EP0716708A1

EP0716708A1 - Medium chain-specific thioesterases

Info

Publication number: EP0716708A1
Application number: EP94928311A
Authority: EP
Inventors: Reinhard TÖPFER; Norbert Martini; Jozef Schell
Original assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Current assignee: Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
Priority date: 1993-09-03
Filing date: 1994-09-02
Publication date: 1996-06-19
Also published as: CA2169094A1; WO1995006740A3; AU688377B2; AU7739894A; WO1995006740A2; US5910631A

Abstract

DNA sequences are disclosed that code for a middle chain-specific acyl-[ACP]-thioesterase, as well as alleles and derivatives of said DNA sequence. This DNA may be used to transform plants capable of forming middle chain-specific acyl-[ACP]-thioesterases.

Description

Medium chain specific thioesterases

The invention relates to DNA sequences for a

encode medium chain specific acyl [ACP] thioesterase, and the alleles and derivatives of these DNA sequences.

The thioesterases are essential in the production of

Fatty acids involved in plant organisms. The fatty acid and triacylglyceride biosynthesis can be due to the compartmentalization as separate biosynthesis, but in

Regarding the end product, see it as a biosynthetic pathway. The de novo biosynthesis of fatty acids takes place in the

Piastiden and is made up of three enzymes or enzyme systems

catalyzed, i.e. the acetyl-CoA carboxylase (ACCase), the fatty acid synthase (FAS) and the acyl [ACP] thioesterase (TE).

The end products of this reaction sequence are in most

Organisms either palmitin (C _{16: 0} ), stearin (C _{18: 0} ) and, after desaturation, oleic acid (Δ9C _{18: 1} ). The acyl [ACP] thioesterase (TE) has the function of chain length termination.

In the cytoplasm, on the other hand, triacylglyceride biosynthesis takes place in the so-called "Kennedy" at the endoplasmic reticulum

Pathway "from glycerin-3-phosphate, which is probably provided by the activity of glycerol-3-phosphate dehydrogenase (G3P-DH), and fatty acids, which are present as acyl-CoA substrates.

In animal systems (eg in rats), acyl [ACP] thioesterase is an integral part of FASI and is responsible for the termination of fatty acid biosynthesis. A second acyl [ACP] thioesterase (TEII), the is expressed in a tissue-specific manner, however, in the milk-producing mammary glands of the rat, the chain extension in the fatty acid biosynthesis is terminated prematurely and C _{10: 0} and C _{12: 0} fatty acids are released. Expression of this TEII in mouse fibroblasts led to the formation of the medium-chain fatty acids mentioned in these cells and thus proves that this enzyme is crucial for termination. the chain length is involved. (SA Bayley et al, Bio / Technology 6, pages 1219-1221 (1988)).

Acyl [ACP] thioesterases were also purified in plants and their activity was examined. Acyl- [ACP] thioesterases with preference for the hydrolysis of long-chain acyl- [ACP] compounds were obtained from Carthamus tinctorius (TA McKeon et al, J.Biol.Chem. 257, pages 12141-12147 (1982)), Cucurbita moschata ( H. Imai et al, Plant.Mol.Biol. 20, pages 199-206

(1992)) and Brassica napus (A. Hellyer et al, Plant.Mol.Biol. 20, pages 763-780 (1992)). Corresponding cDNAs have already been described by Carthamus tinctorius (DS Knutzon et al. Plant Physiol. 100, pages 1751-1758 (1992)) and Brassica napus (ES Loader et al, Plant. Mol.Biol., Vol. 23, pages 769 to 778 (1993)) isolated. Another TE with specificity for the hydrolysis of C _{12: 0} - [ACP] was from Umbellularia californica

(California laurel) isolated and separated from the activity of a C _{18: 0} - [ACP] specific TE (MR Pollard et al,

Art.Biochem.Biophys. 284, pages 306-312 (1991)). In Cuphea lanceolata the activity of a medium- and a long-chain-specific TE was also detected (P. Dörmann et al, Planta 189, pages 425-432 (1993)).

An only partially purified enzyme preparation of a C _{10: 0} -specific acyl [ACP] thioesterase from Cuphea hookeriana is described in WO 91/16421. As measurements of the hydrolysis activities of the enzyme against different substrates show, there are considerable proportions of activities that are not C _{10: 0} -specific. For the TE from Umbellularia californica a cDNA encoding a medium chain specific acyl- [ACP] thioesterase was isolated. In transgenic Arabidopsis thaliana and B. napus plants, it led to the formation of medium-chain fatty acids in the seed, in particular lauric acid (C _{12: 0} ) and small amounts of myristic acid (C _{14: 0} ); (TA Voelker et al, Science 257, pages 72-74 (1992) and HM Davies and TA

Voelker in Murata, N. and C. Somerville (Editor): Current Topics in Plant Physiology: Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, Vol. 9, pages 133-137; American Society of Plant Physiologists, Rockville

(1993)).

There is clearly an increased need to provide medium-chain fatty acids, such as capric acid (C _{10: 0} ), which can be used industrially as raw materials for plasticizers, lubricants, pesticides, surfactants, cosmetics, etc. One way to provide these fatty acids is to isolate (extract) the fatty acids

Plants that have particularly high levels of these fatty acids

exhibit. Increasing levels of medium chain

Fatty acids in the classic way, i.e. by breeding

Plants that produce these fatty acids to an increased extent have so far only been able to be reached to a limited extent.

It is therefore an object of the invention to provide genes or DNA sequences which are used to improve the oil yield and to produce medium-chain fatty acids in plants which are themselves not capable of producing such fatty acids or only to a small extent of these fatty acids

produce, can be used.

This object is achieved with the DNA sequences according to claim 1 or the genes from the genomic clones according to claim 6. The invention relates to DNA sequences which code for a medium-chain-specific acyl [ACP] thioesterase, and to the alleles and derivatives of these DNA sequences.

The invention further relates to genomic clones, the DNA sequences which code for a medium-chain-specific acyl [ACP] thioesterase and contain promoters and regulator sequences, and the alleles and derivatives of these DNA sequences.

The invention also relates to a process for the production of plants, plant parts and plant products in which a DNA sequence which codes for a medium-chain-specific acyl- [ACP] thioesterase is transmitted by genetic engineering.

Finally, the invention also relates to the use of this DNA sequence for the transfer of genes for medium-chain-specific acyl [ACP] thioesterases in plants.

The invention also relates to plants, parts of plants and plant products which are produced by the process mentioned above.

The figures serve to explain the present invention.

Show it:

1 shows the representation of the DNA or amino acid sequence of the degenerate oligonucleotides 3532 and

2740;

FIG. 2 shows the restriction maps of the genomic clones for the acyl [ACP] thioesterase CITEgl,

ClTEg16, ClTEg4 and ClTEg7 from Cuppea

lanceolata;

Figure 3 shows a comparison of the amino acid sequences of

Thioesterases from various plants; Figure 4 functional parts from binary vectors for

Expression of the DNA sequences or genes according to the invention from the genomic clones in

transgenic plants;

FIG. 5 shows the gas chromatogram of fatty acids (pNBM99-2TE) contained in immature rapeseed;

FIG. 6 shows the gas chromatogram of fatty acids (pNBM99-2TE) contained in immature tobacco seeds;

FIG. 7 shows the gas chromatogram of fatty acids contained in rapeseed rapeseed (pNBM99-TEg1); and

Figure 8 shows the gas chromatogram of fatty acids contained in rapeseed rapeseed (pNBM99-TEg16).

It goes without saying that the invention also covers allelic variants and derivatives of the DNA sequences according to the invention, provided that these modified DNA sequences and genes code for medium-chain-specific acyl [ACP] thioesterases. The allelic variants and derivatives include, for example, deletions, substitutions, insertions, inversions or additions of the DNA sequence according to the invention.

The DNA sequences according to the invention and the genes from the genomic clones code for medium-chain-specific acyl [ACP] thioesterases which catalyze the formation of C _{8: 0} to C _{14: 0} fatty acids. The invention relates

in particular on C _{10: 0} -specific acyl [ACP] thioesterases or essentially C _{10: 0} -specific acyl [ACP] thioesterases and C _{14: 0} -specific acyl [ACP] thioesterases or essentially C _{14 : 0} -specific acyl [ACP] thioesterases, which in the

Fatty acid synthesis responsible for the formation of

Capric acid or myristic acid. Any plant material which produces these thioesterases in sufficient quantities is suitable as the starting material for the isolation of genes which code for medium-chain-specific acyl [ACP] thioesterases. As a particularly suitable

In the present invention, the plant Cuphea lanceolata, native to Central America, has proven to be the starting material. The seeds of this plant contain 83% capric acid.

To isolate the DNA sequences according to the invention, a cDNA library from Cuphea lanceolata (wild type) was searched for genes for medium chain-specific acyl [ACP] thioesterases using a hybridization probe, PCR42, produced by PCR (Polymerase Chain Reaction). In this way the cDNA clones ClTE13, ClTE5 and ClTE12 were isolated.

The cDNAs obtained were completely double-stranded sequenced in the usual way. The ClTE13 CDNA comprises 1494 bp as Apal-Eco RI fragment and contains the complete one

Structural gene for a medium chain specific acyl [ACP] thioesterase. The ClTE13 CDNA encodes a 414 amino acid protein including a derived 111 amino acid transit peptide. The complete DNA sequence of the 1494 bp cDNA fragment with the amino acid sequence derived therefrom is reproduced as SEQ ID NO: 1 in the sequence listing. The coding region extends from position 83 to position 1324 of the DNA sequence. The open reading grid begins

Position 83 with the start codon "ATG", which for the

Amino acid encodes methionine and ends at position 1324 with the stop codon "TAG". The derived molecular weight of the mature protein is 34 kDa.

The DNA sequence analysis of the two further cDNAs C1TE5 and ClTE12 has shown that they do not contain the complete structural gene of a medium chain-specific acyl [ACP] thioesterase. The DNA sequences of the cDNAs mentioned with the amino acid sequences derived therefrom are listed in the sequence listing as SEQ ID NO: 2 and SEQ ID NO: 3. The ClTES-cDNA as Eco RO-XhoI fragment has a length of 1404 bp and codes according to the open reading frame for a protein with 375 amino acids, whereby 34 amino acids of the transit peptide are missing compared to the deduced amino acid sequence for ClTE13. The ClTE12 cDNA as Eco Rl-Xhol fragment,

marginal XhoI interface, a length of 1066 bp and encodes according to the open reading frame for a protein with 287 amino acids, wherein 20 amino acids of the mature protein and the transit peptide are missing.

In Table I below, the

Degree of homology or degree of identity of the acyl [ACP] thioesterase amino acid sequences of the mature proteins of the C1TE5 and C1TE13 cDNAs (derived from the DNA sequence) from Cuphea lanceolata, CtTE2-1 and CtTE5-2 from Carthamus tinctorius and UcTE from ombellularia californica compared.

The comparison of the TE amino acid sequence of ClTE13 with the thioesterase from U. californica (UcTE) shows 57.9%

identical amino acids have a fairly high agreement, which is greater than that to the long chain-specific ones

C. tinctorius thioesterases (CtTE2-1 and CtTE5-2). The thioesterase of ClTE5 also shows a very high degree of identity with an identity of 57.0% to UcTE.

FIG. 3 shows an amino acid sequence comparison of thioesterases from plants. The sequences of the mature proteins (exception: ClTE12, -20 amino acids) are from the corresponding thioesterase (TE) cDNAs from Carthamus tinctorius = Ct, Cuphea lanceolata = Cl, Brassica napus = Bn and Umbellularia

californica = Uc derived. PCR42 is the PCR product used to screen the cDNA library. The gap between positions 374 and 393 (approx. 20 amino acids) only occurs with the medium-chain-specific thioesterases and is close to the only cysteine (position 359) conserved across all sequences, the putative active cysteine residue. By changing the sequence sections between the named

Positions and others, see below, can be influenced by the genetic engineering on the chain length specificity of the thioesterases.

Furthermore, genomic clones from Cuphea lanceolata were isolated and characterized which contain the complete structural gene of a medium chain specific acyl [ACP] thioesterase as well as the associated regulatory sequences (such as promoters and

Terminators) included. This means that they form complete transcription units. When screening a Cuphea lanceolata genomic DNA library with the ClTE5 cDNA as a probe, 23 genomic clones were isolated. The genomic clones CITEg1, ClTEg16, ClTEg4 and ClTEg7 are shown in FIG. 2 and characterized on the basis of a restriction analysis using various restriction enzymes. These are DNA fragments with a size of 12.7 kb for CITEg1, 17.4 kb for ClTEglδ, 13.5 kb for ClTEg4 and 14.7 kb for ClTEg7 exhibit. Restriction mapping has shown that the genomic clones shown can be assigned to four different classes of genes. It has been determined on the basis of sequence data that the cDNA ClTE5 the gene from the genomic clone ClTEg4, the ClTE12-CDNA the gene from the genomic clone CITEg1, the ClTE13-CÖNA the gene from the genomic clone ClTEg7 and the PCR product PCR42 Correspond to the gene from the genomic clone CITEg16.

Internal sequence primers located at the 5 'end were derived from the cDNA sequences described. With these, sequence data were obtained on the genomic clones as a template, which provided information about the beginning of the coding region and thus also about the limitation of the promoters of the thioesterase genes. Based on these diagnostic sequence sections of the genomic clones CITEg1, ClTEg16, ClTEg4 and ClTEg7 im

The area of the smallest hybridizing fragment (see black bar in Figure 2) could be next to the

Identity as genes for medium-chain specific thioesterases compared to the amino acid sequence for U. californica thioesterase and the completeness of the thioesterase genes as transcription units can be determined.

The thioesterase genes were identified by DNA sequence analysis of selected sequence sections of the genomic clones CITEg1, ClTEg4, ClTEg7 and ClTEg16. The sequenced areas are recognizable as white bars under the clones shown in FIG. 2. All genes consist of seven exons, the first exon being in the untranslated region of the mRNA. The structural gene of a medium-chain-specific acyl- [ACP] thioestrase is located on a 4098 bp DNA fragment of the clone CITEgl, see SEQ ID NO: 4 in the sequence listing. The coding region begins with exon II at position 1787 and ends with exon VII at position 3941. The structural gene of a medium-chain-specific acyl [ACP] thioesterase is contained on a 4643 bp DNA fragment of the clone ClTEg7. As can be seen from SEQ ID NO: 6 in the sequence listing, the coding region at position 773 with exon II and ends with exon VII at position 3118. The genomic clone ClTEglδ contains the structural gene of a medium-chain-specific acyl- [ACP] thioesterase on a 5467 bp DNA fragment, see SEQ ID NO: 7 in the sequence listing. The coding region begins with exon II at position 3284 and ends with exon VII at position 5275. The coding region for the structural gene of a medium-chain-specific acyl [ACP] thioesterase is for the

genomic clone ClTEg4 only partially available. SEQ ID NO: 5 in the sequence listing shows the exon II at positions 1 to 502 and the incomplete intron II at positions 503 to 928 on a 928 bp DNA fragment.

The structural genes for the medium-chain-specific acyl [ACP] thioesterases detected in the genomic clones CITEg1, ClTEg7 and ClTEg16 each comprise seven exons of almost the same size, with exon II of the

Thioesterase of the clone ClTEg4 falls within the size range of exons II of the other thioesterases. The intron I of all genes may have gene regulatory functions during gene expression.

The genomic clone ClTEg4 was numbered DSM 8493 and the genomic clone ClTEg7 numbered DSM 8494 on August 27, 1993 at DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, D-38124

Braunschweig deposited.

The DNA sequences according to the invention, which code for a medium-chain-specific acyl [ACP] thioesterase, can be introduced or transferred into plants using genetic engineering methods (in the form of anti-sense or overexpression) to produce these fatty acids in these plants. Unless they are available as a complete transcription unit, the DNA sequences according to the invention are preferably used together with suitable promoters, in particular in recombinant vectors, such as binary vectors, introduced into the plants.

The genomic clones CITEg1, ClTEg16, ClTEg4 and ClTEg7 can be used as separate complete transcription units

(containing promoter, structural gene and terminator) for transforming plants, medium-chain-specific fatty acids being accumulated in the storage lipids. The yield of medium-chain fatty acids can be optimized by crossing and thus combining the thioesterase genes. Optimization can take place in such a way that the content of newly introduced fatty acids is increased or various new fatty acids are formed.

All types of plants can be transformed for this purpose. It is preferred to transform plants which are said to have an increased production of medium-chain-specific fatty acids and those which do not naturally synthesize these fatty acids. In this

Oil plants such as rapeseed,

Sunflower, flax, oil palm and soy called.

The genetic engineering introduction of the DNA sequences according to the invention which code for a medium-chain-specific acyl- [ACP] thioesterase can be carried out with the aid of conventional transformation techniques. Such techniques include methods such as direct gene transfer, such as microinjection, electroporation, particle gun, the swelling of plant parts in DNA solutions, pollen or pollen tube transformation, viral vectors and liposome-mediated transfer, and the transfer of corresponding recombinant Ti plasmids or Ri- Plasmids using Agrobacterium tumefaciens and transformation by plant viruses.

In the present invention, for transformation, the ClTE13 cDNA as Apal-Eco RI fragment was firstly behind the constructed double promoter of the 35S RNA from the Cauliflower Mosaic virus (p35S / Δp35S) was introduced into the binary vector pRE9 (pNBM99-3TE); secondly, this fragment was placed behind the seed-specific ACP23 promoter from rapeseed in the binary

Vector pRE1 introduced (pNBM99-2TE). Furthermore, an Xbal fragment (7.3 kb) from the genomic clone CITEg1 and a SalI-Eco RI fragment (6 kb) from the genomic clone ClTEg16 were introduced into pREl. The binary vectors pNBM99-TEgl and pNBM99-TEg16 result from this. The TE gene from CITEgl is in the 3'-5 'orientation and that of ClTEg16 in the 5'-3' orientation in pRE1. The functional parts of the expression vectors thus obtained are shown in FIG. 4.

The abbreviations shown in Figure 4 have the following

Meanings:

RB and LB = right and left border of the transfer DNA.

pACP23 = promoter of the acyl carrier protein gene 23 from rapeseed ClTE13 = cDNA 13 from Cuphea lanceolata

tnos = terminator of the nopaline synthase gene from Agrobacterium tumefaciens

NPTII = neomycin phosphotransferase gene II

p35S = promoter of the 35S RNA of the Cauliflower mosaic virus t35S = terminator of the 35S RNA of the Cauliflower mosaic virus

35s = minimal promoter of the 35S RNA of the Cauliflower Mosaic

virus

The medium-chain specificity of the thioesterases was investigated by transforming the cDNA according to the invention and the genes from the genomic clones. Suitable plant materials are, for example, rapeseed and tobacco, since these plants are only able to produce longer-chain fatty acids from C _{16: 0} .

For this purpose, the expression vectors pNBM99-TEg1 and pNBM99-TEg16 were transformed independently of one another into rape using Agrobacterium. The expression vector pNBM99-TEg1 was numbered DSM 8477 and the expression vector pNBM99-TEg16 numbered DSM 8478 on August 27, 1993 at DSM- German collection of microorganisms and cell cultures GmbH, Mascheroder Weg 1B, D-38124 Braunschweig.

The expression vectors pNBM99-2TE and pNBM99-3TE were transformed with tobacco using Agrobacterium tumefaciens independently of one another.

The transformed rapeseed and tobacco plants were then examined for their content of medium-chain fatty acids. For this purpose, ripening seeds were analyzed by gas chromatography. FIGS. 5 and 6 show the gas chromatogram of fatty acid extracts from transgenic rapeseed or tobacco seeds which have been transformed with the construction pNBM99-2TE.

From the gas chromatograms it can be seen that transgenic oilseed rape as well as transgenic tobacco capric acid (peak C _{10: 0} )

to produce. It follows that the cDNA ClTE13 and the gene from the genomic clone ClTEg7 (see above) encode a thioesterase which is C _{10: 0} -specific or essentially C _{10: 0} -specific.

In further studies on transgenic rapeseed that had been transformed with the expression vectors pNBM99-TEgl or pNBM99-TEgl6, it was found that in the gas chromatogram of the fatty acid extracts in the case of the gene from the genomic clone CITEg1 (FIG. 7) 1.7% Capric acid (C _{10: 0} ) and 0.4% caprylic acid (C _{8: 0} ) and in the case of the gene from the genomic clone ClTEglδ (Figure 8) 5.4% myristic acid (C _{14: 0} ) are formed. The following Table II shows the

Change in the fatty acid pattern of the transgenic oilseed rape plants examined. The information relates to%

Fatty acid in the ripe seed. Table II

Construct C ₈ C ₁₀ C ₁₂ C ₁₄ C ₁₆ C _{18: 0} C _{18: 1} C _{18: 2} C _{18: 3} C _{20: 0} control - - - - 3.0 2.3 76.5 9.9 6, 0 0.8 pNBM99-TEg1 0.4 1.7 - - 3.4 2.2 75.4 8.2 6.5 0.8 pNBM99-TEg16 - - - 5.4 13.4 1.9 56, 6 13.7 7.1 0.7

It follows that the gene from the genomic clone CITEgl and the cDNA C1TE12 (see above) for a C _{10: 0} -specific acyl- [ACP] thioesterase or essentially C _{10: 0} -specific acyl- [ACP] thioesterase and the gene from the genomic clone

Code ClTEg16 for a C _{14: 0} -specific acyl- [ACP] thioesterase or essentially C _{14: 0} -specific acyl- [ACP] thioesterase. When comparing the amino acid sequences in FIG. 3, it is striking that the C ₁₀ / C ₁₄ difference between CITEgl and CITEg16 is due to small sequence differences “RR” (positions 360/361), “M” (position 371), and “KE” (positions 395 / 396) and "D" (position 398), etc. can be traced. There is also a gap (five amino acids) and amino acid exchanges in the range of positions 127 to 135. These regions could have an influence on the chain length limitation.

The DNA sequences according to the invention and the genes from the isolated genomic clones from Cuphea lanceolata are outstandingly suitable for giving transformed plants the ability to form medium-chain-specific fatty acids. This means that a fully functional gene for a medium chain specific acyl [ACP] thioesterase can be transferred. Gas chromatographic studies of transgenic oilseed rape and tobacco have shown the formation of

Capric acid and myristic acid by transfer of genes for a C _{10: 0} or C _{14: 0} -specific acyl- [ACP] thioesterase or essentially C _{10: 0} - or C _{14: 0} -specific acyl- [ACP] -Thioesterase detected. It has been shown that the cDNA according to the invention and the genes from the shown genomic clones as plant genes do not lead to compatibility problems in rapeseed and tobacco. Proper compartmentalization is ensured due to the existing transit peptides. A seed-expressing promoter can be used for regulated expression of the ClTE13-σDNA and the genes from the shown genomic clones are regulated in a tissue-specific manner even by the own promoters. Position effects can be compensated for by the necessary number of transformants. Thus, the DNA sequences according to the invention in the form of the cDNAs presented and in the form of the isolated genes of the genomic clones shown are suitable for the production of medium-chain fatty acids in transgenic plants,

preferably oil plants. An optimization of the content of medium chain specific fatty acids can be achieved by additional transfer of components of the fatty acid synthase system, e.g. DNA sequences for ACP2, a specific KAS,

Ketoreductase and enoyl reductase, for example from Cuphea lanceolata, in rapeseed. In addition, it is expected that the cytoplasmic localized LPA-AT (lysophosphatidic acid acyltransferase) e.g. from Cuphea lanceolata in rapeseed results in a significant increase in the content of medium-chain fatty acids in the triacylglycerides.

The following examples serve to explain the

Invention.

Examples

The plant material used in the context of the present invention consisted of the species Brassica napus (Cruciferae) (oilseed rape), Nicotiana tabacum (Solanaceae) (tobacco) and Cuphea lanceolata (Lythraceae) (lancet-leaved quiver flower or hump flower). The summer rape variety Drakkar and the tobacco line Petit Havanna SRI were used for the transformation.

example 1

Production of acyl- [ACP] thioesterase cDNAs from Cuphea lanceolata

First, a cDNA library from Cuphea lanceolata (wild type) was produced. The cDNA library was produced according to the manufacturer's instructions (Stratagene) using the cDNA ZAP ^® synthesis kit. The starting material for the synthesis of the cDNAs was polyA ⁺ mRNA from isolated, about two to three week old immature embryos. The cDNA obtained in this way Bank has a size of 9.6 × 10 ⁵ recombinant phages with a share of approximately 50% clones, the insertions of which exceed 500 bp.

A specific hybridization probe for acyl [ACP] thioesterase was prepared to screen the cDNA library described above. Suitable degenerate oligonucleotides were first required for this. Voelker et al (1992), Science 257, pages 72-74 show a DNA sequence of a plant acyl [ACP] thioesterase. Oligonucleotide primers were derived and synthesized from some regions of the sequence that are as little degenerate as possible. Primer 3532, which corresponds to amino acids 277 to 284 of the acyl [ACP] thioesterase from Umbellularia californica, is in PCR reactions in connection with primer 2740 (a modified oligo-dT primer with interfaces for the restriction enzymes BstBI, BamHI , HindIII and SalI) suitable for

Amplification of a specific hybridization probe.

Figure 1 shows the sequences for the PCR reactions

used synthetic oligonucleotide primers 3532 and 2740. The orientation of the oligonucleotide primers is from 5 'to 3' for primer 3532, from 3 'to 5' for primer 2740.

Starting from 1 μg poly A ⁺ RNA, reverse transcriptase (Boehringer Mannheim GmbH) from Avian Myeloblastosis Virus (AMV) was used to carry out cDNA synthesis for 30 minutes at a temperature of 37 ° C. For this purpose, the 3'-oligonucleotide primer (2740) shown in FIG. 1 was used to synthesize the specific hybridization probe. After inactivation of the reverse transcriptase by heating for 5

Minutes at a temperature of 95 ° C in the same reaction carried out the PCR reaction with 50 pmol of each primer and 4 units final concentration Ampli Taq ^® polymerase (Perkin Elmer Cetus). The reactions were carried out under the following conditions: a) Buffer conditions: 10 mM Tris-HCl, pH 8.0; 50 mM KCl; 1.5 mM MgCl; 0.01% gelatin and 5 mM dNPPs, 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 denaturation, 2 minutes at 50 ° C 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.

The resulting amplification products were then cloned. For this purpose, protruding single-stranded DNA of the PCR products was filled in using Klenow polymerase and then phosphorylated with polynucleotide kinase (Sambrook et al, A

Laboratory Manual, 2nd edn., (1989)). The PCR products are purified according to standard Sambrook et al (supra) protocols by agarose gel electrophoresis, gel elution, extraction with phenol / chloroform and final precipitation with isopropanol. The thus purified DNA was ligated into Smal ^® cleaved pBluescript vector DNA and cloned.

The cloned PCR fragment was then analyzed using the method of Sanger et al, Proc.Natl.Acad.Sci. 74, pages 5463-5467 sequenced. DNA sequencing was partially radioactive using the Sequencing ^® kit or a Pharmacia Automated Laser Fluorescent ALF ^® DNA sequencer. The sequences were analyzed using the computer software of the University of Wisconsin Genetics Computer Group (Devereux et al, Nucl.Acids Res. 12, pages 387-395.

As from the sequence of the 530 bp acyl [ACP] thioesterase PCR product shown as SEQ ID NO: 8 in the sequence listing,

PCR42, it can be seen, could use a PCR product

significant homology to the starting sequence can be synthesized. Under the DNA sequence is the corresponding one

Amino acid shown.

The 530 bp PCR product described above was used as a probe to isolate acyl [ACP] thioesterase cDNAs. For this purpose, the cDNA library described above was screened with the PCR product and 11 cDNAs were isolated, which can be divided into three classes based on their sequences.

In this context, the cDNA clones C1TE13, ClTE5 and ClTE12 were isolated, which each represent one of the three classes. Your DNA sequences as well as those derived from them

Amino acid sequences are shown as SEQ ID NO: 1,2 and 3 in the sequence listing.

Example 2

Production of αenomic clones of acyl [ACP] thioesterase from Cuphea lanceolata

For this, genomic DNA was isolated from young leaves of Cuphea lanceolata (SL Della Porta, J. Wood and JB Hicks, A plant DNA minipreparation: Version II, Plant.Mol.Biol.Rep., 1, pages 19-21 (1983)) . The DNA was then partially cleaved with the restriction enzyme Sau3A, after which DNA fragments of the order of magnitude between 11000 bp and 19000 bp were cloned into the Xhol-cleaved vector FIX II (Stratagene) after the cleavage sites involved had two each

Nucleotides were partially filled. The non-replicated genomic DNA library represented 5.4 times the Cuphea lanceolata genome. With the C1TE5-CDNA as a probe, 103 hybridizing phages were then generated from this bank

isolated, 40 of them further purified and 23 mapped. These can be divided into four classes. For this purpose, reference is made to FIG. 2, which shows the genomic clones CITEgl, ClTEg16, ClTEg4 and ClTEg7, which are assigned to different classes. Suitable DNA fragments from the genomic clones were sequenced. Their DNA sequences and the amino acid sequences derived from them are shown as SEQ ID NO: 4,5,6 and in the sequence listing. Example 3

Transformation of rapeseed and tobacco

Suitable expression vectors were made. For this purpose, a chimeric gene consisting of the ClTE13-CDNA, the promoter ACP23 and the terminator tnos was inserted into the binary vector pRE1. This results in the vector pNBM99-2TE. The vector pNBM99-3TE was produced by inserting the ClTE13-CDNA into the binary vector pRE9 after the constructed double promoter of the 35S RNA from the Cauliflower Mosaic Virus (p35S / Δp35S). Additional expression vectors were generated using the genomic clones CITEgl and ClTEg16 and the binary vector pREl. The expression vectors thus obtained were designated pNBM99-TEgl and pNBM99-TEgl6.

Rapeseed was transformed using Agrobacterium tumefaciens according to the protocol of De Block et al, Plant

Physiol. 91. Pages 694-701 based on hypocotyl pieces. The Agrobacterium strain GV3101 C58C1 Rif ^r (Van Larebeke et al, Nature 252. Pages 169-170 (1974)) was used with the Ti plasmid pMP90RK (C. Koncz, J. Schell, Mol.Gen.Genet. 204.

Pages 383-396 (1986)) and the expression vectors mentioned above. The selection for kanamycin resistance was carried out with 50 μg (medium A5), later with 15 μg kanamycin

(Monosulphate, Sigma K-4000) per milliliter of medium (medium A6 and A8). The transformation rate was 10%, based on the number of hypocotyl pieces laid out. It is based on the

Verification of the transformation using Southern blot

(Sambrook et al, supra.) And (PCR-Edwards et al, Nucl.Acids Res. 19, page 1349, (1991)).

The transformation of tobacco was carried out with the above-mentioned vector system according to the "leaf-disk" transformation method according to RB Horsch et al, Pant. Mol. Bio. 20, pages 1229-1231, (1985)). The analysis of the fatty acids in the transformed plants was carried out according to the method of W. Thies, Z. Plant Breeding 65, pages 181-202 (1971) and W. Thies, Proc. 4th Int.Rape Seed Con, 4th-8th June, Gießen, pages 275-282 (1974) using a Hewlett-Packard gas chromatograph (model HP5890 series II with FID) and a 10 m long capillary column (FS-FFAP-CB-0.25 from CS-Chromatographie-Service GmbH, Langerwehe) carried out. With hydrogen as the carrier gas, the fatty acid methyl esters were separated in a temperature gradient from 140 ° C to 208 ° C with a temperature increase of 20 ° C per minute. After the final temperature of 208 ° C had been reached, the separation wasotermic for seven minutes. The injector and detector were operated constantly at 250 ° C.

If molecular biological work is not adequately described in any way, this has been done

Standard methods as described in Sambrook et al, supra.

Claims

claims

1. DNA sequences required for a medium chain specific

Encoding acyl [ACP] thioesterase, and the alleles and derivatives of these DNA sequences.

2. DNA sequences according to claim 1, characterized in that they are isolated from plants.

3. DNA sequences according to claim 2, characterized in that they are isolated from Cuphea lanceolata.

4. DNA sequences according to one of claims 1 to 3, characterized in that they code for a C _{10: 0} -specific acyl- [ACP] thioesterase or essentially C _{10: 0} -specific acyl- [ACP] thioesterase .

5. DNA sequences according to one of claims 1 to 3, characterized in that they code for a C _{14: 0} -specific acyl- [ACP] thioesterase or essentially C _{14: 0} -specific acyl- [ACP] thioesterase .

6. Genomic clones that are the complete gene for one

contain medium-chain specific acyl- [ACP] thioesterase and the alleles and derivatives of this gene.

7. Genomic clones according to claim 6, characterized in that the complete gene in addition to the structural gene comprises the promoter sequence and other regulatory sequences.

8. Genomic clones according to claim 6, characterized in that they are isolated from genomic plant DNA.

9. Genomic clones according to claim 8, characterized

characterized in that the plant DNA of Cuphea

lanceolata comes from.

10. Genomic clone according to one of claims 6 to 9, characterized in that the medium chain-specific acyl- [ACP] thioesterase is a C _{10: 0} -specific acyl- [ACP] thioesterase or essentially C _{10: 0} -specific acyl- [ACP] thioesterase.

11. Genomic clone according to one of claims 6 to 9,

characterized in that the medium chain specific acyl [ACP] thioesterase is a C _{14: 0} specific acyl [ACP] thioesterase or essentially C _{14: 0} specific acyl [ACP] thioesterase.

12. ClTEg4 (DSM 8493) and ClTEg7 genomic clones

(DSM 8494).

13. Plasmids pNBM99-TEg1 (DSM 8477) and pNBM99-TEg16

(DSM 8478).

14. A process for the production of plants, plant parts and plant products which produce medium-chain fatty acids, in which a DNA sequence according to one of claims 1 to 5 or one of the genomic

Clones or plasmids according to any one of claims 6 to 13 gene is transferred by genetic engineering.

15. The method according to claim 14, characterized in that the plants, plant parts and plant products

Capric acid (C _{10: 0} ) or essentially capric acid

(C _{10: 0} ) produce.

16. The method according to claim 14, characterized in that the plants, parts of plants and plant products

Myristic acid (C _{14: 0} ) or essentially myristic acid

(C _{14: 0} ) produce.

17. The method according to claim 15 or 16, characterized in that in addition DNA sequences for ACP2, a specific KAS, ketoreductase and

Enoyl reductase and optionally encoding lysophosphatidic acyltransferase are transmitted.

18. The method according to any one of claims 14 to 17, characterized in that the DNA sequence or DNA sequences and the genes by microinjection, electroporation, particle gun, the swelling of plant parts in DNA solutions, pollen or pollen tube transformation, transmission of corresponding recombinant Ti plasmids or Ri plasmids from Agrobacterium

tumefaciens, liposome-mediated transfer or is transmitted by plant viruses.

19. Use of a DNA sequence according to one of claims 1 to 5 or one of the genomic clones or

Plasmids according to one of Claims 6 to 13, derived from genes for transferring genes for medium-chain-specific acyl- [ACP] thioesterases in plants.

20. Use according to claim 19, characterized in that the medium chain-specific acyl [ACP] thioesterase is a C _{10: 0} -specific acyl [ACP] thioesterase or essentially C _{10: 0} -specific acyl [ACP] thioesterase is.

21. Use according to claim 19, characterized in that the medium chain-specific acyl [ACP] thioesterase is a C _{14: 0} -specific acyl [ACP] thioesterase or in

essential C _{14: 0} -specific acyl [ACP] thioesterase.

22. Plants, parts of plants and plant products produced by a process of claims 14 to 18.