WO2003012106A2 - Regulating nucleic acid for expressing a polynucleotide of interest specifically in the endothelium of a plant seed and uses thereof - Google Patents

Regulating nucleic acid for expressing a polynucleotide of interest specifically in the endothelium of a plant seed and uses thereof Download PDF

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Publication number
WO2003012106A2
WO2003012106A2 PCT/FR2002/002784 FR0202784W WO03012106A2 WO 2003012106 A2 WO2003012106 A2 WO 2003012106A2 FR 0202784 W FR0202784 W FR 0202784W WO 03012106 A2 WO03012106 A2 WO 03012106A2
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Prior art keywords
nucleic acid
plant
polynucleotide
nucleotide
expression
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PCT/FR2002/002784
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French (fr)
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WO2003012106A3 (en
Inventor
Loïc LEPINIEC
Isabelle Debeaujon
Martine Devic
Michel Caboche
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Institut National De La Recherche Agronomique (Inra)
Centre National De La Recherche Scientifique (Cnrs)
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Priority to EP02772481A priority Critical patent/EP1414966A2/en
Publication of WO2003012106A2 publication Critical patent/WO2003012106A2/en
Publication of WO2003012106A3 publication Critical patent/WO2003012106A3/en

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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
    • 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
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/829—Female sterility

Definitions

  • the invention relates to the field of the controlled improvement of agronomic quality of various plants, including fruit plants and field crops. It relates to the targeted expression of polynucleotides and / or polypeptides of interest specifically in certain tissues of the plant, and more specifically in the endothelium and / or the albumen of a plant seed.
  • Testa also known as seed coat, is an essential tissue for seed development and viability. When the seed is fully mature, the testa constitutes a protective layer of the internal parts of it. During the development of the seed, the testa is an import fabric of nutrients vital for the development of the embryo. The seed behaves like a “parasite” towards the mother plant, with regard to its nutrition. All raw nutrients necessary for seed growth must be imported from the mother plant. In the seeds of dicotyledonous plants, the vascular tissue enters the seed through the funiculus, then anastomoses in the testa tissue of the seed. There is no connection of vascular tissue between the testa of the seed and the embryo. Thus, all the nutrients released in the developing seed must first be imported into the testa, before being released by diffusion into the embryo.
  • testa the integuments (testa) are involved in many aspects determining the quality of the seed, the most important of which are: - the nutrition of the embryo (Weber et al., 1996)
  • the testa being a protective organ of the embryo from the seed, it may be desirable to affect its development in order to obtain plants that do not have fertile seeds or even plants devoid or practically devoid of seeds.
  • Obtaining plants incapable of producing seeds is of considerable interest in the field of fruit plants. Indeed, fruits devoid of seeds have taste qualities superior to those observed for identical fruits in which the seeds are present.
  • the seeds are often hard and have a bitter taste. In some cases, like grapes, eating the seeds can cause digestive upset.
  • the natural cavities in which they are normally present are replaced by the edible tissue of the fruit, which makes a seedless fruit much more attractive to the consumer.
  • the promoter of the FBP7 gene isolated from the petunia genome has been used to control the expression of the barnase gene in ova and in developing seeds (Colombo et al., 1997). By placing a marker gene under the control of the FBP7 promoter, these authors observed expression of the reporter gene in the testa of the developing seed except for the outer cell layer, as well as in the epidermis of the placenta.
  • the promoter of the FBP7 gene is not specific to testa, since it is also active in the epidermis of the placenta.
  • the applicant has sought to characterize new functional promoter sequences in a plant capable of directing the expression of one or more sequences of interest placed under control, specifically in the testa of the seed.
  • a first object of the invention consists of a nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID N ° 1.
  • the invention also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid as defined above, the polynucleotide of interest being chosen from a polynucleotide coding for a polypeptide and a sense or antisense polynucleotide.
  • the invention also relates to a recombinant cloning and / or expression vector comprising a nucleic acid or an expression cassette as defined above, as well as to a host cell transformed by this nucleic acid or this cassette. expression.
  • the subject of the invention is also the use of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell as defined above for obtaining a plant. transformed.
  • the invention also relates to methods for obtaining a plant transformed with a nucleic acid, an expression cassette, a recombinant vector or a transformed host cell as defined above, as well as to transformed plants. obtained according to these processes and to parts of these transformed plants, in particular fruits and seeds.
  • a nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID - -N ° 1. - - _ _ _ _ _ _ _ _
  • the invention also relates to a nucleic acid of sequence complementary to the nucleic acid as defined above.
  • any conventional technique of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are described for example by Sambrook et al. (1989), Glover et al. (1985), Gait (1984), Hames and Higgins (1985) and Ausubel et al. (1994).
  • a nucleic acid according to the invention is preferably presented in an isolated or purified form.
  • isolated within the meaning of the present invention designates a biological material which has been removed from its original environment
  • a naturally occurring polynucleotide in a plant is not isolated.
  • the same polynucleotide separated from adjacent nucleic acids within from which it is naturally inserted into the genome of the plant is isolated.
  • Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
  • purified does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
  • a polynucleotide or a polypeptide is in the purified state after purification of the starting material or the natural material of at least one order of magnitude, preferably 2 or 3 and preferably four or five orders of magnitude.
  • nucleotide sequence can be used to denote either a polynucleotide or a nucleic acid.
  • nucleotide sequence encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
  • nucleic acid refers to any organic acid
  • polynucleotide refers to any organic compound
  • oligonucleotide or “nucleotide sequence” includes RNA, DNA, cDNA or hybrid sequences
  • RNA / DNA of more than one nucleotide either in the single-stranded form or in the form of duplex.
  • nucleotide refers to both natural nucleotides
  • modified nucleotides which comprise at least one modification such as (i) a purine analog, (ii) a pyrimidine analog, or (iii) a sugar analog, such modified nucleotides being described for example in the application PCT N c WO
  • a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.
  • the complementary bases are A and T (or A and U), and C and G.
  • a first nucleic acid having at least 80% identity with a second reference nucleic acid will have at at least 80%, preferably at least 85%, 90%, 95%, 98%, 99% or 99.5% of nucleotide identity with this second reference polynucleotide, the percentage of identity between two sequences being determined as described below.
  • the "percentage of identity" between two nucleotide or amino acid sequences can be determined by comparing two optimally aligned sequences, through a comparison window.
  • the part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percentage of sequence identity.
  • the optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
  • the percentage of sequence identity is determined using the BLAST software (BLAST version 2.06 of September 1998), using exclusively the default parameters.
  • a nucleic acid having at least 80% nucleotide identity with a nucleic acid according to the invention includes the "variants" of a nucleic acid according to the invention.
  • variant of a nucleic acid according to the invention is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, relative to the nucleic acid of reference.
  • a variant of a nucleic acid according to the invention can be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid may also be an unnatural nucleic acid obtained, for example, by mutagenesis techniques.
  • the differences between the reference nucleic acid and the "variant" nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid have very similar nucleotide sequences and, in many regions , identical.
  • a "Variant" of a regulatory nucleic acid according to the invention retains its ability to direct the expression of a polynucleotide of interest specifically in the endothelium and / or the albumen of a plant seed, if necessary. when this variant is associated with a regulatory sequence with a promoter function.
  • a "variant" of a nucleic acid according to the invention including a nucleic acid having at least 80% identity in nucleotides with the sequence SEQ ID N ° 1 or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1, a person skilled in the art can carry out tests for the specific expression of a reporter or marker gene described in the examples.
  • fragment of a nucleic acid according to the invention is meant a nucleotide sequence of a reduced length compared to the reference nucleic acid, the nucleic acid fragment having a nucleotide sequence identical to the nucleotide sequence of the reference nucleic acid on the common part.
  • Such fragments of a nucleic acid according to the invention have at least 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600 , 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 2000 or 2300 consecutive nucleotides of the reference nucleic acid, the maximum length in nucleotides of a fragment of a nucleic acid according to the invention being indeed understood to be limited by the maximum length in nucleotides of the reference nucleic acid.
  • a "fragment" of a regulatory nucleic acid according to the invention comprises the polynucleotide defined by the sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID No. 1.
  • a "fragment” comprises the polynucleotide defined by the sequence going from the nucleotide at position 2223 to the nucleotide at position 2228, of the sequence SEQ ID No. 1, which corresponds to the motif "CANNTG".
  • variants and fragments of a regulatory nucleic acid according to the invention are “biologically active", because they retain their ability to direct (if they comprise a promoter sequence) or to modulate (for example modulate the activity of a promoter sequence placed nearby), the expression of a polynucleotide of interest, specifically in the endothelium, or in both the endothelium and the aleurone cell layer of a seed of plant, as can be verified by a person skilled in the art using the techniques described in the examples.
  • nucleic acids comprising regulatory signals within the meaning of the invention, is meant a nucleic acid capable of influencing the expression characteristics of a polynucleotide of interest placed under the control of this regulatory nucleic acid, in l occurrence a nucleic acid whose sequence makes it possible to confer a specific expression of tissue, and more particularly capable of conferring a specific expression of the polynucleotide of interest in the endothelium and / or the albumen of a plant seed.
  • Such a nucleic acid may comprise, in addition to regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, also a sequence with a promoter function. .
  • a nucleic acid with a “promoter” function also designated “promoter” or even “promoter sequence”, consists of a nucleic acid which has the recognition patterns of RNA polymerase, and more specifically of a “TATA” box and a “CAAT” box, the structure of which is well known to those skilled in the art.
  • nucleic acid of sequence SEQ ID N ° 1 comprises the regulatory signals necessary for expression of a polynucleotide of interest, specifically in the endothelium of a plant seed, the nucleic acid of sequence SEQ ID No. 1 also comprising a promoter sequence capable of initiating the transcription of a polynucleotide d interest under his control.
  • SEQ ID No. 1 is the first regulatory nucleic acid capable of directing the expression of a polynucleotide of interest specifically in the endothelium of a plant seed. Such specificity of expression had not been observed with the regulatory sequences described by Fobert et al. (1994) or by Colombo et al. (1997) whose expression was not limited to the endothelium but extended to several cell types present in the seed testa.
  • nucleic acid of sequence SEQ ID No 1 is capable of directing the expression d a polynucleotide of interest, for example the reporter gene GUS, optimally as soon as the ovum forms. Consequently, the nucleic acid of sequence SEQ ID No 1 or a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1 as defined above, in addition to its specific activity of the endothelium also allows early expression, from the last stages of ovum development, of a polynucleotide of interest placed under its control.
  • This characteristic of early temporal activity of the regulatory nucleic acid according to the invention makes it possible to express the polynucleotide of interest from the first stages of seed formation, for example to modify the composition or else to alter very early its development, which could not be envisaged with the promoter sequences described in the state of the art.
  • the reporter gene GUS is shown according to the invention as regulatory signals to specifically direct the expression of a polynucleotide of interest in the seed endothelium, and where appropriate also in the aleurone layer of the albumen, were located on the 3 'side of the sequence SEQ ID No. 1.
  • the nucleic acid of sequence SEQ ID No. 1 according to the invention has a length of 2376 nucleotides.
  • each of these DNA constructs comprising a fragment of the nucleic acid ranging from the nucleotide in position 1 to the nucleotide in position 2335 of the sequence SEQ ID No. 1.
  • the DNA constructs also include a promoter-functional nucleic acid.
  • Expression cassettes were produced comprising a polynucleotide of interest coding for a reporter gene, the GUS gene, which was placed under the control of a regulatory nucleic acid as defined above, comprising both the tissue-specific regulatory signals and a promoter-functioning nucleic acid.
  • results of the examples show that the regulatory signals necessary for tissue-specific transcription activity in the plant seed are included in the nucleic acid ranging from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1, which corresponds to the expression of the reporter gene obtained with the construction designated pBAN6.
  • the regulatory nucleic acid present in the construction pBAN6 contains a "CANNTG" motif located from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1.
  • the CANNTG motif constitutes a potential site for binding to transcription factors of the MYC type, as it results from the structural study of the nucleic acid regulating sequence SEQ ID No. 1 using an operating computer. information from the PLACE (Plant cis-acting Regulatory Elements) database data base; HIGO et al., 1999).
  • the inventors believe that the CANNTG motif localized from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1 constitutes an important structural characteristic of the tissue-regulating nucleic acid. specific to the invention.
  • preliminary results indicate the presence of a motif of binding of a transcription factor repressing expression in the albumen, this motif being located in a nucleotide region extending from the nucleotide at position 1919 to the nucleotide at position 2054 of the nucleic acid of sequence SEQ ID No. 1.
  • the promoter-function nucleic acid contained in the nucleic acid of sequence SEQ ID No. 1 is entirely contained in the sequence going from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1.
  • This sequence contains a “TATA” box located from the nucleotide in position 2306 to the nucleotide in position 2312 of the sequence SEQ ID No. 1.
  • This promoter sequence also contains a “CAAT” box located from the nucleotide at position 2172 to the nucleotide at position 2180 of the sequence SEQ ID No. 1.
  • the TATA box constitutes the promoter element proper, which is generally located at a distance of about 30 bases from the site of initiation of transcription.
  • the CAAT box is a cis-acting element which is commonly found in the promoter and activator region ("enhancer").
  • nucleic acid with a promoter function ranging from the nucleotide at position 2172 to the nucleotide at position 2312 of the sequence SEQ ID No. 1, as well as any “biologically active” fragment of this promoter nucleic acid and containing the CAAT and TATA boxes defined above constitutes another object of the present invention.
  • nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% identity in nucleotides with the sequence SEQ ID No 1, or with a fragment of at least 250 consecutive nucleotides of the sequence SEQ ID No 1.
  • such a regulatory nucleic acid will preferably comprise the CANNTG motif ranging from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1.
  • such a nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2273 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is included in particular in the construction pBAN ⁇ of the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2141 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is included in particular in the construction designated pBAN7 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is in particular included in the construction pBAN6 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1919 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is included in particular in the construction pBAN5 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1510 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • Such a regulatory nucleic acid is in particular included in the construction pBAN4 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 101 1 to the nucleotide in position 2335 of the sequence SEQ ID N ° 1.
  • a regulatory nucleic acid is included in the construction designated pBAN3 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 509 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is in particular included in the construction pBAN2 described in the examples.
  • the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
  • a regulatory nucleic acid is included in the construction designated pBAN1 described in the examples.
  • Each of the above nucleic acids can also comprise the sequence going from the nucleotide at position 2336 to the nucleotide at position 2376 of the sequence SEQ ID No. 1.
  • a regulatory nucleic acid comprising at least the sequence going from the nucleotide at position 1919 to the nucleotide at position 2335 of the sequence SEQ ID N ° 1.
  • a shorter regulatory nucleic acid will preferably be used, for example example a regulatory nucleic acid comprising a sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID No 1, devoid of all or part of the sequence 1918-2053 of the sequence SEQ ID No 1.
  • the present invention also relates to a nucleic acid whose sequence is complementary to the sequence of any one of the nucleic acids as defined above.
  • a regulatory nucleic acid as defined above can comprise only the regulatory signals making it possible to modulate the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, without include promoter-function nucleic acid.
  • tissue-specific activity of a regulatory acid according to the invention are exploited by the introduction, into this regulatory nucleic acid, also of a nucleic acid with a promoter function, more specifically of a promoter-functional polynucleotide that is functional in a plant cell.
  • this regulatory nucleic acid is characterized in that it further comprises a polynucleotide with promoter function which is functional in a plant cell.
  • the promoter-functional polynucleotide is the nucleotide sequence going from the nucleotide in position 2172, first nucleotide of the CAAT box, to the nucleotide in position 2312, last nucleotide of the “TATA” box of the sequence SEQ ID N ° 1 or a variant or a biologically active fragment of this sequence.
  • a variant of the nucleic acid with a promoter function defined above comprises a nucleotide sequence having at least 80% identity with the sequence going from the nucleotide in position 2172 to the nucleotide in position 2312 of the sequence SEQ ID N ° 1 and is capable of directing the expression of a polynucleotide of interest in a plant cell.
  • a variant includes the CAAT and TATA boxes defined previously in the present description.
  • a such a nucleic acid fragment can also comprise the nucleotide sequence going from the nucleotide in position 2313 to the nucleotide in position 2376 of the nucleic acid of sequence SEQ ID No. 1.
  • one of the objectives pursued by the present invention is to obtain a controlled expression of a polynucleotide of interest, specifically in the endothelium and / or the aleurone cell layer of the albumen of a plant seed, for example in situations where one seeks to: - modify the composition or the hormonal balance of the testa of the seed;
  • the invention therefore also relates to a nucleic acid comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above.
  • a nucleic acid is also designated “expression cassette” for the purposes of the present description.
  • the invention therefore also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above.
  • an expression cassette according to the invention is characterized in that the polynucleotide of interest codes for a polypeptide.
  • the polynucleotide of interest codes for a polypeptide involved in the regulation of the tannin or fiber content of the seed or also for a polypeptide regulating the hormonal balance in the testa of the seed.
  • the modulation of the metabolism of phenolic compounds can be conceived using a gene coding for transcription factors, for example of the Myc type (Damiani et al., 1999; Nesi et al., 2000) or of the Myb type (Grotewold et al. , 1998; Borevitz et al., 2000), as well as structural genes coding for enzymes of the biosynthetic pathway or for transporters (Debeaujon et al., 2001).
  • the protein BAN (probably a leukocyanidine reductase according to Devic et al., 1999) is a key enzyme leading to the formation of tannins, it is likely that its suppression or over-expression lead to the absence and increase of tannins respectively, this without affecting the levels of other flavonoids; the suppression of CHS (chalcone synthase) leads to the absence of flavonoids; as for LDOX (leucocyanidine deoxygenase) and FLS (flavonol synthase) (Pelletier et al., 1997), their modulation should lead to increasing or suppressing anthocyanins and flavonols, respectively.
  • CHS chalcone synthase
  • FLS flavonol synthase
  • lignin metabolism genes such as those encoding the enzymes F5H (ferulate-5-hydroxylase; Meyer et al., 1998; Franke et al., 2000), cinnamyl-alcohol dehydrogenase and cinnamoyl-CoA reductase (Ralph et al., 1998) are candidates for modification of these compounds.
  • Genes coding for enzymes of the cellulose biosynthetic pathway (Fagard et al., 2000) can be envisaged to modify this compound.
  • the genes coding for testa invertases (Weber et al.,
  • Manipulation of hormone biosynthesis genes to modify the structural characteristics of testa can also be conceived, in particular for gibberellins with GA1 (Sun and Kamiya, 1994), and the gene for GA-20-oxidase (Coles and al., 1999; Kang et al., 1999), for abscissic acid with ABA2 coding for zeaxanthin epoxidase (Marin et al., 1999), for ethylene with the enzyme ACC synthase (Gray et al., 1992), for brassinosteroids with DET2 coding for a steroid 5-alpha-reductase (Noguchi et al., 1999), for auxins with iaaM (Rotino et al., 1997), for cytokinins with ipt (Barry et al., 1984); see the review by Hedden and Phillips (2000) for more details.
  • a polynucleotide of interest whose expression is toxic for the plant cell will be preferred, for example a polynucleotide coding for a polypeptide toxic to the plant cell, or a polynucleotide capable of specifically inhibiting or blocking the function of one or more genes involved in the development of the seed.
  • the sequences coding for the following polypeptides will preferably be chosen:
  • testa such as protease inhibitors for protection against insects (Jouanin et al., 2000), or cellular toxins such as barnase (Colombo et al., 1997; Varoquaux et al., 2000) or diphtheria toxin A (Czaco et al., 1992) to reduce the size of the seed (Koltunow and Brennan, 1998; Koltunow et al., 1998) or to perform genetic ablation of the seed and therefore obtaining fruit without seeds.
  • protease inhibitors for protection against insects
  • cellular toxins such as barnase (Colombo et al., 1997; Varoquaux et al., 2000) or diphtheria toxin A (Czaco et al., 1992)
  • Kunco et al., 1992 diphtheria toxin A
  • the polynucleotide of interest whose expression is toxic to the plant cell can be a sense or antisense polynucleotide capable of inhibiting or blocking respectively the transcription or the translation of a gene required for normal development of the seed, and preferably of a gene required for the normal development of the testa of the seed, especially the testa.
  • an antisense polynucleotide according to the invention hybrid, within the cell, specifically with a region of the target gene comprising the site of initiation of transcription and / or with a sequence important for the correct splicing of RNA messenger.
  • an antisense polynucleotide according to the invention is a nucleic acid complementary to the messenger RNA constituting the transcript of the target gene and comprising the site of initiation of the translation.
  • an antisense polynucleotide can be the DNA complementary to the messenger RNA of the targeted gene.
  • the polynucleotide of interest which is placed under the control of a regulatory nucleic acid according to the invention, codes for a polypeptide consisting of a fusion protein between a glucocorticoid receptor and the protein GAL-4.
  • the invention also relates to recombinant cloning and / or expression vectors comprising a regulatory nucleic acid or an expression cassette as defined above.
  • Another subject of the invention therefore consists of recombinant vectors into which a regulatory nucleic acid according to the invention has been inserted, comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under its control, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment at least 250 consecutive nucleotides of the sequence SEQ ID No. 1.
  • a recombinant cloning and / or expression vector comprises an expression cassette comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above.
  • recombinant vectors for the expression of a polynucleotide of interest in the endothelium and / or the albumen of a plant seed comprising: a) a regulatory nucleic acid as defined in this description; and b) a polynucleotide of interest placed under the control of the regulatory nucleic acid defined in a).
  • the polynucleotide of interest can consist of a polynucleotide coding for a detectable polypeptide, or marker polypeptide such as for example a polypeptide coding for the GUS protein or also for a fluorescent protein, such as the GFP (Green fluorescent protein) or YFP (Yellow fluorescent protein).
  • marker polypeptide such as for example a polypeptide coding for the GUS protein or also for a fluorescent protein, such as the GFP (Green fluorescent protein) or YFP (Yellow fluorescent protein).
  • the polynucleotide of interest encodes a polypeptide involved in the regulation of tannin or fiber content of the seed, or a polypeptide regulating hormonal balance in the testa of grain.
  • the polynucleotide of interest can also be a polynucleotide whose expression is toxic to the plant cell, as defined previously in the present description.
  • a recombinant vector according to the invention corresponding to the above definition is the vector pBAN1 :: GUS / pBIB-Hyg contained in the strain of E. coli deposited at the CNCM on July 17, 2001 under the access number I-2703 .
  • vectors meeting the general definition of a recombinant vector according to the invention are respectively the vectors pBAN2 :: GUS, pBAN3 :: GUS, pBAN4 :: GUS, pBAN5 :: GUS, pBAN6: GUS pBAN7: GUS and PBAN8 : GUS illustrated in the examples.
  • the vector pBAN1 :: GUS comprises all of the regulatory nucleic acid with sequence SEQ ID No. 1.
  • the vector pBAN2 comprises the regulatory nucleic acid going from the nucleotide in position 509 to the nucleotide in position 2376 of the sequence SEQ ID N ° 1, that is to say the nucleic acid SEQ ID N ° 14 .
  • the vector pBAN3 comprises the sequence going from the nucleotide at position 1011 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 13.
  • the vector pBAN4 comprises the regulatory nucleic acid ranging from the nucleotide at position 1510 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 12.
  • the vector pBAN5 comprises a regulatory nucleic acid ranging from the nucleotide at position 1919 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 1 1.
  • the vector pBAN6 comprises a regulatory nucleic acid ranging from the nucleotide in position 2054 to the nucleotide in position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 10.
  • the vector pBAN7 comprises a regulatory nucleic acid ranging from the nucleotide at position 2141 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 9.
  • the vector pBAN ⁇ :: GUS comprises a regulatory nucleic acid ranging from the nucleotide at position 2273 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 8.
  • the nucleic acid of sequence SEQ ID No 1 can be amplified using the primers of sequences SEQ ID No 15 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No. 14 can be amplified using the primers of sequences SEQ ID No. 16 and SEQ ID No. 23.
  • the nucleic acid of sequence SEQ ID No 13 can be amplified using the primers of sequences SEQ ID No 17 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No 12 can be amplified using the primers of sequences SEQ ID No 18 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No 11 can be amplified using the primers of sequences SEQ ID No 19 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No 10 can be amplified using the primers of sequences SEQ ID No 20 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No 9 can be amplified using the primers of sequences SEQ ID No 21 and SEQ ID No 23.
  • the nucleic acid of sequence SEQ ID No. 8 can be amplified using the primers of sequences SEQ ID No. 22 and SEQ ID No. 23.
  • vector in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form.
  • a recombinant vector according to the invention is either a cloning vector, an expression vector, or more specifically a insertion vector, a transformation vector or an integration vector.
  • It can be a vector of bacterial or viral origin.
  • a recombinant vector according to the invention is used in order to amplify the nucleic acid which is inserted therein after transformation or transfection of the desired cellular host.
  • a recombinant vector according to the invention is used to express a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, and in this case specifically in the layer cell aleurone from the albumen of the plant seed.
  • a recombinant vector according to the invention advantageously also comprises sequences for initiating and stopping the appropriate transcription.
  • the recombinant vectors according to the invention may include one or more origins of functional replication in cellular hosts in which their amplification or expression is sought, as well as nucleotide selection marker sequences.
  • a recombinant vector according to the invention is an integrative vector allowing the insertion of multiple copies of the DNA sequence inserted in this vector into the genome of a plant, the transformation of which by a nucleic acid according to l invention is sought.
  • the recombinant vector, or in other cases the expression cassette contained in this vector can also contain 5 'untranslated sequences called "leaders". Such sequences can increase translation.
  • the vectors according to the invention can also comprise so-called "terminator" sequences.
  • terminators which can be used in the constructions of the invention, there may be mentioned in particular:
  • the expression vector may also include sequences of the vacuolar or apoplastic signal peptide type when they are not already present in the sequence of the gene of interest, in order to bring the protein encoded by the heterologous gene into particular compartments of the cells of plant, in particular those comprising the albumen transfer zone.
  • the preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC No. 37 017) or also the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, Wl, United States ).
  • vectors specially adapted for the expression of sequence of interest in plant cells such as the following vectors:
  • the regulatory nucleic acids, the expression cassettes or also the recombinant vectors defined in the present description must be introduced. in a host cell.
  • the introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro, according to the techniques well known to those skilled in the art for transforming or transfecting cells, either in primary culture or in the form of cell lines.
  • the subject of the invention is also a host cell transformed with a regulatory nucleic acid, with an expression cassette or also with a recombinant vector as defined above.
  • Such a transformed host cell is preferably of bacterial, fungal or vegetable origin.
  • bacteria cells of different strains of Escherichia coli or of Agrobacterium tumefaciens can in particular be used.
  • the transformed host cell is a plant cell or also a plant protoplast.
  • the transformed host solution is of plant origin and is chosen from cells of plant species such as Solanacea, Cactaceae, Cucurbitacea, Papillionacea, Actinidiaceae, Cucurbitaceae, Rubiaceae, Moraceae, Passif loraceae, Compositae, Caricaceae, E ⁇ caceae, Gramineae, Cruciferae, Cariofillaceae, Amarylidiaceae, Iridaceae, Leguminosae, Liliaceae, Oleaceae, Paeoniaceae, Papaveraceae, Primulaceae, Rosaceae, Scrophulariaceae, Violaceae, Vitaceae, Malvaceae.
  • plant species such as Solanacea, Cactaceae, Cucurbitacea, Papillionacea, Actinidiaceae, Cucurbitaceae, Rubiaceae, Moraceae, Passif loraceae, Compositae, Caricacea
  • a host cell transformed according to the invention is the Escherichia coli strain containing the plasmid pBAN1 :: GUS / pBIB-Hyg deposited at the CNCM on July 17, 2001 under the access number I-2703.
  • Other preferred transformed host cells according to the invention are respectively host cells transformed with the vectors pBAN2 :: GUS, PBAN3 :: GUS, pBAN4 :: GUS, pBAN5 :: GUS, pBAN6 :: GUS pBAN7: GUS and pBAN8: GUS illustrated in the examples.
  • the invention also relates to a transformed multicellular plant organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by a nucleic acid, an expression cassette or also by a recombinant vector as defined above. above.
  • the invention also relates to a transgenic plant comprising, in a form integrated into its genome, a nucleic acid or an expression cassette as defined in the present description.
  • the invention also relates to the use of a regulatory nucleic acid, an expression cassette, a recombinant vector or also a host cell as defined above for the obtaining a transformed plant.
  • a regulatory nucleic acid in which the expression of the regulatory polynucleotide placed under the control of a nucleic acid according to the invention is toxic for the cell, the transformed plant does not produce mature seeds , or either produces seeds of reduced size, or produces a reduced number of grains or is characterized by the total absence of seeds, particularly the total absence of mature and / or fertile seeds.
  • a first objective pursued according to the present invention is the obtaining of transgenic or transformed plants whose mature seed has improved characteristics, in particular from the point of view of the quality or the quantity of tannins or fibers or even hormonal balances.
  • a second objective pursued according to the invention is the obtaining of transgenic or transformed plants affected in the development of the testa of the seed.
  • transgenic or transformed plants whose seeds contain reduced contents of tannins or anthocyanins compared to normal.
  • Such an objective will be pursued more particularly with regard to obtaining transgenic or transformed rapeseed or maize plants having the above characteristics, and the seeds of which are of a more intense yellow color than normal.
  • the polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention will be chosen in such a way that it modifies the nutritional and / or hormonal quality of the testa de la seed.
  • the polynucleotide of interest will be chosen in such a way that its expression is toxic to the endothelium cell in which it is expressed.
  • the regulatory nucleic acids and the expression cassettes are those described previously in the description.
  • a regulatory nucleic acid preferably comprising a polynucleotide having at least 80% nucleotide identity with the sequence going from the nucleotide at position 1919 will be chosen. up to the nucleotide at position 2335 of the sequence SEQ ID No. 1, as is the case for example for the regulatory nucleic acids contained in the vectors pBAN1 :: GUS, pBAN2 :: GUS, pBAN3 :: GUS, pBAN4: : GUS and pBAN5 :: GUS.
  • a regulatory nucleic acid preferably comprising a polynucleotide having at least 80% identity is chosen. nucleotides with the sequence going from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1, as is the case for the regulatory nucleic acid contained in the vector pBAN6 :: GUS.
  • the invention also relates to any part of a transformed plant as defined in the present description, including its seeds and its fruits.
  • the plants transformed according to the invention preferably belong to the species which have already been listed above concerning the origin of the transformed plant host cells of the invention.
  • the plants transformed according to the invention are chosen from eggplant, tomato, melon or watermelon, cucumber, species of the citrus type, pepper, strawberries, grapes , apples, pears, cherries, rapeseed, cabbage, pepper or olives.
  • the plants can be either dicots or monocots.
  • the subject of the invention is also a process for obtaining transformed plants, characterized in that it comprises the following steps: a) obtaining a recombinant plant host cell according to the invention; b) regeneration of an entire plant from the recombinant host cell obtained in step a); c) selection of the plants obtained in step b) having integrated a nucleic acid or an expression cassette as defined in the present description.
  • the subject of the invention is also a process for obtaining a transformed plant, characterized in that it comprises the following steps: a) obtaining a recombinant host cell of Agrobacterium tumefaciens according to the invention; b) transformation of a plant of interest by infection with the recombinant host cells of Agrobacterium tumefaciens obtained in step a); c) selection of plants having integrated into their genome a nucleic acid or an expression cassette as defined in the present description.
  • Another subject of the invention is a process for obtaining a transformed plant, characterized in that it comprises the following steps: a) transfecting at least one plant cell with a nucleic acid, with an expression cassette or with a recombinant vector according to the invention; b) regenerating an entire plant from the recombinant plant cell obtained in step a); c) selecting the plants which have integrated into their genome a nucleic acid or an expression cassette according to the invention.
  • any of the processes for obtaining a transformed plant described above can also comprise the following additional steps: d) crossing between them of two transformed plants as obtained in step c) with a plant of the same species; e) selection of plants homozygous for the transgene.
  • any of the methods for obtaining a transgenic plant described above can also comprise the following additional steps: f) crossing of a transformed plant obtained in step c) with a plant of the same species; g) selection of the plants resulting from the crossing of step f) having conserved the transgene.
  • Another subject of the invention consists of a transformed or transgenic plant as obtained according to any one of the methods for obtaining a plant defined above.
  • the subject of the invention is also a transformed plant, capable of being obtained by any of the methods described above.
  • the transformation of plant cells can be carried out by techniques known to those skilled in the art. Mention may in particular be made of direct gene transfer methods such as direct microinjection into plant embryoids (NEUHAUS et al., 1987), vacuum infiltration (BECHTOLD et al., 1993) or electroporation (CHUPEAU and al., 1989) or direct precipitation using PEG (SCHOCHER et al., 1986) or bombardment by cannon of particles covered with the plasmid DNA of interest (FROMM et al. 1990).
  • direct gene transfer methods such as direct microinjection into plant embryoids (NEUHAUS et al., 1987), vacuum infiltration (BECHTOLD et al., 1993) or electroporation (CHUPEAU and al., 1989) or direct precipitation using PEG (SCHOCHER et al., 1986) or bombardment by cannon of particles covered with the plasmid DNA of interest (FROMM et al. 1990).
  • the plant can also be infected with a bacterial strain, notably Agrobacterium.
  • the plant cells are transformed by a vector according to the invention, said cell host being capable of infecting said plant cells by allowing the integration into the genome of these latter, sequences nucleotides of interest initially contained in the DNA of the above-mentioned vector.
  • the above-mentioned cell host used is Agrobacterium tumefaciens, in particular according to the method described in the article by Ha and AN (1989), or else Agrobacterium rhizogenes, in particular according to the method described in the article by GUERCHE et al., (1987) or also in PCT application No. WO 00 22,148.
  • the transformation of plant cells can be carried out by the transfer of the T region of the extra-chromosomal circular plasmid inducing Ti tumors of Agrobacterium tumefaciens, using a binary system (WATSON et al. 1994). To do this, two vectors are constructed. In one of these vectors, the T region was deleted, with the exception of the right and left borders, a marker gene being inserted between them to allow selection in plant cells.
  • the other partner of the binary system is a helper Ti plasmid, a modified plasmid which no longer has a T region but still contains the vir virulence genes, necessary for the transformation of the plant cell.
  • ISHIDA et al. Can be used. (1996) for the transformation of Monocotyledons.
  • the transformation is carried out according to the method described by FINER et al. (1992) using the tungsten or gold particle gun.
  • a person skilled in the art is capable of implementing numerous methods of the state of the art in order to obtain plants transformed with a regulatory nucleic acid or an expression cassette according to the invention.
  • a transgenic plant according to the invention can be obtained by biolistic techniques such as those described by FINER et al. (1992) or those described by VAIN et al. (1993).
  • Other preferred techniques for transforming a plant in accordance with the invention with Agrobacterium tumefaciens are those described by ISHIDA et al. (1996) or in the PCT application published under the number WO 95/06 722 in the name of JAPAN TOBACCO.
  • the subject of the invention is also a plant seed whose constituent cells comprise a regulatory nucleic acid or an expression cassette according to the invention, which has been artificially inserted into its genome.
  • the invention also relates to a seed of a transgenic plant as defined above or also a fruit of such a transgenic plant.
  • Another subject of the invention consists in the use of a regulatory nucleic acid as defined in the present description for the expression in vitro or in vivo, preferably in planta, of a polynucleotide of interest, preferably a polynucleotide whose expression is toxic to the plant cell.
  • the invention also relates to a transformed plant affected in the development of these seeds, capable of being obtained by any of the above methods, as well as any part of this transformed plant, including these fruits.
  • FIG. 1 represents a diagram of the different nucleotide constructs or “expression cassettes” respectively containing all or part of the regulatory nucleic acid with sequence SEQ ID No. 1 and a reporter gene placed downstream of the regulatory nucleic acid, the GUS reporter gene.
  • FIG. 2 illustrates the principle of determination of the oligonucleotides used for the amplification of the deletions, FIG. 2 more particularly illustrating the construction of the vector pBAN1 :: GUS.
  • FIG. 3 illustrates the temporal expression profile of the BAN gene (RT-PCR and GUS expression), compared with the activity of the GUS gene placed under the control of p BAN1.
  • FIG. 4 illustrates the construction of the vector pBAN :: GUS
  • FIG. 5 illustrates the protocol for construction of the vector pBan :: Gus / pBIB-HYG.
  • Figure 6 illustrates the expression profile of the GUS reporter gene placed under the control of the pBAN1 promoter.
  • Figure 8 illustrates the construction of the vector pBAN1 :: barnase- barstar / pBIB-HYG
  • Figure 9 illustrates the genetic ablation of the endothelium by the barnase gene placed under the control of the pBAN1 promoter.
  • A Nomarski of immature seed of a transformant pBAN1 :: Barnase revealing the absence of endothelium (compare with the situation in wild seeds, in FIG.
  • B detection of tannins in the endothelium of a wild plant, by the “vanillin” test coloring the tannins in dark red (arrow);
  • C “vanillin” test on immature seeds of a transformant pBAN1 :: Bamase revealing the absence of tannins;
  • D and E mature seeds of wild plants (brown seeds) and of transformant pBAN1 "Barnase (brown seeds) and of transformant p BAN1" Barnase (yellow seeds); the location of the endothelium in the wild.
  • Escherichia coli DH12S (GibcoBRL).
  • This cloning vector contains an ampicillin resistance gene and a multiple cloning site located in the LacZ gene, allowing white / blue selection of transformed / unprocessed colonies.
  • This vector includes the barnase gene under the control of the maize AG promoter as well as the gene coding for Barstar placed under the control of a bacterial promoter to allow the growth of bacteria.
  • This binary vector comprises, in addition to a multicloning site, a gene for resistance to hygromycin (HYG) with expression in planta (BECKER, 1990).
  • HOG hygromycin
  • the seeds are previously disinfected by soaking for 30 min in an alcoholic solution containing 1% of active chlorine, followed by 3 successive rinses in 96% alcohol.
  • the semi is then carried out in Petri dishes on mineral medium from GAMBORG et al. (1968) supplemented with 1% sucrose and 0.8% agar (Bio Mérieux).
  • An antibiotic varying according to the in plant resistance carried by the binary vector (kanamycin or hygromycin at a concentration of 50 mg. 1 "1 ) is added for the selection of transformants.
  • the seeds are subjected to a cold treatment (3 days to 4 ° C) to break the dormancy and homogenize the emergence before being incubated in an air-conditioned culture chamber (60% relative humidity, 16 h of light at 20 ° C / 8 h of darkness at 15 ° C, light intensity 200-250 ⁇ Em “2 ; s " 1 at the boxes).
  • the mini-preparations for rapid verification of the bacterial clones are carried out according to the alkaline lysis method (Birnboim and Doly, 1979).
  • RNAs The extraction of total RNAs was carried out using the “RNeasy Plant Mini Kit” (Qiagen) supplemented by a treatment with Dnase according to the protocol of the kit “RNase-Free Dnase Set” (Qiagen).
  • the 5 'deletions of the BAN promoter were obtained by PCR from the DNA of BAC T13M11 (Accession AC005882). Approximately 150 ng of DNA served as template for the amplification, in the presence of 10 ⁇ l of Pfu 10X buffer, 2 ⁇ l of 5 mM dNTPs, 2 ⁇ l of each of the oligos specific for 10 picoM (Table 2), 0.8 ⁇ l (2 units ) of Pfu TURBO DNA polymerase (Stratagene) and sterile double-distilled water, qs 40 ⁇ l.
  • the sequencing was carried out by the JANGER method using an ABI sequencing device Company Applied BIOSYSTEMS, Inc.
  • Electrocompetent bacteria (Sambrook et al., 1989) are electroporated in the presence of 10 ⁇ l of ligation product. The latter is previously desalted by diafiltration on a 0.025 ⁇ m membrane (SCHLEICHER and SCHUELL). The electroporation is carried out at a voltage of 1.25 kV applied for a period which is a function of the resistance and the capacity (200 ⁇ and 25 ⁇ F, respectively of the circuit of the electroporator of the Gene Incer II System type, marketed by the Bio-Rad Company) ..
  • Single-stranded cDNAs are synthesized from 1 ⁇ g of total RNAs in a volume of 20 ⁇ l containing 20 mM Tris-HCI (pH 8; 4), 50 mM Kcl, 2.5 mM MgCI 2 , 10 mM DIT, 1 mM dNTPs , 500 ng oligo (dT) 12- 18 (GibcoBRL), 25 units of RNase Out (GibcoBRL) and 200 units of MMLV SuperScript II reverse transcriptase (GibcoBRL) for 50 min. at 42 ° C.
  • Two ⁇ l of the 10-fold single-stranded cDNA solution was used for the PCR reaction carried out in a total volume of 50 ⁇ l in the presence of 20 mM Tris-HCl (pH 8.4), 50 mM Kcl, 1.5 mM MgCI 2 , 0.2 mM dNTPs, 0.2 ⁇ M of each gene-specific oligo and 1 unit of Taq DNA polymerase (GibcoBRL).
  • the ban-sense gene-specific oligos (5'-AACAACTAAATCTCTATCTCTGTA-3 'SEQ ID N ° 2) and banantisens (5'-GAATGAGACCAAAGACTCATATAC-3' SEQ ID N ° 3) (Devic et al., 1999) allow d amplify a band of 1.2 kb in the cDNA of BAN and 1.5 kb in the genomic DNA.
  • the gene-specific oligos GBGe306-sense (5'-ACCAGGAGGTTTTCAAAGAC-3 'SEQ ID N ° 4) and GBGe306-antisense (5'-CAACATAACTTGCTCTGTTC-3'SEQ ID N ° 5) amplify a band of 0.9 kg in l CDNA of GBGe306, and 1.1 kb in genomic DNA.
  • the EF1 A4 gene encoding an elongation factor (GenBankX16432; Liboz et al., 1990) was used as a positive control.
  • the gene-specific oligos EF-sense (5'- ATGCCCCAGGACATCGTGATTTCAT-3 'SEQ ID N ° 6) and EF-antisense (5'TTGGCGGCACCCTTAGCTGGATCA-3'SEQ ID N ° 7) amplify a band of 0.7 kb in the EF1 A4 cDNA, and 0.9 kb in genomic DNA.
  • EF-sense 5'- ATGCCCCAGGACATCGTGATTTCAT-3 'SEQ ID N ° 6
  • EF-antisense 5'TTGGCGGCACCCTTAGCTGGATCA-3'SEQ ID N ° 7
  • the amplified samples are separated on 1% agarose gel (w / v) in TAE buffer and are then transferred to a GeneScreen Plus nylon membrane (NEN, USA) positively charged according to the method recommended by Sambrook et al. (1989).
  • NNN GeneScreen Plus nylon membrane
  • the membrane is hybridized at 65 ° C overnight in the same buffer to which is added the ad hoc cDNA probe obtained using the oligos presented above and labeled with ( 32 P) - dCTP using the “Prime-a-Gene Labeling System” kit (Promega).
  • the membrane is then washed for 15 minutes at 65 ° C in LAVI buffer (2 X SSC, 0.5% sarkosyl, 0.2% Na 4 P 2 0 7 , 10 H 2 O) dried and wrapped in cling film (Saran) and then placed at -80 ° C in contact with a photographic film (Hyperfilm MPO, Amersham, GB) under amplifier screen (Amersham).
  • LAVI buffer 2 X SSC, 0.5% sarkosyl, 0.2% Na 4 P 2 0 7 , 10 H 2 O
  • the GUS staining protocol (Jefferson et al., 1987) consists in incubating plant fragments in a sterile buffer containing 100 mM of phosphate buffer pH 7.2 (Sambrook et al., 1989), 10 mM Na 2 - EDTA, 0.1% Triton X-100, 2 mM X-Gluc (Duchefa) and 2.5 mM ferricyanide and ferrocyanide. Incubation takes place in the dark at 37 ° C overnight.
  • the plant organs are incubated for a few minutes in a cell lightening solution, composed of chloral hydrate / glycerol / water (8/1/2, w / v / v) on a microscope slide, before being observed. under coverslip using a microscope equipped with Nomarski-type differential interference contrast optics.
  • the “vanillin” test for the detection of tannins in tissues is carried out according to the method of Aastrup et al. (1984).
  • EXAMPLE 1 Construction of several expression cassettes comprising a polynucleotide coding for the GUS polypeptide. placed under the control of a regulatory nucleic acid according to the invention.
  • - Zone I (415 bp) comprises the 5 'untranslated transcribed region of the BAN gene and the sensu stricto promoter of BAN, as well as the 3' untranslated transcribed region of GBGe306;
  • - zone II (908 bp) corresponds to the genomic region covered by the GBGe306 transcript;
  • Zone III (1323 bp) comprises the 5 'untranslated transcribed region of GBGe306, the promoter sensu stricto of this gene as well as an indefinite intergenic zone.
  • This precut is to analyze the contribution of the region upstream of the sensu stricto promoter on the expression of a polynucleotide of interest, more specifically the polynucleotide coding for the GUS polypeptide.
  • Each zone is also subdivided into 4 (zone I) or 2 parts
  • zones II and III The objective of this subdivision is to facilitate the detection of functional patterns governing the specificities of developmental expression (tissue and time) as well as the response to environmental stimuli such as light, temperature, etc.
  • Each deletion is amplified by PCR from the vector BAC T13M11 containing an insert of genomic DNA from Arabidopsis thaliana, Columbia ecotype.
  • oligonucleotides determined as follows, illustrated in FIG. 2:
  • - 5 'oligo they are specific to the deletion and are modified by the addition of a Salll restriction site preceded by 3 security bases.
  • - 3 'oligo it is common to all deletions. The creation of an Ncol site by modification of the original sequence allows the translational fusion of the deletion and of the reporter gene.
  • the amplification products were then cloned with free edges in the vector pBS-SK at Smal or EcorV sites to be completely sequenced.
  • the cloning of the promoters is carried out in a subcloning vector presenting a cassette rapporteu ⁇ .terminateur. Then the promoter :: reporter :: terminator cassette is transferred into a binary vector.
  • the reporter chose the GUS gene, the product of which is very stable and can accumulate in the tissues, making it possible to detect very weak activities over time.
  • the binary vector pBIB-HYG (Becker, 1990) containing resistance to hygromycin was used.
  • the vector map pBIB-HYG is shown in Figure 5.
  • the construction protocol of the pBAN :: GUS vectors pBIB-HYG is represented in FIG. 5.
  • the protocol for constructing the vector pBAN1-GFP5 / pBIB-HYG is shown in FIG. 6.
  • the expression cassettes in binary vectors are introduced into plants of Arabidopsis ecotype Ws (T 0 , infiltrated plant) by the Silwet infiltration method developed by Clough and Bent
  • the seeds of the transformed plants are harvested in bulk, sterilized and sown on a nutritive medium with hygromycin. Obtaining T1 seedlings (primary transformants) with pods requires an average of 3.5 to 4 months.
  • the GUS staining observations detailed below relate to Arabidopsis transformants expressing the constructions pBAN1 :: GUS, pBAN2 :: GUS, pBAN3 :: GUS, pBAN5 :: GUS and pBAN6 :: GUS. Five independent transformants were studied by built. The transformants containing the constructions pBAN4 :: GUS, pBAN7 :: GUS and pBAN8 :: GUS were also produced.
  • the expression GUS is observed in the endothelium of immature seeds with no measurable difference in the strength of expression between the various cassettes of expression tested.
  • quantitative variability is observed between independent transformants for the same expression cassette, which is probably to be correlated with the number of transgenes expressed per plant, or with effects of position of insertion of T-DNA in the genome.
  • transformants which also show expression during germination localized in the albumen, sometimes at the hypocotyl-root junction or in the root tip.
  • Some expression profiles were also obtained for a particular transformant (ex GUS expression in the whole of the hypocotyle and the root for one of the transformants pBAN5 or expression in the leaves for one of the transformants pBAN6).
  • the expression profile obtained can therefore always vary depending on the transformation event considered: So. whatever the construct considered, it is necessary to test several independent transformants to obtain the desired specific expression profile.
  • the promoter region located between - 457 and - 322 base pairs upstream of the translation initiation site contains the motif (s) necessary and sufficient to repress an expression in the aleurone layer.
  • the promoters of the pBAN5 and pBAN6 type will preferably be used, as necessary, to confer specific expression in the endothelium (pBAN5) or in the endothelium and the albumen (pBAN6).
  • the feasibility of using the barnase placed under the control of the pBAN1 promoter for the genetic ablation of the endothelium was studied on the first two independent transformants.
  • the effect sought for a biotechnological application is the reduction in size of the seed which can go as far as total ablation, as well as the suppression of the flavonoids of the endothelium leading to obtaining yellow seeds.
  • the pBAN1 promoter was placed in front of the barnase :: barstar :: cassette terminator of the vector pWP146 (barnase-barstar) supplied by Pascual Perez (Biogemma).
  • Such an inducible expression system includes:
  • a first expression cassette containing a polynucleotide coding for a fusion protein between a glucocorticoid receptor and the transcription factor GAL4, this polynucleotide being placed under the control of a regulatory nucleic acid derived from the sequence SEQ ID No. 1 according to the invention;
  • the fusion protein between the glucocorticoid receptor and the transcription factor GAL4 is produced constitutively, specifically in the endothelium, or specifically in the endothelium and the albumen of the seeds of this plant, thanks to the first expression cassette.
  • the glucocorticoid When the plant thus transformed is brought into contact with a glucocorticoid, the glucocorticoid binds specifically to the receptor part of the above fusion protein and results in the binding of the GAL4 part of the fusion protein to the regulatory nucleic acid contained in the second expression cassette.
  • the regulatory nucleic acid of the second expression cassette is activated and will induce the expression of the polynucleotide toxic for the cell, specifically in the endothelium, or specifically in the endothelium and the albumen of the seeds of the transformed plant, thus preventing the formation of mature and fertile seeds.
  • the inducible expression system defined in general above allows the controlled obtaining of a viable progeny of the transformed plants or, on the contrary, the controlled obtaining of early seeds abortion events.
  • Chupeau M.C. Bellini C, Guerche, P., Maisonneuve B., Chupeau Y. (1989) Transgenic plants of lettuce (Lactuca sativa) obtained through electroporation of protoplasts. Biotechnology 7, 503-508. Co ⁇ c Y., Lessaint C. (1971). How to ensure good nutrition in water and mineral ions in horticulture. Hortic. Fr. 8, 11-14.
  • Debeaujon I. Léon-Kloosterziel KM, Koornneef M. (2000). Influence of the testa on seed dormancy, germination and longevity in Arabidopsis. Plant Physiol. 122, 403-413. Debeaujon I., Koornnee, M. (2000). Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid. Plant Physiol. 122, 415- 424. Debeaujon I., Peeters AJ, Léon-Kloosterziel KM, Koornneef M.
  • the TRANSPARENT TESTAI 2 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium .
  • Devic M. Guilleminot J., Debeaujon I., Bechtold N., Bensaude E., Koornneef M., Pelletier, G., Delseny M. (1999).
  • the BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development.
  • PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis. Plant Cell 12, 2409-2424.
  • Koncz C Koncz C, Wienaler F., Kalman, Z., Schell, J. (1984). A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors. EMBO J. 3, 1029-1037. Koning A., Jones A., Fillatti JJ, Comai L., Lassner MW (1992). Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A. Plant Mol. Biol. 18, 247-258. Krizek BA (1999) Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs. Dev. Broom. 25, 224-236.
  • ANTHOCYANINLESS2 a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis. Plant Cell 11, 1217-1226. Léon-Kloosterziel K.M., Keijzer C.J., Koornneef M. (1994) A seed shape mutant of Arabidopsis that is affected in integument development. Plant Cell 6, 385-392. Liboz T., Bardet, C, Le Van Thai, A., Axelos, M., Lescure, B. (1990).
  • a chimeric ribonuclease inhibitor gene restores fertility to maie sterile plants. Nature 357, 384-387.
  • Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis Proc.

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Abstract

The invention concerns a nucleic acid comprising regulating signals for expressing a polynucleotide of interest, specifically in the endothelium and/or the albumen of a plant seed, when said polynucleotide of interest is placed under the control of said regulating signals, said nucleic acid having at least 80 % identity in nucleotides with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1.

Description

Acide nucléique régulateur permettant l'expression d'un polynucleotide d'intérêt spécifiquement dans l'endothélium d'une graine de plante, et ses applications Regulatory nucleic acid allowing the expression of a polynucleotide of interest specifically in the endothelium of a plant seed, and its applications
Domaine de l'inventionField of the invention
L'invention se rapporte au domaine de l'amélioration contrôlée de qualité agronomique de plantes variées, incluant les plantes à fruits et les céréales de grande culture. Elle est relative à l'expression ciblée de polynucléotides et/ou de polypeptides d'intérêt spécifiquement dans certains tissus de la plante, et plus spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante.The invention relates to the field of the controlled improvement of agronomic quality of various plants, including fruit plants and field crops. It relates to the targeted expression of polynucleotides and / or polypeptides of interest specifically in certain tissues of the plant, and more specifically in the endothelium and / or the albumen of a plant seed.
Etat de la techniqueState of the art
La testa, aussi désignée téguments de la graine est un tissu essentiel pour le développement et la viabilité de la graine. Lorsque la graine est entièrement mature, la testa constitue une couche protectrice des parties internes de celle-ci. Pendant le développement de la graine, la testa est un tissu d'importation de substances nutritives vitales pour le développement de l'embryon. La graine se comporte comme un « parasite » vis-à-vis de la plante mère, en ce qui concerne sa nutrition. Tous les matériaux nutritifs bruts nécessaires à la croissance de la graine doivent être importés de la plante mère. Dans les graines des plantes dicotylédones, le tissu vasculaire entre dans la graine par le funiculus, puis s'anastomose dans le tissu de la testa de la graine. Il n'existe aucune connexion de tissu vasculaire entre la testa de la graine et l'embryon. Ainsi, toutes les substances nutritives libérées dans la graine en développement doivent être tout d'abord importées à l'intérieur de la testa , avant d'être libérées par diffusion dans l'embryon.Testa, also known as seed coat, is an essential tissue for seed development and viability. When the seed is fully mature, the testa constitutes a protective layer of the internal parts of it. During the development of the seed, the testa is an import fabric of nutrients vital for the development of the embryo. The seed behaves like a “parasite” towards the mother plant, with regard to its nutrition. All raw nutrients necessary for seed growth must be imported from the mother plant. In the seeds of dicotyledonous plants, the vascular tissue enters the seed through the funiculus, then anastomoses in the testa tissue of the seed. There is no connection of vascular tissue between the testa of the seed and the embryo. Thus, all the nutrients released in the developing seed must first be imported into the testa, before being released by diffusion into the embryo.
Il existe un besoin dans l'état de la technique de faire exprimer des gènes d'intérêt spécifiquement dans la testa, en vue d'en modifier la composition, par exemple pour modifier la composition en tannins et en fibres ou encore modifier l'équilibre hormonal. Contextes agronomique et industriel:There is a need in the prior art to express genes of interest specifically in the testa, in order to modify the composition, for example to modify the composition in tannins and in fibers or else to modify the balance. hormone. Agronomic and industrial contexts:
D'un point de vue agronomique, les téguments (testa) interviennent dans de nombreux aspects déterminant la qualité de la graine, dont les plus importants sont: - la nutrition de l'embryon (Weber et al., 1996)From an agronomic point of view, the integuments (testa) are involved in many aspects determining the quality of the seed, the most important of which are: - the nutrition of the embryo (Weber et al., 1996)
- la taille de la graine (Léon-Kloosterziel et al., 1994; Weber et al., 1996; Alonso-Blanco et al., 1999)- the size of the seed (Léon-Kloosterziel et al., 1994; Weber et al., 1996; Alonso-Blanco et al., 1999)
- le contrôle de la dormance, de la germination (obstacle physique à la sortie de la radicule, imperméabilité à l'eau et à l'oxygène) (Werker, 1980; Kelly et al., 1992; Debeaujon et al., 2000; Debeaujon et Koornneef, 2000) et de la qualité de la plantule (Kantar et al., 1996)- control of dormancy, germination (physical obstacle to the exit of the radicle, impermeability to water and oxygen) (Werker, 1980; Kelly et al., 1992; Debeaujon et al., 2000; Debeaujon and Koornneef, 2000) and the quality of the seedling (Kantar et al., 1996)
- la longévité de la graine, par limitation du stress oxydant dû à l'oxygène de l'air et protection contre les pathogènes et les UV (Mohamed- Yasseen et al., 1994). L'influence de la testa sur la dormance / germination et sur la protection de l'embryon est principalement due à la présence de grandes quantités de flavonoïdes (majoritairement tannins et flavonols) localisés dans l'endothélium, qui est la couche cellulaire la plus interne de la testa (Scalbert, 1991 ; Shirley et al., 1995; Winkel-Shirley, 1998; Debeaujon et al., 2000). Mais il est probable que des composés comme la lignine et la cellulose jouent un rôle non négligeable dans la rigidité de ce tissu. Moduler qualitativement et quantitativement la composition de la testa en ces différents composés pourrait présenter un intérêt dans la modification des caractéristiques métaboliques et structurales de ce tissu. Une approche compémentaire consisterait à moduler au niveau de la testa les équilibres des différentes hormones intervenant dans le développement de la graine (gibbérellines ou GAs; acide abscissique ou ABA, éthylène; auxines; cytokinines; brassinostéroïdes; jasmonates, comme montré pour l'acide abscissique dans l'embryon (Phillips et al., 1997). On peut également envisager de faire synthétiser au niveau de la testa des substances qui n'y sont pas naturellement présentes mais qui conféreraient à la graines une protection supplémentaire, en particulier contre les pathogènes. D'un point de vue nutritionel (animaux) et industriel, le contrôle de la composition de la testa représente également un atout majeur. Si l'on prend l'exemple des flavonoïdes, l'obtention de variétés de colza à graines jaunes (sans tannins) permettrait une meilleure valorisation des tourteaux tirés de la graine après extraction de l'huile; ces variétés produisent également plus d'huile et moins de fibres que les variétés à graines noires ou brunes. Ces dernières variétés , riches en tannins, conduisent à des tourteaux plus pauvres en protéines et à digestibilité réduite (Stringam et al., 1974; Theander et al., 1977; Mitaru et al., 1982; Shirzadegan et Rόbbelen, 1985; Simbaya et al., 1995; Jonsson, 1997; Baetzel et al., 1999). Des variétés d'orge sans tannins sont recherchées pour la fabrication de la bière, car ces flavonoïdes causent la précipitation des protéines et donc l'opacification du produit fini (Jende-Strid, 1993). Une seconde alternative à l'absence totale de flavonoïdes est de remplacer les tannins par des anthocyanes et/ou surexprimer les gènes de biosynthèse de flavonols, qui sont deux autres catégories de flavonoïdes, permettrait de bénéficier des propriétés protectrices de flavonoïdes (Rice-Evans et al., 1997) et de s'affranchir des inconvénients des tannins. Finalement, enrichir la testa en anthocyanes permettrait d'augmenter la richesse du produit en antioxydants et ainsi améliorer sa capacité nutritionnelle.- the longevity of the seed, by limiting the oxidative stress due to oxygen in the air and protection against pathogens and UV (Mohamed-Yasseen et al., 1994). The influence of testa on dormancy / germination and on the protection of the embryo is mainly due to the presence of large quantities of flavonoids (mainly tannins and flavonols) located in the endothelium, which is the innermost cell layer de la testa (Scalbert, 1991; Shirley et al., 1995; Winkel-Shirley, 1998; Debeaujon et al., 2000). But it is likely that compounds such as lignin and cellulose play a significant role in the rigidity of this tissue. Qualitatively and quantitatively modulating the composition of testa into these different compounds could be of interest in modifying the metabolic and structural characteristics of this tissue. A complementary approach would consist in modulating at the level of the testa the balances of the various hormones intervening in the development of the seed (gibberellins or GAs; abscissic acid or ABA, ethylene; auxins; cytokinins; brassinosteroids; jasmonates, as shown for abscissic acid in the embryo (Phillips et al., 1997). One could also consider having substances synthesized in the testa which are not naturally present but which would give the seeds additional protection, in particular against pathogens . From a nutritional (animal) and industrial point of view, controlling the composition of the testa is also a major advantage. If we take the example of flavonoids, obtaining varieties of rapeseed with yellow seeds (without tannins) would allow a better valorization of the cakes drawn from the seed after extraction of the oil; these varieties also produce more oil and less fiber than varieties with black or brown seeds. These latter varieties, rich in tannins, lead to oil-poor and protein-reduced meal (Stringam et al., 1974; Theander et al., 1977; Mitaru et al., 1982; Shirzadegan and Rόbbelen, 1985; Simbaya and al., 1995; Jonsson, 1997; Baetzel et al., 1999). Varieties of barley without tannins are sought for the manufacture of beer, because these flavonoids cause the precipitation of proteins and therefore the clouding of the finished product (Jende-Strid, 1993). A second alternative to the complete absence of flavonoids is to replace the tannins with anthocyanins and / or overexpress the biosynthesis genes of flavonols, which are two other categories of flavonoids, would benefit from the protective properties of flavonoids (Rice-Evans and al., 1997) and to overcome the disadvantages of tannins. Finally, enriching the testa with anthocyanins would increase the richness of the product in antioxidants and thus improve its nutritional capacity.
L'intérêt de pouvoir moduler la composition en flavonoïdes (type de produit final) ainsi que leur quantité (augmentation ou réduction) apparaît clairement à travers ces exemples. La même approche peut être envisagée pour d'autres composants comme la lignine, la cellulose ou les équilibres hormonaux. Dans ce contexte, un promoteur spécifique de la testa permettrait de modifier exclusivement cet organe, sans affecter les caractéristiques végétatives de la plante, ce qui est primordial vu le rôle protecteur des flavonoïdes. Enfin, étant donné le rôle important de la testa dans le développment de la graine, on peut penser que sa modification, par exemple par la surexpression de gènes impliqués dans la biosynthèse d'hormones, la production d'une toxine ou controllant la division cellulaire, dans ce tissu d'origine maternelle (Varoquaux et al., 2000) pourrait conduire à une réduction de la taille / nombre voire à une disparition des graines qui est recherchée pour des fruits de type raisin, tomate, concombre, pastèque, melon etc.The advantage of being able to modulate the flavonoid composition (type of final product) as well as their quantity (increase or reduction) is clearly apparent from these examples. The same approach can be considered for other components such as lignin, cellulose or hormonal balances. In this context, a specific promoter of testa would make it possible to modify this organ exclusively, without affecting the vegetative characteristics of the plant, which is essential given the protective role of flavonoids. Finally, given the important role of testa in the development of the seed, we can think that its modification, for example by the overexpression of genes involved in the biosynthesis of hormones, the production of a toxin or controlling cell division , in this tissue of maternal origin (Varoquaux et al., 2000) could lead to a reduction in size / number or even a disappearance of seeds which is sought for grape, tomato, cucumber, watermelon, melon etc. fruits.
La testa étant un organe de protection de l'embryon de la graine, il peut être désirable d'affecter son développement dans le but d'obtenir des plantes ne possédant pas de graines fertiles ou encore de plantes dépourvues ou pratiquement dépourvues de graines. L'obtention de plantes incapables de produire des graines revêt un intérêt considérable dans le domaine des plantes à fruits. En effet, des fruits dépourvus de graines possèdent des qualités gustatives supérieures à celles observées pour des fruits identiques dans lesquels les graines sont présentes. De plus, les graines sont souvent dures et ont un goût amer. Dans certains cas, comme pour le raisin, la consommation des graines peut entraîner des troubles digestifs. En outre, en l'absence de graines, les cavités naturelles dans lesquelles elles sont normalement présentes sont remplacées par le tissu comestible du fruit, qui rend un fruit dépourvu de graines beaucoup plus attractif pour le consommateur.The testa being a protective organ of the embryo from the seed, it may be desirable to affect its development in order to obtain plants that do not have fertile seeds or even plants devoid or practically devoid of seeds. Obtaining plants incapable of producing seeds is of considerable interest in the field of fruit plants. Indeed, fruits devoid of seeds have taste qualities superior to those observed for identical fruits in which the seeds are present. In addition, the seeds are often hard and have a bitter taste. In some cases, like grapes, eating the seeds can cause digestive upset. Furthermore, in the absence of seeds, the natural cavities in which they are normally present are replaced by the edible tissue of the fruit, which makes a seedless fruit much more attractive to the consumer.
Enfin, il a été observé qu'un fruit dépourvu de graines se conserve plus longtemps, du fait que les graines produisent des hormones provoquant la sénescence. Cet effet a pu être observé chez le melon d'eau, dans lequel les graines sont à l'origine de la détérioration du fruit. Selon un autre aspect, l'obtention de plantes ou de fruits dépourvus de graines possède un intérêt supplémentaire dans la culture de plantes transgéniques, car dans ce cas, on évite toute dissémination de graines génétiquement modifiées dans l'environnement. A la connaissance du demandeur, un seul promoteur capable de diriger l'expression d'un gène d'intérêt spécifiquement dans la testa a été isolé à ce jour.Au cours de travaux de sélection à grande échelle de séquences à activité de promoteur dans le génome du tabac, FOBERT et al. (1994) ont isolé et caractérisé un fragment d'ADN d'environ 3 kb capable d'induire l'expression d'un gène rapporteur dans la testa de la graine, dont l'activité maximale a été rapportée à 10 jours après l'anthèse (floraison). Toutefois, selon ces auteurs, ce promoteur originaire du tabac serait un promoteur cryptique, puisque la région génomique dans laquelle il a été localisé ne comporte aucun cadre de lecture ouvert à proximité de celui-ci. Mais surtout, le promoteur décrit par FOBERT et al. n'est pas spécifique de l'endothélium.Finally, it has been observed that a fruit devoid of seeds keeps longer, because the seeds produce hormones causing senescence. This effect has been observed in watermelon, in which the seeds are responsible for the deterioration of the fruit. According to another aspect, obtaining plants or fruits devoid of seeds has an additional interest in the cultivation of transgenic plants, because in this case, any dissemination of genetically modified seeds in the environment is avoided. To the knowledge of the applicant, a single promoter capable of directing the expression of a gene of interest specifically in testa has been isolated to date. During large-scale selection work on sequences with promoter activity in the tobacco genome, FOBERT et al. (1994) isolated and characterized a DNA fragment of approximately 3 kb capable of inducing the expression of a reporter gene in the testa of the seed, the maximum activity of which was reported at 10 days after the anthesis (flowering). However, according to these authors, this promoter originating from tobacco is a cryptic promoter, since the genomic region in which it has been located has no open reading frame close to this one. But above all, the promoter described by FOBERT et al. is not specific to the endothelium.
De même, le promoteur du gène FBP7 isolé à partir du génome du pétunia a été utilisé pour contrôler l'expression du gène de la barnase dans les ovules et dans les graines en développement (Colombo et al., 1997). En plaçant un gène marqueur sous le contrôle du promoteur de FBP7, ces auteurs ont observé une expression du gène rapporteur dans la testa de la graine en développement à l'exception de la couche cellulaire externe, ainsi que dans l'épiderme du placenta. Le promoteur du gène FBP7 n'est pas spécifique de la testa, puisqu'il est actif aussi dans l'épiderme du placenta.Likewise, the promoter of the FBP7 gene isolated from the petunia genome has been used to control the expression of the barnase gene in ova and in developing seeds (Colombo et al., 1997). By placing a marker gene under the control of the FBP7 promoter, these authors observed expression of the reporter gene in the testa of the developing seed except for the outer cell layer, as well as in the epidermis of the placenta. The promoter of the FBP7 gene is not specific to testa, since it is also active in the epidermis of the placenta.
Le demandeur s'est attaché à la caractérisation de nouvelles séquences promotrices fonctionnelles dans une plante et capables de diriger l'expression d'une ou plusieurs séquences d'intérêt placées sous le contrôle, spécifiquement dans la testa de la graine.The applicant has sought to characterize new functional promoter sequences in a plant capable of directing the expression of one or more sequences of interest placed under control, specifically in the testa of the seed.
SOMMAIRE DE L'INVENTIONSUMMARY OF THE INVENTION
Un premier objet de l'invention consiste en un acide nucléique comprenant des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et / ou l'albumen d'une graine de plante, lorsque ce polynucleotide d'intérêt est placé sous le contrôle de ces signaux de régulation, ledit acide nucléique possédant au moins 80% d'identité en nucleotides avec la séquence SEQ ID N°1 , ou avec un fragment d'au moins 200 nucleotides consécutifs de la séquence SEQ ID N°1.A first object of the invention consists of a nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID N ° 1.
L'invention est également relative à une cassette d'expression comprenant un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique tel que défini ci-dessus, le polynucleotide d'intérêt étant choisi parmi un polynucleotide codant pour un polypeptide et un polynucleotide sens ou antisens.The invention also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid as defined above, the polynucleotide of interest being chosen from a polynucleotide coding for a polypeptide and a sense or antisense polynucleotide.
L'invention est également relative à un vecteur recombinant de clonage et/ou d'expression comprenant un acide nucléique ou une cassette d'expression telle que définie ci-dessus, ainsi qu'à une cellule hôte transformée par cet acide nucléique ou cette cassette d'expression. L'invention a encore pour objet l'utilisation d'un acide nucléique, d'une cassette d'expression, d'un vecteur recombinant ou d'une cellule hôte recombinante telle que définie ci-dessus pour l'obtention d'une plante transformée. L'invention a aussi trait à des procédés d'obtention d'une plante transformée par un acide nucléique, une cassette d'expression, un vecteur recombinant ou une cellule hôte transformée telle que définie ci- dessus, ainsi qu'à des plantes transformées obtenues selon ces procédés et à des parties de ces plantes transformées, notamment les fruits et les semences.The invention also relates to a recombinant cloning and / or expression vector comprising a nucleic acid or an expression cassette as defined above, as well as to a host cell transformed by this nucleic acid or this cassette. expression. The subject of the invention is also the use of a nucleic acid, an expression cassette, a recombinant vector or a recombinant host cell as defined above for obtaining a plant. transformed. The invention also relates to methods for obtaining a plant transformed with a nucleic acid, an expression cassette, a recombinant vector or a transformed host cell as defined above, as well as to transformed plants. obtained according to these processes and to parts of these transformed plants, in particular fruits and seeds.
DESCRIPTION DETAILLEE DE L'INVENTIONDETAILED DESCRIPTION OF THE INVENTION
Il est fourni selon l'invention un acide nucléique comprenant des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, lorsque ce polynucleotide d'intérêt est placé sous le contrôle de ces signaux de régulation, ledit acide nucléique possédant au moins 80% d'identité en nucleotides avec la séquence SEQ ID N°1 , ou avec un fragment d'au moins 200 nucleotides consécutifs de la séquence SEQ ID - -N°1. - - _ _ _ _ _A nucleic acid is provided according to the invention comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID - -N ° 1. - - _ _ _ _ _
L'invention concerne aussi un acide nucléique de séquence complémentaire à l'acide nucléique tel que défini ci-dessus.The invention also relates to a nucleic acid of sequence complementary to the nucleic acid as defined above.
Selon l'invention, toute technique classique de biologie moléculaire, de microbiologie et d'ADN recombinant connue de l'homme du métier peut être utilisée. De telles techniques sont décrites par exemple par Sambrook et al. (1989), Glover et al. (1985), Gait (1984), Hames et Higgins (1985) et Ausubel et al. (1994).According to the invention, any conventional technique of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are described for example by Sambrook et al. (1989), Glover et al. (1985), Gait (1984), Hames and Higgins (1985) and Ausubel et al. (1994).
Un acide nucléique selon l'invention se présente préférentiellement sous une forme isolée ou purifiée.A nucleic acid according to the invention is preferably presented in an isolated or purified form.
Le terme " isolé " au sens de la présente invention désigne un matériel biologique qui a été soustrait à son environnement originelThe term "isolated" within the meaning of the present invention designates a biological material which has been removed from its original environment
(l'environnement dans lequel il est localisé naturellement). Par exemple, un polynucleotide présent à l'état naturel dans une plante n'est pas isolé. Le même polynucleotide séparé des acides nucléiques adjacents au sein desquels il est naturellement inséré dans le génome de la plante est isolé. Un tel polynucleotide peut être inclus dans un vecteur et/ou un tel polynucleotide peut être inclus dans une composition et demeurer néanmoins à l'état isolé du fait que le vecteur ou la composition ne constitue pas son environnement naturel.(the environment in which it is naturally located). For example, a naturally occurring polynucleotide in a plant is not isolated. The same polynucleotide separated from adjacent nucleic acids within from which it is naturally inserted into the genome of the plant is isolated. Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.
Le terme " purifié " ne nécessite pas que le matériel soit présent sous une forme de pureté absolue, exclusive de la présence d'autres composés. Il s'agit plutôt d'une définition relative.The term "purified" does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition.
Un polynucleotide ou un polypeptide est à l'état purifié après purification du matériel de départ ou du matériel naturel d'au moins un ordre de grandeur, de préférence 2 ou 3 et préférentiellement quatre ou cinq ordres de grandeur.A polynucleotide or a polypeptide is in the purified state after purification of the starting material or the natural material of at least one order of magnitude, preferably 2 or 3 and preferably four or five orders of magnitude.
Aux fins de la présente description, l'expression " séquence nucléotidique " peut être employée pour désigner indifféremment un polynucleotide ou un acide nucléique. L'expression " séquence nucléotidique " englobe le matériel génétique lui-même et n'est donc pas restreinte à l'information concernant sa séquence.For the purposes of the present description, the expression "nucleotide sequence" can be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not limited to information regarding its sequence.
Les termes " acide nucléique ", " polynucleotide ",The terms "nucleic acid", "polynucleotide",
" oligonucléotide " ou encore " séquence nucléotidique " englobent des séquences d'ARN, d'ADN, d'ADNc ou encore des séquences hybrides"oligonucleotide" or "nucleotide sequence" includes RNA, DNA, cDNA or hybrid sequences
ARN/ADN de plus d'un nucléotide, indifféremment sous la forme simple brin ou sous la forme de duplex.RNA / DNA of more than one nucleotide, either in the single-stranded form or in the form of duplex.
Le terme " nucléotide " désigne à la fois les nucleotides naturelsThe term "nucleotide" refers to both natural nucleotides
(A, T, G, C) ainsi que des nucleotides modifiés qui comprennent au moins une modification telle que (i) un analogue d'une purine, (ii) un analogue d'une pyrimidine, ou (iii) un sucre analogue, de tels nucleotides modifiés étant décrits par exemple dans la demande PCT NcWO(A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (i) a purine analog, (ii) a pyrimidine analog, or (iii) a sugar analog, such modified nucleotides being described for example in the application PCT N c WO
95/04064.95/04064.
Aux fins de la présente invention, un premier polynucleotide est considéré comme étant " complémentaire " d'un second polynucleotide lorsque chaque base du premier nucléotide est appariée à la base complémentaire du second polynucleotide dont l'orientation est inversée.For the purposes of the present invention, a first polynucleotide is considered to be "complementary" to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.
Les bases complémentaires sont A et T (ou A et U), et C et G.The complementary bases are A and T (or A and U), and C and G.
Selon l'invention, un premier acide nucléique ayant au moins 80% d'identité avec un second acide nucléique de référence, possédera au moins 80%, de préférence au moins 85%, 90%, 95%, 98%, 99% ou 99,5% d'identité en nucleotides avec ce second polynucleotide de référence, le pourcentage d'identité entre deux séquences étant déterminé comme décrit ci-dessous. Le " pourcentage d'identité " entre deux séquences de nucleotides ou d'acides aminés, au sens de la présente invention, peut être déterminé en comparant deux séquences alignées de manière optimale, à travers une fenêtre de comparaison.According to the invention, a first nucleic acid having at least 80% identity with a second reference nucleic acid, will have at at least 80%, preferably at least 85%, 90%, 95%, 98%, 99% or 99.5% of nucleotide identity with this second reference polynucleotide, the percentage of identity between two sequences being determined as described below. The "percentage of identity" between two nucleotide or amino acid sequences, within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window.
La partie de la séquence nucléotidique ou polypeptidique dans la fenêtre de comparaison peut ainsi comprendre des additions ou des délétions (par exemple des " gaps ") par rapport à la séquence de référence (qui ne comprend pas ces additions ou ces délétions) de manière à obtenir un alignement optimal des deux séquences.The part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences.
Le pourcentage est calculé en déterminant le nombre de positions auxquelles une base nucléique ou un résidu d'aminoacide identique est observé pour les deux séquences (nucléique ou peptidique) comparées, puis en divisant le nombre de positions auxquelles il y a identité entre les deux bases ou résidus d'aminoacides par le nombre total de positions dans la fenêtre de comparaison, puis en multipliant le résultat par cent afin d'obtenir le pourcentage d'identité de séquence.The percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percentage of sequence identity.
L'alignement optimal des séquences pour la comparaison peut être réalisé de manière informatique à l'aide d'algorithmes connus.The optimal alignment of the sequences for the comparison can be carried out by computer using known algorithms.
De manière préférée, le pourcentage d'identité de séquences est déterminé à l'aide du logiciel BLAST (version BLAST 2.06 de Septembre 1998), en utilisant exclusivement les paramètres par défaut.Preferably, the percentage of sequence identity is determined using the BLAST software (BLAST version 2.06 of September 1998), using exclusively the default parameters.
Un acide nucléique possédant au moins 80% d'identité en nucleotides avec un acide nucléique selon l'invention englobe les " variants " d'un acide nucléique selon l'invention.A nucleic acid having at least 80% nucleotide identity with a nucleic acid according to the invention includes the "variants" of a nucleic acid according to the invention.
Par " variant " d'un acide nucléique selon l'invention, on entend un acide nucléique qui diffère de l'acide nucléique de référence par une ou plusieurs substitutions, additions ou délétions d'un nucléotide, par rapport à l'acide nucléique de référence. Un variant d'un acide nucléique selon l'invention peut être d'origine naturelle, tel qu'un variant allélique qui existe naturellement. Un tel acide nucléique variant peut être également un acide nucléique non naturel obtenu, par exemple, par des techniques de mutagenèse.By "variant" of a nucleic acid according to the invention is meant a nucleic acid which differs from the reference nucleic acid by one or more substitutions, additions or deletions of a nucleotide, relative to the nucleic acid of reference. A variant of a nucleic acid according to the invention can be of natural origin, such as an allelic variant which exists naturally. Such a variant nucleic acid may also be an unnatural nucleic acid obtained, for example, by mutagenesis techniques.
En général, les différences entre l'acide nucléique de référence et l'acide nucléique " variant " sont réduites de telle sorte que l'acide nucléique de référence et l'acide nucléique variant ont des séquences nucléotidiques très similaires et, dans de nombreuses régions, identiques.In general, the differences between the reference nucleic acid and the "variant" nucleic acid are reduced so that the reference nucleic acid and the variant nucleic acid have very similar nucleotide sequences and, in many regions , identical.
Un " Variant " d'un acide nucléique régulateur selon l'invention conserve sa capacité à diriger l'expression d'un polynucleotide d'intérêt spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, le cas échéant lorsque ce variant est associé à une séquence régulatrice à fonction de promoteur. Pour vérifier le caractère fonctionnel d'un " variant " d'un acide nucléique selon l'invention, y compris d'un acide nucléique possédant au moins 80% d'identité en nucleotides avec la séquence SEQ ID N°1 ou avec un fragment d'au moins 200 nucleotides consécutifs de la séquence SEQ ID N°1 , l'homme du métier peut mettre en œuvre les tests d'expression spécifique d'un gène rapporteur ou marqueur décrits dans les exemples.A "Variant" of a regulatory nucleic acid according to the invention retains its ability to direct the expression of a polynucleotide of interest specifically in the endothelium and / or the albumen of a plant seed, if necessary. when this variant is associated with a regulatory sequence with a promoter function. To verify the functional nature of a "variant" of a nucleic acid according to the invention, including a nucleic acid having at least 80% identity in nucleotides with the sequence SEQ ID N ° 1 or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1, a person skilled in the art can carry out tests for the specific expression of a reporter or marker gene described in the examples.
Par " fragment " d'un acide nucléique selon l'invention, on entend une séquence nucléotidique d'une longueur réduite par rapport à l'acide nucléique de référence, le fragment d'acide nucléique possédant une séquence nucléotidique identique à la séquence nucléotidique de l'acide nucléique de référence sur la partie commune. De tels fragments d'un acide nucléique selon l'invention possèdent au moins 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 2000 ou 2300 nucleotides consécutifs de l'acide nucléique de référence, la longueur maximale en nucleotides d'un fragment d'un acide nucléique selon l'invention étant bien entendu limitée par la longueur maximale en nucleotides de l'acide nucléique de référence.By “fragment” of a nucleic acid according to the invention is meant a nucleotide sequence of a reduced length compared to the reference nucleic acid, the nucleic acid fragment having a nucleotide sequence identical to the nucleotide sequence of the reference nucleic acid on the common part. Such fragments of a nucleic acid according to the invention have at least 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 500, 600 , 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 2000 or 2300 consecutive nucleotides of the reference nucleic acid, the maximum length in nucleotides of a fragment of a nucleic acid according to the invention being indeed understood to be limited by the maximum length in nucleotides of the reference nucleic acid.
Préférentiellement, un " fragment " d'un acide nucléique régulateur selon l'invention comprend le polynucleotide défini par la séquence allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. De manière tout à fait préférée, un tel " fragment " comprend le polynucleotide défini par la séquence allant du nucléotide en position 2223 jusqu'au nucléotide en position 2228, de la séquence SEQ ID N°1 , qui correspond au motif « CANNTG ». Les "variants " et les " fragments " d'un acide nucléique régulateur selon l'invention sont " biologiquement actifs ", du fait qu'ils conservent leur capacité à diriger (s'ils comprennent une séquence promotrice) ou à moduler (par exemple moduler l'activité d'une séquence promotrice placée à proximité), l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium, ou à la fois dans l'endothélium et la couche cellulaire à aleurone d'une graine de plante, comme cela peut être vérifié par l'homme du métier mettant en œuvre les techniques décrites dans les exemples.Preferably, a "fragment" of a regulatory nucleic acid according to the invention comprises the polynucleotide defined by the sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID No. 1. Most preferably, such a "fragment" comprises the polynucleotide defined by the sequence going from the nucleotide at position 2223 to the nucleotide at position 2228, of the sequence SEQ ID No. 1, which corresponds to the motif "CANNTG". The "variants" and "fragments" of a regulatory nucleic acid according to the invention are "biologically active", because they retain their ability to direct (if they comprise a promoter sequence) or to modulate (for example modulate the activity of a promoter sequence placed nearby), the expression of a polynucleotide of interest, specifically in the endothelium, or in both the endothelium and the aleurone cell layer of a seed of plant, as can be verified by a person skilled in the art using the techniques described in the examples.
Par « acides nucléique comprenant des signaux de régulation » au sens de l'invention, on entend un acide nucléique capable d'influencer les caractéristiques d'expression d'un polynucleotide d'intérêt placé sous le contrôle de cet acide nucléique régulateur, en l'occurrence un acide nucléique dont la séquence permet de conférer une expression spécifique de tissu, et plus particulièrement capable de conférer une expression spécifique du polynucleotide d'intérêt dans l'endothélium et/ou l'albumen d'une graine de plante. Un tel acide nucléique peut comprendre, en plus des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, également une séquence à fonction de promoteur.By “nucleic acids comprising regulatory signals” within the meaning of the invention, is meant a nucleic acid capable of influencing the expression characteristics of a polynucleotide of interest placed under the control of this regulatory nucleic acid, in l occurrence a nucleic acid whose sequence makes it possible to confer a specific expression of tissue, and more particularly capable of conferring a specific expression of the polynucleotide of interest in the endothelium and / or the albumen of a plant seed. Such a nucleic acid may comprise, in addition to regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, also a sequence with a promoter function. .
Aux fins de la présente description, un acide nucléique à fonction de « promoteur » aussi désigné « promoteur » ou encore « séquence promotrice », consiste en un acide nucléique qui possède les motifs de reconnaissance de l'ARN polymérase, et plus spécifiquement d'une boîte « TATA » et d'une boîte « CAAT », dont la structure est bien connue de l'homme du métier.For the purposes of the present description, a nucleic acid with a “promoter” function, also designated “promoter” or even “promoter sequence”, consists of a nucleic acid which has the recognition patterns of RNA polymerase, and more specifically of a “TATA” box and a “CAAT” box, the structure of which is well known to those skilled in the art.
Il a été montré selon l'invention que l'acide nucléique de séquence SEQ ID N°1 comprend les signaux de régulation nécessaires à l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium d'une graine de plante, l'acide nucléique de séquence SEQ ID N°1 comprenant également une séquence promotrice capable d'initier la transcription d'un polynucleotide d'intérêt placé sous son contrôle. A la connaissance du demandeur, l'acide nucléique de séquenceIt has been shown according to the invention that the nucleic acid of sequence SEQ ID N ° 1 comprises the regulatory signals necessary for expression of a polynucleotide of interest, specifically in the endothelium of a plant seed, the nucleic acid of sequence SEQ ID No. 1 also comprising a promoter sequence capable of initiating the transcription of a polynucleotide d interest under his control. To the knowledge of the applicant, the nucleic acid of sequence
SEQ ID N°1 est le premier acide nucléique régulateur capable de diriger l'expression d'un polynucleotide d'intérêt spécifiquement dans l'endothélium d'une graine de plante. Une telle spécificité d'expression n'avait pas été observée avec les séquences régulatrices décrites par Fobert et al. (1994) ou par Colombo et al. (1997) dont l'expression n'était pas limitée à l'endothélium mais s'étendait plusieurs types cellulaires présents dans la testa de la graine.SEQ ID No. 1 is the first regulatory nucleic acid capable of directing the expression of a polynucleotide of interest specifically in the endothelium of a plant seed. Such specificity of expression had not been observed with the regulatory sequences described by Fobert et al. (1994) or by Colombo et al. (1997) whose expression was not limited to the endothelium but extended to several cell types present in the seed testa.
Il a aussi été montré selon l'invention que l'acide nucléique de séquence SEQ ID N°1 , ou encore un fragment d'au moins 250 nucleotides consécutifs de la séquence SEQ ID N°1 , est capable de diriger l'expression d'un polynucleotide d'intérêt, par exemple le gène rapporteur GUS, de manière optimale dès la formation de l'ovule En conséquence, l'acide nucléique de séquence SEQ ID N°1 ou un fragment d'au moins 200 nucleotides consécutifs de la séquence SEQ ID N°1 telle que définie ci-dessus, outre son activité spécifique de l'endothélium permet aussi une expression précoce, dès les derniers stades du développement de l'ovule, d'un polynucleotide d'intérêt placé sous son contrôle. Cette caractéristique d'activité temporelle précoce de l'acide nucléique régulateur selon l'invention rend possible l'expression du polynucleotide d'intérêt dès les premiers stades de formation de la graine, par exemple pour en modifier la composition ou encore pour altérer très précocement son développement, ce qui ne pouvait pas être envisagé avec les séquences promotrices décrites dans l'état de la technique. Grâce à la réalisation de constructions d'ADN comprenant la totalité ou des parties de plus en plus restreintes de la séquence SEQ ID N°1 , placées en aval d'un polynucleotide d'intérêt, le gène rapporteur GUS, est montré selon l'invention que les signaux de régulation permettant de diriger spécifiquement l'expression d'un polynucleotide d'intérêt dans l'endothélium de la graine, et le cas échéant également dans la couche à aleurone de l'albumen, étaient localisés du côté 3' de la séquence SEQ ID N°1.It has also been shown according to the invention that the nucleic acid of sequence SEQ ID No 1, or else a fragment of at least 250 consecutive nucleotides of the sequence SEQ ID No 1, is capable of directing the expression d a polynucleotide of interest, for example the reporter gene GUS, optimally as soon as the ovum forms. Consequently, the nucleic acid of sequence SEQ ID No 1 or a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1 as defined above, in addition to its specific activity of the endothelium also allows early expression, from the last stages of ovum development, of a polynucleotide of interest placed under its control. This characteristic of early temporal activity of the regulatory nucleic acid according to the invention makes it possible to express the polynucleotide of interest from the first stages of seed formation, for example to modify the composition or else to alter very early its development, which could not be envisaged with the promoter sequences described in the state of the art. By virtue of the production of DNA constructs comprising all or more and more and more restricted parts of the sequence SEQ ID No. 1, placed downstream of a polynucleotide of interest, the reporter gene GUS, is shown according to the invention as regulatory signals to specifically direct the expression of a polynucleotide of interest in the seed endothelium, and where appropriate also in the aleurone layer of the albumen, were located on the 3 'side of the sequence SEQ ID No. 1.
L'acide nucléique de séquence SEQ ID N°1 selon l'invention a une longueur de 2376 nucleotides.The nucleic acid of sequence SEQ ID No. 1 according to the invention has a length of 2376 nucleotides.
Acides nucléiques comportant les signaux de régulation spécifiques de tissus.Nucleic acids with specific tissue regulatory signals.
Différentes constructions d'ADN ont été réalisées, chacune de ces constructions d'ADN comprenant un fragment de l'acide nucléique allant du nucléotide en position 1 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Les constructions d'ADN comprennent également un acide nucléique à fonction de promoteur.Different DNA constructs have been produced, each of these DNA constructs comprising a fragment of the nucleic acid ranging from the nucleotide in position 1 to the nucleotide in position 2335 of the sequence SEQ ID No. 1. The DNA constructs also include a promoter-functional nucleic acid.
Il a été réalisé des cassettes d'expression comprenant un polynucleotide d'intérêt codant pour un gène rapporteur, le gène GUS, qui a été placé sous le contrôle d'un acide nucléique régulateur tel que défini ci-dessus, comprenant à la fois les signaux de régulation spécifiques de tissu et un acide nucléique à fonction de promoteur.Expression cassettes were produced comprising a polynucleotide of interest coding for a reporter gene, the GUS gene, which was placed under the control of a regulatory nucleic acid as defined above, comprising both the tissue-specific regulatory signals and a promoter-functioning nucleic acid.
Ces cassettes d'expression ont été utilisées pour transformer des plantes, plus spécifiquement des plantes de l'espèce Arabidopsis thaliana, après quoi l'expression du gène rapporteur a été suivie dans les différents tissus de la plante au cours du temps.These expression cassettes were used to transform plants, more specifically plants of the Arabidopsis thaliana species, after which the expression of the reporter gene was monitored in the various tissues of the plant over time.
Les résultats des exemples montrent que les signaux de régulation nécessaires à l'activité de transcription tissu-spécifique dans la graine de plante sont compris dans l'acide nucléique allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 , ce qui correspond à l'expression du gène rapporteur obtenu avec la construction désignée pBAN6.The results of the examples show that the regulatory signals necessary for tissue-specific transcription activity in the plant seed are included in the nucleic acid ranging from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1, which corresponds to the expression of the reporter gene obtained with the construction designated pBAN6.
Il a été montré selon l'invention que l'acide nucléique régulateur présent dans la construction pBAN6 contenait un motif « CANNTG » localisé du nucléotide en position 2223 jusqu'au nucléotide en position 2228 de la séquence SEQ ID N°1. Le motif CANNTG constitue un site potentiel de liaison à des facteurs de transcription du type MYC, ainsi qu'il résulte de l'étude structurale de l'acide nucléique régulateur de séquence SEQ ID N°1 à l'aide d'un ordinateur exploitant les informations de la base de données PLACE (Plant cis-acting Regulatory Eléments data base; HIGO et al., 1999). Sans vouloir être liés par une quelconque théorie, les inventeurs pensent que le motif CANNTG localisé du nucléotide en position 2223 jusqu'au nucléotide en position 2228 de la séquence SEQ ID N°1 constitue une caractéristique structurelle importante de l'acide nucléique régulateur tissu-spécifique de l'invention.It has been shown according to the invention that the regulatory nucleic acid present in the construction pBAN6 contains a "CANNTG" motif located from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1. The CANNTG motif constitutes a potential site for binding to transcription factors of the MYC type, as it results from the structural study of the nucleic acid regulating sequence SEQ ID No. 1 using an operating computer. information from the PLACE (Plant cis-acting Regulatory Elements) database data base; HIGO et al., 1999). Without wishing to be bound by any theory, the inventors believe that the CANNTG motif localized from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1 constitutes an important structural characteristic of the tissue-regulating nucleic acid. specific to the invention.
De plus, des résultats préliminaires indiquent la présence d'un motif de liaison d'un facteur de transcription réprimant l'expression dans l'albumen, ce motif étant localisé dans une région nucléotidique allant du nucléotide en position 1919 jusqu'au nucléotide en position 2054 de l'acide nucléique de séquence SEQ ID N°1.In addition, preliminary results indicate the presence of a motif of binding of a transcription factor repressing expression in the albumen, this motif being located in a nucleotide region extending from the nucleotide at position 1919 to the nucleotide at position 2054 of the nucleic acid of sequence SEQ ID No. 1.
Description de l'acide nucléique à fonction de promoteur contenu dans la séquence SEQ ID N°1Description of the nucleic acid with promoter function contained in the sequence SEQ ID N ° 1
L'acide nucléique à fonction de promoteur contenu dans l'acide nucléique de séquence SEQ ID N°1 est entièrement contenu dans la séquence allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Cette séquence contient une boîte « TATA » localisée du nucléotide en position 2306 jusqu'au nucléotide en position 2312 de la séquence SEQ ID N°1. Cette séquence promotrice contient également une boîte « CAAT » localisée du nucléotide en position 2172 jusqu'au nucléotide en position 2180 de la séquence SEQ ID N°1. La boîte TATA constitue l'élément promoteur proprement dit, qui est localisé en général à une distance d'environ 30 bases du site d'initiation de la transcription. La boîte CAAT est un élément agissant en cis qui est retrouvée communément dans la région promotrice et activatrice (« enhancer »).The promoter-function nucleic acid contained in the nucleic acid of sequence SEQ ID No. 1 is entirely contained in the sequence going from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1. This sequence contains a “TATA” box located from the nucleotide in position 2306 to the nucleotide in position 2312 of the sequence SEQ ID No. 1. This promoter sequence also contains a “CAAT” box located from the nucleotide at position 2172 to the nucleotide at position 2180 of the sequence SEQ ID No. 1. The TATA box constitutes the promoter element proper, which is generally located at a distance of about 30 bases from the site of initiation of transcription. The CAAT box is a cis-acting element which is commonly found in the promoter and activator region ("enhancer").
L'acide nucléique à fonction de promoteur allant du nucléotide en position 2172 jusqu'au nucléotide en position 2312 de la séquence SEQ ID N°1 , ainsi que tout fragment « biologiquement actif » de cet acide nucléique promoteur et contenant les boîtes CAAT et TATA définies ci- dessus constitue un autre objet de la présente invention.The nucleic acid with a promoter function ranging from the nucleotide at position 2172 to the nucleotide at position 2312 of the sequence SEQ ID No. 1, as well as any “biologically active” fragment of this promoter nucleic acid and containing the CAAT and TATA boxes defined above constitutes another object of the present invention.
Acides nucléiques régulateurs selon l'inventionRegulatory nucleic acids according to the invention
Il s'agit en premier lieu d'un acide nucléique comprenant des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, lorsque ce polynucleotide d'intérêt est placé sous le contrôle de ces signaux de régulation, ledit acide nucléique possédant au moins 80% d'identité en nucleotides avec la séquence SEQ ID N°1, ou avec un fragment d'au moins 250 nucleotides consécutifs de la séquence SEQ ID N°1.It is firstly a nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under the control of these regulatory signals, said nucleic acid having at least 80% identity in nucleotides with the sequence SEQ ID No 1, or with a fragment of at least 250 consecutive nucleotides of the sequence SEQ ID No 1.
Comme déjà précisé, un tel acide nucléique régulateur comprendra préférentiellement le motif CANNTG allant du nucléotide en position 2223 jusqu'au nucléotide en position 2228 de la séquence SEQ ID N°1.As already specified, such a regulatory nucleic acid will preferably comprise the CANNTG motif ranging from the nucleotide at position 2223 to the nucleotide at position 2228 of the sequence SEQ ID No. 1.
Selon un premier aspect, un tel acide nucléique est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 2273 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est compris notamment dans la construction pBANδ des exemples.According to a first aspect, such a nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2273 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is included in particular in the construction pBANδ of the examples.
Selon un second aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 2141 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est compris notamment dans la construction désignée pBAN7 décrite dans les exemples.According to a second aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2141 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is included in particular in the construction designated pBAN7 described in the examples.
Selon un troisième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est notamment compris dans la construction pBAN6 décrite dans les exemples.According to a third aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is in particular included in the construction pBAN6 described in the examples.
Selon un quatrième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 1919 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est compris notamment dans la construction pBAN5 décrite dans les exemples. Selon un cinquième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 1510 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est notamment compris dans la construction pBAN4 décrite dans les exemples.According to a fourth aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1919 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is included in particular in the construction pBAN5 described in the examples. According to a fifth aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1510 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is in particular included in the construction pBAN4 described in the examples.
Selon un sixième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 101 1 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 . Un tel acide nucléique régulateur est compris dans la construction désignée pBAN3 décrite dans les exemples.According to a sixth aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 101 1 to the nucleotide in position 2335 of the sequence SEQ ID N ° 1. Such a regulatory nucleic acid is included in the construction designated pBAN3 described in the examples.
Selon un septième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 509 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 . Un tel acide nucléique régulateur est notamment compris dans la construction pBAN2 décrite dans les exemples.According to a seventh aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 509 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is in particular included in the construction pBAN2 described in the examples.
Selon un huitième aspect, l'acide nucléique régulateur est caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 1 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. Un tel acide nucléique régulateur est compris dans la construction désignée pBAN1 décrite dans les exemples. Chacun des acides nucléiques ci-dessus peut aussi comprendre la séquence allant du nucléotide en position 2336 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 .According to an eighth aspect, the regulatory nucleic acid is characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1 to the nucleotide in position 2335 of the sequence SEQ ID # 1. Such a regulatory nucleic acid is included in the construction designated pBAN1 described in the examples. Each of the above nucleic acids can also comprise the sequence going from the nucleotide at position 2336 to the nucleotide at position 2376 of the sequence SEQ ID No. 1.
Pour obtenir l'expression d'un polynucleotide d'intérêt uniquement dans l'endothélium de la testa de la graine, on utilisera un acide nucléique régulateur comprenant au moins la séquence allant du nucléotide en position 1919 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 . Lorsque l'on recherchera l'expression du polynucleotide d'intérêt à la fois dans l'endothélium de la testa de la graine et dans la couche de cellules à aleurone de l'albumen, on utilisera préférentiellement un acide nucléique régulateur plus court, par exemple un acide nucléique régulateur comprenant une séquence allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 , dépourvu de tout ou partie de la séquence 1918- 2053 de la séquence SEQ ID N°1. La présente invention est également relative à un acide nucléique dont la séquence est complémentaire à la séquence de l'un quelconque des acides nucléiques tels que définis ci-dessus.To obtain the expression of a polynucleotide of interest only in the endothelium of the seed testa, a regulatory nucleic acid will be used comprising at least the sequence going from the nucleotide at position 1919 to the nucleotide at position 2335 of the sequence SEQ ID N ° 1. When the expression of the polynucleotide of interest is sought both in the endothelium of the seed testa and in the aleurone layer of the albumen cells, a shorter regulatory nucleic acid will preferably be used, for example example a regulatory nucleic acid comprising a sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID No 1, devoid of all or part of the sequence 1918-2053 of the sequence SEQ ID No 1. The present invention also relates to a nucleic acid whose sequence is complementary to the sequence of any one of the nucleic acids as defined above.
Un acide nucléique régulateur tel que défini ci-dessus peut comprendre uniquement les signaux de régulation permettant de moduler l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, sans inclure d'acide nucléique à fonction de promoteur.A regulatory nucleic acid as defined above can comprise only the regulatory signals making it possible to modulate the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, without include promoter-function nucleic acid.
Dans ce cas, les caractéristiques fonctionnelles d'activité tissu- spécifique d'un acide régulateur selon l'invention sont exploitées par l'introduction, dans cet acide nucléique régulateur, également d'un acide nucléique à fonction de promoteur, plus spécifiquement d'un polynucleotide à fonction de promoteur qui est fonctionnel dans une cellule de plante.In this case, the functional characteristics of tissue-specific activity of a regulatory acid according to the invention are exploited by the introduction, into this regulatory nucleic acid, also of a nucleic acid with a promoter function, more specifically of a promoter-functional polynucleotide that is functional in a plant cell.
Ainsi, dans un mode de réalisation particulier d'un acide régulateur selon l'invention, cet acide nucléique régulateur est caractérisé en ce qu'il comprend de plus un polynucleotide à fonction de promoteur qui est fonctionnel dans une cellule de plante.Thus, in a particular embodiment of a regulatory acid according to the invention, this regulatory nucleic acid is characterized in that it further comprises a polynucleotide with promoter function which is functional in a plant cell.
Dans un mode de réalisation particulier, le polynucleotide à fonction de promoteur est la séquence nucléotidique allant du nucléotide en position 2172, premier nucléotide de la boîte CAAT, jusqu'au nucléotide en position 2312, dernier nucléotide de la boîte « TATA » de la séquence SEQ ID N°1 ou encore un variant ou un fragment biologiquement actif de cette séquence.In a particular embodiment, the promoter-functional polynucleotide is the nucleotide sequence going from the nucleotide in position 2172, first nucleotide of the CAAT box, to the nucleotide in position 2312, last nucleotide of the “TATA” box of the sequence SEQ ID N ° 1 or a variant or a biologically active fragment of this sequence.
Un variant de l'acide nucléique à fonction de promoteur défini ci- dessus comprend une séquence nucléotidique ayant au moins 80% d'identité avec la séquence allant du nucléotide en position 2172 jusqu'au nucléotide en position 2312 de la séquence SEQ ID N°1 et est capable de diriger l'expression d'un polynucleotide d'intérêt dans une cellule de plante. De manière tout à fait préférée, un tel variant comprend les boîtes CAAT et TATA définies précédemment dans la présente description. Un tel fragment d'acide nucléique peut aussi comprendre la séquence nucléotidique allant du nucléotide en position 2313 jusqu'au nucléotide en position 2376 de l'acide nucléique de séquence SEQ ID N°1.A variant of the nucleic acid with a promoter function defined above comprises a nucleotide sequence having at least 80% identity with the sequence going from the nucleotide in position 2172 to the nucleotide in position 2312 of the sequence SEQ ID N ° 1 and is capable of directing the expression of a polynucleotide of interest in a plant cell. Most preferably, such a variant includes the CAAT and TATA boxes defined previously in the present description. A such a nucleic acid fragment can also comprise the nucleotide sequence going from the nucleotide in position 2313 to the nucleotide in position 2376 of the nucleic acid of sequence SEQ ID No. 1.
Comme cela a déjà été précisé, l'un des objectifs poursuivis par la présente invention est l'obtention d'une expression contrôlée d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou la couche cellulaire à aleurone de l'albumen d'une graine de plante, par exemple dans les situations où l'on cherche à: - modifier la composition ou l'équilibre hormonal de la testa de la graine;As has already been specified, one of the objectives pursued by the present invention is to obtain a controlled expression of a polynucleotide of interest, specifically in the endothelium and / or the aleurone cell layer of the albumen of a plant seed, for example in situations where one seeks to: - modify the composition or the hormonal balance of the testa of the seed;
- obtenir des plantes avec des graines de taille réduite ou encore avec un nombre réduit de graines , voire une absence pratiquement totale ou totale de graines, et plus spécifiquement de graines matures ou de graines fertiles.- obtain plants with seeds of reduced size or with a reduced number of seeds, or even an almost total or total absence of seeds, and more specifically of mature seeds or fertile seeds.
L'invention a donc encore pour objet un acide nucléique comprenant un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique régulateur tel que défini ci-dessus. Un tel acide nucléique est aussi désigné « cassette d'expression » aux fins de la présente description.The invention therefore also relates to a nucleic acid comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above. Such a nucleic acid is also designated “expression cassette” for the purposes of the present description.
L'invention a donc encore pour objet une cassette d'expression comprenant un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique régulateur tel que défini précédemment.The invention therefore also relates to an expression cassette comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above.
CASSETTES D'EXPRESSION SELON L'INVENTIONEXPRESSION CASSETTES ACCORDING TO THE INVENTION
Selon un premier mode de réalisation, une cassette d'expression selon l'invention est caractérisée en ce que le polynucleotide d'intérêt code pour un polypeptide.According to a first embodiment, an expression cassette according to the invention is characterized in that the polynucleotide of interest codes for a polypeptide.
Préférentiellement, le polynucleotide d'intérêt code pour un polypeptide impliqué dans la régulation du contenu en tannins ou en fibres de la graine ou encore pour un polypeptide régulant l'équilibre hormonal dans la testa de la graine.Preferably, the polynucleotide of interest codes for a polypeptide involved in the regulation of the tannin or fiber content of the seed or also for a polypeptide regulating the hormonal balance in the testa of the seed.
Au vu des exemples cités ci-dessus, on peut envisager d'augmenter (surexpression) ou de supprimer par cosuppression, antisens (Vaucheret et Fagard, 2001), ARN interférence (Smith et al., 2000) ou immunomodulation (Phillips et al., 1997) l'expression de divers gènes du métabolisme placés sous le contrôle du promoteur pBAN.In view of the examples cited above, one can consider increasing (overexpression) or suppressing by cosuppression, antisense (Vaucheret and Fagard, 2001), RNA interference (Smith et al., 2000) or immunomodulation (Phillips et al., 1997) the expression of various metabolic genes under the control of the pBAN promoter.
La modulation du métabolisme des composés phénoliques peut se concevoir en utilisant un gène codant pour des facteurs de transcription, par exemple de type Myc (Damiani et al., 1999; Nesi et al., 2000) ou de type Myb (Grotewold et al., 1998; Borevitz et al., 2000), ainsi que des gènes de structure codant pour des enzymes de la voie de biosynthèse ou pour des transporteurs (Debeaujon et al., 2001). La protéine BAN (probablement une leucocyanidine reductase selon Devic et al., 1999) est une enzyme clé conduisant à la formation des tannins, il est probable que sa suppression ou sa surexpression conduisent à l'absence et à l'augmentation de tannins respectivement, cela sans affecter les niveaux des autres flavonoïdes; la suppression de la CHS (chalcone synthase) conduit à l'absence de flavonoïdes; quant à la LDOX (leucocyanidine déoxygenase) et la FLS (flavonol synthase) (Pelletier et al., 1997), leur modulation devrait conduire à augmenter ou supprimer les anthocyanes et les flavonols, respectivement. L'utilisation de gènes du métabolisme des lignines tels que ceux codant pour les enzymes F5H (ferulate-5-hydroxylase; Meyer et al., 1998; Franke et al., 2000), cinnamyl-alcool dehydrogenase et cinnamoyl-CoA reductase (Ralph et al., 1998) sont des candidats pour la modification de ces composés. Des gènes codant pour des enzymes de la voie de biosynthèse de la cellulose (Fagard et al., 2000) peuvent être envisagés pour modifier ce composé. Les gènes codant pour des invertases de la testa (Weber et al.,The modulation of the metabolism of phenolic compounds can be conceived using a gene coding for transcription factors, for example of the Myc type (Damiani et al., 1999; Nesi et al., 2000) or of the Myb type (Grotewold et al. , 1998; Borevitz et al., 2000), as well as structural genes coding for enzymes of the biosynthetic pathway or for transporters (Debeaujon et al., 2001). The protein BAN (probably a leukocyanidine reductase according to Devic et al., 1999) is a key enzyme leading to the formation of tannins, it is likely that its suppression or over-expression lead to the absence and increase of tannins respectively, this without affecting the levels of other flavonoids; the suppression of CHS (chalcone synthase) leads to the absence of flavonoids; as for LDOX (leucocyanidine deoxygenase) and FLS (flavonol synthase) (Pelletier et al., 1997), their modulation should lead to increasing or suppressing anthocyanins and flavonols, respectively. The use of lignin metabolism genes such as those encoding the enzymes F5H (ferulate-5-hydroxylase; Meyer et al., 1998; Franke et al., 2000), cinnamyl-alcohol dehydrogenase and cinnamoyl-CoA reductase (Ralph et al., 1998) are candidates for modification of these compounds. Genes coding for enzymes of the cellulose biosynthetic pathway (Fagard et al., 2000) can be envisaged to modify this compound. The genes coding for testa invertases (Weber et al.,
1996) sont des cibles potentielles pour modifier les modalités de nutrition de l'embryon.1996) are potential targets for modifying the nutrition modalities of the embryo.
La manipulation de gènes de biosynthèse d'hormones pour modifier les caractéristiques structurales de la testa peut également se concevoir, en particulier pour les gibbérellines avec GA1 (Sun et Kamiya, 1994), et le gène de la GA-20-oxidase (Coles et al., 1999; Kang et al., 1999), pour l'acide abscissique avec ABA2 codant pour la zéaxanthine epoxidase (Marin et al., 1996), pour l'éthylène avec l'enzyme ACC synthase (Gray et al., 1992), pour les brassinostéroïdes avec DET2 codant pour une stéroïde 5-alpha-réductase (Noguchi et al., 1999), pour les auxines avec iaaM (Rotino et al., 1997), pour les cytokinines avec ipt (Barry et al., 1984); voir la revue de Hedden et Phillips (2000) pour plus de détails.Manipulation of hormone biosynthesis genes to modify the structural characteristics of testa can also be conceived, in particular for gibberellins with GA1 (Sun and Kamiya, 1994), and the gene for GA-20-oxidase (Coles and al., 1999; Kang et al., 1999), for abscissic acid with ABA2 coding for zeaxanthin epoxidase (Marin et al., 1996), for ethylene with the enzyme ACC synthase (Gray et al., 1992), for brassinosteroids with DET2 coding for a steroid 5-alpha-reductase (Noguchi et al., 1999), for auxins with iaaM (Rotino et al., 1997), for cytokinins with ipt (Barry et al., 1984); see the review by Hedden and Phillips (2000) for more details.
Dans la situation où l'on cherche à obtenir une plante pour laquelle le développement d'une graine mature est affecté, on préférera un polynucleotide d'intérêt dont l'expression est toxique pour la cellule de plante, par exemple un polynucleotide codant pour un polypeptide toxique pour la cellule de plante, ou encore un polynucleotide capable d'inhiber ou de bloquer spécifiquement la fonction d'un ou plusieurs gènes impliqués dans le développement de la graine. De manière préférée, lorsque le polynucleotide d'intérêt code pour un polypeptide toxique pour la cellule de plante, les séquences codant pour les polypeptides suivants seront choisies de préférence:In the situation where it is sought to obtain a plant for which the development of a mature seed is affected, a polynucleotide of interest whose expression is toxic for the plant cell will be preferred, for example a polynucleotide coding for a polypeptide toxic to the plant cell, or a polynucleotide capable of specifically inhibiting or blocking the function of one or more genes involved in the development of the seed. Preferably, when the polynucleotide of interest codes for a polypeptide toxic for the plant cell, the sequences coding for the following polypeptides will preferably be chosen:
- la Barnase (Paddon et Hartley, 1987 ; Hartley, 1989)- Barnase (Paddon and Hartley, 1987; Hartley, 1989)
- la toxine diphtérique (YAMAIZUMI et al., 1987); II peut ainsi s'agir des gènes dont on sait qu'ils s'expriment chez les plantes, en se référant à la revue de Day et Irish (1997):- diphtheria toxin (YAMAIZUMI et al., 1987); It may thus be a question of genes which we know to express in plants, with reference to the review by Day and Irish (1997):
- barnase de Bacillus amyloliquefaciens (Paddon et Hartley, 1987; Hartley, 1989)- barnase of Bacillus amyloliquefaciens (Paddon and Hartley, 1987; Hartley, 1989)
- toxine diphtérique chaîne A de Corynebacterium diphteria (Yamaizumi et al., 1978; Czaco et al., 1992).- Corynebacterium diphteria A chain diphtheria toxin (Yamaizumi et al., 1978; Czaco et al., 1992).
- RNase T1 de Aspergillus oryzae (Mariani et al., 1990)- RNase T1 from Aspergillus oryzae (Mariani et al., 1990)
- exotoxine A de Pseudomonas aeruginosa (Koning et al., 1992)- Pseudomonas aeruginosa exotoxin A (Koning et al., 1992)
On peut aussi faire exprimer des gènes conférant de nouvelles propriétés à la testa comme des inhibiteurs de protéases pour la protection contre les insectes (Jouanin et al., 2000), ou des toxines cellulaires telles que la barnase (Colombo et al., 1997; Varoquaux et al., 2000) ou la toxine A diphtérique (Czaco et al., 1992) pour réduire la taille de la graine (Koltunow et Brennan, 1998; Koltunow et al., 1998) ou pour effectuer l'ablation génétique de la graine et donc l'obtention de fruits sans graines. Pour cette dernière approche, l'utilisation d'un système inductible comme ceux décrits par Aoyama et Chua (1997) ainsi que Zuo et al. (2000) peut être envisagée. Tirer profit du système barnase / barstar, comme décrit par Mariani et al. (1990, 1992) est une deuxième possibilité. L'ablation génétique de la graine peut aussi être atteinte en utilisant des antisens de gènes du cycle cellulaire comme CDC2 (Ferreira et al., 1991 ; Hemerly et al., 1992). On peut penser aussi à des gènes tels que AINTEGUMENTA dont le mutant est embryon-léthal (Elliot et al., 1996; Klucher et al., 1996). Des gènes intervenant dans le métabolisme d'hormones telles que les gibbérellines et les auxines sont des candidats potentiels pour l'obtention d'une parthénocarpie (Vivian-Smith et Koltunow, 1999; Varoquaux et al., 2000).It is also possible to express genes conferring new properties on testa such as protease inhibitors for protection against insects (Jouanin et al., 2000), or cellular toxins such as barnase (Colombo et al., 1997; Varoquaux et al., 2000) or diphtheria toxin A (Czaco et al., 1992) to reduce the size of the seed (Koltunow and Brennan, 1998; Koltunow et al., 1998) or to perform genetic ablation of the seed and therefore obtaining fruit without seeds. For this last approach, the use of an inducible system like those described by Aoyama and Chua (1997) as well as Zuo et al. (2000) can be considered. Take advantage of the barnase / barstar system, as described by Mariani et al. (1990, 1992) is a second possibility. Genetic ablation of the seed can also be achieved by using antisense genes from the cell cycle such as CDC2 (Ferreira et al., 1991; Hemerly et al., 1992). We can also think of genes such as AINTEGUMENTA whose mutant is embryo-lethal (Elliot et al., 1996; Klucher et al., 1996). Genes involved in the metabolism of hormones such as gibberellins and auxins are potential candidates for obtaining parthenocarpy (Vivian-Smith and Koltunow, 1999; Varoquaux et al., 2000).
Selon un autre aspect, le polynucleotide d'intérêt dont l'expression est toxique pour la cellule de plante peut être un polynucleotide sens ou antisens capable d'inhiber ou de bloquer respectivement la transcription ou la traduction d'un gène requis pour le développement normal de la graine, et de manière préférée d'un gène requis pour le développement normal de la testa de la graine, tout particulièrement la testa. 1 ) Pour modifier les produits finaux de voies biochimiques, comme celle des flavonoïdes, des lignines ou de la celluloseAccording to another aspect, the polynucleotide of interest whose expression is toxic to the plant cell can be a sense or antisense polynucleotide capable of inhibiting or blocking respectively the transcription or the translation of a gene required for normal development of the seed, and preferably of a gene required for the normal development of the testa of the seed, especially the testa. 1) To modify the end products of biochemical pathways, such as that of flavonoids, lignins or cellulose
- gènes de biosynthèses de flavonoïdes, particulièrement BAN (pour suppression des tannins)- flavonoid biosynthesis genes, particularly BAN (for suppression of tannins)
- gènes du métabolisme des lignines - gènes de biosynthèse de la cellulose- genes for lignin metabolism - genes for cellulose biosynthesis
- Gènes du métabolisme hormonal de la graine- Genes of the hormonal metabolism of the seed
2) Pour réduire la taille de la graine voire conduire à sa disparition:2) To reduce the size of the seed or even lead to its disappearance:
- gènes du cycle cellulaire - gènes de développement de la testa (Schneitz, 1999), particulièrement AINTEGUMENTA (Krizek, 1999)- cell cycle genes - testa development genes (Schneitz, 1999), particularly AINTEGUMENTA (Krizek, 1999)
- gènes du métabolisme hormonal- hormone metabolism genes
De préférence, un polynucleotide antisens selon l'invention hybride, au sein de la cellule, spécifiquement avec une région du gène cible comprenant le site d'initiation de la transcription et/ou avec une séquence importante pour l'épissage correct de l'ARN messager.Preferably, an antisense polynucleotide according to the invention hybrid, within the cell, specifically with a region of the target gene comprising the site of initiation of transcription and / or with a sequence important for the correct splicing of RNA messenger.
De préférence, un polynucleotide antisens selon l'invention est un acide nucléique complémentaire de l'ARN messager constituant le produit de transcription du gène cible et comprenant le site d'initiation de la traduction. Un tel polynucleotide antisens peut être l'ADN complémentaire de l'ARN messager du gène visé.Preferably, an antisense polynucleotide according to the invention is a nucleic acid complementary to the messenger RNA constituting the transcript of the target gene and comprising the site of initiation of the translation. Such an antisense polynucleotide can be the DNA complementary to the messenger RNA of the targeted gene.
Selon encore un autre mode de réalisation, le polynucleotide d'intérêt, qui est placé sous le contrôle d'un acide nucléique régulateur selon l'invention, code pour un polypeptide constitué d'une protéine de fusion entre un récepteur aux glucocorticoïdes et la protéine GAL-4.According to yet another embodiment, the polynucleotide of interest, which is placed under the control of a regulatory nucleic acid according to the invention, codes for a polypeptide consisting of a fusion protein between a glucocorticoid receptor and the protein GAL-4.
VECTEURS RECOMBINANTS SELON L'INVENTIONRECOMBINANT VECTORS ACCORDING TO THE INVENTION
L'invention concerne aussi des vecteurs recombinants de clonage et/ou d'expression comprenant un acide nucléique régulateur ou une cassette d'expression telle que définis ci-dessus.The invention also relates to recombinant cloning and / or expression vectors comprising a regulatory nucleic acid or an expression cassette as defined above.
Un autre objet de l'invention consiste donc en des vecteurs recombinants dans lequel a été inséré un acide nucléique régulateur selon l'invention, comprenant des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, lorsque ce polynucleotide d'intérêt est placé sous son contrôle, ledit acide nucléique possédant au moins 80% d'identité en nucléotide avec la séquence SEQ ID N°1 , ou avec un fragment d'au moins 250 nucleotides consécutifs de la séquence SEQ ID N°1.Another subject of the invention therefore consists of recombinant vectors into which a regulatory nucleic acid according to the invention has been inserted, comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under its control, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment at least 250 consecutive nucleotides of the sequence SEQ ID No. 1.
Avantageusement, un vecteur recombinant de clonage et/ou d'expression selon l'invention comprend une cassette d'expression comprenant un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique régulateur tel que défini ci-dessus. Font ainsi partie de l'invention des vecteurs recombinants pour l'expression d'un polynucleotide d'intérêt dans l'endothélium et/ou l'albumen d'une graine de plante, comprenant: a) un acide nucléique régulateur tel que défini dans la présente description; et b) un polynucleotide d'intérêt placé sous le contrôle de l'acide nucléique régulateur défini en a).Advantageously, a recombinant cloning and / or expression vector according to the invention comprises an expression cassette comprising a polynucleotide of interest placed under the control of a regulatory nucleic acid as defined above. Thus part of the invention are recombinant vectors for the expression of a polynucleotide of interest in the endothelium and / or the albumen of a plant seed, comprising: a) a regulatory nucleic acid as defined in this description; and b) a polynucleotide of interest placed under the control of the regulatory nucleic acid defined in a).
Le polynucleotide d'intérêt peut consister en un polynucleotide codant pour un polypeptide détectable, ou polypeptide marqueur tel que par exemple un polypeptide codant pour la protéine GUS ou encore pour une protéine fluorescente, telle que la protéine GFP (Green fluorescent protein) ou YFP (Yellow fluorescent protein).The polynucleotide of interest can consist of a polynucleotide coding for a detectable polypeptide, or marker polypeptide such as for example a polypeptide coding for the GUS protein or also for a fluorescent protein, such as the GFP (Green fluorescent protein) or YFP (Yellow fluorescent protein).
Selon un autre aspect, le polynucleotide d'intérêt code pour un polypeptide impliqué dans la régulation du contenu en tannins ou en fibres de la graine, ou pour un polypeptide régulant l'équilibre hormonal dans la testa du grain. Le polynucleotide d'intérêt peut être aussi un polynucleotide dont l'expression est toxique pour la cellule de plante, tel qu'il est défini précédemment dans la présente description.In another aspect, the polynucleotide of interest encodes a polypeptide involved in the regulation of tannin or fiber content of the seed, or a polypeptide regulating hormonal balance in the testa of grain. The polynucleotide of interest can also be a polynucleotide whose expression is toxic to the plant cell, as defined previously in the present description.
Un vecteur recombinant selon l'invention répondant à la définition ci-dessus est le vecteur pBAN1 ::GUS/pBIB-Hyg contenu dans la souche de E.coli déposée à la CNCM le 17 juillet 2001 sous le numéro d'accès I- 2703.A recombinant vector according to the invention corresponding to the above definition is the vector pBAN1 :: GUS / pBIB-Hyg contained in the strain of E. coli deposited at the CNCM on July 17, 2001 under the access number I-2703 .
D'autres vecteurs répondant à la définition générale d'un vecteur recombinant selon l'invention sont respectivement les vecteurs pBAN2::GUS, pBAN3::GUS, pBAN4::GUS, pBAN5::GUS, pBAN6:GUS pBAN7 :GUS et PBAN8 : GUS illustrés dans les exemples.Other vectors meeting the general definition of a recombinant vector according to the invention are respectively the vectors pBAN2 :: GUS, pBAN3 :: GUS, pBAN4 :: GUS, pBAN5 :: GUS, pBAN6: GUS pBAN7: GUS and PBAN8 : GUS illustrated in the examples.
Le vecteur pBAN1 ::GUS comprend la totalité de l'acide nucléique régulateur de séquence SEQ ID N°1.The vector pBAN1 :: GUS comprises all of the regulatory nucleic acid with sequence SEQ ID No. 1.
Le vecteur pBAN2::GUS comprend l'acide nucléique régulateur allant du nucléotide en position 509 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 , c'est-à-dire l'acide nucléique SEQ ID N°14. Le vecteur pBAN3::GUS comprend la séquence allant du nucléotide en position 1011 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 c'est-à-dire l'acide nucléique SEQ ID N°13. Le vecteur pBAN4::GUS comprend l'acide nucléique régulateur allant du nucléotide en position 1510 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 c'est-à-dire l'acide nucléique SEQ ID N°12.The vector pBAN2 :: GUS comprises the regulatory nucleic acid going from the nucleotide in position 509 to the nucleotide in position 2376 of the sequence SEQ ID N ° 1, that is to say the nucleic acid SEQ ID N ° 14 . The vector pBAN3 :: GUS comprises the sequence going from the nucleotide at position 1011 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 13. The vector pBAN4 :: GUS comprises the regulatory nucleic acid ranging from the nucleotide at position 1510 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 12.
Le vecteur pBAN5::GUS comprend un acide nucléique régulateur allant du nucléotide en position 1919 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 c'est-à-dire l'acide nucléique SEQ ID N°1 1.The vector pBAN5 :: GUS comprises a regulatory nucleic acid ranging from the nucleotide at position 1919 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 1 1.
Le vecteur pBAN6::GUS comprend un acide nucléique régulateur allant du nucléotide en position 2054 jusqu'au nucléotide en position 2376 de la séquence SEQ ID N°1 , c'est-à-dire l'acide nucléique SEQ ID N°10.The vector pBAN6 :: GUS comprises a regulatory nucleic acid ranging from the nucleotide in position 2054 to the nucleotide in position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 10.
Le vecteur pBAN7 ::GUS comprend un acide nucléique régulateur allant du nucléotide en position 2141 jusqu'au nucléotide en position 2376 de la séquence SEQ ID n°1 , c'est-à-dire l'acide nucléique SEQ ID n°9.The vector pBAN7 :: GUS comprises a regulatory nucleic acid ranging from the nucleotide at position 2141 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 9.
Le vecteur pBANδ ::GUS comprend un acide nucléique régulateur allant du nucléotide en position 2273 jusqu'au nucléotide en position 2376 de la séquence SEQ ID n°1 , c'est-à-dire l'acide nucléique SEQ ID n°8.The vector pBANδ :: GUS comprises a regulatory nucleic acid ranging from the nucleotide at position 2273 to the nucleotide at position 2376 of the sequence SEQ ID No. 1, that is to say the nucleic acid SEQ ID No. 8.
L'acide nucléique de séquence SEQ ID N°1 peut être amplifié à l'aide des amorces de séquences SEQ ID N°15 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No 1 can be amplified using the primers of sequences SEQ ID No 15 and SEQ ID No 23.
L'acide nucléique de séquence SEQ ID N°14 peut être amplifié à l'aide des amorces de séquences SEQ ID N°16 et SEQ ID N°23. L'acide nucléique de séquence SEQ ID N°13 peut être amplifié à l'aide des amorces de séquences SEQ ID N°17 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No. 14 can be amplified using the primers of sequences SEQ ID No. 16 and SEQ ID No. 23. The nucleic acid of sequence SEQ ID No 13 can be amplified using the primers of sequences SEQ ID No 17 and SEQ ID No 23.
L'acide nucléique de séquence SEQ ID N°12 peut être amplifié à l'aide des amorces de séquences SEQ ID N°18 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No 12 can be amplified using the primers of sequences SEQ ID No 18 and SEQ ID No 23.
L'acide nucléique de séquence SEQ ID N°1 1 peut être amplifié à l'aide des amorces de séquences SEQ ID N°19 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No 11 can be amplified using the primers of sequences SEQ ID No 19 and SEQ ID No 23.
L'acide nucléique de séquence SEQ ID N°10 peut être amplifié à l'aide des amorces de séquences SEQ ID N°20 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No 10 can be amplified using the primers of sequences SEQ ID No 20 and SEQ ID No 23.
L'acide nucléique de séquence SEQ ID N°9 peut être amplifié à l'aide des amorces de séquences SEQ ID N°21 et SEQ ID N°23. L'acide nucléique de séquence SEQ ID N°8 peut être amplifié à l'aide des amorces de séquences SEQ ID N°22 et SEQ ID N°23.The nucleic acid of sequence SEQ ID No 9 can be amplified using the primers of sequences SEQ ID No 21 and SEQ ID No 23. The nucleic acid of sequence SEQ ID No. 8 can be amplified using the primers of sequences SEQ ID No. 22 and SEQ ID No. 23.
Caractéristiques générales des vecteurs recombinants selon l'invention. Par " vecteur " au sens de la présente invention, on entendra une molécule d'ADN ou d'ARN circulaire ou linéaire qui est indifféremment sous forme simple brin ou double brin.General characteristics of the recombinant vectors according to the invention. By "vector" in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form.
Un vecteur recombinant selon l'invention est indifféremment un vecteur de clonage, un vecteur d'expression, ou plus spécifiquement un vecteur d'insertion, un vecteur de transformation ou un vecteur d'intégration.A recombinant vector according to the invention is either a cloning vector, an expression vector, or more specifically a insertion vector, a transformation vector or an integration vector.
Il peut s'agir d'un vecteur d'origine bactérienne ou virale.It can be a vector of bacterial or viral origin.
Selon un premier mode de réalisation, un vecteur recombinant selon l'invention est utilisé afin d'amplifier l'acide nucléique qui y est inséré après transformation ou transfection de l'hôte cellulaire désiré.According to a first embodiment, a recombinant vector according to the invention is used in order to amplify the nucleic acid which is inserted therein after transformation or transfection of the desired cellular host.
Selon le mode de réalisation préféré, un vecteur recombinant selon l'invention est utilisé pour exprimer un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, et dans ce cas spécifiquement dans la couche cellulaire à aleurone de l'albumen de la graine de plante.According to the preferred embodiment, a recombinant vector according to the invention is used to express a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, and in this case specifically in the layer cell aleurone from the albumen of the plant seed.
Un vecteur recombinant selon l'invention comprend avantageusement aussi des séquences d'initiation et d'arrêt de la transcription appropriée. En outre, les vecteurs recombinants selon l'invention pourront inclure une ou plusieurs origines de réplication fonctionnelle chez les hôtes cellulaires dans lesquels leur amplification ou leur expression est recherchée, ainsi que des séquences nucléotidiques marqueurs de sélection. Selon un aspect avantageux, un vecteur recombinant selon l'invention est un vecteur intégratif permettant l'insertion de multiples copies de la séquence d'ADN insérée dans ce vecteur dans le génome d'une plante, dont la transformation par un acide nucléique selon l'invention est recherchée. De manière avantageuse, le vecteur recombinant, ou dans d'autres cas la cassette d'expression contenue dans ce vecteur, peut aussi contenir des séquences 5' non traduites dites " leaders ". De telles séquences peuvent augmenter la traduction. Parmi celles connues de l'homme du métier, on peut citer: - le leader EMCV (EncephaloMyoCarditis VIRUS 5' non coding région) (ELROY-STEIN et al., 1989); - le leader TEV (Tobacco Etch Virus) (CARRINGTON AND FREED, 1990);A recombinant vector according to the invention advantageously also comprises sequences for initiating and stopping the appropriate transcription. In addition, the recombinant vectors according to the invention may include one or more origins of functional replication in cellular hosts in which their amplification or expression is sought, as well as nucleotide selection marker sequences. According to an advantageous aspect, a recombinant vector according to the invention is an integrative vector allowing the insertion of multiple copies of the DNA sequence inserted in this vector into the genome of a plant, the transformation of which by a nucleic acid according to l invention is sought. Advantageously, the recombinant vector, or in other cases the expression cassette contained in this vector, can also contain 5 'untranslated sequences called "leaders". Such sequences can increase translation. Among those known to those skilled in the art, there may be mentioned: - the leader EMCV (EncephaloMyoCarditis VIRUS 5 'non coding region) (ELROY-STEIN et al., 1989); - the leader TEV (Tobacco Etch Virus) (CARRINGTON AND FREED, 1990);
- le leader du gène BiP codant pour la protéine de liaison à la chaîne lourde de l'immunoglobuline humaine (MACEJACK et al., 1991); - le leader AMV RNA 4 provenant de l'ARNm de la protéine du virus de la mosaïque de la luzerne (JOBLING et Gehrky, 1987);- the leader of the BiP gene coding for the heavy chain binding protein of human immunoglobulin (MACEJACK et al., 1991); - the leader AMV RNA 4 originating from the mRNA of the protein of the alfalfa mosaic virus (JOBLING and Gehrky, 1987);
- le leader du virus de la mosaïque du tabac (GALLIE et al., 1989). Les vecteurs selon l'invention peuvent aussi comprendre des séquences dites " terminateurs ". Parmi les terminateurs utilisables dans les constructions de l'invention, on peut citer notamment:- the leader of the tobacco mosaic virus (GALLIE et al., 1989). The vectors according to the invention can also comprise so-called "terminator" sequences. Among the terminators which can be used in the constructions of the invention, there may be mentioned in particular:
- le polyA 35S du virus de la mosaïque du chou-fleur (CaMV), décrit dans l'article de FRANCK et al. (1980);- the polyA 35S of the cauliflower mosaic virus (CaMV), described in the article by FRANCK et al. (1980);
- le terminateur correspondant à la région 3' non-codante du gène de la nopaline synthase du plasmide Ti d'Agrobacteήum tumefaciens- the terminator corresponding to the 3 'non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacteήum tumefaciens
(DEPICKER et al., 1982),(DEPICKER et al., 1982),
- le terminateur du gène histone (EP 0 633 317).- the terminator of the histone gene (EP 0 633 317).
Le vecteur d'expression peut aussi comprendre des séquences de type peptides signaux vacuolaire ou apoplastique lorsqu'elles ne sont pas déjà présentes dans la séquence du gène d'intérêt, pour amener la protéine codée par le gène hétérologue dans des compartiments particuliers des cellules de plante, en particulier celles comprenant la zone de transfert de l'albumen.The expression vector may also include sequences of the vacuolar or apoplastic signal peptide type when they are not already present in the sequence of the gene of interest, in order to bring the protein encoded by the heterologous gene into particular compartments of the cells of plant, in particular those comprising the albumen transfer zone.
Les vecteurs bactériens préférés selon l'invention sont par exemple les vecteurs pBR322 (ATCC n°37 017) ou encore les vecteurs tels que pAA223-3 (Pharmacia, Uppsala, Suède) et pGEM1 (Promega Biotech, Madison, Wl, Etats-Unis).The preferred bacterial vectors according to the invention are for example the vectors pBR322 (ATCC No. 37 017) or also the vectors such as pAA223-3 (Pharmacia, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, Wl, United States ).
On peut encore citer d'autres vecteurs commercialisés tels que les vecteurs pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI et pSG (Stratagene). Il peut s'agir également de vecteurs du type Baculovirus tel que le vecteur pVL1392/1393 (Pharmingen) utilisé pour transfecter les cellules de la lignée Sf9 (ATCC N°CRL 1711 ) dérivée de Spodoptera frugiperda.Mention may also be made of other commercially available vectors such as the vectors pQE70, pQE60, pQE9 (Quiagen), psiX174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSV2CAT, pOG44, pXTI and pSG (Stratagene). They may also be vectors of the Baculovirus type such as the vector pVL1392 / 1393 (Pharmingen) used to transfect the cells of the line Sf9 (ATCC No. CRL 1711) derived from Spodoptera frugiperda.
Préférentiellement, on aura recours à des vecteurs spécialement adaptés pour l'expression de séquence d'intérêt dans des cellules de plantes, tels que les vecteurs suivants:Preferably, use will be made of vectors specially adapted for the expression of sequence of interest in plant cells, such as the following vectors:
- vecteur pBluescript SK+ commercialisé par la Société Stratagène ;- vector pBluescript SK + marketed by the Company Stratagène;
- vecteur pRTL2 - GUS décrit par Carrington et al. (1990) ; - vecteur pAVA 393, illustré dans les exemples et qui dérive du vecteur pAVA321 décrit par Von Amhim et al. (1998) ;- vector pRTL2 - GUS described by Carrington et al. (1990) ; - vector pAVA 393, illustrated in the examples and which derives from the vector pAVA321 described by Von Amhim et al. (1998);
- les vecteurs pBIB-HYG et pBIB-KAN décrits par Becker et al.(1990) ; - vecteur pBIN19 (BEVAN et al. 1984), commercialisé par la- the vectors pBIB-HYG and pBIB-KAN described by Becker et al. (1990); - vector pBIN19 (BEVAN et al. 1984), marketed by the
Société CLONTECH (Palo Alto, Californie, USA);CLONTECH Company (Palo Alto, California, USA);
- vecteur pB1 101 (JEFFERSON, 1987), commercialisé par la Société CLONTECH ;- vector pB1 101 (JEFFERSON, 1987), sold by the company CLONTECH;
- vecteur pBI121 (JEFFERSON, 1987), commercialisé par la Société CLONTECH;- vector pBI121 (JEFFERSON, 1987), sold by the company CLONTECH;
- vecteur pEGFP; Yang et al. (1996a et 1996b ), commercialisé par la Société CLONTECH;- vector pEGFP; Yang et al. (1996a and 1996b), marketed by the CLONTECH Company;
- vecteur pCAMBIA 1302 (HAJDUKIIEWICZ et al., 1994)- vector pCAMBIA 1302 (HAJDUKIIEWICZ et al., 1994)
CELLULES HOTES TRANSFORMEES SELON L'INVENTIONHOST CELLS TRANSFORMED ACCORDING TO THE INVENTION
Pour permettre l'expression d'un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique régulateur selon l'invention, les acides nucléiques régulateurs, les cassettes d'expression ou encore les vecteurs recombinants définis dans la présente description doivent être introduits dans une cellule hôte. L'introduction des polynucléotides selon l'invention dans une cellule hôte peut être réalisée in vitro, selon les techniques bien connues de l'homme du métier pour transformer ou transfecter des cellules, soit en culture primaire, soit sous la forme de lignées cellulaires.To allow the expression of a polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention, the regulatory nucleic acids, the expression cassettes or also the recombinant vectors defined in the present description must be introduced. in a host cell. The introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro, according to the techniques well known to those skilled in the art for transforming or transfecting cells, either in primary culture or in the form of cell lines.
L'invention a en outre pour objet une cellule hôte transformée par un acide nucléique régulateur, par une cassette d'expression ou encore par un vecteur recombinant tel que défini ci-dessus.The subject of the invention is also a host cell transformed with a regulatory nucleic acid, with an expression cassette or also with a recombinant vector as defined above.
Une telle cellule hôte transformée est préférentiellement d'origine bactérienne, fongique ou végétale.Such a transformed host cell is preferably of bacterial, fungal or vegetable origin.
Ainsi, peuvent être notamment utilisées des cellules bactériennes de différentes souches de Escherichia coli ou encore d'Agrobacterium tumefaciens .Thus, bacteria cells of different strains of Escherichia coli or of Agrobacterium tumefaciens can in particular be used.
De manière préférée, la cellule hôte transformée est une cellule de plante ou encore un protoplaste de plante.Preferably, the transformed host cell is a plant cell or also a plant protoplast.
De manière tout à fait préférée, la solution hôte transformée est d'origine végétale et est choisie parmi les cellules des espèces végétales telles que Solanacea, Cactaceae, Cucurbitacea, Papillionacea, Actinidiaceae, Cucurbitaceae, Rubiaceae, Moraceae, Passif loraceae, Compositae, Caricaceae, Eήcaceae, Gramineae, Cruciferae, Cariofillaceae, Amarylidiaceae, Iridaceae, Leguminosae, Liliaceae, Oleaceae , Paeoniaceae, Papaveraceae, Primulaceae, Rosaceae, Scrophulariaceae, Violaceae, Vitaceae, Malvaceae.Most preferably, the transformed host solution is of plant origin and is chosen from cells of plant species such as Solanacea, Cactaceae, Cucurbitacea, Papillionacea, Actinidiaceae, Cucurbitaceae, Rubiaceae, Moraceae, Passif loraceae, Compositae, Caricaceae, Eήcaceae, Gramineae, Cruciferae, Cariofillaceae, Amarylidiaceae, Iridaceae, Leguminosae, Liliaceae, Oleaceae, Paeoniaceae, Papaveraceae, Primulaceae, Rosaceae, Scrophulariaceae, Violaceae, Vitaceae, Malvaceae.
On préfère tout particulièrement les plantes dérivées de l'aubergine, de la tomate, du melon ou du melon d'eau, du concombre, des espèces du type citrus, du poivre, des fraises, du raisin, de la pomme, de la poire, de la cerise, l'olive , du colza, du choux ou du poivron.Especially preferred are plants derived from eggplant, tomato, melon or watermelon, cucumber, citrus-like species, pepper, strawberries, grapes, apple, pear , cherry, olive, rapeseed, cabbage or pepper.
Il peut s'agir indifféremment d'une cellule végétale originaire d'une plante dicotylédone ou monocotylédone.It can equally be a plant cell originating from a dicotyledonous or monocotyledonous plant.
Une cellule hôte transformée selon l'invention est la souche de Escherichia coli contenant le plasmide pBAN1 ::GUS/pBIB-Hyg déposé à la CNCM le 17 juillet 2001 sous le numéro d'accès I-2703. D'autres cellules hôtes transformées préférées selon l'invention sont respectivement des cellules hôtes transformées avec les vecteurs pBAN2::GUS, PBAN3::GUS, pBAN4::GUS, pBAN5::GUS, pBAN6::GUS pBAN7 :GUS et pBAN8 :GUS illustrés dans les exemples.A host cell transformed according to the invention is the Escherichia coli strain containing the plasmid pBAN1 :: GUS / pBIB-Hyg deposited at the CNCM on July 17, 2001 under the access number I-2703. Other preferred transformed host cells according to the invention are respectively host cells transformed with the vectors pBAN2 :: GUS, PBAN3 :: GUS, pBAN4 :: GUS, pBAN5 :: GUS, pBAN6 :: GUS pBAN7: GUS and pBAN8: GUS illustrated in the examples.
PLANTES TRANSFORMEES SELON L'INVENTIONPLANTS TRANSFORMED ACCORDING TO THE INVENTION
L'invention concerne aussi un organisme multicellulaire végétal transformé, caractérisé en ce qu'il comprend une cellule hôte transformée ou une pluralité de cellules hôtes transformées par un acide nucléique, une cassette d'expression ou encore par un vecteur recombinant tel que défini ci-dessus.The invention also relates to a transformed multicellular plant organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by a nucleic acid, an expression cassette or also by a recombinant vector as defined above. above.
L'invention a encore pour objet une plante transgénique comprenant, sous une forme intégrée dans son génome, un acide nucléique ou une cassette d'expression telle que définie dans la présente description.The invention also relates to a transgenic plant comprising, in a form integrated into its genome, a nucleic acid or an expression cassette as defined in the present description.
De manière générale, l'invention est aussi relative à l'utilisation d'un acide nucléique régulateur, d'une cassette d'expression, d'un vecteur recombinant ou encore d'une cellule hôte telle que définie ci- dessus pour l'obtention d'une plante transformée. Dans un mode de réalisation particulier de l'utilisation ci-dessus, dans lequel l'expression du polynucleotide régulateur placé sous le contrôle d'un acide nucléique selon l'invention est toxique pour la cellule, la plante transformée ne produit pas de graines matures, ou bien produit des graines de taille réduite, ou bien produit un nombre réduit de grains ou se caractérise par l'absence totale de graines, particulièrement l'absence totale de graines matures et/ou fertiles.In general, the invention also relates to the use of a regulatory nucleic acid, an expression cassette, a recombinant vector or also a host cell as defined above for the obtaining a transformed plant. In a particular embodiment of the above use, in which the expression of the regulatory polynucleotide placed under the control of a nucleic acid according to the invention is toxic for the cell, the transformed plant does not produce mature seeds , or either produces seeds of reduced size, or produces a reduced number of grains or is characterized by the total absence of seeds, particularly the total absence of mature and / or fertile seeds.
Un premier objectif poursuivi selon la présente invention est l'obtention de plantes transgéniques ou transformées dont la graine mature possède des caractéristiques améliorées, notamment du point de vue de la qualité ou de la quantité en tannins ou en fibres ou encore des équilibres hormonaux . Un second objectif poursuivi selon l'invention est l'obtention de plantes transgéniques ou transformées affectées dans le développement de la testa de la graine.A first objective pursued according to the present invention is the obtaining of transgenic or transformed plants whose mature seed has improved characteristics, in particular from the point of view of the quality or the quantity of tannins or fibers or even hormonal balances. A second objective pursued according to the invention is the obtaining of transgenic or transformed plants affected in the development of the testa of the seed.
Conformément à un troisième objectif, on cherche à obtenir des plantes transgéniques ou transformées dont les graines comprennent des teneurs réduites en tannins ou en anthocyanes par rapport à la normale. Un tel objectif sera poursuivi plus particulièrement concernant l'obtention de plantes transgéniques ou transformées de colza ou de maïs ayant les caractéristiques ci-dessus, et dont les graines sont d'une couleur jaune plus intense que la normale.In accordance with a third objective, it is sought to obtain transgenic or transformed plants whose seeds contain reduced contents of tannins or anthocyanins compared to normal. Such an objective will be pursued more particularly with regard to obtaining transgenic or transformed rapeseed or maize plants having the above characteristics, and the seeds of which are of a more intense yellow color than normal.
Selon le premier objectif ci-dessus, le polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique régulateur selon l'invention sera choisi de telle manière à ce qu'il modifie la qualité nutritionnelle et/ou hormonale de la testa de la graine. Selon un second objectif ci-dessus, le polynucleotide d'intérêt sera choisi de telle manière à ce que son expression soit toxique pour la cellule de l'endothélium dans laquelle il est exprimé.According to the first objective above, the polynucleotide of interest placed under the control of a regulatory nucleic acid according to the invention will be chosen in such a way that it modifies the nutritional and / or hormonal quality of the testa de la seed. According to a second objective above, the polynucleotide of interest will be chosen in such a way that its expression is toxic to the endothelium cell in which it is expressed.
Les acides nucléiques régulateurs et les cassettes d'expression sont ceux décrits précédemment dans la description. Lorsqu'une expression du polynucleotide d'intérêt sera recherchée exclusivement dans l'endothélium de la testa, on choisira de préférence un acide nucléique régulateur comprenant un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 1919 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 , comme c'est le cas par exemple pour les acides nucléiques régulateurs contenus dans les vecteurs pBAN1 ::GUS, pBAN2::GUS, pBAN3::GUS, pBAN4::GUS et pBAN5::GUS.The regulatory nucleic acids and the expression cassettes are those described previously in the description. When an expression of the polynucleotide of interest is sought exclusively in the endothelium of the testa, a regulatory nucleic acid preferably comprising a polynucleotide having at least 80% nucleotide identity with the sequence going from the nucleotide at position 1919 will be chosen. up to the nucleotide at position 2335 of the sequence SEQ ID No. 1, as is the case for example for the regulatory nucleic acids contained in the vectors pBAN1 :: GUS, pBAN2 :: GUS, pBAN3 :: GUS, pBAN4: : GUS and pBAN5 :: GUS.
Lorsqu'au contraire on cherchera à obtenir une expression du polynucleotide d'intérêt également dans la couche à aleurone de l'albumen, on choisira alors de préférence un acide nucléique régulateur comprenant un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 2054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1 , comme c'est le cas de l'acide nucléique régulateur contenu dans le vecteur pBAN6::GUS. L'invention a également trait à toute partie d'une plante transformée telle que définie dans la présente description, y compris ses graines et ses fruits.When, on the contrary, it is sought to obtain an expression of the polynucleotide of interest also in the aleurone layer of the albumen, then a regulatory nucleic acid preferably comprising a polynucleotide having at least 80% identity is chosen. nucleotides with the sequence going from the nucleotide at position 2054 to the nucleotide at position 2335 of the sequence SEQ ID No. 1, as is the case for the regulatory nucleic acid contained in the vector pBAN6 :: GUS. The invention also relates to any part of a transformed plant as defined in the present description, including its seeds and its fruits.
Les plantes transformées selon l'invention appartiennent préférentiellement aux espèces qui ont déjà été listées ci-dessus concernant l'origine des cellules hôtes végétales transformées de l'invention.The plants transformed according to the invention preferably belong to the species which have already been listed above concerning the origin of the transformed plant host cells of the invention.
De manière tout à fait préférée, les plantes transformées selon l'invention sont choisies parmi l'aubergine, la tomate, le melon ou le melon d'eau, le concombre, les espèces du type citrus, le poivre, les fraises, les raisins, les pommes, les poires, les cerises, le colza , le choux, le poivron ou encore les olives.Most preferably, the plants transformed according to the invention are chosen from eggplant, tomato, melon or watermelon, cucumber, species of the citrus type, pepper, strawberries, grapes , apples, pears, cherries, rapeseed, cabbage, pepper or olives.
Les plantes peuvent être indifféremment des dicotylédones ou des monocotylédones.The plants can be either dicots or monocots.
L'invention a encore pour objet un procédé d'obtention de plantes transformées caractérisé en ce qu'il comprend les étapes suivantes: a) obtention d'une cellule hôte recombinante végétale selon l'invention; b) régénération d'une plante entière à partir de la cellule hôte recombinante obtenue à l'étape a); c) sélection des plantes obtenues à l'étape b) ayant intégré un acide nucléique ou une cassette d'expression telle que définie dans la présente description.The subject of the invention is also a process for obtaining transformed plants, characterized in that it comprises the following steps: a) obtaining a recombinant plant host cell according to the invention; b) regeneration of an entire plant from the recombinant host cell obtained in step a); c) selection of the plants obtained in step b) having integrated a nucleic acid or an expression cassette as defined in the present description.
L'invention a encore pour objet un procédé d'obtention d'une plante transformée caractérisé en ce qu'il comprend les étapes suivantes: a) obtention d'une cellule hôte recombinante de Agrobacterium tumefaciens selon l'invention; b) transformation d'une plante d'intérêt par infection avec les cellules hôtes recombinantes de Agrobacterium tumefaciens obtenues à l'étape a); c) sélection des plantes ayant intégré dans leur génome un acide nucléique ou une cassette d'expression telle que définie dans la présente description.The subject of the invention is also a process for obtaining a transformed plant, characterized in that it comprises the following steps: a) obtaining a recombinant host cell of Agrobacterium tumefaciens according to the invention; b) transformation of a plant of interest by infection with the recombinant host cells of Agrobacterium tumefaciens obtained in step a); c) selection of plants having integrated into their genome a nucleic acid or an expression cassette as defined in the present description.
L'invention a encore pour objet un procédé d'obtention d'une plante transformée caractérisé en ce qu'il comporte les étapes suivantes : a) transfecter au moins une cellule de plante avec un acide nucléique, avec une cassette d'expression ou avec un vecteur recombinant selon l'invention; b) régénérer une plante entière à partir de la cellule de plante recombinante obtenue à l'étape a); c) sélectionner les plantes ayant intégré dans leur génome un acide nucléique ou une cassette d'expression selon l'invention.Another subject of the invention is a process for obtaining a transformed plant, characterized in that it comprises the following steps: a) transfecting at least one plant cell with a nucleic acid, with an expression cassette or with a recombinant vector according to the invention; b) regenerating an entire plant from the recombinant plant cell obtained in step a); c) selecting the plants which have integrated into their genome a nucleic acid or an expression cassette according to the invention.
L'un quelconque des procédés d'obtention d'une plante transformée décrit ci-dessus peut en outre comporter les étapes additionnelles suivantes: d) croisement entre elles de deux plantes transformées telles qu'obtenues à l'étape c) avec une plante de la même espèce; e) sélection des plantes homozygotes pour le transgène. Dans un second mode de réalisation particulier, l'un quelconque des procédés d'obtention d'une plante transgénique décrit ci-dessus peut en outre comporter les étapes additionnelles suivantes: f) croisement d'une plante transformée obtenue à l'étape c) avec une plante de la même espèce; g) sélection des plantes issues du croisement de l'étape f) ayant conservé le transgène. Un autre objet de l'invention consiste en une plante transformée ou transgénique telle qu'obtenue selon l'un quelconque des procédés d'obtention d'une plante définie ci-avant.Any of the processes for obtaining a transformed plant described above can also comprise the following additional steps: d) crossing between them of two transformed plants as obtained in step c) with a plant of the same species; e) selection of plants homozygous for the transgene. In a second particular embodiment, any of the methods for obtaining a transgenic plant described above can also comprise the following additional steps: f) crossing of a transformed plant obtained in step c) with a plant of the same species; g) selection of the plants resulting from the crossing of step f) having conserved the transgene. Another subject of the invention consists of a transformed or transgenic plant as obtained according to any one of the methods for obtaining a plant defined above.
L'invention a également pour objet une plante transformée, susceptible d'être obtenue par l'un quelconque des procédés décrits ci- dessus.The subject of the invention is also a transformed plant, capable of being obtained by any of the methods described above.
Elle est également relative à une semence ou un fruit d'une plante transformée susceptible d'être obtenue par l'un quelconque des procédés ci-dessus. Elle est également relative à toute partie, notamment, tige, feuille, fleur, fruit d'une plante transformée susceptible d'être obtenue par l'un quelconques des procédés ci-dessus.It also relates to a seed or a fruit of a transformed plant capable of being obtained by any of the above processes. It also relates to any part, in particular, stem, leaf, flower, fruit of a transformed plant capable of being obtained by any of the above processes.
La transformation de cellules végétales peut être réalisée par les techniques connues de l'homme du métier. On peut citer notamment les méthodes de transfert direct de gènes telles que la microinjection directe dans des embryoïdes de plante (NEUHAUS et al., 1987), l'infiltration sous vide (BECHTOLD et al., 1993) ou l'électroporation (CHUPEAU et al., 1989) ou encore la précipitation directe au moyen de PEG (SCHOCHER et al., 1986) ou le bombardement par canon de particules recouvertes de l'ADN plasmidique d'intérêt (FROMM et al. 1990).The transformation of plant cells can be carried out by techniques known to those skilled in the art. Mention may in particular be made of direct gene transfer methods such as direct microinjection into plant embryoids (NEUHAUS et al., 1987), vacuum infiltration (BECHTOLD et al., 1993) or electroporation (CHUPEAU and al., 1989) or direct precipitation using PEG (SCHOCHER et al., 1986) or bombardment by cannon of particles covered with the plasmid DNA of interest (FROMM et al. 1990).
On peut également infecter la plante par une souche bactérienne notamment d'Agrobacterium. Selon un mode de réalisation du procédé de l'invention, les cellules végétales sont transformées par un vecteur selon l'invention, ledit hôte cellulaire étant susceptible d'infecter lesdites cellules végétales en permettant l'intégration dans le génome de ces dernières, des séquences nucléotidiques d'intérêt initialement contenues dans l'ADN du vecteur susmentionné. Avantageusement, l'hôte cellulaire susmentionné utilisé est Agrobacterium tumefaciens, notamment selon la méthode décrite dans l'article de Ha and AN (1989), ou encore Agrobacterium rhizogenes, notamment selon la méthode décrite dans l'article de GUERCHE et al., (1987) ou encore dans la demande PCT N°WO 00 22.148.The plant can also be infected with a bacterial strain, notably Agrobacterium. According to one embodiment of the method of the invention, the plant cells are transformed by a vector according to the invention, said cell host being capable of infecting said plant cells by allowing the integration into the genome of these latter, sequences nucleotides of interest initially contained in the DNA of the above-mentioned vector. Advantageously, the above-mentioned cell host used is Agrobacterium tumefaciens, in particular according to the method described in the article by Ha and AN (1989), or else Agrobacterium rhizogenes, in particular according to the method described in the article by GUERCHE et al., (1987) or also in PCT application No. WO 00 22,148.
Par exemple, la transformation des cellules végétales peut être réalisée par le transfert de la région T du plasmide circulaire extra- chromosomique inducteur de tumeurs Ti d'Agrobacterium tumefaciens, en utilisant un système binaire (WATSON et al. 1994). Pour ce faire, deux vecteurs sont construits. Dans l'un de ces vecteurs, la région T a été éliminée par délétion, à l'exception des bordures droite et gauche, un gène marqueur étant inséré entre eux pour permettre la sélection dans les cellules de plantes. L'autre partenaire du système binaire est un plasmide Ti auxiliaire, plasmide modifié qui n'a plus de région T mais contient toujours les gènes de virulence vir, nécessaires à la transformation de la cellule végétale.For example, the transformation of plant cells can be carried out by the transfer of the T region of the extra-chromosomal circular plasmid inducing Ti tumors of Agrobacterium tumefaciens, using a binary system (WATSON et al. 1994). To do this, two vectors are constructed. In one of these vectors, the T region was deleted, with the exception of the right and left borders, a marker gene being inserted between them to allow selection in plant cells. The other partner of the binary system is a helper Ti plasmid, a modified plasmid which no longer has a T region but still contains the vir virulence genes, necessary for the transformation of the plant cell.
Selon un mode préféré, on peut utiliser la méthode décrite par ISHIDA et al. (1996) pour la transformation des Monocotylédones.According to a preferred mode, the method described by ISHIDA et al. Can be used. (1996) for the transformation of Monocotyledons.
Selon un autre protocole, la transformation est réalisée selon la méthode décrite par FINER et al. (1992) utilisant le canon à particules de tungstène ou d'or.According to another protocol, the transformation is carried out according to the method described by FINER et al. (1992) using the tungsten or gold particle gun.
L'homme du métier est capable de mettre en oeuvre de nombreux procédés de l'état de la technique afin d'obtenir des plantes transformées par un acide nucléique régulateur ou une cassette d'expression selon l'invention.A person skilled in the art is capable of implementing numerous methods of the state of the art in order to obtain plants transformed with a regulatory nucleic acid or an expression cassette according to the invention.
L'homme du métier pourra se référer avantageusement à la technique décrite par BECHTOLD et al. (1993) afin de transformer une plante à l'aide de la bactérie Agrobacterium tumefaciens. Des techniques utilisant d'autres types de vecteurs peuvent également être utilisées, telles que les techniques décrites par BOUCHEZ et al. (1993) ou encore par HORSCH et al. (1994).Those skilled in the art may advantageously refer to the technique described by BECHTOLD et al. (1993) in order to transform a plant using the bacterium Agrobacterium tumefaciens. Techniques using other types of vectors can also be used, such as the techniques described by BOUCHEZ et al. (1993) or by HORSCH et al. (1994).
A titre illustratif, une plante transgénique selon l'invention peut être obtenue par des techniques de biolistique telles que celles décrites par FINER et al. (1992) ou encore celles décrites par VAIN et al. (1993). D'autres techniques préférées de transformation d'une plante conformément à l'invention par Agrobacterium tumefaciens sont celles décrites par ISHIDA et al. (1996) ou encore dans la demande PCT publiée sous le n°WO 95/06 722 au nom de JAPAN TOBACCO. L'invention a encore pour objet une semence de plante dont les cellules constitutives comprennent un acide nucléique régulateur ou une cassette d'expression selon l'invention, qui a été artificiellement inséré dans son génome.By way of illustration, a transgenic plant according to the invention can be obtained by biolistic techniques such as those described by FINER et al. (1992) or those described by VAIN et al. (1993). Other preferred techniques for transforming a plant in accordance with the invention with Agrobacterium tumefaciens are those described by ISHIDA et al. (1996) or in the PCT application published under the number WO 95/06 722 in the name of JAPAN TOBACCO. The subject of the invention is also a plant seed whose constituent cells comprise a regulatory nucleic acid or an expression cassette according to the invention, which has been artificially inserted into its genome.
L'invention a encore pour objet une semence d'une plante transgénique telle que définie ci-avant ou encore un fruit d'une telle plante transgénique.The invention also relates to a seed of a transgenic plant as defined above or also a fruit of such a transgenic plant.
Un autre objet de l'invention consiste en l'utilisation d'un acide nucléique régulateur tel que défini dans la présente description pour l'expression in vitro ou in vivo, de préférence in planta, d'un polynucleotide d'intérêt, préférentiellement un polynucleotide dont l'expression est toxique pour la cellule végétale.Another subject of the invention consists in the use of a regulatory nucleic acid as defined in the present description for the expression in vitro or in vivo, preferably in planta, of a polynucleotide of interest, preferably a polynucleotide whose expression is toxic to the plant cell.
L'invention a encore pour objet une plante transformée affectée dans le développement de ces graines, susceptible d'être obtenue par l'un quelconque des procédés ci-dessus, ainsi que toute partie de cette plante transformée, y compris ces fruits.The invention also relates to a transformed plant affected in the development of these seeds, capable of being obtained by any of the above methods, as well as any part of this transformed plant, including these fruits.
L'invention est en outre illustrée, sans pour autant être limitée, par les figures et les exemples suivants.The invention is further illustrated, without being limited, by the following figures and examples.
Description des figuresDescription of the figures
La figure 1 représente un schéma des différentes constructions nucléotidiques ou « cassettes d'expression » contenant respectivement tout ou partie de l'acide nucléique régulateur de séquence SEQ ID N°1 et un gène rapporteur placé en aval de l'acide nucléique régulateur, le gène rapporteur GUS. Schéma des constructions contenant le gène rapporteur GUS placé sous le contrôle de différentes délétions réalisées en 5' de la région promotrice du gène BAN. + 1 et ATG représentent les sites d'initiation de la traduction respectivement ; RNT, région non traduite ; Myc et Myb, boîte cis reconnues par des facteurs de transcription de type Myc (bHLH) et de type Myb, respectivement ; les chiffres représentent des paires de bases.FIG. 1 represents a diagram of the different nucleotide constructs or “expression cassettes” respectively containing all or part of the regulatory nucleic acid with sequence SEQ ID No. 1 and a reporter gene placed downstream of the regulatory nucleic acid, the GUS reporter gene. Diagram of the constructions containing the reporter gene GUS placed under the control of different deletions carried out 5 'from the promoter region of the BAN gene. + 1 and ATG represent the sites of initiation of translation respectively; RNT, region not translated; Myc and Myb, cis box recognized by Myc-like (bHLH) and Myb-like transcription factors, respectively; numbers represent base pairs.
La figure 2 illustre le principe de détermination des oligonucléotides utilisés pour l'amplification des délétions, la figure 2 illustrant plus particulièrement la construction du vecteur pBAN1 ::GUS.FIG. 2 illustrates the principle of determination of the oligonucleotides used for the amplification of the deletions, FIG. 2 more particularly illustrating the construction of the vector pBAN1 :: GUS.
En haut : Addition d'un site de restriction SAN à l'extrémité 5'gta GTCGAC ATTTAGGCCAAGATTTTAGGAG.Top: Addition of a SAN restriction site at the 5'gta end GTCGAC ATTTAGGCCAAGATTTTAGGAG.
En bas : Modification de la région ATG pour créer un site de restriction Ncol.Bottom: Modification of the ATG region to create an Ncol restriction site.
La figure 3 illustre le profil d'expression temporelle du gène BAN (RT-PCR et expression GUS), comparée à l'activité du gène GUS placé sous le contrôle de p BAN1.FIG. 3 illustrates the temporal expression profile of the BAN gene (RT-PCR and GUS expression), compared with the activity of the GUS gene placed under the control of p BAN1.
La figure 4 illustre la construction du vecteur pBAN::GUS La figure 5 illustre le protocole de construction du vecteur pBan::Gus/pBIB-HYG.FIG. 4 illustrates the construction of the vector pBAN :: GUS FIG. 5 illustrates the protocol for construction of the vector pBan :: Gus / pBIB-HYG.
La Figure 6 illustre le profil d'expression du gène rapporteur GUS placé sous le contrôle du promoteur pBAN1.Figure 6 illustrates the expression profile of the GUS reporter gene placed under the control of the pBAN1 promoter.
A, bouton floral ; B, fleur non pollinisée ; C, silique de 3 jours après pollinisation (jap) ; D, gros plan de graines immatures dans une silique de 3 JAP. E, Nomarski de graine immature d'une plante sauvage (sans GUS, montrant la structure de la testa, particulièrement la localisation de l'endothélium ; F, coupe transversale dans une graine ; G, Nomarski de graine immature (3 jap). H, fragment de feuille de rosette ; I, fragments de tiges ; J. germination de 4 jours. Les flèches des figures D ? E, F et G désignent l'endothélium ; en, endothélium ; t, testa ; emb, embryon ; alb, albumen. La Figure 7 illustre la construction du vecteur pBAN1 ::barnase- barstarA, flower bud; B, non-pollinated flower; C, pod 3 days after pollination (jap); D, close-up of immature seeds in a pod of 3 JAP. E, Nomarski of immature seed from a wild plant (without GUS, showing the structure of the testa, particularly the location of the endothelium; F, cross section in a seed; G, Nomarski of immature seed (3 jap). H , fragment of rosette leaf; I, fragments of stems; J. 4-day germination. The arrows in Figures D? E, F and G denote the endothelium; en, endothelium; t, testa; emb, embryo; alb, Figure 7 illustrates the construction of the vector pBAN1 :: barnase- barstar
La Figure 8 illustre la construction du vecteur pBAN1::barnase- barstar/pBIB-HYG La Figure 9 illustre l'ablation génétique de l'endothélium par le gène barnase placé sous le contrôle du promoteur pBAN1. A, Nomarski de graine immature d'un transformant pBAN1 ::Barnase révélant l'absence d'endothélium (comparer avec la situation chez des graines sauvages, à la figure 6E) ; B, détection de tannins au niveau de l'endothélium chez une plante sauvage, par le test « vanilline » colorant les tannins en rouge sombre (flèche) ; C, test « vanilline » sur graines immatures d'un transformant pBAN1 ::Bamase révélant l'absence de tannins ; D et E , graines matures de plantes sauvages (graines brunes) et de graines de transformant pBAN1 "Barnase (graines brunes) et de graines de transformant p BAN1 "Barnase (graines jaunes); le, localisation de l'endothélium chez le sauvage.Figure 8 illustrates the construction of the vector pBAN1 :: barnase- barstar / pBIB-HYG Figure 9 illustrates the genetic ablation of the endothelium by the barnase gene placed under the control of the pBAN1 promoter. A, Nomarski of immature seed of a transformant pBAN1 :: Barnase revealing the absence of endothelium (compare with the situation in wild seeds, in FIG. 6E); B, detection of tannins in the endothelium of a wild plant, by the “vanillin” test coloring the tannins in dark red (arrow); C, “vanillin” test on immature seeds of a transformant pBAN1 :: Bamase revealing the absence of tannins; D and E, mature seeds of wild plants (brown seeds) and of transformant pBAN1 "Barnase (brown seeds) and of transformant p BAN1" Barnase (yellow seeds); the location of the endothelium in the wild.
EXEMPLES MATERIEL ET METHODES GENERAUX UTILISES DANS LES EXEMPLES. a) Matériel végétalMATERIAL EXAMPLES AND GENERAL METHODS USED IN THE EXAMPLES. a) Plant material
Les essais ont été réalisés exclusivement sur Arabidopsis thaliana (L.) Heynh. génotype Wassilevskija-2 (Ws-2), qui est également utilisé pour générer la collection de transformants à ADN-T de l'INRA de Versailles (BECHTOLD et al., 1993).The tests were carried out exclusively on Arabidopsis thaliana (L.) Heynh. Wassilevskija-2 genotype (Ws-2), which is also used to generate the collection of T-DNA transformants from INRA Versailles (BECHTOLD et al., 1993).
b) Souches bactériennesb) Bacterial strains
Pour les clonages moléculaires: Escherichia coli DH12S (GibcoBRL).For molecular cloning: Escherichia coli DH12S (GibcoBRL).
Pour la transformation de plantes: Agrobacte um tumefaciens C58CI souche GV3101 contenant le plasmide pMP90 (KONCZ et al., 1984).For the transformation of plants: Agrobacte um tumefaciens C58CI strain GV3101 containing the plasmid pMP90 (KONCZ et al., 1984).
c) Plasmidesc) Plasmids
- pBluescript SK + abrégé pBS (Strataqène)- pBluescript SK + abbreviated pBS (Strataqene)
Ce vecteur de clonage contient un gène de résistance à l'ampicilline et un site multiple de clonage situé dans le gène LacZ, permettant une sélection blanc/bleu des colonies transformées/non transformées.This cloning vector contains an ampicillin resistance gene and a multiple cloning site located in the LacZ gene, allowing white / blue selection of transformed / unprocessed colonies.
- PRTL2-GUS Ce vecteur de clonage a été construit par James Carrington- PRTL2-GUS This cloning vector was built by James Carrington
(Washington State University, Pullman, USA). Il dérive du vecteur pRTL2 (Carrington et al., 1990) auquel a été ajoutée la cassette suivante: le gène rapporteur GUS (JEFFERSON et al., 1987) précédé d'une courte séquence activatrice de traduction (TL) et placé entre un double promoteur 35S et le terminateur 35S (TOPFER et al., 1987). Un site N col créé à l'ATG du gène GUS permet la réalisation de fusions traductionnelles.(Washington State University, Pullman, USA). It is derived from the vector pRTL2 (Carrington et al., 1990) to which the following cassette has been added: the reporter gene GUS (JEFFERSON et al., 1987) preceded by a short translation activating sequence (TL) and placed between a double 35S promoter and the 35S terminator (TOPFER et al., 1987). An N col site created at the ATG of the GUS gene allows the realization of translational fusions.
- PWP146- PWP146
Ce vecteur comporte le gène de la barnase sous le contrôle du promoteur AG du maïs ainsi que le gène codant pour la Barstar placé sous le contrôle d'un promotur bactérien pour permettre la croissance des bactéries.This vector includes the barnase gene under the control of the maize AG promoter as well as the gene coding for Barstar placed under the control of a bacterial promoter to allow the growth of bacteria.
- pBIB-HYG- pBIB-HYG
Ce vecteur binaire comporte, outre un site de multiclonage, un gène de résistance à l'hygromycine (HYG) à expression in planta (BECKER, 1990).This binary vector comprises, in addition to a multicloning site, a gene for resistance to hygromycin (HYG) with expression in planta (BECKER, 1990).
B) METHODESB) METHODS
B.1) Culture de plantes.B.1) Cultivation of plants.
- En serre climatisée:- In an air-conditioned greenhouse:
Les plantules provenant soit d'un semi préalable sur papier filtre humide, soit de culture in vitro sont repiquées en pots individuelsThe seedlings coming either from a semi preliminary on wet filter paper, or from in vitro culture are transplanted in individual pots
(multiplication de génotypes) ou en barquettes (transformations, à raison de 30 plantules/barquette), sur terreau (Stender, Allemagne). La croissance des plantes est soutenue par un arrosage régulier avec une solution nutritive (Coïc et Lessaint, 1971). La température est maintenue aux environs de 25°C le jour et 20°C la nuit. D'autre part, un éclairage d'appoint à l'aide de lampes à vapeur de mercure assure quotidiennement 16h de jour tout au long de l'année. Dans ces conditions, il faut compter environ 3 mois entre la plantation et la récolte de graines matures. Le battage des transformants s'effectue en serre, où les eaux usagées sont filtrées puis traitées à l'eau de JAVEL. Equipées d'un sas d'entrée et d'un filtre de mailles inférieures à 1 mm au niveau des aérations, les serres sont conformes aux normes de sécurité concernant la manipulation d'organismes génétiquement modifiés.(multiplication of genotypes) or in trays (transformations, at the rate of 30 seedlings / tray), on soil (Stender, Germany). Plant growth is supported by regular watering with a nutrient solution (Coïc and Lessaint, 1971). The temperature is maintained at around 25 ° C during the day and 20 ° C at night. On the other hand, a supplemental lighting using mercury vapor lamps ensures daily 16h of day throughout the year. Under these conditions, it takes about 3 months between planting and harvesting mature seeds. The threshing of the transformants takes place in a greenhouse, where the used water is filtered and then treated with JAVEL water. Equipped with an entry airlock and a mesh filter of less than 1 mm at the air vents, the greenhouses comply with safety standards concerning the handling of genetically modified organisms.
- En culture in vitro (conditions axéniques)- In vitro culture (axenic conditions)
Les graines sont préalablement désinfectées par trempage 30 min dans une solution alcoolique à 1 % de chlore actif suivi de 3 rinçages successifs dans l'alcool 96%. Le semi s'effectue alors en boîtes de Pétri sur milieu minéral de GAMBORG et al. (1968) additionné de 1 % de saccharose et de 0,8% d'agar (Bio Mérieux). Un antibiotique variant selon la résistance in planta portée par le vecteur binaire (kanamycin ou hygromycin à une concentration de 50 mg.1"1) est ajouté pour la sélection de transformants. Les graines sont soumises à un traitement au froid (3 jours à 4°C) pour lever la dormance et homogénéiser la levée avant d'être incubées en chambre de culture climatisée (60% d'humidité relative, 16 h de lumière à 20°C/8 h d'obscurité à 15°C, intensité lumineuse 200-250μ E.m"2;s"1 au niveau des boîtes).The seeds are previously disinfected by soaking for 30 min in an alcoholic solution containing 1% of active chlorine, followed by 3 successive rinses in 96% alcohol. The semi is then carried out in Petri dishes on mineral medium from GAMBORG et al. (1968) supplemented with 1% sucrose and 0.8% agar (Bio Mérieux). An antibiotic varying according to the in plant resistance carried by the binary vector (kanamycin or hygromycin at a concentration of 50 mg. 1 "1 ) is added for the selection of transformants. The seeds are subjected to a cold treatment (3 days to 4 ° C) to break the dormancy and homogenize the emergence before being incubated in an air-conditioned culture chamber (60% relative humidity, 16 h of light at 20 ° C / 8 h of darkness at 15 ° C, light intensity 200-250μ Em "2 ; s " 1 at the boxes).
B.2. Extractions d'acides nucléiquesB.2. Nucleic acid extraction
- ADN plasmidique- plasmid DNA
Les minipréparations pour vérification rapide des clones bactériens sont réalisées suivant la méthode de lyse alcaline (Birnboim et Doly, 1979).The mini-preparations for rapid verification of the bacterial clones are carried out according to the alkaline lysis method (Birnboim and Doly, 1979).
Les maxipréparations de plasmides et de BACs pour clonages sont effectuées à l'aide du kit « Plasmid Purification » (Qiagen), suivant les recommandations du fournisseur. - ADN végétalThe maxi-preparations of plasmids and BACs for cloning are carried out using the “Plasmid Purification” kit (Qiagen), according to the supplier's recommendations. - plant DNA
Le protocole adopté est celui préconisé par KUBO et al. (1999). L'ADN est isolé à partir de feuilles de rosettes âgées de 3 semaines.The protocol adopted is that recommended by KUBO et al. (1999). DNA is isolated from 3 week old rosette leaves.
- ARNs totaux végétaux- Total plant RNAs
L'ARN a été isolé à partir de tissus pour la plupart récoltés sur plantes sauvages Ws cultivées en serre. Seules les racines et les germinations de 4 jours ont été obtenues in vitro. Les stades de développement des siliques immatures ont été numérotés de 1 à 10 du sommet à la base des hampes florales, chaque stade incluant 4 siliques successives. Le stade moyen de développement de l'embryon a été déterminé pour chaque catégorie de siliques par observation d'un échantillon de graines sous microscope après incubation dans une solution d'hydrate de chloral (cf g) NOMARSKI).The RNA was isolated from tissues mostly collected from wild plants Ws grown in greenhouses. Only the 4-day roots and germinations were obtained in vitro. The stages of development of immature pods were numbered from 1 to 10 from the top to the base of the flower stalks, each stage including 4 successive pods. The average stage of development of the embryo was determined for each category of pods by observation of a sample of seeds under a microscope after incubation in a chloral hydrate solution (cf g NOMARSKI).
L'extraction d'ARNs totaux a été réalisée à l'aide du « RNeasy Plant Mini Kit » (Qiagen) complété par un traitement à la Dnase suivant le protocole du kit « RNase-Free Dnase Set » (Qiagen).The extraction of total RNAs was carried out using the “RNeasy Plant Mini Kit” (Qiagen) supplemented by a treatment with Dnase according to the protocol of the kit “RNase-Free Dnase Set” (Qiagen).
B.3 Clonages moléculaires:B.3 Molecular cloning:
- Amplification des promoteurs par PCR- Amplification of promoters by PCR
Les délétions en 5' du promoteur de BAN ont été obtenues par PCR à partir de l'ADN du BAC T13M11 (Accession AC005882). Approximativement 150 ng d'ADN ont servi de matrice pour l'amplification, en présence de 10 μl tampon Pfu 10X, 2μl dNTPs 5 mM, 2 μl de chacun des oligos spécifiques à 10 picoM (Tableau 2), 0,8μl (2 unités) d'ADN polymérase Pfu TURBO (Stratagène) et de l'eau bidistillée stérile, q.s.p. 40 μl. Après une dénaturation préalable de 3 min à 94°C, 35 cycles d'amplification sont réalisés, 1 cycle comprenant 30 sec à 94°C, 30 sec à 60°C et 2 min 30 sec à 72°C; la PCR se termine par 10 min à 72°C. Les PCRs sont réalisées à l'aide d'un thermocycleur PTC- 100 (programmable Thermal Controller, MJ Research, INC.). - Vérification des construitsThe 5 'deletions of the BAN promoter were obtained by PCR from the DNA of BAC T13M11 (Accession AC005882). Approximately 150 ng of DNA served as template for the amplification, in the presence of 10 μl of Pfu 10X buffer, 2 μl of 5 mM dNTPs, 2 μl of each of the oligos specific for 10 picoM (Table 2), 0.8 μl (2 units ) of Pfu TURBO DNA polymerase (Stratagene) and sterile double-distilled water, qs 40 μl. After a prior denaturation of 3 min at 94 ° C, 35 amplification cycles are carried out, 1 cycle comprising 30 sec at 94 ° C, 30 sec at 60 ° C and 2 min 30 sec at 72 ° C; the PCR ends with 10 min at 72 ° C. PCRs are carried out using a PTC-100 thermal cycler (Programmable Thermal Controller, MJ Research, INC.). - Verification of constructions
Elle est effectuée par analyse de restriction et par séquençage à l'aide d'oligos situés au niveau des jonctions vecteur/insert en utilisant des oligos universels de pBS (M 13 et reverse) ou d'oligos spécifiques.It is performed by restriction analysis and by sequencing using oligos located at the vector / insert junctions using universal pBS oligos (M 13 and reverse) or specific oligos.
- Digestions et ligatures- Digestions and ligatures
Elles sont réalisées en suivant les conditions préconisées par le' fournisseur des enzymes de restriction (GIBCOBRL) et de la T4 ADN ligase (GIBCOBRL). - Séguençage d'ADNThey are carried out according to the conditions recommended by the supplier of restriction enzymes (GIBCOBRL) and of T4 DNA ligase (GIBCOBRL). - DNA sequencing
Le séquençage a été réalisé par la méthode de JANGER à l'aide d'un appareil séquenceur ABI Société Applied BIOSYSTEMS, Inc.The sequencing was carried out by the JANGER method using an ABI sequencing device Company Applied BIOSYSTEMS, Inc.
- Transformation de bactéries Des bactéries électrocompétentes (Sambrook et al., 1989) sont électroporées en présence de 10μl de produit de ligation. Ce dernier est préalablement dessalé par diafiltration sur membrane de 0,025 μm (SCHLEICHER et SCHUELL). L'électroporation est réalisée sous une tension de 1 ,25 kV appliquée pendant une durée qui est fonction de la résistance et de la capacité (200 Ω et 25 μF, respectivement du circuit de l'électroporateur du type Gène Puiser II System, commercialisé par la Société Bio-Rad)..- Transformation of bacteria Electrocompetent bacteria (Sambrook et al., 1989) are electroporated in the presence of 10 μl of ligation product. The latter is previously desalted by diafiltration on a 0.025 μm membrane (SCHLEICHER and SCHUELL). The electroporation is carried out at a voltage of 1.25 kV applied for a period which is a function of the resistance and the capacity (200 Ω and 25 μF, respectively of the circuit of the electroporator of the Gene Puiser II System type, marketed by the Bio-Rad Company) ..
B.4. Production de plantes transgénigues La transformation est réalisée par incubation de plantes en début de floraison pendant 5 min dans un milieu d'infiltration contenant Agrobacterium (voir BECHTOLD et al., 1993, pour les détails de la procédure) ainsi que de l'agent mouillant Silwet (WILCO, Belgique) à 15μl/250 ml, selon la méthode de Clough and Bent (1998).B.4. Production of transgenic plants The transformation is carried out by incubating plants at the start of flowering for 5 min in an infiltration medium containing Agrobacterium (see BECHTOLD et al., 1993, for the details of the procedure) as well as the wetting agent Silwet (WILCO, Belgium) at 15μl / 250ml, according to the method of Clough and Bent (1998).
B.5. RT-PCR SEMI-QUANTITATIVEB.5. SEMI-QUANTITATIVE RT-PCR
Les ADNcs simples brins sont synthétisés à partir de 1 μg d'ARNs totaux dans un volume de 20 μl contenant 20 mM Tris-HCI (pH 8;4), 50 mM Kcl, 2.5 mM MgCI2, 10 mM DIT, 1 mM dNTPs, 500 ng oligos (dT)12- 18 (GibcoBRL), 25 unités de Rnase Out (GibcoBRL) et 200 unités de transcriptase réverse MMLV SuperScript II (GibcoBRL) pendant 50 min. à 42°C.Single-stranded cDNAs are synthesized from 1 μg of total RNAs in a volume of 20 μl containing 20 mM Tris-HCI (pH 8; 4), 50 mM Kcl, 2.5 mM MgCI 2 , 10 mM DIT, 1 mM dNTPs , 500 ng oligo (dT) 12- 18 (GibcoBRL), 25 units of RNase Out (GibcoBRL) and 200 units of MMLV SuperScript II reverse transcriptase (GibcoBRL) for 50 min. at 42 ° C.
Deux μl de la solution d'ADNcs simples brins préalablement diluée 10 fois a été utilisée pour la réaction de PCR réalisée dans un volume total de 50 μl en présence de 20 mM Tris-HCI (pH 8.4), 50 mM Kcl, 1.5 mM MgCI2, 0.2 mM dNTPs, 0.2 μM de chaque oligo gène-spécifique et 1 unité de Taq DNA polymérase (GibcoBRL). Les oligos gène-spécifique ban-sens (5'-AACAACTAAATCTCTATCTCTGTA-3' SEQ ID N°2) et ban- antisens (5'-GAATGAGACCAAAGACTCATATAC-3' SEQ ID N°3) (Devic et al., 1999) permettent d'amplifier une bande de 1 ,2 kb dans l'ADNc de BAN et de 1 ,5 kb dans l'ADN génomique. Les oligos gène-spécifique GBGe306-sens (5'-ACCAGGAGGTTTTCAAAGAC-3' SEQ ID N°4) et GBGe306-antisens (5'-CAACATAACTTGCTCTGTTC-3'SEQ ID N°5) amplifient une bande de 0,9 kg dans l'ADNc de GBGe306, et de 1 ,1 kb dans l'ADN génomique. Le gène EF1 A4 codant pour un facteur d'élongation (GenBankX16432; Liboz et al., 1990) a été utilisé comme contrôle positif. Les oligos gène-spécifique EF-sens (5'- ATGCCCCAGGACATCGTGATTTCAT-3' SEQ ID N°6) et EF-antisens (5'TTGGCGGCACCCTTAGCTGGATCA-3'SEQ ID N°7) amplifient une bande de 0,7 kb dans l'ADNc de EF1 A4, et de 0,9 kb dans l'ADN génomique. Pour s'assurer de la linéarité de l'amplification des PCRs comportant 18, 21 et 24 cycles ont été réalisées. Chaque cycle comprend 30 sec à 94°C, 30 sec à 60°C et 2 min 30 sec à 72°C. Les trois séries ont satisfait les exigences de linéarité d'amplification, mais seulement la série à 21 cycles est présentée. Les échantillons amplifiés sont séparés sur gel d'agarose à 1% (p/v) en tampon TAE puis sont transférés sur membrane de nylon GeneScreen Plus (NEN, USA) chargée positivement selon la méthode préconisée par Sambrook et al. (1989). Après préhybridation dans un tampon à 7% SDS, 2 mM EDTA et 0,25 M Na2HPθ4/NaH2P04 pH 7,4 durant 1 heure à 65°C, la membrane est hybridée à 65°C durant une nuit dans le même tampon auquel est ajoutée la sonde cDNA ad hoc obtenue en utilisant les oligos présentés ci-dessus et marquée au (32P)- dCTP à l'aide du kit « Prime-a-Gene Labeling System » (Promega). La membrane est ensuite lavée durant 15 minutes à 65°C dans le tampon LAVI (2 X SSC, 0,5% sarkosyl, 0,2% Na4P207, 10 H2O) séchée et enveloppées dans un film alimentaire (Saran) puis placée à -80°C au contact d'un film photographique (Hyperfilm MPO, Amersham, GB) sous écran amplificateur (Amersham).Two μl of the 10-fold single-stranded cDNA solution was used for the PCR reaction carried out in a total volume of 50 μl in the presence of 20 mM Tris-HCl (pH 8.4), 50 mM Kcl, 1.5 mM MgCI 2 , 0.2 mM dNTPs, 0.2 μM of each gene-specific oligo and 1 unit of Taq DNA polymerase (GibcoBRL). The ban-sense gene-specific oligos (5'-AACAACTAAATCTCTATCTCTGTA-3 'SEQ ID N ° 2) and banantisens (5'-GAATGAGACCAAAGACTCATATAC-3' SEQ ID N ° 3) (Devic et al., 1999) allow d amplify a band of 1.2 kb in the cDNA of BAN and 1.5 kb in the genomic DNA. The gene-specific oligos GBGe306-sense (5'-ACCAGGAGGTTTTCAAAGAC-3 'SEQ ID N ° 4) and GBGe306-antisense (5'-CAACATAACTTGCTCTGTTC-3'SEQ ID N ° 5) amplify a band of 0.9 kg in l CDNA of GBGe306, and 1.1 kb in genomic DNA. The EF1 A4 gene encoding an elongation factor (GenBankX16432; Liboz et al., 1990) was used as a positive control. The gene-specific oligos EF-sense (5'- ATGCCCCAGGACATCGTGATTTCAT-3 'SEQ ID N ° 6) and EF-antisense (5'TTGGCGGCACCCTTAGCTGGATCA-3'SEQ ID N ° 7) amplify a band of 0.7 kb in the EF1 A4 cDNA, and 0.9 kb in genomic DNA. To ensure the linearity of the amplification of the PCRs comprising 18, 21 and 24 cycles were carried out. Each cycle includes 30 sec at 94 ° C, 30 sec at 60 ° C and 2 min 30 sec at 72 ° C. All three series met the amplification linearity requirements, but only the 21-cycle series is presented. The amplified samples are separated on 1% agarose gel (w / v) in TAE buffer and are then transferred to a GeneScreen Plus nylon membrane (NEN, USA) positively charged according to the method recommended by Sambrook et al. (1989). After prehybridization in a buffer at 7% SDS, 2 mM EDTA and 0.25 M Na 2 HPθ 4 / NaH 2 P0 4 pH 7.4 for 1 hour at 65 ° C, the membrane is hybridized at 65 ° C overnight in the same buffer to which is added the ad hoc cDNA probe obtained using the oligos presented above and labeled with ( 32 P) - dCTP using the “Prime-a-Gene Labeling System” kit (Promega). The membrane is then washed for 15 minutes at 65 ° C in LAVI buffer (2 X SSC, 0.5% sarkosyl, 0.2% Na 4 P 2 0 7 , 10 H 2 O) dried and wrapped in cling film (Saran) and then placed at -80 ° C in contact with a photographic film (Hyperfilm MPO, Amersham, GB) under amplifier screen (Amersham).
B.6 Test GUSB.6 GUS test
Le protocole de coloration GUS (Jefferson et al., 1987) consiste à incuber des fragments de plantes dans un tampon stérile contenant 100 mM de tampon phosphate pH 7,2 (Sambrook et al., 1989), 10 mM Na2- EDTA, 0,1 % Triton X-100, 2 mM X-Gluc (Duchefa) et 2,5 mM de ferricyanide et de ferrocyanide. L'incubation a lieu à l'obscurité à 37°C pendant une nuit.The GUS staining protocol (Jefferson et al., 1987) consists in incubating plant fragments in a sterile buffer containing 100 mM of phosphate buffer pH 7.2 (Sambrook et al., 1989), 10 mM Na 2 - EDTA, 0.1% Triton X-100, 2 mM X-Gluc (Duchefa) and 2.5 mM ferricyanide and ferrocyanide. Incubation takes place in the dark at 37 ° C overnight.
B.7 MicroscopieB.7 Microscopy
Les organes végétaux sont incubés pendant quelques minutes dans une solution d' éclaircissement de cellules, composée d'hydrate de chloral/glycérol/eau (8/1/2, p/v/v) sur lame de microscopie, avant d'être observés sous lamelle à l'aide d'un microscope équipé d'optiques à contraste d'interférence différentielle de type Nomarski. Le test « vanilline » pour la détection des tannins dans les tissus est réalisé selon la méthode de Aastrup et al. (1984).The plant organs are incubated for a few minutes in a cell lightening solution, composed of chloral hydrate / glycerol / water (8/1/2, w / v / v) on a microscope slide, before being observed. under coverslip using a microscope equipped with Nomarski-type differential interference contrast optics. The “vanillin” test for the detection of tannins in tissues is carried out according to the method of Aastrup et al. (1984).
EXEMPLE 1 : Construction de plusieurs cassettes d'expression comprenant un polynucleotide codant pour le polypeptide GUS. placé sous le contrôle d'un acide nucléigue régulateur selon l'invention.EXAMPLE 1 Construction of several expression cassettes comprising a polynucleotide coding for the GUS polypeptide. placed under the control of a regulatory nucleic acid according to the invention.
1. a) Plan des délétions de la partie 5' du gène BAN1. a) Plan of the deletions of the 5 'part of the BAN gene
Des délétions sur une région de 2.4 kb en amont du site d'initiation de la traduction ont été réalisées. Ces délétions sont illustrées sur la figure 1.Deletions on a 2.4 kb region upstream of the translation initiation site have been carried out. These deletions are illustrated in Figure 1.
Un découpage en trois zones majeures a été effectué.A division into three major zones was carried out.
- la zone I (415 pb) comprend la région 5' transcrite non traduite du gène BAN et le promoteur sensu stricto de BAN, ainsi que la région 3' transcrite non traduite de GBGe306; - la zone II (908 pb) correspond à la région génomique couverte par le transcrit de GBGe306;- Zone I (415 bp) comprises the 5 'untranslated transcribed region of the BAN gene and the sensu stricto promoter of BAN, as well as the 3' untranslated transcribed region of GBGe306; - zone II (908 bp) corresponds to the genomic region covered by the GBGe306 transcript;
- la zone III (1323 pb) comprend la région 5' transcrite non traduite de GBGe306, le promoteur sensu stricto de ce gène ainsi qu'une zone intergénique indéfinie.- Zone III (1323 bp) comprises the 5 'untranslated transcribed region of GBGe306, the promoter sensu stricto of this gene as well as an indefinite intergenic zone.
Ce prédécoupage a pour but d'analyser la contribution de la région en amont du promoteur sensu stricto sur l'expression d'un polynucleotide d'intérêt, plus spécifiquement le polynucleotide codant pour le polypeptide GUS. Chaque zone est également subdivisée en 4 (zone I) ou 2 partiesThe purpose of this precut is to analyze the contribution of the region upstream of the sensu stricto promoter on the expression of a polynucleotide of interest, more specifically the polynucleotide coding for the GUS polypeptide. Each zone is also subdivided into 4 (zone I) or 2 parts
(zones II et III). L'objectif de cette subdivision est de faciliter la détection de motifs fonctionnels gouvernant les spécificités d'expression développementales (tissulaire et temporelle) ainsi que la réponse à des stimuli environnementaux comme la lumière, la température etc.(zones II and III). The objective of this subdivision is to facilitate the detection of functional patterns governing the specificities of developmental expression (tissue and time) as well as the response to environmental stimuli such as light, temperature, etc.
1.b) Sous-clonage des délétions1.b) Subcloning of deletions
Chaque délétion est amplifiée par PCR à partir du vecteur BAC T13M11 contenant un insert d'ADN génomique de Arabidopsis thaliana, écotype Columbia. L'ADN polymérase employée et la Pfu, pour son activité d'autocorrection.Each deletion is amplified by PCR from the vector BAC T13M11 containing an insert of genomic DNA from Arabidopsis thaliana, Columbia ecotype. The DNA polymerase used and Pfu, for its self-correcting activity.
L'amplification est effectuée à l'aide d'oligonucléotides déterminés de la manière suivante, illustrée sur la figure 2:The amplification is carried out using oligonucleotides determined as follows, illustrated in FIG. 2:
- oligo 5': ils sont spécifiques de la délétion et sont modifiés par l'addition d'un site de restriction Salll précédé de 3 bases de sécurité. - oligo 3': il est commun à toutes les délétions. La création d'un site Ncol par modification de la séquence d'origine permet la mise en fusion traductionnelle de la délétion et du gène rapporteur.- 5 'oligo: they are specific to the deletion and are modified by the addition of a Salll restriction site preceded by 3 security bases. - 3 'oligo: it is common to all deletions. The creation of an Ncol site by modification of the original sequence allows the translational fusion of the deletion and of the reporter gene.
Les produits d'amplification ont ensuite été clones bords francs dans le vecteur pBS-SK aux sites Smal ou EcorV pour être séquences intégralement.The amplification products were then cloned with free edges in the vector pBS-SK at Smal or EcorV sites to be completely sequenced.
1.c) Gène rapporteur.1.c) Reporter gene.
La mise en fusion traductionnelle nécessite la présence d'un siteTranslational fusion requires the presence of a site
Ncol à l'ATG du gène rapporteur. Le clonage des promoteurs s'effectue dans un vecteur de sous-clonage présentant une cassette rapporteuπ.terminateur. Ensuite la cassette promoteur::rapporteur::terminateur est transférée dans un vecteur binaire.Ncol at the ATG of the reporter gene. The cloning of the promoters is carried out in a subcloning vector presenting a cassette rapporteuπ.terminateur. Then the promoter :: reporter :: terminator cassette is transferred into a binary vector.
Le choix du rapporteur s'est porté sur le gène GUS dont le produit est très stable et peut s'accumuler dans les tissus, permettant de détecter des activités très faibles avec le temps.The reporter chose the GUS gene, the product of which is very stable and can accumulate in the tissues, making it possible to detect very weak activities over time.
L'intérêt du gène GUS provenant du plasmide pRTL2 (fig.4) construit par James Carrington (Washington State University, Pullman,The value of the GUS gene from the plasmid pRTL2 (fig. 4) constructed by James Carrington (Washington State University, Pullman,
USA) est qu'il présente au niveau de son codon d'initiation de la traduction (ATG) un site de restriction NeOI qui permet sa mise en fusion traductionnelle avec les promoteurs.USA) is that it has a NeOI restriction site at its translation initiation codon (ATG) which allows its translational fusion with the promoters.
1.d) Introduction des promoteurs BAN dans les vecteurs de sous-clonage. La cassette TL::GUS::terminateur 35S a été transférée dans le plasmide pBS-SK pour construire le plasmide pBS-GUS. Ce plasmide intermédiaire est plus adéquat que pRTL2 pour le clonage ultérieur des promoteurs. La construction des plasmides pBAN ::GUS est illustrée sur la figure 4. Les promoteurs, excisés de pBS par digestion avec Sali et Ncol, sont ligaturés dans pBS-GUS préalablement digéré par Xhol (compatible Sali) et Ncol pour construire les plasmides pBAN::GUS (voir figure 3).1.d) Introduction of BAN promoters into subcloning vectors. The TL :: GUS :: 35S terminator cassette was transferred into the plasmid pBS-SK to construct the plasmid pBS-GUS. This intermediate plasmid is more suitable than pRTL2 for the subsequent cloning of promoters. The construction of the plasmids pBAN :: GUS is illustrated in FIG. 4. The promoters, excised from pBS by digestion with Sali and Ncol, are ligated in pBS-GUS previously digested with Xhol (compatible Sali) and Ncol to construct the plasmids pBAN: : GUS (see Figure 3).
1.e) Transfert des cassettes d'expression pBAN::rapporteur::terminateur 35S dans un vecteur binaire.1.e) Transfer of the expression cassettes pBAN :: reporter :: 35S terminator into a binary vector.
Le vecteur binaire pBIB-HYG (Becker, 1990) comportant une résistance à l'hygromycine a été utilisé . La carte du vecteur pBIB-HYG est représentée sur la figure 5.The binary vector pBIB-HYG (Becker, 1990) containing resistance to hygromycin was used. The vector map pBIB-HYG is shown in Figure 5.
- Construction du vecteur binaire pBAN:GUS- Construction of the binary vector pBAN: GUS
La cassette pBAN::GUS::term35S, sortie du vecteur pBAN-GUS par digestion Kpnl/Smal est ligaturée dans le vecteur pBIB-HYG digéré par Kpnl/Smal, pour former le vecteur pBAN-GUS/pBIB-HYG. Le protocole de construction des vecteurs pBAN ::GUS pBIB-HYG est représenté sur la figure 5. - Construction du vecteur binaire pBAN:GFP5The cassette pBAN :: GUS :: term35S, output from the vector pBAN-GUS by Kpnl / Smal digestion is ligated into the vector pBIB-HYG digested with Kpnl / Smal, to form the vector pBAN-GUS / pBIB-HYG. The construction protocol of the pBAN :: GUS vectors pBIB-HYG is represented in FIG. 5. - Construction of the binary vector pBAN: GFP5
La cassette pBAN::GFP5::term35S, sortie du vecteur pBAN-GFP5 par digestion Kpnl/Smal est ligaturée dans le vecteur pBIB-HYG digéré par Kpnl/Smal, pour former le vecteur pBAN-GUS/pBIB-HYG. Le protocole de construction du vecteur pBAN1-GFP5/pBIB-HYG est représenté sur la figure 6.The cassette pBAN :: GFP5 :: term35S, output from the vector pBAN-GFP5 by Kpnl / Smal digestion is ligated into the vector pBIB-HYG digested with Kpnl / Smal, to form the vector pBAN-GUS / pBIB-HYG. The protocol for constructing the vector pBAN1-GFP5 / pBIB-HYG is shown in FIG. 6.
1.fl Construction du vecteur pBAN1 ::Barnase-Barstar (BN-BS)/pBIB- HYG Le promoteur pBAN1 excisé de pBS par une digestion Sall/Ncol est ligaturé dans le vecteur pWP146 préalablement digéré par Sali et Ncol (Figure 7).1.fl Construction of the vector pBAN1 :: Barnase-Barstar (BN-BS) / pBIB-HYG The pBAN1 promoter excised from pBS by SalI / Ncol digestion is ligated into the vector pWP146 previously digested with SalI and Ncol (FIG. 7).
La cassette d'expression pBAN1 ::BN-BS::tCaMV est excisée de pBAN1 ::BN-BS par digestion Xhol et ligaturée dans pBIB-HYG préalablement digéré par Sali, qui est compatible avec Xhol (Figure 8).The expression cassette pBAN1 :: BN-BS :: tCaMV is excised from pBAN1 :: BN-BS by Xhol digestion and ligated into pBIB-HYG previously digested with Sali, which is compatible with Xhol (Figure 8).
EXEMPLE 2- Transformation in planta.EXAMPLE 2- Transformation in planta.
Les cassettes d'expression en vecteurs binaires sont introduites dans des plantes d'Arabidopsis écotype Ws (T0, plante infiltrée) par la méthode d'infiltration au Silwet mise au point par Clough and BentThe expression cassettes in binary vectors are introduced into plants of Arabidopsis ecotype Ws (T 0 , infiltrated plant) by the Silwet infiltration method developed by Clough and Bent
(1998). Le Silwet est un agent mouillant très puissant introduit dans la solution d'Agrobacterium juste avant trempage des plantes.(1998). Silwet is a very powerful wetting agent introduced into the Agrobacterium solution just before soaking the plants.
Les graines des plantes transformées sont récoltées en bulk, stérilisées et semées sur milieu nutritif avec hygromycine. L'obtention de plantules T1 (transformants primaires) avec siliques nécessite en moyenne 3,5 à 4 mois.The seeds of the transformed plants are harvested in bulk, sterilized and sown on a nutritive medium with hygromycin. Obtaining T1 seedlings (primary transformants) with pods requires an average of 3.5 to 4 months.
EXEMPLE 3: Transformation de plantes avec diverses cassettes d'expression contenant un acide nucléigue régulateur dérivé de la séquence SEQ ID N°1 selon l'invention.EXAMPLE 3 Transformation of plants with various expression cassettes containing a regulatory nucleic acid derived from the sequence SEQ ID No. 1 according to the invention.
Les observations de coloration GUS détaillées ci-après concernent des transformants d'Arabidopsis exprimant les constructions pBAN1 ::GUS, pBAN2::GUS, pBAN3::GUS, pBAN5::GUS et pBAN6::GUS. Cinq transformants indépendants ont été étudiés par construit. Les transformants contenant les constructions pBAN4 ::GUS, pBAN7 ::GUS et pBAN8 ::GUS ont également été réalisés.The GUS staining observations detailed below relate to Arabidopsis transformants expressing the constructions pBAN1 :: GUS, pBAN2 :: GUS, pBAN3 :: GUS, pBAN5 :: GUS and pBAN6 :: GUS. Five independent transformants were studied by built. The transformants containing the constructions pBAN4 :: GUS, pBAN7 :: GUS and pBAN8 :: GUS were also produced.
D'une façon générale, dans la majorité des transformants considérés, l'expression GUS est observée dans l'endothélium de graines immatures sans différence mesurable dans la force d'expression entre les différentes cassettes d'expression expérimentées. Cependant, une variabilité quantitative est observée entre transformants indépendants pour une même cassette d'expression, ce qui est probablement à mettre en corrélation avec le nombre de transgènes exprimés par plante, ou avec des effets de position d'insertion du T-DNA dans le génome.In general, in the majority of transformants considered, the expression GUS is observed in the endothelium of immature seeds with no measurable difference in the strength of expression between the various cassettes of expression tested. However, quantitative variability is observed between independent transformants for the same expression cassette, which is probably to be correlated with the number of transgenes expressed per plant, or with effects of position of insertion of T-DNA in the genome.
Quelle que soit la cassette d'expression utilisée, il existe des transformants montrant également une expression durant la germination localisée dans l'albumen, parfois à la jonction hypocotyle-racine ou bien dans la pointe racinaire. Quelques profils d'expression ont également été obtenus pour un transformant particulier ( ex expression GUS dans la totalité de l'hypocotyle et de la racine pour un des transformants pBAN5 ou expression dans les feuilles pour l'un des transformants pBAN6). Le profil d'expression obtenu peut donc toujours varier en fonction de l'événement de transformation considéré: Donc. quel que soit lé construit considéré, il est nécessaire de tester plusieurs transformants indépendants pour obtenir le profil d'expression spécifique souhaité.Whatever expression cassette is used, there are transformants which also show expression during germination localized in the albumen, sometimes at the hypocotyl-root junction or in the root tip. Some expression profiles were also obtained for a particular transformant (ex GUS expression in the whole of the hypocotyle and the root for one of the transformants pBAN5 or expression in the leaves for one of the transformants pBAN6). The expression profile obtained can therefore always vary depending on the transformation event considered: So. whatever the construct considered, it is necessary to test several independent transformants to obtain the desired specific expression profile.
L'observation des colorations GUS effectuées sur les plantes exprimant la cassette d'expression pBAN5 montre que l'utilisation d'un promoteur de 457 (415 + 42) paires de base en amont du site d'initiation de la traduction permet d'obtenir une expression forte et endothélium spécifique. Cependant, l'observation des plantes exprimant la cassette d'expression pBAN6 montre qu'un promoteur plus petit, de 322 (280 + 42) paires de base en amont du site d'initiation de la traduction, confère toujours une forte expression graine spécifique, dans l'endothélium, mais également dans la couche à aleurone.Observation of the GUS stains carried out on the plants expressing the pBAN5 expression cassette shows that the use of a promoter of 457 (415 + 42) base pairs upstream of the translation initiation site makes it possible to obtain strong expression and specific endothelium. However, observation of the plants expressing the pBAN6 expression cassette shows that a smaller promoter, of 322 (280 + 42) base pairs upstream of the translation initiation site, always confers a strong specific seed expression. , in the endothelium, but also in the aleurone layer.
En conséquence, la région promotrice située entre - 457 et - 322 paires de base en amont du site d'initiation de la traduction contient le (s) motif(s) nécessaire(s) et suffisant(s) pour réprimer une expression dans la couche à aleurone. CONCLUSION:Consequently, the promoter region located between - 457 and - 322 base pairs upstream of the translation initiation site contains the motif (s) necessary and sufficient to repress an expression in the aleurone layer. CONCLUSION:
Bien que les différents promoteurs testés soient fonctionnels, les promoteurs de type pBAN5 et pBAN6 seront utilisés préférentiellement, selon les besoins, pour conférer une expression spécifique dans l'endothélium (pBAN5) ou dans l'endothélium et l'albumen (pBAN6).Although the various promoters tested are functional, the promoters of the pBAN5 and pBAN6 type will preferably be used, as necessary, to confer specific expression in the endothelium (pBAN5) or in the endothelium and the albumen (pBAN6).
Enfin, une analyse in silico de la séquence du promoteur pBAN6 (confrontation à la base de données PLACE à l'aide du serveur Web Signal Scan; http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan.html) a permis de dresser une carte de motifs de régulation putatifs présents sur ce promoteur. Une importante information est la localisation d'un site de fixation de facteur de transcription de type MYB, à proximité de celui d'un facteur de type MYC, au début de pBAN7. Or NESI et al. (2000) ont montré que le gène BAN est régulé positivement par les gènes TT2 (spécifique graine, facteur de typeMYB, séquence non publiée) et TT8 (codant pour un facteur MYC). Ces résultats suggèrent que ces boîtes seraient à l'origine de l'activité endothélium spécifique du promoteur.Finally, an in silico analysis of the pBAN6 promoter sequence (confrontation with the PLACE database using the Signal Scan web server; http://www.dna.affrc.go.jp/htdocs/PLACE/signalscan. html) made it possible to draw up a map of putative regulatory reasons present on this promoter. An important piece of information is the location of a MYB-type transcription factor binding site, close to that of a MYC-type factor, at the start of pBAN7. NESI et al. (2000) have shown that the BAN gene is upregulated by the TT2 (seed-specific, typeMYB factor, unpublished sequence) and TT8 (coding for a MYC factor) genes. These results suggest that these boxes are at the origin of the promoter's specific endothelium activity.
EXEMPLE 4 : Construction d'une cassette d'expression contenant un polynucleotide- dont~l'expression ~est toxique pouf" la cellule; placé sous le contrôle d'un acide nucléigue régulateur selon l'invention.EXAMPLE 4 Construction of an Expression Cassette Containing a Polynucleotide- Whose ~ Expression ~ Is Toxic Puff " the Cell; Placed Under the Control of a Regulatory Nucleic Acid According to the Invention
La faisabilité de l'utilisation de la barnase placée sous le contrôle du promoteur pBAN1 pour l'ablation génétique de l'endothélium a été étudiée sur les deux premiers transformants indépendants. L'effet recherché pour une application biotechnologique est la réduction de taille de la graine pouvant aller jusqu'à l'ablation totale, ainsi que la suppression des flavonoïdes de l'endothélium conduisant à l'obtention de graines jaunes. Le promoteur pBAN1 a été placé devant la cassette barnase::barstar::terminateur du vecteur pWP146 (barnase-barstar) fourni par Pascual Perez (Biogemma). La cassette pBAN1 ::bamase::bastar:term a ensuite été transférée dans pBIB-HYG et le vecteur obtenu utilisé pour transformer des plantes sauvages d'Arabidopsis. Des observations histologiques (Nomarski) ont révélé une absence totale d'endothélium chez les graines produites par les deux transformants obtenus (Figure 9A). D'autre part, aucun tannin n'a été détecté dans ces graines à l'aide du test vanilline (Figures 9B et 9C), ce qui conduit à la production de graines matures jaunes (Figure 9D), contrairement aux graines sauvages dont la couleur brune est due à la présence de tannins dans l'endothélium (Figure 9). Les graines des transformants sont d'autre part plus petites que celles de plante sauvage et présentent parfois une forme de cœur. Leur capacité à germer n'est pas affectée. Ces résultats préliminaires observés sur deux transformants indépendants confirment l'intérêt de l'approche d'ablation génétique de l'endothélium pour l'obtention de graines viables jaunes et de plus petite taille. Ils démontrent également que la suppression de l'endothélium seul n'est pas suffisante pour conduire à l'avortement de la graine. L'utilisation de pBAN6, qui permettrait d'affecter également l'albumen, est une possibilité à tester. Parallèlement on peut penser à modifier le présent système d'expression en ajoutant une séquence d'adressage de la toxine à l'extérieur de l'endothélium pour détruire l'albumen et l'embryon, la préservation de l'endothélium permettant la production d'une plus grande quantité de toxine (Martine Devic, communication personnelle).The feasibility of using the barnase placed under the control of the pBAN1 promoter for the genetic ablation of the endothelium was studied on the first two independent transformants. The effect sought for a biotechnological application is the reduction in size of the seed which can go as far as total ablation, as well as the suppression of the flavonoids of the endothelium leading to obtaining yellow seeds. The pBAN1 promoter was placed in front of the barnase :: barstar :: cassette terminator of the vector pWP146 (barnase-barstar) supplied by Pascual Perez (Biogemma). The cassette pBAN1 :: bamase :: bastar: term was then transferred into pBIB-HYG and the vector obtained used to transform wild Arabidopsis plants. Histological observations (Nomarski) revealed a total absence of endothelium in the seeds produced by the two transformants obtained (Figure 9A). On the other hand, no tannin was detected in these seeds using the vanillin test (Figures 9B and 9C), which leads to the production of mature yellow seeds (Figure 9D), unlike wild seeds whose brown color is due to the presence of tannins in the endothelium (Figure 9). The seeds of the transformants are on the other hand smaller than those of wild plant and sometimes present a shape of heart. Their ability to germinate is not affected. These preliminary results observed on two independent transformants confirm the interest of the genetic ablation approach of the endothelium for obtaining viable yellow seeds and of smaller size. They also demonstrate that the removal of the endothelium alone is not sufficient to lead to the abortion of the seed. The use of pBAN6, which would also affect the endosperm, is one possibility to test. At the same time we can think of modifying the present expression system by adding a sequence of addressing of the toxin outside the endothelium to destroy the endosperm and the embryo, the preservation of the endothelium allowing the production of '' a greater amount of toxin (Martine Devic, personal communication).
EXEMPLE 5: Construction d'un système d'expression inductible d'un polynucleotide toxigue pour la cellule sous le contrôle d'un acide régulateur dérivé de la séguence SEQ ID N°1 selon l'invention.EXAMPLE 5 Construction of an Inducible Expression System of a Toxic Polynucleotide for the Cell Under the Control of a Regulating Acid Derived from Sequence SEQ ID No. 1 According to the Invention
Un tel système d'expression inductible comprend:Such an inducible expression system includes:
- une première cassette d'expression contenant un polynucleotide codant pour une protéine de fusion entre un récepteur aux glucocorticoïdes et le facteur de transcription GAL4, ce polynucleotide étant placé sous le contrôle d'un acide nucléique régulateur dérivé de la séquence SEQ ID N°1 selon l'invention; eta first expression cassette containing a polynucleotide coding for a fusion protein between a glucocorticoid receptor and the transcription factor GAL4, this polynucleotide being placed under the control of a regulatory nucleic acid derived from the sequence SEQ ID No. 1 according to the invention; and
- une seconde cassette d'expression contenant un polynucleotide dont l'expression est toxique pour une cellule de plante, ce polynucleotide étant placé sous le contrôle d'un acide nucléique régulateur susceptible d'être activé par le facteur de transcription GAL4. Lorsqu'une plante est transformée par le système d'expression inductible ci-dessus, la protéine de fusion entre le récepteur aux glucocorticoïdes et le facteur de transcription GAL4 est produite de manière constitutive, spécifiquement dans l'endothélium, ou spécifiquement dans l'endothélium et l'albumen des graines de cette plante, grâce à la première cassette d'expression.- A second expression cassette containing a polynucleotide whose expression is toxic to a plant cell, this polynucleotide being placed under the control of a regulatory nucleic acid capable of being activated by the transcription factor GAL4. When a plant is transformed by the above inducible expression system, the fusion protein between the glucocorticoid receptor and the transcription factor GAL4 is produced constitutively, specifically in the endothelium, or specifically in the endothelium and the albumen of the seeds of this plant, thanks to the first expression cassette.
Lorsque la plante ainsi transformée est mise en contact avec un glucocorticoïde, le glucocorticoïde se fixe spécifiquement sur la partie récepteur de la protéine de fusion ci-dessus et entraîne la fixation de la partie GAL4 de la protéine de fusion sur l'acide nucléique régulateur contenu dans la seconde cassette d'expression. L'acide nucléique régulateur de la seconde cassette d'expression est activé et va induire l'expression du polynucleotide toxique pour la cellule, spécifiquement dans l'endothélium, ou spécifiquement dans l'endothélium et l'albumen des graines de la plante transformée, empêchant ainsi la formation de graines matures et fertiles.When the plant thus transformed is brought into contact with a glucocorticoid, the glucocorticoid binds specifically to the receptor part of the above fusion protein and results in the binding of the GAL4 part of the fusion protein to the regulatory nucleic acid contained in the second expression cassette. The regulatory nucleic acid of the second expression cassette is activated and will induce the expression of the polynucleotide toxic for the cell, specifically in the endothelium, or specifically in the endothelium and the albumen of the seeds of the transformed plant, thus preventing the formation of mature and fertile seeds.
Le système d'expression inductible défini de manière générale ci- dessus permet l'obtention contrôlée d'une descendance viable des plantes transformées ou au contraire l'obtention contrôlée d'événements d'avortement précoce des graines. The inducible expression system defined in general above allows the controlled obtaining of a viable progeny of the transformed plants or, on the contrary, the controlled obtaining of early seeds abortion events.
TABLEAU 1 : SEQUENCESTABLE 1: SEQUENCES
SEQ ID N° DésignationSEQ ID N ° Description
1 Insert d'ADN de Pbanl1 DNA insert from Pbanl
2 Amorce ban-sens2 Ban-sense primer
3 Amorce ban-antisens3 Ban-antisense primer
4 Amorce GBGe 306 sens4 GBGe 306 sense primer
5 Amorce GBGe 306 antisens5 GBGe 306 antisense primer
6 Amorce EF-sens6 EF-sense primer
7 Amorce EF-antisens7 EF-antisense primer
8 Insert d'ADN de pBAN88 pBAN8 DNA insert
9 Insert d'ADN de pBAN79 pBAN7 DNA insert
10 Insert d'ADN de pBAN610 pBAN6 DNA insert
11 Insert d'ADN de pBAN511 pBAN5 DNA insert
12 Insert d'ADN de pBAN412 pBAN4 DNA insert
13 Insert d'ADN de pBAN313 DNA insert of pBAN3
14 Insert d'ADN de pBAN214 DNA insert of pBAN2
15 Amorce pBAN 1-5'15 1-5 'pBAN primer
16 Amorce pBAN2-5'16 primer pBAN2-5 '
17 Amorce pBAN3-5'17 primer pBAN3-5 '
18 Amorce pBAN4-5'18 primer pBAN4-5 '
19 Amorce pBAN5-5'19 Primer pBAN5-5 '
20 Amorce pBAN6-5'20 Primer pBAN6-5 '
21 Amorce pBAN7-521 primer pBAN7-5
22 Amorce pBAN8-5'22 Primer pBAN8-5 '
23 Amorce pBAN-3' 23 pBAN-3 'primer
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Claims

Revendications claims
1. Acide nucléique comprenant des signaux de régulation permettant l'expression d'un polynucleotide d'intérêt, spécifiquement dans l'endothélium et/ou l'albumen d'une graine de plante, lorsque ce polynucleotide d'intérêt est placé sous le contrôle de ces signaux de régulation, ledit acide nucléique possédant au moins 80% d'identité en nucleotides avec la séquence SEQ ID N°1 , ou avec un fragment d'au moins 200 nucleotides consécutifs de la séquence SEQ ID N°1.1. Nucleic acid comprising regulatory signals allowing the expression of a polynucleotide of interest, specifically in the endothelium and / or the albumen of a plant seed, when this polynucleotide of interest is placed under control of these regulatory signals, said nucleic acid having at least 80% nucleotide identity with the sequence SEQ ID No. 1, or with a fragment of at least 200 consecutive nucleotides of the sequence SEQ ID No. 1.
2. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 2273 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.2. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2273 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
3. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 2141 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.3. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 2141 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
4. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 2 054 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.4. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% nucleotide identity with the sequence going from the nucleotide in position 2054 to the nucleotide in position 2335 of the sequence SEQ ID # 1.
5. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 1919 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.5. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% of nucleotides identity with the sequence going from the nucleotide in position 1919 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
6. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 1510 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1. 6. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% of nucleotides identity with the sequence going from the nucleotide in position 1510 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
7. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80 % d'identité en nucleotides avec la séquence allant du nucléotide en position 1011 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.7. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% of nucleotides identity with the sequence going from the nucleotide in position 1011 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
8. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 509 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.8. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 509 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
9. Acide nucléique selon la revendication 1 , caractérisé en ce qu'il comprend un polynucleotide possédant au moins 80% d'identité en nucleotides avec la séquence allant du nucléotide en position 1 jusqu'au nucléotide en position 2335 de la séquence SEQ ID N°1.9. Nucleic acid according to claim 1, characterized in that it comprises a polynucleotide having at least 80% identity in nucleotides with the sequence going from the nucleotide in position 1 to the nucleotide in position 2335 of the sequence SEQ ID N 1.
10. Acide nucléique de séquence complémentaire à un acide nucléique selon l'une des revendications 1 à 9.10. Nucleic acid of sequence complementary to a nucleic acid according to one of claims 1 to 9.
11. Cassette d'expression comprenant un polynucleotide d'intérêt placé sous le contrôle d'un acide nucléique selon l'une des revendications 1 à 9.11. Expression cassette comprising a polynucleotide of interest placed under the control of a nucleic acid according to one of claims 1 to 9.
12. Cassette d'expression selon la revendication 11 , caractérisée en ce que le polynucleotide d'intérêt code pour un polypeptide.12. Expression cassette according to claim 11, characterized in that the polynucleotide of interest codes for a polypeptide.
13. Cassette d'expression selon la revendication 12, caractérisé en ce que le polynucleotide d'intérêt code pour un polypeptide impliqué dans la régulation du contenu en tannins ou en fibres de la graine ou pour un polypeptide régulant l'équilibre hormonal dans la testa de la graine.13. Expression cassette according to claim 12, characterized in that the polynucleotide of interest codes for a polypeptide involved in the regulation of the tannin or fiber content of the seed or for a polypeptide regulating the hormonal balance in the testa from the seed.
14. Cassette d'expression selon la revendication 13, caractérisée en ce que le polynucleotide d'intérêt code pour un polypeptide choisi parmi les protéines suivantes : - une protéine de fusion entre un récepteur aux glucocorticoïdes et la protéine GAL4 ;14. Expression cassette according to claim 13, characterized in that the polynucleotide of interest codes for a polypeptide chosen from the following proteins: - a fusion protein between a glucocorticoid receptor and the GAL4 protein;
- la toxine diphtérique ; et- diphtheria toxin; and
- la barnase.- the barnase.
15. Cassette d'expression selon la revendication 11 , caractérisée en ce que le polynucleotide d'intérêt est un polynucleotide sens ou antisens.15. Expression cassette according to claim 11, characterized in that the polynucleotide of interest is a sense or antisense polynucleotide.
16. Vecteur recombinant de clonage et/ou d'expression comprenant un acide nucléique selon l'une des revendications 1 à 9 ou une cassette d'expression selon l'une des revendications 11 à 15..16. Recombinant cloning and / or expression vector comprising a nucleic acid according to one of claims 1 to 9 or an expression cassette according to one of claims 11 to 15.
17. Vecteur recombinant selon la revendication 16, caractérisé en ce qu'il s'agit du vecteur pBAN1 : :GUS contenu dans la souche de E. coli déposée à la CNCM le 17 Juillet 2001 sous le numéro d'accès I- 2703.17. Recombinant vector according to claim 16, characterized in that it is the vector pBAN1:: GUS contained in the strain of E. coli deposited at the CNCM on July 17, 2001 under the access number I-2703.
18. Cellule hôte transformée par un acide nucléique selon l'une des revendications 1 à 9 par une cassette d'expression selon l'une des revendications 11 à 15 ou par un vecteur recombinant selon l'une des revendications 16 et 17.18. Host cell transformed with a nucleic acid according to one of claims 1 to 9 by an expression cassette according to one of claims 11 to 15 or by a recombinant vector according to one of claims 16 and 17.
19. Cellule hôte selon la revendication 18, caractérisée en ce qu'il s'agit de la la souche de E coli déposée à la CNCM le 27 Juillet 2001 sous le numéro d'accès I-2703..19. Host cell according to claim 18, characterized in that it is the E coli strain deposited at the CNCM on July 27, 2001 under the access number I-2703 ..
20 Cellule hôte selon la revendication 18, caractérisée en ce qu'il s'agit d'une cellule de Agrobacterium tumefaciens.20 Host cell according to claim 18, characterized in that it is a Agrobacterium tumefaciens cell.
21. Cellule hôte selon la revendication 18, caractérisée en ce qu'il s'agit d'une cellule de plante, ou d'un protoplaste de plante.21. Host cell according to claim 18, characterized in that it is a plant cell, or a plant protoplast.
22. Utilisation d'un acide nucléique selon l'une des revendications 1 à 9, d'une cassette d'expression selon l'une des revendications 11 à 15, d'un vecteur recombinant selon l'une des revendications 16 et 17 ou d'une cellule hôte selon l'une des revendications 18, 20 ou 21 pour l'obtention d'une plante transformée22. Use of a nucleic acid according to one of claims 1 to 9, of an expression cassette according to one of claims 11 to 15, a recombinant vector according to one of claims 16 and 17 or a host cell according to one of claims 18, 20 or 21 for obtaining a transformed plant
23. Utilisation selon la revendication 22, caractérisée en ce que la plante transformée produit des graines affectées dans le développement de la testa de la graine..23. Use according to claim 22, characterized in that the transformed plant produces seeds affected in the development of the testa of the seed.
24. Plante transformée par un acide nucléique selon l'une des revendications 1 à 9, par une cassette d'expression selon l'une des revendications 11 à 15 ou par un vecteur recombinant selon l'une des revendications 16 et 17.24. Plant transformed by a nucleic acid according to one of claims 1 to 9, by an expression cassette according to one of claims 11 to 15 or by a recombinant vector according to one of claims 16 and 17.
25. Plante transformée selon la revendication 24, caractérisée en ce qu'elle produit des graines affectées dans le développement de la testa de la graine.25. A transformed plant according to claim 24, characterized in that it produces seeds affected in the development of the testa of the seed.
26. Partie d'une plante transformée selon l'une des revendications 24 ou 25.26. Part of a transformed plant according to one of claims 24 or 25.
27. Procédé d'obtention d'une plante transformée caractérisé en ce qu'il comprend les étapes suivante : a) obtention d'une cellule hôte recombinante végétale selon la revendication 21 ; b) Régénération d'une plante entière à partir de la cellule hôte recombinante obtenue à l'étape a) ; c) Sélection des plantes obtenues à l'étape b) ayant intégré un acide nucléique selon l'une des revendications 1 à 9 ou une cassette d'expression selon l'une des revendications 11 à 15.27. A method of obtaining a transformed plant characterized in that it comprises the following steps: a) obtaining a recombinant plant host cell according to claim 21; b) Regeneration of an entire plant from the recombinant host cell obtained in step a); c) Selection of the plants obtained in step b) having integrated a nucleic acid according to one of claims 1 to 9 or an expression cassette according to one of claims 11 to 15.
28. Procédé d'obtention d'une plante transformée caractérisé en ce qu'il comprend les étapes suivantes : a) Obtention d'une cellule hôte recombinante de Agrobacterium tumefaciens selon la revendication 20 ; b) Transformation d'une plante d'intérêt par infection avec la cellule hôte recombinante de Agrobacterium tumefaciens obtenue à l'étape a) ; c) Sélection des plantes ayant intégré dans leur génome un acide nucléique selon l'une des revendications 1 à 9 ou une cassette d'expression selon l'une des revendications 11 à 15.28. A method of obtaining a transformed plant characterized in that it comprises the following steps: a) obtaining a recombinant host cell of Agrobacterium tumefaciens according to claim 20; b) Transformation of a plant of interest by infection with the recombinant host cell of Agrobacterium tumefaciens obtained in step a); c) Selection of plants having integrated into their genome a nucleic acid according to one of claims 1 to 9 or an expression cassette according to one of claims 11 to 15.
29. Procédé d'obtention d'une plante transformée caractérisé en ce qu'il comporte les étapes suivantes : a) Transfecter au moins une cellule de plante avec un acide nucléique selon l'une des revendications 1 à 9, avec une cassette d'expression selon l'une des revendications 14 à 15 ou avec un vecteur recombiant selon l'une des revendications 16 et 17 ; b) régénérer une plante entière à partir de la cellule de plante recombinante obtenue à l'étape a) ; c) Sélectionner les plantes ayant intégré dans leur génome un acide nucléique selon l'une des revendications 1 à 9 ou une cassette d'expression selon l'une des revendications 1 1 à 15.29. Method for obtaining a transformed plant characterized in that it comprises the following steps: a) transfect at least one plant cell with a nucleic acid according to one of claims 1 to 9, with a cassette of expression according to one of claims 14 to 15 or with a recombinant vector according to one of claims 16 and 17; b) regenerating an entire plant from the recombinant plant cell obtained in step a); c) Select the plants having integrated into their genome a nucleic acid according to one of claims 1 to 9 or an expression cassette according to one of claims 1 1 to 15.
30. Procédé d'obtention d'une plante transformée selon l'une des revendications 27 à 29, caractérisé en ce qu'il comporte en outre les étapes suivantes : d) croisement entre elles de deux plantes transformées telles qu'obtenues à l'étape c) avec une plante de la même espèce ; e) sélection des plantes homozygotes pour le transgène.30. Process for obtaining a transformed plant according to one of claims 27 to 29, characterized in that it further comprises the following steps: d) crossing between them of two transformed plants as obtained from step c) with a plant of the same species; e) selection of plants homozygous for the transgene.
31. Procédé d'obtention d'une plante transformée selon l'une des revendications 27 à 29, caractérisé en ce qu'il comporte en outre les étapes suivantes : f) croisement d'une plante transformée obtenue à l'étape c) avec une plante de la même espèce ; g) sélection des plantes issues du croisement de l'étape f) ayant conservé le transgène. 31. Method for obtaining a transformed plant according to one of claims 27 to 29, characterized in that it further comprises the following steps: f) crossing of a transformed plant obtained in step c) with a plant of the same species; g) selection of the plants resulting from the crossing of step f) having preserved the transgene.
32. Plante transformée, telle qu'obtenue par le procédé selon l'une des revendications 27 à 31.32. Processed plant, as obtained by the process according to one of claims 27 to 31.
33. Semence ou fruit d'une plante transformée selon la revendications 32.33. Seed or fruit of a plant transformed according to claims 32.
34. Partie d'une plante transformée selon la revendication 32. 34. Part of a transformed plant according to claim 32.
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AU2005265356B2 (en) * 2004-07-09 2010-10-07 The Samuel Roberts Noble Foundation, Inc. Genetic manipulation of condensed tannins

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