Technical Field

[0001] The present invention relates to plants, such as borage, with enhanced gamma linolenic acid (GLA) production.

Background

[0002] Borago officinalis, commonly known as borage, is an annual herb from Boraginaceae family native to the Mediterranean region and now naturalized worldwide. The seed oil of borage is known as a rich source of gamma-linolenic acid (GLA).

[0003] GLA is an 18-carbon polyunsaturated fatty acid containing three double bonds, as shown in FIG. 1. GLA is biosynthesized in the metabolization of linolenic acid (LA) by the enzymatic activity of delta-6-desaturase. Delta-6-desaturase is highly conserved across plant species.

[0004] GLA content in borage seed oil is typically well below 23% for most cultivated borage varieties. The variation in GLA content is believed to be due to multiple factors including genetics, geographical location, length of the light period during growing season, average temperature and diurnal temperature difference

[0005] GLA is an essential fatty acid important for normal health functions and regulating metabolism. Due to its anti-inflammatory and antioxidant properties, borage oil containing GLA has been used to treat a wide range of both short- and long-term illnesses such as autoimmune disorders (e.g. multiple sclerosis), skin disorders, premenstrual syndrome and cramps, diabetic neuropathy, and other inflammatory conditions. Because of its health benefits, many nutraceutical supplements, functional foods and body care products are enriched with borage oil.

[0006] Triacylglycerol fatty acid stereospecific positional distribution can affect the efficiency of fatty acid bio-absorption. GLA is known to be more bioavailable and bio-absorptive when present predominantly at the sn-2 position, rather than the sn-1 and sn-3 positions, of triacylglycerols.

[0007] Borage and other plants with seed oil having a high GLA content, including in particular at the sn-2 position of triacylglycerols, are desirable, for example for nutraceutical and pharmaceutical applications.

Summary

[0008] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above- described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

[0009] One aspect of the invention provides a plant or plant cell comprising a mutant allele at a gene locus for delta-6-desaturase, wherein the mutant allele produces delta-6- desaturase having increased activity resulting in increased gamma linolenic acid (GLA) production relative to a corresponding wild-type delta-6-desaturase, wherein the plant or plant cell produces seeds yielding an oil having a GLA content of greater than 25%, wherein the GLA content at the sn-2 position is at least 1 .45 times higher than the sum of the GLA content at the sn-1 and sn-3 positions. The plant may be borage and the plant cell may be a borage plant cell.

[0010] Another aspect of the invention provides a plant, plant cell or seed comprising a mutant allele at a gene locus for delta-6-desaturase, wherein the mutant allele comprises a nucleic acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:1 and/or (ii) comprising a G to T nucleotide substitution at the 852nd base pair from the start codon of the open reading frame of the delta-6-desaturase gene. The plant may be borage, the plant cell may be a borage plant cell, and the seed may be a borage seed.

[0011] Another aspect of the invention provides a plant, plant cell or seed comprising a mutant allele at a gene locus for delta-6-desaturase, wherein the mutant allele comprises an amino acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:2 and/or (ii) an amino acid sequence comprising a Gin to His substitution at the 284th amino acid of the delta-6-desaturase enzyme. The plant may be borage, the plant cell may be a borage plant cell, and the seed may be a borage seed.

[0012] Another aspect of the invention provides a delta-6-desaturase enzyme comprising a nucleic acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:1 and/or (ii) comprising a G to T nucleotide substitution at the 852nd base pair from the start codon of the open reading frame of the delta-6-desaturase gene. The delta-6-desaturase enzyme may be a borage delta-6-desaturase enzyme.

[0013] Another aspect of the invention provides a delta-6-desaturase enzyme comprising an amino acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:2 and/or (ii) comprising a Gin to His substitution at the 284th amino acid of the delta-6-desaturase enzyme. The delta-6-desaturase enzyme may be a borage delta-6-desaturase enzyme.

[0014] Another aspect of the invention provides an isolated polynucleotide sequence encoding a polypeptide having delta-6-desaturase activity, wherein the polynucleotide comprises a nucleic acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:1 or (ii) comprising a G to T nucleotide substitution at the 852nd base pair from the start codon of the open reading frame of the polynucleotide.

[0015] Another aspect of the invention provides an isolated polypeptide sequence having delta-6-desaturase activity comprising an amino acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:2 or (ii) comprising a Gin to His substitution at the 284th amino acid of the polypeptide.

[0016] Another aspect of the invention provides a method of producing oil comprising: (a) crushing or pressing seeds of the plant of any one of claims 1 to 6; and (b) extracting the oil from the crushed or pressed seeds.

[0017] Another aspect of the invention provides a method of producing a plant comprising crossing plants wherein at least one of the plants comprises a mutant allele at a gene locus for delta-6-desaturase, wherein the mutant allele comprises a nucleic acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:1 and/or (ii) comprising a G to T nucleotide substitution at the 852nd base pair from the start codon of the open reading frame of the delta-6-desaturase gene. The plant may be borage.

[0018] Another aspect of the invention provides a method of producing a plant comprising crossing plants wherein at least one of the plants comprises an amino acid sequence: (i) with a sequence identity of at least 95% with SEQ ID NO:2 and/or (ii) an amino acid sequence comprising a Gin to His substitution at the 284th amino acid of the delta-6- desaturase enzyme. The plant may be borage.

[0019] Another aspect of the invention provides a use of the plant, plant cell or seed of any one of claims 1 to 6 to produce seed oil. The seed oil may be borage seed oil.

[0020] Another aspect of the invention provides a use of an enzyme of as described herein, or a isolated polynucleotide as described herein, or an isolated polypeptide as described herein, to increase the GLA content in seed oil of a plant relative to a wild-type plant. The plant may be borage.

[0021] Another aspect of the invention provides a seed oil comprising GLA content at the sn-2 position at least 1.45 times higher than the sum of the GLA content at the sn-1 and sn-3 positions. The seed oil may be borage seed oil

[0022] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.

Brief Description of the Drawings [0023] In drawings which illustrate non-limiting embodiments of the invention:

[0024] FIG. 1 shows the chemical structure of gamma-linolenic acid (GLA).

[0025] FIG. 2 shows the single nucleotide polymorphism (SNP) identified at the 852nd bp of the open reading frame of the delta-6-desaturase gene between low GLA and high GLA borage lines. The delta-6-desaturase DNA primers (D6-F and D6-R) are underlined. The nucleotides of the amplified delta-6-desaturase DNA sequence are in upper case letters.

[0026] FIG. 3 shows the amino acid substitution identified at the 284 ^th amino acid of the delta-6-desaturase protein sequence between low GLA and high GLA borage lines.

Description

[0027] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0028] The term “allele" refers to an alternative form of a nucleic acid sequence. Alleles result from a mutation, a change or an alternative reading of the genetic code. Any given gene may have none, one, or many allelic forms. Mutations which give rise to alleles include deletions, additions, or substitutions of nucleotides. Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence.

[0029] The term "amino acid sequence" refers to a peptide, a polypeptide, or a protein of either natural or synthetic origin. The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, variant, or derivative of a protein expressed by a nucleic acid sequence.

[0030] The term "isolated polynucleotide sequence" refers to a nucleic acid sequence that is no longer in its natural environment, for example in vitro or in a recombinant plant host cell.

[0031] The term "isolated polypeptide sequence" refers to an amino acid sequence that is no longer in its natural environment, for example in vitro or in a recombinant plant host cell.

[0032] The term "naturally occurring" refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells.

[0033] The term "nucleic acid sequence" refers to a DNA, cDNA or RNA molecule in single or double stranded form.

[0034] The term "recombinant nucleic acid" refers to a nucleic acid sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination may be accomplished by chemical synthesis or artificial manipulation of isolated segments of nucleic acids, e.g., by conventional genetic engineering techniques. The term recombinant nucleic acid includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid sequence. A recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector, such as a viral vector that is used, for example, to transform a plant cell.

[0035] Percentage sequence identities may determined by the Basic Local Alignment Search tool (BLAST™) program set to default parameters available from the National Center for Biotechnology Information (NCBI), or by the CLUSTAL V™ algorithm as incorporated into the MEGALIGN™ sequence alignment program set to default parameters available as part of the LASERGENE™ software package, a suite of molecular biological analysis programs (DNASTAR™, Madison WT).

[0036] The inventors have discovered mutant alleles at the gene locus for delta-6- desaturase. As described herein, plants containing such alleles can produce seed oil with higher GLA content and/or a greater proportion of GLA content at the sn-2 position, than wild-type varieties. The resulting high GLA content lines can be used for pharmaceutical, nutraceutical, and food purposes, for example. Plants referred to herein include borage, other members of Boraginaceae, as well as plants known to produce oil with relatively high GLA content including but not limited to plant species in families Ranunculaceae, Primulaceae, Cannabaceae (e.g. hemp), and Lamiaceae (e.g. perilla).

[0037] Some embodiments relate to a single nucleotide polymorphism (SNP) of G to T at the 852 ^nd bp of the open reading frame of the delta-6-desaturase enzyme as shown in FIG. 2. The SNP corresponds to a Gin to His substitution at the 284 ^th amino acid of the delta-6- desaturase enzyme as shown in FIG. 3. The SNP was identified through comparative genotypic analyses of high GLA and low GLA borage varieties and identifying markers of high GLA content. Trait stability was verified over multiple generations.

[0038] Some embodiments relate to borage plants and parts thereof, the seed oil of which yields a GLA content of greater than wild-type borage plants, for example greater than 25%, and/or wherein the GLA content at the sn-2 position is higher than the sum of the GLA content at the sn-1 and sn-3 positions compared to wild-type borage plants, for example at least 1 .45 times higher than the sum of the GLA content at the sn-1 and sn-3 positions.

[0039] Some embodiments relate to borage plants and seeds, in addition to those derived from cross-breeding as described in the examples herein, that are derived from mutations, recombinant variants and genetically engineered derivatives derived by conventional means which possess the enhanced GLA characteristics described herein. Some embodiments also extends to plant parts other than seeds, including plant cells, plant tissues and plant organs whether produced by plant tissue culture or otherwise.

[0040] Some embodiments relate to nucleic acid sequences, including but not limited to isolated polynucleotides, coding a delta-6-desaturase enzyme having an identity of at least 60% with the sequence specified in SEQ ID NO 1 , in particular of at least 70%, preferably of at least 80% and particularly preferably of at least 90% and especially preferably of at least 95%. The delta-6-desaturase enzyme may be a borage delta-6-desaturase enzyme or a delta-6-desaturase enzyme of another plant species.

[0041] Some embodiments relate to amino acid sequences, including but not limited to isolated polypeptides, coding a delta-6-desaturase enzyme having an identity of at least 60% with the sequence specified in SEQ ID NO 2, in particular of at least 70%, preferably of at least 80% and particularly preferably of at least 90% and especially preferably of at least 95%. The delta-6-desaturase enzyme may be a borage delta-6-desaturase enzyme or a delta-6-desaturase enzyme of another plant species.

[0042] Some embodiments relate to crushing or pressing seeds of high GLA plants described herein to extract seed oil by using conventional methods to separate the seed oil from other seed constituents such as seed proteins and carbohydrates. The plant may be borage or another plant species.

[0043] Some embodiments relate to using conventional transgenic and other conventional genetic engineering methods that use a recombinant nucleic acid including the sequence specified in SEQ ID NO 2 and/or a recombinant nucleic acid that codes for an amino acid sequence including the amino acid sequence specified in SEQ ID NO 3, to increase the GLA content in seed oil in a plant, such as borage plant.

[0044] Some embodiments relate to producing plants having seed oil with high GLA content by crossing other plants with the high GLA borage plants described herein. Other plants may be wild-type plants or plants having seed oil with high GLA content, including but not limited to those described herein. The plants may be borage plants or other plant species.

[0045] Some embodiments relate to a method of growing a crop comprising sowing seeds of high GLA plants described herein, and cultivating the seeds and resultant plant under suitable conditions to produce a crop. The crop may be a borage crop or crop of another plant species.

Examples [0046] Specific embodiments of the invention are described with reference to the following examples, which are intended to be illustrative and not limiting in nature.

Example 1 .0 - High GLA borage breeding [0047] 15,00 borage lines were grown from germplasm collections. After GLA screening with gas chromatography (GO), a few borage lines with GLA contents between 23.1% and 24.9% were determined, as shown in Table 1.

Table 1 : [0048] Those high GLA borage lines were used as female parents to cross with other good agronomic borage varieties or lines. Single plant pedigree selected through a large screening of offspring from those crosses in the F4 generation. High GLA plants with GLA content as high as 27.3% were found, as shown in Table 2.

Table 2:

[0049] More crosses were made between the selected high GLA borage plants. Then a couple of recurrent populations were created by bulking those new high GLA crosses. Using recurrent selection, high GLA borage plants with record breaking GLA content as high as 32.8% (ST-70-2-7-E) were found, as shown in Table 3.

Table 3:

[0050] Similar results were observed a further generation, as shown in Table 4. The inventor believes high GLA genes were pyramided by the current selection, assembling high GLA genes from different parents into a single genotype. Table 4:

Example 2.0 - Position distribution analysis of borage triacylqlycerols by hydrolysis with pancreatic lipase

[0051] Total lipids from borage seeds were extracted using chloroform/methanol. Triacylglycerols (TAG) were isolated from total lipids by thin layer chromatography (TLC). TAGs exist in enantiomeric forms. Their fatty acid positions are defined by a stereospecific numbering' (sn) system as sn-1 , sn-2 and sn-3. The composition of fatty acid position at sn-2 of TAGs can be determined by incubating them with the enzyme pancreatic lipase in an appropriate buffer. The fatty acids are hydrolysed from the primary positions leaving a 2- monoacyl-sn-glycerol (2-MAG), which can be isolated by TLC for determination of its fatty acid composition. The samples for total lipids, TAG and 2-MAG were transmethylated with methanolic sulfuric acid and then analyzed with gas chromatography (GC).

[0052] Fatty acid distribution analyses were performed on the high GLA borage varieties from Example 1.0. GLA was determined to be high at the sn-2 position of these borage TAGs, a shown in Table 5.

Table 5:

[0053] In particular, GLA content at the sn-2 position was found to be 1 .45 to 1.64 times higher than the sum of GLA at the sn-1 and sn-3 positions. The relatively high GLA at the sn- 2 position indicates the GLA in the oil of these high GLA borage lines are more bio-available and better absorbed when ingested.

Example 3.0 - Determination of functional single nucleotide polymorphism marker for high GLA borage lines

Selection of borage lines

[0054] Thirteen of borage lines including 7 high GLA lines and 6 low GLA lines were selected for molecular marker development, as shown in Table 6. Low and high GLA borage genotypes were raised in the greenhouse under a 16 hour light (23 3) and 8 hour dark (1713) cycle. Young leaf tissues were collected an d stored in -8013 for further utilization.

Table 6:

Genomic DNA extraction

[0055] The total genomic DNA was extracted from the collected leaf tissues using DNeasy™ Plant mini kit (Qiagen, Germany) following the manufacturer’s protocol. Extracted DNA was utilized for further PCR application.

Primer design

[0056] GLA is synthesised by a D6-desaturase using linolenic as a substrate. The open reading frame of the borage D6-desaturase gene is 1344 base pairs long and it encodes 448 amino acids. After referring to the full length of the borage delta-6-desaturase mRNA in GenBank (GenBank accession number EF495160), a pair of primers were designed for DNA amplification of the borage delta-6-desaturase using PCR: (D6-F: 5'- TTTCATCAATGGCTGCTCAAAT-3’at 36 bp and D6-R: 5'- AAAGACAAGTTGCAATGACTCC-3' 22, at 1487 bp).

PCR amplification

[0057] After synthesis of DNA primers, PCR amplification was performed according to the program in Table 7. able 7:

[0058] One 1452 bp fragment was amplified with PCR program above.

DNA fragment sequencing, assembly and comparison

[0059] After PCR amplification, the forward and reverse sequencing were performed with the Sanger sequencing platform. The beginning portion of the DNA sequence was resequenced with the Illumina™ platform to ensure DNA sequencing was correct. Then the forward sequence and the reverse sequence were assembled. After the DNA sequences from all thirteen borage samples were assembled, the assembled sequences were aligned together.

[0060] After comparing these 13 sequences, a single nucleotide polymorphism (SNP) was identified between the high GLA and low GLA borage lines. The SNP is present at the 852th bp from the start codon (ATG) of the open reading frame for the borage delta-6-desaturase gene. The SNP was a change from nucleotide G for low GLA lines to nucleotide T for the high GLA lines, as shown in FIG. 2. This results in an amino acid substitution from Q (glutamine) to H (histidine) at the 284th amino acid for the delta-6-desaturase protein as shown in FIG. 3.

[0061] The single amino acid change results in protein configuration changes and therefore enzyme activity changes. In particular, this substitution results in higher activities for the delta-6-desaturase enzyme, and consequently higher GLA content in the high GLA borage lines. [0062] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.