RU2847186C1 - Sunflower grains - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: problem solved in accordance with present invention consists in production of sunflower line with high content of stearic acid. This problem can be solved using sunflower grains containing an insertion sequence mutation on 3'-side of the TATA box and on 5'-side from the start codon of stearoyl acyl carrying protein desaturase 17 (SAD17).

EFFECT: sunflower grain with high content of stearic acid is obtained.

12 cl, 6 dwg, 2 ex

Description

Field of technology

[0001]

The present invention relates to the field of genetics and agricultural biotechnology.

Prerequisites for the creation of the invention

[0002]

Sunflower is an important and valuable agricultural crop that provides food for humans and livestock.

[0003]

Sunflower is commonly grown for oil production. The resulting fats and oils consist primarily of unsaturated fatty acids such as linoleic acid (18:2) and oleic acid (18:1), and also include palmitic acid (16:0), stearic acid (18:0), arachidonic acid (20:0), and behenic acid (22:0). The stearic acid content of the fatty acid composition of sunflower fats and oils is typically 10% or less, and typically ranges from 3 to 7%.

[0004]

Sunflower can be classified as an oilseed crop; fats and oils obtained from sunflower seeds contain linoleic acid, which accounts for 50 to 70% of the fatty acid composition. Currently, many mutants have been obtained by modifying the fatty acid composition through chemical mutagenesis using ethyl methanesulfonate or sodium azide, or by mutagenesis using X-rays or gamma rays.

[0005]

Currently, the most common sunflower mutants include high-oleic sunflowers, which contain 75 to 90% oleic acid and 2 to 10% linoleic acid. High-oleic sunflower oil is a liquid fat with excellent stability, ideal for frying and storage.

[0006]

On the other hand, since most fats and oils derived from plants are liquid fats rich in unsaturated fatty acids, there is a worldwide shortage of solid fats. Solid fats are essential as raw materials for the production of many food products, such as margarine, shortening, fillings, and fats and oils for confectionery production.

[0007]

Although solid fats can be produced by hydrogenation to harden vegetable fats and oils rich in unsaturated fatty acids, the problem is that this process produces trans fatty acids as a byproduct, which can cause health problems. Therefore, the food industry currently demands natural vegetable solid fats to replace hydrogenated fats and oils.

List of references

Non-patent documents

[0008]

Non-patent document 1: Osorio J, Fernandez-Martinez J, Mancha M, Garces R. Mutant sunflowers with high concentration of saturated fatty acids in the oil; Crop Sci 1995; 35: 739-742.

Non-patent document 2: Hongtrakul V, Slabaugh MB, Knapp SJ. DFLP, SSCP, and SSR markers for Δ9-stearoyl-acyl carrier protein desaturases strongly expressed in developing seeds of sunflower: Intron lengths are polymorphic among elite inbred lines; Mol Breed 1998; 4: 195-203.

Brief description of the invention

Technical problem

[0009]

The present inventors focused on the use of sunflower oil with high stearic acid content for the efficient production of solid lipids.

[0010]

Non-patent document (NPD) 1 reports sunflower with high stearic acid content (CAS-3). The high stearic acid trait in CAS-3 is partially regulated by recessive alleles (es1, es2) at two independent loci, Es1 and Es2. However, their specific genes and mutation pathway are unclear, making it difficult to effectively transmit this trait to future generations or effectively develop lines with other traits in addition to this trait through improved breeding.

[0011]

The aim of the present invention is to create a sunflower line with a high content of stearic acid.

Solution to the problem

[0012]

The present inventors conducted extensive research and found that the above objective can be achieved using sunflower seeds containing a mutation in the form of a sequence insertion on the 3' side of the TATA box and on the 5' side of the start codon of the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene. The present inventors conducted additional research based on the data obtained, which formed the basis for the implementation of the present invention. More specifically, the present invention encompasses the following embodiments.

[0013]

Option 1. Sunflower seed for obtaining lipid containing an insertional mutation of the sequence on the 3' side of the TATA box and on the 5' side of the start codon of the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene, while the length of the sequence is from 100 to 1200 bases.

[0014]

Option 2. Sunflower seed according to item 1, wherein the length of the sequence is from 200 to 1000 bases

[0015]

Option 3. Sunflower seed according to item 1, wherein the length of the sequence is from 400 to 800 bases.

[0016]

Option 4: Sunflower seed according to any one of paragraphs 1-3, in which the sequence includes the following sequences (a)

or (b):

(a) the base sequence represented by SEQ ID NO: 1, or

(b) a base sequence that is at least 80% identical to the base sequence represented by SEQ ID NO: 1. [0017]

Option 5. Sunflower seed according to claim 4, wherein the identity is at least 90%.

[0018]

Option 6. Sunflower seed according to any one of claims 1-5, wherein the insertional sequence mutation is introduced into a sunflower line with a high oleic acid content.

[0019]

Option 7. Sunflower grain according to any of paragraphs 1-6, having a fatty acid composition with a stearic acid content of 11% or more.

[0020]

Option 8. Sunflower seed according to any of paragraphs 1-7, containing an insertional mutation of the sequence in both pairs of chromosomes.

[0021]

Variant 9. A sunflower seed according to any one of claims 1 to 8, comprising an insertional mutation of the sequence on the 3'-side of the TATA box and on the 5'-side of the start codon of the base sequence represented by SEQ ID NO: 2 stearoyl-acyl transfer protein desaturase 17 (SAD17), wherein the sequence has a length of 400 to 800 bases, wherein the sunflower seed has a higher stearic acid content than the stearic acid content in sunflower seeds without the insertional mutation of the sequence.

[0022]

Option 10. Sunflower grain according to any of paragraphs 1-9, which is grain of the FO-HS43 line (registration number in the International Patent Depository of Organisms: IPOD FERM BP 22390) or grain of its derivative line.

[0023]

Option 11. A sunflower plant for obtaining lipid, containing sunflower grain according to any one of paragraphs 1-10.

[0024]

Option 12. A method for obtaining a lipid, including collecting a lipid from a sunflower seed according to any of paragraphs 1-10.

Preferred effects of the invention

[0025]

The present invention relates to a sunflower line with a high stearic acid content.

Brief description of the drawings

[0026]

Fig. 1 shows a graph of the frequency distribution of stearic acid content in the fatty acid composition of grains from the M2 population of the FO-HS43 line. The vertical axis shows the number of grains, and the horizontal axis shows the stearic acid content in the fatty acids of the grains.

Fig. 2 shows the results of mapping the high stearic acid (Hs) locus based on linkage analysis. LG1 represents the first chain group.

Figure 3 shows a portion of the gene sequences near the 5'-UTR of stearoyl-acyl transfer protein desaturase 17 (SAD17) of the high-oleic acid sunflower line FO-HO38 and the high-stearic acid sunflower line FO-HS43 (FO-HO38: SEQ ID NO: 2, FO-HS43: SEQ ID NO: 3). The region boxed in blue shows sequences that are putative TATA boxes, and the sequence boxed in red shows the translation start codon of SAD17. The region underlined in red is the sequence of the insertion in FO-HS43.

Figure 4 shows an electropherogram showing the results of PCR using a primer set flanking the insertion sequence upstream of the 5'-UTR of SAD17 in FO-HS43. Bands were confirmed for wild-type SAD17 (lower arrow) and for the mutant SAD17 containing the insertion sequence (upper arrow). Hs represents the dominant mutant (with the insertion sequence) allele, and hs represents the wild-type allele.

Figure 5 shows a graph illustrating the fatty acid content and Hs genotypes in each mature seed. The vertical axis shows the arachidonic acid content of the fatty acids, and the horizontal axis shows the stearic acid content of the fatty acids. Hs/Hs is homozygous for the dominant mutant (with a sequence insertion) allele, Hs/hs is heterozygous for the dominant mutant allele, and hs/hs is homozygous for the wild-type allele.

Figure 6 shows graphs illustrating the results of assessing the amount of accumulated SAD gene transcripts in grains at the developmental stage (10 days after pollination). Hs/Hs is homozygous for the dominant mutant (with sequence insertion) allele, Hs/hs is heterozygous for the dominant mutant allele, and hs/hs is homozygous for the wild-type allele. The vertical axis shows the relative values of the accumulation sum. The graphs on the left show the assessment results for the SAD6 gene, the SAD17 gene (both wild-type SAD17 and mutant SAD17), and the mutant SAD17 gene (only mutant SAD17).

Description of embodiments of the invention

[0027]

In this description, the terms “comprise,” “include,” and “contains” encompass the concepts of “comprising,” “including,” “consisting essentially of…” and “consisting of…”.

[0028]

1. Sunflower seed and sunflower plant

In one embodiment, the present invention relates to sunflower seeds containing an insertional mutation of the sequence at the 3'-side of the TATA box and at the 5'-side of the start codon of the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene (also referred to herein as "sunflower seeds according to the invention"). A more detailed description is provided below.

[0029]

Sunflower ( Helianthus annuus ) kernel oil contains approximately 5% stearic acid, and stearic acid is desaturated to oleic acid by Δ9-stearoyl-acyl-transfer protein desaturases (SADs). Attempts have been made to identify sunflower SAD genes, and two SAD gene sequences (SAD6 and SAD17) have been isolated as full-length cDNAs, which are highly expressed during the kernel development stage (NPL 2).

[0030]

In various sunflower species, the base sequence and amino acid sequence of the SAD17 gene are known, or they can be easily determined based on the known base sequence or amino acid sequence of the SAD17 gene (e.g., by analyzing for identity with these sequences). For example, the SAD17 gene is the gene identified in NCBI Gene ID: U91340, the amino acid sequence of the SAD17 gene is the amino acid sequence (SEQ ID NO: 12) identified under the accession number NCBI RefSeq: XP_021982111, and the mRNA sequence of the SAD17 gene is the base sequence (SEQ ID NO: 13) identified under the accession number NCBI RefSeq: XM_022126419.

[0031]

The SAD17 gene, which is the subject of the present invention, includes naturally occurring variants. The SAD17 gene, which is the subject of the present invention, may have major mutations such as substitutions, deletions, additions, and insertions, for example, in the promoter and/or coding regions, provided that the desaturase activity of the protein encoded by the target gene is not significantly impaired. The mutations preferably represent a mutation that does not result in an amino acid substitution in the protein translated from mRNA, or, preferably, a mutation that causes a conservative amino acid substitution.

[0032]

As used herein, the term "conservative substitution" means the replacement of an amino acid residue with another amino acid residue having a similar side chain. Thus, for example, a substitution with amino acid residues, each of which has a basic side chain, such as lysine, arginine, or histidine, is a conservative substitution. The following substitutions are also considered conservative: substitution of amino acid residues with an acidic side chain, such as aspartic acid or glutamic acid; substitution of amino acid residues with an uncharged polar side chain, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine; substitution of amino acid residues with a non-polar side chain, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; substitution of amino acid residues with a β-branched side chain, such as threonine, valine, or isoleucine; and substitution of amino acid residues with an aromatic side chain, such as tyrosine, phenylalanine, tryptophan, or histidine.

[0033]

For example, the amino acid sequence of the protein encoded by the SAD17 gene, which is the subject of the present invention, is, for example, at least 95%, preferably at least 98%, and more preferably at least 99% identical to the amino acid sequence of the protein encoded by the wild-type SAD17 gene of the same line. Furthermore, for example, the amino acid sequence of the protein encoded by the SAD17 gene, which is the subject of the present invention, is identical to the amino acid sequence of the protein encoded by the wild-type SAD17 gene of the same line; or has one, two or more (for example, from 2 to 10, preferably from 2 to 5, more preferably from 2 to 3, and even more preferably 2) substitutions, deletions, additions or insertions in this amino acid sequence.

[0034]

"Identity" of amino acid sequences refers to the degree to which two or more distinct amino acid sequences match. Thus, the greater the degree of match between two amino acid sequences, the greater the identity or similarity of these sequences. The degree of amino acid sequence identity can be determined using, for example, FASTA, a sequence analysis program with default parameters. Alternatively, the degree of amino acid sequence identity can be determined using the BLAST program, the algorithm of Karlin and Altschul (Karlin S, Altschul SF. Methods for assessing statistical significance of molecular sequence features by using general scoring schemes. Proc Natl Acad Sci USA. 87, pp. 2264–2268 (1990), and Karlin S, Altschul SF. Applications and statistics for multiple high-scoring segments in molecular sequences. Proc Natl Acad Sci USA. 90: 5873–7 (1993)). A program called "BLASTX" has been developed based on the BLAST algorithm. Specific methods for performing these analyses are known to those skilled in the art and can be found on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). The "identity" of base sequences is also determined as described above.

[0035]

The sunflower grain according to the invention contains an insertional mutation of the sequence on the 3' side of the TATA box and on the 5' side of the start codon of the SAD17 gene, which is the subject of the present invention, described above.

[0036]

The insertion sequence preferably has a length of 100 to 1200 bases, more preferably 200 to 1000 bases, even more preferably 400 to 800 bases, even more preferably 500 to 700 bases, and most preferably 550 to 650 bases.

[0037]

The sequence for insertion preferably includes, for example, the following sequences (a) or (b):

(a) the base sequence represented by SEQ ID NO: 1, or

(b) a base sequence that is at least 80% identical to the base sequence represented by SEQ ID NO: 1.

[0038]

The identity of the base sequence (b) is preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and particularly preferably at least 99%.

[0039]

The site into which the insertion sequence is inserted is not particularly limited, provided that it is located on the 3' side of the TATA box and on the 5' side of the start codon of the SAD17 gene. For example, the TATA box is the sequence (TATATAAAA) between the first base and the ninth base from the 5' end of the base sequence represented by SEQ ID NO: 2, which is a partial sequence of the SAD17 gene. The expression "on the 3' side of the TATA box" means being present on the 3' side from the base at the 3' end of the TATA box. For example, the start codon is the sequence between the 122nd base and the 124th base from the 5' base sequence represented by SEQ ID NO: 2, which is a partial sequence of the SAD17 gene. The term "upstream of the initiation codon" means a location 5' of the base at the 5' end of the initiation codon. In one embodiment, the site into which the insertion sequence is inserted is 3' of the TATA box and 5' of the start codon of the base sequence represented by SEQ ID NO: 2 of the SAD17 gene. For example, in the base sequence represented by SEQ ID NO: 2, which is a partial sequence of the SAD17 gene, the site into which the sequence is inserted is preferably a site from the 15th base to the 102nd base from the 5'-end of the sequence, more preferably a site from the 25th to the 82nd base from the 5'-end of the sequence, even more preferably a site from the 30th to the 62nd base from the 5'-end of the sequence, and even more preferably a site from the 35th base to the 52nd base from the 5'-end of the sequence. Various mutations (e.g., a substitution, deletion, addition or insertion of 1 to 30 (preferably 1 to 20, and more preferably 1 to 10) bases in length) introduced as a result of such an insertion may be present near the inserted sequence. Preferred examples of such a mutation include the insertion of a sequence consisting of GCTACTCT.

[0040]

In the sunflower seed according to the invention, it is preferable for both pairs of chromosomes to have an insertional sequence mutation. However, the goal can be achieved even if only one pair of chromosomes has an insertional sequence mutation.

[0041]

In the sunflower grain according to the invention, the introduction of a mutation reduces the accumulation of SAD17 gene transcripts. In the sunflower grain according to the invention, the amount of accumulated SAD17 gene transcripts (including both genes with an insertion sequence and genes without an insertion sequence) is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, and even more preferably 10% or less relative to the same amount of accumulation in the sunflower grains of the line before the introduction of the insertion mutation of the sequence. In a preferred embodiment of the sunflower grain according to the invention, the introduction of a mutation reduces the amount of accumulated SAD6 gene transcripts. In sunflower grains according to the invention, the level of SAD6 gene transcript accumulation is, for example, 90% or less, preferably 80% or less, and more preferably 70% or less, relative to the same amount of accumulation in the grains of the sunflower line before the introduction of the insertional mutation of the sequence. The amount of accumulated gene transcripts can be quantified by quantitative RT-PCR analysis using RNA isolated from grains at the developmental stage (e.g., 10 days after flowering (DAF)).

[0042]

The sunflower grain according to the invention has a fatty acid composition containing stearic acid, for example, 11% or more, preferably 15% or more, more preferably 20% or more, and even more preferably 25% or more. The upper limit is not particularly limited and is, for example, 40%, 35%, or 33%. In the present description, % fatty acids refers to the weight percentage of fatty acids as a component of the lipid. "Fatty acid composition" means the composition of fatty acid residues that make up the lipid.

[0043]

The sunflower grain according to the invention may also have a mutation different from the insertional mutation of the sequence described above. For example, in a preferred embodiment of the invention, the sunflower grain according to the invention is a sunflower grain obtained by introducing an insertional mutation of the sequence described above into a sunflower line with a high oleic acid content. Sunflower lines with a high oleic acid content are not particularly limited and are sunflower lines having an oleic acid content in the grains of, for example, 60% or more, preferably 70% or more, and more preferably 80% or more.

[0044]

Specific examples of sunflower grain according to the invention include grains of the FO-HS43 line (registration number in the International Patent Depository of Organisms: IPOD FERM BP-22390) and grains of their derivative lines. The grain of their derivative lines is not particularly limited, provided that it is grain of lines obtained by crossing grains of the FO-HS43 line. Examples include grains of lines obtained by self-crossing grains of the FO-HS43 line; grains obtained by crossing grains of the FO-HS43 line with grains of other lines; and grains of generations from n+1 to m (where each n and m represents any integer, for example, from 1 to 50), where the above-mentioned grains represent generation n.

[0045]

The sunflower grain according to the invention has a higher content of stearic acid in the fatty acid composition than sunflower grains (control sunflower grains) without an insertional sequence mutation (an insertional sequence mutation on the 3'-side of the TATA box and on the 5'-side of the start codon of the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene). Thus, for example, the content of stearic acid in the fatty acid composition of the sunflower grains according to the invention is, for example, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more higher than the content of stearic acid in the fatty acid composition of the control sunflower grains.

[0046]

In one embodiment, the present invention relates in part to sunflower plants containing sunflower seeds according to the invention or intended for the production of such seeds, and to sunflower plants obtained by germination and cultivation from sunflower seeds according to the invention (hereinafter they may be referred to by the general term "sunflower plant according to the invention"). The expression "containing sunflower seeds according to the invention" means that they contain seeds of grown sunflower according to the invention in a flower, and the expression "intended for the production of sunflower seeds according to the invention" means that they are intended for the production of sunflower seeds according to the invention by cultivation.

[0047]

A sunflower plant is defined as not only a plant at any stage of its development but also any part of a plant that can be attached to or separated from the whole plant. Such plant parts include, but are not limited to, plant organs, tissues, and cells. Specific examples of plant parts include a stem, leaf, root, inflorescence, flower, individual flowers, fruit, peduncle, petiole, stamen, anther, stigma, style, ovary, petal, sepal, carpel, root tip, root cap, root hair, leaf hair, seed hair, pollen particle, microspore, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, germ albumen, companion cell, and guard cell; and any other known plant organs, tissues, and cells.

[0048]

The sunflower seeds and sunflower plants of the invention may be produced using known methods or in accordance with known methods. For example, the sunflower seeds and sunflower plants of the invention may be produced using agrobacterial methods, particle "shooting" methods, protoplast methods, genome editing methods, and the like. Alternatively, the sunflower seeds and sunflower plants of the invention may be created by crossing the FO-HS43 line or a derivative thereof.

[0049]

The sunflower seeds and sunflower plants of the invention exhibit a dominantly inherited trait of high stearic acid content. As used herein, the term "dominance" denotes a non-recessive inheritance pattern; and as used herein, the term "dominance" includes incomplete dominance.

[0050]

2. Method for obtaining lipids

In one embodiment, the present invention relates to a method for producing a lipid comprising collecting the lipid from sunflower seed according to the invention (this method may be referred to hereinafter as the "production method according to the invention"). A detailed description is provided below.

[0051]

In the production method according to the invention, sunflower seeds are used as oilseeds. The sunflower seeds according to the invention can be obtained after pre-processing, such as drying and crushing, if necessary.

[0052]

The collection method can be any, as long as the lipid can be collected from the sunflower seeds. Examples of collection methods include pressing, solvent extraction, and methods that combine these methods. In the pressing method, the seeds are physically pressed under pressure, resulting in the collection of the lipid. In the solvent extraction method, a solvent is added to the seeds to transfer the oil contained in the seeds into the solvent. The solvent, in which this oil is dissolved, is separated into the volatile solvent and the oil using a distillation apparatus, thus collecting the lipid.

[0053]

The collected lipid (crude oil) can then undergo further purification. Impurities contained in the crude oil, such as resins, free fatty acids, pigments, odorants, and finely dispersed foreign substances, can be removed as needed. Examples of purification processes include refining, deacidification, bleaching, dewaxing, and deodorization.

[0054]

The resulting lipid can be used as sunflower oil for various purposes. Furthermore, the resulting lipid can be used as a raw material for producing SOS lipid sources as a cocoa butter substitute.

[0055]

3. Other options

In one embodiment, the present invention relates to a method for producing sunflower seeds in which the distribution of saturated fatty acids, namely, the various types of triglyceride molecules, is altered compared to wild-type seeds. This method comprises the step of treating the parent seeds for a period of time sufficient to induce one or more mutations in genetic traits involved in triglyceride biosynthesis, using a sufficiently powerful mutagenesis method. This method increases the stearic acid content. Examples of treatment by mutagenesis include mutagenesis using a mutagenic agent such as sodium azide or an alkylating agent, and mutagenesis using X-ray or gamma radiation. Alternatively, other mutagenesis methods that produce the same or similar effects as the above-mentioned agents may also be used.

Examples

[0056]

The present invention is described in detail below with reference to Examples; however, the present invention is not limited to these Examples.

[0057]

Test Example 1: Creation of a Sunflower Mutant with High Stearic Acid Content

A total of 14,000 kernels of the high-oleic acid sunflower line FO-HO38 were irradiated with a carbon ion beam ( ¹² C ⁶⁺ , energy: 320 MeV, dose: 5 Gy) to obtain first-generation mutant (M1) kernels. The M1 kernels were grown and self-pollinated to produce M2 kernels of at least 3,000 lines. The M2 kernels of each line were subjected to gas chromatography to analyze the fatty acid composition of individual kernels, and one kernel line with high stearic acid content was selected. Figure 1 shows a graph of the frequency distribution of stearic acid content in the M2 kernel population of this line. Figure 1 shows two distinct peaks, indicating that approximately 28% of the kernels (25/88 kernels) had low stearic acid content (less than 11%), which is almost the same as the stearic acid content of the parent line (FO-HO38) (approximately 5%), while approximately 72% of the kernels (63/88 kernels) had high stearic acid content (11% or more). Kernels with high stearic acid content were then selected, and the progeny were grown for five generations; and the results confirmed stable stearic acid content. This line was named “FO-HS43 line” and was deposited with the International Patent Organism Depository of the National Institute of Technology and Evaluation (IPOD, Room 120, 2-5-8, Kazusakamatari, Kisarazu, Chiba, 292-0818 Japan) (deposit number: IPOD FERM BP-22390, deposit date: April 23, 2020).

[0058]

According to Mendelian inheritance, if a mutation that arose in the grains of the M1 generation (probably in only one of the chromosome pairs) is inherited in the M2 generation through self-pollination, then in the grains of the M2 generation, the ratio of grains without mutation in both pairs of chromosomes (+/+): grains with mutation in only one of the chromosome pairs (+/mt): grains with mutation in both pairs of chromosomes (mt/mt) is 1:2:1. In this case, if the trait obtained as a result of mutation is a dominant trait, then the grains with this trait should make up approximately 75% (3 (the sum of +/mt and mt/mt)/4 (the sum of +/+, +/mt and mt/mt)). The above results showed that the high stearic acid content of the grains accounted for approximately 72%, and thus it was suggested that the high stearic acid content trait caused by the mutation in the grains was a dominant mutant trait.

[0059]

If this is true, it may be possible to create isogenic hybrids with different fatty acid composition groups using this genetic mutation in either heterozygous or homozygous form, which can be further expanded by using suitable genetic resources.

[0060]

To confirm this, the FO-HS43 line was crossed with a low-stearic acid line (the FO-HO13 line, with a stearic acid content of approximately 3 to 5%). Fatty acid analysis of the resulting F1 grains revealed that all grains had a stearic acid content of 15% or more. These results indicated that the high-stearic acid trait in the FO-HS43 line was dominant; the dominant mutant allele with high stearic acid content was named "Hs."

[0061]

Test Example 2: Identification of the Hs gene with high stearic acid content

To identify the Hs locus in FO-HS43, FO-HS43 was crossed with the HA89 line and linkage analysis was performed using the F2 population. As a result, the Hs locus was mapped at one end of the first linkage group (LG1) and located near the SSR markers ORS959 and ORS970 (Fig. 2).

[0062]

Since LG1 contains the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene, the sequence near the SAD17 gene of FO-HS43 was analyzed in detail. The results confirmed the insertion of a 586-bp sequence (SEQ ID NO: 1), which was not found in FO-HO38, on the 3' side of the TATA box and on the 5' side of the start codon of the SAD17 gene (Fig. 3).

[0063]

The fact that the insertion was detected downstream of the TATA box sequence (TATATAAAA) suggests that the insertion sequence can also be transcribed. Thus, SAD17 transcripts were isolated in FO-HS43, confirming the formation of a long SAD17 transcript (mutant SAD17) containing part of the insertion sequence.

[0064]

Confirmation was done by PCR using a primer set (forward: SEQ ID NO: 4; reverse: SEQ ID NO: 5) to flank the above-mentioned SAD17 insertion sequence in FO-HS43. The result revealed the presence of the insertion sequence (Hs allele) and the absence of the insertion sequence (hs allele), indicating the possibility of using this primer set as a codominant marker to facilitate the determination of Hs gene genotypes (homozygous Hs mutant, heterozygous Hs mutant, or homozygous hs wild-type) (Fig. 4). Furthermore, the Hs genotypes determined by this method showed a high correlation with the stearic acid content (Fig. 5).

[0065]

Grains at the development stage (10 days after pollination) were subjected to quantitative RT-PCR to determine the amount of accumulated SAD gene transcripts. The following primer sets were used: primers for detecting the SAD6 gene (forward: SEQ ID NO: 6, reverse: SEQ ID NO: 7), primers for detecting the SAD17 gene (both wild-type SAD17 and mutant SAD17) (forward: SEQ ID NO: 8, reverse: SEQ ID NO: 9), and primers for detecting the mutant SAD17 gene (only mutant SAD17) (forward: SEQ ID NO: 10, reverse: SEQ ID NO: 11). The results confirmed that the accumulation of SAD17 gene transcripts was almost undetectable in homozygous individuals with the mutant Hs (Hs/Hs), and that the accumulation amount in heterozygous individuals with the mutant Hs (Hs/hs) was also reduced to half or less than that of the wild-type individuals (hs/hs) (Fig. 6). For the homozygous Hs mutant individuals, the results also showed a trend towards a decrease in the accumulation of SAD6 transcripts, which is a homologous gene of SAD17. These results correlated with the results for the Hs genotypes and the stearic acid content of the grains, indicating that the expression of the high stearic acid trait in FO-HS43 was due to the effective inhibition of SAD gene expression achieved by inserting a sequence 3' to the TATA box and 5' to the start codon of the SAD17 gene.

List of sequences

P21-066WO_PCT_ _20210511_113354_0.txt

SEQUENCE LIST

--->

<110> FUJI OIL HOLDINGS INC.

<120> Sunflower seeds

<130> P21-066WO

<150> JP 2020-127157

<151> 2020-07-28

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 586

<212> DNA

<213> Artificial sequence

<220>

<223> insert

<400> 1

taggggtgtt caaaaaactc gtggctcgca gctcgctcga aactcgctcg aaaaaagctc 60

gaagaaagct cggctcgaaa ttggctcggt tgtaaacgag ccagctcggc tcggctcggt 120

ttgtaaacga gccgagctcg agcttggcct ggctcggctc gtgagctcgt gagctggctc 180

gataaggctt acatacttca tttttttttt tgtcccacat cgcttagaaa acaaaaacta 240

agggagtatc tcccctataa aagaaggtaa agacaatgga gaaagtgaat tacctattta 300

tacttgcata tttagccatg tggttaaatt aaatggcaac tatggccctt aggctttgaa 360

gaaattcatt tttaagggct ttttaagtaa taaattttgc ggtactttgt tagttcgagc 420

taaatcgagc cgagccgagc tagctcgaat tcaaatcgag ccgagctcga gcctcaaatc 480

tcagctcgat ttgaaattcg agctcgagcc gagccagctc gaatcgagtt cgagccgagc 540

tcgagctggg tctagctcgg ctcgactcgg ctcgtttaca gcccta 586

<210> 2

<211> 124

<212> DNA

<213> Helianthus annuus

<400> 2

tatataaaac atagcaaaat ataagaccat ctgtgctact ctcttctttc tcacttgtgt 60

ggaggaaaac acaactcaaa aaactgtgca acaacaagca cacacaagaa caacatcaac 120

aatg 124

<210> 3

<211> 718

<212> DNA

<213> Artificial sequence

<220>

<223> built-in 5'UTR

<400> 3

tatataaaac atagcaaaat ataagaccat ctgtgctact cttaggggtg ttcaaaaaac 60

tcgtggctcg cagctcgctc gaaactcgct cgaaaaaagc tcgaagaaag ctcggctcga 120

aattggctcg gttgtaaacg agccagctcg gctcggctcg gtttgtaaac gagccgagct 180

cgagcttggc ctggctcggc tcgtgagctc gtgagctggc tcgataaggc ttacatactt 240

catttttttt tttgtcccac atcgcttaga aaacaaaaac taagggagta tctcccctat 300

aaaagaaggt aaagacaatg gagaaagtga attacctatt tatacttgca tatttagcca 360

tgtggttaaa ttaaatggca actatggccc ttaggctttg aagaaattca tttttaaggg 420

ctttttaagt aataaatttt gcggtacttt gttagttcga gctaaatcga gccgagccga 480

gctagctcga attcaaatcg agccgagctc gagcctcaaa tctcagctcg atttgaaatt 540

cgagctcgag ccgagccagc tcgaatcgag ttcgagccga gctcgagctg ggtctagctc 600

ggctcgactc ggctcgttta cagccctagc tactctcttc tttctcactt gtgtggagga 660

aaacacaact caaaaaactg tgcaacaaca agcacacaca agaacaacat caacaatg 718

<210> 4

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 4

aacataagtc taagggcata tataaaaca 29

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 5

aatttgggag atctgagagg tttcggttga 30

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 6

gagtccggtg acgcttcaac gggaga 26

<210> 7

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 7

gtggtggggt gaagggcttc ttggta 26

<210> 8

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 8

caatacggcg acgtttcaat cagacc 26

<210> 9

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 9

ttggaggggt aaagggcttt ttggtg 26

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 10

tcgactcggc tcgtttacag 20

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> Primer

<400> 11

ccattgttga tgttgttctt gt 22

<210> 12

<211> 396

<212> PRT

<213> Helianthus annuus

<400> 12

Met Ala Ile Arg Ile Asn Thr Ala Thr Phe Gln Ser Asp Leu Tyr Arg

1 5 10 15

Ser Phe Ala Phe Pro Gln Pro Lys Pro Leu Arg Ser Pro Lys Phe Ala

20 25 30

Met Ala Ser Thr Ile Gly Ser Ala Thr Thr Lys Val Glu Ser Thr Lys

35 40 45

Lys Pro Phe Thr Pro Pro Arg Glu Val His Gln Gln Val Leu His Ser

50 55 60

Met Pro Pro Gln Lys Ile Glu Ile Phe Lys Ser Met Glu Gly Trp Ala

65 70 75 80

Glu Asp Asn Ile Leu Val His Leu Lys Pro Val Glu Lys Cys Trp Gln

85 90 95

Ala Gln Asp Phe Leu Pro Asp Pro Ala Ser Asp Gly Phe Met Glu Gln

100 105 110

Val Glu Glu Leu Arg Ala Arg Ala Lys Glu Ile Pro Asp Asp Tyr Phe

115 120 125

Val Val Leu Val Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr

130 135 140

Gln Thr Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala

145 150 155 160

Ser Pro Thr Ser Trp Ala Ile Trp Thr Arg Ala Trp Thr Ala Glu Glu

165 170 175

Asn Arg His Gly Asp Leu Leu His Gln Tyr Leu Tyr Leu Ser Gly Arg

180 185 190

Val Asp Met Arg Gln Ile Gln Lys Thr Ile Gln Tyr Leu Ile Gly Ser

195 200 205

Gly Met Asp Pro Arg Thr Glu Asn Ser Pro Tyr Leu Gly Phe Ile Tyr

210 215 220

Thr Ser Phe Gln Glu Arg Ala Thr Phe Ile Ser His Gly Asn Thr Ala

225 230 235 240

Arg His Ala Lys Glu His Gly Asp Val Lys Leu Ala Gln Met Cys Gly

245 250 255

Ile Ile Ala Ala Asp Glu Lys Arg His Glu Thr Ala Tyr Thr Lys Ile

260 265 270

Val Glu Lys Leu Phe Glu Ile Asp Pro Asp Gly Thr Val Leu Ala Phe

275 280 285

Ala Asp Met Met Arg Lys Lys Ile Ser Met Pro Ala His Leu Met Tyr

290 295 300

Asp Gly Arg Asp Asp Asn Leu Phe Glu Asn Phe Ser Ala Val Ala Gln

305 310 315 320

Arg Leu Gly Val Tyr Thr Ala Lys Asp Tyr Ala Asp Ile Leu Glu Phe

325 330 335

Leu Val Gly Arg Trp Lys Val Ala Asp Leu Thr Gly Leu Ser Gly Glu

340 345 350

Gly Arg Lys Ala Gln Asp Tyr Val Cys Gly Leu Ala Pro Arg Ile Arg

355 360 365

Arg Leu Glu Glu Arg Asn Ser Ala Arg Ala Lys Glu Ser Val Asn Val

370 375 380

Pro Phe Ser Trp Ile Phe Asp Arg Glu Val Lys Leu

385 390 395

<210> 13

<211> 1459

<212> DNA

<213> Helianthus annuus

<400> 13

ctcttctttc tcacttgtgt ggaggaaaac acaactcaaa aaactgtgca acaacaagca 60

cacacaagaa caacatcaac aatggcgatt cgcatcaata cggcgacgtt tcaatcagac 120

ctgtatcgtt cattcgcgtt tcctcaaccg aaacctctca gatctcccaa attcgccatg 180

gcttccacca ttggatccgc tacaacgaag gttgaaagca ccaaaaagcc ctttacccct 240

ccaagggagg ttcaccaaca ggtgctacac tcaatgccgc cacaaaagat cgaaatcttc 300

aaatccatgg agggttgggc cgaagataac atattggttc acctaaagcc cgtcgaaaaa 360

tgctggcaag cacaggattt cctaccagat cccgcatctg acggatttat ggaacaagtg 420

gaggaactac gagctcgggc taaggagatt ccggatgatt actttgttgt tttggtggga 480

gatatgatta ctgaagaagc actgcctact taccaaacaa tgcttaatac tcttgatggt 540

gtgcgtgatg agaccggggc tagccctact tcttgggcta tctggactcg ggcttggacc 600

gctgaagaaa acaggcacgg tgatcttcta catcagtatc tgtatcttag tgggcgggtc 660

gacatgaggc agattcagaa gacaattcag tacctcattg ggtctggaat ggacccccgg 720

actgaaaaca gtccttacct tgggttcatc tacacttcat ttcaagagcg tgccaccttc 780

atctctcacg gaaacacagc ccggcacgca aaggagcatg gtgacgtgaa gctggctcaa 840

atgtgcggta taattgcagc tgatgaaaaa aggcacgaaa ccgcctacac aaaaatagta 900

gaaaaactct tcgaaattga cccggacggc actgttctcg cttttgctga catgatgagg 960

aaaaagatct ccatgcctgc acacttgatg tacgatgggc gtgacgataa cctcttcgaa 1020

aatttctcag ctgttgccca aaggctcggt gtgtacactg caaaggacta tgcagacatt 1080

ctggagtttc tggtgggccg gtggaaggtg gcggatttaa ccgggctttc tggtgaaggg 1140

cgtaaagccc aagactatgt gtgcgggctg gccccaagaa tcagaaggct tgaggagagg 1200

aactcggcaa gggcgaagga aagtgtgaac gttccgttca gctggatctt tgatagagaa 1260

gtgaagctct gaaagttgct atagtaagtt gttaaaacct atattagatt gtcgtttctt 1320

tcgtgtagtg ctgatatgtg ttttcttctc gactttgtaa tgtgtctcga gttcgggtgt 1380

ttgtaaactt gttggacata ttataaaact tgttatgcca gttttgatga cgattatcgt 1440

ctcatcggtc ggttgcaga 1459

<---

Claims (17)

1. Sunflower seed for obtaining lipid containing an insertional mutation of the sequence on the 3' side of the TATA box and on the 5' side of the start codon of the stearoyl-acyl transfer protein desaturase 17 (SAD17) gene, wherein the length of the sequence is from 100 to 1200 bases. 2. A sunflower seed according to claim 1, wherein the length of the sequence is from 200 to 1000 bases. 3. A sunflower seed according to claim 1, wherein the length of the sequence is from 400 to 800 bases. 4. Sunflower seed according to any one of paragraphs 1-3, wherein the sequence comprises the following sequences (a) or (b): (a) the base sequence represented by SEQ ID NO: 1, or (b) a sequence of bases that is at least is 80% identical to the base sequence shown in SEQ ID NO: 1. 5. Sunflower seed according to claim 4, wherein the identity is at least 90%. 6. Sunflower seed according to any one of claims 1 to 5, wherein the insertional sequence mutation is introduced into a high oleic acid sunflower line. 7. Sunflower grain according to any one of paragraphs 1-6, having a fatty acid composition with a stearic acid content of 11% or more. 8. Sunflower seed according to any one of claims 1-7, containing an insertional mutation of a sequence in both pairs of chromosomes. 9. Sunflower seed according to any one of claims 1 to 8, containing an insertional mutation of the sequence on the 3'-side of the TATA box and on the 5'-side of the start codon of the base sequence represented by SEQ ID NO: 2 stearoyl-acyl transfer protein desaturase 17 (SAD17), and the sequence has a length of 400 to 800 bases, Moreover, the sunflower grain has a higher stearic acid content than the stearic acid content in sunflower grains without the insertional sequence mutation. 10. Sunflower grain according to any one of claims 1 to 9, which is grain of the FO-HS43 line (registration number in the International Patent Depository of Organisms: IPOD FERM BP-22390) or grain of a derivative line thereof. 11. A sunflower plant for obtaining a lipid, containing a sunflower seed according to any one of claims 1-10. 12. A method for obtaining a lipid, including collecting lipid from sunflower grain according to any one of claims 1-10.