloned into pBluescript and clones were sequenced as described above. A clone containing the 3' half of a Dm- AMP 1 gene was identified.
- the 5' and 3' sections of the mature gene were combined to assemble the sequence of the mature Dm-AMPl gene (see Figure 1 SEQ ID No.5) which is comprised of exon 1, 64 bp encoding part of the leader peptide, 92 bp intron and exon 2 encoding the end of the leader sequence, Dm-AMPl core and C-terminal extension.
- oligonucleotides were designed based on the DNA sequence of the mature Dm-AMPl gene: DMVEC-1 top strand priming at the 5' end of the mature DmAMP-l gene incorporating a
- DMVEC-3 top strand priming at the 5' end of the mature DmAMP-l gene incorporating a Nco I site at the translation start of DmAMP-l allowing cloning into pMJBl also encoding complete signal peptide (minus intron).
- a PCR was carried out with Dahlia genomic DNA and oligonucleotides DMVEC-1 and DMVEC-2 using the following conditions; 94°C, 60 seconds, 60°C, 12 seconds and 72°C, 60 seconds for 45 cycles.
- a PCR product of approximately 450 bp spanning the mature Dm- AMP 1 gene was obtained, this PCR product was isolated from an agarose gel and used as a template for PCRs described in vector construction below.
- a PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-1 and DMVEC-4 using the following conditions, 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles.
- the PCR product was cut with Nco I and Sac I the 60 bp Nco I/Sac I fragment was isolated and ligated with pMJBlcut with Nco I and Sac I.
- the ligation mix was used to transform competent E. coli MCI 022 cells and plasmid DNA of ampicillin resistant transformants was obtained as described above.
- pDmAMPA The identity of the fragment in one of the resulting transformants was confirmed by sequencing, the clone was termed pDmAMPA.
- a PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-3 and DMVEC-4 using the following conditions; 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles.
- the PCR product was cut with Nco I, the resulting 150 bp Nco I fragment isolated and cloned into pDmAMPA cut with Nco I.
- DNA sequencing confirmed that one transformant termed pDmAMPD, contained DNA encoding Dm-AMP leader and core region.
- the PCR product obtained with DMVEC-1 and DMVEC-2 was cut with Nco I and Sac I the 180 bp Nco I/Sac I fragment was isolated and cloned into pMJBl cut with Nco I and Sac I as described above. The identity of the fragment in one of the resulting transformants was confirmed by sequencing, the clone was termed pDmAMPB.
- a PCR was carried out with the DMVEC-1 and DMVEC-2 450 bp PCR product and oligonucleotides DMVEC-3 and DMVEC-4 using the following conditions; 94°C, 48 seconds, 58°C, 12 seconds and 72°C, 90 seconds for 33 cycles.
- the PCR product was cut with Nco I and the resulting 150 bp Nco I fragment isolated and cloned into pDmAMPB cut with Nco I.
- DNA sequencing confirmed that one transformant termed pDmAMPE, contained
- Both pDmAMPD and pDmAMPE vector sequences contained PCR derived base substitutions with respect to Dm-AMPl gene DNA sequence however the base changes were silent having no effect on the expected amino acid sequence.
- pDmAMPD and pDmAMPE are both digested with the restriction endonucleases Hindlll and EcoRI and the appropriate restriction fragment isolated and purified. Each fragment is ligated into a binary vector (a pBIN19 derivative named pBinl9i) which has also been digested with Hindlll and EcoRI.
- the resulting constructs named pDmAMPLC and pDmAMPLCC, incorporate the expression cassettes from pDmAMPD and pDmAMPE respectively.
- pDmAMPLC and pDmAMPLCC are subsequently introduced into Agrobacterium tumefaciens strain LBA4404 and introduced into tobacco and oil seed rape using standard plant transformation methodology.
- Plants are regenerated from callus tissue resistant to the selective agent kanamycin and expression of the Dm-AMPl product is monitored by standard Western blot or ELISA methods using antibody which had been raised against Dm-AMPl protein. A range of expression levels are detected.
- the Dm-AMPl expressed in selected transgenic is further characterised following extraction and partial purification from leaves of such lines.
- the product is of the predicted mass, as indicted by mass spectrometry. It is also demonstrated to retain biological activity after extraction as demonstrated by retention of antifungal activity in in-vitro (micro-titre plate) assays.
- Primers OWB175 (5'AGGAAGTTCATTTCATTTGG) and OWB279 (5'- GCCTTTGGCACAACTTCTGCCTCTTTCCGATGAGTTGTTCGGCTTTAAGTTTGTC); were used in a first PCR reaction with plasmid pDMAMPE (see above) as a template.
- the second PCR reaction was done using as a template plasmid pFRG4 (Terras F.R.G. et al., 1995, Plant Cell 7, 573-588) and as primers a mixture of the PCR product of the first PCR reaction, primer OWB175 and primer OWB172 (5 TAGAGCTCCTATTAACAAGGAAAGTAGC, Sad site underlined).
- Plasmid pFAJ3109 was constructed by cloning the Hindlll-EcoRI fragment of plasmid pDMAMPD (see above) into the corresponding sites of plant transformation vector pGPTVbar (see above). Plant transformation
- Arabidopsis thaliana ecotype Columbia-O was transformed using recombinant Agrobacterium tumefaciens by the inflorescence infiltration method of Bechtold N. et al. (1993, CR. Acad. Sci. 316, 1194-1199). Transformants were selected on a sand/perlite mixture subirrigated with water containing the herbicide Basta (Agrevo) at a final concentration of 5 mg/1 for the active ingredient phosphinothricin. Elisa assays and protein assays Antisera were raised in rabbits injected with either RsAFP2 (purified as described in Terras F.R.G. et al., 1992, J. Biol. Chem.
- ELISA assays were set up as competitive type assays essentially as described by Penninckx I.A.M.A. et al. (1996, Plant Cell 8, 2309-2323). Coating of the ELISA microtiter plates was done with 50 ng/ml RsAFP2 or DmAMPl in coating buffer. Primary antisera were used as 1000- and 2000-fold diluted solutions (DmAMPl and RsAFP2, respectively) in 3 % (w/v) gelatin in PBS containing 0.05 % (v/v) Tween 20.
- Total protein content was determined according to Bradford (1976, Anal. Biochem. 72, 248- 254) using bovine serum albumin as a standard. Purification and characterisation of expressed proteins Arabidopsis leaves were homogenized under liquid nitrogen and extracted with a buffer consisting of 10 mM NaH 2 PO 4 , 15 mM Na ⁇ PO ⁇ 100 mM KCl, 1.5 M NaCl. The homogenate was heated for 10 min at 85°C and cooled down on ice.
- the heat-treated extract was centrifuged for 15 min at 15 000 x g and was injected on a reserved phase high pressure liquid chromatography column (RP-HPLC) consisting of C8 silica (0,46 cm x 25 cm; Rainin) equilibrated with 0.1 % (v/v) trifluoroacetic acid (TFA).
- RP-HPLC high pressure liquid chromatography column
- the column was eluted at 1 ml/min in a linear gradient in 35 min. from 15 % to 50 % (v/v) acetonitrile in 0.1 % (v/v) TFA.
- the eluate was monitored for absorbance at 214 nm, collected as 1 ml fractions, evaporated and finally redissolved in water. The fractions were tested by ELISA assays.
- Intercellular fluid was collected from Arabidopsis leaves by immersing the leaves in a beaker containing extraction buffer (10 mM NaH 2 PO 4 , 15 mM Na 2 HPO 4 , 100 mM KCl, 1.5 M NaCl).
- extraction buffer (10 mM NaH 2 PO 4 , 15 mM Na 2 HPO 4 , 100 mM KCl, 1.5 M NaCl).
- the beaker with the leaves was placed in a vacuum chamber and subjected to six consecutive rounds of vacuum for 2 min followed by abrupt release of vacuum.
- the infiltrated leaves were gently placed in a centrifuge tube on a grid separated from the tube bottom.
- the intercellular fluid was collected from the bottom after centrifugation of the tubes for 15 min at 1800 x g.
- the leaves were resubjected to a second round of vacuum infiltration and centrifugation and the resulting (extracellular) fluid was combined with that obtained after the first vacuum infiltration. After this step the leaves were extracted in a Phastprep (BlOlOl/Savant) reciprocal shaker and the extract clarified by centrifugation (10 min at 10,000 x g) and the resulting supernatant considered as the intracellular extract. Characterization of transgenic plants and expression analysis
- Construct 3106 has a propeptide consisting of a part of the DmAMP 1 propeptide and a putative subtilisin-like protease processing site (IGKR) at its C- terminus.
- C-terminal cleavage of the internal propeptide in these constructs should be executed by a subtilisin-like protease, a member of which in yeast (Kex2) is known to occur in the Golgi apparatus (Wilcox CA. and Fuller R.S., 1991, J. Cell. Biol. 115, 297- ), while a member in tomato occurs in the apoplast (Tornero P. et al., 1997, J. Biol. Chem. 272, 14412-14419).
- Proteins deposited in the apoplast, the preferred deposition site for antimicrobial proteins engineered in transgenic plants are normally synthesized via the secretory pathway, encompassing the Golgi apparatus.
- glucose-6-phosphate dehydrogenase was used as a marker to detect contamination of the extracellular fluid fraction with intracellular components.
- glucose-6-phosphate dehydrogenase was partitioned in a ratio of about 80/20 between intracellular extract fractions and extracellular fluid fractions.
- DmAMPl and RsAFP2 content in all transgenic plants tested was found in the extracellular fluid fractions.
- Constructs were prepared containing the Dahlia (Dahlia merckii) antimicrobial protein signal peptide fused with the Brazzein gene under the transcriptional control of the Arabidopsis polyubiquitin extension protein promoter (UBQ) or the Polygalacturonase promoter (PG). Constructs were also prepared which encoded Brazzein without a signal peptide but with an N-terminal methionine by the insertion of ATG nucleotides upstream of the Brazzein gene under the expressional control of either the UBQ promoter or the PG promoter. These were prepared as follows:
- a synthetic DNA was produced which coded for the Dahlia signal peptide fused to Brazzein.
- the codons were optimised for expression in tomato.
- the coding sequence was cloned into a plasmid vector.
- the coding region was excised from the plasmid and cloned between the promoter in question and the terminator in the correct orientation for expression.
- the vector was transferred to Agrobacterium tumefaciens LBA4404 (a microorganism widely available to plant biotechnologists) and used to transform tomato plants. Transformation of tomato stem segments followed standard protocols (e.g. Bird et al Plant Molecular Biology 11, 651-662, 1988). Transformed plants were identified by their ability to grow on media containing the antibiotic kanamycin. Up to 30 individual plants were regenerated with each construct and grown to maturity. The presence of the construct in all of the plants was confirmed by polymerase chain reaction analysis. DNA Southern blot analysis on all plants indicated that the insert copy number was between 1 and 10. Northern blot analysis on fruit from one plant indicated that the Brazzein gene was expressed.
- Brazzein production in the fruit of all plants was measured by ELISA (enzyme linked imunoabsorption assay) using a polyclonal and a monoclonal antibody raised against native Brazzein protein isolated from the fruit of the plant Pentadiplandra brazzeana Baillon Two fruit were collected from each transgenic plant at 7 days post breaker (the term breaker is used to indicate when the tomato fruit first show signs of the orange colouration characteristic of most mature tomato fruit). Total fruit protein was extracted from a sample of the pericarp of each of the fruit. The amount of Brazzein protein in the total protein extract was measured by ELISA and calculated as the amount of Brazzein per gram fresh weight of the fruit. For each plant the average Brazzein content of the two fruits was calculated.
- pALD-MR7 The vector chosen for these experiments was pALD-MR7.
- pALD-MR7 was constructed using the commercially available cloning vector pNEB193
- Example 5a Vector DNA was prepared by digesting plasmid DNA of pAIDMR7 with Xba I and the ends filled in with Klenow DNA polymerase. The linear vector was isolated from an agarose gel by electroelution and ethanol precipitation. The DNA pellet was air dried and taken up in a small volume of water.
- Plasmid DNA of a cDNA clone containing a Dm2.1 ORF was digested with Eco Rl and Sea I and the ends filled in with Klenow DNA polymerase. Insert DNA containing the Dm2.1 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.
- the DNA pellet was air dried and taken up in a small volume of water.
- Vector and insert DNA were ligated together using T4 DNA ligase and transformed into competent E. coli MCI 022 cells. Tranformation mixes were plated onto L-agar plates containing 100 ⁇ g/ml ampicillin and incubated at 37°C for 16 hours. Colonies were picked and cells shaken for 16 hours in 3 ml L-broth containing 100 ⁇ g/ml ampicillin at 37°C.
- Plasmid DNA was prepared from several colonies and used in DNA sequencing reactions to identify transformants containing the Dm2.1 coding region in the appropriate orientation with respect to the MR7 promoter. One such clone was identified and named pAIDMR721.
- Vector DNA was prepared as in Example 5a.
- Plasmid DNA of a cDNA clone containing a Dm2.3 ORF was digested with Eco Rl and Sea
- Insert DNA containing the Dm2.3 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.
- the DNA pellet was air dried and taken up in a small volume of water.
- Vector and insert DNA were ligated together, transformed into E. coli MCI 022 and colonies characterised by DNA sequencing as described in Example 5a.
- a clone was identified containing the Dm2.3 ORF in the desired orientation and named pAIDMR723.
- Vector DNA was prepared as in Example 5a.
- Plasmid DNA of a cDNA clone containing a Dm2.5 ORF was digested with Eco Rl and Dra
- Insert DNA containing the Dm2.5 coding region was isolated from an agarose gel by electroelution and ethanol precipitation.
- the DNA pellet was air dried and taken up in a small volume of water.
- Vector and insert DNA were ligated together, transformed into E. coli MCI 022 and colonies characterised by DNA sequencing as described in Example 5a.
- a clone was identified containing the Dm2.5 ORF in the desired orientation and named pAIDMR725.
- Plasmid DNA of clones pAIDMR721 pAIDMR723, pAIDMR725 and pAIDMR7 is used to transform cultured maize BMS cells using the PEG method. Protoplast preparation and transformation
- Protoplasts are isolated from a maize suspension of Black Mexican Sweet Corn suspension culture (BMS) [Green, Hort. Sci., 12 (1977) 131 ; Smith et al., Plant Sci. Lett., 36 (1984) 67] subcultured in BMS medium (MS medium supplemented with 2% sucrose, 2 mg/1 2,4-D, pH5.8).
- BMS Black Mexican Sweet Corn suspension culture
- Cells from suspensions two days post subculture are digested in enzyme mixture (2.0% cellulase RS (Yakult Honsha Co., Ltd), 0.2% pectolyase Y23 (Yakult Honsha Co., Ltd), 0.5M mannitol, 5mM CaCl 2 2H 2 O, 0.5% MES, pH5.6, ⁇ 660mmol/kg) using ⁇ 10ml/g cells, incubating at 25°C, rotating gently for 2 hours.
- the digestion mixture is sieved through 250 ⁇ m and 38 ⁇ m sieves, and the filtrate centrifuged at 700rpm for 3.5 minutes.
- Protoplasts are resuspended in wash buffer (0.358M KCl, l .OmM NH 4 NO 3 , 5.0mM CaCl 2 2H 2 O, 0.5mM KH 2 PO 4 , pH4.8, ⁇ 670mmol/kg) and pelleted twice prior to resuspending in wash buffer and counting. Transformation is achieved using PEG (PEG 3350, Sigma Co) mediated uptake (Negrutiu et al., 1987) employing plasmid DNA prepared using Qiagen midi plasmid preparation kit (Qiagen Ltd, Crawl ey, UK).
- Protoplasts are resuspended at 2 x lOVml in MaMg medium (0.4M mannitol, 15mM MgCl 2 , 0.1% MES, pH5.6, ⁇ 450mmol/kg) aliquotting 0.5ml / treatment (i.e. lxlO 6 protoplasts/treatment). Samples are heat shocked at 45°C for 5 minutes then cooled to room temperature. Each transformation is carried out with lO ⁇ g of pAID-MR7 alone or lO ⁇ g of each construct pAIDMR721, pAIDMR723 or pAIDMR725.
- Each protoplast treatment is resuspended in 1.5ml culture medium (MS medium, 2% sucrose, 2mg/l 2,4-D, 9% mannitol, pH5.6, ⁇ 700mmol/kg). Samples are incubated in 3cm dishes at 25°C, in the dark, for 48 hours prior to harvesting.
- MS medium 2% sucrose, 2mg/l 2,4-D, 9% mannitol, pH5.6, ⁇ 700mmol/kg.
- Cells are osmotically lysed by the addition of water. Cell debris is pelleted by centrifugation and the proteins remaining in solution are freeze dried. The freeze dried proteins are taken up in a small volume of water and the concentration of protein determined using Bradford reagent.
- Culture media removed from cells is freeze dried and taken up in a small volume of water, the concentration of protein is determined as above.
- Protein samples isolated from all BMS transformations are assayed for spore germination inhibition in a bioassay against Fusarium culmorum spores as described in Published International Patent Application No. WO 93/05153.
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Abstract
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Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU54344/99A AU772284B2 (en) | 1998-08-18 | 1999-08-17 | Polynucleotide sequences |
EP99940349A EP1104480A1 (en) | 1998-08-18 | 1999-08-17 | Polynucleotide sequences |
BR9913042-4A BR9913042A (en) | 1998-08-18 | 1999-08-17 | Polynucleotide, polynucleotide sequence, plant transformation vector, plant tissue, morphologically normal fertile complete plants, plant progeny, plant production process that are substantially tolerant or substantially resistant to microbial infection, use of polynucleotide, translation product, and, process to selectively control microorganisms in a location containing plants, progeny and / or seeds |
JP2000566448A JP2002523052A (en) | 1998-08-18 | 1999-08-17 | Polynucleotide sequence |
CA002335652A CA2335652A1 (en) | 1998-08-18 | 1999-08-17 | Polynucleotide sequences |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9818003.7A GB9818003D0 (en) | 1998-08-18 | 1998-08-18 | Polynucleotide sequences |
GB9818003.7 | 1998-08-18 |
Publications (1)
Publication Number | Publication Date |
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WO2000011196A1 true WO2000011196A1 (en) | 2000-03-02 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1999/002720 WO2000011196A1 (en) | 1998-08-18 | 1999-08-17 | Polynucleotide sequences |
Country Status (8)
Country | Link |
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EP (1) | EP1104480A1 (en) |
JP (1) | JP2002523052A (en) |
CN (1) | CN1316007A (en) |
AU (1) | AU772284B2 (en) |
BR (1) | BR9913042A (en) |
CA (1) | CA2335652A1 (en) |
GB (1) | GB9818003D0 (en) |
WO (1) | WO2000011196A1 (en) |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000068405A2 (en) * | 1999-05-07 | 2000-11-16 | E.I. Du Pont De Nemours And Company | Plant defensins |
WO2000078983A2 (en) * | 1999-06-23 | 2000-12-28 | Pioneer Hi-Bred International, Inc. | Sunflower anti-pathogenic proteins and genes and their uses |
US6667427B1 (en) | 1999-10-14 | 2003-12-23 | Pioneer Hi-Bred International, Inc. | Sclerotinia-inducible promoters and their uses |
US6855865B2 (en) | 1999-05-07 | 2005-02-15 | E.I. Du Pont De Nemours And Company | Nucleic acids encoding plant defensins and methods of use thereof |
US6911577B2 (en) | 2001-06-22 | 2005-06-28 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
WO2013056309A1 (en) | 2011-10-19 | 2013-04-25 | Hexima Limited | Novel plant defensins and use in the treatment of proliferative diseases |
EP2762498A1 (en) | 2007-04-20 | 2014-08-06 | Hexima Limited | Modified plant defensin |
US9497908B2 (en) | 2011-02-07 | 2016-11-22 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
US9848603B2 (en) | 2008-02-01 | 2017-12-26 | Hexima Limited | Methods for protecting plants with antifungal compositions |
US9889184B2 (en) | 2008-08-05 | 2018-02-13 | Hexima Limited | Anti-pathogen systems |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008018488A1 (en) * | 2006-08-09 | 2008-02-14 | Takara Bio Inc. | Antibacterial agent |
CN114480475B (en) * | 2021-12-29 | 2023-05-30 | 中国科学院海洋研究所 | Recombinant Porphyra yezoensis strain rich in flavor substances, construction method and application |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993005153A1 (en) * | 1991-08-29 | 1993-03-18 | Zeneca Limited | Biocidal proteins |
WO1994016076A1 (en) * | 1993-01-08 | 1994-07-21 | Zeneca Limited | Antimicrobial-protein-producing endosymbiotic microorganisms |
-
1998
- 1998-08-18 GB GBGB9818003.7A patent/GB9818003D0/en not_active Ceased
-
1999
- 1999-08-17 WO PCT/GB1999/002720 patent/WO2000011196A1/en not_active Application Discontinuation
- 1999-08-17 CA CA002335652A patent/CA2335652A1/en not_active Abandoned
- 1999-08-17 BR BR9913042-4A patent/BR9913042A/en not_active IP Right Cessation
- 1999-08-17 EP EP99940349A patent/EP1104480A1/en not_active Withdrawn
- 1999-08-17 CN CN 99810392 patent/CN1316007A/en active Pending
- 1999-08-17 AU AU54344/99A patent/AU772284B2/en not_active Ceased
- 1999-08-17 JP JP2000566448A patent/JP2002523052A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993005153A1 (en) * | 1991-08-29 | 1993-03-18 | Zeneca Limited | Biocidal proteins |
WO1994016076A1 (en) * | 1993-01-08 | 1994-07-21 | Zeneca Limited | Antimicrobial-protein-producing endosymbiotic microorganisms |
Non-Patent Citations (2)
Title |
---|
DOMON,C., ET AL.: "nucleotide sequence of two anther-specific cDNAs from sunflower (Helianthus annuus L.)", PLANT MOLECULAR BIOLOGY, vol. 15, 1990, pages 643 - 646, XP002125194 * |
EVRARD, J-L., ET AL.: "anther-specific, developmentaly regulated expression of genes encoding a new class of proline-rich proteins in sunflower", PLANT MOLECULAR BIOLOGY, vol. 16, 1991, pages 271 - 281, XP002125195 * |
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US7238781B2 (en) | 1999-05-07 | 2007-07-03 | E. I. Dupont De Nemours And Company | Plant defensin polynucleotides and methods of use thereof |
WO2000068405A3 (en) * | 1999-05-07 | 2001-02-08 | Du Pont | Plant defensins |
US6855865B2 (en) | 1999-05-07 | 2005-02-15 | E.I. Du Pont De Nemours And Company | Nucleic acids encoding plant defensins and methods of use thereof |
WO2000068405A2 (en) * | 1999-05-07 | 2000-11-16 | E.I. Du Pont De Nemours And Company | Plant defensins |
EP1849868A3 (en) * | 1999-05-07 | 2008-02-20 | E.I.Du pont de nemours and company | Plant defensins |
WO2000078983A2 (en) * | 1999-06-23 | 2000-12-28 | Pioneer Hi-Bred International, Inc. | Sunflower anti-pathogenic proteins and genes and their uses |
WO2000078983A3 (en) * | 1999-06-23 | 2001-08-16 | Pioneer Hi Bred Int | Sunflower anti-pathogenic proteins and genes and their uses |
US6677503B1 (en) | 1999-06-23 | 2004-01-13 | Pioneer Hi-Bred International, Inc. | Sunflower anti-pathogene proteins and genes and their uses |
US6667427B1 (en) | 1999-10-14 | 2003-12-23 | Pioneer Hi-Bred International, Inc. | Sclerotinia-inducible promoters and their uses |
US7396980B2 (en) | 2001-06-22 | 2008-07-08 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US8026415B2 (en) | 2001-06-22 | 2011-09-27 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US6911577B2 (en) | 2001-06-22 | 2005-06-28 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US8710296B2 (en) | 2001-06-22 | 2014-04-29 | Pioneer Hi Bred International Inc | Defensin polynucleotides and methods of use |
US7855327B2 (en) | 2001-06-22 | 2010-12-21 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US7855328B2 (en) | 2001-06-22 | 2010-12-21 | Pioneer Hi-Bred Int'l., Inc. | Defensin polynucleotides and methods of use |
US7897847B2 (en) | 2001-06-22 | 2011-03-01 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US7910806B2 (en) | 2001-06-22 | 2011-03-22 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
US7919686B2 (en) | 2001-06-22 | 2011-04-05 | Pioneer Hi-Bred International, Inc. | Defensin polynucleotides and methods of use |
EP1534829A4 (en) * | 2002-06-21 | 2007-08-22 | Du Pont | Plant defensins |
EP1534829A2 (en) * | 2002-06-21 | 2005-06-01 | E.I. Du Pont De Nemours And Company | Plant defensins |
EP2762498A1 (en) | 2007-04-20 | 2014-08-06 | Hexima Limited | Modified plant defensin |
US9848603B2 (en) | 2008-02-01 | 2017-12-26 | Hexima Limited | Methods for protecting plants with antifungal compositions |
US9889184B2 (en) | 2008-08-05 | 2018-02-13 | Hexima Limited | Anti-pathogen systems |
US9497908B2 (en) | 2011-02-07 | 2016-11-22 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
US10174339B2 (en) | 2011-02-07 | 2019-01-08 | Hexima Limited | Modified plant defensins useful as anti-pathogenic agents |
WO2013056309A1 (en) | 2011-10-19 | 2013-04-25 | Hexima Limited | Novel plant defensins and use in the treatment of proliferative diseases |
Also Published As
Publication number | Publication date |
---|---|
CA2335652A1 (en) | 2000-03-02 |
AU5434499A (en) | 2000-03-14 |
BR9913042A (en) | 2001-05-08 |
EP1104480A1 (en) | 2001-06-06 |
CN1316007A (en) | 2001-10-03 |
JP2002523052A (en) | 2002-07-30 |
GB9818003D0 (en) | 1998-10-14 |
AU772284B2 (en) | 2004-04-22 |
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