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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)
  • C12N15/8241—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
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development

Definitions

• the present invention relates to a method for destroying the germinative power of a plant seed by the introduction, into said seed, of a nucleic acid sequence coding for an enzyme capable of rapidly metabolizing an energy reserve essential for the germination of said seed.
• the invention is based on the fact that it can happen when cultivating in the open field that a non-negligible quantity of newly formed seeds falls on the ground during cultivation and / or harvesting. These seeds, involuntarily disseminated, will be able to germinate and multiply as soon as the climatic conditions are suitable. Controlling the non-multiplication of plants resulting from this dissemination requires binding monitoring of cultivated plots, consisting for example of a treatment with herbicides, a crop rotation, sometimes for several years for certain plants such as rapeseed, whose seeds can survive a decade.
• the present invention aims precisely to destroy the germinative power of seeds remaining in the field in order to prevent their multiplication without resorting to the use of a toxic substance.
• This goal is achieved thanks to the presence in the seeds of enzymes capable of rapidly burning their energy reserve before germination is initiated. By rapidly, it is meant that the energy reserves are burned within a period of between approximately 4 weeks and approximately 12 weeks.
• This modification gives the seeds of the invention a very clear disadvantage which considerably limits, or even prohibits, their involuntary multiplication. In fact, when the climatic conditions suitable for germination occur, the seeds modified according to the invention will no longer have the energy necessary for their germination and will therefore be unable to multiply.
• the semen is stored, from harvest, under controlled and regular conditions, the enzymatic activity introduced will be inhibited and the semen will retain its germinative power and may be multiplied.
• the energy reserves essential for the germination of a seed are, most often, in the form of lipid or starch.
• Normal conditions suitable for allowing germination, are characterized by sufficient humidity, oxygenation and temperature, depending on the species concerned.
• a sufficient temperature for rapeseed and tobacco is around 4 ° C to 10 ° C.
• the subject of the invention is therefore plant seeds whose germinative power is destroyed by the total or partial absence of at least one energy reserve essential for germination, in which the destruction of the germinative power is imparted by an enzyme present in the seed at a level allowing the rapid consumption of said energy reserve.
• the enzyme is expressed in the plant with an expression rate of at least 0.3% relative to the total of the proteins expressed in the plant.
• the invention particularly relates to transgenic seeds and plants whose germination power is destroyed by the total or partial absence of at least one energy reserve essential for germination.
• a first embodiment of a seed of the invention is a transgenic seed which contains at least one heterologous gene coding for an enzyme which confers the destruction of the germinative power of said seed by rapid consumption of at least one energy reserve essential for germination. More particularly, the invention relates to a transgenic seed which contains at least one heterologous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination.
• a transgenic seed contains in its cells at least one chimeric gene successively comprising:
• a promoter for example the double 35S constitutive promoter or more particularly a specific seed promoter
• the pCRU promoter of the radish cruciferin gene allowing the expression of proteins or polypeptides only in seeds or in the seeds (Depigny-This et al., : Plant Mol. Biol., 20, 467-479, 1992).
• the three sequences above are described in the articles by Murakami et al. (Plant Mol. Biol., 7, 343-355, 1986) and Matsuoka and Nakamura (Pro. Natl. Acad. Sci. USA, 88, 834-838, 1991).
• the heterologous sequence coding for the enzyme can be, for example, that of dog gastric lipase described in the PCT patent application published under the number WO 96/33277 or that of human pancreatic lipase (Lowe et al., J. Biol Biochem., 264, 20042-2008, 1989).
• lipase in the seeds of plants according to the invention leads, when it is not stopped or slowed down by the storage conditions, a reduction in the triglyceride content without modification of the fatty acid composition. Indeed, the action of lipase on the composition of the seed must be identical to that of the enzymes of germination and / or digestion.
• a second embodiment of a seed according to the invention is a transgenic seed which contains at least one endogenous gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination, placed under the control of a promoter chosen from those allowing the overexpression and activation of said gene before the initiation of the germination process, such as a more or less early specific seed promoter.
• the invention also relates to a transgenic plant or part of this plant, such as cells, characterized in that it contains:
• promoters can be those already described above.
• the seeds and plants or parts of plants according to the invention may also contain one or more heterologous genes of interest, such as genes capable of conferring on the plant an agronomic character or a resistance to a predator or to a herbicide or a gene encoding a protein of pharmaceutical interest.
• heterologous genes of interest can be introduced into seeds at the same time or before the chimeric genes making it possible to destroy the germinative power of said seeds in accordance with the invention.
• the seeds of the invention are then remarkable in that they make it possible to limit the involuntary dissemination of seeds carrying these genes of interest.
• the seeds and plants according to the invention can be dicotyledonous or monocotyledonous seeds and plants, such as those selected from the group consisting of nightshades (tomatoes, tobacco), crucifers (rapeseed), cereals (corn, wheat).
• the method according to the invention has advantages over techniques coupling male cytoplasmic sterility and a gene of interest. Indeed, the destruction of the germinative power of the seed prohibits the multiplication of seeds from male and female gametes. While male sterility does not prevent the obtaining of seeds from female gametes. In addition, obtaining seeds homozygous for the heterologous gene is easy whereas it is complex in the case of a male sterility technique, since it is necessary to have a technique of restoring fertility.
• the invention therefore also relates to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination to prepare a transgenic plant whose germination is controlled.
• the invention relates to the use of a gene coding for an enzyme responsible for the consumption of an energy reserve essential for germination to prepare a transgenic plant whose germinative power is destroyed. It is particularly preferred to use, according to the invention, a gene which codes for an enzyme chosen from the group of lipases, proteases, amylases, the action of which on the metabolism of semen is identical to that of the enzymes of germination and / or digestion.
• FIG. 1 represents the conservation of transgenic rapeseed seeds expressing a lipase under a specific seed promoter.
• the promoter used is the double 35S promoter. Upstream of the gene coding for dog lipase is placed a sequence coding for a secretory signal peptide.
• Tobacco plants used for processing experiments ⁇ Nicotiana tabacum var. Xanthi NC and PBD6) are cultured in vitro on the basic medium of Murashige and Skoog (1962) supplemented with vitamins from Gamborg et al. (1968, Sigma reference M0404) sucrose at 20 g / l and agar (Merk) at 8 g / 1.
• the pH of the medium is adjusted to 5.8 with a potassium hydroxide solution before autoclaving at 120 ° C for 20 min.
• Tobacco seedlings are transplanted by cutting internodes every 30 days on this MS20 multiplication medium.
• thermoperiod 26 ° C during the day, 24 ° C at night.
• the transformation technique used is derived from that of Horsch et al. (Science, 227, 1229-1231, 1985).
• a preculture of Agrobacterium tumefaciens strain LBA4404 containing the plasmids pBIOC29 or pBIOC26 or pBI0C25 is carried out for 48 h at 28 ° C with stirring, in LB medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press , 1989) supplemented with appropriate antibiotics (rifampicin and tetracycline).
• the preculture is then diluted to 50th in the same medium and cultivated under the same conditions. After one night, the culture is centrifuged (10 min., 3000 g), the bacteria are taken up in an equivalent volume of liquid MS30 medium (30 g / 1 sucrose) and this suspension is diluted with 10.
• Explants of about 1 cm 2 are cut from the leaves of the seedlings described above. They are then brought into contact with the bacterial suspension for one hour, then quickly dried on filter paper and placed on a coculture medium (solid MS30). After 2 days, the explants are transferred to petri dishes on the MS30 regeneration medium, containing a selective agent, kanamycin (200 mg / 1), a bacteriostatic, augmentin (400 mg / 1) and the necessary hormones. to the induction of buds (BAP, 1 mg / 1 and ANA, 0.1 mg / 1). The explants are subcultured on the same medium after 2 weeks of culture.
• the buds are subcultured in petri dishes on the development medium composed of the MS20 medium supplemented with kanamycin and augmentin. After 15 days, the buds are transplanted in half. Rooting takes about 20 days, at the end of which the seedlings can be cloned by cutting internodes or taken out in the greenhouse. According to the same method, it is possible to obtain tobacco seeds expressing human pancreatic lipase under the control of a constitutive promoter.
• the promoter used is the cruciferin promoter. Upstream of the gene coding for lipase is placed a sequence coding for a secretory signal peptide.
• the spring rapeseeds (Brassica napus cv WESTAR or Limagrain lines) are disinfected for 40 minutes in a 15% Domestos solution. After 4 rinses with sterile water, the seeds are put to germinate, at a rate of 20 seeds per pot of 7 cm in diameter and 10 cm high, on mineral medium of Murashige and Skoog (Sigma M 5519) with 30g / 1 of sucrose and solidified with agargel at 5 g / 1. These pots are placed in a culture chamber at 26 ° C with a photoperiod of 16h / 8h and under a light intensity of the order of 80 ⁇ E m ⁇ 2 s ⁇ ⁇ .
• the cotyledons are removed sterile by cutting each petiole about 1mm above the cotyledon node.
• a preculture of Agrobacterium tumefaciens strain LBA4404, containing the plasmid pGAZE into which the sequence coding for a PPS (or PS) addressing signal has been inserted under the control of the pCRU promoter (or pd35S), is carried out in Erlenmeyer flask of 50 ml, for 36 h at 28 ° C in 10 ml of 2YT bacterial medium (Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) supplemented with antibiotics useful for the selection of the strain used .
• This preculture is used to seed at 1% a new bacterial culture carried out under the same conditions. After 14 h the culture is centrifuged for 15 min at 3000 g and the bacteria are taken up in an equivalent volume of liquid germination medium. This suspension is distributed in petri dishes 5 cm in diameter at a rate of 5 ml / box.
• the severed end of the petiole is immersed for a few seconds in the solution of agrobacteria thus prepared, then the petiole is pressed a few millimeters into the regeneration medium.
• This medium has the same basic composition as the germination medium with an additional 4 mg / l of benzylamino-purine (BAP), a phytohormone promoting the new formation of buds.
• BAP benzylamino-purine
• Ten explants (cotyledon with petiole) are cultured per Petri dish 9 cm in diameter (Greiner reference 664102).
• the explants Twice in succession, 3 weeks apart, the explants are transplanted sterile onto new medium under the same conditions.
• the green buds that appear at the end of the second or third transplanting are separated from the explant and cultured individually in transparent pots 5cm in diameter and 10cm high containing a medium identical to the previous one but devoid of BAP.
• the stem of the transformed bud is cut off and the bud is transplanted into a pot of fresh medium.
• the roots are sufficiently developed to allow acclimatization in phytotron (temperature 21 ° C, photoperiod 16h / 8h and 84% relative humidity), the seedlings are repotted in pots 12 cm in diameter filled with the same potting soil enriched with late fertilizer (Osmote, at the rate of 4 g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at 18 ° C, with two daily waterings of 2 minutes.
• phytotron temperature 21 ° C, photoperiod 16h / 8h and 84% relative humidity
• the seedlings are repotted in pots 12 cm in diameter filled with the same potting soil enriched with late fertilizer (Osmote, at the rate of 4 g / 1 of potting soil) then transported to a greenhouse (class S2) regulated at 18 ° C, with two daily waterings of 2 minutes.
• the pods When the pods have reached maturity, they are harvested, dried and then beaten. The seeds obtained are used for assaying biochemical activity.
• the selection of the transgenic progeny is done by germination on a medium containing kanamycin sulfate at a rate of 100 to 150 mg / 1 (depending on the genotypes).
• the operating conditions are identical to those described above except that the germinations are carried out in glass tubes with only one seed per tube. Only the seedlings developing secondary roots during the first three weeks are acclimatized in a phytotron before being transferred to the greenhouse. According to the same process, it is possible to obtain rapeseed expressing human pancreatic lipase under the control of a specific seed promoter.
• the activity of the lipase when it is not slowed down or stopped by conditions of conservation, cold of 4 ° C and reduced hygrometry, involves a reduction in the content of triglyceride without modification of the fatty acid composition.
• the lipase activity is determined using a pH-STAT by the titrimetric method of Gargouri et al. (Gastroenterology, 91, 919-925, 1986) in which the substrate used is tributyrin.
• the tributyrin emulsion (4 ml for 30 ml of emulsion) is produced using a vortex in the presence of bile salts (1.04 g / 1) of bovine albumin (0.1 g / 1) and NaCl (9 g / 1).
• the assay consists in neutralizing the butyric acid released under the action of the lipase with a sodium hydroxide solution at a set pH of 5.5 to 37 ° C.
• One lipase unit corresponds to the quantity of enzyme which causes the release of a micromole of fatty acid in 1 minute at 37 ° C and under optimal pH conditions (5.5).
• Purified dog gastric lipase has a specific activity of 570 units / mg of protein. The lipase activity can be measured on the total ground material, on the pellet or on the centrifugation supernatant. Measurements of lipase activity confirm the expression, in the seeds of transformed plants, of the lipase encoded by the heterologous gene. If we follow the evolution of the germination power of these seeds to lipase activity over time, we observe a rapid deterioration in the capacity of these seeds to germinate until disappearance after 16 weeks.
• transgenic plants according to the invention for example rapeseed expressing dog gastric lipase under the control of the cruciferin promoter radish, and non-transgenic lines, each on identified spaces, and harvest the plants under the same conditions.
• a part of the seeds produced will fall into the plot during the cultivation or during the harvest, and the following year, the value of the seeds and plants according to the invention is noted by comparing the number of regrowths on each of the plots.
• transgenic colza homozygous for the transgene cDNA coding for gastric lipase under constitutive promoter was carried out. This field of transgenic rapeseed was surrounded by borders of non-transgenic rapeseed. This test made it possible to confirm in real conditions the potential for re-germination of transgenic seeds expressing the cDNA coding for gastric lipase. Observation of the number of regrows on each plot.
• the extraction buffer (0.2 M Tris-HCl; 0.25 mM Na-EDTA; 0.25 M NaCl; 0.5% SDS; pH 8.0). Then, the reaction medium is subjected to the vortex for 5 sec. followed by extraction with phenol- chloroform-isoamyl alcohol (25/24/1 v / v / v). The aqueous phase is then precipitated by adding isopropanol at -80 ° C for 15 min. After centrifugation at 4000 g for 15 min. the pellet is washed in 70% ethanol, then recentrifuged and dried. The pellet is taken up in 100 ⁇ l of H20.
• the PCR amplification reaction of the DNA is carried out in a GeneAmp PCR System 9700 thermocycler in the presence of 0.36 ⁇ l of each of the oligodeoxynucleotide primers 5 'TTCTACCCACACCACTTC 3' and 5 'ACATGCATGTCTGTCAGG 3' at 50 pmol / ⁇ l, 0, 5 ⁇ l lNm dNTP, 3 ⁇ l 98% glycerol, 1.8 ⁇ l MgC12 25mM, 0.36 ⁇ l BSA 10 mg / ml, 3 ⁇ l of Promega xlO DNA polymerase buffer and 0.36 ⁇ l of Taome DNA polymerase 5 U / ⁇ l in a reaction medium of 30 ⁇ l final.
• a GeneAmp PCR System 9700 thermocycler in the presence of 0.36 ⁇ l of each of the oligodeoxynucleotide primers 5 'TTCTACCCACACCACTTC 3' and 5 'ACATG
• the DNA is denatured for 5 min at 94 ° C, subjected to 35 cycles each consisting of 30 denaturation at 94 ° C, 1 min. hybridization at 50 ° C, and 1 min. elongation at 72 ° C., then the elongation is continued for 5 min.
• the regrowth potential of the transgenic seeds is approximately 12 times less important than for non-transgenic seeds - the transgenic seeds which germinated within the framework of this test express half the recombinant lipase than the initial seeds. It therefore seems that when the seeds express lipase at least up to 120 UTC4 / g, they have no potential for re-germination in open field conditions.
• Rapeseed lipase-expressing rapeseed (T2 and T3) has a fat content of the order of 22 to 24% instead of 40% on average, a drop of around 40%.

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• 1998
  • 1998-04-09 FR FR9804495A patent/FR2777155B1/fr not_active Expired - Fee Related
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