WO2001051637A1 - Promoteur de plante fruit specifique - Google Patents
Promoteur de plante fruit specifique Download PDFInfo
- Publication number
- WO2001051637A1 WO2001051637A1 PCT/FR2001/000069 FR0100069W WO0151637A1 WO 2001051637 A1 WO2001051637 A1 WO 2001051637A1 FR 0100069 W FR0100069 W FR 0100069W WO 0151637 A1 WO0151637 A1 WO 0151637A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- promoter
- sequence
- gene
- nucleic acid
- fruit
- Prior art date
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)
- C12N15/8216—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
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8235—Fruit-specific
-
- 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
Definitions
- the present invention relates to a DNA sequence having a transcriptional promoter activity directing the expression of the gene or genes placed under its control in the fruits of a plant.
- the invention also relates to a recombinant nucleic acid molecule as a vector comprising said promoter, as well as plant cells and transgenic plants which have incorporated this promoter into their genome.
- the work carried out within the framework of the present invention has made it possible to isolate a new promoter from the wild strawberry genome (Fragaria vesca cv Reine des vallées) having remarkable specificity properties in the fruit, effectiveness, inducibility from the first stages of fruit ripening, as well as adaptability to other fruit species, such as tomatoes or grapes.
- the subject of the invention is a promoter which selectively directs the expression of the DNA sequences placed under its control in the fruits of plants, characterized in that it comprises all or part of the sequence No. 1 of the attached sequence list, its complementary strand or a derivative thereof. More particularly, the invention relates to the promoter sequence comprised between the nucleotides at positions 1 and 1393, its complementary strand or a derivative thereof exhibiting promoter activity which selectively directs the expression of DNA sequences placed under his control in the fruits of plants.
- derivative of a promoter sequence according to the invention is intended to mean the above sequences modified by deletion, substitution or insertion of one or more nucleotides, and retaining a promoter activity which selectively directs the expression of DNA sequences. placed under their control in the fruits of plants. These are for example sequences having at least 60% and preferably at least 80% homology with the sequence No. 1 of the sequence list in the appendix. Such derivatives are more particularly the promoter sequences of other rosacea homologs to that of the wild strawberry genome (Fragaria vesca cv Reine des vallées) isolated in the context of the present invention.
- a person skilled in the art is able to determine, from tests of the type of those reported in the experimental part below, whether said derivatives exhibit a promoter activity which selectively directs the expression of the DNA sequences placed under their control. in the fruits of plants according to the present invention.
- a promoter according to the invention opens up very important prospects in the industrial field by the creation of new varieties but also in the field of research both fundamental and applied.
- the creation of transgenic strawberries carrying the construction of this promoter associated with a gene either of agronomic interest or of ill-defined function makes it possible to understand the role of these genes in the processes of fruit ripening. It is the only current tool that allows us to understand the role of genes and their product in vivo and their effects on fruit ripening.
- the genes known to influence the organoleptic qualities of fruits can be integrated into strawberry plants downstream of this promoter in order to exacerbate ( gene in the sense position) or reduce (gene in the antisense position) the effects associated with these genes.
- Genes supposed to influence the quality are currently known and their impact on the maturation processes can be analyzed in vivo like certain parietal hydrolases implied in the loss of firmness of the fruits (cellulase, Pectin Methyl Transferase, Polygalacturonase, ⁇ -galactosidase, expansins, etc. .), carotenoid and anthocyanin biosynthetic enzymes
- the promoter according to the invention makes it possible to target the expression of a gene spatio-temporally in the fruit from the turning stage (corresponding to the initiation of the maturation processes) and gradually during the maturation. It differs from the promoters currently used as: - the 35S CAMV promoter which directs the expression of genes constitutively in the plant, or
- the promoter according to the invention directs the expression of genes in the fruit at a marketable stage (stage turning to the mature stage).
- the invention therefore also relates to a recombinant nucleic acid molecule comprising a promoter sequence as defined above linked operatively to a DNA sequence comprising one or more genes, optionally a selection gene placed under the control of its own promoter or of the same promoter as said nucleic acid sequence, and advantageously a terminator sequence placed downstream of said DNA sequence.
- the said gene (s), other than the selection gene are advantageously genes, in the sense or antisense position, encoding a protein of agronomic interest. As indicated previously, it is for example a protein capable of influencing the organoleptic qualities of fruits, preferably strawberries.
- a recombinant nucleic acid molecule according to the invention can be a cassette or a vector for the expression of one or more genes of interest in a plant cell.
- a vector useful for the transformation of a plant-like host includes:
- the invention therefore also relates to plant cells in the genome of which is stably incorporated a recombinant nucleic acid molecule comprising the previously defined promoter operably linked to a DNA sequence comprising at least one gene which is thus expressed only under the control of the promoter.
- the invention also relates to a plant which has stably incorporated into its genome a recombinant nucleic acid molecule comprising the previously defined promoter operably linked to a DNA sequence comprising at least one gene which is thus expressed only in fruits. of the plant.
- Transgenic plants according to the invention can be prepared in a conventional manner by transforming a plant cell with said nucleic acid molecule and then by regenerating a plant from the transformed cell.
- the gus gene has the property of coding for the ⁇ -glucuronidase enzyme, the activity of which is easily detectable either by direct staining of the tissues (histochemical technique) or by enzymology (fluorimetric assay technique).
- the binary plasmid used is the plasmid pBilOl, derived from the vector pBIN19 (2). This vector has
- an origin of RK2 replication with a low copy number (i) an origin of RK2 replication with a low copy number, (ii) an np tll gene allowing the selection of bacteria transformed on kanamycin, (iii) the RB and LB terminals ensuring the transfer of the DNA placed between these terminals.
- This transfer DNA consists of an nptll gene framed by the promoter and terminator NOS (NOS-Pr and NOS-Ter) allowing the selection of transformed plants, a gus gene of 1.87 Kb without promoter (gene derived from the plasmid pRAJ260) inserted at the Kpn1 site in the multicloning region, the 260pb NOS terminator clones Sstl-EcoRI in the multicloning region (2).
- 3 plasmids pBIlOl were constructed distinct from 1 bp (pBHOl.l, pBil01.2, pBil01.3).
- the plasmid pBil01.2 was used to achieve the fusion between the FPl promoter and the gus gene.
- the promoter sequence was amplified with specific primers to which the restriction site sequences were added:
- AGL1 chromosome C58 (resistance to Rifampicin), plasmid Ti of the strain B0542 (resistance to ampicillin).
- C58GV2260 chromosome C58 (resistance to Rifampicin), plasmid Ti of the strain B6806 (resistance to ampicillin).
- strains were named A and B respectively.
- Leaf explants of about 0.5 cm 2 are cut from leaves about 6 to 8 weeks old on strawberry plants grown in vitro. They are transferred to a regeneration medium (Murashige and Skoog medium, 3% sucrose, 0.1% Gamborg vitamin (B5), 18 ⁇ M of Benzyl Amino Purine, 2.5 ⁇ M of Indol-3 butyric acid). Twenty-four hours later, the explants are brought into contact with solutions of Agrobacteria (A or B). The concentration of Agrobacteria is evaluated at 1.10 9 cells / ml. Co-culture is carried out at 28 ° C for 1 hour with stirring at 100 rpm.
- the explants are then drained on sterile filter paper and placed on the regeneration medium in the presence of 100 ⁇ M of acetosyringone. Forty-eight hours later, the explants are transferred to the regeneration medium with 500 mg / l of timentin. The explants with swelling and callous formations are subcultured every 3 weeks on new regeneration medium with 500 mg / l of timentin.
- the first shoots appear 2 months after transformation and are transplanted individually on elongation medium (Murashige and Skoog medium, 3% sucrose, 0.1% Gamborg vitamin (B5), 2.5 ⁇ M of Benzyl Amino Purine, 1 , 25 ⁇ M of Indol-3 butyric acid) in the presence of 500 mg / 1 of timentin and 25 mg / 1 of kanamycin.
- elongation medium Merashige and Skoog medium, 3% sucrose, 0.1% Gamborg vitamin (B5), 2.5 ⁇ M of Benzyl Amino Purine, 1 , 25 ⁇ M of Indol-3 butyric acid
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP01905853A EP1248845A1 (fr) | 2000-01-10 | 2001-01-10 | Promoteur de plante fruit specifique |
AU2001233829A AU2001233829A1 (en) | 2000-01-10 | 2001-01-10 | Specific fruit- plant promoter |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR00/00237 | 2000-01-10 | ||
FR0000237A FR2803600B1 (fr) | 2000-01-10 | 2000-01-10 | Promoteur de plante fruit specifique |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001051637A1 true WO2001051637A1 (fr) | 2001-07-19 |
Family
ID=8845741
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2001/000069 WO2001051637A1 (fr) | 2000-01-10 | 2001-01-10 | Promoteur de plante fruit specifique |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1248845A1 (fr) |
AU (1) | AU2001233829A1 (fr) |
FR (1) | FR2803600B1 (fr) |
WO (1) | WO2001051637A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003029399A2 (fr) * | 2001-10-03 | 2003-04-10 | Universidad De Malaga | Sequence regulatrice de l'expression specifique en fruit d'un gene et applications correspondantes |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4943674A (en) * | 1987-05-26 | 1990-07-24 | Calgene, Inc. | Fruit specific transcriptional factors |
US5608150A (en) * | 1993-07-12 | 1997-03-04 | Monsanto Company | Fruit specific promoters |
WO1997027295A1 (fr) * | 1996-01-23 | 1997-07-31 | Horticulture Research International | Genes associes au murissement des fruits |
WO1998031812A1 (fr) * | 1997-01-21 | 1998-07-23 | Monsanto Company | Promoteurs et genes de fraises |
-
2000
- 2000-01-10 FR FR0000237A patent/FR2803600B1/fr not_active Expired - Fee Related
-
2001
- 2001-01-10 EP EP01905853A patent/EP1248845A1/fr not_active Withdrawn
- 2001-01-10 WO PCT/FR2001/000069 patent/WO2001051637A1/fr not_active Application Discontinuation
- 2001-01-10 AU AU2001233829A patent/AU2001233829A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4943674A (en) * | 1987-05-26 | 1990-07-24 | Calgene, Inc. | Fruit specific transcriptional factors |
US5608150A (en) * | 1993-07-12 | 1997-03-04 | Monsanto Company | Fruit specific promoters |
WO1997027295A1 (fr) * | 1996-01-23 | 1997-07-31 | Horticulture Research International | Genes associes au murissement des fruits |
WO1998031812A1 (fr) * | 1997-01-21 | 1998-07-23 | Monsanto Company | Promoteurs et genes de fraises |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003029399A2 (fr) * | 2001-10-03 | 2003-04-10 | Universidad De Malaga | Sequence regulatrice de l'expression specifique en fruit d'un gene et applications correspondantes |
ES2188400A1 (es) * | 2001-10-03 | 2003-06-16 | Univ Malaga | Secuencia reguladora de la expresion especifica en fruto de un gen y sus aplicaciones. |
WO2003029399A3 (fr) * | 2001-10-03 | 2004-03-04 | Univ Malaga | Sequence regulatrice de l'expression specifique en fruit d'un gene et applications correspondantes |
Also Published As
Publication number | Publication date |
---|---|
EP1248845A1 (fr) | 2002-10-16 |
FR2803600B1 (fr) | 2002-04-05 |
AU2001233829A1 (en) | 2001-07-24 |
FR2803600A1 (fr) | 2001-07-13 |
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