FR2785911A1 - GENE ENCODING COLZA ACYLTRANSFERASE, AND USES THEREOF - Google Patents

Abstract

The invention concerns a nucleic acid sequence coding for a lysophosphatidic acyltransferase of oil seed rape. Said sequence is useful in particular for transforming plants, so as to regulate the fatty acid content of the triglycerides produced by them.

Description

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GENE ENCODING COLZA ACYLTRANSFERASE,
AND ITS USES
The invention relates to the identification and cloning of a gene coding for an acyltransferase, and to its uses.

 Glycerolipids (glycolipids, phospholipids, and triacylglycerides) constitute in plants the major part of the lipids. Their common precursor is sn-1,2-diacylglycerol-3-phosphate or phosphatidic acid (PA), resulting from the esterification of the sn-1 and sn-2 positions of glycerol-3-phosphate (G3P) by fatty acids. . Sn-glycerol-3-phosphate acyltransferase (GPAT) (E.C. 3.1.15) catalyzes the acylation of the sn-1 position of G3P to form sn-1-acylglycerol-3-phosphate or lysophosphatidic acid ( LPA). The LPA is then used as substrate with 1-acyl-sn-glycerol-3-phosphate acyltransferase or lysophosphatidic acyltransferase (LPAAT) (E.C.2.3.1.51) which acylates the sn-2 position of glycerol. In the synthesis of triacylglycerides, which constitute the bulk of the storage lipids, is involved a third enzyme, sn-1,2-diacylglycerol acyltransferase or diacylglycerol acyltransferase (DAGAT), which catalyzes the acylation of the sn-3 position.

 Plant lipids are currently used in a wide variety of fields, from food to the chemical industry, and it is desirable to have lipid-producing plants specifically adapted to the intended use. For this purpose, one seeks in particular to modify the fatty acid composition of glycerolipids, and in particular triacylglycerides.

 For example, in the case of rapeseed (Brassica napus), plants with the lowest possible erucic acid content are used for cooking oil. On the other hand, a high level of erucic acid is sought in plants producing oils intended for industrial use.

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 The composition of the glycerolipids in fatty acids depends essentially on the one hand on the quantitative and qualitative distribution of the fatty acids produced by the plant, and on the other hand on the substrate specificity of the acyltransferases with respect to these fatty acids. To control this composition, it has been proposed to act separately or jointly on these two factors, by intervening: at the level of the biosynthesis of fatty acids, to promote or otherwise inhibit the production of one or more specific fatty acids, and optionally to induce the synthesis of new fatty acids; at the acylation level of G3P, to modify its specificity in the desired direction.

 In the case of rapeseed, the most erucic acid varieties currently available produce an oil in which erucic acid represents at most 50 to 60% of the total fatty acids. The analysis of the triacylglycerides of seeds from these varieties has shown that this acid is present almost exclusively at positions sn-1 and sn-3; this selective distribution was attributed to the substrate specificity of rapeseed LPAAT, which would exclude very long-chain (> C20) fatty acids; this limits the erucic acid content of rapeseed triacylglycerides to a theoretical maximum of 66% of total fatty acids.

 In order to overcome this limitation, the LPAAT gene of Limnanthes alba capable of incorporating erucic acid at the sn-2 position was expressed in the seeds of a high acid rapeseed variety. erucic. However, although erucic acid incorporation at the sn-2 position was indeed observed in the triacylglycerides of the seeds of this transgenic rapeseed, this incorporation remained low; furthermore, the total amount of erucic acid incorporated in these triacylglycerides was not greater than

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 greater than that of the non-transformed control plants [LASSNER et al., Plant Physiol. 109: 1389-1394 (1995)].

 This result could be explained, apart from the possibility of a limiting production of erucic acid, by a low specificity of the exogenous LPPAT of Limnanthes, which, in addition to erucic acid, could also incorporate oleic acid. and also by the existence of competition between exogenous LPAAT activity and endogenous LPAAT rapeseed activity, which would need to be inhibited to increase erucic acid incorporation.

 Until now, there was little information on the enzyme or enzymes responsible for LPAAT activity in rapeseed. LPAAT are indeed membrane enzymes, difficult to purify in active form.

 With the exception of coconut LPAAT [KNUTZON et al., Plant Physiol. 109: 999-1006, (1995)], which has been purified from the membranes of albumen, and whose gene was subsequently isolated by screening a cDNA library, the plant LPAATs already identified have for the most have been characterized by molecular genetic techniques. This is corn LPAAT [BROWN et al., Plant Mol. Biol., 26: 211-223, (1994)], LPAAT of Limnanthes [BROWN et al., Plant Mol. Biol., 29: 267-278 ,. (1995); HANKE et al., Eur. J. Biochem.

232: 806-810, (1995)].

 In order to improve the control of acylation at the sn-2 position, the inventors have undertaken to characterize the enzyme (s) involved in the LPAAT activity in rapeseed.

 They have thus succeeded in isolating a DNA sequence of Brassica napus coding for a functional plastidial LPAAT; this LPAAT will be hereinafter BAT2 (Brassica Acyl-Transferase 2).

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 A nucleic acid sequence comprising the BAT2 coding sequence is represented in the attached sequence listing under the number SEQ ID NO: 1, and the deduced polypeptide sequence is represented under the number SEQ ID NO: 2.

 2 ATG codons that may constitute potential translation initiation sites are present on the sequence SEQ ID NO: 1; the 344 amino acid polypeptide starting at the 16-position methionine residue of the sequence SEQ ID NO: 2 is sufficient for the LPAAT activity.

 The analysis of the amino acid sequence of BAT2 by the pSORT software suggests the presence of a signal sequence comprised in the 95 N-terminal amino acids of the sequence SEQ ID NO: 2. This signal sequence is involved in the addressing of the LPPAT BAT2 in the plastid membrane.

 The sequence of the mature active protein is included in the 279 C-terminal amino acids.

 The comparison, using the BLASTX2 software [GISH et al., Nat. Genet., 31: 266-272, (1994)] between the peptide sequence of BAT2, and the previously known LPAAT peptide sequences, shows a very low homology (at most 20% identity) when the comparison is made on the whole sequence.

 In certain regions of the sequence, greater homology is observed. FIG. 1 represents the alignment of the sequence 187-302 of BAT2 with the LPAAT sequences presenting the strongest homology. The most important scores are observed with: the SLC1 gene product of S. cerevisiae (P33333) [NAGIAC et al. J. Biol. Chem., 268: 22145-22163, (1993)]: 32% identity and 51% equivalence, on an alignment of 204 amino acids; microsomal LPAAT of meadowfoam seeds (Q42870) [HANKE et al., Eur. J. Biochem., 232: 806-810,

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 (1995); LASSNER et al., Plant Physiol. 109: 1389-1394, (1995); BROWN et al., Plant Mol. Biol., 29: 267-278, (1995)]: 30% identity and 54% equivalence, on an alignment of 182 amino acids; coconut endosperm LPAAT (Q42670) [KNUTZON et al., Plant Physiol. 109: 999-1006, (1995)]: 31% identity and 47% equivalence, on an alignment of 229 amino acids; the hypothetical LPAAT of Synechocystis, (P74498): 30% identity and 55% equivalence, on an alignment of 143 amino acids; the E. coli plsC protein (P26647): 31% identity and 50% equivalence, on an alignment of 115 amino acids.

 The subject of the present invention is a nucleic acid fragment comprising: a) a sequence encoding a plant LPAAT whose peptide sequence exhibits at least 20%, preferably at least 30% and advantageously at least 50 to 95% identity with the sequence SEQ ID NO: 2; and / or b) a sequence complementary to coding sequence a) above.

 According to a preferred embodiment of the present invention, said coding sequence codes for the polypeptide of sequence SEQ ID NO: 2.

The invention also encompasses fragments of greater than 20 bp, and preferably greater than 30 bp, of a coding sequence as defined above, or capable of specifically hybridizing under stringent conditions, with said sequence. This includes in particular the fragments of any sequence coding for the polypeptide SEQ ID NO: 2, or of its complementary sequence, with the exception of fragments constituted by an oligonucleotide coding for one of the following peptide sequences (code 1 letter):
FPEGTRS;

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PFKKGA; which are common to LPAATs of previously known sequences, or complementary fragments of said oligonucleotide.

 Nucleic acid fragments according to the invention may in particular be used as primers and / or probes, for detecting and cloning sequences encoding plastid, rapeseed or other plant LPAATs, as well as sequences coding for LPAATs of rapeseed or other species, in particular Cruciferae, expressed in cell compartments other than plastids, in particular the endoplasmic reticulum.

 The Southern blot and RFLP analyzes using the 32 P-labeled BAT2 cDNA as stringent hybridization probe show the presence in the rapeseed genome as well as in the A genome. thaliana, of at least 2 homologous copies of the BAT2 gene, and make it possible to suppose that this gene belongs to a multigene family comprising 4 members.

 The present invention also relates to: the recombinant vectors resulting from the insertion of at least one nucleic acid fragment according to the invention into a suitable vector; These are expression vectors in which the nucleic acid fragment according to the invention is inserted under transcriptional control of regulatory sequences (such as promoter and / or terminator) functional in a host cell. in which it is desired to express said fragment.

 host cells, prokaryotic or eukaryotic, and multicellular organisms, in particular plant cells and plants, transformed with at least one nucleic acid fragment according to the invention.

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 The invention also encompasses recombinant LPAAT or recombinant LPAAT fragments, resulting from the expression in a host cell, of the sequence encoding said LPAAT or said fragment carried by a nucleic acid fragment according to the invention. . The recombinant LPAAT according to the invention, or fragments thereof, may for example be used to produce anti-LPAAT antibodies, allowing the screening of cDNA expression libraries as part of the detection and cloning of other LPAATs. .

 Nucleic acid fragments according to the invention may advantageously be used, in orientation or antisense, to produce transgenic plants, in particular from rapeseed or other oleaginous plants, in order to regulate the LPAAT activity in the plant. thus transformed, and act on the fatty acid composition of lipids, and in particular triacylglycerides, produced by this plant.

 The present invention also encompasses the transgenic plants produced in this way.

 These plants can be obtained by the usual techniques, known in themselves, of plant transgenesis. Depending on the intended use, a nucleic acid sequence according to the invention may be placed under the control of an inducible promoter or a constitutive promoter, a ubiquitous promoter, or a tissue-specific promoter. These plants may also contain other transgenes, preferably transgenes derived from genes involved in lipid biosynthesis.

 In particular, it is possible to obtain: transgenic plants, expressing at least one sequence according to the invention coding for a functional LPAAT, in place of or in addition to one or more sequences coding for endogenous LPAATs;

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 transgenic plants expressing at least one sequence according to the invention in antisense orientation, in order to inhibit the expression of homologous endogenous LPAATs, and thus to promote the activity of other LPAATs, of endogenous or exogenous origin.

 For example: to produce high erucic acid transgene rapeseed rapeseed can be co-transformed with, on the one hand, a DNA sequence coding for an LPAAT preferably incorporating erucic acid at the sn-2 position, such as LPAAT of Limnanthes alba [LASSNER et al., (1995), publication cited above], and secondly a nucleic acid sequence according to the invention in antisense orientation, in order to inhibit, at least in part, the production of endogenous LPAAT competing with the activity of the exogenous LPAAT of Limnanthes; to increase the overall seed content of triglycerides, the rapeseed can be converted with a DNA sequence according to the invention, coding for a plastid LPAAT deleted from its plastid addressing sequence; to increase the content of saturated fatty acids, in particular palmitic acid, the rapeseed can be cotransformed with a DNA sequence according to the invention, coding for a plastid LPAAT deleted from its addressing sequence in chloroplasts, and with one or more ACP-thioesterase genes preferentially using palmitoyl-ACP as substrates.

 The present invention will be better understood using the additional description which follows, which refers to non-limiting examples illustrating the identification and cloning of a gene encoding LPAAT BAT2 Brassica napus.

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EXAMPLE 1 ISOLATION AND CHARACTERIZATION OF THE cDNA OF A COLZA LPAAT
To search for the presence of genes encoding LPAAT, a cDNA library of immature rapeseed embryos was screened by heterologous complementation of the plsC gene mutation of E. coli strain JC201. Coli [COLEMAN, J. Biol. Chem., 265: 17215-17221, (1990)]. This point mutation confers JC201 mutants a thermosensitive phenotype, due to the high temperature inactivation of LPAAT encoded by the plsC gene. These mutants grow well at 30 C, hardly at 37 C and not at all at 42-44 C.

 For screening, a phagemid library derived from 2 x 10 6 clones taken from a cDNA library of immature rapeseed embryos cloned into the lambda vector ZAPII (STRATAGEN), was constructed using the ExAssist kit (STRATAGEN).

 The bacteria are transformed by electroporation with these phagemids and then cultured on LB agar, in the presence of ampicillin and IPTG (isopropyl-ss-D-galactothiopyranoside). Bacteria growing at 42 C are selected. The plasmid DNA of the clones capable of growing at 42 ° C. was analyzed by PCR, to determine the size of the insert. After 3 cycles of transformation followed by selection, about 85% of the clones contain an insert of about 1.2 kb. Sequencing the ends of the inserts of 4 of these clones shows that they are identical. One of these clones, called pBAT2, was completely sequenced.

EXAMPLE 2: BAT2 NUCLEOTIDE SEQUENCE AND PEPTIDE SEQUENCE DEDUCTED

 The cDNA of clone pBAT2 comprises a sequence of 1155 bp followed by a tail poly (A) 18 residues. This sequence comprises a single open reading phase, corresponding to a polypeptide of 351 amino acids, which represents a fusion protein, comprising 344 acids.

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 amines of the pBAT2 sequence, and a portion of the β-galactosidase sequence of the cloning vector. The sequence which is represented on the list of sequences in the appendix under the number SEQ ID NO: 1, furthermore comprises a part of the genomic sequence (nucleotides 1 to 79 of the sequence SEQ ID NO: 1) situated upstream of the sequence cDNA of pBAT2.

 2 ATG codons that may constitute potential translation initiation sites have been located on the sequence SEQ ID NO: 1; if the first of them (position 58 of the sequence SEQ ID NO: 1) is used, the translation product of the sequence SEQ ID NO: 1 is a polypeptide of 359 amino acids, whose theoretical molecular weight and pI are respectively 39.6 kDa and about 9.8; polypeptide is represented on the list of sequences in the appendix under the number SEQ ID NO: 2. If the initiation of the translation takes place at the 2nd ATG codon (position 103 of the sequence SEQ ID NO: 1), the product translation is a polypeptide of 344 amino acids, whose theoretical molecular weight is approximately 37.9 kDa.

 The analysis of the amino acid sequence of BAT2 by the pSORT software suggests the presence of a signal peptide of about 80 to 95 residues. This potential signal sequence is rich in serine, alanine, valine, and basic amino acids, which is characteristic of the membrane addressing sequences of chloroplasts.

 The analysis of the polypeptide sequence also indicates the presence of two potential transmembrane domains situated respectively between amino acids 124 to 140 and 219 to 235.

 The consensus sequences of the LPAATs (FPEGTRS and PFKKGA) are located respectively at positions 273-279 and 286-291 of the sequence SEQ ID NO: 2; a sequence corresponding to the consensus sequence NHXXXXD, which is

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 preserved in all membrane acyltransferases known hitherto, is located at positions 202-208 of the sequence SEQ ID NO: 2.

EXAMPLE 3: ENZYMATIC ACTIVITY OF THE PROTEIN CODED BY THE cDNA INSERT OF pBAT2
To verify that the protein encoded by the pBAT2 insert actually had LPAAT activity, the ability of this protein to incorporate oleic acid or palmitic acid at the sn-2 position of LPA was tested.

 E. coli strain JC201 transformed with pBAT2, and as controls, the strain E. coli JC201 untransformed, or transformed with the vector (pBSK) lacking the cDNA insert of BAT2, are cultured at 30 C up to an optical density of 0.5.

 After induction with IPTG, and culture for 3 h at 30 ° C., the bacteria are lysed and fractionated and the LPAAT specific activity is measured, on the crude membrane extracts, in the presence of oleoyl-CoA (1-14C) or palmitoyl-CoA (1-14C), according to the protocol of CAO et al.

[Plant Physiol., 9: 1199-1206, (1990)].

The results are shown in Table I below, which indicates the specific activity, in pmol of phosphatidic acid formed / mg protein / hour.

<Tb>
<Tb>

TABLE <SEP> I
<tb> CULTURE <SEP> SUBSTRATE
<tb> Oleoyl-CoA <SEP> (1-14C) <SEP> Palmitoyl-CoA (1-14C)
<tb> JC201 <SEP> 1.86 <SEP> 3.74
<tb> JC201 + pBSK <SEP> 1.65 <SEP> 1.59
<tb> JC201 + pBAT2 <SEP> 5.8 <SEP> 11.06
<Tb>

The membrane extracts of the culture transformed with pBAT2 exhibit an LPAAT activity greater than that of the membrane extracts obtained from the non-transformed culture, or transformed with the pBSK vector, which shows that the LPAAT activity is effectively restored by the product. of translation of the pBAT2 insert.

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EXAMPLE 4 CELL LOCATION OF BAT2
The plastid localization suggested by sequence analysis of BAT2 was verified by testing the ability of isolated pea chloroplasts to import BAT2.

 For this purpose, the cDNA of clone pBAT2 was transcribed in vitro using T3 RNA polymerase; transcribed is translated into acellular system of wheat germ, in the presence of 35S-labeled methionine. This produces a translation product of about 40 kDa. This product is incubated with chloroplasts isolated from peas.

After incubation, the chloroplasts are protease treated and fractionated according to the protocol described by BROCK et al. [Plant Mol. Biol. 23 (4), 717, (1993)], and the various fractions analyzed by electrophoresis to look for the 35S-labeled product.

 The results of this analysis show that the translation product of BAT2 is imported into pea chloroplasts, and cleaved into a 32 kDa protein which is essentially located in the membrane fraction, and an 8 kDa signal peptide.

 These results confirm that the BAT2 protein is well synthesized with a signal peptide whose role is to import the protein into the chloroplast membrane. The precursor has an apparent mass of about 40 kDa and the signal peptide of about 8 kDa.

EXAMPLE 5: LOCALIZATION OF THE EXPRESSION OF BAT2 GENE
The expression of the BAT2 gene has been studied in different organs of B. napus and A. thaliana.

 The study was carried out by Northern blotting, using a probe corresponding to the coding sequence of BAT2, on total RNAs of stems, roots, leaves, flowers, seeds under development [28 DAYS (days after pollination), and dry seeds.

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 In each of the B. napus and A. thaliana tissues tested, hybridization of the probe was observed with a transcript of approximately 1.3 kb in B. napus, and with a transcript of approximately 1 kb in A thaliana. The intensity of the hybridization signal is similar in all tissues, including non-photosynthetic tissues containing plasts other than chloroplasts. The observation of hybridization in the mature seeds, further indicates that the messenger of BAT2 remains stable during the maturation of the seed.

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SEQUENCE LIST NUMBER OF SEQUENCES: (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: base pairs (B) TYPE: (C) NUMBER OF STRANDS: double (D) ) CONFIGURATION: linear (ix) CHARACTERISTIC: (A) NAME / KEY: (B) LOCATION: 58..1134 (xi) DESCRIPTION OF THE SEQUENCE: ID NO: 1: TAAAAACAGC AGAGAAAAGA GTCAAGAGAT AAAAGCAATG AAGATGGAGA GATAAGC 57 ATG AGC AAA TCT CAC GGA CGA TGT TTT AGC TCG CGA GAT TCC GCC ATG 105 Met Ser Lys Ser His Gly Arg Cys Phe Ser Ser Arg Asp Ser Ser Ala Met
1 5 10 15 GAT GTC GCT TCT GCT CGG GGG GTC TCC TCA CAT CCT CCA TAT TAT AGC 153 Asp Val Ala Ser Ala Arg Gly Val Ser Ser His Pro Pro Tyr Tyr Ser
20 25 30 AAA CCC ATT TGT TCA TCA CAG TCA TCG TTG ATT CG CGC ATC ATC AGT 201 Lys Pro Ile Cys Ser Ser Gln Ser Ser Leu Ile Arg Ile Pro Ile Ser
35 40 45 AAA GGA TGT TGC TT GCT CGT TCT TCG AAC TTG ATT ACT TCC CTT CAT 249 Lys Gly Cys Cys Phe Ala Arg Ser Ser Asn Leu Ile Thr Ser Leu His
50 55 60 GCT GCT TCG AGA GGG GTG ACA AGG CGT ACT AGT GGT GTA CAA TGG TGT 297 Ala Ala Ser Arg Gly Val Thr Arg Arg Ser Ser Gly Val Gln Trp Cys
65 70 75 80 TAC CGT TCT ATT AGA TT GAC CCT TTC AAA GTT AAT GAT AAG AAC TCA 345 Tyr Arg Ser Ile Arg Phe Asp Pro Phe Lys Val Asn Asp Lys Asn Ser
85 90 95 AGA ACT GTG ACT GTG AGA TCG GAT CTT TCA GGA GCT GCA ACC CCT GAA 393 Arg Thr Val Thr Val Arg Ser Asp Leu Ser Gly Ala Ala Thr Pro Glu
100 105 110 TCT ACT TAT CCA GAA CCA GAG AT ATT TTG AGC TCA AGA CTC AGA GGG 441 Ser Thr Tyr Pro Glu Pro Glu Ile Lys Leu Ser Ser Arg Leu Arg Gly
115 120 125 ATA TGC TTC TGT CTC GTT GCT GGC ATC TCC GCC ATT GTT CTC ATC GTC 489 Ile Cys Phe Cys Leu Val Ala Gly Ile Ser Ala Ile Val Leu Ile Val
130 135 140 CTG ATG ATC ATT GGC CAT CCC TTC GTC CTT CTA TTT GAT CGT TAC AGG 537 Leu Met Island Ile Gly His Pro Phe Val Leu Leu Phe Asp Arg Tyr Arg 145 150 155 160

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AGA AAG TTC CAT CAC TTC ATT GCT AAG CTT TGG GCT TCC ATA AGC ATC 585 Arg Lys Phe His His Phe Ile Ala Lys Leu Trp Ala Ser Ile Ser Ile
165 170 175 TAC CCG TTT TAC AAA ACA GAC ATC CAA GGT TTG GAG AAT CTG CCG TCG 633 Tire Pro Phe Tyr Lys Thr Asp Gln Island Gly Leu Glu Asn Leu Pro Ser
180 185 190 TCA GAC ACT CCT TGT GTA TAC GTT TCG AAC CAC CAA AGC TTT CTG GAT 681 Ser Asp Thr Pro Cys Val Tyr Val Ser Asn His Gln Ser Phe Leu Asp
195 200 205 ATA TAC ACA CTT CTC AGC CTT GGC CAA AGC TAT AAG TTC ATC AGC AAG 729 Tyr Island Leu Leu Leu Ser Leu Gly Gln Tyr Ser Lys Phe Ile Ser Lys
210 215 220 ACA GGG ATA TTC GTT ATT CCT GTC ATC GGT TGG GCT ATG TCC ATG ATG 777 Thr Gly Ile Phe Val Ile Pro Val Ile Gly Trp Ala Met Ser Met Met 225 230 235 240 GGG GTT GTT CCC TTG AAG AGG ATG GAC CCA AGA AGC CAA GTG GAT TGC 825 Val Val Pro Gly Val Lol Arg Met Arg Pro Arg Arg Asp Gln Val Asp Cys
245 250 255 TTA AAA CGC TGC ATG GAA CTA GTG AAG GGA AAG GCT TCC GTC TT TTC 873 Leu Lys Arg Cys Met Glu Leu Val Lys Lys Gly Ala Ser Val Phe Phe
260 265 270 TTC CCA GAG GGA AGG AGG AGT AGGAG GAT GGT GGT TTA GGT AGC TTC AAG 921 Phe Glu Pro Gly Thr Arg Ser Gly Arg Gly Arg Lly Gly Pro Phe Lys
275 280 285 AAA GGG GCT TT ACG ATA GCA GCT AAG ACA GGA GTT CCA GTG GTG CCA 969 Lys Gly Ala Phe Thr Ala Ala Lys Thr Gly Val Pro Val Val Pro
290 295 300 ATA ACG CTG ATG GGA ACA GGG AAG ATC ATG CCG ACG GGT AGT GAA GGT 1017 Thr Ile Leu Met Gly Thr Gly Lys Ile Met Pro Thr Gly Ser Glu Gly 305 310 315 320 ATA CTG AAT CAT GGG GAT GTG AGA GTG ATC ATC CAC AGC CCG ATA TAT 1065 Island Leu Asn His Gly Asp Val Arg Val Ile Island His Lys Pro Island Tyr
325 330 335 GGA AGC AAA GCT GAT GTT CTT TGC GAA GAG GCG AGA AAC AAG ATA GCT 1113 Gly Ser Lys Ala Asp Val Leu Cys Glu Glu Ala Arg Asn Lily Ala Island
340 345 350 GAA TCT ATG AAT CTC TTG AGT TGAAACGTTT GTTTTTTAAG CAGTGTCTCT 1164 Glu Ser Met Asn Leu Leu Ser
355 ATGAACAATG AGAAGGCTAA ACCATTTTTA CATGTCAGTT TTATTGTTTA AAATAAAATT 1224 TAGGCTTTTC AAAAAAAAAA AAAAAAAA 1252 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: amino acids (B) TYPE: amino acid (D) CONFIGURATION : linear

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(ii) TYPE OF MOLECULE: (xi) DESCRIPTION OF THE SEQUENCE: ID NO: 2: Met Ser Lys Ser His Gly Arg Ser Cys Phe Ser Ser Arg Asp Ser Ala Met
1 5 10 15 Asp Val Ala Ser Ala Arg Ser Gly Val Ser Ser Pro Pro Tyr Tyr Ser
20 25 30 Lys Pro Ile Cys Ser Ser Gin Ser Ser Leu Ile Arg Ile Pro Ile Ser
35 40 45 Lys Gly Cys Cys Phe Ala Arg Ser Ser Asn Leu Thr Island Ser Ser Leu His
50 55 60 Ala Ala Ser Arg Gly Val Thr Arg Arg Ser Ser Gly Val Gin Trp Cys
65 70 75 80 Tyr Arg Ser Isle Arg Phe Asp Pro Phe Lys Val Asn Asp Lys Asn Ser
85 90 95 Arg Thr Val Thr Val Arg Ser Asp Leu Ser Gly Ala Ala Thr Pro Glu
100 105 110 Ser Thr Tyr Pro Glu Pro Glu Ile Lys Leu Ser Ser Arg Leu Arg Gly
115 120 125 Ile Cys Phe Cys Leu Val Ala Gly Ile Ser Ala Ile Val Leu Ile Val
130 135 140 Leu Met Ile Ile Gly His Pro Phe Val Leu Leu Phe Asp Arg Tyr Arg 145 150 155 160 Arg Lys Phe His His Phe Ile Ala Lys Leu Trp Ala Ser Ile Ser Ile
165 170 175 Tyr Pro Phe Tyr Lys Asp Asp Gin Island Gly Leu Glu Asn Leu Pro Ser
180 185 190 Ser Asp Thr Pro Cys Val Tyr Ser Val Asn His Gin Ser Phe Leu Asp
195 200 205 Tire Island Tyr Leu Leu Leu Ser Leu Gly Gin Ser Tyr Lys Phe Ile Ser Lys
210 215 220 Thr Gly Ile Phe Val Ile Val Val Ile Gly Trp Ala Met Ser Met Met 225 230 235 240 Gly Val Val Pro Leu Lys Arg Met Asp Pro Arg Ser Gin Val Asp Cys
245 250 255 Leu Lys Arg Cys Met Glu Leu Val Lys Lilies Gly Ala Ser Val Phe Phe
260 265 270 Phe Pro Glu Gly Thr Arg Ser Lys Asp Gly Arg Leu Gly Pro Phe Lys
275 280 285 Lys Gly Ala Phe Thr Ala Ala Lys Thr Island Gly Val Val Val Val Pro Pro
290 295 300 Thr Island Leu Met Gly Thr Gly Lys Met Pro Thr Gly Ser Glu Gly 305 310 315 320

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Ile Leu Asn His Gly Asp Val Val Val Ile Ile His Lys Pro Ile Tire
325 330 335 Gly Ser Lys Ala Asp Val Leu Cys Glu Glu Ala Arg Asn Lily Ala Island
340 345 350 Glu Ser Asn Met Leu Leu Ser Ser
355

Claims (12)

1) Nucleic acid fragment comprising: a) a sequence encoding a plant LPAAT whose peptide sequence has at least 20% identity with the sequence SEQ ID NO: 2; and / or b) a sequence complementary to coding sequence a) above.  2) nucleic acid fragment according to claim 1, characterized in that said coding sequence codes for the polypeptide of sequence SEQ ID NO: 2.  3) Nucleic acid fragment of more than 20 bp, capable of hybridizing specifically under stringent conditions with a coding sequence as defined in any one of claims 1 or 2, with the exception of fragments consisting of an oligonucleotide coding for 1 one of the following peptide sequences: FPEGTRS; PFKKGA; or by its complementary.  4) Recombinant vector containing a nucleic acid fragment according to any one of claims 1 to 3.  5) Cell transformed with at least one nucleic acid fragment according to any one of claims 1 to 3.  6) transformed cell according to claim 5, characterized in that it is a plant cell.  7) transgenic plant transformed with at least one nucleic acid fragment according to any one of claims 1 to 3.  8) Use of a nucleic acid fragment according to any one of claims 1 to 3 for regulating LPAAT activity in a plant.  9) Use according to claim 8, characterized in that said plant is rapeseed. <Desc / Clms Page number 19>  10) Use according to any one of claims 8 or 9, characterized in that said nucleic acid fragment is used in antisense orientation.  11) Use according to claim 10, characterized in that said nucleic acid fragment is used to express a functional LPAAT.  12) Use according to claim 11, characterized in that said LPAAT is deleted from its addressing sequence in the plastids.