WO2001090387A2 - Gene regulatory region that promotes root-specific transcription and its uses - Google Patents
Gene regulatory region that promotes root-specific transcription and its uses Download PDFInfo
- Publication number
- WO2001090387A2 WO2001090387A2 PCT/IB2001/001137 IB0101137W WO0190387A2 WO 2001090387 A2 WO2001090387 A2 WO 2001090387A2 IB 0101137 W IB0101137 W IB 0101137W WO 0190387 A2 WO0190387 A2 WO 0190387A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nucleic acid
- sequence
- plants
- isolated nucleic
- roots
- Prior art date
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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
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- 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
- C12N15/8227—Root-specific
Definitions
- This invention relates to a nucleic acid sequence which directs root-specific transcription of contiguous genes in plants.
- the nucleic acid sequence of the present invention can be used in transgenic plants to promote expression of endogenous and foreign genes in roots to enhance plant resistance to diseases and pests, stress (salt) tolerance, and improve nutritive value of edible root plants.
- the nucleic acid sequence of the present invention can be useful for the production of recombinant proteins in roots, with applications in molecular farming and phytoremediation.
- Plant improvement using genetic engineering techniques requires the availability of highly tissue-specific promoters, so that the expression of genes of interest may be targeted to appropriate tissues.
- the two principal reasons for that are as follows. First, high levels of expression in the desired tissue, rather than constitutive expression, is likely to be less metabolically demanding. Second, it is desirable to direct expression of foreign genes to tissues which are not used for human or animal consumption, whenever the introduced gene does not encode a nutrient protein. Despite their important role in plant development, relatively little work has been done on the regulation of gene expression in roots in comparison to shoots, and as a result regulatory sequences that promote root-specific expression are not abundant.
- the present invention involves the isolation and use of a nucleic acid sequence which directs root-specific transcription of contiguous genes in plants.
- the inventors have isolated a nucleic acid sequence which acts as a promoter from gene encoding a root-specific condensing enzyme of very long chain fatty acid biosynthesis in Lesquerellafendleri, L/KCS45.
- the present invention demonstrates that the L ⁇ CS45 5' regulatory sequence is useful in promoting transcription of heterologous genes in Arabidopsis roots.
- the nucleic acid sequence of the present invention can be used in transgenic plants to promote expression of endogenous and foreign genes in roots to enhance plant resistance to diseases and pests, stress (salt) tolerance, and improve nutritive value of edible root plants.
- the nucleic acid sequence of the present invention can be useful for the production of recombinant proteins in roots, with applications in molecular farming and phytoremediation.
- Figure 1 shows the sequence of the insert in the plasmid pLfKCS3-GUS.
- the present invention provides a nucleic acid sequence which directs root-specific transcription of contiguous genes in plants.
- the present invention shows that an isolated transcription regulatory region from the Lf CS45 gene is capable of directing expression of desired genes in a root-specific manner.
- this regulatory sequence can also promote transcription in roots of a different plant species, it is useful for a number of different applications in a variety of dicotyledonous plants. Examples of applications wherein the nucleic acid sequence of the present invention can be useful includes for example:
- roots against root-feeding insects expression of genes encoding insecticidal toxin of Bacillus thuringiensis, chitinase, proteinase inhibitor, an antibody, etc.
- nematodes expression of genes encoding a collagenase, a lectin, a proteinase inhibitor, an antibody, etc.
- disease expression of Pto gene, viral coat protein, an antibody, etc.
- a Lesquerella fendleri genomic DNA library was obtained from Dr. Chris Somerville of the Carnegie Institution of Washington, Stanford, CA.
- the genomic library was plated on E. coli LE392 (Promega) and about 150,000 clones were screened using Arabidopsis FAE1 gene (James et al., 1995) as a probe.
- the probe was prepared by PCR using ⁇ GEM-7Zf(+)-FAEl (Millar and Kunststoff, 1997) as a template with FAE1 upstream primer, 5'CCGAGCTCAAAGAGGATACATAC-3' and EJEl downstream primer, 5'GATACTCGAGAACGTTGGCACTCAGATAC-3 ⁇ PCR was performed in a lO ⁇ l
- the sequence of the insert in the plasmid pLfKCS3-GUS is shown in Figure 1.
- the pLfKCS45-GUS construct was introduced into Agrobacterium tumefaciens strain GN3101 (pMP90) (Koncz and Schell, 1986) by heat shock and selected for resistance to kanamycin (50 [ ⁇ g/mL).
- A. thaliana ecotype Columbia was transformed with A. tumefaciens harbouring the pLfKCS45-GUS construct using floral dip method (Clough and Bent, 1998). Screening for transformed seed was done on 50 [ ⁇ g/mL kanamycin as described previously (Katavic et al., 1994).
- transgenic lines were generated and examined for GUS activity.
- Histochemical localization of GUS activity in transgenic plants was done in different tissues as follows. Tissue sections were incubated in 50 mM sodium phosphate, pH 7.0, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, 0.05%(w/v) triton X-100, and 0.35 mg/ml 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronide (X-Gluc) for 4 to 7 hours at 37°C (Jefferson, 1987). Following staining the blue-stained samples were fixed in 70% ethanol.
- LfKCS45 promoter is useful for root-specific expression of foreign genes in transgenic plants and is capable of promoting transcription in plants other than Lesquerella fendleri.
- LTKCS45 promoter root-specific expression conferred by the LTKCS45 promoter is independent of the native terminator at the LJKCS45 gene 3' end.
- a terminator derived from the Agrobacterium nopaline synthase gene was used.
- sequence in the 1025 bp promoter construct is sufficient for the desired expression profile independent of ancillary sequences.
- T L -DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204: 383-396.
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002409881A CA2409881A1 (en) | 2000-05-24 | 2001-05-24 | Gene regulatory region that promotes root-specific transcription and its uses |
EP01940917A EP1283893A2 (en) | 2000-05-24 | 2001-05-24 | Gene regulatory region that promotes root-specific transcription and its uses |
BR0111081-0A BR0111081A (en) | 2000-05-24 | 2001-05-24 | Gene regulatory region that specifically promotes root transcription and uses |
AU2001274405A AU2001274405A1 (en) | 2000-05-24 | 2001-05-24 | Gene regulatory region that promotes root-specific transcription and its uses |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US20678800P | 2000-05-24 | 2000-05-24 | |
US60/206,788 | 2000-05-24 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001090387A2 true WO2001090387A2 (en) | 2001-11-29 |
WO2001090387A3 WO2001090387A3 (en) | 2002-06-27 |
Family
ID=22767963
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB2001/001137 WO2001090387A2 (en) | 2000-05-24 | 2001-05-24 | Gene regulatory region that promotes root-specific transcription and its uses |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1283893A2 (en) |
AU (1) | AU2001274405A1 (en) |
BR (1) | BR0111081A (en) |
CA (1) | CA2409881A1 (en) |
WO (1) | WO2001090387A2 (en) |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995007357A2 (en) * | 1993-09-04 | 1995-03-16 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Promoters |
WO1997005261A1 (en) * | 1995-07-28 | 1997-02-13 | North Carolina State University | Root cortex specific gene promoter |
EP0824150A2 (en) * | 1996-08-12 | 1998-02-18 | Sumitomo Chemical Company, Limited | Plant promoter and utilization thereof |
WO1998046766A1 (en) * | 1997-04-14 | 1998-10-22 | The University Of British Columbia | Nucleic acids encoding a plant enzyme involved in very long chain fatty acid synthesis |
WO1999053016A2 (en) * | 1998-04-09 | 1999-10-21 | Arizona Board Of Regents, Acting On Behalf Of Arizona State University | Expression of alfin1 and methods for producing transgenic plants having increased root growth and root specific gene activation |
WO1999054471A1 (en) * | 1998-04-20 | 1999-10-28 | Zeneca Limited | Polynucleotide sequences and their use in a method of producing plants with an increased number of stomata |
WO2001090364A2 (en) * | 2000-05-24 | 2001-11-29 | The University Of British Columbia | Nucleic acid encoding a plant very long chain fatty acid biosynthetic enzyme |
-
2001
- 2001-05-24 EP EP01940917A patent/EP1283893A2/en not_active Withdrawn
- 2001-05-24 AU AU2001274405A patent/AU2001274405A1/en not_active Abandoned
- 2001-05-24 CA CA002409881A patent/CA2409881A1/en not_active Abandoned
- 2001-05-24 WO PCT/IB2001/001137 patent/WO2001090387A2/en not_active Application Discontinuation
- 2001-05-24 BR BR0111081-0A patent/BR0111081A/en not_active Application Discontinuation
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995007357A2 (en) * | 1993-09-04 | 1995-03-16 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Promoters |
WO1997005261A1 (en) * | 1995-07-28 | 1997-02-13 | North Carolina State University | Root cortex specific gene promoter |
EP0824150A2 (en) * | 1996-08-12 | 1998-02-18 | Sumitomo Chemical Company, Limited | Plant promoter and utilization thereof |
WO1998046766A1 (en) * | 1997-04-14 | 1998-10-22 | The University Of British Columbia | Nucleic acids encoding a plant enzyme involved in very long chain fatty acid synthesis |
WO1999053016A2 (en) * | 1998-04-09 | 1999-10-21 | Arizona Board Of Regents, Acting On Behalf Of Arizona State University | Expression of alfin1 and methods for producing transgenic plants having increased root growth and root specific gene activation |
WO1999054471A1 (en) * | 1998-04-20 | 1999-10-28 | Zeneca Limited | Polynucleotide sequences and their use in a method of producing plants with an increased number of stomata |
WO2001090364A2 (en) * | 2000-05-24 | 2001-11-29 | The University Of British Columbia | Nucleic acid encoding a plant very long chain fatty acid biosynthetic enzyme |
Non-Patent Citations (3)
Title |
---|
DATABASE EMBL [Online] ACCESSION NO: AJ011628 , 26 September 1999 (1999-09-26) CARDON, G.H.: "Arabidopsis thaliana (ecotype Landsberg erecta) mRNA for squamosa promoter binding protein-like 1" XP002195417 * |
DEVIC MARTINE ET AL: "Efficient PCR walking on plant genomic DNA" PLANT PHYSIOLOGY AND BIOCHEMISTRY, GAUTHIER-VILLARS, PARIS, FR, vol. 35, no. 4, 1997, pages 331-339, XP000926417 ISSN: 0981-9428 * |
SCHRODER-PONTOPPIDAN M ET AL: "Very long chain and hydroxylated fatty acids in offspring of somatic hybrids between Brassica napus and Lesquerella fendleri." THEORETICAL AND APPLIED GENETICS, vol. 99, no. 1-2, July 1999 (1999-07), pages 108-114, XP002195215 ISSN: 0040-5752 * |
Also Published As
Publication number | Publication date |
---|---|
BR0111081A (en) | 2003-04-08 |
AU2001274405A1 (en) | 2001-12-03 |
EP1283893A2 (en) | 2003-02-19 |
WO2001090387A3 (en) | 2002-06-27 |
CA2409881A1 (en) | 2001-11-29 |
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