EP0716707A1 - Promoteurs - Google Patents

Promoteurs

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Publication number
EP0716707A1
EP0716707A1 EP94926238A EP94926238A EP0716707A1 EP 0716707 A1 EP0716707 A1 EP 0716707A1 EP 94926238 A EP94926238 A EP 94926238A EP 94926238 A EP94926238 A EP 94926238A EP 0716707 A1 EP0716707 A1 EP 0716707A1
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EP
European Patent Office
Prior art keywords
regulatory elements
untranslated region
possibly
dsm
promoter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94926238A
Other languages
German (de)
English (en)
Inventor
Reinhard TÖPFER
Jaqueline Bautor
Hendrick Bothmann
Elke Filsak
Christa HÖRICKE-GRANDPIERRE
Barbara Klein
Norbert Martini
Andreas MÜLLER
Wolfgang Schulte
Michael Voetz
Josef Walek
Jeff Schell
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften eV filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Publication of EP0716707A1 publication Critical patent/EP0716707A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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)
    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/01009—Enoyl-[acyl-carrier-protein] reductase (NADH) (1.3.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/01—Oxidoreductases acting on the CH-CH group of donors (1.3) with NAD+ or NADP+ as acceptor (1.3.1)
    • C12Y103/0101—Enoyl-[acyl-carrier-protein] reductase (NADPH, B-specific)(1.3.1.10)

Definitions

  • the present invention relates to promoters and possibly or other regulatory elements in the 5'-untranslated region of genes which code for proteins of de novo fatty acid biosynthesis, and the alleles and derivatives thereof.
  • the fatty acid and triacylglyceride biosynthesis can be regarded as separate biosynthetic pathways due to the compartmentalization, but with regard to the end product, as a biosynthetic pathway.
  • the de novo biosynthesis of fatty acids takes place in the plastids and is catalyzed by three enzymes or enzyme systems, these are (1) the acetyl-CoA carboxylase (ACCase), (2) the fatty acid synthase (FAS) and (3) the acyl - [ACP] thioesterase (TE).
  • ACCase acetyl-CoA carboxylase
  • FAS fatty acid synthase
  • TE acyl - [ACP] thioesterase
  • the end products of this reaction sequence in most organisms are either palmitic, stearic and, after desaturation, oleic acid.
  • the fatty acid synthase consists of an enzyme complex of dissociable individual enzymes with the individual enzymes acetyl- [ACP] -transacylase, malonyl- [ACP] -transacylase, ß-ketoacyl- [ACP] synthases I, II, III, ß-ketoacyl- [ACP] -Reductase,
  • HMW- Plant Cell 1, pages 569-578 (1989), V. Colot et al, Mol.Gen.Genet. 216, pages 81-90 (1989)
  • Bäumlein et. al The Plant Journal, 2, pages 233 to 239, 1992
  • Zein- Zein-
  • Lectin- Lectin- (P. Guerche et al, Mol.Gen.Genet. Pages 306-314 (1990))
  • USP- H.
  • the object of the present invention is primarily to provide promoters with which foreign genes can be expressed in plants with high efficiency or can be specifically expressed in certain tissues or cell types.
  • the object is achieved with the promoters and, if appropriate, or other regulatory elements in the 5 'untranslated region according to claim 1.
  • the invention relates to promoters and possibly or other regulatory elements in the 5 'untranslated region of genes which code for proteins of de novo fatty acid biosynthesis, and the alleles and derivatives of these promoters.
  • the invention further relates to genomic clones which contain a gene which codes for a protein of de novo fatty acid biosynthesis, and the alleles and derivatives of this gene, the gene comprising the promoter, the structural gene or at least parts thereof and other regulatory sequences.
  • the invention also relates to a process for the production of transgenic plants, plant parts and plant products, in which a promoter and possibly or other regulatory elements in the 5 'untranslated region of genes which code for proteins of de novo fatty acid biosynthesis with a desired one expressing gene is coupled and then transferred in a suitable vehicle.
  • the invention also relates to plants, parts of plants and plant products which have been produced by the above process.
  • the invention relates to the use of a promoter and, if appropriate, or other regulatory elements in the 5'-untranslated region of genes which code for proteins of de novo fatty acid biosynthesis, for the production of plants with modified gene expression.
  • Figure 1 shows the restriction maps of the genomic clones
  • Figure 2 shows the restriction map of the genomic clone
  • Figure 3 shows the restriction maps of the genomic clones
  • Figure 4 shows the restriction maps of the genomic clones
  • Figure 5 shows the restriction maps of the genomic clones
  • Figure 6 shows the restriction maps of the genomic clones
  • Figure 7 shows a Northern blot with RNAs from different
  • Plant tissues hybridized with a gene-specific probe for CITEgl
  • Figure 8 shows a Northern blot with RNAs from different
  • Plant tissues hybridized with the C1TE13 cDNA corresponding to the gene from ClTEg7;
  • Figure 9 shows a Northern blot with RNAs from different
  • Plant tissues hybridized with a specific ACP cDNA probe.
  • allelic variants and derivatives of the invention are also within the scope of the invention Promoters and other regulatory elements are detected in the 5 'untranslated area, provided that these modified units have the desired activity.
  • the allelic variants and derivatives include, for example, deletions, substitutions, insertions, inversions or additions of the promoters according to the invention. The same applies to the genomic clones that contain the above-mentioned units.
  • rapeseed Brass napus
  • lanceolate leaved quiver flower or hump flower Cincosa lanceolata
  • genes from fatty acid biosynthesis was carried out with specific hybridization probes. Based on polyA + RNA, these were prepared from approximately two to three weeks old, immature seeds from Brassica napus or from approximately two weeks old embryos from Cuphea lanceolata, after a cDNA first strand synthesis by means of polymerase chain reaction (PCR). The synthetic oligonucleotide primers required for this will be described later.
  • the promoters of the gene families of the acetyl-CoA carboxylase (ACCase) of the acyl carrier protein (ACP), the ß-ketoacyl [ACP] synthasel (KASI), the ß-ketoacyl [ACP] reductase ( KR), the enoyl [ACP] reductase (ER) and the acyl [ACP] thioesterase (TE) are isolated.
  • the promoters according to the invention and other regulatory units in the 5 'untranslated region are described as follows.
  • the DNA sequences are taken into account, which are in front of the start codon, i.e. before the start of translation of the respective structural genes.
  • the specified transcription start points describe only one of several transcription start points. It is generally known that several transcription start points can be determined for one gene.
  • the DNA sequence of the promoter region and parts of the DNA sequence of the structural gene of the ACC gene from the genomic clone BnACCaseg3 are shown as SEQ ID NO: 1 in the sequence listing. This sequence comprises 2505 bp of the promoter region and approximately 700 bp of the structural gene.
  • the start codon "ATG" of the ACCase gene is located at position 2506 of the DNA sequence. The start codon in position 2506 with the neighboring nucleotides shows good agreement with the plant consensus motif for translation initiation areas (G. Heidecker and J. Messing, Annual Review Plant Physiology 3_7, pages 439-466 (1986))
  • Position 2456 If the adenine in position 2456 is assumed to be the first nucleotide of an mRNA based on 5'-RACE experiments, then there is a possible TATA box at a suitable distance of 36 nucleotides (positions 2416 to 2422). A CAAT box (positions 2283 to 2286) is further 130 nucleotides away. Thus the most important elements of a promoter and 5 'untranslated region are present.
  • the DNA fragments described below from the two other genomic clones BnACCaseglO and BnACCasegl also contain the promoter region of the ACC gene and parts of the structural gene.
  • a 4450 bp DNA fragment from the BnACCl clone contains the before the start of translation with the start codon "ATG" (position 4089) Promoter sequence and other 5 'regulatory units of the ACC gene with 4088 bp.
  • the protein-coding region of the ACC extends to position 4421.
  • the 5 'untranslated region is interrupted by an intron and, based on 5'-RACE data, begins at position 3367 (start of transcription). This intron ranges from position 3493 to 4078.
  • the promoter sequence of the ACC gene is located in a 3350 bp DNA fragment of the BnACCaseglO clone (SEQ ID NO: 3 in the sequence listing) before the start of translation with the start codon at position 2611 protein-coding sequence of the ACC goes up to position 3341 and is interrupted by an untranslated region (intron), positions 2909 to 3000.
  • genomic clone BnACCasegl and the genomic clone BnACCaseglO were deposited under number DSM 8480 and DSM 8481 on August 27, 1993 at the DSM-German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig.
  • DNA sequence analysis of a 1200 bp DNA fragment of the subcloned DNA fragment from ClACPgl has shown that the promoter region of the ACP gene is contained in this fragment. It is located in front of the protein-coding sequence of the ACP gene, which begins at position 1160 with the start codon "ATG".
  • the TATA signal typical of the promoter region is located at positions 1051 to 1054.
  • the GUS gene Under the control of the ACP promoter from the genomic clone ClACPgl from Cuphea lanceolata, the GUS gene was expressed in oilseed rape. Measurements of the ⁇ -glucuronidase expression of a fusion of the ACP promoter from ClACPgl as a 1.2 kb Pstl-PvuII part. Fragment with the GUS gene showed promoter activity in the examined leaf, flower and immature seed tissues. Northern blot analyzes (see FIG. 9) showed that the corresponding ACP gene in Cuphea lanceolata is expressed in leaf, flower, root and preferably in embryonic tissue.
  • a 2450 bp partial sequence from the genomic clone ClKASIg4 (SEQ ID NO: 6 in the sequence listing) encompasses the promoter region at 1962 bp, before the putative "ATG” at position 1963 putative intron ranges from positions 2053 to 2242. The beginning of the mature protein is at position 2402.
  • Parts of the promoter region are contained in a 1350 bp partial sequence from the genomic clone ClKASIgl3 (SEQ ID NO: 8 in the sequence listing). It covers 472 bp up to the start codon "ATG" (positions 473 to 475). The start of the mature protein is at position 1075. In front of it is the transit peptide, which is interrupted by an intron that cannot be precisely defined.
  • An 1141 bp fragment from the genomic clone ClKASIgl9 (SEQ ID NO: 9 in the sequence listing) contains the promoter region in a 520 bp fragment.
  • the putative ATG is located at position 521.
  • the start of the mature protein is at position 956.
  • the transit peptide which is interrupted by an intron which cannot be precisely defined, is located in front of this.
  • the 3750 bp partial sequence from the genomic clone ClKASIg20 contains the promoter region as a 3067 bp DNA fragment.
  • the putative "ATG” is at position 3068.
  • the mature protein begins at position 3661. In front of it is the transit peptide, which is interrupted by an intron that cannot be precisely defined.
  • the sequencing of the clone ClKASIg ⁇ showed that the nucleotide sequence of the structural gene to ClKASIg4 has an identity of 98%.
  • the derived protein to ClKASIg4 also has an identity of 98%.
  • the ClKASIg2 promoter region is also very similar to ClKASIg4.
  • the close relationship between ClKASIg2, ClKASIg4 and ClKASIg ⁇ among themselves is not only clear at the sequence level, but also at the restriction map level. These three genes could be alleles, each with significant sequence differences in the promoter region.
  • the specific PCR product listed in Table 1 was initially used to isolate cDNAs from Cuphea lanceolata.
  • two types of cDNAs, C1KR10 and C1KR27, which are also different from one another at the amino acid level, were identified (B. Klein et al., Plant Lipids, pages 156-59 (1992)).
  • the cDNAs were 1295 and 1276 bp in size (with polyA residue) and each code for an open reading frame of 326 and 320 amino acids including the transit peptides of 69 and 63 amino acids.
  • the proteins derived from the DNA sequence have a molecular weight of 27 kDa.
  • cDNA C1KR27 from nucleotide 210 as a fusion with the glutathione-S-transferase in vector pGEX-KG resulted in the purification of a fusion protein of 53 kDa.
  • This fusion protein was used for enzyme determination for ß-ketoacyl- [ACP] reductase with acetoacetyl-CoA.
  • the measured values showed that the cDNA C1KR27 codes for a NADPH-dependent KR, which can be specifically inhibited by phenylglyoxal (Klein, supra).
  • the promoter region of the ClKRg2 gene is located on a 1570 bp DNA fragment (SEQ ID NO: 11 in the sequence listing). It comprises 1511 bp with a non-translatable area.
  • the protein coding sequence of the KR gene begins with the start codon "ATG" from position 1512.
  • the TATA signal is located at positions 1412 to 1429, the putative transcription start at position 1445 (see above).
  • the promoter region of the ClKRg3 gene is located on a 926 bp DNA fragment (SEQ ID NO: 12 in the sequence listing). At 915 bp, it encompasses the area in front of the start codon "ATG" at position 916.
  • the region of the TATA box is at positions 827 to 838, the putative transcription start is at position 864 (see above).
  • the complete gene is contained in ClKRgl2. It was sequenced in double strands and the exon and intron regions were determined.
  • the promoter region of this gene is located on a 1450 bp DNA fragment (SEQ ID NO: 13 in the sequence listing). It is in a range of 1420 bp before the start codon "ATG" at position 1421.
  • the range of the TATA box extends from positions 1327 to 1343 and the putative transcription start is at position 1369 (see above).
  • the promoter regions of the three KR genes show TATA box motifs which correspond to the consensus sequence for plants according to Joshi (1987), supra, TCACTATATATAG: ClKRg2 is correct in l ⁇ positions (items 1412-1429), ClKRgl2 in 16 positions (item . 1327-1343) and ClKRg3 in 12 positions (pos. 827-836).
  • the translation initiation sequence also shows high homo- on the known consensus sequence motifs (Kozak (1984), supra, Joshi (1987), supra, Lütcke et al (1987), sup ⁇ ra).
  • the promoters of the ClKRgl2 and ClKRg3 genes show very high agreement with one another, with the exception of an approximately 500 bp insertion in the ClKRgl2 promoter. This insertion has numerous "inverted repeats" with unknown function.
  • a cDNA, CLER18 is 1533 bp in length and encodes a 391 amino acid protein including 75 amino acids for a transit peptide.
  • the mature protein has a calculated molecular weight of 33.4 kDa and shows 83.3% identical amino acids with the ER from Brassica napus.
  • the cDNA C1ER7 from nucleotide 297 was fused with the glutathione S-transferase, expressed in E.
  • genomic ER clones Five genomic ER clones were isolated from a ⁇ genomic bank of Cuphea lanceolata DNA using the PCR product as a probe. 5 shows the restriction maps of the genomic clones ClERg ⁇ (12.5 kb), ClERg7 (14.4 kb), ClERg9 (16.4 kb), ClERglO (12 kb) and ClERg20 (11.8 kb). The black bars show the one hybridizing with the probe Area, the white bars the DNA sections sequenced for the promoter area.
  • the gene ClERg9 could be assigned to type B by hybridization with specific oligonucleotides that go back to cDNA C1ER8. Sequencing of the hybridizing Sall fragment of the ClERg5 gene revealed differences in the deduced amino acid sequence from the two identified classes of enoyl [ACP] reductases and thus represents the third class of E genes, type C.
  • the genomic structure of the coding region for the mature protein from ClERg5 was identified.
  • the mature protein has 11 exons.
  • a 1800 bp partial sequence of the gene from ClERg5 (SEQ ID NO: 14 in the sequence listing) shows parts of the promoter with regulatory units other than 1763 bp DNA sequence.
  • the CAAT box (1335 to 1338) and the TATA box (1362 to 1367) are located in this area.
  • the start of transcription is at position 1415, based on 5'RACE.
  • An intron in the non-coding 5 'area is located at positions 1560 to 1741. Translation begins with the start codon "ATG" at position 1764.
  • Fusions of the promoter region with the GUS gene showed marked activity in the examined tissues (leaf and flower) of transgenic oilseed rape plants.
  • Fusions of the promoter region with the GUS gene showed marked activity in the examined tissues (leaf and flower) of transgenic oilseed rape plants.
  • An approximately 870 bp DNA fragment from ClERg9 contains, as an approximately 690 bp DNA segment, other regulatory elements in the 5 'untranslated region.
  • the transcription start is assumed at position 1 after 5'RACE.
  • An incomplete intron in the untranslated area extends to position 329 (SEQ ID NO: 16b in the sequence listing).
  • the translated area begins with the start codon "ATG" at position 367 (SEQ ID NO: 16b in the sequence listing).
  • a non-sequenced area of approximately 160 bp is between SEQ ID NO: 16a and 16b.
  • Parts of the promoter and other regulatory elements are located on an approx. 2800 bp DNA fragment from ClERglO (SEQ ID NO: 17a and 17b in the sequence listing). This region comprises approximately 2709 bp and contains an intron in the 5 'untranslated region at positions 251 to 448 (SEQ ID NO: 17b in the sequence listing).
  • the translation start begins with the start codon "ATG" at position 472 (SEQ ID NO: 17b in the sequence listing).
  • a non-sequenced area of approximately 78 bp is between SEQ ID NO: 17a and 17b.
  • a part of the promoter and other regulatory elements are contained in an approx. 1060 bp DNA fragment from ClERg20 (SEQ ID NO: 18a and 18b in the sequence listing).
  • This area comprises approx. 912 bp and contains the CAAT box (items 159 to 162) (SEQ ID NO: 18a in the sequence listing) and the TATA box (positions 211 to 215) (SEQ ID NO: 18a in the sequence listing) - an intron at positions 309 (SEQ ID NO: 18a) to 567 (SEQ ID NO: 18b).
  • Translation begins with "ATG" at position 598 (SEQ ID NO: 18b in the sequence listing).
  • a short, non-sequenced range of approximately 5 bp lies between SEQ ID NO: 17a and 18b.
  • the genomic clone ClERg7 under the number DSM 8489, the genomic clone ClERg9 under the number DSM 8490, the genomic clone ClERglO under the number DSM 8491 and the genomic clone ClERg20 under the number DSM 8492 at the DSM-German Collection by microorganisms and cell cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig.
  • Corresponding cDNAs from maturing embryos from Cuphea lanceolata were used with the help of the PCR product listed in Table 1.
  • One of the cDNAs obtained, C1TE13 has a length of 1404 bp and codes for a protein with 414 amino acids including a transit peptide with 111 amino acids. The molecular weight of the mature protein is 34 kDa.
  • other but incomplete cDNAs were isolated.
  • One of these cDNAs, C1TE5 which lacks 34 amino acids of the transit peptide, has been included in the comparison of derived sequences of mature proteins from previously known plant TEs.
  • the C1TE5 also shows a greater similarity to medium-chain-specific TEs than to long-chain-specific TEs.
  • Figure 6 shows restriction maps of the genomic clones CITEgl, ClTEg4, ClTEg7 and ClTEgl6.
  • the black bars show the areas hybridizing with the probe, the white bars mark the DNA sections sequenced for the promoter area.
  • the clones presented contain the full gene of acyl [ACP] thioesterase.
  • a 3350 bp partial sequence of the gene with the promoter region from CITEgl6 shows the promoter region with other regulatory elements as a DNA sequence with 3290 bp.
  • the areas of the CAAT box and the TATA box are at positions 2914 to 2918 and 3035 to 3038, respectively.
  • the transcription start is probably at position 3068 (see above). Exon and intron areas are located at positions 3068 to 3107 (exon I), 310 ⁇ to 3280 (intron I) and 3281 to 3350 (exon II, incomplete).
  • the legumin box can be recognized at positions 3120 to 3132.
  • the translation begins at position 3291 with the start codon "ATG".
  • An 1850 bp partial sequence of the gene from CITEgl (SEQ ID NO: 19 in the sequence listing) comprises the promoter and other 5 'regulatory units of the TE gene in the untranslated region as a DNA sequence with 1796 bp.
  • the CAAT box and the TATA box are located in the promoter area at positions 1428 to 1432 and 1553 to 1556.
  • the mapped transcription start is at position 1585. This is followed by exon and intron areas at positions 1585 to 1629 (exon I ), 1630 to 1786 (intron I) and 1787 to 1850 (exon II, incomplete).
  • the legumin box is located at position 1642 to 1657.
  • the translation start begins with the start codon "ATG" at position 1797.
  • a 2750 bp partial sequence of the gene from ClTEg4 contains the promoter and other 5 'regulatory units in the untranslated region of the TE gene as a DNA sequence with 2636 bp.
  • An exon (exon I) ends at position 2193 and an intron (intron I) and another exon (exon II, incomplete) are located at positions 2194 to 2626 and 2627 to 2750.
  • the translation start begins with the start codon "ATG" at position 2637.
  • An 850 bp partial sequence of the gene from ClTEg7 shows the promoter and other 5 'regulatory units in the untranslated region of the TE gene as a DNA sequence with 782 bp. Exon and intron regions are at positions 143 to 190 (exon I, possibly incomplete), 191 to 772 (intron I) and 773 to 850 (exon II, incomplete).
  • the translation start begins with the start codon "ATG" at position 783.
  • the promoters of the genes from the clones CITEgl and CITEgl6 according to the invention are therefore suitable, for example, for the directed expression of chimeric genes in embryo-specific plant tissue, and the promoters of the genes from the clones ClTEg4 and ClTEg7 according to the invention, for example, for an extraordinarily strong expression of chimeric genes in flowers.
  • the genomic clone ClTEg4 under the number DSM 8493 and the genomic clone ClTEg7 under the number DSM 8494 was deposited with the DSM-German Collection of Microorganisms and Cell Cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig.
  • the two other genomic clones CITEgl and ClTEgl6 were plasmids in which parts of these genomic clones are present under the numbers DSM 8477 (pNBM99-TEgl) and DSM 8478 (pNBM99-TEgl6) on August 27, 1993 at the DSM-Deutsche Sammlung von Microorganisms and cell cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig.
  • clonable 5 'regulatory DNA fragments can be produced from the genomic clones, which, in combination with any desired gene, specifically induce their expression in any plants.
  • the following Table 2 shows examples of clonable fragments from the investigated genomic clones with the possible fusions. Table 2
  • the cloning of regulatory important parts of the promo- tors and other regulatory elements, for example the introns in the 5 'untranslated region, can be used to construct chimeric promoter / expression units.
  • the promoters according to the invention can be transferred with any desired gene to form chimeric genes in a suitable vehicle in plants with the formation of transgenic plants by genetic engineering.
  • the genes in question can be expressed constitutively or induced.
  • the induced expression can be development-specific, externally induced (biotic / abiotic) or cell type-specific.
  • Such genes include, in particular, selectable marker genes for the transformation of plants, resistance genes (herbicide resistance, pathogen resistance), regulatory genes and genes which are used for the seed-specific expression of genes of fatty acid metabolism, carbohydrate metabolism, amino acid metabolism, secundum metabolism, such as for example polyhydroxybutyrate. Synthesis are responsible.
  • Suitable gene transfer vehicles are, for example, binary vectors of the pPCV 002 series (Konz and Schell, Mol.Gen.Genet. 204. Pages 383-396 (1986)) and vectors of the pRT series for direct DNA transfer (Töpfer et al, Methods in Enzymology ed. R. Wu, Academic Press Inc., New York 217, pages 66-78 (1993)), and viral vectors.
  • the expression of foreign genes in transgenic plants can thus be controlled with the promoters according to the invention. This means that either the gene expression can be significantly increased or inhibited (by endogenous genes) or a targeted expression can be caused in certain tissues of plants.
  • the examples serve to explain the invention.
  • Restriction endonucleases and nucleic acid modifying or synthesizing enzymes were developed by Boehringer Mannheim GmbH (Mannheim), GIBCO-BRL (Eggenstein), New England Biolabs GmbH (Schwalbach), Perkin Elmer Cetus (Norwalk, USA), Pharmacia Biotech GmbH (Freiburg) and Stratagene GmbH ( Heidelberg) related.
  • kits A number of methods for cleaning, analysis or synthesis are facilitated and accelerated by prefabricated or compiled materials with specific application protocols, so-called “kits”. The following list provides an overview of the kits used, which were used according to the manufacturer's protocols:
  • Hybond N ® membrane filters and Amersham Buchler GmbH & Co. KG were used for Southern and Northern blots as well as for the screening of cDNA and genomic DNA banks.
  • X-Omat X-ray films from Kodak (Rockland, USA) for autoradiographs, Sephadex G 50 columns and NAP 25 columns from Pharmacia Biotech GmbH (Freiburg) for the cleaning of radioactively labeled hybridization probes and for cleaning synthetic oligonucleotides, Dynabeads ® 01igo (dT) 25 from Dynal (Oslo, Norway) for the polyA * insulation, films type 52 and type 55 for Polaroid flat film cassettes model 545 from Polaroid (Cambridge, USA), Quiagen-tip 100 from Diagen GmbH (Hilden) for DNA isolations and 3MM paper from Whatman (Maidstone, USA) in the screening of cDNA and genomic DNA banks.
  • Plasmid pBluescript ® II SK (-) (. No. 212206 Stratagene), pUC19, pUC18 (C. Yanisch-Perron et al, Gene 4J, pages 103-119 (1985).).
  • PKl ⁇ RD Pridmore, Gene 56, pages 309- 312 (1987)
  • pGEX-KG KL Guan et al, Anal. Biochem. 192, pages 262-267 (1991)
  • a cDNA library from C. lanceolata (wild type) was produced using the cDNA ZAP ® synthesis kit according to the manufacturer's instructions.
  • the starting material for the synthesis of the cDNAs was mRNA from isolated, immature embryos about two to three weeks old.
  • the cDNA library obtained has a size of 9.6 ⁇ 10 5 recombinant phages with a proportion of approximately 50% of clones, the insertions of which exceed 500 bp.
  • the genomic DNA libraries were created with the Lambda FIX ® II vector system from DNA of B. napus (cv AKELA) and C. lanceolata K ⁇ .
  • the size of the rapeseed genomic DNA bank is 7.5 x 10 5 recombinant phages (with Insertions of 15 kb on average) and thus represents 3.6 times the rapeseed genome (the size of the rapeseed genome is 3.1 x 10 6 kb; C. Hallden et al, J. Mol. Vol. 25, pages 318-323 (1987 )).
  • the size of the C is 7.5 x 10 5 recombinant phages (with Insertions of 15 kb on average) and thus represents 3.6 times the rapeseed genome (the size of the rapeseed genome is 3.1 x 10 6 kb; C. Hallden et al, J. Mol. Vol. 25, pages 318-323 (1987 )).
  • lanceolata genomic DNA library is 3.5 ⁇ 10 5 recombinant phages (with insertions of about 15 kb on average) and thus comprises about 17 times the genome of this plant, the genome of which is 3 ⁇ 10 5 kb owns.
  • ⁇ -glucuronidase activity was determined fluorometrically with 4-methylumbelliferylglucuronide or histochemically with 5-bromo-4-chloro-3-indolyl- ⁇ -D-glucuronic acid (X-gluc, Clontech Laboratories, Palo Alto) (RA Jefferson et al., EMBO J 6, pages 3901-3907 (1987)).
  • Acetyl-CoA carboxylase Specific primers for acetyl-CoA carboxylase were compared by comparing different biotin-containing proteins, including the ACCase from chicken and the ACCase (more precisely the biotin carboxylase) from E. coli in the publication by Kondo et al, Proc. Natl.Acad.Sci. 8 . , Pages 9730-9733 (1991) derived from conserved sections of the sequences.
  • oligonucleotide primers are based on the amino acids of the regions 304 to 311 and 383 to 390 based on the amino acid sequence of the rat ACCase (Kondo et. Al, supra).
  • Acyl Carrier Protein Degenerate oligonucleotides for the N-terminus of the C. lanceolata acyl carrier protein were derived from N-terminal amino acid sequence data, kindly provided by F. Spener (Munster) prior to publication (Kopka ⁇ t al, Planta 191, pages 102 -111 (1993)). This amino acid sequence together with the conserved VMGLEEEF motif from acyl carrier proteins (e.g. Souciet and Weil 1992, Kopka et al 1993) were used to synthesize the primers listed in Table 2 (3098 and 3240).
  • ⁇ -ketoacyl [ACP] synthase I A comparison of ⁇ -ketoacyl [ACP] synthase I from barley with that from E. coli shows only a few areas with greater homology (Siggaard-Anderson et al, Proc.Ntl. Acad. Sci .88., Pages 4114-4118 (1991)). A was used to synthesize a specific pair of primers (Table 2) Sequence section located at the N-terminal (item 13 to 21 for primer 2763) and the region around the cysteine binding the inhibitor cerulenin (item 71 to 79 for primer 2762) (Siggaard-Anderson et aj-, supra). The KAS sequence from barley was kindly provided by P. von Wettstein-Knowles prior to publication (Siggaard-Anderson e_t al, supra).
  • ⁇ -Ketoacyl [ACP] reductase A sequence comparison of two typical fragments of the ⁇ -ketoacyl [ACP] reductase from avocado with the sequence of the nodG protein from Rhizobium meliloti in the publication by Sheldon et al, Biochem.J. 271, pages 713-729 (1990) has short areas of high homology between the two proteins.
  • the two oligonucleotide primers 2189 and 2187 were synthesized based on the fragmentary sequences of the ⁇ -ketoacyl- [ACP] reductase from avocado and the homology to nodG given in this publication (Table 2).
  • Enoyl [ACP] reductase To obtain a specific primer pair (Table 2), amino acid sequence sections of the enoyl [ACP] reductase from rapeseed (Kater et al. Plant. Mol. Biol. 37, pages 895-909 (1991)) selected with a relatively little degenerate genetic code. The selected sequences correspond to amino acid positions 101 to 108 (primer 3389) and 153 to 160 (primer 3391). (Table 2)
  • Acyl- [ACP] thioesterase The publication by Voelker et al, Science 257, pages 72-74 (1992) shows the first sequence of a plant acyl- [ACP] thioesterase. Since it is also the sequence of a medium-chain-specific enzyme, some areas of the sequence whose derived DNA sequence is as little as possible degenerate. Oligonucleotide primers derived and synthesized.
  • the primer 3532 (Table 2), which is the amino acids 277 to 264 of the acyl [ACP] thioesterase from Umbellularia California, was found to be suitable for amplification in PCR reactions in connection with primer number 2740 (a modified oligo-dT primer with interfaces for the restriction endonucleases BstBI, BamHI, Hindlll and Sall) a specific hybridization probe.
  • reverse transcriptase (Boehringer Mannheim GmbH) from Avian Myeloblastosis Virus (AMV) was used to carry out a cDNA synthesis at 37 ° C. for 30 minutes.
  • AMV Avian Myeloblastosis Virus
  • the 3'-oligonucleotide primers shown in Table 3 were used for the synthesis of a specific hybridization probe.
  • Ampli Taq ® polymerase (Perkin Elmer Cetus) was dissolved in the same reaction mixture, the PCR reaction with 50 pmol final concentration each primer (3 see Table) is performed.
  • the reactions were carried out under the following conditions: a) Buffer conditions: 10 mM Tris-HCl, pH 8.0; 50 mM KC1; 1.5 mM MgCl 2 ; 0.01% gelatin and 5 mM dNTPs, b) reaction time and temperatures: 3 minutes at 92 ° C for the first denaturation, then 25 to 30 temperature cycles with: 2 minutes at 92 ° C for the denaturation, 2 minutes for the in Table 4 specified temperature for annealing the oligonucleotides and 2.5 minutes at 72 ° C for amplification of the DNA and finally 7 minutes at 72 ° C to achieve a complete synthesis of the last synthesis products.
  • Buffer conditions 10 mM Tris-HCl, pH 8.0; 50 mM KC1; 1.5 mM MgCl 2 ; 0.01% gelatin and 5 mM dNTPs
  • reaction time and temperatures 3 minutes at 92 ° C for the first denaturation, then 25 to 30 temperature cycles with:
  • the PCR products described in Example 1 were used for the isolation of the genomic clones. This was done either directly by using the PCR product as a probe for searching a bank of genomic DNA or via a cDNA as a probe, which was used by using the PCR product as a suitable probe for searching cDNA banks.
  • the genomic clones found were sequenced in a conventional manner and characterized with regard to the promoter regions.

Abstract

L'invention concerne des promoteurs et le cas échéant d'autres éléments régulateurs dans la région 5' non traduite de gènes qui codent des protéines de la biosynthèse de novo d'acides gras, ainsi que les allèles et dérivés de ces promoteurs. Ces promoteurs et le cas échéant d'autres éléments régulateurs dans la région 5' non traduite peuvent par exemple être couplés à des gènes étrangers, afin de former des gènes chimères, et être transmis à des végétaux dans des systèmes vecteurs appropriés.
EP94926238A 1993-09-04 1994-09-05 Promoteurs Withdrawn EP0716707A1 (fr)

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US5910631A (en) * 1993-09-03 1999-06-08 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Middle chain-specific thioesterase genes from Cuphea lanceolata
GB9414622D0 (en) * 1994-07-20 1994-09-07 Zeneca Ltd Beta-ketoacylacp reductase genes from brassica napus
AU4858897A (en) * 1996-10-31 1998-05-22 Ravinder Kumar Jain Flax promoters for manipulating gene expression
FR2768747B1 (fr) * 1997-09-19 2000-12-01 Pasteur Institut Acides nucleiques, cellules recombinantes, et procede de preparation de compositions immunogenes
DE19926456A1 (de) * 1999-06-10 2000-12-14 Norddeutsche Pflanzenzucht Han Verfahren zur Erhöhung des Fettsäuregehalts in Pflanzensamen
DE19950589A1 (de) * 1999-10-20 2001-05-23 Gvs Ges Fuer Erwerb Und Verwer Elongasepromotoren für gewebespezifische Expression von Transgenen in Pflanzen
BR0111081A (pt) * 2000-05-24 2003-04-08 Univ British Columbia Região reguladora de gene que promove transcrição especìfica em raiz e usos da mesma
AU2001274402A1 (en) * 2000-05-24 2001-12-03 The University Of British Columbia Gene regulatory region that promotes early seed-specific transcription
GB0031558D0 (en) 2000-12-22 2001-02-07 Biogemma Uk Ltd Elongase promoters

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US6133506A (en) 2000-10-17
WO1995007357A2 (fr) 1995-03-16
AU695775B2 (en) 1998-08-20

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