WO1998018948A1 - Flax promoters for manipulating gene expression - Google Patents
Flax promoters for manipulating gene expression Download PDFInfo
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- WO1998018948A1 WO1998018948A1 PCT/CA1997/000812 CA9700812W WO9818948A1 WO 1998018948 A1 WO1998018948 A1 WO 1998018948A1 CA 9700812 W CA9700812 W CA 9700812W WO 9818948 A1 WO9818948 A1 WO 9818948A1
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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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8226—Stem-specific, e.g. including tubers, beets
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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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/823—Reproductive tissue-specific promoters
- C12N15/8234—Seed-specific, e.g. embryo, endosperm
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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
- C12N15/8247—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
Definitions
- This invention relates to gene promoters useful for the genetic manipulation of plants. More particularly, the invention relates to gene promoters isolated from flax useful, for example, for manipulating the expression of indigenous genes or transgenes in flax and other plants to modify endogenous characteristics or to introduce new ones.
- BACKGROUND ART ART
- Flax ⁇ Linum usi ta tissimum is the second most important oilseed crop in Canada and an important crop worldwide.
- the use of flax seed oil is limited by the narrow range of natural fatty acids present in it. Therefore, there is a need to create new cultivars with a wider range of fatty acid composition to supplement the existing food and confections markets (Rowland et al., 1995 - please refer to the "References" section below for full reference identification details) .
- flax as a vehicle for biofarming of pharmaceutical-related products by molecular genetic manipulation of appropriate transgenes (Moloney and van Rooijen, 1996) .
- flax varieties tolerant to various abiotic and biotic stresses has also been recognized (Rowland et al., 1995) .
- herbicide-tolerant flax varieties would be very useful in crop rotation programs.
- promoters useful for expressing foreign genes in various other plants Molecular genetic manipulation of flax seed composition or other characteristics, such as stress tolerance, can be achieved by expressing appropriate transgenes using seed-specific or constitutive gene promoters. While a cDNA sequence corresponding to a flax gene has been reported (Singh et al., 1994), no promoter has yet been characterized from flax.
- An object of the invention is to identify and isolate one or more genes and promoter sequences from flax and to utilize such sequences in the genetic manipulation of plants.
- Another object of the invention is to provide a vector containing a promoter sequence from flax for introducing an indigenous gene or a transgene into flax or other plants.
- Another object of the invention is to provide a method of modifiying flax and other plants to change characteristics thereof.
- the present invention is based on the isolation, purification and characterization by the inventors of the present invention of two genes from flax and two promoters from those genes.
- the sequences obtained are used for regulating the expression of a heterologous gene (foreign, reporter or transgene) in flax and other plant species.
- This can result in flax plants having different range of fatty acids than natural flax and can result in the development of transgenic plants suitable for the production of specific products or having new and useful characteristics.
- Such plants and products are of commercial and industrial interest.
- genes are useful in themselves for making antisense or sense constructs based on the derived sequences. Both types of contruct can be used to reduce the levels of similar mRNA during expression of the natural genes. This would result in an increase in 18:0 fatty acid in membrane or storage lipids in flax and other plant species. Sense constructs may also be used in enhancing the levels of mRNA. Such enhancement will result in the increase of 16:1 or 18:1 fatty acids in membranes or storage lipids in flax and other plant species. Such plants will be of increased commercial interest and value.
- a method of changing fatty acids of membrane and storage lipids of plants characterized by making an antisense or sense construct based on SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, ligating the constuct into a plant transformation vector, using the vector to transform the genome of a plant or plant seed, and then growing the plant or plant seed and extracting membrane or storage lipids from the plants.
- isolated and purified deoxyribonucleic acid of SEQ ID NO: 3 or SEQ ID NO: 4 (deposited as plasmids ATCC 98193 and 98192, respectively, see details below) . These are the promoters that are useful for enhancing or enabling the expression of genes introduced into flax or other plants .
- a gene expression cassette comprising a sequence according to SEQ ID N0:1, SEQ ID N0:2, SEQ ID NO: 3 or SEQ ID NO: 4.
- the gene expression cassette is useful in itself as this part of the plasmids mentioned above can be used to construct other plasmid suitable to transform other plant species.
- a vector for introduction of a gene into a plant cell comprising a promotor of SEQ ID NO: 3 or SEQ ID NO: 4.
- the invention also relates to transgenic plants and plant seeds having a genome containing an introduced promoter sequence of SEQ ID NO: 3 or SEQ ID NO: 4 regulating the expression of an introduced gene, and a method of producing such plants and plant seeds.
- the invention also relates to substantially homologous DNA sequences (e.g. greater than or equal to 40% homology, more preferably greater than or equal to 70% homology) isolated and/or characterized by known methods using the sequence information of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and to parts of reduced length of promoter sequences SEQ ID NO: 3 or SEQ ID NO: 4 that are still able to function as promoters of gene expression.
- substantially homologous DNA sequences e.g. greater than or equal to 40% homology, more preferably greater than or equal to 70% homology
- promoter sequences include the core promoter elements (TATA box and initiator) and upstream regulatory elements (enhancers) (Datla et al., 1997).
- the promoters of the invention are beneficial in manipulating the expression of genes in flax and other crops .
- Fig. 1 shows genomic DNA sequence of the SAD1 [SEQ ID NO:l; identified in Fig. 1 as LUSADl.SEQ] and SAD2 [SEQ ID NO: 2; identified in Fig. 1 as LUSAD2.SEQ] genes and the corresponding SAD cDNA sequence [SEQ ID NO: 5; identified in Fig. 1 as LUCDNA] .
- Nucleotides (nt) are represented by capital letters. Nucleotides different from the cDNA sequence are shaded, including those of introns . Differences between SADl and SAD2 are shown in shaded lower case letters. Gaps in the sequences are presented by dashes. The start and stop codons on the cDNA sequence are boxed.
- Fig. 2A is a partial restriction map of the SADl gene
- Fig. 2B shows the result of a DNA blot analysis identifying the regulatory sequences of SADl and SAD2.
- Fig. 3 shows an outline of the scheme employed to isolate the promoter regions of the two SAD genes.
- Fig. 4 discloses nucleotide sequences [SEQ ID NO: 3 (SADl) and SEQ ID NO: 4 (SAD2)] of the 5'- regulatory regions of the two SAD genes. Homologous nt are represented by a dash (-) , gaps by a dot (.), and additions by lower case letters. A putative transcriptional site is indicated by +1, and a TATA box is overlined. Key restriction sites are also shown.
- Fig. 5 shows salient features of the plasmids CDC214 and pCDC220.
- Various abbreviations are as follows: flax promoter I, SADl gene promoter; flax promoter II, SAD2 gene promoter; GUS ( uidA) , gene for ⁇ -glucuronidase enzyme; nos-T, transcriptional terminator of the nopaline synthase gene; nptll, neomycin phospho-transferase expression cassette.
- the arrowheads indicate the direction of transcription. Key restriction sites are shown. Regions outside the left and right border (LB and RB) are that of a previously described binary plant transformation vector, pRD410 (Datla et al . , 1992).
- FIG. 6 shows the expression of a heterologous gene ( uidA) by the two SAD gene promoters in various tissues of flax.
- Different tissues are abbreviated as YL+A, young leaves and apices; ML, mature leaves: S, stems; R, roots; B, buds; 1/2 OF, half open flower; FI, Flower; and MS, seeds at about mid-development.
- Data presented are from one generation of two plants transformed with a tandem 35s promoter (2x35s) , two generations of two plants transformed with pCDC214 (SADl), and one generation of two plants transformed with pCDC220 (SAD2) .
- Fig. 7 shows the expression of a heterologous gene ( uidA) by the two SAD gene promoters during flax seed development and in relation to .fatty acid and protein biosyntheses .
- uidA heterologous gene
- Fig. 9 shows the expression of a heterologous gene (uidA) by the two SAD gene promoters during tobacco seed development.
- Various developmental stages of tobacco seeds were identified according to de Silva et al. (1992) and are abbreviated as W, white; LB, light brown; B, brown; DB, dark brown; and M, mature.
- Data represent 5 to 8 transgenic plants transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and pRD420 ( uidA alone) .
- Fig. 10 shows the expression of a heterologous gene ( uidA) by the two SAD gene promoters in canola leaves and mature seeds.
- Data represent 2 to 5 plants transformed with pCDC214 (SADl), pCDC220 (SAD2), pRD410 (35s), and untransformed plants (UT) .
- SADl pCDC214
- SAD220 pCDC220
- pRD410 35s
- UT untransformed plants
- the promoters developed according to the present invention can be used to modify an endogenous characteristic of flax or another plant species, or to to add a new characteristic.
- An example of a modification of an endogenous characteristic of flax is, for example, the alteration of levels of different types of fatty acids in the seed oils.
- the introduction of a new characteristic is, for example, the production of a thermoplastic polymer in plants that normally do not produce thermoplastics. While it is normally easy to detect added characteristics, it is sometimes difficult to detect altered characteristics because of natural variation of characteristics in plants.
- the alterations can, however, be detected by comparing the average characteristics of a statistically significant number of the plants under examination with a statistically significant number of genomically-unmodified plants of the same genotype, grown under identical environmental conditions at the same time. If there is an appreciable difference in the measured characteristic, then it can be said that there has been an alteration of that characteristic and that the alteration is a result of the genomic- modification.
- the promoters of the present invention belong to a two-member gene family encoding the enzyme ⁇ 9 desaturase (Stearoyl-acyl carrier protein desaturase; SAD; EC 1.14.99.6).
- Stearoyl-acyl carrier protein desaturase is the first enzyme in the fatty acid desaturation pathway, and it catalyzes the conversion of stearoyl-ACP(18:0-ACP) to oleoyl-ACP (18 : 1 ⁇ 9-ACP) .
- the promoters were isolated using the inverse polymerase chain reaction (IPCR) technique. They are capable of expressing a foreign gene, e.g.
- uidA which encodes ⁇ -glucuronidase : GUS
- GUS ⁇ -glucuronidase
- both promoters showed a similar temporal expression pattern for uidA (measured as GUS activity) .
- the GUS activity could be detected as early as 4 dap in developing seeds and in desiccated seeds ( ⁇ 50 dap) of transgenic flax.
- the ability of the promoters to effect uidA gene expression correlated well with both fatty acid and protein biosyntheses and the maximum activity of GUS preceded the maximal accumulation of fatty acids and proteins.
- the promoters of the invention are useful in manipulating transgene expression in a variety of tissues including seeds.
- Some of the products which are possible using these promoters include, but are not limited to, the following: plants with enhanced herbicide, pest, pathogen, and stress resistance; plants containing oil, protein, and carbohydrate of altered composition and content; plants with reduced anti-nutritional substances; plants producing pharmaceutical compounds such as antibodies, neuropeptides, recombinant proteins, and biodegradable thermoplastics (Bennett, 1993; Moloney and van Rooijen, 1996; Datla et al., 1997).
- the effectiveness of the promoters of the present invention is predictable from the effectiveness of known promoters. For example, it is well established that promoters such as cauliflower mosaic virus (CaMV) are capable of expressing a wide variety of genes in a wide varity of plant species.
- CaMV cauliflower mosaic virus
- Napin promoter from rapeseed has been used to express a variety of genes in canola/rapeseed (Knutzon et al., 1992; Jones et al . , 1995; Dahesh et al., 1996).
- Phaseolin gene promoter from bean
- Phaseolin gene promoter has also been used to express several genes in rapeseed (Hitz et al., 1995).
- the ⁇ -conglycinin promoter from soyabean has been used to express genes not only in soyabean but also in Petunia (Kinney, 1997; Chen et al. , 1986) .
- oligodeoxy- ribonucleotides used in nucleotide sequencing, and PCR techniques were synthesised using a phosphoramidate synthesis procedure in a Biosearch 8750 DNA synthesizer (New Brunswick Scientific Co.), and purified by HPLC- based protocols (Gait, 1984) .
- IPCR was done according to Ochman et al. (1993) and Warner et al . (1993). Plant DNA was extracted using the protocol of
- RNAase B (Sigma) was removed by adding 100 ⁇ g of RNAase B (Sigma) followed by incubation at 65°C for 20 min. The DNA was extracted once with an equal volume of phenol: chloroform (1:1, v/v) and once with an equal volume of chloroform: isoamyl alcohol (24:1, v/v).
- DNA Five ⁇ g of DNA was digested with the appropriate restriction enzyme, fractionated on a 0.8% agarose gel, and pressure-blotted onto Hybond-NTM nylon membranes (Amersha ) using the PosiBlotTM apparatus (Stratagene) after depurination, denaturation and neutralization of the DNA (Sambrook et al., 1989).
- the blotting solution contained 0.02 M NaOH and 1 M NH 4 -acetate.
- the DNA was immobilized on the membrane by baking the membranes at 80°C for 1 h.
- a radioactive probe for identifying promoters was prepared by annealing 10 ng of oligo-29A and 30A (Table 1 below) and then filling in the ends using the Klenow fragment of DNA polymerase and random primer kit solutions (GIBCO BRL) .
- oligo-29A corresponded to nt 120-
- the SADl and SAD2 gene 5' regulatory elements were cloned into pRD420 as a Sall-Smal fragment in front of the uidA.
- the plasmid pRD420 was obtained from Dr. R.S.S. Datla, NRC Plant Biotechnology Institute, 110
- Flax seeds were surface sterilized by stirring in 70% ethanol for 2 minutes, followed by three 10 minute washes in 0.5% sodium hypochlorite (freshly diluted from the commercial product) , and 5 rinses in sterile distilled water. Seeds were germinated on basal medium consisting of Murashige and Skoog (MS) major and minor salts and Gamborg vitamins (Sigma 0404), 3% sucrose and 0.8% agar. The pH of the medium was adjusted to 5.8 before autoclaving. About 10 surface-sterilized seeds were placed in each 100x15 mm plate. The plates were sealed with parafilm and placed in the dark at 22°C for 5 to 7 days.
- Derivatives of Agrobacteria tumefaciens strain GV3101/pMP90 carrying pCDC214 and pCDC220 were grown on solidified 2x YT medium (Sambrook et al. 1989) supplemented with 50 ⁇ g/ml kanamycin and 50 ⁇ g/ml gentamycin sulfate. Single colonies from 2 to 3 day- old culture plates were used to inoculate 10 ml liquid 2x YT medium containing antibiotics as above and 20 ⁇ M acetosyringone. Cultures were grown at 28°C with rotary agitation for about 24 hours. Prior to inoculation of flax tissues, the cell concentration of the suspension was adjusted to lxlO 9 cells/ml.
- hypocotyls of 5-7 day aseptic flax seedlings were cut into segments 3-4 mm long. To avoid dehydration, the segments were maintained in a small Volume of liquid basal medium until all the hypocotyls were cut.
- the hypocotyl segments were immersed in bacterial suspension (lxlO 9 cells/ml) for 30 minutes with occasional swirling. The suspension was removed by aspiration and the hypocotyl segments were transferred to sterile filter paper to remove excess liquid.
- the segments were placed on agar-solidified (0.8%) basal medium supplemented with 4.44 ⁇ M 6-benzylaminopurine and 0.54 ⁇ M naphthaleneacetic acid (MSD4x2 medium; Basiran et al . , 1987). Maltose (3%) replaced sucrose as the carbohydrate source. About 25 explants were placed in each 100x15 mm Petri dish and maintained at 22-24°C, with a 16 h photoperiod and photon density of approximately 50 ⁇ mol/m 2 /s. After 2 days the segments were transferred to the same medium supplemented with 100 ⁇ g/ml kanamycin for selection of transformed cells and 200 ⁇ g/ml cefotaxime to eliminate Agrobacteria . The explants were maintained under the same growth conditions for 3 weeks. As a control, non-inoculated segments were treated in the same way.
- Green callus formed at the cut ends of most of the inoculated hypocotyl segments, whereas little or no callus appeared on non-inoculated segments and they were completely bleached after 3 weeks on the selection medium. Callus was excised and transferred to basal medium (3% maltose) supplemented with 5 ⁇ M zeatin and antibiotics as above. Shoots regenerated from some of the calli within 3-4 weeks.
- the shoots When the shoots had elongated to 0.5 to 1.0 cm, they were removed from the callus and placed in capped glass tubes (100x25 mm) containing 8 ml rooting medium: 1/2 strength MS salts, 3% sucrose, 0.1 ⁇ M IAA, 0.8% agar, pH 5.8, and 30 ⁇ g/ml kanamycin for selection of transformed shoots.
- the shoots were maintained under low light ( ⁇ 25 ⁇ mol/m 2 /s) for 6-8 days by which time some of the shoots had roots about 2-3 mm long.
- the plantlets were transferred to pots in the growth chamber within 10-14 days, when roots had elongated to about 2 cm and the shoots were 3-5 cm tall.
- Transgenic plants were grown under 18 h of light (300-500 ⁇ mol/m 2 /s) and day/night temperature of 20/17°C.
- the plants were fertilized just before flowering with a solution containing 27 g of 15N:30P:15K supplemented with 0.9 g CuS0 4 in 9 liters of water.
- Fluorimetric GUS assay was done essentially according to Jefferson (1987) . The assays were done in a micro well titer plate and fluorescence of the reactions was measured by CytoFluorTM II multi-well fluorescence plate reader (PerSeptive Biosystems). Determination of Fatty Acid and Protein Content in Seeds
- the fatty acid content of seeds of different ages was determined by fatty acid methyl ester analysis of seed homogenates as described previously (Taylor et al., 1992).
- the genomic sequences of the two SAD genes were amplified by PCR.
- oligonucleotide primers were synthesized based on the nucleotide sequence of the published SAD cDNA sequence (Singh et al., 1994). These primers were used in all possible combinations with flax genomic DNA as the template to amplify different segments of SAD genes.
- the molecular size of the PCR products was determined by agarose gel electrophoresis; in most reactions two products of very similar molecular size were detected, suggesting the possibility of two SAD genes in flax.
- Amplification with oligo-25 and 24 yielded a fragment of about 2.6 kb. This fragment contained the whole SAD gene as determined by sequence data.
- the amplified SAD gene fragments were cloned into pCRII vector (Invitrogen Corp.). The identity of the amplified gene products was confirmed by comparison of their nucleotide sequences with the SAD cDNA sequence (Singh et al., 1994). Sequence analyses indicated that the SADl and SAD2 genes have 97.2% similarity with each other in the coding region and 96.2% and 93.7% with the published flax cDNA sequence, respectively (Fig. 1) . It is clear that the mRNA for SAD cDNA, reported by Singh et al. (1994), was transcribed from the SADl gene. Some general features of the flax SAD genes have been deduced from sequence analysis.
- the coding region of the gene is 1191 bps. This consists of three exons interrupted by two introns of approximately 0.6 to 0.7 kb. Exon 1 consists of 123 bp, whereas exons 2 and 3 are 507 bp and 561 bp long, respectively. Verification for the presence of two SAD genes in flax comes from the analyses of two independent clones, each containing the full length gene. Although the nucleotide sequences of the coding regions are almost identical, there are several base changes. One of these has altered a restriction enzyme site, Ncol , resulting in the observation that the two clones have different restriction digestion patterns. The two clones also differ significantly in their intron sequences (Fig. 1) . The different intron sequences are presumably responsible for the slight difference in the molecular size of the two PCR products generated by the same primer combination.
- Genomic DNA was extracted from 7-10 days old seedlings of flax var. McGregor (obtained from Dr. G. Rowland, Crop Development Centre, 51 Campus Dr., Saskatoon, Saskatchewan S7N 5A8 ) , digested with restriction enzyme, BamRI , Bell , Bglll , Ndel or Sstl, gel-fractionated and blotted onto nylon membrane for probing. These restriction enzymes would cut within the flax SAD genomic sequence as indicated in Figure 2A and elsewhere in the flax genome. When the DNA blot was hybridized with the probe, DNA fragments containing the 5'- upstream region and a part of the 5'- untranslated and coding region of the SAD gene were expected to hybridize ( Figure 2A) .
- Flax genomic DNA was digested with the restriction enzyme Sstl and gel fractionated. DNA fragments were isolated from a region of the agarose gel where the two promoter fragments that hybridized with the SAD probe were expected (Fig. 2B and 3) . These DNA fragments were ligated at a concentration favoring the circularization of single DNA molecules (Ochman et al., 1993; Warner et al., 1993). The circularized DNA was then used as a template in the IPCR with two primers (oligo-39 and oligo 110; Table 1) . The orientation of the each member of the primer set used in the IPCR is opposite to that normally used in a regular PCR (Fig. 3).
- promoters were capable of expressing the uidA gene in various tissues, with high level of expression in seeds (Fig. 6). In developing seeds, both the promoters showed similar temporal expression patterns for GUS (Fig. 7) .
- the GUS activity could be detected as early as 4 dap in developing seeds and in desiccated seeds (approximately 50 dap) of transgenic flax with higher activities around mid-development (14 to 28 dap) .
- flax promoters The utility of the flax promoters disclosed here is demonstrated by comparing their effect on uidA gene expression with both lipid and protein biosynthesis in developing flax seeds. In developing seeds, uidA expression correlated well with both fatty acid and protein biosynthesis (Fig. 7) . In seeds, maximum expression of the uidA gene controlled by the SAD gene promoters preceded the maximum accumulation of fatty acids and proteins. Also, in tobacco the temporal pattern of uidA gene expression correlated well with the lipid biosynthesis (de Silva et al., 1992). Therefore, these promoters are useful in manipulating gene expression in seeds. Since these promoters are also active in other tissues they are useful in manipulating gene expression in a variety of tissues. Utility of SAD Genes
- antisense or sense constructs are made using the disclosed or other promoters.
- these genes or their parts can be ligated into a Smal restriction site of pCDC 214 or 220 (Fig. 5) or any other convenient cloning site of another plant transformation vector.
- These recombinant plasmids can then be mobilized, for example, into an Agrobacterium strain which can then be used to transform a variety of plant species. Any changes in fatty acids of membrane and storage lipids can be evaluated by routine methods described in this application.
- Both type of constructs are expected to reduce the levels of similar mRNA during expression of the natural genes resulting in an increase of 18:0 fatty acid in membrane or storage lipids.
- Sense constructs can also be used in enhancing the levels of mRNA. Such enhancement will likely result in the increase of 16:1 or 18:1 fatty acids in membranes or storage lipids of plants. Such plants will be of increased commercial interest and value.
- MOLECULE TYPE DNA (genomic)
- AATCAATTCC TTTGTGTTTC ACGATTCTGG GTTTTGCGCT GTAATTGATT ⁇ GTCAGTGTTT 480 GCACAGGTTT CCCCTTCTCC TCCTCCGTCC ATCAAATGCA TGTTATTACC ATTTCAATTT 540
- GGCAGCCACA GGATTTCCTG CCCGAACCTG AGTCGGATGG GTTCGAGGAG CAAGTGAAGG 1080
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- TCATATTCAT TTTGACGACT ATGTATAGTC GTACAAACTA TTCGGTTAAC TAATCTACAT 600
- AAATGCCTCC AAAGTGCTCT CTACTTGCGG TTGGCCTGGT TCAATGGCGA ATCATTGAAT 840
- MOLECULE TYPE DNA (genomic)
- ATGACAATGT AACATCAATG TCAATCTCTG CAGATTTTTG TTAGCAGCAG -GTCATGATTC 420 TTTTTTGGTT GATTCTTGTG AATGTAAGCT ATTTGTTGTT GTAATATATG CATTGATTGT 480
- CAGTCGCTCA ACGCATCGGG GATACTGCCA AGGATTATGC CGATATCCTG GAGTTCCTGG 1140
- ORGANISM Linum usitatissimum (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTGGTGGAGG TGGAACTGAA 20
- TL- DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204:383-396.
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Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002268745A CA2268745A1 (en) | 1996-10-31 | 1997-10-30 | Flax promoters for manipulating gene expression |
AU48588/97A AU4858897A (en) | 1996-10-31 | 1997-10-30 | Flax promoters for manipulating gene expression |
GB9908939A GB2332908B (en) | 1996-10-31 | 1997-10-30 | Flax promoter for manipulating gene expression |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US2941696P | 1996-10-31 | 1996-10-31 | |
US60/029,416 | 1996-10-31 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998018948A1 true WO1998018948A1 (en) | 1998-05-07 |
Family
ID=21848902
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA1997/000812 WO1998018948A1 (en) | 1996-10-31 | 1997-10-30 | Flax promoters for manipulating gene expression |
Country Status (4)
Country | Link |
---|---|
AU (1) | AU4858897A (en) |
CA (1) | CA2268745A1 (en) |
GB (1) | GB2332908B (en) |
WO (1) | WO1998018948A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001016340A1 (en) * | 1999-08-27 | 2001-03-08 | Sembiosys Genetics Inc. | Flax seed specific promoters |
WO2002102970A2 (en) * | 2001-06-06 | 2002-12-27 | Bioriginal Food & Science Corporation | Flax (linum usitatissimim l.) seed-specific promoters |
US6777591B1 (en) * | 1999-08-27 | 2004-08-17 | Sembiosys Genetics Inc. | Legume-like storage protein promoter isolated from flax and methods of expressing proteins in plant seeds using the promoter |
EP1846560A1 (en) * | 2005-02-09 | 2007-10-24 | Bioriginal Food & Science Corp. | Novel omega-3 fatty acid desaturase family members and uses thereof |
EP1925671A1 (en) * | 2001-06-06 | 2008-05-28 | Bioriginal Food & Science Corp. | Flax (Linum usitatissimum L.) seed-specific promoters |
US7642346B2 (en) * | 1999-08-27 | 2010-01-05 | Sembiosys Genetics Inc. | Flax seed specific promoters |
US9745362B2 (en) | 2009-12-09 | 2017-08-29 | Shengjun An | Seed-specific expression vector and its construction methods and applications |
Citations (6)
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WO1991013972A1 (en) * | 1990-03-16 | 1991-09-19 | Calgene, Inc. | Plant desaturases - compositions and uses |
WO1991018985A1 (en) * | 1990-05-25 | 1991-12-12 | E.I. Du Pont De Nemours And Company | Nucleotide sequence of soybean stearoyl-acp desaturase gene |
NL9002130A (en) * | 1990-09-28 | 1992-04-16 | Stichting Tech Wetenschapp | Cruciferous stearoyl-ACP-desaturase coding sequences - for modifying lipid biosynthesis in plants, esp. oilseed rape |
EP0561569A2 (en) * | 1992-03-13 | 1993-09-22 | The Lubrizol Corporation | Modification of vegetable oils using desaturase |
WO1995007357A2 (en) * | 1993-09-04 | 1995-03-16 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Promoters |
EP0736598A1 (en) * | 1993-12-28 | 1996-10-09 | Kirin Beer Kabushiki Kaisha | Gene for fatty acid desaturase, vector containing said gene, plant containing said gene transferred thereinto, and process for creating said plant |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5475099A (en) * | 1990-08-15 | 1995-12-12 | Calgene Inc. | Plant fatty acid synthases |
-
1997
- 1997-10-30 CA CA002268745A patent/CA2268745A1/en not_active Abandoned
- 1997-10-30 AU AU48588/97A patent/AU4858897A/en not_active Abandoned
- 1997-10-30 WO PCT/CA1997/000812 patent/WO1998018948A1/en active Application Filing
- 1997-10-30 GB GB9908939A patent/GB2332908B/en not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1991013972A1 (en) * | 1990-03-16 | 1991-09-19 | Calgene, Inc. | Plant desaturases - compositions and uses |
WO1991018985A1 (en) * | 1990-05-25 | 1991-12-12 | E.I. Du Pont De Nemours And Company | Nucleotide sequence of soybean stearoyl-acp desaturase gene |
NL9002130A (en) * | 1990-09-28 | 1992-04-16 | Stichting Tech Wetenschapp | Cruciferous stearoyl-ACP-desaturase coding sequences - for modifying lipid biosynthesis in plants, esp. oilseed rape |
EP0561569A2 (en) * | 1992-03-13 | 1993-09-22 | The Lubrizol Corporation | Modification of vegetable oils using desaturase |
WO1995007357A2 (en) * | 1993-09-04 | 1995-03-16 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | Promoters |
EP0736598A1 (en) * | 1993-12-28 | 1996-10-09 | Kirin Beer Kabushiki Kaisha | Gene for fatty acid desaturase, vector containing said gene, plant containing said gene transferred thereinto, and process for creating said plant |
Non-Patent Citations (8)
Title |
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BIOLOGICAL ABSTRACTS, vol. 101, Philadelphia, PA, US; abstract no. 15165, ROWLAND G G ET AL: "The application of chemical mutagenesis and biotechnology to the modification of linseed (Linum usitatissimum L.)." XP002055122 * |
CSIRO, DIVISION OF PLANT INDUSTRY, REPORT 1985-86, (1986) PP. 12-16. PUBLISHER: CSIRO. MELBOURNE * |
DATABASE CAB CAB INTERNATIONAL, WALLINGFORD, OXON, GB; GREEN, A.G., ET AL.: "The development of edible-oil flax - a potential new polyunsaturated oilseed crop.", XP002055123 * |
EUPHYTICA 85 (1-3). 1995. 317-321. ISSN: 0014-2336 * |
KNUTZON D S ET AL: "MODIFICATION OF BRASSICA SEED OIL BY ANTISENSE EXPRESSION OF A STEAROYL-ACYL CARRIER PROTEIN DESATURASE GENE", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 89, April 1992 (1992-04-01), pages 2624 - 2628, XP002018311 * |
POLASHOCK, J.J., ET AL.: "Expression of the yeast delta-9 fatty acid desaturase in Nicotiana tabacum", PLANT PHYSIOLOGY, vol. 100, 1992, pages 894 - 901, XP002055086 * |
SINGH, S., ET AL.: "Sequence of a cDNA from linum usitatissimum encoding the stearoyl-acyl carrier protein desaturase", PLANT PHYSIOLOGY, vol. 104, 1994, pages 1075, XP002055265 * |
SINGH, S.P., ET AL.: "L.usitatissimum mRNA for stearoyl-(acyl-carrier-protein)-desaturase", EMBL SEQUENCE DATABASE, ACCESSION NO. X70962, 26-FEB-1993, XP002055085 * |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
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US7642346B2 (en) * | 1999-08-27 | 2010-01-05 | Sembiosys Genetics Inc. | Flax seed specific promoters |
JP2003525030A (en) * | 1999-08-27 | 2003-08-26 | セムバイオシス ジェネティックス インコーポレイテッド | Flax seed-specific promoter |
WO2001016340A1 (en) * | 1999-08-27 | 2001-03-08 | Sembiosys Genetics Inc. | Flax seed specific promoters |
KR100719629B1 (en) | 1999-08-27 | 2007-05-18 | 셈비오시스제네틱스인코포레이티드 | Flax seed specific promoters |
US6777591B1 (en) * | 1999-08-27 | 2004-08-17 | Sembiosys Genetics Inc. | Legume-like storage protein promoter isolated from flax and methods of expressing proteins in plant seeds using the promoter |
AU782218B2 (en) * | 1999-08-27 | 2005-07-14 | Commonwealth Scientific And Industrial Research Organisation | Flax seed specific promoters |
US7193134B2 (en) | 2001-06-06 | 2007-03-20 | Bioriginal Food & Science Corp. | Flax (Linum usitatissimum L.) fatty acid desaturase |
US7473820B2 (en) | 2001-06-06 | 2009-01-06 | Bioriginal Food & Science Corp. | Flax (Linum usitatissimum L.) seed-specific promoters |
WO2002102970A2 (en) * | 2001-06-06 | 2002-12-27 | Bioriginal Food & Science Corporation | Flax (linum usitatissimim l.) seed-specific promoters |
WO2002102970A3 (en) * | 2001-06-06 | 2004-03-18 | Bioriginal Food & Science Corp | Flax (linum usitatissimim l.) seed-specific promoters |
EP1925671A1 (en) * | 2001-06-06 | 2008-05-28 | Bioriginal Food & Science Corp. | Flax (Linum usitatissimum L.) seed-specific promoters |
EP2184359A1 (en) * | 2001-06-06 | 2010-05-12 | Bioriginal Food & Science Corp. | Flax (Linum usitatissimum L.) seed-specific promoters |
US8841512B2 (en) | 2001-06-06 | 2014-09-23 | Bioriginal Food & Science Corporation | Flax (Linum usitatissimum L.) seed-specific promotors |
EP1846560A4 (en) * | 2005-02-09 | 2009-02-11 | Bioriginal Food & Science Corp | Novel omega-3 fatty acid desaturase family members and uses thereof |
EP2365086A1 (en) * | 2005-02-09 | 2011-09-14 | Bioriginal Food & Science Corporation | Novel omega-3 fatty acid desaturase family members and uses thereof |
US8088978B2 (en) | 2005-02-09 | 2012-01-03 | Bioriginal Food & Science Corp. | Omega-3 fatty acid desaturase family members and uses thereof |
EP1846560A1 (en) * | 2005-02-09 | 2007-10-24 | Bioriginal Food & Science Corp. | Novel omega-3 fatty acid desaturase family members and uses thereof |
US9745362B2 (en) | 2009-12-09 | 2017-08-29 | Shengjun An | Seed-specific expression vector and its construction methods and applications |
Also Published As
Publication number | Publication date |
---|---|
CA2268745A1 (en) | 1998-05-07 |
GB2332908A (en) | 1999-07-07 |
AU4858897A (en) | 1998-05-22 |
GB9908939D0 (en) | 1999-06-16 |
GB2332908B (en) | 2001-03-28 |
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