CA2382444A1 - Dna construct and its use - Google Patents
Dna construct and its use Download PDFInfo
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- CA2382444A1 CA2382444A1 CA002382444A CA2382444A CA2382444A1 CA 2382444 A1 CA2382444 A1 CA 2382444A1 CA 002382444 A CA002382444 A CA 002382444A CA 2382444 A CA2382444 A CA 2382444A CA 2382444 A1 CA2382444 A1 CA 2382444A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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Abstract
A DNA construct comprising in the 5' to 3' direction of transcription operably linked a promoter region directing transcription to the seed of an oilseed plant, a nucleotide sequence coding for at least one peptide with enzyme activity necessary for keto group containing xanthophyll production and esterification in an oilseed plant and a transcriptional termination region is disclosed. The DNA construct may additionally comprise a nucleotide sequence coding for a transit peptide directing the translated fusion polypeptide to the chloroplast of the oilseed plant. The peptide with enzyme activity is preferably a peptide with .beta.-carotene C-4-oxygenase activity, e.g. from the alga <aematococcus pluvialis. Comprised by the invention are also a transgenic oilseed plant cell, e.g. of rape, sunflower, soybean or mustard origin, and a transgenic oilseed plant-produced xanthophyll, such as canthaxanthin or astaxanthin, and also astaxanthin esters.
Description
W~ ~l/20~11 CA 02382444 2002-02-19 PCT/SE00/01767 DNA construct and its use.
The present invention relates to a new DNA construct for transformation into oilseed plants. The DNA construct comprises nucleotide sequences encoding peptides with enzyme activities necessary for the high-level production and esterification of keto group-containing xanthophylls in oilseed plants.
Background of the invention Carotenoids are produced de novo by plants, fungi, algae and some bacteria. A
number of biosynthetic steps are needed for the biological production of the carotenoids.
There are two chemically different groups of carotenoids, namely carotenes containing only carbon and hydrogen molecules and xanthophylls containing oxygen in the molecule in addition to carbon and hydrogen.
The xanthophylls, and particularly astaxanthin (3,3'-dihydroxy-(3-~3-carotene-4,4'-dione), are often colored pigments and are used as such or as anti-oxidants.
Carotenes are biological precursors for the production of the oxygen-containing xanthophylls. There are two types of enzymes responsible for the introduction of hydroxy groups and keto groups into the carotenes, namely hydroxylases and ketolases, respectively.
The keto group-containing xanthophyll astaxanthin, which has keto and hydroxy groups, is biosynthetically produced from beta-carotene.
Large-scale production of xanthophylles from natural sources is at present performed by AstaCarotene AB, Gustavsberg, Sweden, by cultivation of the alga Haematococcus pluvialis for the production of astaxanthin in esterified form.
It would be desirable to be able to produce keto group-containing xanthophylls particularly astaxanthin, in oilseed plants. Oilseed plants have naturally (3-carotene hydroxylases but lack (3-carotene C-4-oxygenase enzymes or ketolases.
Description of the invention The present invention provides DNA constructs enabling and promoting production of keto group containing xanthophylls, especially astaxanthin, in oilseed plants, such as rape, sunflower, soybean and mustard. The DNA construct is transformed into the oilseed plant cell for expression of a protein or fused protein which has an enzyme activity enabling keto group insertion into a carotene or hydroxy carotene for the biosynthetic production of a keto group containing xanthophyll, such as cantaxanthin (~3,[3-carotene-4,4'-dione) and/or astaxanthin. Use is thus made of the biosynthetic pathway of the oilseed plant to w~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 produce carotenoids. The naturally occurring synthesis of carotenoids involves a number of enzymes, namely 1-D-deoxyxylulose S-phosphate synthase, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, lycopene beta-s cyclase, (3-carotene hydroxylase, and (3-carotene C-4-oxygenase. Genes coding for peptides having these enzymatic activities may be inserted into the DNA construct of the invention, one or several per construct, to promote high-level production in the transgenic oilseed plant.
In case only one enzyme coding gene is inserted per plant, two or more plants may be sexually interbred to produce plants containing all the desired enzyme activities.
Thus, the present invention is directed to a DNA construct comprising in the 5' to 3' direction of transcription operably linked a promoter region directing transcription to the seed of an oilseed plant, a nucleotide sequence coding for at least one peptide with enzyme activity necessary for keto group containing xanthophyll production and esterification in an oilseed plant and a transcriptional termination region.
In a preferred embodiment of the invention the DNA construct additionally comprises between the promoter region and the nucleotide sequence coding for at least one peptide with enzyme activity a nucleotide sequence coding for a transit peptide directing the translated fusion polypeptide to the chloroplast of the oilseed plant.
The DNA construct is preferably such that the promoter is a napin promoter, the peptide with enzyme activity necessary for keto group containing xanthophyll production is selected from the group consisting of peptides with 1-D-deoxyxylulose 5-phosphate synthase, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, lycopene beta-cyclase, (3-carotene hydroxylase, and (3-carotene C-4-oxygenase activity. To promote esterification of astaxanthin a nucleotide sequence coding for a peptide with acyl transferase activity may be included in the group.
In a preferred embodiment of the DNA construct according to the invention the nucleotide sequence coding for a peptide with enzyme activity is a nucleotide sequence coding for a N-terminally truncated (3-carotene C-4-oxygenase gene from the alga Haematococcus pluvialis.
An example of the DNA construct of the invention is presented in the sequence listing as SEQ ID NO:1 and in Fig.l.
W~ ~1/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 The present invention is also directed to a transgenic oilseed plant cell comprising the DNA construct of the invention, and preferably the oilseed plant is selected from the group consisting of rape, sunflower, soybean and mustard.
The invention is additionally directed to transgenic oilseed plant-produced xanthophyll, e.g. canthaxanthin and astaxanthin.
A preferred aspect of the invention is directed to transgenic oilseed plant-produced astaxanthin esters.
The present invention will now be illustrated with reference to the DNA
construct disclosed in the sequence listing and in Fig.l, and the following description of embodiments. However, the invention is not limited to these exemplifications.
Short description of the drawings Fig.l illustrates the nucleotide sequence of the DNA construct comprising the napin promoter, the chloroplast localization signal, the N-terminally truncated (3-carotene C-4-oxygenase gene and the termination sequence, and the deduced amino acid sequences of the transit peptide and the (3-carotene C-4-oxygenase.
Description of embodiments The invention is illustrated by production of astaxanthin in the seed of oilseed rape. The astaxanthin produced in the seed of the transgenic plant is extracted as part of the extracted oil. By use of conventionally used protocols for Agrobacterium tumefaciens mediated transformation such as described by (Hoekema et a1.1983, An et al.
1986, Fry et al.
1987, DeBlock et al. 1988, Radke et a1.1988, or Moloney et al. 1989) transgenic plants are produced having a chimeric DNA construct that is genetically inherited and is able to produce astaxanthin. The nucleotide sequence of the chimeric DNA construct consist of four parts of different genetic origin namely: (1) a promoter, (2) a localization signal, (3) a (3-carotene C-4-oxygenase coding region and (4) a termination sequence.
The napin promoter directs transcription to the seed of oilseed rape (Stalberg et al 1996). This promoter was coupled to a localization signal similar but not identical to a transit peptide (TP) of Rbcsla (Krebbers, 1988) that directs the translated product of a fused gene to the chloroplast. The promoter and the TP sequence were ligated to a part of the coding sequence of a ketolase gene BCK (Kajiwara et al. 1995). This enzyme oxygenates (3-carotene to canthaxanthin, (Fraser et al. 1997). The chimeric DNA construct was then coupled to a suitable termination sequence, e.g. that of the Agrobacterium tumefaciens nopaline synthase gene (the nos 3' end)(Bevan et al. 1983), as illustrated in Fig.l.
VV0 ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 Cellular storage of Astaxantin The storage of large amounts of free astaxanthin in plants will be difficult due to toxic effects of the molecule as it intercalates in the plant membranes. An effective esterification of astaxanthin to fatty acids enables storage of the esterified molecules in triacylglycerol containing oleosomes. Thus, an acyl transferase can be claimed to be of fundamental importance for the process, as is proteins that can mediate transport of different forms of astaxanthin from the chloroplast to the vesicles.
Seauences and oligonucleotides used in the construction of the DNA construct 1. Napin promoter (GeneBankACCESSIONNo. J02798) This promoter sequence, a 1145 base pair fragment including the 5' leader sequence has a unique HindIII site at the 5' end. The 3' end was synthesized with an additionally 6 nucleotide BamHI site.
2. Transit peptide similar to RBCSI a (GeneBank ACCESSION No. X13611, X14565) The transit peptide (TP) was amplified by PCR from -28 to the end of the transit cleavage aa=54/55 site of the Rbcsla gene. The 5' end was synthesized with a Barnl-iI site and similarly the 3' sequence was synthesized with a XbaI site. The two following oligonucleotides were used for the PCR amplification.
BamHI
The present invention relates to a new DNA construct for transformation into oilseed plants. The DNA construct comprises nucleotide sequences encoding peptides with enzyme activities necessary for the high-level production and esterification of keto group-containing xanthophylls in oilseed plants.
Background of the invention Carotenoids are produced de novo by plants, fungi, algae and some bacteria. A
number of biosynthetic steps are needed for the biological production of the carotenoids.
There are two chemically different groups of carotenoids, namely carotenes containing only carbon and hydrogen molecules and xanthophylls containing oxygen in the molecule in addition to carbon and hydrogen.
The xanthophylls, and particularly astaxanthin (3,3'-dihydroxy-(3-~3-carotene-4,4'-dione), are often colored pigments and are used as such or as anti-oxidants.
Carotenes are biological precursors for the production of the oxygen-containing xanthophylls. There are two types of enzymes responsible for the introduction of hydroxy groups and keto groups into the carotenes, namely hydroxylases and ketolases, respectively.
The keto group-containing xanthophyll astaxanthin, which has keto and hydroxy groups, is biosynthetically produced from beta-carotene.
Large-scale production of xanthophylles from natural sources is at present performed by AstaCarotene AB, Gustavsberg, Sweden, by cultivation of the alga Haematococcus pluvialis for the production of astaxanthin in esterified form.
It would be desirable to be able to produce keto group-containing xanthophylls particularly astaxanthin, in oilseed plants. Oilseed plants have naturally (3-carotene hydroxylases but lack (3-carotene C-4-oxygenase enzymes or ketolases.
Description of the invention The present invention provides DNA constructs enabling and promoting production of keto group containing xanthophylls, especially astaxanthin, in oilseed plants, such as rape, sunflower, soybean and mustard. The DNA construct is transformed into the oilseed plant cell for expression of a protein or fused protein which has an enzyme activity enabling keto group insertion into a carotene or hydroxy carotene for the biosynthetic production of a keto group containing xanthophyll, such as cantaxanthin (~3,[3-carotene-4,4'-dione) and/or astaxanthin. Use is thus made of the biosynthetic pathway of the oilseed plant to w~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 produce carotenoids. The naturally occurring synthesis of carotenoids involves a number of enzymes, namely 1-D-deoxyxylulose S-phosphate synthase, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, lycopene beta-s cyclase, (3-carotene hydroxylase, and (3-carotene C-4-oxygenase. Genes coding for peptides having these enzymatic activities may be inserted into the DNA construct of the invention, one or several per construct, to promote high-level production in the transgenic oilseed plant.
In case only one enzyme coding gene is inserted per plant, two or more plants may be sexually interbred to produce plants containing all the desired enzyme activities.
Thus, the present invention is directed to a DNA construct comprising in the 5' to 3' direction of transcription operably linked a promoter region directing transcription to the seed of an oilseed plant, a nucleotide sequence coding for at least one peptide with enzyme activity necessary for keto group containing xanthophyll production and esterification in an oilseed plant and a transcriptional termination region.
In a preferred embodiment of the invention the DNA construct additionally comprises between the promoter region and the nucleotide sequence coding for at least one peptide with enzyme activity a nucleotide sequence coding for a transit peptide directing the translated fusion polypeptide to the chloroplast of the oilseed plant.
The DNA construct is preferably such that the promoter is a napin promoter, the peptide with enzyme activity necessary for keto group containing xanthophyll production is selected from the group consisting of peptides with 1-D-deoxyxylulose 5-phosphate synthase, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, lycopene beta-cyclase, (3-carotene hydroxylase, and (3-carotene C-4-oxygenase activity. To promote esterification of astaxanthin a nucleotide sequence coding for a peptide with acyl transferase activity may be included in the group.
In a preferred embodiment of the DNA construct according to the invention the nucleotide sequence coding for a peptide with enzyme activity is a nucleotide sequence coding for a N-terminally truncated (3-carotene C-4-oxygenase gene from the alga Haematococcus pluvialis.
An example of the DNA construct of the invention is presented in the sequence listing as SEQ ID NO:1 and in Fig.l.
W~ ~1/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 The present invention is also directed to a transgenic oilseed plant cell comprising the DNA construct of the invention, and preferably the oilseed plant is selected from the group consisting of rape, sunflower, soybean and mustard.
The invention is additionally directed to transgenic oilseed plant-produced xanthophyll, e.g. canthaxanthin and astaxanthin.
A preferred aspect of the invention is directed to transgenic oilseed plant-produced astaxanthin esters.
The present invention will now be illustrated with reference to the DNA
construct disclosed in the sequence listing and in Fig.l, and the following description of embodiments. However, the invention is not limited to these exemplifications.
Short description of the drawings Fig.l illustrates the nucleotide sequence of the DNA construct comprising the napin promoter, the chloroplast localization signal, the N-terminally truncated (3-carotene C-4-oxygenase gene and the termination sequence, and the deduced amino acid sequences of the transit peptide and the (3-carotene C-4-oxygenase.
Description of embodiments The invention is illustrated by production of astaxanthin in the seed of oilseed rape. The astaxanthin produced in the seed of the transgenic plant is extracted as part of the extracted oil. By use of conventionally used protocols for Agrobacterium tumefaciens mediated transformation such as described by (Hoekema et a1.1983, An et al.
1986, Fry et al.
1987, DeBlock et al. 1988, Radke et a1.1988, or Moloney et al. 1989) transgenic plants are produced having a chimeric DNA construct that is genetically inherited and is able to produce astaxanthin. The nucleotide sequence of the chimeric DNA construct consist of four parts of different genetic origin namely: (1) a promoter, (2) a localization signal, (3) a (3-carotene C-4-oxygenase coding region and (4) a termination sequence.
The napin promoter directs transcription to the seed of oilseed rape (Stalberg et al 1996). This promoter was coupled to a localization signal similar but not identical to a transit peptide (TP) of Rbcsla (Krebbers, 1988) that directs the translated product of a fused gene to the chloroplast. The promoter and the TP sequence were ligated to a part of the coding sequence of a ketolase gene BCK (Kajiwara et al. 1995). This enzyme oxygenates (3-carotene to canthaxanthin, (Fraser et al. 1997). The chimeric DNA construct was then coupled to a suitable termination sequence, e.g. that of the Agrobacterium tumefaciens nopaline synthase gene (the nos 3' end)(Bevan et al. 1983), as illustrated in Fig.l.
VV0 ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 Cellular storage of Astaxantin The storage of large amounts of free astaxanthin in plants will be difficult due to toxic effects of the molecule as it intercalates in the plant membranes. An effective esterification of astaxanthin to fatty acids enables storage of the esterified molecules in triacylglycerol containing oleosomes. Thus, an acyl transferase can be claimed to be of fundamental importance for the process, as is proteins that can mediate transport of different forms of astaxanthin from the chloroplast to the vesicles.
Seauences and oligonucleotides used in the construction of the DNA construct 1. Napin promoter (GeneBankACCESSIONNo. J02798) This promoter sequence, a 1145 base pair fragment including the 5' leader sequence has a unique HindIII site at the 5' end. The 3' end was synthesized with an additionally 6 nucleotide BamHI site.
2. Transit peptide similar to RBCSI a (GeneBank ACCESSION No. X13611, X14565) The transit peptide (TP) was amplified by PCR from -28 to the end of the transit cleavage aa=54/55 site of the Rbcsla gene. The 5' end was synthesized with a Barnl-iI site and similarly the 3' sequence was synthesized with a XbaI site. The two following oligonucleotides were used for the PCR amplification.
BamHI
5' primer: TP1 5'AGAC GGATCC TCAGTCACACAAAGAGTA 3' SacI XbaI
3' primer: TP2 5'GTTC GAGCTC TCTAGA CATGCAGTTAACGC 3' 3. BCK (carotene C-4 oxygenase) (GenebankACCESSIONNo. D45881) The BCK fragment was amplified by PCR including a 5' XbaI site and was ligated to the TP already described. The 5' primer (BCK1) used for PCR, is homologous to the BCK sequence from nucleotide 264 and the 3' oligonucleotide (Ax40) ends with a stop codon and was synthesized with a SacI restriction site for cloning. The synthesized fragment was fused to the TP as shown in Fig 1.
Oligonucleotides used for PCR:
XbaI
5' primer: BCK1 5'ACAG TCTAGA ATGCCATCCGAGTCGTCA 3' SacI
3'primer: AX40 5'CACCGAGCTCCATGACACTCTTGTGCAGA 3' V~~ ~l/2~011 CA 02382444 2002-02-19 PCT/SE00/01767 Description of SEQ ID NO:1 and SEQ ID N0:2 The sequences shown i Fig.l are the same as the two sequences which are shown in the sequence listing.
The SEQ ID NO:1 is a nucleotide sequence composed of the following features:
S Nucleotide No.
Cloning site HindIII 1-6 Napin Promoter 1-1145 Cloning site BamHI 1146-1151 Transit peptide leader 1152-1178 Transit peptide coding 1179-1347 Cloning site XbaI 1348-1353 ~i-carotene C-4-oxygenase 1354-2217 (3-carotene C-4-oxygense 3' untranslated 2218-2266 Cloning site SacI 2267-2272 Nopaline synthetase termination 2273-2536 Cloning site EcolZI 2538-2543 The SEQ ID NO: 2 is a deduced amino acid sequence of the fusion protein of the transit peptide and the peptide with (3-carotene C-4-oxygenase activity.
W~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 References An G, Watson BD, Chiang CC (1986), Transformation of tobacco, tomato, potato and S Arabidopsis-thaliana using a binary vector system. Plant Physiology 81 (1) 301-305.
Bevan M, BarnesWM and Chilton MD (1983). Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 11 (2), 369-385 .
DeBlock M, DeBrouwer D, Terming P (1989). Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the BAR and NEO
genes in transgenic plants Plant Physiology 91:2, 694-701.
Fraser PD, Miura Y, Misawa N, (1997). In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem. Mar 7;272(10):6128-35.
Fry J, Barnason A, and Horsch RB, (1987). Transformation of Brassica napus with Agrobacteriium tumefaciens based vectors. Plant Cell Reports 6:321-325.
Hoekema A, Hirsch PR, Hooykas PJJ Schilperoort, (1983). A binary vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid.
Nature vol 303, 179-180.
Josefsson LG, Lenman M, Ericson ML and Rask L, (1987). Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus. J. Biol. Chem. 262 (25), 12196-12201.
Kajiwara S, KakizonoT, Saito T, Kondo K, OhtaniT, Nishio N, Nagai S and Misawa N.
(1995). Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli Plant Mol. Biol.
29 (2), 343-352.
W~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 Krebbers E, Seurinck J, Herdies L, Cashmore AR and Timko MP, (1988). Four genes in two diverged subfamilies encode the rubulose-l, S-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana Plant Mol. Biol. 11, 745-759.
Moloney M, Walker JM and Sharma KK, (1989). High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242.
Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988), Transformation of Brassica napus using Agrobacterium tumefaciens -Developmentally regulated Expression of a reintroduced napin gene. TAG, 75: (5) 685-694 .
Pua E-C, Mehra-Palta A, Nagy F and Chua N-H, (1987). Transgenic plants of Brassica napus.
Biotechnology vol 5, 81 S-817.
Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L (1996). Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199(4):515-9.
SEQUENCE LISTING
<110> AstaCarotene AB
<120> DNA construct and its use <130> 29295-AstaCarotene <140>
<141>
<160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 2543 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: napin promoter + choroplast localization signal + beta-carotene C-4 oxygenase coding sequence + termination sequence <220>
<221> promoter <222> (1)..(1145) <220>
<221> transit-peptide <222> (1179)..(1347) <220>
<221> CDS
<222> (1179)..(2217) <220>
<221> terminator <222> (2273)..(2536) <400> 1 aagctttctt catcggtgat tgattccttt aaagacttat gtttcttatc ttgcttctga 60 ggcaagtatt cagttaccag ttaccactta tattctggac tttctgactg catcctcatt 120 tttccaacat tttaaatttc actattggct gaatgcttct tctttgagga agaaacaatt 180 cagatggcag aaatgtatca accaatgcat atatacaaat gtacctcttg ttctcaaaac 240 atctatcgga tggttccatt tgctttgtca tccaattagt gactacttta tattattcac 300 tcctctttat tactattttc atgcgaggtt gccatgtaca ttatatttgt aaggattgac 360 gctattgagc gtttttcttc aattttcttt attttagaca tgggtatgaa atgtgtgtta 420 gagttgggtt gaatgagata tacgttcaag tgaagtggca taccgttctc gagtaaggat 480 gacctaccca ttcttgagac aaatgttaca ttttagtatc agagtaaaat gtgtacctat 540 aactcaaatt cgattgacat gtatccattc aacataaaat taaaccagcc tgcacctgca 600 tccacatttc aagtattttc aaaccgttcg gctcctatcc accgggtgta acaagacgga 660 ttccgaattt ggaagatttt gactcaaatt cccaatttat attgaccgtg actaaatcaa 720 ctttaacttc tataattctg attaagctcc caatttatat tcccaacggc actacctcca 780 aaatttatag actctcatcc ccttttaaac caacttagta aacgtttttt tttttaattt 840 tatgaagtta agtttttacc ttgtttttaa aaagaatcgt tcataagatg ccatgccaga 900 acattagcta cacgttacac atagcatgca gccgcggaga attgtttttc ttcgccactt 960 gtcactccct tcaaacacct aagagcttct ctctcacagc acacacatac aatcacatgc 1020 gtgcatgcat tattacacgt gatcgccatg caaatctcct ttatagccta taaattaact 1080 catccgcttc actctttact caaaccaaaa ctcatcaata caaacaagat taaaaacata 1140 cacgaggatc ctcagtcaca caaagagtaa agaagaaca atg get tcc tct atg 1194 Met Ala Ser Ser Met ctc tct tcc get act atg gtt gcc tct ccg get cag gcc act atg gtc 1242 Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala Gln Ala Thr Met Val get cct ttc aac gga ctt aag tcc tcc get gcc ttc cca gcc acc cgc 1290 Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala Phe Pro Ala Thr Arg aag get aac aac gac att act tcc atc aca agc aac ggc gga cgc gtt 1338 Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val aac tgc atg tct aga atg cca tcc gag tcg tca gac gca get cgt cct 1386 Asn Cys Met Ser Arg Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro gcg cta aag cac gcc tac aaa cct cca gca tct gac gcc aag ggc atc 1434 Ala Leu Lys His Ala Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile acg atg gcg ctg acc atc att ggc acc tgg acc gca gtg ttt tta cac 1482 Thr Met Ala Leu Thr Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His gca ata ttt caa atc agg cta ccg aca tcc atg gac cag ctt cac tgg 1530 Ala Ile Phe Gln Ile Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp ttg cct gtg tcc gaa gcc aca gcc cag ctt ttg ggc gga agc agc agc 1578 Leu Pro Val Ser Glu Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser cta ctg cac atc get gca gtc ttc att gta ctt gag ttc ctg tac act 1626 Leu Leu His Ile Ala Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr ggt cta ttc atc acc aca cat gac gca atg cat ggc acc ata get ttg 1674 Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile Ala Leu agg cac agg cag ctc aat gat ctc ctt ggc aac atc tgc ata tca ctg 1722 Arg His Arg Gln Leu Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu tac gcc tgg ttt gac tac agc atg ctg cat cgc aag cac tgg gag cac 1770 Tyr Ala Trp Phe Asp Tyr Ser Met Leu His Arg Lys His Trp Glu His cac aac cat act ggc gaa gtg ggg aaa gac cct gac ttc cac aag gga 1818 His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp Phe His Lys Gly aat ccc ggc ctt gtc ccc tgg ttc gcc agc ttc atg tcc agc tac atg 1866 Asn Pro Gly Leu Val Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met tcc ctg tgg cag ttt gcc cgg ctg gca tgg tgg gca gtg gtg atg caa 1914 Ser Leu Trp Gln Phe Ala Arg Leu Ala Trp Trp Ala Val Val Met Gln atg ctg ggg gcg ccc atg gca aat ctc cta gtc ttc atg get gca gcc 1962 Met Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala cca atc ttg tca gca ttc cgc ctc ttc tac ttc ggc act tac ctg cca 2010 Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro cac aag cct gag cca ggc cct gca gca ggc tct cag gtg atg gcc tgg 2058 His Lys Pro Glu Pro Gly Pro Ala Ala Gly Ser Gln Val Met Ala Trp ttc agg gcc aag aca agt gag gca tct gat gtg atg agt ttc ctg aca 2106 Phe Arg Ala Lys Thr Ser Glu Ala Ser Asp Val Met Ser Phe Leu Thr tgc tac cac ttt gac ctg cac tgg gag cac cac aga tgg ccc ttt gcc 2154 Cys Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro Phe Ala ccc tgg tgg cag ctg ccc cac tgc cgc cgc ctg tcc ggg cgt ggc ctg 2202 Pro Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu gtg cct gcc ttg gca tgacctggtc cctccgctgg tgacccagcg tctgcacaag 2257 Val Pro Ala Leu Ala agtgtcatgg agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag 2317 attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 2377 gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 2437 agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 2497 taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattc 2543 <210> 2 <211> 346 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: deduced fusion protein of transit peptide + peptide with beta-carotene C-4 oxygenase activity <400> 2 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val Asn Cys Met Ser Arg Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Ala Leu Lys His Ala Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Ser Met Leu His Arg Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala Arg Leu Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly Pro Ala Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr Ser Glu Ala Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala
3' primer: TP2 5'GTTC GAGCTC TCTAGA CATGCAGTTAACGC 3' 3. BCK (carotene C-4 oxygenase) (GenebankACCESSIONNo. D45881) The BCK fragment was amplified by PCR including a 5' XbaI site and was ligated to the TP already described. The 5' primer (BCK1) used for PCR, is homologous to the BCK sequence from nucleotide 264 and the 3' oligonucleotide (Ax40) ends with a stop codon and was synthesized with a SacI restriction site for cloning. The synthesized fragment was fused to the TP as shown in Fig 1.
Oligonucleotides used for PCR:
XbaI
5' primer: BCK1 5'ACAG TCTAGA ATGCCATCCGAGTCGTCA 3' SacI
3'primer: AX40 5'CACCGAGCTCCATGACACTCTTGTGCAGA 3' V~~ ~l/2~011 CA 02382444 2002-02-19 PCT/SE00/01767 Description of SEQ ID NO:1 and SEQ ID N0:2 The sequences shown i Fig.l are the same as the two sequences which are shown in the sequence listing.
The SEQ ID NO:1 is a nucleotide sequence composed of the following features:
S Nucleotide No.
Cloning site HindIII 1-6 Napin Promoter 1-1145 Cloning site BamHI 1146-1151 Transit peptide leader 1152-1178 Transit peptide coding 1179-1347 Cloning site XbaI 1348-1353 ~i-carotene C-4-oxygenase 1354-2217 (3-carotene C-4-oxygense 3' untranslated 2218-2266 Cloning site SacI 2267-2272 Nopaline synthetase termination 2273-2536 Cloning site EcolZI 2538-2543 The SEQ ID NO: 2 is a deduced amino acid sequence of the fusion protein of the transit peptide and the peptide with (3-carotene C-4-oxygenase activity.
W~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 References An G, Watson BD, Chiang CC (1986), Transformation of tobacco, tomato, potato and S Arabidopsis-thaliana using a binary vector system. Plant Physiology 81 (1) 301-305.
Bevan M, BarnesWM and Chilton MD (1983). Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 11 (2), 369-385 .
DeBlock M, DeBrouwer D, Terming P (1989). Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the BAR and NEO
genes in transgenic plants Plant Physiology 91:2, 694-701.
Fraser PD, Miura Y, Misawa N, (1997). In vitro characterization of astaxanthin biosynthetic enzymes. J Biol Chem. Mar 7;272(10):6128-35.
Fry J, Barnason A, and Horsch RB, (1987). Transformation of Brassica napus with Agrobacteriium tumefaciens based vectors. Plant Cell Reports 6:321-325.
Hoekema A, Hirsch PR, Hooykas PJJ Schilperoort, (1983). A binary vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid.
Nature vol 303, 179-180.
Josefsson LG, Lenman M, Ericson ML and Rask L, (1987). Structure of a gene encoding the 1.7 S storage protein, napin, from Brassica napus. J. Biol. Chem. 262 (25), 12196-12201.
Kajiwara S, KakizonoT, Saito T, Kondo K, OhtaniT, Nishio N, Nagai S and Misawa N.
(1995). Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli Plant Mol. Biol.
29 (2), 343-352.
W~ ~l/2~~11 CA 02382444 2002-02-19 PCT/SE00/01767 Krebbers E, Seurinck J, Herdies L, Cashmore AR and Timko MP, (1988). Four genes in two diverged subfamilies encode the rubulose-l, S-bisphosphate carboxylase small subunit polypeptides of Arabidopsis thaliana Plant Mol. Biol. 11, 745-759.
Moloney M, Walker JM and Sharma KK, (1989). High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Reports 8:238-242.
Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1988), Transformation of Brassica napus using Agrobacterium tumefaciens -Developmentally regulated Expression of a reintroduced napin gene. TAG, 75: (5) 685-694 .
Pua E-C, Mehra-Palta A, Nagy F and Chua N-H, (1987). Transgenic plants of Brassica napus.
Biotechnology vol 5, 81 S-817.
Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L (1996). Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta 199(4):515-9.
SEQUENCE LISTING
<110> AstaCarotene AB
<120> DNA construct and its use <130> 29295-AstaCarotene <140>
<141>
<160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 2543 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: napin promoter + choroplast localization signal + beta-carotene C-4 oxygenase coding sequence + termination sequence <220>
<221> promoter <222> (1)..(1145) <220>
<221> transit-peptide <222> (1179)..(1347) <220>
<221> CDS
<222> (1179)..(2217) <220>
<221> terminator <222> (2273)..(2536) <400> 1 aagctttctt catcggtgat tgattccttt aaagacttat gtttcttatc ttgcttctga 60 ggcaagtatt cagttaccag ttaccactta tattctggac tttctgactg catcctcatt 120 tttccaacat tttaaatttc actattggct gaatgcttct tctttgagga agaaacaatt 180 cagatggcag aaatgtatca accaatgcat atatacaaat gtacctcttg ttctcaaaac 240 atctatcgga tggttccatt tgctttgtca tccaattagt gactacttta tattattcac 300 tcctctttat tactattttc atgcgaggtt gccatgtaca ttatatttgt aaggattgac 360 gctattgagc gtttttcttc aattttcttt attttagaca tgggtatgaa atgtgtgtta 420 gagttgggtt gaatgagata tacgttcaag tgaagtggca taccgttctc gagtaaggat 480 gacctaccca ttcttgagac aaatgttaca ttttagtatc agagtaaaat gtgtacctat 540 aactcaaatt cgattgacat gtatccattc aacataaaat taaaccagcc tgcacctgca 600 tccacatttc aagtattttc aaaccgttcg gctcctatcc accgggtgta acaagacgga 660 ttccgaattt ggaagatttt gactcaaatt cccaatttat attgaccgtg actaaatcaa 720 ctttaacttc tataattctg attaagctcc caatttatat tcccaacggc actacctcca 780 aaatttatag actctcatcc ccttttaaac caacttagta aacgtttttt tttttaattt 840 tatgaagtta agtttttacc ttgtttttaa aaagaatcgt tcataagatg ccatgccaga 900 acattagcta cacgttacac atagcatgca gccgcggaga attgtttttc ttcgccactt 960 gtcactccct tcaaacacct aagagcttct ctctcacagc acacacatac aatcacatgc 1020 gtgcatgcat tattacacgt gatcgccatg caaatctcct ttatagccta taaattaact 1080 catccgcttc actctttact caaaccaaaa ctcatcaata caaacaagat taaaaacata 1140 cacgaggatc ctcagtcaca caaagagtaa agaagaaca atg get tcc tct atg 1194 Met Ala Ser Ser Met ctc tct tcc get act atg gtt gcc tct ccg get cag gcc act atg gtc 1242 Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala Gln Ala Thr Met Val get cct ttc aac gga ctt aag tcc tcc get gcc ttc cca gcc acc cgc 1290 Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala Phe Pro Ala Thr Arg aag get aac aac gac att act tcc atc aca agc aac ggc gga cgc gtt 1338 Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val aac tgc atg tct aga atg cca tcc gag tcg tca gac gca get cgt cct 1386 Asn Cys Met Ser Arg Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro gcg cta aag cac gcc tac aaa cct cca gca tct gac gcc aag ggc atc 1434 Ala Leu Lys His Ala Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile acg atg gcg ctg acc atc att ggc acc tgg acc gca gtg ttt tta cac 1482 Thr Met Ala Leu Thr Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His gca ata ttt caa atc agg cta ccg aca tcc atg gac cag ctt cac tgg 1530 Ala Ile Phe Gln Ile Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp ttg cct gtg tcc gaa gcc aca gcc cag ctt ttg ggc gga agc agc agc 1578 Leu Pro Val Ser Glu Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser cta ctg cac atc get gca gtc ttc att gta ctt gag ttc ctg tac act 1626 Leu Leu His Ile Ala Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr ggt cta ttc atc acc aca cat gac gca atg cat ggc acc ata get ttg 1674 Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile Ala Leu agg cac agg cag ctc aat gat ctc ctt ggc aac atc tgc ata tca ctg 1722 Arg His Arg Gln Leu Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu tac gcc tgg ttt gac tac agc atg ctg cat cgc aag cac tgg gag cac 1770 Tyr Ala Trp Phe Asp Tyr Ser Met Leu His Arg Lys His Trp Glu His cac aac cat act ggc gaa gtg ggg aaa gac cct gac ttc cac aag gga 1818 His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp Phe His Lys Gly aat ccc ggc ctt gtc ccc tgg ttc gcc agc ttc atg tcc agc tac atg 1866 Asn Pro Gly Leu Val Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met tcc ctg tgg cag ttt gcc cgg ctg gca tgg tgg gca gtg gtg atg caa 1914 Ser Leu Trp Gln Phe Ala Arg Leu Ala Trp Trp Ala Val Val Met Gln atg ctg ggg gcg ccc atg gca aat ctc cta gtc ttc atg get gca gcc 1962 Met Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala cca atc ttg tca gca ttc cgc ctc ttc tac ttc ggc act tac ctg cca 2010 Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro cac aag cct gag cca ggc cct gca gca ggc tct cag gtg atg gcc tgg 2058 His Lys Pro Glu Pro Gly Pro Ala Ala Gly Ser Gln Val Met Ala Trp ttc agg gcc aag aca agt gag gca tct gat gtg atg agt ttc ctg aca 2106 Phe Arg Ala Lys Thr Ser Glu Ala Ser Asp Val Met Ser Phe Leu Thr tgc tac cac ttt gac ctg cac tgg gag cac cac aga tgg ccc ttt gcc 2154 Cys Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro Phe Ala ccc tgg tgg cag ctg ccc cac tgc cgc cgc ctg tcc ggg cgt ggc ctg 2202 Pro Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu gtg cct gcc ttg gca tgacctggtc cctccgctgg tgacccagcg tctgcacaag 2257 Val Pro Ala Leu Ala agtgtcatgg agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag 2317 attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa 2377 gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag 2437 agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga 2497 taaattatcg cgcgcggtgt catctatgtt actagatcgg gaattc 2543 <210> 2 <211> 346 <212> PRT
<213> Artificial Sequence <223> Description of Artificial Sequence: deduced fusion protein of transit peptide + peptide with beta-carotene C-4 oxygenase activity <400> 2 Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala Phe Pro Ala Thr Arg Lys Ala Asn Asn Asp Ile Thr Ser Ile Thr Ser Asn Gly Gly Arg Val Asn Cys Met Ser Arg Met Pro Ser Glu Ser Ser Asp Ala Ala Arg Pro Ala Leu Lys His Ala Tyr Lys Pro Pro Ala Ser Asp Ala Lys Gly Ile Thr Met Ala Leu Thr Ile Ile Gly Thr Trp Thr Ala Val Phe Leu His Ala Ile Phe Gln Ile Arg Leu Pro Thr Ser Met Asp Gln Leu His Trp Leu Pro Val Ser Glu Ala Thr Ala Gln Leu Leu Gly Gly Ser Ser Ser Leu Leu His Ile Ala Ala Val Phe Ile Val Leu Glu Phe Leu Tyr Thr Gly Leu Phe Ile Thr Thr His Asp Ala Met His Gly Thr Ile Ala Leu Arg His Arg Gln Leu Asn Asp Leu Leu Gly Asn Ile Cys Ile Ser Leu Tyr Ala Trp Phe Asp Tyr Ser Met Leu His Arg Lys His Trp Glu His His Asn His Thr Gly Glu Val Gly Lys Asp Pro Asp Phe His Lys Gly Asn Pro Gly Leu Val Pro Trp Phe Ala Ser Phe Met Ser Ser Tyr Met Ser Leu Trp Gln Phe Ala Arg Leu Ala Trp Trp Ala Val Val Met Gln Met Leu Gly Ala Pro Met Ala Asn Leu Leu Val Phe Met Ala Ala Ala Pro Ile Leu Ser Ala Phe Arg Leu Phe Tyr Phe Gly Thr Tyr Leu Pro His Lys Pro Glu Pro Gly Pro Ala Ala Gly Ser Gln Val Met Ala Trp Phe Arg Ala Lys Thr Ser Glu Ala Ser Asp Val Met Ser Phe Leu Thr Cys Tyr His Phe Asp Leu His Trp Glu His His Arg Trp Pro Phe Ala Pro Trp Trp Gln Leu Pro His Cys Arg Arg Leu Ser Gly Arg Gly Leu Val Pro Ala Leu Ala
Claims (11)
1. A DNA construct comprising in the 5' to 3' direction of transcription operably linked a promoter region directing transcription to the seed of an oilseed plant, a nucleotide sequence coding for at least one peptide with enzyme activity necessary for keto group containing xanthophyll production and esterification in an oilseed plant and a transcriptional termination region.
2. The DNA construct according to claim 1, which between the promoter region and the nucleotide sequence coding for at least one peptide with enzyme activity additionally comprises a nucleotide sequence coding for a transit peptide directing the translated fusion polypeptide to the chloroplast of the oilseed plant.
3. The DNA construct according to claim 1 or 2, wherein the promoter is a napin promoter, the peptide with enzyme activity necessary for keto group containing xanthophyll production and esterification is selected from the group consisting of peptides with, 1-D-deoxyxylulose 5-phosphate synthase, isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase, geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, zeta-carotene desaturase, lycopene beta-cyclase, .beta.-carotene hydroxylase, .beta.-carotene C-4-oxygenase, and acyl transferase activity.
4. The DNA construct according to any one of claims 1 - 3, wherein the nucleotide sequence coding for a peptide with enzyme activity is a nucleotide sequence coding for a N-terminally truncated .beta.-carotene C-4-oxygenase gene from the alga Haematococcus pluvialis.
5. The DNA construct according to claim 4, wherein the nucleotide sequence is SEQ ID NO:1.
6. Transgenic oilseed plant cell comprising the DNA construct of any one of claims 1-5.
7. Transgenic oilseed plant cell according to claim 6, wherein the oilseed plant is selected from the group consisting of rape, sunflower, soybean and mustard.
8. Transgenic oilseed plant-produced xanthophyll.
9. Transgenic oilseed plant-produced xanthophyll according to claim 8, wherein the xanthophyll is canthaxanthin
10. Transgenic oilseed plant-produced xanthophyll according to claim 8, wherein the xanthophyll is astaxanthin.
11. Transgenic oilseed plant-produced xanthophyll according to claim 8, wherein the xanthophyll is astaxanthin esters.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9903336A SE9903336D0 (en) | 1999-09-17 | 1999-09-17 | DNA construct and its use |
| SE9903336-7 | 1999-09-17 | ||
| PCT/SE2000/001767 WO2001020011A1 (en) | 1999-09-17 | 2000-09-13 | Dna construct and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2382444A1 true CA2382444A1 (en) | 2001-03-22 |
Family
ID=20417035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002382444A Abandoned CA2382444A1 (en) | 1999-09-17 | 2000-09-13 | Dna construct and its use |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP1212444A1 (en) |
| JP (1) | JP2003509057A (en) |
| AU (1) | AU7693600A (en) |
| CA (1) | CA2382444A1 (en) |
| NO (1) | NO20021305L (en) |
| SE (1) | SE9903336D0 (en) |
| WO (1) | WO2001020011A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1303614A2 (en) | 2000-07-18 | 2003-04-23 | National Research Council Of Canada | Cloning, sequencing and expression of a comamonas cyclopentanone 1,2-monooxygenase-encoding gene in escherichia coli |
| US7385123B2 (en) | 2002-08-20 | 2008-06-10 | Sungene Gmbh & Co. Kgaa | Process for preparing ketocarotenoids in genetically modified organisms |
| DE10321963A1 (en) * | 2003-05-15 | 2004-12-02 | Icon Genetics Ag | Process for the production of a plastid-directed protein in plant cells |
| CN103589650A (en) | 2005-03-18 | 2014-02-19 | 米克罗比亚公司 | Production of carotenoids in oleaginous yeast and fungi |
| US20070293568A1 (en) | 2006-06-16 | 2007-12-20 | Yamaha Hatsudoki Kabushiki Kaisha | Neurocyte Protective Agent |
| US8691555B2 (en) | 2006-09-28 | 2014-04-08 | Dsm Ip Assests B.V. | Production of carotenoids in oleaginous yeast and fungi |
| EP2456894A4 (en) | 2009-07-23 | 2013-09-25 | Chromatin Inc | Sorghum centromere sequences and minichromosomes |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5618988A (en) * | 1990-03-02 | 1997-04-08 | Amoco Corporation | Enhanced carotenoid accumulation in storage organs of genetically engineered plants |
| US5916791A (en) * | 1995-11-24 | 1999-06-29 | Hirschberg; Joseph | Polynucleotide molecule from Haematococcus pluvialis encoding a polypeptide having a β--C--4--oxygenase activity for biotechnological production of (3S,3S)astaxanthin |
| US6429356B1 (en) * | 1996-08-09 | 2002-08-06 | Calgene Llc | Methods for producing carotenoid compounds, and specialty oils in plant seeds |
| BR9713462A (en) * | 1996-08-09 | 2000-03-28 | Calgene Inc | Methods for producing carotenoid compounds and special oils in plant seeds. |
| SE522246C2 (en) * | 1997-02-27 | 2004-01-27 | Astacarotene Ab | Oral preparation for prophylactic and therapeutic treatment of Helicobacter Sp. infection |
| SE511237C2 (en) * | 1997-12-16 | 1999-08-30 | Astacarotene Ab | Use of at least one type of xanthophyll for the preparation of a human or veterinary drug for the prophylactic treatment of mastitis in mammalian mothers |
-
1999
- 1999-09-17 SE SE9903336A patent/SE9903336D0/en unknown
-
2000
- 2000-09-13 AU AU76936/00A patent/AU7693600A/en not_active Abandoned
- 2000-09-13 WO PCT/SE2000/001767 patent/WO2001020011A1/en not_active Application Discontinuation
- 2000-09-13 JP JP2001523782A patent/JP2003509057A/en active Pending
- 2000-09-13 EP EP00966616A patent/EP1212444A1/en not_active Withdrawn
- 2000-09-13 CA CA002382444A patent/CA2382444A1/en not_active Abandoned
-
2002
- 2002-03-15 NO NO20021305A patent/NO20021305L/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| SE9903336D0 (en) | 1999-09-17 |
| NO20021305D0 (en) | 2002-03-15 |
| JP2003509057A (en) | 2003-03-11 |
| AU7693600A (en) | 2001-04-17 |
| EP1212444A1 (en) | 2002-06-12 |
| NO20021305L (en) | 2002-05-15 |
| WO2001020011A1 (en) | 2001-03-22 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued |