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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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
• • 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
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

Definitions

• Nucleic acids coding for a plant phosphatase of the MIPP type with phytase activity and applications are provided.
• the present invention relates to nucleic acid sequences coding for a plant phosphatase of the MIPP type with phytase activity, the isolated proteins coded by these sequences, as well as their applications.
• This invention relates more specifically to nucleic acid sequences of corn, rice and wheat encoding an MIPP.
• Phosphorus is essential for the growth of the animal; 80% of the phosphorus is located in the skeleton. The rest of the phosphorus is contained in the soft tissues where it is involved in many biochemical reactions such as the synthesis of nucleic acids, phospholipids and certain B vitamins. Phosphorus is supplied to animals by food, mainly by plants . Most of the phosphate present in plants, in particular. in seeds is found in the form of phytin, complex salt of myo-inositol-hexakisphosphoric acid or phytic acid.
• Phytin therefore constitutes a reserve of phosphorus, of sugars, but also of various cations (Ca 2+ , Zn 2+ , Mg 2+ , Fe 3+ ).
• Phytin, salt of phytic acid complexed with various cations therefore represents the major form of storage of phosphate in seeds but it is also present in pollen, storage organs such as tubers or in the roots. Its distribution in seeds varies from one species to another:
• dicots in dicots (soybeans, castor), it is mainly present in the cotyledons and the endosperm;
• phytin and phytic acid from dry seed meal are not digested by monogastric animals (such as pork and chicken) in the absence of exogenous phosphatase, and are therefore excreted as such. They thus contribute to the pollution of soil and water, by phosphate, in areas of intensive farming.
• phytic acid is considered to be an anti-nutritional factor because it chelates the mineral elements and causes protein aggregation. Consequently, these minerals and proteins will not be correctly assimilated during intestinal transit by monogastric animals (Graf, 1986).
• the authors of the present invention are interested in another family of enzymes, which do not exhibit significant homology with Phyt I or Phyt II: MLPP - Multiple Inositol Polyphosphate Phosphatases, related to the histidine phosphatases listed only in the animal kingdom. They play an important role in the processes of ossification, in particular in the development and differentiation of chondrocytes (Romano et al, 1998). These phosphatases are also known to be the only animal enzymes which hydrolyze the molecules of inositol-tetra, -penta- and - hexa ⁇ hos ⁇ hate (Chi and ⁇ /., 1999).
• the present invention therefore relates to an acid. isolated nucleic acid coding for a plant MIPP enzyme with phytase activity. Cereal MIPP enzymes are particularly targeted, in particular those of corn, rice and wheat.
• the invention relates more particularly to an isolated nucleic acid comprising a sequence chosen from SEQ ID No. 1 (corn cDNA), SEQ ID No. 3 (rice cDNA) or SEQ ID No. 17 (corn cDNA).
• the subject of the invention is an isolated nucleic acid comprising the sequence SEQ ID No. 17 or a sequence homologous to the sequence SEQ ID No. 17.
• sequences complementary to these nucleic acid sequences also form part of the present invention.
• a subject of the invention is also fragments of the above sequences coding for peptides retaining the activity mentioned, or the complementary sequences of these fragments.
• the invention also relates to the homologous sequences of the sequences mentioned above.
• homologous refers to any nucleic acid having one or more sequence modification (s) from all or part of a given sequence.
• homologous sequences are, preferably, defined as: i) sequences similar to at least 70% of the sequence SEQ ID No. 1, No. 3 or No. 17, preferably at least 80%, more preferably at least minus 90%; or ii) sequences hybridizing with the sequence SEQ ID No. 1, No. 3 or No. 17, or its complementary sequence, under stringent hybridization conditions, or iii) sequences coding for a plant MIPP enzyme comprising the sequence amino acid SEQ ID No. 2, No. 4 or No. 18, or a homologous amino acid sequence.
• a homologous nucleotide sequence specifically hybridizes to the sequences complementary to the sequence SEQ ID No. 1, No. 3 or No. 17 under stringent conditions.
• the parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
• Tm 81.5 + 0.41 (% G + C) + 16.6 Log (cation concentration) - 0.63 (% formamide) - ( 600 / number of bases) (Sambrook et al, 1989).
• Tm 4 (G + C) + 2 (A + T).
• the hybridization temperature can preferably be 5 to 10 ° C below Tm, and the hybridization buffers used are preferably force solutions high ionic content such as 6xSSC solution for example.
• similar sequences used above refers to the perfect resemblance or identity between the nucleotides compared but also to the non-perfect resemblance which is called similarity. This search for similarities in the nucleic sequences distinguishes, for example, purines and pyrimidines.
• a nucleotide sequence homologous to the ORF represented in SEQ ID No. 1, No. 3 or No. 17 therefore includes any nucleotide sequence which differs from the sequence SEQ ID No. 1, No. 3 or No. 17 by mutation, insertion, deletion or substitution of one or more bases, or by degeneration of the genetic code, provided that it codes for a polypeptide exhibiting the phytase activity of plant MTPP.
• homologous sequences are included the sequences of cereal genes other than corn or rice, as well as allelic variants.
• the present invention also relates to an isolated polypeptide, called
• Plant MIPP preferably comprising the amino acid sequence SEQ ID No. 2, No. 4 or No. 18. More particularly, the polypeptide comprises the amino acid sequence SEQ ID No. 18. It is understood that are also understood the homologous sequences, advantageously defined as i) sequences similar to at least 70% of the sequence SEQ ID No. 2, No. 4 or No. 18, preferably at least 80%, more preferably at least 90 %; or ii) the sequences encoded by a homologous nucleic acid sequence as defined above, that is to say a nucleic acid sequence hybridizing with the sequence SEQ ID No. 1, No. 3 or No. 17 or its complementary sequence, under stringent hybridization conditions.
• similar refers to the perfect resemblance or identity between the amino acids compared but also to the non-perfect resemblance which is termed similarity.
• This search for similarities in a polypeptide sequence takes into account conservative substitutions which are amino acid substitutions of the same class, such as amino acid substitutions for the uncharged side chains (such as asparagine, glutamine, serine, threo ine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acid side chains (such as aspartic acid and glutamic acid); amino acids with apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine).
• amino acid substitutions for the uncharged side chains such as asparagine, glutamine, serine, threo ine, and tyrosine
• homologous amino acid sequence any amino acid sequence which differs from the sequence SEQ ID No. 2, No. 4 or No. 18 by substitution, deletion and / or insertion of an amino acid or a reduced number of amino acids, in particular by substitution of natural amino acids with non-natural amino acids or pseudo-amino acids at positions such that these modifications do not significantly affect the biological activity of the Vegetable MIPP.
• Homology is usually determined using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e. identity or similarity, as defined above).
• the degree of homology is established by recording all the positions for which the amino acids of the two sequences compared are identical, relative to the total number of positions.
• the biological activity of plant MIPP refers in particular to its phytase activity, which can be determined by the determination of the phosphate released by the enzyme from sodium phytate.
• polypeptide of the present invention can be synthesized by any method well known to those skilled in the art.
• the polypeptide of the invention can for example be synthesized by the techniques of synthetic chemistry, such as the Merrifield type synthesis which is advantageous for reasons of purity, absence of unwanted side products and for its ease of production. .
• a recombinant protein can also be produced by a method, in which a vector containing a nucleic acid according to the invention, such as a nucleic acid comprising the sequence SEQ ID No. 1, No. 3 or No. 17 or a homologous sequence is transferred to a prokaryotic or eukaryotic host cell which is cultured under conditions allowing expression of the corresponding polypeptide.
• the protein produced can then be recovered and purified.
• the purification methods used are known to those skilled in the art.
• the recombinant polypeptide obtained can be purified from cell lysates and extracts, supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, etc.
• the present invention therefore also relates to a method for producing vegetable MIPP protein, characterized in that it comprises the steps of: a) transformation of a cell, in particular of a plant or of a microorganism, with a expression cassette comprising regulatory sequences capable of controlling the expression of an increased amount of MIPP in said host cell; b) optionally culturing said host cell or, in the case where this host cell is a plant cell, growing the transformed plant; c) extraction of the MIPP protein from the cell culture or from the transformed plant.
• the present invention therefore also relates to an expression cassette, characterized in that it comprises at least one of the nucleic acid sequences or fragments as defined above, placed under the control of at least one regulatory sequence capable of controlling expression of the vegetable MTPP protein.
• promoters include promoters, activators and introns and transcription terminators.
• a large number of promoters can be used for the transformation of plants according to the present invention.
• the specific promoters of an organ or a tissue of the plant and more particularly the specific grain promoters (Datla et al, 1997), such as the promoters of genes coding for reserve proteins such as: a glutenin of high molecular weight HMWG (High Molecular Weight Glutenin) of wheat or barley specific for albumen (Roberts et al, 1989; Anderson OD et al, 1989), napine, phaseoline, helianthinine, albumin, oleosin, GEAI and GEA6 from Arabidopsis thaliana (Gaubier et al, 1993), corn ⁇ -zein, the promoter of the pHyPRP gene coding for a hybrid protein rich in corn proline (HyPRP: Hybrid Proline Rich Protein) which is specific for the embryo and more particularly for scutellum (Josè-Estanyol et al, 1992), the Npl promoter of the gene Niviparousl (transcription activator -
• promoter of the gene coding for the small subunit of RUBISCO Ribulose 1.5 BISphosphate Carboxylase Oxygenase, promoter of the gene coding for chlorophyll a / b,
• genes coding for enzymes preferably chosen from these: corn alcohol dehydrogenase 1 (Adhl), phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG), chitinase, glucanase , protease inhibitors, genes of the PR1 family, the vspB gene (US 5,670,349), HMG2 (US 5,670,349), apple beta-galactosidase (Abgl), or amino-cyciopropane carboxylate synthase (WO 98/45445 ).
• tissue-specific or organ-specific ectopic expression allows the production of seeds rich in inorganic phosphate or enriched in MTPP protein capable of degrading phytin while limiting any physiological damage due to a variation in the phytin content.
• a constitutive promoter will preferably be used such as the pAct 1 promoter of the rice Act 1 gene contained in the plasmid pActl-F4 (Me Elroy et al, 1991) or the p35S promoter (Kay et al, 1987), or a tissue specific promoter such as the HMWG wheat or barley promoter, or the pCRU promoter of the radish cruciferin gene, which all allow expression of the protein of interest in the seeds (Roberts et al, 1989; Anderson OD et al, 1989; Depigny-This et al, 1992).
• elements such as activators and introns can also be inserted into the expression cassette in order to amplify the expression of the gene of interest.
• an activator is the TEN (Tobbaco Etch Nirus) translation activator described by Carrington and Freed (1990).
• the first intron of the Adhl S gene from corn which can be placed between the promoter and the coding sequence. This intron, when included in a genetic construction, increases the expression of the desired protein in corn cells (Callis et al, 1987).
• the expression cassette 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 transcription terminator is the nos terminator of the nopaline synthase gene from Agrobacterium tumefaciens (Depicker et al, 1992) or the polyA 35S sequence of the cauliflower mosaic virus (CaMN) , described in the article by Franck et al. (1980).
• said regulatory sequences also include addressing signals capable of directing the expression specifically in a particular type of cell compartment, for example the extracellular space or the apoplasm.
• chloroplast addressing signals mention may be made of the sequence coding for the transit peptide of the precursor of the small subunit of ribulose 1,5-bis-phosphate carboxylase oxygenase from Pisum sativum.
• mitochondrial addressing signals mention may be made of the sequence coding for the transit peptide of the precursor of the ⁇ subunit of the mitochondrial ATP-ase FI of Nicotiana plumbaginifolia.
• the addressing sequences can be sequences coding for an N-terminal signal peptide ("prepeptide"), possibly in association with a signal responsible for the retention of the protein in the endoplasmic reticulum (signal of the KDEL type or KTEL), or a vacuolar or "propeptide” addressing signal.
• the presence of the N-terminal signal peptide or prepeptide allows the introduction of the neopolypeptide into the endoplasmic reticulum where a number of post-translational modifications take place, in particular the cleavage of the signal peptide, the N-glycosylations, if the protein in question presents N-glycosylation sites, and the formation of disulfide bridges.
• the prepeptide responsible for addressing the protein in the endoplasmic reticulum, is very useful. It is normally a hydrophobic N-terminal signal peptide having between 10 and 40 amino acids and being of animal or vegetable origin. Preferably, it is a prepeptide of plant origin, for example that of sporamine, barley lectin, plant extensin, protein PR1 or PR2.
• the signal peptide is cleaved by a signal-peptidase upon the co-translational introduction of the neopolypeptide into the lumen of the RER (Rough Endoplasmic Reticulum).
• the mature protein no longer has this N-terminal extension.
• the addressing sequences can, in addition to the prepeptide, also include an endoplasmic retention signal, consisting of the peptides KDEL, KTEL, SEKDEL or HEKDEL. These signals are normally found at the C-terminus of the protein and can remain on the mature protein. The presence of this signal tends to increase the yields of recombinant proteins.
• KDEL or KTEL type sequences at the C-terminal end of proteins leads to their retention in the endosplasmic reticulum and therefore to a cellular compartmentalization of these proteins.
• This compartmentalization can in certain cases block the accessibility of the enzyme to its substrate present in a different cellular compartment, leading to inactivity of the protein.
• the elimination of these sequences can lead to an apoplastic expression of the protein which can allow better accessibility of the protein to its substrate.
• Elimination can also lead to expression of the protein in a compartment other than that in which its substrate is expressed, thus avoiding rapid degradation of phytic acid capable of altering the development of the seed.
• the activity of the enzyme vis-à-vis its substrate can therefore be controlled in space and time.
• a subsequent step of grinding the seed can be carried out to allow the enzyme to access its substrate.
• the elimination of the KTEL or KDEL sequence can be carried out in particular by the PCR amplification technique using primers specific for the C-terminal part of the protein just upstream of the KTEL or KDEL sequence and by adding on the initiates a stop codon.
• the addressing signals may, in addition to the prepeptide, also include a vacuolar or "propeptide" addressing signal.
• a vacuolar or "propeptide” addressing signal In the presence of such a signal, after passing through the RER, the protein is addressed to the vacuoles of the aqueous tissues, for example the leaves, as well as to the protein bodies of the reserve tissues, for example the seeds, tubers and roots.
• the addressing of the protein towards the protein bodies of the seed is particularly interesting because of the capacity of the seed to accumulate proteins, up to 40% of the proteins relative to the dry matter, in cellular organelles derived from vacuoles. , called protein bodies and because of the possibility of storing for several years the seeds containing the recombinant proteins in the dehydrated state.
• propeptide a signal of animal or plant origin can be used, plant signals being particularly preferred, for example pro-sporamine.
• the propeptide can be N-terminal ("N-terminal targeting peptide" or NTTP), or C-terminal (CTTP). Since propeptides are normally cleaved upon entry of the protein into the vacuole, they are not present in the mature protein.
• signal peptide or prepeptide can lead to the glycosylation of the protein.
• the invention also relates to a vector into which is inserted the expression cassette as defined above.
• This vector can be a plasmid which may further comprise a marker gene for distinguishing a transformed plant from a plant which does not contain the transferred foreign DNA.
• the vector can include, as a marker gene, both selection genes which confer resistance to an antibiotic or a herbicide, as well as reporter genes. the selection genes which can be used, there may be mentioned:
• the chloramphenicol acetyl transferase (CAT) gene which detoxifies chloramphenicol.
• the following reporter genes can be mentioned: the gene coding for the ⁇ -glucuronidase (GUS) enzyme,
• vectors comprising a DNA sequence containing at least one origin of replication such as plasmids, cosmids, bacteriophages, viruses, etc. .. Plasmids are nevertheless preferred.
• the invention also relates to cellular hosts, in particular bacterial hosts, containing the above-mentioned vectors.
• the invention also provides a method for producing transgenic plants comprising, inter alia, the steps of: transformation of plant cells with an expression vector containing a nucleic acid fragment of the present invention, selection of the transformed cells, generation plants transformed from these cells, expressing the inserted nucleic acid fragment.
• the transformation of the plants according to the invention can be carried out by different methods, known to the skilled person. We can cite, first of all, direct gene transfer methods such as micro-injection into plant embryo cells (Neuhaus et al., 1987) or electroporation (Chupeau et al., 1989 ), direct precipitation with polyethylene glycol (Schocher et al., 1986) or bombardment with a particle gun (Me Cabe et al., 1988).
• the bacterial strain may contain the gene coding for the plant MLPP enzyme, under the control of elements ensuring the expression of said gene.
• the bacterial strain can be transformed by a vector into which is inserted a sequence encoding said enzyme under the control of elements ensuring its expression.
• This sequence is inserted, for example, into a binary vector such as pBLN19 (Bevan, 1984) or pMON 505 (Horsch and Klee, 1986) or any other binary vector derived from the plasmids pTi and pRi. It can also be usefully introduced by homologous recombination into a disarmed pTi or pRi plasmid, such as pGN 3850 (Zambryski et Coll., 1983) before the transformation of the plant.
• the nucleic acid fragments are introduced into the cells by bombardment of particles covered by said fragments.
• Particle bombardment offers the advantage of rapid transformation. Generally immature embryos undergo only one bombardment. However, repeated bombardment can increase the frequency of transformation (WO 98/49316).
• the transformed cells are selected according to the phenotypic markers used in the vector. This selection is carried out on a medium containing an appropriate selective agent. The transformed cells thus selected are then cultivated and the plants are regenerated. DNA extraction and a Southern blot with probes specific for the gene of interest confirm the transformation.
• the methods making it possible to isolate DNA from biological materials in culture and to verify the presence of the insert are well known to those skilled in the art, they are, among others, described by Southern et a, 1975 and Mullis et a, 1987.
• a more particular subject of the present invention is a method for increasing the phytase activity of a plant, characterized in that an overexpression of a MIPP, more particularly of corn, rice or wheat, is induced in said plant. using an expression cassette as described above and according to the preceding method.
• overexpression is meant here both an increase in the level of MIPP compared to the level expressed in a normal plant, and an ectopic expression of this enzyme, in a tissue or a compartment and / or at a stage of development where that -this is not normally expressed.
• the foreign sequence can be heterologous, that is to say that it comes from a plant different from the host cell. It can also be a sequence of MIPP naturally produced by the plant.
• the present invention also relates to plant hosts, consisting of plants or plant organs, transformed with one or more nucleic acids of the invention and according to the method defined above. If the transformation involves several genes of interest, then these can be inserted into the same expression cassette or else into different expression cassettes.
• plant hosts includes plants as well as plant cells or different parts of the plant, such as seed, fruit or leaf.
• transgenic plants in particular corn, wheat, barley, sorghum, rye, rice, preferentially corn, as well as peas, soybeans and potato.
• transgenic plants of the invention include both first generation plants and their descendants (lines or hybrids, in particular).
• the present invention more particularly relates to transgenic plant hosts in which a plant MIPP, more particularly of corn, wheat or rice, can be expressed in a part of the plant or a cellular compartment where this enzyme is not produced naturally or only in small quantities.
• These plant hosts are characterized by a genome into which an expression cassette according to the invention has been introduced comprising regulatory sequences which induce specific expression in said part of the plant or said cell compartment.
• the plant host in question is advantageously a cereal and more advantageously still corn, wheat and rice, or their organs.
• the present invention relates more precisely to a transgenic plant, according to the invention, characterized in that it produces transgenic seeds expressing an increased quantity of enzymes with phytase activity.
• the present invention also comprises the seeds of this transgenic plant and in particular the seeds comprising an increased vegetable MIPP content, obtained by specific expression of one of the nucleic acid sequences according to the present invention, in the seed.
• the subject of the present invention is a composition for human or animal consumption comprising seeds, a seed meal or a vegetable MIPP, produced according to the present invention.
• the nucleic acids defined by the invention can be used as a marker for the phenotype linked to the expression of MIPP proteins.
• the invention allows the use of the nucleotide fragments as molecular markers in culture programs, in particular in the selection of varieties expressing a MIPP protein or a plant transformed with a sequence coding for this MIPP.
• these sequences make it possible to obtain genomic DNAs by methods known to those skilled in the art, for example by screening genomic libraries according to the indications of Sambrook et al. (1989, Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, New York).
• degenerate probes they allow the identification of genes coding for proteins homologous to plant MIPPs.
• the transgenic plants expressing the plant MIPP according to the invention or the enzyme itself can be used for various purposes, in any situation where the phytase activity of this enzyme is necessary or desired.
• Plant MIPP is thus useful in food preparation processes or in starch extraction processes (soaking process).
• this enzyme it is possible to increase the extractability of the starches of the grains where it is expressed.
• Phytate precipitates proteins with starch so that if you reduce the phytate level, the extraction of starch is easier.
• Addition of an active enzyme phytase to the grains of plants from which it is desired to extract the starch makes it possible to compensate for the precipitation of proteins by phytates.
• a plant transformed with a sequence coding for a plant MIPP according to the process defined by the invention, or a part thereof, can be used to improve the digestibility of phytate, in the food ration of monogastric animals, and therefore , reduce the level of phytate in manure or slurry.
• the phytase activity of the vegetable MIPP can also be used to make the most of recovered soaking water or brewing water, during food preparations.
• the increased availability of phosphorus makes these waters very useful as additives for culture media or fermentation supports.
• the phytase activity generated allows better recovery of inositol and its derivatives from soaking or brewing water.
• the invention also relates to the use of a plant MIPP according to the invention or the use of all or part of plants containing an MLPP according to the invention, for therapeutic or dietetic purposes, in particular for the production of myo-inositol and / or for the reduction of phytic acid.
• Phytic acid has been held responsible for certain impacts on human health such as bone malformation, osteosporosis and anemia caused by iron deficiency, or interference with the assimilation of calcium, magnesium and zinc (Berlyne et al., 1973; Shan et al., 1979).
• Myo-inositol the active nutritional form of inositol, is a constituent of the phospholipid phosphatidylinositol. He is known for his virtues in the health field. It has often been attributed to effects on the decrease in the concentration of triglycerides and cholesterol in the blood, and more generally for protection against cardiovascular diseases. In addition, it is recognized that compounds derived from a phospholipid such as myo-inositol have beneficial effects on insomnia and anxiety. In addition, the peripheral neuropathy of the diabetic is one of the most paralyzing complications of diabetes.
• the invention relates to the use of the vegetable MIPP, according to the invention, for releasing inorganic phosphate directly assimilable by monogastrics (Pointillard, 1994) and / or improving the availability of cations chelated by phytin such as iron, calcium, magnesium or zinc. This avoids the addition of supplements in animal rations and increases the nutritional value of the seed.
• Figure 1 represents an alignment of the nucleic sequences coding for the MLPP of corn and rice.
• Figure 2 shows an alignment of the protein sequences of MIPP in rice and maize.
• Figure 3 shows a comparison of the amino acid sequences of MIPP in rice and corn with animal MIPP, acid phophatases and fungal phytases.
• Figure 4 is a restriction map of plasmid pRD-257.
• Figure 5 is a restriction map of plasmid p3214.
• Figure 6 is a restriction map of plasmid pBIOS 421.
• Figure 7 is a restriction map of plasmid pBIOS 422.
• SEQ ID NO: 1 Nucleic sequence of cDNA isolated from maize
• SEQ ID N ° 2 Amino acid sequence of a corn MLPP protein (ZmMIPP)
• SEQ ID N ° 3 Nucleic sequence of cDNA isolated from rice
• SEQ ID N ° 4 Amino acid sequence of a rice MIPP protein (OsMIPP)
• SEQ ID N ° 5 Oligonucleotide 5'olZMP1 used as primer 5 'for obtaining the ZmMIPP cDNA.
• SEQ ID N ° 7 Internal oligonucleotide olMIPPl.
• SEQ TD No. 8 Internal oligonucleotide olMIPP2.
• SEQ ID No. 10 Oligonucleotide 3'olOSP used as primer 3 'for obtaining the OsMIPP cDNA.
• SEQ ID NO: 1 Oligonucleotide 5'olOSP1 used as primer 5 'for obtaining the OsMIPP cDNA.
• SEQ TD N ° 12 oligonucleotide olMIPP9 used as a 3 'primer for obtaining the cDNA coding for a wheat MIPP.
• SEQ ID N ° 13 oligonucleotide olMIPPlO used as a 3 'primer for obtaining cDNA encoding a wheat MIPP.
• SEQ TD N ° 14 Nucleic sequence of EST 3'TaMIPP.
• SEQ ID N ° 17 Nucleic sequence of cDNA isolated from corn
• SEQ ID N ° 18 Amino acid sequence of a corn MTPP protein (ZmMIPP)
• EXAMPLE 1 Isolation of the cDNA clones coding for a plant MIPP protein.
• the plant MLPP sequences can be obtained by carrying out a bioanalysis study from sequences coding for animal MTPPs, presented by Caffrey et al. (1999). These sequences thus make it possible to define ho ologies with cDNA sequences referenced, for example, in databases specific to corn, wheat and rice. By identifying ESTs specific to the homologous sequences of said animal sequences, oligonucleotides can then be defined and used as primers to isolate by PCR these cDNAs of interest from cDNA or cDNA libraries.
• Immature leaves 40 seeds B73 are put in potting soil and placed in a greenhouse. For the harvest of immature leaves, we await the stage where 7-8 leaves are visible, that is to say approximately 3-4 weeks in the greenhouse. The immature leaves are still in the cornet. A sample at 3 weeks and 4 weeks was taken. Immature leaves were also collected at 2 weeks after germination. Obtaining type II calluses
• the ear samples are taken when the immature embryos have reached a size of 1.5 mm to 2 mm, that is to say 10 days after fertilization.
• the harvested ears are stripped of their husks and their bristles then are disinfected with Domestos® 20% (v / v) for 15 minutes with stirring.
• the ears are rinsed three times with sterile water.
• the upper part of the grain is cut to reveal the albumen, then a light pressure on the grain allows to release the albumen.
• the immature embryo which is still in the grain is extracted and then deposited on the callogenesis medium N6P6 (Salts N6 3.98% (w / v) (Sigma C1416); vitamins N6 5ml / L; L-proline 0.7 % (w / v); casein hydrolyzate 0.1% (w / v); sucrose 20% (w / v); 2,4-D 0.001% (w / v); phytagel 2.5% (w / v), pH 5.8) by orienting it flat side on the agar.
• the embryos are cultured for 15 days in a culture chamber at
• the embryos are separated from their radicle and then subcultured on a new N6P6 medium.
• the proliferating callus is subcultured every 2 to 3 weeks on N6P6 medium (box of 16 calluses).
• the calluses are multiplied in a culture chamber at 26 ° C and in the dark.
• oligonucleotides used as PCR primers for the screening of cDNAs are presented in the appendix.
• the oligonucleotides were determined from the nucleic acid sequences of the ESTs identified in bioanalysis as being highly homologous to the sequences coding for animal MIPPs.
• the oligonucleotides which surround the coding sequence of the ZmMIPP gene have restriction sites in 5 ′: Ncol or NJel and in 3 ′: Bam ⁇ l, this to allow cloning oriented in the bacterial expression vector pET-14b ( ⁇ ovagen).
• the internal oligonucleotides olMTPP1 and olMIPP2 SEQ LD ⁇ ° 7 and 8), which are used to verify the identity of the sequences, do not have any.
• PCR amplification The manipulation was repeated on two different pools of total RNA for the same species and the same tissue.
• the amplification reaction was carried out directly on the first strand of cDNA synthesized from the total RNAs.
• CDNA from immature leaves and calluses allowed amplification of a 1.6 kb fragment.
• a PCR was carried out with the pairs of oligonucleotides 5'olZMP1-olMIPP2 (SEQ LD N ° 5 and 8), and olMIPPl-3'olZMP (SEQ ID N ° 7 and 6), olMTPPl and olMIPP2 being internal oligonucleotides specific for the RHGXRXP consensus sequence, generating respectively an amplification at 0.2 kb and 1.4 kb.
• This result shows the presence of the RHGXRXP site in the 1.6 kb fragment. Therefore, it was cloned into pGEM-T (Promega) and sequenced.
• MGMAAPRAPLPLPQLLLLLNAALLA (SEQ . LD ⁇ ° 15) - a putative retention signal in the endoplasmic reticulum at the C-terminal end of the protein: KTEL
• RNA extraction is carried out using rice calluses.
• Cultivation conditions Induction of rice calluses. Ripe rice grains are stripped of their outer husks, disinfected 1 min in 70% ethanol (v / v) and 30 min in 50% domestos (v / v). The grains are rinsed in sterile distilled water and deposited on the N6P6 medium contained in petri dishes. The boxes are sealed and placed for 21 days in the oven at 28 ° C in the dark. From the scutellum of the embryo, a primary callus develops. Around the primary callus, small embryogenic units are individualized, they are transferred to petri dishes containing the N6P6 medium and incubated for 14 days at 28 ° C and in the dark. During this period, their size will increase. The calluses are then removed for the extraction of RNA.
• the oligonucleotides used were determined from the ESTs identified in bioanalysis as being highly homologous to the sequences coding for animal MTPP.
• the oligonucleotides which should frame the coding sequence of the MIPP gene have restriction sites in 5 ′: Nc ⁇ l or Ndel and in 3 ′: BamHl, this to allow cloning oriented in the bacterial expression vector pET-14b.
• the internal oligonucleotides which are used to verify the identity of the sequences do not have any.
• PCR amplification The manipulation was repeated on two different pools of total RNA for the same species and the same tissue.
• the amplification reaction was carried out directly on the first strand of cDNA synthesized from the total RNAs.
• the amplification reaction with the pairs of oligonucleotides 5O1OSP-3O1OSP (SEQ ID N ° 9 and 10) or 5O1OSP1-3O1OSP (SEQ ID N ° 11 and 10) on the callus cDNAs generates a 1.6 kb fragment.
• MAAPRTPLPLVLLLVSAA SEQ LD n ° 16
• the authors of the invention carried out kinetics of the phytase activity of a fraction of the grain enriched in bran during development at 10, 20, 30 and 40 JAA. This phytase activity increases during grain development. This increase can be explained by an activation of enzymes and / or by an accumulation of enzymes in the bran during grain development.
• RNA extracted from grain bran taken at 10, 20, 30, 40 JAA.
• expression kinetics i.e. Northern blots, with the OsMP probe (nucleotide 105 to nucleotide 1506 - sequence corresponding to 467 amino acids of the C-terminal part of the protein) or the ZmMP probe nucleotide 1 to nucleotide 1572 - (sequence corresponding to 524 amino acids of the protein), were carried out on Total RNA extracted from grain bran taken at 10, 20, 30 and 40 JAA. Furthermore, the cDNAs corresponding to the total RNAs extracted from its sample taken from
• the wheat MIPP cDNA is isolated by RT-PCR or by RACE-PCR.
• the oligonucleotides used for the amplification reactions are determined from the nucleic sequences of the rice and maize MIPP cDNA clones in the areas where the sequence homologies are strongest. It is also possible to use degenerate oligonucleotides, determined from the nucleic acid sequences, in particular that of rice and corn, corresponding to the N and C-terminal regions of plant MIPPs.
• the nucleic sequence of the OsMIPP cDNA was used to search for homologous sequences in EST bases of wheat Triticum aestivum.
• a TSE from wheat cDNA has been retained, it is named 3'TaMIPP (SEQ TD N ° 14).
• the protein sequence deduced from the EST has a strong homology with the 58 amino acids of the C-terminal part of the protein OsMIPP. This EST was used to define two primers olMIPP9 and olMIPPlO (SEQ ID No. 12, SEQ LD No. 13).
• the Ndel restriction site brought by the oligonucleotides 5'olZMPl and 5'olOSPl and the BamHI restriction site, brought by the oligonucleotides 3'olZMPl and 3'olOSPl, frame the CDNA ZmMIPP and OsMIPP. They allow the oriented and phase cloning of cDNAs at the NdeI and BamHI restriction sites of the bacterial expression vector pET-14b (Novagen).
• the vectors obtained pET-OsMTPP and pET-ZrnMTPP were verified by sequencing.
• Protein production is done according to the recommendations of the supplier Novagen. E. coli strain BL21 (D ⁇ 3) pLysS bacteria were used.
• the vector pLysS makes it possible to avoid the expression of the recombinant protein when the production has not been induced and facilitates the lysis of the cells because it carries the gene coding for the lysozyme of phage T7.
• E. coli strain BL21 (D ⁇ 3) pLysS bacteria are transformed with the vectors p ⁇ T-OsMTPP and p ⁇ T-ZmMIPP; these bacterial transformants are used respectively for the production of the proteins OsMIPP and ZmMIPP.
• E. coli strain BL21 (D ⁇ 3) pLysS bacteria are transformed with p ⁇ T-14b; these bacterial transformants serve as a negative production control.
• E. coli strain BL21 (D ⁇ 3) pLysS bacteria are also used as a negative production control.
• Each bacterial transformant is cultured in 5 ml of LB medium (Luria-Bertani medium) in the presence of suitable antibiotics at 37 ° C. with stirring (175 rpm) for 15 hours.
• suitable antibiotics Two antibiotics are used: carbenicillin and chloramphenicol. Resistance to chloramphenicol is provided by the vector pLysS (concentration used: 30 ⁇ g / ml). Resistance to carbenicillin is provided by the vector p ⁇ T-14b (concentration used: 50 ⁇ g / ml).
• the appropriate use of one or both antibiotics makes it possible to select the bacterial transformants which contain the vectors pLysS and / or p ⁇ T-14b.
• a volume of 1 ml of each preculture is used to inoculate a volume of 100 ml of LB medium with the appropriate antibiotics contained in a 500 ml Erlenmeyer flask.
• DOeoonm 0.6
• the 100 ml of LB medium are distributed equitably in two 250 ml Erlenmeyer flasks.
• the first is used for protein production.
• the production is induction by adding 500 ⁇ l of 100 mM IPTG, ie a final concentration of 1 mM.
• the production is carried out for 6 h at 30 ° C. with stirring (175 rpm).
• the second Erlenmeyer flask serves as an uninduced witness. It is incubated for 6 h at 30 ° C. with shaking (175 rpm).
• the bacterial culture is centrifuged at 3000 rpm for 5 min at 4 ° C.
• the bacterial pellet is taken up in a volume of T ⁇ buffer pH 8.0 (10 mM Tris-HCl, ⁇ DTA 1 mM) such that the T volume volume / culture volume ratio is 1/20.
• the bacteria are stored at -80 ° C.
• Bacteria are lysed in two stages: a freezing stage - defrosting (two freezing cycles at -80 ° C / defrosting at 37 ° C), an ultrasound step: 3 times 15 s, 30%, 4 draws / s. Between each cycle, the sample is placed in ice.
• a cocktail of protease inhibitors is added to the bacterial lysate, which contains bacterial proteins and heterologous proteins, in order to preserve them.
• the concentration of total proteins in the extracts is evaluated according to the method of Bradford (1976).
• 2 ⁇ l of bacterial lysate are added to 200 ⁇ l of Coomassie reagent (Bio-Rad protein assay).
• the protein extract is replaced by 2 ⁇ l of TE buffer pH 8.0.
• the absorbances are read at 595 nm with a fluorimeter (Labsystems Genesis N2.00) assisted by the software (Labsystems).
• the amount of protein is determined according to a standard curve made with bovine serum albumin (1 mg / ml).
• the separation gel is 10% polyacrylamide (acrylamide / bisacrylamide,
• the concentration gel is 4% polyacrylamide containing 0.12 M Tris-HCl pH 6.8 and 0.1% SDS (w / v).
• the polymerization of the gel is carried out in the presence of ammonium persulfate 0.05% (w / v) and Temed 0.078% (v / v).
• 40 ⁇ g of protein is used for the SDS-PAGE analysis.
• the proteins are taken up in the deposition buffer composed of 30 mM Tris-HCl, pH 6.8, 12.5% glycerol (v / v), 1% SDS (w / v), 0.005% bromophenol blue.
• the migration buffer is composed of 25 mM Tris-base, 0.2 M glycine and 0.1% SDS (w / v).
• the electrophoresis is carried out at constant voltage 150 volts for 1 hour at room temperature. After migration, the gel is rinsed three times 10 minutes in water, the proteins are stained with a solution of Coomassie blue G250 (Bio-Safe Coomassie, Bio-Rad) for 1 hour, the gel is then rinsed in water, at room temperature.
• heterologous protein SDS-PAGE analysis was performed on crude bacterial extracts.
• the presence of the heterologous protein in the bacterial culture after induction of production is difficult to determine.
• Western blot analysis with an appropriate antibody confirms the presence of the heterologous protein.
• the heterologous protein has the advantage of being synthesized with a peptide at its N-terminal end.
• Antibodies specific for this peptide are commercially available and can be used to demonstrate the heterologous protein. This is in line with the work of Craxton et al. (1997): during production trials in E. coli of an animal MIPP, the protein could only be detected by immunodetection.
• the phytase activity is determined by the dosage of phosphate released by the enzyme from sodium phytate. The measurement of phytase activity is carried out directly on the bacterial lysates. .
• the determination of free phosphate is carried out on the supernatant.
• 750 ⁇ l of supernatant 750 ⁇ l of the following reagent is added: FeSO 0.38 M, H SO 0.16 N / ammonium molybdate 12 mM, H 2 SO 1 N, 1/4, v / v.
• the absorbance is measured at 690 nm.
• the amount of phosphate is determined from a standard curve.
• the phytase activity is expressed in rrmoles of phosphate released per hour, per mg of total protein at 55 ° C.
• the phytase activity was carried out on bacterial extracts.
• a step of purification of the heterologous protein from the crude bacterial extracts is nevertheless preferable to improve the interpretation of the results. This is easily possible due to the presence of the peptide at the N-terminal end of the heterologous protein which makes it possible to purify it by affinity chromatography.
• the pCsNMV promoter (Nerdaguer et al, 1996, 1998) of the cassava rib mosaic virus is used.
• the construct is carried out as follows:
• the 556 bp ClaI-Sacll fragment of the vector pRD 257 contains the promoter pCsVMV. This fragment is cloned into the vector p3214 (FIG. 5) digested with ClaI and EcoRI.
• the vector obtained is named vector pBIOS 366. It contains the promoter pCsVMN and the terminator ter nos.
• the OsMIPP cDNA was cloned into the vector pG ⁇ M-T (Promega) giving the vector pBIOS 367.
• the vector pBIOS 367 is digested with NotI generating a fragment of 1563 bp containing the complete OsMIPP cDNA.
• This fragment is cloned into the vector pBIOS 366 digested with Pstl.
• the vector pBIOS 368 which contains the cassette: pCsVMV promoter, OsMIPP cDNA, ter nos terminator.
• the vector pBIOS 368 is digested with Xho, which allows the cassette to be released: pCsVMV promoter, OsMIPP cDNA, ter nos terminator. It is cloned at the Xhol site of the binary vector pBIOS 273.
• the vector pBIOS 273 contains the T-DNA of Arabidopsis thaliana in which are found the promoter pActl and the first intron of the rice Actl gene, the coding sequence of the bar gene and the terminator nos.
• the vector obtained is the vector pBIOS 369.
• the vector pBIOS 369 comprises firstly the T-DNA in which the cassette for expression of the OsMIPP gene is found, inserted between the promoter pCsVMV and the terminator Nos, the cassette for expression of the selection gene, the Bar gene inserted between the pActl promoter and the first intron of the rice Actl gene and the nos terminator.
• pBIOS 369 also contains the gene for resistance to spectinomycin and an origin of functional replication in E. coli
• the construct allowing the expression of OsMIPP in the cytoplasm of the albumen cells is carried out as follows:
• vector pBIOS 370 10 - Digestion of the vector pBIOS 370 with the enzyme Xhol generates a fragment of 2287 bp which is cloned at the Xhol site of the binary vector pBIOS 273.
• the vector obtained is named pBIOS 271.
• the vector pBIOS 371 comprises on the one hand the T-DNA in which the expression cassette for the OsMLPP gene is located, inserted between the promoter pHMWG and the terminator Nos, the expression cassette for the selection gene, the Bar gene inserted between the pActl promoter and the first intron of the rice Actl gene and the nos terminator. On the other hand, pBIOS 371 also contains the gene for resistance to spectinomycin and an origin of replication in E. coli.
• the promoter pHMWG of the gene coding for a high molecular weight wheat glutenin (HMWG: High Molecular Weight Glutenin) is used.
• ZmMIPP in the vector pG ⁇ M-T easy generates a 1600 bp fragment containing the complete ZmMIPP cDNA. This fragment is cloned at the EcoRI and BamHI sites of the vector P3214 (FIG. 5) between the promoter pHMWG and the terminator ter NOS. The vector obtained is called vector pBIOS 421 and described in FIG. 6.
• the pHMWG-ZmMTPP-JT vector comprises, on the one hand, the T-DNA in which the expression cassette for the ZmMIPP gene is found, inserted between the promoter pHMWG and the ter NOS terminator, the expression cassette for the selection gene , the Bar gene (White et al, 1990) inserted between the pActlet promoter the first Actl rice intron and the ter NOS terminator.
• the selection gene expression cassette is also inserted between the transposable elements Ac / Ds (Lechelt et al, 1989) which allow excision of the selection cassette after the action of a transposase.
• the vector pHMWG-ZmMIPP-JT also contains the gene for resistance to spectinomycin and an origin of replication in E. coli.
• the pHyPRP promoter of the gene coding for a hybrid protein rich in corn proline (HyPRP: Hybrid Proline Rich Protein) is used.
• the construct allowing the expression of ZmMIPP in the cells of the embryo is carried out as follows:
• vector pBIOS 413 Digestion by the enzymes Apal and NdeI of the vector pBIOS 413 (fragment pHyPRP in vector pBluescript) generates a fragment of 2162 bp containing the promoter of the pHyPRP gene described by Josè- ⁇ stanyol et al (1992). This fragment is cloned at the Apal and NJel sites of the vector pBIOS 421 in front of the complete ZmMIPP cDNA and the ter NOS terminator. The vector obtained is called vector pBIOS 422 described in FIG. 7.
• the pHyPRP-ZmMLPP-JT vector comprises, on the one hand, the T-DNA in which the expression cassette for the ZmMIPP gene is found, inserted between the promoter pHyPRP and the ter NOS terminator, the expression cassette for the selection gene , the Bar gene inserted between the pActlet promoter the first Actl rice intron and the ter NOS terminator.
• the expression cassette the selection gene is also inserted between the transposable elements Ac / Ds which allow excision of the selection cassette after the action of a transposase.
• the vector pHyPRP-ZmMIPP-JT also contains the gene for resistance to spectinomycin and an origin of replication in E. coli.
• embryogenic callus cells or type II calluses The genetic transformation of corn by bombardment of particles takes place on embryogenic callus cells or type II calluses. These calluses are obtained from immature embryos of genotype HiU or A188 x B73 according to the method described by Armstrong (1994). The calluses thus obtained can be multiplied and maintained by successive subcultures every 15 days on the initiation medium. Seedlings are regenerated from these calluses by modification of the hormonal and osmotic balance of the cells according to the method described by Vain et al. (1989).
• the transfer of the genes of interest and selection by bombardment of particles in type II calluses is done according to the following protocol. Four hours before the bombardment, fragments of type II calluses with an area of 10 to 20 mm 2 are placed in the center of a petri dish containing the initiation medium supplemented with 0.2 M mannitol and sorbitol 0 , 2 M, 16 fragments per box.
• the vectors carrying the genes of interest and selection are prepared using the CONCERT system according to the supplier's instructions (GIBCO BRL). They are then precipitated on tungsten particles (M10) following the protocol described by Klein (1987). The particles thus coated are projected towards the target cells using a particle gun. Optimization of the bombardment conditions may depend on the type of aircraft used and is one of the techniques normally mastered by those skilled in the art.
• the boxes are sealed using fresh Scello® and placed in the dark at 27 ° C.
• the calluses are transferred to an initiation medium supplemented with a selective agent 24 h after the bombardment.
• the calluses are maintained on this medium for 3 months, the medium is changed every
• Calluses whose growth is not inhibited by the selective agent are usually and predominantly composed of cells resulting from the division of a cell having integrated in its genetic heritage one or more copies of the selection gene.
• the frequency of obtaining such calluses is approximately 0.8 calluses per bombarded box.
• calluses are identified, individualized, multiplied and then cultivated so as to regenerate seedlings. In order to avoid any interference with untransformed cells, all of these operations are carried out on culture media containing the selective agent.
• the plants thus regenerated are acclimatized and then cultivated in a greenhouse where they can be crossed or self-fertilized.
• the superbinary vector used for the transformation of corn comes from the homologous recombination between two vectors: the vector pBIOS 371 and the vector pSB1.
• the vector pBIOS 371 constructed as above (example 3.2), comprises on the one hand the T-DNA in which the cassette for expression of the OsMIPP gene is found, inserted between the promoter pHMWG and the terminator Nos, the cassette of expression of the selection gene, the Bar gene.
• pBIOS 371 also contains the gene for resistance to spectinomycin and an origin of replication in E. coli.
• the vector pSB1 contains the virB, virC and virG genes of the plasmid pTiBo542 present in Agrobacterium strain A281 (ATCC 37349), the gene for resistance to tetracycline, an origin of functional replication in E. coli and Agrobacterium.
• the vectors pSB1 and pBIOS 371 have a homologous region which allows them to recombine and generate the superbinary vector pRec 371. The homologous recombination between the two vectors takes place in Agrobacterium.
• the vector pBIOS 371 is introduced into Agrobacterium strain LBA4404 containing the vector pSBl by electroporation using the device C ⁇ LL PORATOR Voltage Booster (GIBCO BRL) according to the method described by Mattanovitch et al. (1989) and the protocol given by the supplier (Life Technologies, USA).
• the agrobacteria containing the superbinary vector pRec 371 are selected on YT medium in the presence of CaC 2, rifampicin and spectinomycin.
• the rifampicin resistance gene is carried by the bacterial chromosome. Resistance to spectinomycin, carried by the vector pBIOS 371 (origin of functional replication in E. coli), can only be expressed after homologous recombination with the vector pSB1 (origin of functional replication in Agrobacterium and E. coli).
• the superbinary vector pRec 371 has the T-DNA in which the expression cassettes for the Bar gene and for the sequence OsMIPPs are found (under the control of the pHMWG promoter for specific tissue expression), origins of both functional replication in E. coli and Agrobacterium, the tetracycline and spectinomycin resistance genes, and the virulence genes virB, virC and virG of the plasmid pTiBo542.
• the transformed calluses are selected on a culture medium containing the selective agent and the bacteriostatic agent, cefotaxime. Calluses of type I are obtained, from these, seedlings will be regenerated on a culture medium containing the selective agent and the bacteriostatic agent, cefotaxime. The regenerated seedlings are then transferred to a development medium containing the selective agent.
• the plants obtained are acclimatized to the phytotron, then cultivated in a greenhouse where they can be crossed or self-fertilized.
• the nature and concentration of the selective agent may vary depending on the gene used.
• the selective agents which can be used are generally active compounds of certain herbicides (Basta®, Round up®) or certain antibiotics (Hygromycin, Kanamycin,).
• the Bar gene from Streptomyces hygroscopicus codes for a phosphinothricin acetyl transferase (PAT) which inactivates phosphinotricin - the active molecule of the herbicide Basta® - by acetylation.
• PAT phosphinothricin acetyl transferase
• the coding sequence of the Bar gene is under the control of a regulatory region allowing a strong and constitutive expression in plant cells.
• a regulatory region can advantageously consist of the promoter and the first intron of the rice Actin 1 gene (Me ⁇ lroy et al, 1991).
• the chimeric gene composed of the promoter and the first intron of the rice Actin 1 gene and of the Bar gene, is cloned in the plasmid pDM 302 allowing its multiplication in E. coli (Cao et a, 1992). Culture media intended for the selection of transformed cells are supplemented with phosphinothricin 2 mg / 1.
• cotransformation For the introduction of the OsMIPP constructs which should lead to an ectopic expression of the proteins derived from the OsMIPP genes, a technique called cotransformation can advantageously be used.
• the two plasmids (the Bar gene plasmid and the OsMIPP gene plasmid) are coprecipitated on the tungsten particles, the total amount of DNA precipitated on the particles remaining identical to that in the protocol. standard (5 ⁇ g of DNA for 2.5 mg of particles), each plasmid will represent approximately half of the total DNA used.
• the OsMIPP gene can thus advantageously be followed in the progenies thanks to the resistance to the herbicide which is closely associated with it.
• the superbinary vector pRec 371 has the T-DNA in which the expression cassettes of the Bar and OsMIPP genes are found, origins of functional replication both in E. coli and Agrobacterium, the tetracycline and spectinomycin resistance genes, and the virulence genes virB, virC and virG of the plasmid pTiBo542.
• Immature embryos are co-cultivated with A. tumefaciens strain LBA 4404, containing the superbinary vector pRec 371 for 5 minutes, then placed on an initiation medium for callogenesis for 5 days in the dark and at 25 ° C.
• the selection of transformed type I calluses and the regeneration of seedlings is carried out on a medium containing phosphinotricin 5 to 10 mg / 1 and a bacteriostatic agent, cefotaxime. Seedling development takes place on a medium containing only phosphinotricin. Calluses and plants which have integrated into their genome, the T-DNA which contains the OsMIPP gene and the Bar gene, can be followed in the progenies thanks to the resistance to the herbicide.
• the final objective of the production of corn transformed with the OsMIPP gene being the obtaining of an increased phytase activity measurements of enzymatic activity are planned.
• the dosage of phytase activity is carried out according to the method described above in Example 2, paragraph 2.5.
• the phytase activity is measured on the protein extracts of different organs of transgenic corn and non-transgenic corn: leaves, seeds, young seedlings in the process of germination (5 or 6 days of germination).
• the extraction of total proteins from these tissues is carried out according to the method described below.
• the tissues, taken and frozen at -80 ° C, are ground in the form of powder in liquid nitrogen.
• 100 mg of ground vegetable powder is transferred to 1 ml of extraction buffer (100 mM sodium acetate, pH 4.8, 2 mM CaCl 2 , 1 mM DTT, cocktail of protease inhibitors 0.5 to 1 mM (Roche)).
• the samples are homogenized for 1 hour at 4 ° C and the extracts are centrifuged at 8000 rpm for 20 min at 4 ° C. The supernatant containing the total proteins is used for the measurement of phytase activity.
• the production of processed rice can be envisaged according to two methods: via
• Example 371 described in Example 3, are used for the transformation of rice via Agrobacterium tumefaciens.
• the tissues used for gene transfer were prepared according to the method described in patent EP 0674 715 B1. These are type M calluses which are used for bombardment. They can be obtained from immature embryos taken from immature wheat grains and placed on an MS medium described by Murashige and Skoog (1962) containing maltose.
• the vectors used for the transformation of wheat are purified on a Cesium gradient and then concentrated to 1 mg / ml in TE buffer pH 8.0 (10 mM Tris-HCl; 1 mM EDTA) (Sambrook et al, 1989).
• Gold particles of 1 ⁇ are coated with DNA to be transferred according to the method of Daines (1990).
• the particles are taken up in absolute ethanol, 60 mg of particles per ml of ethanol.
• a volume of 35 ⁇ l of this suspension is transferred to a 1.5 ml micro-centrifuge tube, the particles are recovered by centrifugation at 14,000 g for
• the bombardment of the cells was carried out as described in patent application EP 0 674 715.
• the Petri dish containing the explants to be bombarded is placed on the platform in the center of the opening from which the gold particles coated with DNA or microprojectiles are projected.
• the microprojectiles are placed on a support above the explants.
• the chamber is closed and sealed, the chamber is vacuumed and the helium tank filled with an appropriate amount of gas.
• the bombardment is triggered allowing the projection of microprojectiles onto the explants.
• the explants are bombarded twice with microprojectiles. After bombardment, the calluses are kept in the dark for 15 hours and then placed on the MS medium for 15 days.
• the selection of transformed calluses is carried out in the MS medium containing the selective agent, methotrexate for 4 months.
• calluses are transferred to MS medium containing 2,4-D in the dark.
• the calluses are transferred to an MS medium containing hormones (auxins and gibberellins) in the light for 15 days, this step allows the induction of stems.
• the explants are then transferred to a hormone-free MS medium in order to allow induction of the roots.
• the seedlings are acclimatized in phytotron before being grown in the greenhouse.

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