Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0083—Miscellaneous (1.14.99)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—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 fatty acid desaturases and nucleic acids encoding desaturase proteins . More particularly, the invention relates to nucleic acids encoding delta-12 and delta-15 fatty acid desaturase proteins that affect fatty acid composition in plants, polypeptides produced from such nucleic acids and plants expressing such nucleic acids.
• Alterations in fatty acid composition of vegetable oils is desirable for meeting specific food and industrial uses.
• Brassica varieties with increased monounsaturate levels (oleic acid) in the seed oil, and products derived from such oil, would improve lipid nutrition.
• Canola lines which are low in polyunsaturated fatty acids and high in oleic acid tend to have higher oxidative stability, which is a useful trait for the retail food industry.
• Delta-12 fatty acid desaturase also known as oleic desaturase is involved in the enzymatic conversion of oleic acid to linoleic acid.
• Delta-15 fatty acid desaturase (also known as linoleic acid desaturase) is involved in the enzymatic conversion of linoleic acid to ⁇ -linolenic acid.
• a microsomal delta-12 desaturase has been cloned and characterized using T-DNA tagging. Okuley, et al . , Plant Cell 6:147-158 (1994).
• the nucleotide sequences of higher plant genes encoding microsomal delta-12 fatty acid desaturase are described in Lightner et al . , W094/11516. Sequences of higher plant genes encoding microsomal and plastid delta-15 fatty acid desaturases are disclosed in Yadav, N.
• the mutant sequence may be derived from, for example, a Brassica napus, Brassica rapa, Brassica juncea or Helianthus delta-12 or delta-15 desaturase gene.
• Another embodiment of the invention involves a method of producing a Brassicaceae or Helianthus plant line comprising the steps of: (a) inducing mutagenesis in cells of a starting variety of a Brassicaceae or Helianthus species; (b) obtaining progeny plants from the mutagenized cells; (c) identifying progeny plants that contain a mutation in a delta-12 or delta-15 fatty acid desaturase gene; and (d) producing a plant line by selfing or crossing. The resulting plant line may be subjected to mutagenesis in order to obtain a line having both a delta-12 desaturase mutation and a delta-15 desaturase mutation.
• Yet another embodiment of the invention involves a method of producing plant lines containing altered fatty acid composition
• a method of producing plant lines containing altered fatty acid composition comprising: (a) crossing a first plant with a second plant having a mutant delta-12 or delta-15 fatty acid desaturase; (b) obtaining seeds from the cross of step (a) ; (c) growing fertile plants from such seeds; (d) obtaining progeny seed from the plants of step (c) ; and (e) identifying those seeds among the progeny that have altered fatty acid composition.
• Suitable plants are soybean, rapeseed, sunflower, safflower, castor bean and corn.
• Preferred plants are rapeseed and sunflower.
• the invention is also embodied in vegetable oil obtained from plants disclosed herein, which vegetable oil has an altered fatty acid composition.
• SEQ ID NO:l shows a hypothetical DNA sequence of a Brassica Fad2 gene.
• SEQ ID NO : 2 is the deduced amino acid sequence of SEQ ID NO:l.
• SEQ ID NO: 3 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 316.
• SEQ ID NO: is the deduced amino acid sequence of SEQ ID NO: 3.
• SEQ ID NO: 5 shows a hypothetical DNA sequence of a Brassica Fad2 gene.
• SEQ ID NO: 6 is the deduced amino acid sequence of SEQ ID NO : 5.
• SEQ ID NO: 7 shows a hypothetical DNA sequence of a Brassica Fad2 gene having a mutation at nucleotide 515.
• SEQ ID NO : 8 is the deduced amino acid sequence of SEQ ID NO: 7.
• SEQ ID NO : 9 shows the DNA sequence for the coding region of a wild type Brassica Fad2-D gene.
• SEQ ID NO: 10 is the deduced amino acid sequence for SEQ ID NO: 9.
• SEQ ID NO: 11 shows the DNA sequence for the coding region of the IMC 129 mutant Brassica Fad2-D gene.
• SEQ ID NO: 12 is the deduced amino acid sequence for SEQ ID NO: 11.
• SEQ ID NO: 13 shows the DNA sequence for the coding region of a wild type Brassica Fad2-F gene.
• SEQ ID NO: 14 is the deduced amino acid sequence for SEQ ID NO: 13.
• SEQ ID NO: 15 shows the DNA sequence for the coding region of the Q508 mutant Brassica Fad2-F gene.
• SEQ ID NO: 16 is the deduced amino acid sequence for SEQ ID NO: 15.
• SEQ ID NO: 17 shows the DNA sequence for the coding region of the Q4275 mutant Brassica Fad2-F gene.
• SEQ ID NO: 18 is the deduced amino acid sequence for SEQ ID NO:17.
• SEQ ID NOS: 19-27 show oligonucleotide sequences.
• SEQ ID NO: 28 shows the genomic DNA sequence for the Fad2-U gene from Brassica .
• SEQ ID NOS: 30-31 show genomic sequences located upstream from the start codon of Brassica Fad2-D genes.
• Figure 1 is a histogram showing the frequency distribution of seed oil oleic acid (C 18:1 ) content in a segregating population of a Q508 X Westar cross.
• the bar labeled WSGA 1A represents the C 18;1 content of the Westar parent.
• the bar labeled Q508 represents the C 18:1 content of the Q508 parent.
• Figure 2 shows the nucleotide sequences for a Brassica Fad2-D wild type gene (Fad2-D wt) , IMC129 mutant gene (Fad2-D GA316 IMC129) , Fad2-F wild type gene (Fad2-F wt) , Q508 mutant gene (Fad2-F TA515 Q508) and Q4275 mutant gene (Fad2-F GA908 Q4275) .
• Figure 3 shows the deduced amino acid sequences for the polynucleotides of Figure 2.
• fatty acids herein are percent by weight of the oil of which the fatty acid is a component.
• a "line” is a group of plants that display little or no genetic variation between individuals for at least one trait. Such lines may be created by several generations of self-pollination and selection, or vegetative propagation from a single parent using tissue or cell culture techniques.
• the term "variety" refers to a line which is used for commercial production.
• mutagenesis refers to the use of a mutagenic agent to induce random genetic mutations within a population of individuals. The treated population, or a subsequent generation of that population, is then screened for usable trait (s) that result from the mutations.
• a “population” is any group of individuals that share a common gene pool.
• M 0 is untreated seed.
• M- is the seed (and resulting plants) exposed to a mutagenic agent
• M 2 is the progeny (seeds and plants) of self-pollinated M x plants
• M 3 is the progeny of self-pollinated M 2 plants
• M 4 is the progeny of self-pollinated M 3 plants.
• M 5 is the progeny of self-pollinated M 4 plants.
• M 6 is the progeny of self-pollinated plants of the previous generation.
• selfed as used herein means self -pollinated.
• Applicants have discovered plants with mutations in a delta-12 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid compositions of the seed oil. Such mutations confer, for example, an elevated oleic acid content, a decreased, stabilized linoleic acid content, or both elevated oleic acid and decreased, stabilized linoleic acid content. Applicants have further discovered plants with mutations in a delta-15 fatty acid desaturase gene. Such plants have useful alterations in the fatty acid composition of the seed oil, e.g., a decreased, stabilized level of c_-linolenic acid.
• a nucleic acid fragment of the invention may comprise a portion of the coding sequence, e.g., at least about 10 nucleotides, provided that the fragment contains at least one mutation in the coding sequence.
• the length of a desired fragment depends upon the purpose for which the fragment will be used, e.g., PCR primer, site- directed mutagenesis and the like.
• the invention relates to an isolated nucleic acid fragment of at least 50 nucleotides in length that has at least 70% sequence identity to the nucleotide sequences of SEQ ID NO: 30 or SEQ ID NO -.31. In some embodiments, such nucleic acid fragments have at least 80% or 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 31. Sequence identity for these and other nucleic acids disclosed herein can be determined, for example, using Blast 2.0.4 (Feb. 24, 1998) to search the nr database (non-redundant GenBank, EMBL, DDBT and PDB) .
• the nucleotide sequences of SEQ ID NO: 30 and SEQ ID NO: 31 are located upstream of the ATG start codon for the fad2-D gene and can be isolated from Bridger and Westar canola plants, respectively. These upstream elements contain intron-like features.
• the invention also relates to an isolated nucleic acid fragment that includes a sequence of at least 200 nucleotides.
• the fragment has at least 70% identity to nucleotides 1 to about 1012 of SEQ ID NO: 28.
• the fragment has 80% or at least 90% sequence identity to nucleotides 1 to about 1012 of SEQ ID NO: 28.
• This portion of SEQ ID NO: 28 is located upstream of the ATG start codon and has intron-like features .
• a mutation in a nucleic acid fragment of the invention may be in any portion of the coding sequence that renders the resulting gene product non-functional.
• Suitable types of mutations include, without limitation, insertions of nucleotides, deletions of nucleotides, or transitions and transversions in the wild-type coding sequence. Such mutations result in insertions of one or more amino acids, deletions of one or more amino acids, and non-conservative amino acid substitutions in the corresponding gene product.
• the sequence of a nucleic acid fragment may comprise more than one mutation or more than one type of mutation.
• Insertion or deletion of amino acids in a coding sequence may, for example, disrupt the conformation of essential alpha-helical or beta-pleated sheet regions of the resulting gene product. Amino acid insertions or deletions may also disrupt binding or catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non- functional , compared to a smaller number of inserted or deleted amino acids.
• Non-conservative amino acid substitutions may replace an amino acid of one class with an amino acid of a different class.
• Non-conservative substitutions may make a substantial change in the charge or hydrophobicity of the gene product.
• Non-conservative amino acid substitutions may also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanyl residue for a isoleucyl residue.
• Examples of non-conservative substitutions include the substitution of a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid. Because there are only 20 amino acids encoded in a gene, substitutions that result in a non- functional gene product may be determined by routine experimentation, incorporating amino acids of a different class in the region of the gene product targeted for mutation.
• Preferred mutations are in a region of the nucleic acid encoding an amino acid sequence motif that is conserved among delta-12 fatty acid desaturases or delta- 15 fatty acid desaturases, such as a His-Xaa-Xaa-Xaa-His motif (Tables 1-3) .
• An example of a suitable region has a conserved HECGH motif that is found, for example, in nucleotides corresponding to amino acids 105 to 109 of the Arabidopsis and Brassica delta-12 desaturase sequences, in nucleotides corresponding to amino acids 101 to 105 of the soybean delta-12 desaturase sequence and in nucleotides corresponding to amino acids 111 to 115 of the maize delta-12 desaturase sequence.
• An illustrative embodiment of a mutation in a nucleic acid fragment of the invention is a Glu to Lys substitution in the HECGH motif of a Brassica microsomal delta-12 desaturase sequence, either the D form or the F form.
• This mutation results in the sequence HECGH being changed to HKCGH as seen by comparing SEQ ID NO: 10 (wild- type D form) to SEQ ID NO: 12 (mutant D form) .
• a similar mutation in other Fad-2 sequences is contemplated to result in a non-functional gene product. (Compare SEQ ID NO: 2 to SEQ ID NO : 4 ) .
• a similar motif may be found at amino acids 101 to 105 of the Arabidopsis microsomal delta-15 fatty acid desaturase, as well as in the corresponding rape and soybean desaturases (Table 5). See, e.g., WO 93/11245; Arondel, V. et al . , Science, 258:1153-1155 (1992); Yadav, N. et al., Plant Physiol . , 103:467-476 (1993). Plastid delta-15 fatty acids have a similar motif (Table 5) .
• non-conservative substitutions is substitution of a glycine residue for either the first or second histidine. Such a substitution replaces a charged residue
• glycine with a non-polar residue (glycine)
• Another type of mutation that renders the resulting gene product non- functional is an insertion mutation, e.g., insertion of a glycine between the cysteine and glutamic acid residues in the HECGH motif.
• Other regions having suitable conserved amino acid motifs include the HRRHH motif shown in Table 2, the HRTHH motif shown in Table 6 and the HVAHH motif shown in Table 3. See, e.g., WO 94/115116; Hitz, W. et al . , Plant Physiol., 105:635-641 (1994); Okuley, J., et al . , supra; and Yadav, N. et al .
• An illustrative example of a mutation in the region shown in Table 3 is a mutation at nucleotides corresponding to the codon for glycine (amino acid 303 of B . napus) .
• a non-conservative Gly to Glu substitution results in the amino acid sequence
• Another region suitable for a mutation in a delta- 12 desaturase sequence contains the motif KYLNNP at nucleotides corresponding to amino acids 171 to 175 of the Brassica desaturase sequence.
• An illustrative example of a mutation is this region is a Leu to His substitution, resulting in the amino acid sequence (Table 4) KYHNN (compare wild-type Fad2-F SEQ ID NO: 14 to mutant SEQ ID NO: 16) .
• a similar mutation in other Fad-2 amino acid sequences is contemplated to result in a nonfunctional gene product. (Compare SEQ ID NO : 6 to SEQ ID NO: 8) .
• Oil composition typically is analyzed by crushing and extracting fatty acids from bulk seed samples (e.g., 10 seeds) .
• Fatty acid triglycerides in the seed are hydrolyzed and converted to fatty acid methyl esters .
• Those seeds having an altered fatty acid composition may be identified by techniques known to the skilled artisan, e.g., gas-liquid chromatography (GLC) analysis of a bulked seed sample or of a single half-seed.
• GLC gas-liquid chromatography
• Half-seed analysis is well known in the art to be useful because the viability of the embryo is maintained and thus those seeds having a desired fatty acid profile may be planted to from the next generation.
• half-seed analysis is also known to be an inaccurate representation of genotype of the seed being analyzed.
• Fatty acid composition can also be determined on larger samples, e.g., oil obtained by pilot plant or commercial scale refining, bleaching and deodorizing of endogenous oil in the seeds .
• the nucleic acid fragments of the invention can be used as markers in plant genetic mapping and plant breeding programs. Such markers may include restriction fragment length polymorphism (RFLP) , random amplification polymorphism detection (RAPD) , polymerase chain reaction (PCR) or self-sustained sequence replication (3SR) markers, for example. Marker-assisted breeding techniques may be used to identify and follow a desired fatty acid composition during the breeding process.
• RFLP restriction fragment length polymorphism
• RAPD random amplification polymorphism detection
• PCR polymerase chain reaction
• 3SR self-sustained sequence replication
• Marker-assisted breeding techniques may be used in addition to, or as an alternative to, other sorts of identification techniques.
• An example of marker-assisted breeding is the use of PCR primers that specifically amplify a sequence containing a desired mutation in delta-12 desaturase or delta-15 desaturase.
• Methods according to the invention are useful in that the resulting plants and plant lines have desirable seed fatty acid compositions as well as superior agronomic properties compared to known lines having altered seed fatty acid composition.
• Superior agronomic characteristics include, for example, increased seed germination percentage, increased seedling vigor, increased resistance to seedling fungal diseases (damping off, root rot and the like) , increased yield, and improved standabilit .
• M 2 seed from individual plants were individually catalogued and stored, approximately 15,000 M 2 lines was planted in a summer nursery in Carman, Manitoba.
• the seed from each selfed plant were planted in 3 -meter rows with 6 -inch row spacing.
• Westar was planted as the check variety. Selected lines in the field were selfed by bagging the main raceme of each plant. At maturity, the selfed plants were individually harvested and seeds were catalogued and stored to ensure that the source of the seed was known.
• the selected M 3 seeds were planted in the greenhouse along with Westar controls. The seed was sown in 4-inch pots containing Pro-Mix soil and the plants were maintained at 25°C/15°C, 14/10 hr day/night cycle in the greenhouse. At flowering, the terminal raceme was self-pollinated by bagging. At maturity, selfed M 4 seed was individually harvested from each plant, labelled, and stored to ensure that the source of the seed was known. The M 4 seed was analyzed in 10-seed bulk samples. Statistical thresholds for each fatty acid component were established from 259 control samples using a Z- distribution of 1 in 800. Selected M 4 lines were planted in a field trial in Carman, Manitoba in 3 -meter rows with 6-inch spacing.
• dihaploid populations were made from the microspores of the F- L hybrids. Self-pollinated seed from dihaploid plants were analyzed for fatty acid analysis using methods described previously.
• the gas chromatograph was set at 180°C for 5.5 minutes, then programmed for a 2°C/minute increase to 212 °C, and held at this temperature for 1.5 minutes. Total run time was 23 minutes. Chromatography settings were: Column head pressure - 15 psi, Column flow (He) - 0.7 mL/min., Auxiliary and Column flow - 33 mL/min. , Hydrogen flow - 33 mL/min., Air flow - 400 mL/min., Injector temperature - 250°C, Detector temperature - 300°C, Split vent - 1/15.
• EXAMPLE 2 High Oleic Acid Canola Lines In the studies of Example 1, at the M 3 generation, 31 lines exceeded the upper statistical threshold for oleic acid (> 71.0%) . Line W7608.3 had 71.2% oleic acid. At the M 4 generation, its selfed progeny (W7608.3.5, since designated A129.5) continued to exceed the upper statistical threshold for C 18:1 with 78.8% oleic acid. M 5 seed of five self -pollinated plants of line A129.5 (ATCC 40811) averaged 75.0% oleic acid. A single plant selection, A129.5.3 had 75.6% oleic acid.
• the fatty acid composition of this high oleic acid mutant which was stable under both field and greenhouse conditions to the M 7 generation, is summarized in Table 9. This line also stably maintained its mutant fatty acid composition to the M 7 generation in field trials in multiple locations. Over all locations the self-pollinated plants (A129) averaged 78.3% oleic acid. The fatty acid composition of the A129 for each Idaho trial location are summarized in Table 10. In multiple location replicated yield trials, A129 was not significantly different in yield from the parent cultivar Westar.
• the canola oil of A129 after commercial processing, was found to have superior oxidative stability compared to Westar when measured by the Accelerated Oxygen Method (AOM) , American Oil Chemists' Society Official Method Cd 12-57 for fat stability; Active Oxygen Method (revised 1989) .
• AOM Accelerated Oxygen Method
• Westar was 18 AOM hours and for A129 was 30 AOM hours.
• Oleic Acid Canola Line Produced by Seed Mutagenesis
• Genotype C 16 0 C 18 : 0 C 18 : 1 C 18 : 2 C 18 : 3 Sat s
• Linoleic Acid Canol a Line Produced by Seed Mutagenesis
• Linolenic Acid Canola Line Produced by Seed Mutagenesis
• Seeds of the B . napus line IMC-129 were mutagenized with methyl N-nitrosoguanidine (MNNG) .
• MNNG methyl N-nitrosoguanidine
• the MNNG treatment consisted of three parts: pre-soak, mutagen application, and wash.
• a 0.05M Sorenson's phosphate buffer was used to maintain pre-soak and mutagen treatment pH at 6.1.
• Two hundred seeds were treated at one time on filter paper (Whatman #3M) in a petri dish (100mm x 15mm) .
• the seeds were pre-soaked in 15 mis of 0.05M Sorenson's buffer, pH 6.1, under continued agitation for two hours. At the end of the pre-soak period, the buffer was removed from the plate.
• the seeds were washed with three changes of distilled water at 10 minute intervals. The fourth wash was for thirty minutes. This treatment regime produced an LD60 population.
• Treated seeds were planted in standard greenhouse potting soil and placed into an environmentally controlled greenhouse. The plants were grown under sixteen hours of light. At flowering, the racemes were bagged to produce selfed seed. At maturity, the M2 seed was harvested. Each M2 line was given an identifying number. The entire MNNG-treated seed population was designated as the Q series.
• Harvested M2 seeds was planted in the greenhouse. The growth conditions were maintained as previously described. The racemes were bagged at flowering for selfing. At maturity, the selfed M3 seed was harvested and analyzed for fatty acid composition. For each M3 seed line, approximately 10-15 seeds were analyzed in bulk as described in Example 1.
• Fatty acid composition of A129 is the average of 50 self -pollinated plants grown with the M3 population M 4 generation Q508 plants had poor agronomic qualities in the field compared to Westar. Typical plants were slow growing relative to Westar, lacked early vegetative vigor, were short in stature, tended to be chlorotic and had short pods. The yield of Q508 was very low compared to Westar.
• the M 4 generation Q508 plants in the greenhouse tended to be reduced in vigor compared to Westar. However, Q508 yields in the greenhouse were greater than Q508 yields in the field.
• M4 high-oleic low-linoleic lines were also identified: Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676. Some of these lines had good agronomic characteristics and an elevated oleic acid level in seeds of about 80% to about 84%.
• Q4275 was crossed to the variety Cyclone. After selfing for seven generations, mature seed was harvested from 93GS34-179, a progeny line of the Q4275 Cyclone cross. Referring to Table 17, fatty acid composition of a bulk seed sample shows that 93GS34 retained the seed fatty acid composition of Q4275. 93GS34-179 also maintained agronomically desirable characteristics.
• F 2 seed of the 92EF population was planted in the greenhouse to analyze the genetics of the Q508 line.
• F 3 seed was analyzed from 380 F2 individuals.
• the C 18:1 levels of F 3 seed from the greenhouse experiment is depicted in Figure 1.
• the data were tested against the hypothesis that Q508 contains two mutant genes that are semi -dominant and additive: the original IMC 129 mutation as well as one additional mutation.
• the hypothesis also assumes that homozygous Q508 has greater than 85% oleic acid and homozygous Westar
• the fatty acid composition of representative F3 individuals having greater than 85% oleic acid in seed oil is shown in Table 18.
• the levels of saturated fatty acids are seen to be decreased in such plants, compared to Westar. TABLE 18
• the mutation in the D gene of IMC 129 and Q508 mapped to a region having a conserved amino acid motif (His-Xaa-Xaa-Xaa-His) found in cloned delta-12 and delta- 15 membrane bound-desaturases (Table 20) .
• the primers used to specifically amplify delta-12 desaturase F gene RNA from the indicated tissues were sense primer 5 ' -GGATATGATGATGGTGAAAGA-3 ' and antisense primer 5 ' -TCTTTCACCATCATCATATCC-3 ' .
• the primers used to specifically amplify delta-12 desaturase D gene RNA from the indicated tissues were sense primer 5' -GTTATGAAGCAAAGAAGAAAC-3' and antisense primer 5'- GTTTCTTCTTTGCTTCATAAC-3' .
• RNA of both the D and F gene was expressed in seed and leaf tissues of IMC 129, Q508 and wild type Westar plants .
• vi tro transcription and translation analysis showed that a peptide of about 46 kD was made. This is the expected size of both the D gene product and the F gene product, based on sum of the deduced amino acid sequence of each gene and the cotranslational addition of a microsomal membrane peptide.
• PCR primers Based on the single base change in the mutant D gene of IMC 129 described in above, two 5' PCR primers were designed. The nucleotide sequence of the primers differed only in the base (G for Westar and A for IMC 129) at the 3' end. The primers allow one to distinguish between mutant fad2-D and wild-type Fad2-D alleles in a DNA-based PCR assay. Since there is only a single base difference in the 5' PCR primers, the PCR assay is very sensitive to the PCR conditions such as annealing temperature, cycle number, amount, and purity of DNA templates used. Assay conditions have been established that distinguish between the mutant gene and the wild type gene using genomic DNA from IMC 129 and wild type plants as templates.
• Conditions may be further optimized by varying PCR parameters, particularly with variable crude DNA samples.
• a PCR assay distinguishing the single base mutation in IMC 129 from the wild type gene along with fatty acid composition analysis provides a means to simplify segregation and selection analysis of genetic crosses involving plants having a delta-12 fatty acid desaturase mutation.
• the first plasmid, pIMCHO was prepared by inserting into a disarmed Ti vector the full length wild type Fad3 gene in sense orientation (nucleotides 208 to 1336 of SEQ ID 6 in WO 93/11245) , flanked by a napin promoter sequence positioned 5' to the Fad3 gene and a napin termination sequence positioned 3' to the Fad3 gene.
• the rapeseed napin promoter is described in EP 0255378.
• the second plasmid, pIMC205 was prepared by inserting a mutated Fad3 gene in sense orientation into a disarmed Ti vector.
• the mutant sequence contained mutations at nucleotides 411 and 413 of the microsomal Fad3 gene described in W093/11245, thus changing the sequence for codon 96 from GAC to AAG.
• the amino acid at codon 96 of the gene product was thereby changed from aspartic acid to lysine. See Table 20.
• the phaseolin sequence is described in Doyle et al . , (1986) J. Biol. Chem. 261:9228-9238) and Slightom et al . , (1983) Proc. Natl. Acad. Sci. USA 80:1897-1901.
• the appropriate plasmids were engineered and transferred separately to Agrobacterium strain LBA4404. Each engineered strain was used to infect 5 mm segments of hypocotyl explants from Westar seeds by cocultivation. Infected hypocotyls were transferred to callus medium and, subsequently, to regeneration medium. Once discernable stems formed from the callus, shoots were excised and transferred to elongation medium. The elongated shoots were cut, dipped in RootoneTM, rooted on an agar medium and transplanted to potting soil to obtain fertile TI plants. T2 seeds were obtained by selfing the resulting TI plants.
• Fad2-D and Fad2-F High molecular weight genomic DNA was isolated from leaves of Q4275 plants (Example 5) and from Westar and Bridger canola plants. This DNA was used as template for amplification of Fad2-D and Fad2-F genes by polymerase chain reaction (PCR) . PCR amplifications were carried out in a total volume of 100 ⁇ l and contained 0.3 ⁇ g genomic DNA, 200 ⁇ M deoxyribonucleoside triphosphates, 3 mM MgS0 4 , 1-2 Units DNA polymerase and IX Buffer (supplied by the DNA polymerase manufacturer) . Cycle conditions were: 1 cycle for 1 min at 95°C, followed by 30 cycles of 1 min at 94°C, 2 min at 55°C and 3 min at 73°C. The Fad2-D gene was amplified once using Elongase ®
• CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG SEQ ID NO: 23
• CUACUACUACUATCATAGAAGAGAAAGGTTCAG SEQ ID NO: 24
• the Fad2-F gene was independently amplified 4 times, twice with Elongase ® and twice with Taq polymerase (Boehringer Mannheim) .
• the PCR primers used were: 5 ' CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3 ' (SEQ ID NO: 25) and 5 ' CUACUACUACUATCTTTCACCATCATCATATCC3 ' (SEQ ID NO: 26) for the 5' and 3' ends of the gene, respectively.
• Fad2-U High molecular weight genomic DNA was isolated from the leaves of Bridger and Westar Brassica plants by standard methods.
• the Fad2-U gene was amplified in a 100 ⁇ l total reaction containing 1 ⁇ M of each primer, 0.3 ⁇ g genomic DNA, 200 ⁇ M dNTP, 3 mM MgS0 4 , lx Buffer (supplied by the manufacturer of the DNA polymerase) , and 1-2 units of Elongase DNA polymerase (BRL) .
• the amplification conditions included one cycle for 1 min at 95°C, 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and elongation at 72°C for 3 min. Subsequently, the reaction was incubated at 72 °C for an additional 10 min. Fad2U gene was amplified twice from Westar and twice from Bridger genomic DNAs using the following primers:
• ADDRESSEE Fish & Richardson P.C., P. A.
• MOLECULE TYPE Genomic DNA
• FEATURE :
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Genomic DNA
• FEATURE
• MOLECULE TYPE protein
• FRAGMENT TYPE internal
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO: 19: GGATATGATG ATGGTGAAAG A 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO: 20: TCTTTCACCA TCATCATATC C 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:21: GTTATGAAGC AAAGAAGAAA C 21
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:22: GTTTCTTCTT TGCTTTGCTT CATAAC 26
• MOLECULE TYPE Other Nucleic Acid
• SEQUENCE DESCRIPTION SEQ ID NO:24: CUACUACUAC UATCATAGAA GAGAAAGGTT CAG 33
• MOLECULE TYPE Genomic DNA
• FEATURE

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Biotechnology (AREA)
• Organic Chemistry (AREA)
• Wood Science & Technology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Molecular Biology (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
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• Plant Pathology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Medicinal Chemistry (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (1)

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Family Applications (1)

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Cited By (30)

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Citations (3)

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• 1998
  • 1998-06-11 JP JP50329899A patent/JP4209949B2/ja not_active Expired - Fee Related
  • 1998-06-11 AU AU80715/98A patent/AU750363B2/en not_active Ceased
  • 1998-06-11 CA CA002293810A patent/CA2293810C/en not_active Expired - Lifetime
  • 1998-06-11 WO PCT/US1998/012332 patent/WO1998056239A1/en not_active Ceased
  • 1998-06-11 EP EP98929058A patent/EP0987936A4/en not_active Withdrawn

Patent Citations (3)

Non-Patent Citations (3)

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