KR102369344B1 - Composition for modifying the ratio of unsaturated fatty acids in seeds and seeds prepared by the same - Google Patents

Abstract

The present invention relates to a composition for producing a seed having a controlled unsaturated fatty acid ratio and a seed using the same, and by overexpressing or inhibiting HAT2, a plant transcription regulator, the ratio of oleic acid, linoleic acid or linolenic acid in the seed of a plant can be adjusted.

Description

A composition for producing a seed having a controlled unsaturated fatty acid ratio and a seed prepared therefrom {Composition for modifying the ratio of unsaturated fatty acids in seeds and seeds prepared by the same}

The present invention relates to a composition for producing a seed with a controlled unsaturated fatty acid ratio and a seed using the same.

Fatty acids in plants are important constituents of cell membranes and oil storage in seeds. Plant seed oil contains saturated fatty acids and unsaturated fatty acids in a certain ratio. Saturated fatty acids and unsaturated fatty acids are used in various industries according to their structure and properties. For example, the saturated fatty acids palmitic acid (16:0) and stearic acid (18:0) are solid at room temperature, so they are used for the manufacture of margarine, and the monounsaturated fatty acid oleic acid (18:0) Because it is stable against oxidation, it is suitable for use in salads or frying, and is useful as a fatty acid for biodiesel. Polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3) are known as health functional fatty acids.

Accordingly, research for producing plants and plant oils having selected fatty acids in order to increase industrial utilization is being conducted. For example, there is a study on a transgenic plant transformed with the perilla-derived microsomal linoleic acid desaturase gene of Korean Patent Registration No. 0914454, and a method of regulating the fatty acid composition in the seed oil of the transgenic plant using this gene. However, there have been no studies on increasing the content of specific fatty acids in plant seeds by introducing a gene encoding HAT2, a transcriptional regulator of Arabidopsis thaliana, into a plant.

Korean Patent No. 0914454 (2009.08.21.)

An object of the present invention is to provide a composition for preparing seeds in which the ratio of unsaturated fatty acids is controlled.

An object of the present invention is to provide a seed having a controlled unsaturated fatty acid ratio and a method for producing the same.

An object of the present invention is to provide a plant for producing seeds in which the ratio of unsaturated fatty acids is controlled, and a method for producing the same.

1. A composition for producing a seed in which the ratio of unsaturated fatty acids is controlled, including a substance that promotes expression of a gene encoding HAT2 protein.

2. The composition for preparing seeds according to the above 1, wherein the unsaturated fatty acid is oleic acid, linoleic acid or linolenic acid.

3. The composition for preparing seeds according to the above 1, wherein the content of oleic acid and linoleic acid in the total fatty acids in the seed is increased and the content of linolenic acid is decreased, the unsaturated fatty acid ratio is controlled.

4. The composition for preparing seeds according to 1 above, wherein the seed is a seed of Arabidopsis thaliana or an oil plant, the ratio of unsaturated fatty acids being controlled.

5. The above 4, wherein the oil and fat plant includes any one or more selected from the group consisting of camelina, sesame, rapeseed, sunflower, castor, peanut, soybean, coco palm, oil palm and jojoba, unsaturated A composition for preparing seeds with a controlled fatty acid ratio.

6. The composition for producing a seed according to 1 above, wherein the seed is a seed of Arabidopsis thaliana or Camelina, and the gene consists of the nucleotide sequence of SEQ ID NO: 1, the ratio of unsaturated fatty acids is controlled.

7. The composition according to 1 above, wherein the substance promoting the expression of the gene encoding the HAT2 protein is a vector into which the gene encoding the HAT2 protein is introduced, the composition for producing a seed with a controlled unsaturated fatty acid ratio.

8. A method for producing a seed in which the ratio of unsaturated fatty acids is controlled, comprising transforming the plant with a recombinant vector containing a gene encoding the HAT2 protein.

9. The method according to 8 above, wherein the content of oleic acid and linoleic acid in the total fatty acids in the seed is increased, and the content of linolenic acid is decreased, the unsaturated fatty acid ratio is controlled.

10. constructing a recombinant vector containing a gene encoding the HAT2 protein; transforming the plant with the recombinant vector; and selecting a transgenic plant; A method for producing a plant for seed production in which the ratio of unsaturated fatty acids is controlled, comprising:

11. Plants for seed production in which the ratio of unsaturated fatty acids prepared by the method according to 10 above is controlled.

12. Seeds in which the ratio of unsaturated fatty acids produced by the plant according to 11 above is controlled.

13. Culturing a seed in which the gene encoding the HAT2 protein is attenuated or deleted; and selecting a mutant plant in which the attenuated or deleted gene is homogenized from among plants obtained by culturing the seed; A method for producing a plant for producing seeds with a controlled unsaturated fatty acid ratio comprising a.

14. A plant for producing seeds in which the ratio of unsaturated fatty acids produced by the method according to claim 13 is controlled.

15. Seed in which the ratio of unsaturated fatty acids produced by the plant according to claim 14 is controlled.

The present invention relates to a composition for producing a seed having a controlled unsaturated fatty acid ratio and a seed using the same, and by overexpressing or inhibiting HAT2, an Arabidopsis transcriptional regulator, the ratio of oleic acid, linoleic acid or linolenic acid in the seed of a plant can be controlled. .

Figure 1a is a diagram confirming the structure by analyzing the amino acid sequence of the HAT2 protein, Figure 1b is a diagram confirming the location in the nucleus of the HAT2 protein.
Figure 2a is a diagram analyzing the expression of each Arabidopsis thaliana tissue by qRT-PCR of the HAT2 gene, Figure 2b is a diagram analyzing the expression of each seed development stage, and Figure 2c is Arabidopsis thaliana expressing the GUS gene under the control of the HAT2 promoter. It is a diagram analyzing the expression of the GUS reporter gene for each tissue of the transformant.
Figure 3a is a diagram showing the gene structure of the hat2-1 mutant and hat2-2 mutant, Figure 3b is a diagram analyzing the genotype of the mutant, Figure 3C is analysis of the expression of the HAT2 gene in the wild-type and mutant. It is also
Figure 4 is a diagram comparing the fatty acid composition of wild-type and mutant Arabidopsis thaliana seeds.
5 is a diagram comparing and analyzing the composition of seed fatty acids between the wild-type and Arabidopsis thaliana transformants overexpressing the HAT2 gene.
6 is a diagram illustrating the production of a Camelina transformant into which the Arabidopsis HAT2 gene is introduced.
7 is an analysis of the expression of Arabidopsis HAT2 in the seeds of HAT2 gene overexpression Camelina transformants.
8 is a diagram illustrating comparative analysis of the composition of seed fatty acids between wild-type and HAT2 gene overexpressing Camelina transformants.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preparing seeds in which the ratio of unsaturated fatty acids is controlled, including a substance that promotes expression of a gene encoding HAT2 protein.

The HAT2 protein is a plant transcriptional regulator expressed in all plants, and is a transcriptional regulator encoded by the HAT2 gene belonging to the HD-Zip II subfamily (Homeodomain-leucine zipper II subfamily).

In the present invention, promoting gene expression means improving, inducing, stimulating, or increasing the expression of a gene by acting directly or indirectly on the gene.

The substance is any substance that can promote the expression of HAT2 gene, and is not limited to the type or type of substance, for example, a high molecular compound such as a peptide, protein, nucleic acid, carbohydrate and lipid, a natural product, or a chemically synthesized compound All of these may be used.

In addition, according to an embodiment of the present invention, the material may be a recombinant vector including a gene encoding the protein, and, if necessary, the vector may be one into which a promoter overexpressing the gene is inserted.

The recombinant vector is preferably for plant transformation, but is not limited thereto. By 'recombinant vector' is meant a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expression of an operably linked coding sequence in a particular host organism. The appropriate nucleic acid sequence may be a promoter, and may further include an enhancer, a transcription terminator and a polyadenylation signal. Promoters, enhancers, transcription terminators and polyadenylation signals available in eukaryotes are known.

The recombinant vector may be a plant expression vector that may be directly introduced into a plant cell by inserting the nucleotide sequence of the gene, or may be introduced into a microorganism causing an infection in a plant. The vector system of the present invention can be constructed through various methods known in the art. The vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. On the other hand, vectors that can be used in the present invention include plasmids (eg, pSK349, pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series and pUC19, etc.), phage (eg, λgt ㆍλ4B, λ-Charon, λΔz1 and M13, etc.) or virus (eg SV40, etc.) can be manufactured.

For example, a recombinant vector is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host such as Agrobacterium tumefaciens . There are many strains of Agrobacterium that can be used for such manipulation and are known in the art. Other types of Ti-plasmid vectors can now be used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant. Other types of Ti-plasmid vectors can now be used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant. A particularly preferred form of the Ti-plasmid vector is a binary vector. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors such as those that can be derived from double-stranded plant viruses (eg CaMV) and single-stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The vector can be advantageously used when it is difficult to properly transform a plant host.

The vector of the present invention may include a resistance gene commonly used in the art as a selection marker. Examples include resistance genes to basta herbicides, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

The promoter may be a 35S CaMV, Glycinin, actin, ubiquitin, pEMU, MAS, or histone promoter, but is not limited thereto. 'Promoter' refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.

As a specific example when the substance promoting the expression of the gene encoding the HAT2 protein is a recombinant vector carrying the gene encoding the HAT2 protein, the gene may be a gene of a target plant species. For example, when the target plant is Arabidopsis thaliana, the gene consisting of the sequence of SEQ ID NO: 1 encoding the Arabidopsis HAT2 protein may be used. For the HAT2 gene sequence of each plant species, a sequence known from NCBI genbank or the like may be used. In addition, the gene may use the HAT2 gene of a plant species different from the target plant species. For example, when the target plant is camelina, the gene consisting of the sequence of SEQ ID NO: 1 encoding the Arabidopsis HAT2 protein may be used.

In addition, variants of the nucleotide sequence may be included within the scope of the present invention. Specifically, the gene includes a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to the base sequence of SEQ ID NO: 1, respectively. can

The '% sequence homology' is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the sequence in the comparison region is located in a reference sequence (not including additions or deletions) to the optimal alignment of the two sequences. It may include additions or deletions compared to.

The HAT2 protein encoded by the HAT gene consisting of the nucleotide sequence of SEQ ID NO: 1 may consist of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto. The HAT2 protein of the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the protein. "Functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more of the amino acid sequence of SEQ ID NO: 2 as a result of amino acid addition, substitution or deletion. As having sequence homology, it refers to a protein that exhibits substantially the same physiological activity as the protein consisting of the amino acid sequence of SEQ ID NO: 2. In the present invention, 'substantially homogeneous physiological activity' refers to the activity of regulating the ratio of unsaturated fatty acids in the seed of a plant.

Although the base sequence of the HAT2 gene present in plants is different for each plant, overexpression or suppression of the HAT2 gene of each plant can control the ratio of unsaturated fatty acids in the seeds of the plant.

In the present invention, the unsaturated fatty acid may be oleic acid, linoleic acid, or linolenic acid, but is not limited thereto. Oleic acid is an unsaturated fatty acid of 18:1 (CH ₃ (CH ₂ ) ₇ CH=CH(CH ₂ ) ₇ COOH), and is a main component of oils and fats such as olive oil and camellia oil. In some cases, it is used as a bone agent instead. Linoleic acid is an unsaturated fatty acid of 18:2 (CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH(CH ₂ ) ₇ COOH). fatty acids. Linolenic acid is an unsaturated fatty acid of 18:3 (CH ₃ (CH ₂ CH=CH) ₃ CH ₂ (CH ₂ ) ₆ COOH), flaxseed oil, walnut oil, evening primrose, blackcurrant seed oil, barley. It is contained in borage oil or vegetable oils such as perilla, and is effective in lowering blood cholesterol levels. do.

According to an embodiment of the present invention, the composition may increase the content of oleic acid and linoleic acid among the total fatty acids in the seed, and decrease the content of linolenic acid.

The present invention can control only the ratio of specific fatty acids including oleic acid, linoleic acid or linolenic acid among the total fatty acids in the seed without significant change in the total fatty acid content per seed.

The composition of the present invention includes a substance that promotes expression of a gene encoding the HAT2 protein, and the substance may be a recombinant vector containing the gene, but is not limited thereto.

The gene may be a gene consisting of the nucleotide sequence of SEQ ID NO: 1 derived from Arabidopsis, but is not limited thereto, and may be a HAT2 gene of each plant species. For the HAT2 gene sequence of each plant species, a sequence known from NCBI genbank or the like may be used.

In addition, the composition may further include any material that can be used for the production of seeds with the ratio of unsaturated fatty acids adjusted. The composition of the present invention may include a microorganism transformed with the recombinant vector. The microorganism may be used without limitation as long as it causes an infection in a plant.

In an embodiment of the present invention, Agrobacterium tumefaciens may be used. The method for introducing the recombinant vector of the present invention into Agrobacterium may be carried out through various methods known to those skilled in the art, for example, particle bombardment, electroporation, transfection. ), a lithium acetate method, a heat shock method, and a freeze-thaw method.

The seeds whose unsaturated fatty acid ratio is adjusted according to the present invention may be seeds of Arabidopsis thaliana or oilseed plants, but is not limited thereto. The oil and fat plant may be a plant that obtains oil from seeds or fruits, for example, any one or more selected from the group consisting of camelina, sesame, rapeseed, sunflower, castor, peanut, soybean, coco palm, oil palm, and jojoba. may include

The plant according to the present invention includes food crops selected from the group consisting of rice, wheat, barley, corn, soybean, potato, wheat, red bean, oat and sorghum; vegetable crops selected from the group consisting of Arabidopsis thaliana, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; fruit trees selected from the group consisting of apple trees, pear trees, jujube trees, peaches, poplars, grapes, tangerines, persimmons, plums, apricots and bananas; flowers selected from the group consisting of roses, gladiolus, gerbera, carnations, chrysanthemums, lilies and tulips; And it may be forage crops selected from the group consisting of ryegrass, red clover, orchard grass, alpha alpha, tall fescue and perennial ryegrass.

The present invention provides a method for producing a seed having a controlled unsaturated fatty acid ratio, comprising transforming a plant with a recombinant vector comprising a gene encoding HAT2 protein.

As the gene encoding the HAT2 protein, a gene of a target plant species may be used. For example, when the target plant is Arabidopsis thaliana, the gene consisting of the sequence of SEQ ID NO: 1 encoding the Arabidopsis HAT2 protein may be used. For the HAT2 gene sequence of each plant species, a sequence known from NCBI genbank or the like may be used. In addition, the gene encoding the HAT2 protein may be a gene of a plant species different from the target plant species. For example, when the target plant is camelina, the gene consisting of the sequence of SEQ ID NO: 1 encoding the Arabidopsis HAT2 protein may be used.

The method of transforming a plant with a recombinant vector containing the gene means any method of transferring DNA to a plant. The method of the present invention may use microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, Agrobacterium-mediated transfection method, DEAE-dextran treatment method, and gene bambadment, etc. It is not limited thereto, and various known methods may be used.

Plants produced by the method of the present invention can produce seeds in which the ratio of unsaturated fatty acids is controlled. The seeds increase the content of oleic acid and linoleic acid among the total fatty acids in the seed, and decrease the content of linolenic acid.

The present invention can control only the ratio of specific fatty acids including oleic acid, linoleic acid or linolenic acid among the total fatty acids in the seed without significant change in the total fatty acid content per seed.

The present invention comprises the steps of constructing a recombinant vector comprising a gene encoding the HAT2 protein; transforming the plant with the recombinant vector; and selecting a transgenic plant; It provides a method for producing a plant for seed production in which the ratio of unsaturated fatty acids containing a control is adjusted.

As the gene encoding the HAT2 protein, a gene of a target plant species may be used. For example, when the target plant is Arabidopsis thaliana, the gene consisting of the sequence of SEQ ID NO: 1 encoding the Arabidopsis HAT2 protein may be used. For the HAT2 gene sequence of each plant species, a sequence known from NCBI genbank or the like may be used.

The recombinant vector may be within the range described above, and may be manufactured by a method within the range described above.

A method for transforming a plant may use a method within the above-described range.

Selection of transgenic plants can be carried out by exposing the transgenic culture to a selection marker. Plants transformed and stably containing a marker gene conferring selection agent resistance are grown and divided in the above-mentioned culture. The selection mark may be within the range described above, but is not limited thereto.

The transgenic plant is one in which the gene encoding the HAT2 protein is overexpressed.

In an embodiment of the present invention, when the transgenic plant is Arabidopsis thaliana, the gene consisting of the nucleotide sequence of SEQ ID NO: 1 may be overexpressed. The transgenic plant can produce seeds in which the ratio of unsaturated fatty acids is controlled, and according to an embodiment of the present invention, the seeds have an increased content of oleic acid and linoleic acid, and a reduced content of linolenic acid.

At this time, only the ratio of specific fatty acids including oleic acid, linoleic acid or linolenic acid among the total fatty acids in the seed can be controlled without a significant change in the content of total fatty acids per seed.

The method of the present invention may use any method known in the art outside the above-mentioned scope.

The present invention comprises the steps of culturing a seed in which the gene encoding the HAT2 protein is attenuated or deleted; and selecting a mutant plant in which the attenuated or deleted gene is homogenized from among plants obtained by culturing the seed; It provides a method for producing a plant for producing seeds in which the ratio of unsaturated fatty acids is controlled, including a.

In an embodiment of the present invention, when the transgenic plant is Arabidopsis thaliana, the gene consisting of the nucleotide sequence of SEQ ID NO: 1 may be attenuated or deleted. This may indicate that the function of the gene is lost due to the insertion of T-DNA into the corresponding gene, but is not limited thereto.

According to an embodiment of the present invention, the mutant plant in which the gene is attenuated or deleted can produce seeds having an increased linolenic acid content and a reduced oleic acid and linoleic acid content.

At this time, only the ratio of specific fatty acids including oleic acid, linoleic acid or linolenic acid among the total fatty acids in the seed can be controlled without a significant change in the content of total fatty acids per seed.

The method of the present invention may use any method known in the art.

The present invention provides a plant produced according to the method of the present invention. The plant can produce a seed in which the ratio of unsaturated fatty acids is controlled.

The present invention also provides a seed produced by a plant according to the method of the present invention. The seed is one in which the ratio of unsaturated fatty acids is controlled. According to an embodiment of the present invention, the seeds produced by the transgenic plant overexpressing the HAT2 gene have increased oleic acid and linoleic acid content and reduced linoleic acid content Žc. According to another embodiment of the present invention, the seed produced by the mutant plant in which the HAT2 gene is attenuated or deleted has an increased content of linolenic acid and a reduced content of oleic acid and linoleic acid.

Hereinafter, examples will be given to describe the present invention in detail.

Example

1. Analysis of the structure of HAT2 protein and the location of organelles

The amino acid sequence of the HAT2 protein translated from the HAT2 gene (At5g47370) nucleotide sequence was analyzed to confirm the function and location of the electronic regulator of the protein encoded by the HAT2 gene.

As a result, HAT2 had an EAR motif involved in the repressor role among transcriptional regulators in the 10-14 amino acid sequence. The 127-186 amino acid sequence was assumed to be involved in DNA binding to the homeobox domain. Amino acids 194-215 are the leucine zipper domains, and were presumed to be regions involved in homo- or hetero-dimer formation. HAT2 was found to be a domain that serves as the above-mentioned three transcriptional regulators (FIG. 1A). As a result of transforming the HAT2-GFP fusion protein in which the GFP gene was bound to HAT2 into Arabidopsis protoplasts under the control of the CaMV 35S promoter, the HAT2:GFP protein was detected with green fluorescence in the nucleus of the cell, and a control RFP protein located in the nucleus of red fluorescence and HAT2:GFP protein were expressed in the nucleus overlapping each other, showing yellow color (Fig. 1B). From this result, it was confirmed that the HAT2 protein is a transcriptional regulator and is located in the nucleus.

2. Expression analysis of HAT2 gene

The gene expression of HAT2 was analyzed by qRT-PCR method in various tissues of Arabidopsis plants. Arabidopsis rosette leaf (RL: rosette leaf), cauline leaf (CL: cauline leaf), stem (St:stem), open flower (OF:open flower), unopened flower (UF:unopened flower) seeds Total RNA was isolated from pods (Sq:silique), roots (R:root), and seedlings (Sd:seedling), and the expression of the transcriptome of HAT2 was analyzed.

As a result, it was possible to confirm the expression of the HAT2 gene in all tissues (FIG. 2A). Since HAT2 is also expressed in siliques containing seeds, the expression of HAT2 was analyzed by dividing the silique into 8 stages according to the seed development stage. increased and the expression was decreased in the S8 stage, which is the later stage of seed germination (FIG. 2B).

As a result of analyzing the expression of HAT2 using Arabidopsis transformants expressing the GUS report gene under the control of the HAT2 promoter, it was confirmed that HAT2 was expressed in all tissues in the seedlings and strongly expressed in the seeds and embryos inside the seeds (Fig. 2C). . a, c, e, g, i and k are control plants, and b, d, f, h, j, and l are transformants. Through these results, it was confirmed that HAT2 protein plays an important role in seed formation because it is expressed in all tissues but also strongly expressed in seeds.

3. HAT2 loss-of-function mutant construction and assay

3-1. Construction and selection of HAT2 loss-of-function mutants

Hat2-1 (sail_87_E06) and hat2-2 (salk_055288C) seeds with T-DNA inserted into the HAT2 gene were purchased from ABRC (Arabidopsis Biological Resource Center), and the T-DNA fragment of Arabidopsis thaliana obtained by culturing the seeds was HAT2 In order to obtain a hat2 mutant plant that has been inserted into the gene and has completely lost its function, in the case of hat2-1, LB1 (SEQ ID NO: 3), which is a T-DNA sequence specific primer, and the T-DNA insertion site of the Hat2 gene are placed in the center, and from the left to the hat2- 1LP primer (SEQ ID NO: 4), hat2-1RP primer (SEQ ID NO: 5) was prepared at the right position, and in the case of hat2-2, LBb1.3 (SEQ ID NO: 6), a T-DNA sequence-specific primer, and T- of the HAT2 gene With the DNA insertion site in the center, the hat2-2 LP primer (SEQ ID NO: 7) from the left and the hat2-2 RP primer (SEQ ID NO: 8) from the right position were prepared and PCR was performed on plant genomic DNA using each primer set. Independent mutant plants of homogenized hat2-1 and hat2-2 were selected (FIGS. 3A, 3B)

To confirm whether the expression of the HAT2 gene is detected in the seeds of the homogenized hat2-1 and hat2-2 mutant plants, the N-terminal (N-ter) part primer HAT2F (SEQ ID NO: 9) and C-terminal ( C-ter) part HAT2-R primer (SEQ ID NO: 10) was prepared and gene expression was analyzed by RT-PCR (Reverse transcriptase-polymerase chain reaction) method using these two primer sets.

As a result, the HAT2 gene was expressed in the control wild-type (WT) plant seeds, but the HAT2 gene was not expressed in the seeds of the homogenized hat2-1 and hat2-2 mutant plants (FIG. 3C).

3-2. Analysis of fatty acids in seeds of HAT2 loss-of-function mutants

The fatty acids of the seeds harvested from the HAT2 homo mutant hat2-1 and hat2-2 plants selected in Example 3-1 and the control wild-type (WT) plants were analyzed using Gas Chromatography (GC). The seed fatty acid composition was analyzed.

As a result, when the fatty acid composition of hat2-1 and hat2-2 mutant seeds was compared with that of the wild type, a difference was observed in the composition ratio. In the hat2-1 and hat2-2 mutant seeds, the ratio of oleic acid (18:1) and linoleic acid (18:2) fatty acids was slightly reduced statistically significantly by about 1.2% and 1,7% compared to the wild type. In contrast, palmitic acid (16:0), stearic acid (18:0), linolenic acid (18:3), and eicosenic acid (20:1) fatty acids increased to a significant level (FIG. 4).

4. Construction and assay of HAT2 overexpressing transformants

4-1. Production and selection of HAT2 overexpressing transformants

In order to construct a plant transformation vector capable of overexpressing the HAT2 gene in plants, HAT2_F (SEQ ID NO: 11) and HAT2_R (SEQ ID NO: 12) containing the HAT2 gene from RNA isolated from Arabidopsis seeds were used to RT- HAT2 cDNA was amplified by PCR. The amplified cDNA was cloned into the pCR8 vector to construct a pCR8-HAT2 entry vector. LR clonase reaction with the prepared pCR8-HAT2 entry vector and pEarleyGate101 (11.7kb), a plant expression vector containing the 35S CaMV promoter, was performed to produce a recombinant plant expression vector OX-HAT2 vector in which the HAT2 gene is overexpressed under the control of the 35S CaMV promoter. produced. After transforming the prepared OX-HAT2 vector into Agrobacterium GV3101, Agrobacterium was used for plant transformation of Arabidopsis thaliana. Thereafter, the OX-HAT2 expression vector was inserted into the plant genome, and 27 transformed T1 Arabidopsis plants showing resistance to herbicides (BASTA) were selected.

4-2. Analysis of fatty acids in seeds of HAT2 overexpressing transformants

Gas chromatograph (GC) analysis was performed to analyze the fatty acid composition of T2 seeds obtained from T1 transgenic plants of 27 individuals selected in Example 4-1.

As a result, the OX-HAT2 transformant seed fatty acids of each of the 27 individuals showed a significant difference in the composition of the non-transformant control wild-type (WT) seed fatty acids. Oleic acid (18: 1) and linoleic acid (18: 2) were significantly increased in OX-HAT compared to wild-type WT seeds. It also increased by 20:1. In the case of oleic acid (18:1), WT was 16.7%, but OX-HAT2 was further enhanced by 9.3% to a maximum of 26.0%. In the case of linoleic acid (18:2), WT was 30.5%, but OX-HAT2 was up to 34%, which was further enhanced by 3.5%. Conversely, the ratio of palmitic acid (16:0), stearic acid (18:0) and linolenic acid (18:3) fatty acids was decreased compared to WT. Among them, in the case of linolenic acid (18:3), WT was 19.2%, but OX-HAT2 was further reduced by 8.7% to a maximum of 10.5% (FIG. 5).

5. Analysis of Camelina Plants Transformed with a Recombinant Vector Inducing Arabidopsis HAT2 Expression

A vector capable of overexpressing Arabidopsis HAT2 (AtHAT2) under the control of the Glycinin promoter was constructed. In the vector production process, the AtHAT2 cDNA gene was amplified by PCR using cDNA synthesized from total RNA isolated from the developing seeds of Arabidopsis thaliana, and subcloned into pEASY-Blunt3 (pBlunt3) vector. After EcoR I After cutting AtHAT2 cDNA from the pBlunt3 vector using restriction enzymes, the AtHAT2 cDNA gene was cloned into the EcoRI site of the pBinGlyRed3 (pBGR3) vector having a seed-specific Glycinin promoter. The pBGR3_HAT2 vector prepared in this way was transformed into Agrobacterium GV3101, and the Camelina plant was transformed by the floral dipping method (FIG. 6). An expression vector (pBGR3_AtHAT2) cloning AtHAT2 under the control of the seed-specific Glycinin promoter was inserted into the genome of Camelina, and T1 plant seeds showing fluorescence by DsRed3 in the seeds were visually selected through a red filter under green light irradiation. The 15 selected seeds were germinated to obtain 15 Camelina transformants, and genomic DNA PCR and RT-PCR were performed to select AtHAT2-transformed Camelina plants. Genomic DNA PCR was performed using a set of primers (SEQ ID NO: 13) binding to the Glycinin promoter present in the pBinGlyRed3 vector and primers (SEQ ID NO: 14) binding to the C-terminal portion of AtHAT2 for DNA extracted from camelina leaves. As a result, it was shown that the AtHAT2 gene was inserted into the remaining Camelina individuals except for wild-type (WT), Gly_HAT2-9, and Gly_HAT2-12 (FIG. 7A).

After that, total RNA was extracted from immature seeds and synthesized into cDNA except for two individuals (Gly_HAT2-9, Gly_HAT2-12) into which the AtHAT2 gene was not inserted. Then, RT-PCR was performed to confirm the expression of the transformed AtHAT2 gene. carried out. On the mRNA of AtHAT2 synthesized from the pBGR3_AtHAT2 vector inserted into the Camelina genome, PCR was performed with a set of 5'UTR partial primers (SEQ ID NO: 15) and C-terminal partial primers (SEQ ID NO: 16). For the accuracy of the RT-PCR experiment, RT-PCR of the Camelina ACTIN gene was performed on all samples as a control experiment. As a result, AtHAT2 was not expressed in the wild type (WT), but it was confirmed that it was expressed in the transformed Camelina seeds (FIG. 7B).

6. Analysis of Seed Fatty Acids of HAT2 Overexpressed Transgenic Camelina

Gas chromatography (GC) analysis was performed on T2 seeds obtained from T1 transgenic plants. As a result of analyzing the fatty acids in the seeds, 13 different Gly_HAT2 transformant seed fatty acids showed a significant difference in the composition of the seed fatty acids of the non-transformant control wild-type (WT). Compared to wild-type WT seeds, Gly_HAT2 increased significantly in 18:1 and 18:2. In the case of 18:1, WT was 14.5%, but Gly_HAT2 was up to 21.3%, and the content increased by 6.8%. In the case of 18:2, WT was 15.1%, but Gly_HAT2 was increased by 9% to a maximum of 26.1%. Conversely, the ratios of 16:0, 18:0, and 18:3 fatty acids were decreased compared to WT. Among them, in the case of 18:3, WT was 38.2%, but in the case of Gly_HAT2, it was reduced by 8.3% to at least 29.9% (FIG. 8).

This result showed the same trend as the fatty acid composition change of OX-HAT2 preceded in Arabidopsis. Through Camelina transformation overexpression experiments, overexpression of Arabidopsis HAT2 gene changed the composition of seed fatty acids not only in Arabidopsis thaliana but also in other plants, and it was shown that it can be used to enhance 18:1 and 18:2 fatty acids in particular.

<110> SEJONG UNIVERSITY INDUSTRY ACADEMY COOPERATION FOUNDATION <120> Composition for modifying the ratio of unsaturated fatty acids in seeds and seeds prepared by the same <130> 19P12042 <150> KR 10-2019-0109991 <151> 2019-09-05 <160> 16 <170> KoPatentIn 3.0 <210> 1 <211> 852 <212> DNA <213> Arabidopsis thaliana <400> 1 atgatgatgg gcaaagaaga tctaggtttg agcctaagct tagggttttc acaaaatcac 60 aatcctcttc agatgaatct gaatcctaac tcttcattat caaacaatct ccagagactc 120 ccatggaacc aaacattcga tcctacatca gatcttcgca agatagacgt gaacagtttt 180 ccatcaacgg ttaactgcga ggaagacaca ggagtttcgt caccaaacag tacgatctca 240 agcaccatta gcgggaagag aagtgagaga gaaggaatct ccggaaccgg cgttggctcc 300 ggcgacgatc acgacgagat cactccggat cgagggtact cacgtggaac ctcagatgaa 360 gaagaagacg ggggcgaaac gtcgaggaag aagctcaggt tatcaaaaga tcagtctgct 420 tttctcgaag agactttcaa agaacacaac actctcaatc ccaaacagaa gctagctttg 480 gctaagaagc tgaacttgac ggcaagacaa gtggaagtgt ggttccaaaa cagaagagct 540 agaaccaagt taaagcaaac ggaggtagat tgcgaatact tgaaacggtg cgtagagaag 600 ctaacggaag agaaccggag acttcagaaa gaggctatgg agcttcgaac tctcaagctg 660 tctccacaat tctacggtca gatgactcca ccaactacac tcatcatgtg tccttcgtgc 720 gagcgtgtgg gtggcccatc atcatcgaac catcaccaca atcacaggcc cgtttctatc 780 aatccgtggg ttgcttgtgc tggtcaggtg gctcatgggc tgaattttga agccttgcgt 840 ccacgatcgt ga 852 <210> 2 <211> 283 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Met Met Gly Lys Glu Asp Leu Gly Leu Ser Leu Ser Leu Gly Phe 1 5 10 15 Ser Gln Asn His Asn Pro Leu Gln Met Asn Leu Asn Pro Asn Ser Ser 20 25 30 Leu Ser Asn Asn Leu Gln Arg Leu Pro Trp Asn Gln Thr Phe Asp Pro 35 40 45 Thr Ser Asp Leu Arg Lys Ile Asp Val Asn Ser Phe Pro Ser Thr Val 50 55 60 Asn Cys Glu Glu Asp Thr Gly Val Ser Ser Pro Asn Ser Thr Ile Ser 65 70 75 80 Ser Thr Ile Ser Gly Lys Arg Ser Glu Arg Glu Gly Ile Ser Gly Thr 85 90 95 Gly Val Gly Ser Gly Asp Asp His Asp Glu Ile Thr Pro Asp Arg Gly 100 105 110 Tyr Ser Arg Gly Thr Ser Asp Glu Glu Glu Asp Gly Gly Glu Thr Ser 115 120 125 Arg Lys Lys Leu Arg Leu Ser Lys Asp Gln Ser Ala Phe Leu Glu Glu 130 135 140 Thr Phe Lys Glu His Asn Thr Leu Asn Pro Lys Gln Lys Leu Ala Leu 145 150 155 160 Ala Lys Lys Leu Asn Leu Thr Ala Arg Gln Val Glu Val Trp Phe Gln 165 170 175 Asn Arg Arg Ala Arg Thr Lys Leu Lys Gln Thr Glu Val Asp Cys Glu 180 185 190 Tyr Leu Lys Arg Cys Val Glu Lys Leu Thr Glu Glu Asn Arg Arg Leu 195 200 205 Gln Lys Glu Ala Met Glu Leu Arg Thr Leu Lys Leu Ser Pro Gln Phe 210 215 220 Tyr Gly Gln Met Thr Pro Pro Thr Thr Leu Ile Met Cys Pro Ser Cys 225 230 235 240 Glu Arg Val Gly Gly Pro Ser Ser Ser Asn His His His Asn His Arg 245 250 255 Pro Val Ser Ile Asn Pro Trp Val Ala Cys Ala Gly Gln Val Ala His 260 265 270 Gly Leu Asn Phe Glu Ala Leu Arg Pro Arg Ser 275 280 <210> 3 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LB1 <400> 3 gccttttcag aaatggataa atagccttgc ttcc 34 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LP primer1 <400> 4 tggccatctt atttgttttg g 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RP primer1 <400> 5 ctgttttgga accacacttc c 21 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LBb1.3 <400> 6 attttgccga tttcggaac 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LP Primer2 <400> 7 tttccatcaa cggttaactg c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> RP Primer2 <400> 8 cggaaaagca attgaagtca g 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> N-terminal primer <400> 9 atgatgatgg gcaaagaaga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C-terminal primer <400> 10 cgatcgtgga cgcaaggctt 20 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F primer <400> 11 aacatgatga tgggcaaaga ag 22 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer <400> 12 tcacgatcgt ggacgcaagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 13 tctccgcttc acaactcaaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 14 tcacgatcgt ggacgcaagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 15 aattcgattg gatcgccctt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 4 <400> 16 gattgtggtg atggttcgat 20

Claims (15)

A composition for producing seeds with a controlled unsaturated fatty acid ratio comprising a substance that promotes expression of a gene encoding HAT2 protein,
The control is an increase in the ratio of oleic acid and linoleic acid, and the ratio of linolenic acid is decreased, the unsaturated fatty acid ratio is controlled seed production composition.
delete delete The method according to claim 1, wherein the seed is Arabidopsis or oil plant (oil plant) seed, unsaturated fatty acid ratio is controlled seed production composition.
The method according to claim 4, wherein the oil and fat plant includes any one or more selected from the group consisting of camelina, sesame, rapeseed, sunflower, castor, peanut, soybean, coco palm, oil palm and jojoba, unsaturated fatty acid ratio A composition for making this conditioned seed.
The method according to claim 1, The seed is Arabidopsis thaliana or Camelina seed, the gene is composed of the nucleotide sequence of SEQ ID NO: 1, unsaturated fatty acid ratio is controlled seed production composition.
The composition for preparing seeds according to claim 1, wherein the substance promoting the expression of the gene encoding the HAT2 protein is a vector into which the gene encoding the HAT2 protein is introduced.
A method for producing a seed with a controlled unsaturated fatty acid ratio, comprising transforming a plant with a recombinant vector comprising a gene encoding HAT2 protein,
The control is an increase in the ratio of oleic acid and linoleic acid, and the ratio of linolenic acid is decreased, the unsaturated fatty acid ratio is controlled seed production method.
delete constructing a recombinant vector comprising a gene encoding the HAT2 protein;
transforming the plant with the recombinant vector; and
selecting a transgenic plant; As a method for producing a plant for seed production in which the ratio of unsaturated fatty acids is controlled, comprising:
The control is an increase in the ratio of oleic acid and linoleic acid, and the ratio of linolenic acid is decreased.
delete delete Culturing a seed in which the expression of the gene encoding the HAT2 protein is attenuated or the gene is deleted; and
selecting a mutant plant in which the attenuated or deleted gene is homologized among plants obtained by culturing the seed; As a method for producing a plant for producing seeds with a controlled unsaturated fatty acid ratio comprising:
The control is the ratio of oleic acid and linoleic acid is decreased, and the ratio of linolenic acid is increased. delete delete

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