CA2150678A1 - Acetyl-coa-carboxylase-gen - Google Patents

Abstract

A DNA sequence is described that codes for acetyl-CoA-carboxylase and the alleles and derivatives of this DNA sequence. The acetyl-CoA-carboxylase gene sequence can be used to achieve herbicide resistances by heterologous expression, for example, in grains relative to gramicidal herbicides or to alter by homologous or heterologous expression the quality and quantity of vegetable oils and fats.

Description

Your reference Our reference Munich 2 Patent application M 7101 January 3 21, 1994 7 zur Forderung der Wissenschaften e.V.
8 [Max-Planck Institute for Scientific Research]
9 37073 Gottingen 12 Acetyl-CoA-Carboxylase Gene 14 The invention concerns a DNA sequence that codes for acetyl-CoA-carboxylase and 15 the alleles and derivatives of this DNA sequence.

17 The enzyme acetyl-CoA-carboxylase (EC 6.4.1.2) represents an important key 18 enzyme in fatty acid metabolism of prokaryotes and eukaryotes. It catalyzes ATP-19 dependent carboxylation of acetyl-CoA to malonyl-CoA in a two-step reaction (A.
W. Alberts and P. R. Vagelos, The Enzymes (Boyer, P. D., editor), Vol. 6, pp. 37-82, 21 3rd edition, Academic Press, New York, 1972) according to the following reaction 22 equations:

24 BCCP + HCO - biotin carboxylase BCCP-COO- + ADP + Pi 26 BCCP-COO- +3 acetyl-CoA transcarboxylase BCCP + malonyl-CoA

28 Acetyl-CoA-carboxylase (ACC) has been investigated biochemically especially in 29 animal systems and E. coli, in which molecular biological studies have also been 30 recently conducted in a wide variety of organs, for example, in the rat (F. Lopez-31 Casillas, D. H. Bai, X. Luo, I. S. Kong, M. S. Hermodson and K. H. Kim, PNAS, 85, 32 pp. 5784-5788 (1988)), the chicken (T. Takai, C. Yokoyama, K. Wada and T.
33 Tanabe, J. Biol. Soc., 263, pp. 2651-2657 (1988)), yeast (W. Al-Feel, S. S. Chirala 34 and S. J. Wakil, PNAS, 89, pp. 4534-4538 (1984)) and E. coli (J.-H. Aliz, DNA, 8, pp. 779-789 (1989); H. Kondo, K. Shiratuchi, T. Yoshimoto, T. Masuda, A.
36 Kitazono, D. Truru, M. Anai, M. Sekiguchi and T. Tanabe, PNAS, 88, pp. 9730-9733 37 (1991); S.-J. Li and J. E. Cronan, Jr., J. Biol. Chem., 267, pp. 855-863 (1992a)).

39 The ACC enzyme in bacteria consists of three different polypeptide chains assembled 40 from three functional units consisting of biotin carboxylase (BC), biotin carboxy 41 carrier protein (BCCP) and carboxyl transferase (CT) (H. G. Wood and R. E. Barden, Annu. Rev. Biochem., 46, pp. 385-413 (1977)). Parts of the amino acid sequence of 2 E. coli ACC enzyme in the region of the biotin domains were identified by M. R.
3 Sutton, R. R. Fall, A. M. Nervi, A. W. Alberts, P. R. Vagelos and R. A. Bradshaw, J.

4 Biol. Chem., 252, pp. 3934-3940 (1977)). The genes for BCCP and BC from E. coli (J.-H. Alix, supra), as well as for CT (S.-J. Li and J. E. Conan, supra) were recently 6 described. The molecular weights of these proteins derived from the nucleic acid 7 sequence are 17 kD for BCCP, 49 kD for BC and 35 kD for the~ subunit of CT or 8 33 kD for the,~subunit of CT.

The three functional units or domains just mentioned are combined in ~nim~l~, yeast 11 and plants in a polypeptide (D. G. Hardie and P. Cohen, FEBS Letters, 91, pp. 1-7 12 (1978); M. Mishina, R. Roggenkamp and E. Schweizer, Eur. J. Biochem., 111, 13 pp. 79-87 (1980); B. Egin-Buhler, R. Loyal and J. Ebel, Arch. Biochem. Biophys., 14 203, pp. 90-100 (1979); A. R. Slabas and A. Hellyer, Plant Sci., 39, pp. 177-182 (1985); Hellyer et al., J. L. Harwood, Ann. Rev. Plant. Physiol., 39, pp. 101-138 16 (1988)). The molecular weights is of a multifunctional subunit lie above 200 kD.
17 The ACC of the rat has a molecular weight of 265 kD (Lopez-Casillas et al., supra), 18 that of the yeast a molecular weight of 251 kD (Al-Feel et al., supra) and that from 19 plants varies between 210 and 240 kD (Hellyer et al., supra).
21 Table 1 reviews the homologies of the known ACC enzymes.

23 Table 1.

Identity 26 Chicken Rat Yeast E. coli 27 Chicken - 93.2 46.1 25.8 28 Rat . 96.4 - 46.1 25.8 29 Yeast 65.7 65.8 - 27.5 E. coli 53.6 53.7 51.5 31 Homology 33 Table 1 shows the percentages of identical amino acids or degree of homology in 34 acetyl-CoA-carboxylases of the chicken, rat, yeast and E. coli. It is readily apparent 35 that the ACC enzymes also exhibit a relatively high degree of affinity over the 36 different organisms. Despite the significant evolutionary distance between the 37 rat/chicken, on the one hand, and yeast, on the other, about 66% homology over the 38 entire amino acid sequence still exists. If we pick out individual regions, homologies of about 80 to 100% can be found in some sections (Al-Feel et al., supra). The same 2 form of org~ni7~tion of eukaryotic ACCs with respect to the sequence of domains 3 BC-BCCP-CT is worthy of note. This suggests early fusion of individual genes of 4 prokaryotes in the course of evolution of eukaryotes. The high conservation of5 ACCs is experimentally confirmed by the high cross reactivity of antibodies between 6 the rat, chicken and yeast (Al-Feel et al., supra).

8 Regulation of acetyl-CoA-carboxylase is still largely unexplained in various 9 organisms. Enzyme activity in plants has thus far been studied on two different 10 experimental systems, namely in chloroplasts and developing rape seeds. It turned 11 out that ACC activity in developing rape seeds is induced before lipid incorporation, 12 but then drops quickly when complete lipid incorporation has been achieved (E.
13 Turnham and D. H. Northcote (1983), Biochem. J., 212, pp. 223-229). This means 14 that regulation occurs via the end product. Moreover, ACC appears to regulate the 15 entire de novo fatty acid biosynthesis as the enzyme that limits conversion rate (P. D.
16 Simcox, W. Garland, V. De Lica, D. T. Canvin and D. T. Dennis, Can. J. Bot., 57, 17 pp. 1008-1014 (1979); Turnham and Northcote, supra). These experimental findings 18 make acetyl-CoA-carboxylase in plants an interesting object for intervention into 19 fatty acid metabolism with a view toward increasing yield or altering the fatty acid pattern during appl.,pliate overproduction of ACC in seeds.

22 Studies concerning the mechanism of action of different herbicides used to control 23 weeds, for example grasses, in stands of dicotyledonous crops have shown that 24 certain herbicides intervene in the metabolism of grasses by inhibiting ACC.Substances of three different classes have thus far been described that exhibit a 26 herbicidal effect by interaction with ACC. Thus, derivatives of 27 aryloxyphenoxypropionic acid (for example, diclofop, enoxaprop, fluazifop and 28 haloxyfop).(K. Kobek et al., Z. Naturforschung, 43c, pp. 47-54 (1988)), cyclohexane-29 1,3-dione (for example cycloxydim, clethodim and setoxydim) (M. Focke and H. K.
Lichtenthaler, Z. Naturforschung, 42c, pp. 1361-1363 (1987)) or PP600 (3-isopropyl-31 6-(N-[2,2-dimethylpropyl]acetamido- 1,3,5-triazine-2,4-(1 H,3H)-dione) (K. A.
32 Walker, S. M. Ridley and J. L. Lewis Harwood, Phytochem., 29, pp. 3743-3747 33 (1990)) inhibit ACC of sensitive plants. At the moment it is still unclear how the 34 inhibitor effect occurs in detail and why ACCs of dicotyledonous plants are not inhibited.

37 A biotin-containing polypeptide with a molecular weight of 50 kD representing a 38 subunit of a plant acetyl-CoA-carboxylase is described in EP-A-0 469 810.

However, it has been established, among other things, that the 229 bp clone CC 8 of 2 Figure 8 has no amino acid reading frame that exhibits a meaningful homology to 3 one of the known ACC amino acid sequences. This inevitably leads to the 4 conclusion that the antibody used in EP-A-0 469 810 is not specific to ACC or at least a subunit of ACC.

7 It is the task of the invention to offer a DNA sequence with which, on the one hand, 8 the quality and quantity of vegetable oils or fats can be altered by homologous or 9 heterologous expression in plant systems and, on the other hand, herbicide resistances to a wide variety of herbicides can be imparted or transferred to economic 11 plans by heterologous expression.

13 This task is solved with a DNA sequence according to Claim 1.

The invention concerns a DNA sequence that codes for acetyl-CoA-carboxylase and 16 the alleles and derivatives of this DNA sequence.

18 The invention also concerns genomic clones that contain a DNA sequence that codes 19 for the acetyl-CoA-carboxylase and the alleles and derivatives of this DNA sequence.
21 The invention further concerns a process for production of plants, plant parts and 22 plant products in which a DNA sequence that codes for acetyl-CoA-carboxylase is 23 transferred by genetic engineering.

The invention finally also concerns the use of this DNA sequence to impart or 26 transfer herbicide resistances or to alter the quality and quantity of vegetable oils and 27 fats.

29 The figures serve to explain the present invention. In them:
31 Figure 1 shows a sequence comparison of amino acid sequences of biotin-32 dependent and related enzymes in their BC domains;

34 Figure 2 shows the DNA or amino acid sequence of degenerated oligonucleotides 3455 and 3464;

37 Figure 3a shows the DNA sequence and the amino acid sequence derived from it in 38 the one-letter code of the 260 bp PCR fragment as specific hybridization probe;

3 Figure 3b shows a comparison of the amino acid sequence of ACC of the rat (top 4 line) with the amino acid sequence from Figure 3a (bottom line);
s 6 Figure 4 shows the restriction maps of DNA sequences inserted into the genomic7 clones BnACC3, BnACC8, BnACC10 and BnACC1;

9 Figure 5 shows the DNA sequence of the acetyl-CoA-carboxylase gene;
11 Figure 6 shows the functional regions in the DNA sequence from Figure 5 and the 12 amino acid sequences derived from the DNA sequences in the one-letter13 code;

15 Figure 7 shows a schematic representation of the functional regions of the DNA
16 sequence from Figure 6; and 18 Figure 8 shows a Southern blot hybridization (cross-hybridization) of different 19 genomic plant DNA with part of the ACC gene of the genomic clone BnACC8.

22 It goes without saying that allelic variants and derivatives of the DNA sequence 23 according to the invention are encompassed by the invention, provided that these 24 modified DNA sequences code for acetyl-CoA-carboxylase. Allelic variants andderivatives include, for example, deletions, substitutions, insertions, inversions or 26 additions of the DNA sequence according to the invention.

28 The gene for acetyl-CoA-carboxylase is present in all plants and therefore can be 29 isolated from them in a variety of ways. For example, the gene can be isolated by means of oligonucleotide probes or specific antibodies from genomic plant DNA
31 banks or its cDNA from cDNA banks. Rape (Brassica napus) of the Akela variety 32 proved to be a particularly suitable plant material.

34 A gene bank of the rape genome (Brassica napus) of the Akela variety inserted in a phage was used in the present invention as starting material for isolation of genomic 36 clones that contain the gene for ACC. This gene bank was searched thoroughly for 37 ACC genes with a hybridization probe produced by means of PCR (polymerase chain 38 reaction). In this fashion a genomic clone with the designation BnACC8 was isolated that contains the complete structural gene (protein coding region (exons and 2 introns)) of ACC from rape on a 13.7 kB XbaI fragment. This genomic clone has 3 been filed under the number DSM 7384.

Moreover, the genomic clones BnACC3, BnACC10 and BnACC1 were isolated, 6 which also contain the structural gene of ACC from rape or at least parts of it on 7 roughly 20 kb, 15 kb or 15 kb DNA fragments.

9 The 13.7 kb DNA fragment was subcloned and sequenced in the form of XbaI/SmaI
fragments in appropliate vectors. The amino acid sequences derived from the DNA
11 sequences were compared with the ACC amino acid sequence of the rat from 12 Figure 2 of the article of F. Lopez-Casillas (supra) by computer analysis. It was 13 established on the basis of amino acid sequence homologies that the 13.7 kb DNA
14 fragment contains the acetyl-CoA-carboxylase gene.
16 In addition, a roughly 2 kb DNA fragment of the roughly 20 kb DNA sequence from 17 BnACC3 was also sequenced.

19 Figure 4 shows the restriction maps of the DNA fragments inserted into the genomic clones BnACC3, BnACC8, BnACC10 and BnACC1. Except for the overlapping 21 clones BnACC3 and BnACC8, which belong to one class of genes, only one 22 representative (BnACC10 and BnACCl) of two additional classes of genomic clones 23 were prepared. The regions marked in black showed DNA regions that hybridize24 with the employed probe. The DNA fragments are delimited by the cleavage sites of the cloning vector Lambda Fix II, which are marked with the symbol "Y": XbaI, 26 SacI, NotI, SacI and SalI on one side or SalI, SacI, NotI, SacI and XbaI on the other 27 side. The sequenced regions of the 13.7 kb DNA fragment from BnACC8, as well as 28 the 2 kb DN. A fragment from BnACC3, were marked with white bars.

11.9 kb of the 13.7 kb DNA fragment from BNACC8 was sequenced using the usual 31 method. Moreover, about 2 kb of the second SalI cleavage site of the roughly 20 kb 32 DNA fragment from BnACC3 was sequenced in the 3 direction up to overlapping 33 with the clone BnACC8. Both clones overlap in the 5 region. A DNA sequence 34 with a length of 13.753 kb is obtained by sequence comparison.

36 The complete DNA sequence of the 13.753 kb from the 11.9 kb of the 13.7 kb DNA
37 fragment from BnACC8 and 1904 bp of the roughly 20 kb DNA fragment from 38 BnACC3 is show in Figure 5. The DNA sequence on the 5 end includes the sequence from BnACC3 beginning from the second SalI cleavage site and extends on2 the 3 end with 678 bp over the EcoRI cleavage site of BnACC8. The DNA
3 sequence of 11.9 kb of the 13.7 kb DNA fragment from BnACC8 begins at position 4 1905 in Figure 5.

6 The acetyl-CoA-carboxylase structural gene, as well as the promoter, are contained 7 in the 17.753 kb DNA fragment, in which the structural gene is already situated on 8 the 11.9 kb of the 13.7 kb DNA fragment from BnAAC8. For this purpose reference 9 is made to Figure 6, which shows the DNA sequence from Figure 5 with its functional regions. Regulator elements, like the CAAT box (positions 2283-2286),11 the TATA box (positions 2416-2419), as well as a polyadenylation signal (positions 12 13284-13289) are underlined. The first roughly 600 bp from position 1905 (11.9 kb 13 from BnACC8) already represent part of the promoter; the preceding 1904 bp contain 14 the entire promoter and come from BnACC3. The ATG start codon of ACC is situated in position 2506 and the corresponding TGA stop codon in position 13253.
16 The exon/intron boundaries are marked in black.

18 The corresponding amino acid sequences are shown in the black exon regions in the 19 sequence. The exon/intron boundaries were established based on similarity to acetyl-CoA-carboxylases from other org~ni.sm.s (rat) (F. Lopez-Casillas, supra), if these 21 boundaries were not determined by means of PCR. The first exon of the gene begins 22 at ATC at a start codon with an open reading frame. The 5 untranslated regions are 23 therefore not marked in black. Marking of the last exon ends at the corresponding 24 stop codon so that the 3 -untranslated region is also llnm~rked.
26 The functional regions of the ACC sequence of the clone BnACC8 are schematically 27 depicted in Figure 7. The exons are marked in black. ATG denotes the start codon, 28 MKM the conserved biotin-binding site and TGA the stop codon. Moreover the 29 three domains can be clearly coordinated on the sequence: BC = biotin carboxylase, BCCP = biotin carboxy carrier protein, CT = carboxyl transferase. The three 31 domains were determined by homology to ACC of other org~nisms.

33 It was established in cross hybridizations with genomic DNA from Arabidopsis34 th~ qn~, Brassica napus, Avena sativa, Hordeum vulgare, Oryza sativa, Triticum aestivum and Zea mays that parts of the ACC sequence according to the invention 36 are suitable for isolation of ACC genes for other plants. An SmaI/SacI fragment (see 37 Figure 4) from th clone BnACC8 was used in Figure 8 as DNA probe. The genomic 38 DNAs of the different plants were cleaved with EcoRI and subjected to a Southern blot. The cross-reactivity of the probe is observed in mono- and dicotyledonous 2 plants.

4 The DNA sequence according to the invention that codes for acetyl-CoA-5 carboxylase, the alleles and derivatives of this DNA sequence can be introduced or 6 transferred to plants to control fatty acid metabolism (in the form of antisense or 7 overexpression) by means of genetic engineering methods.

9 Antisense constructions, for example, with sequences from positions 1905 to 3187, 3188 to 8108 and 11039 to 12846 of the DNA sequence according to the invention of l l Figure 6 can be used to inhibit the activity of ACC in a plant. This can occur in 12 particular by controlling fragments of the ACC gene by controlling fragments of the 13 ACC gene [sic] by applupflate regulatory elements (promoters) in seeds. In this 14 fashion blockage of acetyl-CoA can be achieved, since this intermediate can no longer enter into fatty acid metabolism and thus influence metabolism of, say, a plant 16 cell:

18 1. A "suicide gene" can thus be produced with applopliate regulatory elements 19 when an antisense construction leads to a situation in which formation of fatty acids in a cell does not occur. A hypersensitive reaction can be triggered in this fashion in 21 controlling plant diseases.

23 2. By incorporation of additional genes whose gene products employ acetyl-24 CoA, blockage of acetyl-CoA can be hampered. For example, the genes for synthesis of, say, polyhydroxybutyrate (PHB) (Piorier et al., 1992, Science, 256, pp. 520-523) 26 can be expressed specifically in certain tissues/organs/cell types of a plant, preferably 27 storage tissues, like seeds (endosperm, cotyledon); roots; various types of tubers). If 28 an ACC an,tisense construction is simultaneously expressed in the same parts of the 29 plant, the unemployed acetyl-CoA can then be used for synthesis of PHBs.
31 Oligonucleotides can be derived from the DNA sequence according to the invention 32 in order to synthesize a cDNA or pieces of a cDNA. This cDNA or pieces of it can 33 be used alone or in conjunction with parts of the genomic clone in order to isolate a 34 complete cDNA. These cDNA or cDNA pieces can also be used for antisense expression.

37 Thus, individual cDNA fragments or the entire cDNA can be used for 38 complementation of mutants of ACC, for example, in microorganisms. In this fashion the microorganisms (mutants from E. coli fabE; Silbert et al., 1976, J.
2 Bakteriol., 126, pp. 1351-1354; Harder et al., 1972, PNAS, 69, pp. 3105-3109) and 3 from yeast (Schweizer et al., about 1980) are functionally complemented under 4 nonpermissive conditions by the plant ACC and are directly dependent on the plant enzyme. This offers the possibility of selection for the plant enzyme and a test6 system for development and optimization of inhibitors for ACC. In addition to better 7 active agents for use as herbicides, resistant forms of the ACC enzyme can also be 8 developed or selected on this account after mutagenesis of the gene (or regions of the 9 gene).
11 The cDNA can also be used to recover larger amounts of protein or parts of the 12 protein. This produced protein can be used for studies on the reaction mechanism 13 and regulation or in order to clarify the three--lim~.n.~ional structure of the enzyme of 14 parts of the enzyme. The last named aspect is particularly significant for protein modeling, since it permits adjustment of, for example, inhibitors to the structure of 16 the protein.

18 The ACC gene sequence, the alleles and derivatives of this sequence are preferably 19 introduced to the plants together with applupriate promoters, especially in recombinant vectors.

22 All types of plants can be transformed for this purpose. Useful plants, garden plants 23 and ornamental plants can be cited in this connection. Among the useful plants, 24 Brassica napus, B. rapa, coconut and oil palms, sunflowers and flax are particularly preferred.

27 The DNA sequence according to the invention codes for ACC can be used in28 particular tQ achieve herbicide resistances in useful plants, especially grains, against 29 specific herbicides. Corn, wheat, barley, rice and rye can be mentioned as preferred plants for transformation.

32 Incorporation by genetic engineering of the ACC DNA sequence, the alleles and 33 derivatives of this sequence can be carried out by means of usual transformation 34 techniques. Such techniques include methods like direct gene transfer, for example, microinjection, electroporation, particle gun, viral vectors and liposome-m~ ing36 transfer, as well as transfer of corresponding recombinant Ti plasmids or Ri plasmids 37 and transformation by plant viruses.

2150~78 '() Demonstration of transformation can be carried out in a cell culture of the 2 monocotyledonous plant, like barley, wheat or corn, by selection with an appropriate 3 herbicide. Moreover, demonstration can be achieved by Southern blot with, for 4 example, intron sequences of rape ACC DNA as hybridization probe.

6 Thus, the invention also concerns plants, plant parts and plant products that have 7 been produced or transformed according to one of the above processes.

9 The following examples serve to explain the invention.

13 Example 1: Production of hybridization probe for acetyl-CoA-carboxylase (ACC) (a) Production of degenerated oligonucleotide 17 Starting from a sequence comparison of different biotin-containing proteins, 18 synthetic oligonucleotides were derived from conserved sections of the ACC
19 sequences. For this purpose Figure 1 is referred to, which shows a sequencecomparison of the amino acid sequence of biotin-dependent and related enzymes in21 their BC domains. This figure refers back to Figure 3 from the publication of Kondo 22 et al. (supra). The abbreviations in the left column have the following me~nin~s:

24 EACC = ACC from E. coli;
26 cACC = ACC from chicken;

28 rPCCA = A subunit of propionyl-CoA-carboxylase from the rat;

yPC = pyruvate carboxylase from yeast and 32 ECPSN = n-terminal half of carbamoyl phosphate synthetase.

34 Identical amino acids are framed and strongly conserved residues are marked by points. In addition, the conserved sequences used for production or derivation of the 36 degenerated oligonucleotides 3455 and 3464 were additionally emphasized by arrows 37 and numbers in Figure 1.

2150678 "
The oligonucleotides were synthesized on an Applied Biosystems DNA synthesizer 2 (model 380B) and are shown in Figure 2. Both oligonucleotides are shown in the 5 -3 3 -orientation so that during comparison with Figure 1 the amino acid sequence of 4 the oligonucleotide 3464 must be read in the opposite direction.
s 6 Different bases were incorporated into the oligonucleotides based on the degenerated 7 genetic code and the possible variability of the amino acid sequence in individual 8 positions, for example, C or T and A or G in oligonucleotide 3464. Moreover, I was 9 introduced, which can interact with all nucleotides and therefore be viewed as an unspecific base.

12 (b) Polymerase chain reaction (PCR) 14 cDNA synthesis with oligonucleotide 3464 as primer was carried out for 30 minutes at a temperature of 37C starting from 1 llg of polyA + RNA with avian 16 myeloblastosis virus (AMV) reverse transcriptase. After inactivation of the reverse 17 transcriptase by heating for 5 minutes at a temperature of 95C the PCR reaction was 18 run in the same reaction charge with 50 pmol final concentration of each primer 19 (3455 and 3464) and four units of Ampli-Taq polymerase (Perkin Elmer Cetus).
The reactions were run under the following conditions:

22 a) 10 mM Tris HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl, 0.01% gelatin and 5 23 mM dNTPs b) Reaction temperatures: 3 minutes at a temperature of 92C to first 26 denaturation, then 30 telllpeldlllre cycles of 2 minutes each at a temperature of 92C
27 to denaturation, 2 minutes at a temperature of 51 C for anne~ling of the 28 oligonucleotide and 2.5 minutes at a temperature of 72C for amplification of DNA
29 and then 2.5 minutes at a temperature of 72C in order to achieve complete synthesis of the last synthesis product.

32 c) Cloning of the amplification product 34 The rem~ining single-stranded DNA of the PCR products was filled in by means of Klenow polymerase (Sambrook et al., Molecular Cloning - A laboratory manual, 2nd36 edition, Cold Spring Harbor Laboratory Press, New York (1989)) and then 37 phosphorylated with polynucleotide kinase (Sambrook et al., supra). Purification of 38 the PCR products occurred according to the standard protocols after Sambrook et al.

(supra) by agarose gel electrophoresis, gel elution, purification with 2 phenol/chloroform and subsequent precipitation with isopropanol. The DNA
3 purified in this manner was ligated in Smal cleaved pBluescript vector-DNA and4 cloned.

6 d) DNAsequencing 8 To determine the DNA sequence of subclones produced in pBluescript and those 9 DNA sequences in which deletions were produced by exonuclease III (see also 10 example 2) (Sarnbrook et al., supra) sequencing was carried out according to the 11 method of Sanger et al., Proc. Natl. Acad. Sci., 74, pp. 5463-5467 (1977). The 12 sequence data were analyzed with the computer software of the University of 13 Wisconsin Genetics Computer Group (Devereux et al., Nucl. Acids Res., 12, pp. 387-14 395 (1984)). The homology studies occurred with the "Bestfit" program.
16 e) Synthesis of a specific hybridization probe by PCR

18 Starting from polyA+-RNA from imm~tllre rape seeds (Brassica napus) (about 2 to 3 19 weeks old) DNA fragments were amplified after a cDNA first-strand synthesis by PCR reactions. The oligonucleotides required for this purpose were synthesized 21 based on homology comparison, among other things, between the ACC of the 22 chicken and from E. coli (Figure 1). A product of 260 bp length was to be amplified 23 according to calculation with these (degenerated) oligonucleotides, which codes for 24 86 amino acids. Amplification products of this order of m~gnitucle were therefore isolated from the obtained product mixture, cloned in pBluescript and identified by 26 DNA sequencing. In addition to other unspecific PCR products, one that has the 27 expected the size of 260 bp could be cloned and has an open reading frame of 86 28 amino acids (Figure 3a). This product, also counting the homology in the region of 29 the oligonucleotide, exhibits 77.9% identical amino acids in comparison with ACC
of the rat and a homology of 88.4% (Figure 3b). If we consider the identity or 31 homology relative only to the amplified sequence, i.e., without the region of the 32 oligonucleotides, which also permit incorrect pairings, we still obtain values of 33 73.2% identical amino acids or 85.9% homology between the protein sequences of 34 ACC of the rat and rape. These numbers show that the cloned PCR product codes a part of the ACC from rape and can therefore be used as a specific hybridization 36 probe. It was to be expected based on the position of the homology to ACC of the rat 37 between the amino acids of positions 304 and 389 (Figure 3b) that the cloned PCR
38 fragment only recognizes cDNAs that are more than 6000 bp long.

2 Example 2: Characterization of a genomic clone with a DNA sequence that codes for 5 Ten genomic clones were isolated and characterized from a gene bank of rape 6 (Brassica napus) of the Akela variety, which had been constructed in the vector 7 Lambda FIX II (Stratagene) by means of the cloned PCR fragment described under8 example 2. They can be divided into three classes based on their restriction maps.
9 Figure 4 shows the restriction maps of the genomic clones BnACC3, BnACC8, 10 BnACC10 and BnACC1. The clone BnACC8, which belongs to the most commonly 11 represented class, contains a DNA fragment with a size of 13.7 kb. This DNA
12 fragment includes the complete structure of ACC from rape. The DNA fragment was 13 subcloned and sequenced in the form of XbaI-SmaI fragments in pBluescript . In 14 addition, a roughly 3.2 kb SalI-SmaI fragment of the DNA fragment of the roughly 15 20 kb DNA fragment from BnACC3 was subcloned and about 2 kb of the SalI

16 cleavage site from sequenced from it in the 3 direction.

2150678 '~
Registration number of applicant or representative M 7101 3 International Registration No PCT/EP94/00150 s 8 (Rule 13bis PCT) 11 A. The following information concerns the microorganism mentioned in the12 specification on page 8, line 19.

14 B. Characterization of filing Additional filings are to be characterized on an additional page.

17 Name of filing location 18 DSM - German Collection of Microorg~ni.cm~ and Cell Cultures GmbH

Address of filing location (including P.O. Box and country) 21 Mascheroder Weg lB
22 D-38124 Braunschweig 24 Date of filing: File No.:
January 8, 1993 DSM 7384 27 C. Additional information (leave blank if not applicable) 28 The information is continued on a separate page.

D. Signatory states for which the information is made (if the information 31 does not apply to all signatories) 32 EP, Canada, US, Japan 34 E. Submission of information (leave blank if not applicable) The following inforrnation will be submitted later to the International 36 Office (please state the type of information, for example, "File number of 37 registration").
38 Only for use in the application office Only for use in the International 39 Office 2150678 ~

-2 [ ] This page is submitted with the [ ] This page is submitted to the 3 international application International Off1ce Authorized individual Authorized individual 2 1 5 0 6 7 ~ I L

The microorganism BnACC8 mentioned on page 8, line 19 and in Claim 9 was 2 filed on January 8, 1993 with the DSM German Collection of Microorganisms and 3 Cell Cultures GmbH, Mascheroder Weg lB, D-38124 Braunschweig under the 4 file number DSM 7384.

Claims (17)

New Claims 1 to 17

1. DNA sequence that codes for acetyl-CoA-carboxylase and the alleles as well as derivatives of this DNA sequence.

2. DNA sequence according to Claim 1, characterized by the fact that it is isolated from rape (Brassica napus).

3. DNA sequence according to Claim 1 or 2, characterized by the fact that it hasa size of 13.7 kb and contains the complete structural gene, as well as at least parts of the promoter of acetyl-CoA-carboxylase.

4. Genomic clone containing a plant DNA sequence that codes for acetyl-CoA-carboxylase and the alleles as well as derivatives of this DNA sequence.

5. Genomic clone according to Claim 4, characterized by the fact that the DNA
sequence is isolated from rape (Brassica napus).

6. Genomic clone according to one of the Claims 4 or 5, characterized by the fact that the DNA sequence has a size of 13.7 kb and contains the complete structural gene, as well as at least parts of the promoter of acetyl-CoA-carboxylase.

7. Genomic clone BnACC8 (DSM 7384).

8. Genomic clones BnACC1, BnACC3 and BnACC10 that contain the DNA
sequence for the complete structural gene of acetyl-CoA-carboxylase or at least parts of it and the alleles as well as derivatives of this DNA sequence.

9. Genomic clones according to Claim 8, characterized by the fact that the DNA
sequence for the structural gene is isolated from rape (Brassica napus).

10. Genomic clones according to Claim 9, characterized by the fact that at leastthe genomic clone BnACC3 contains the DNA sequences for the promoter for the structural gene of acetyl-CoA-carboxylase.

11. DNA sequence that contains the structural gene and promoter for acetyl-CoA-carboxylase according to Figure 5.

12. Process for production of plants, plant parts and plant products that exhibit herbicide resistance in which a DNA sequence according to one of the Claims 1 to 3 or Claim 11, or a DNA sequence originating from the genomic clones according to one of the Claims 4 to 10 is transferred by genetic engineering.

13. Process for production of plants, plant parts and plant products whose quality and quantity with respect to oil and fatty acid production are altered, in which a DNA
sequence according to one of the Claims 1 to 3 or Claim 11 or a DNA sequence originating from the genomic clones according to one of the Claims 4 to 10 is transferred by genetic engineering.

14. Process according to Claim 12 or 13, characterized by the fact that the DNA
sequence is transferred by microinjection, electroporation, particle gun, transfer of corresponding recombinant Ti plasmids or Ri plasmids, liposome-mediated transferor by plant viruses.

15. Use of a DNA sequence according to one of the Claims 1 to 3 or Claim 11 or a DNA sequence originating from the genomic clones according to one of the Claims 4 to 10 for transfer of herbicide resistances to plants.

16. Use of a DNA sequence according to one of the Claims 1 to 3 or Claim 11 or a DNA sequence originating from the genomic clones according to one of the Claims 4 to 10 to alter the quality and quantity with respect to oil and fatty acid production in plants.

17. Plants, plant parts and plant products produced according to a method of Claim 12 or 13 and 14.