KR102675413B1 - IbFAD8 gene from sweetpotato for improving cold storage stability and increasing omega-3 fatty acid content in tuberous root of sweetpotato and uses thereof - Google Patents

Abstract

The present invention relates to the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) gene, which increases the low-temperature storage capacity and omega-3 fatty acid content of sweet potato tubers, and its use. The IbFAD8 gene is used to reduce environmental stress in unfavorable areas. By improving tolerance and low-temperature storage ability, it can be usefully used to improve the storage properties of sweet potatoes and to develop sweet potatoes with high linolenic acid content, which are effective for high blood pressure and cardiovascular diseases.

Description

IbFAD8 gene from sweetpotato for improving cold storage stability and increasing omega-3 fatty acid content in tuberous root of sweetpotato and uses thereof}

The present invention relates to the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) gene, which increases the low-temperature storage capacity and omega-3 fatty acid content of sweet potato tubers, and its use.

Omega (ω) is a word meaning 'last, end', and unsaturated fatty acids in which a double bond is formed from the third carbon from the end carbon of the carbon chain that makes up the fatty acid molecule are called omega-3 fatty acids.

Alpha-linolenic acid (omega-3 fatty acid) is an essential fatty acid that cannot be synthesized in the body and must be consumed through food. It is a precursor that is converted into EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) in the body. It is known to be effective in preventing heart disease by lowering blood cholesterol and reducing vascular inflammation indicators.

Omega-3 fatty acids protect cells in the human body, maintain cell structure, and help with smooth metabolism. It also has the effect of suppressing blood film formation and promoting and strengthening bone formation. It is known that a lack of omega-3 fatty acids leads to a lack of DHA required for the brain and retina, which reduces learning ability and visual function. According to recent research results, clinical research has reported that alpha-linolenic acid reduces C-reactive protein, a marker of inflammation. Consuming alpha-linolenic acid lowers blood cholesterol, reduces the risk of cardiovascular disease, and reduces arterial disease. You can achieve the effect of protecting functions, etc.

Sweet potato ( Ipomoea batatas ) is a perennial plant of the Convolvulaceae family. It grows roots from the base of the petiole at the bottom of the stem, and a part of it grows underground to become a sweet potato, a tuberous root. Sweet potatoes are a representative root crop that can be grown even on relatively barren land and are used as food and livestock feed, with a production volume of about 30 tons per hectare. The components of the tuber include 69.39% moisture, 27.7% sugar, and 1.3% protein, and the main component is starch. Before World War II, most people consumed it for food, but recently, only about 40% of it is edible, and it is mainly used as side dishes or snacks. For industrial purposes, about 30% is used for starch, and it is widely used as a raw material for taffy, glucose, confectionery, processed edible products, medicine, cosmetics, alcohol, whiskey, and soju. It is also used as feed for livestock such as pigs, and the leaves and stems are used as green manure to maintain the productivity of the land.

Meanwhile, Korean Patent Publication No. 2018-0073430 discloses 'FAD2 genetically engineered oleic acid-enhanced plant and method of manufacturing same', and Korean Patent No. 2038481 discloses 'Yield transformed with PfFAD3-1 gene derived from Resquerella'. Although this 'enhanced soybean plant and method for producing the same' is disclosed, there is no description of the 'sweet potato-derived IbFAD8 gene that increases the low-temperature storage capacity and omega-3 fatty acid content of sweet potato tubers and its use' of the present invention.

The present invention was derived from the above needs. In order to develop sweet potatoes with improved low-temperature storage and high content of omega-3 fatty acids, the present inventors used the omega-6 fatty acid linoleic acid (C18:2) as an omega-3 fatty acid. The FAD8 (Fatty acid desaturase 8) gene, which has the function of changing the fatty acid composition in plant cells by desaturating it with linolenic acid (C18:3), was isolated from sweet potato ( Ipomoea batatas ), and a transgenic sweet potato overexpressing the IbFAD8 gene was prepared and characterized. As a result of the analysis, the linolenic acid/linoleic acid ratio of the transgenic sweet potato overexpressing the IbFAD8 gene was increased in the tubers, the low-temperature storage ability of the tubers was improved, and the low temperature and drying stress tolerance of the transgenic sweet potato plants was increased compared to the non-transgenic sweet potato plants. By confirming that this was done, the present invention was completed.

In order to solve the above problem, the present invention transforms sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2, thereby generating the IbFAD8 gene. A method for increasing low-temperature storage and omega-3 fatty acid content of sweet potato tubers compared to the wild type, including the step of overexpressing, is provided.

In addition, the present invention includes the steps of transforming sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato-derived IbFAD8 protein consisting of the amino acid sequence of SEQ ID NO: 2; and redifferentiating the transformed sweet potato plant from the transformed sweet potato plant cell. It provides a method for producing a transformed sweet potato plant with increased low-temperature storage capacity and omega-3 fatty acid content of the tuber compared to the wild type.

In addition, the present invention provides transgenic sweet potato plants and tubers thereof with increased low-temperature storage and omega-3 fatty acid content of the tubers prepared by the above method.

In addition, the present invention provides a composition for improving low-temperature storage and omega-3 fatty acid content of sweet potato tubers, which contains as an active ingredient a gene encoding the sweet potato-derived IbFAD8 protein consisting of the amino acid sequence of SEQ ID NO: 2.

Transgenic sweet potatoes overexpressing the IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) gene according to the present invention were confirmed to have improved low-temperature storage of tubers, significantly increased linolenic acid in tubers compared to the wild type, and increased low-temperature and drying stress tolerance. It has been done. Therefore, by using the IbFAD8 gene, it is possible to improve the environmental stress tolerance and low-temperature storage ability of sweet potatoes in unfavorable areas, and develop sweet potatoes with high linoleic acid content that are effective against high blood pressure and cardiovascular diseases, which can be used industrially.

Figure 1(a) is the result of amino acid and base sequence analysis of the IbFAD8 protein derived from sweet potato, (b) and (c) are the results of comparing the relationship with FAD of other plants based on the amino acid sequence, and (d) is the result of IbFAD8 This is the result of temporary expression by inoculating tobacco leaves with a vector combining GFP to the base sequence and observation using a confocal microscope.
Figure 2 is an analysis of the expression of the IbFAD8 gene in each sweet potato tissue, including leaves (L), stems (S), fibrous roots (FR), pencil roots (PR), and tuberous roots. This is the result of analyzing the expression of the IbFAD8 gene in roots (TR) by qRT-PCR.
Figure 3 shows the results of analyzing the expression and fatty acid levels of the IbFAD8 gene in sweet potato leaves. Each number represents the 2nd, 4th, ... 12th leaf from the shoot of the sweet potato plant.
Figure 4 shows the results of analyzing changes in IbFAD8 gene expression under environmental stress conditions. (a) shows the results of analyzing changes in expression of the IbFAD8 gene in sweet potato leaves under low temperature (4°C) and high temperature (42°C) conditions, ( b) is the result of analyzing the expression change of the IbFAD8 gene in tubers stored at low temperature (4℃), and (c) is the expression change of the IbFAD8 gene in leaf petioles after treatment under dry stress (30% PEG) conditions. This is the result of analysis.
Figure 5 is a schematic diagram of a recombinant vector for producing a transgenic sweet potato overexpressing IbFAD8 (a), a PCR result testing the transformation of a sweet potato plant transformed with the recombinant vector (b), and IbFAD8 in the leaves of the selected transformant. This is the result (c) of confirming gene expression using qRT-PCR.
Figure 6 shows the results of environmental stress tolerance analysis of IbFAD8 -overexpressing transgenic sweet potatoes. (a) is the result of tolerance to cold stress, and (b) is the result of tolerance to drying stress.
Figure 7 shows the root yield results of sweet potato. Roots were harvested from non-transformant (NT) and IbFAD8- overexpressing transgenic sweet potato plants (TF) grown for 5 months, and pencil roots (PR) and tuberous roots were harvested. , TR).
Figure 8 shows the results of analysis of low-temperature storage changes in IbFAD8 -overexpressing transgenic sweet potato tubers. (a) shows harvested tubers of NT and TF sweet potatoes under appropriate storage temperature (13°C) and low temperature (4°C) conditions for 6 weeks. This is a photo observing the condition after storage, and (b) is the result of MDA (malondialdehyde) content analysis.

In order to achieve the object of the present invention, the present invention transforms sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2 to produce IbFAD8. A method for increasing low-temperature storage and omega-3 fatty acid content of sweet potato tubers compared to the wild type is provided, comprising the step of overexpressing the gene.

In the present invention, a tuberous root is a plant root that has become large and fat to store nutrients, and is also called a tuber. The root is spindle-shaped or spherical in shape and has multiple buds for vegetative reproduction. The roots of sweet potatoes, turnips, and melons fall into this category, and they usually store a large amount of starch or sugar.

The scope of the IbFAD8 protein derived from sweet potato according to 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, and even more preferably 90% or more, as a result of addition, substitution or deletion of amino acids, compared to the amino acid sequence shown in SEQ ID NO: 2. refers to a protein that has more than 95% sequence homology and exhibits substantially the same physiological activity as the protein represented by SEQ ID NO: 2. “Substantially the same physiological activity” refers to the activity of increasing the low-temperature storage capacity and omega-3 fatty acid content of sweet potato tubers.

Additionally, the present invention includes a gene encoding the IbFAD8 protein, and the scope of the gene includes all of genomic DNA, cDNA, and synthetic DNA encoding the IbFAD8 protein. Preferably, the gene encoding the IbFAD8 protein of the present invention may include the base sequence represented by SEQ ID NO: 1. Additionally, homologs of the above base sequence are included within the scope of the present invention. Specifically, the gene includes a base sequence having sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% with the base sequence of SEQ ID NO: 1. can do. The “% sequence homology” for a polynucleotide is determined by comparing two optimally aligned sequences, wherein a portion of the polynucleotide sequence in the region of comparison is a reference sequence (not containing additions or deletions) for the optimal alignment of the two sequences. may contain additions or deletions (i.e. gaps) compared to

In the method for low-temperature storage and increasing the content of omega-3 fatty acids according to an embodiment of the present invention, the omega-3 fatty acid may preferably be linolenic acid, but is not limited thereto.

The term “gene overexpression” means causing the gene to be expressed above the level expressed in wild-type plants. A method of introducing the gene into a plant includes transforming the plant using a recombinant expression vector containing the gene under the control of a promoter.

The term “recombinant” refers to a cell that replicates a heterologous nucleic acid, expresses a heterologous nucleic acid, or expresses a peptide, heterologous peptide, or protein encoded by a heterologous nucleic acid. Recombinant cells can express genes or gene segments that are not found in the natural form of the cell, either in sense or antisense form. Additionally, recombinant cells can express genes found in cells in their natural state, but the genes have been modified and reintroduced into the cells by artificial means.

The term “vector” is used to refer to a DNA fragment(s) or nucleic acid molecule that is delivered into a cell. Vectors replicate DNA and can reproduce independently in host cells. The term “vector” is often used interchangeably with “vector”. The term “expression vector” refers to a recombinant DNA molecule containing a coding sequence of interest and an appropriate nucleic acid sequence necessary to express the operably linked coding sequence in a particular host organism.

Vectors of the invention can typically be constructed as vectors for cloning or expression. Additionally, the vector of the present invention can be constructed using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of advancing transcription (e.g., pLλ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.), It typically includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When Escherichia coli is used as a host cell, the promoter and operator regions of the E. coli tryptophan biosynthesis pathway and the left-handed promoter of phage λ (pLλ promoter) can be used as control regions.

Meanwhile, vectors that can be used in the present invention include plasmids often used in the art (e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB) , λ-Charon, λΔz1, and M13, etc.) or viruses (e.g., SV40, etc.). On the other hand, when the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus) Viral late promoters, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and typically have a polyadenylation sequence as the transcription termination sequence.

The recombinant vector of the present invention is preferably a plant expression vector.

A preferred example of a plant expression vector is the Ti-plasmid vector, which is capable of transferring part of itself, the so-called T-region, into plant cells when present in a suitable host such as Agrobacterium tumefaciens . Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced by appropriately inserting the hybrid DNA into the plant's genome. there is. A particularly preferred form of Tiplasmid vectors are the so-called binary vectors as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the genes according to the invention into a plant host include viral vectors such as those that may be derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc. For example, it may be selected from non-intact plant virus vectors. The use of such vectors can be particularly advantageous when it is difficult to properly transform plant hosts.

The expression vector preferably contains one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. There is a resistance gene, but it is not limited to this.

In the plant expression vector according to the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to the region of DNA upstream from a structural gene and refers to the DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. A “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of transformants can be accomplished at various stages and by various tissues. Therefore, constitutive promoters do not limit selection possibilities.

In the plant expression vector according to the present invention, the terminator can be a conventional terminator, examples of which include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, and Agrobacterium terminator. These include, but are not limited to, the terminator of the Octopine gene of Rhium tumefaciens. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly preferred in the context of the present invention.

The “plant cell” used for plant transformation can be any plant cell. The plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably cultured cell, cultured tissue or cultured organ and more preferably cultured cell. The term “plant tissue” refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used in culture, i.e., single cells, Includes protoplast, bud and callus tissue. Plant tissue may be in planta or in organ culture, tissue culture, or cell culture.

Methods for transporting the vector of the present invention into a host cell include, when the host cell is a prokaryotic cell, the CaCl ₂ method or the Hanahan method (Hanahan, D., (1983) J. Mol. Biol., 166:557-580) and electroporation. In addition, when the host cell is a eukaryotic cell, the vector can be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment. there is.

The present invention also,

Transforming sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2; and

Provided is a method for producing transgenic sweet potato plants with increased low-temperature storage properties and omega-3 fatty acid content of the tubers compared to the wild type, including the step of redifferentiating the transformed sweet potato plants from the transformed sweet potato plant cells.

Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily require a regeneration and/or tissue culture period. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant elements, particle bombardment (DNA or RNA-coated) of various plant elements, invasion of plants or characterization of mature pollen or spores. It can be appropriately selected from Agrobacterium tumefaciens-mediated gene transfer by conversion, infection by (non-complete) virus, etc. A preferred method according to the invention involves Agrobacterium mediated DNA transfer. Particular preference is given to using the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The production method of the present invention includes the step of transforming plant cells with the recombinant vector according to the present invention, and the transformation may be mediated by Agrobacterium tumefaciens ( A. tumefaciens ). Additionally, the method of the present invention includes the step of redifferentiating a transformed plant from the transformed plant cell. Any method known in the art can be used to redifferentiate a transgenic plant from a transgenic plant cell.

In the production method according to one embodiment of the present invention, increasing the expression of the sweet potato-derived IbFAD8 protein coding gene in the transformed plant cells results in increased low-temperature storage of the tuber and increased omega-3 fatty acid content compared to the wild type. Converted sweet potato plants can be produced. The omega-3 fatty acid may preferably be linolenic acid, but is not limited thereto.

The present invention also provides transgenic sweet potato plants and tubers thereof with increased low-temperature storage and omega-3 fatty acid content of the tubers prepared by the above method.

As described above, the transgenic sweet potato plant according to the present invention has increased low-temperature storage and omega-3 fatty acid content of the tubers compared to the wild type. In particular, the content of omega-3 fatty acid increased due to overexpression of the IbFAD8 gene. It is characterized by a marked increase in tubers rather than leaves. The omega-3 fatty acid may preferably be linolenic acid, but is not limited thereto.

In addition, the transgenic sweet potato plant with increased low-temperature storage capacity and omega-3 fatty acid content of the tubers according to the present invention is characterized by increased tolerance to low temperature and drying stress compared to the wild type.

The present invention also provides a method for improving low-temperature storage and omega-3 fatty acid content of sweet potato tubers, which contains as an active ingredient a gene encoding the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2. A composition is provided.

The composition of the present invention contains, as an active ingredient, IbFAD8 protein derived from sweet potato consisting of the amino acid sequence of SEQ ID NO: 2; Alternatively, the low-temperature storability and omega-3 fatty acid content of sweet potato tubers can be increased by transforming plant cells with the gene or a recombinant vector containing the gene.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Sweet potato origin Fatty acid desaturase 8 gene ( IbFAD8 ) Cloning and sequence analysis

cDNA was synthesized from RNA extracted from the leaves of sweet potato ( Ipomoea batatas cv. Xushu 29) plants using Trizol (Invitrogen) using the TOPscript™ cDNA Synthesis kit (Enzynomics, Korea). Afterwards, PCR was performed using a forward primer (5'-ATGGCGAGTTGGGTGTTATC-3', SEQ ID NO: 3) and a reverse primer (5'-TCACTTCTCGGTTCCAGCAA-3', SEQ ID NO: 4) specific for IbFAD8 , and the obtained product was electrophoresed. The IbFAD8 gene was isolated by purifying the PCR product.

As a result of analyzing the sequence of the isolated gene through sequencing analysis, it was confirmed that the IbFAD8 gene consists of 1,338 bases (Figure 1a), and the amino acid sequence could be inferred. As a result of comparing the relationship with FAD proteins (AtFAD8, OsFAD8, AtFAD7, OsFAD7) of other plants based on the above amino acid sequence, three histidine clusters (HDCGH, HGWRISHRTHH, HHDIGTHVIHH) specific to FAD protein were identified ( Figure 1b and c).

In addition, a vector combining the GFP coding sequence with the IbFAD8 protein coding sequence was inoculated into tobacco leaves and transiently expressed. As a result of observation using confocal microscopy, the IbFAD8 protein appeared in a form surrounding chlorophyll a and was located in the chloroplast membrane. The location could be seen (Figure 1d).

Example 2. Sweet potato tissue type IbFAD8 Gene expression levels and fatty acid analysis

The expression level of IbFAD8 in leaves, stems, thread roots, hard roots, and tubers of sweet potato was analyzed using qRT-PCR. qRT-PCR was performed with the CFX real-time PCR system (Bio-Rad™) using a 96-well plate. Each reaction (final 20 μl) contained 2 μl of diluted cDNA, 10 μl of EvaGreen PCR Master Mix (Biofact), and forward primer (5'-CCCCCATTTAAGCTGTCTGA-3', SEQ ID NO: 5) and reverse primer (5'-ACATGGTTCCTTGAGCCAAC). -3', SEQ ID NO: 6) was performed by mixing 1 ㎕ of each. As a result, the IbFAD8 gene showed relatively high expression in leaves (L) and stems (S) of sweet potato tissues (Figure 2).

In addition, as a result of analyzing changes in IbFAD8 expression according to sweet potato leaf development, it was confirmed that IbFAD8 expression was high in young and mature leaves, and expression was decreased in aged leaves (Figures 3a and b). Fatty acid analysis was performed by gas chromatography (GC) analysis using an Agilent series 7890A (Agilent Technologies Inc.) equipped with a flame ionization detector. Extracts were obtained from freeze-dried sweet potato samples using a mixture of chloroform: hexane: methanol (8:5:2, v/v/v). After mixing with methylation reagent (0.25 M methanolic sodium methoxide: petroleum ether: ethyl ether, 1:5:2, v/v/v), DB-FFAP capillary column (30 mx 0.25 mm, 0.25 μm, Agilent) Technologies Inc.), fatty acids were separated. As a result of analysis of fatty acid composition (%) according to leaf development, linolenic acid (18:3) fatty acid was found to be the highest in young and aged leaves, and linoleic acid (18:2) was found to be the highest in mature leaves (Figure 3c).

Example 3. Under environmental stress conditions IbFAD8 Gene expression change analysis

To analyze changes in IbFAD8 expression due to environmental stress, sweet potato plants grown for 4 weeks at 25 ± 2℃ were grown at low (4℃) and high (42℃) temperatures at 0, 1, 3, 6, 12, 24, and 48. After letting it stand for some time, qRT-PCR was performed using leaf tissue. As a result, it was confirmed that the expression of IbFAD8 tended to decrease after 6 hours after being rapidly induced by low-temperature treatment, and that the expression was rapidly decreased under high-temperature conditions (Figure 4a). Additionally, as a result of analyzing the expression of IbFAD8 in tubers stored at low temperature (4°C), it was confirmed that the expression of IbFAD8 increased after 6 weeks of low temperature storage (Figure 4b).

Additionally, for drying stress treatment, leaf petioles were immersed in a 1.5 ml tube containing 30% PEG (polyethylene glycol) and then gene expression was analyzed over time. As a result, it was confirmed that the expression of IbFAD8 was drastically reduced by drying stress treatment (Figure 4c).

Example 4. IbFAD8 Production of overexpression transgenic sweet potatoes and evaluation of environmental stress tolerance

To analyze the function of IbFAD8 in sweet potato, an IbFAD8 overexpression vector was constructed and introduced into the embryogenic callus of Xushu 29, a sweet potato variety that grows well in high latitudes in China, using Agrobacterium (Figure 5a). The embryonic callus is made by adding meristem (meristem) extracted from sweet potato plant nodes to which Thiamine-HCl (vitamin B2), myo-inositiol (vitamin B8), and plant hormone auxin are added at concentrations of 0.4, 100, and 1 mg/l, respectively. The cells were incubated in MS medium and incubated in dark conditions at 25°C for 3 months to induce induction. gDNA was extracted from the leaves of sweet potato plants differentiated in antibiotic condition medium, and transformation was tested by PCR using forward primer (5'-AGTGAGCGCAACGCAATTAATGTG-3', SEQ ID NO: 7) and reverse primer (SEQ ID NO: 4) ( Figure 5b), among the selected transformants, lines 5 and 6 with high expression of the IbFAD8 gene in leaves were selected using qRT-PCR, named TF5 and TF6, respectively, and used in the next experiment (Figure 5c) .

Low temperature (4°C) and drying stress tolerance evaluations were performed using TF line plants. Non-transgenic sweet potato (NT) and TF line plants grown for 4 weeks were treated with cold stress, and tolerance was evaluated through Fv/Fm measurement, MDA content measurement, and chlorophyll content analysis. The Fv/Fm value was calculated by measuring the fluorescence emitted from the 3rd and 4th leaves of plants dark-treated for 20 minutes using a Handy PEA fluorometer (Hansatech), which measures chlorophyll fluorescence emitted from photosystem 2, and MDA The content is determined by mixing the extract extracted with 0.1% trichloroacetic acid (TCA) from 50 mg of fresh weight sweet potato with 0.5% thiobarbituric acid (TBA) and reacting it in water at about 90°C. The maximum measured wavelength of the TBA-MDA complex in the mixture is The MDA content was calculated by measuring the absorbance at 532 nm and 600 nm, which is the non-specific measurement wavelength. The chlorophyll content was measured using SPAD-502 (Konica Minolta), and the chlorophyll content of the 3rd and 4th leaves of sweet potato plants was measured in SPAD units. Measured. As a result of the analysis, it was confirmed that the low temperature tolerance of the TF line plants was increased (Figure 6a). In addition, a clear increase in drying stress tolerance was confirmed in the TF line plants as a result of drying stress treatment through suspension of water supply (Figure 6b).

Roots were harvested from NT and TF line plants grown for 5 months, and classified into pencil roots (PR) and tuberous roots (TR). As a result of confirming root development per plant, there was no significant difference in root weight and development rate between NT and TF lines, confirming that the IbFAD8 gene had no effect on sweet potato root development (Figure 7).

The tubers of harvested NT and TF line plants were stored under appropriate storage temperature (13°C) and low temperature (4°C) conditions for 6 weeks, and low-temperature storage was evaluated. As a result, the NT showed severe root rot and softness due to low temperature, whereas the TF line showed only slight browning symptoms in the cortex of the root cross section (Figure 8a). As a result of analyzing the content of MDA (malondialdehyde), an indicator of lipid peroxidation, by biochemical analysis, the MDA content was found to be higher in the NT line than in the TF line under low temperature conditions (Figure 8b). The above results showed that the low-temperature storage properties of the tubers of the TF line plants were improved. This suggests the possibility of improving storage management after harvesting sweet potatoes by increasing the low-temperature storage of sweet potatoes.

Example 5. Analysis of changes in fatty acid composition of transgenic sweet potatoes

To analyze changes in sweet potato fatty acid composition due to IbFAD8 overexpression, gas chromatography (GC) was used to analyze palmitic acid (16:0), stearic acid (18:0), and oleic acid ( The composition (%) of fatty acids (18:1), linoleic acid (18:2), and linolenic acid (18:3) was analyzed. Fatty acid composition was analyzed by GC (gas chromatography) using an Agilent series 7890A (Agilent Technologies Inc.) equipped with a flame ionization detector, and the detailed method was the same as that of Example 2.

As a result, it was confirmed that in the leaves, the composition of linoleic acid (18:2) was decreased in the TF line compared to NT, and the composition of linolenic acid (18:3) was slightly increased (Table 1). On the other hand, in tubers, the composition of linoleic acid (18:2) is reduced by about 2 times in the TF line compared to NT, and the composition of linolenic acid (18:3) is increased by about 4 times, so that the linolenic acid/linoleic acid ratio is about 8 compared to NT. It was confirmed that it had increased to a very high level of ~9-fold (Table 2).

The above results confirmed the possibility of developing sweet potatoes high in plant-derived omega-3 linolenic acid, which are known to be effective in high blood pressure and cardiovascular diseases, using the IbFAD8 gene.

<110> Korea Research Institute of Bioscience and Biotechnology <120> IbFAD8 gene from sweetpotato for improving cold storage stability and increasing omega-3 fatty acid content in tuberous root of sweetpotato and uses it <130>PN21044 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 1338 <212> DNA <213> Ipomoea batatas <400> 1 atggcgagtt gggtgttatc agaatgtggt ctcagaccat tccccaaaat ctaccctaag 60 ccaagaactg ggatgggttc tctcagttgt ggcagcccct caaggctgac agttgcaggg 120 acagatctgg ggtcttgttc tttgatggg attagggaga ggaattgggc attgagggtt 180 agtgccccag tgagggttcc ctcagttggg gaggaagagg aagaagagag ggagagtatc 240 aatgttgtca atggggatgg ggatgagttt tttgaccctg gggcaccacc cccatttaag 300 ctgtctgata ttagagcagc cattcctaag cattgctggg ttaaggaccc ttggaagtcc 360 atgagctttg tggttaggga tgttgcaatt gtgtttggat tggcagctgt ggctgcttct 420 ctcaataatt ggattgtttg gcctctgtat tggttggctc aaggaaccat gttctgggct 480 ctctttgttc ttggccatga ctgtggccat ggaagctttt ctaatgaccc aaagctgaac 540 agtgttgctg gccatctcct ccactcttca attcttgtcc cttaccatgg atggagaatt 600 agccatagga ctcatcacca gaaccatggc catgtcgaaa atgatgaatc ttggcaccct 660 ctgcctgaga agatattcaa gagccttgac aatgtcacga gaaagttgag gttcaccata 720 ccgttcccac tgcttgcata ccccgtctat ctgtggagta gaagccctgg gaagaaaggc 780 tctcattttg atccaaacag cgatttgttt gtgccaaatg agaagaaaga cgtgattacc 840 tcaaccgtgt gttggtcagc catggttgct atccttgctg gtttatcgtt cgtgatgggg 900 cccgtccaat tgctcaaact ctatggagtt ccatatgcga tctttgttat gtggctggat 960 ttagtcacct acttacatca tcatggacat gaagacaaac ttccatggta tcgtggaaag 1020 gaatggagtt accttagggg tggactgacc actcttgatc gcgactatgg atggataaac 1080 aacatacacc acgatattgg gacccatgtc atacatcatc ttttccccca aatccctcat 1140 tatcacttga tcgaagctac cgaggctgct aagccagtgc tcgggaagta ttacagagag 1200 ccgaagaaat catggcctct tccattgcac ctgttgggag acctcgtgag aagcatgaag 1260 aaggatcact acgtgagtga tgacggggat gttgtatatt accaaaccga ccccaagctt 1320 gctggaaccg agaagtga 1338 <210> 2 <211> 445 <212> PRT <213> Ipomoea batatas <400> 2 Met Ala Ser Trp Val Leu Ser Glu Cys Gly Leu Arg Pro Phe Pro Lys 1 5 10 15 Ile Tyr Pro Lys Pro Arg Thr Gly Met Gly Ser Leu Ser Cys Gly Ser 20 25 30 Pro Ser Arg Leu Thr Val Ala Gly Thr Asp Leu Gly Ser Cys Ser Leu 35 40 45 Ile Gly Ile Arg Glu Arg Asn Trp Ala Leu Arg Val Ser Ala Pro Val 50 55 60 Arg Val Pro Ser Val Gly Glu Glu Glu Glu Glu Glu Arg Glu Ser Ile 65 70 75 80 Asn Val Val Asn Gly Asp Gly Asp Glu Phe Phe Asp Pro Gly Ala Pro 85 90 95 Pro Pro Phe Lys Leu Ser Asp Ile Arg Ala Ala Ile Pro Lys His Cys 100 105 110 Trp Val Lys Asp Pro Trp Lys Ser Met Ser Phe Val Val Arg Asp Val 115 120 125 Ala Ile Val Phe Gly Leu Ala Ala Val Ala Ala Ser Leu Asn Asn Trp 130 135 140 Ile Val Trp Pro Leu Tyr Trp Leu Ala Gln Gly Thr Met Phe Trp Ala 145 150 155 160 Leu Phe Val Leu Gly His Asp Cys Gly His Gly Ser Phe Ser Asn Asp 165 170 175 Pro Lys Leu Asn Ser Val Ala Gly His Leu Leu His Ser Ser Ile Leu 180 185 190 Val Pro Tyr His Gly Trp Arg Ile Ser His Arg Thr His His Gln Asn 195 200 205 His Gly His Val Glu Asn Asp Glu Ser Trp His Pro Leu Pro Glu Lys 210 215 220 Ile Phe Lys Ser Leu Asp Asn Val Thr Arg Lys Leu Arg Phe Thr Ile 225 230 235 240 Pro Phe Pro Leu Leu Ala Tyr Pro Val Tyr Leu Trp Ser Arg Ser Pro 245 250 255 Gly Lys Lys Gly Ser His Phe Asp Pro Asn Ser Asp Leu Phe Val Pro 260 265 270 Asn Glu Lys Lys Asp Val Ile Thr Ser Thr Val Cys Trp Ser Ala Met 275 280 285 Val Ala Ile Leu Ala Gly Leu Ser Phe Val Met Gly Pro Val Gln Leu 290 295 300 Leu Lys Leu Tyr Gly Val Pro Tyr Ala Ile Phe Val Met Trp Leu Asp 305 310 315 320 Leu Val Thr Tyr Leu His His His Gly His Glu Asp Lys Leu Pro Trp 325 330 335 Tyr Arg Gly Lys Glu Trp Ser Tyr Leu Arg Gly Gly Leu Thr Thr Leu 340 345 350 Asp Arg Asp Tyr Gly Trp Ile Asn Asn Ile His His Asp Ile Gly Thr 355 360 365 His Val Ile His His Leu Phe Pro Gln Ile Pro His Tyr His Leu Ile 370 375 380 Glu Ala Thr Glu Ala Ala Lys Pro Val Leu Gly Lys Tyr Tyr Arg Glu 385 390 395 400 Pro Lys Lys Ser Trp Pro Leu Pro Leu His Leu Leu Gly Asp Leu Val 405 410 415 Arg Ser Met Lys Lys Asp His Tyr Val Ser Asp Asp Gly Asp Val Val 420 425 430 Tyr Tyr Gln Thr Asp Pro Lys Leu Ala Gly Thr Glu Lys 435 440 445 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atggcgagtt gggtgttatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tcacttctcg gttccagcaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cccccattta agctgtctga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acatggttcc ttgagccaac 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agtgagcgca acgcaattaa tgtg 24

Claims (7)

Sweet potato compared to the wild type, including the step of overexpressing the IbFAD8 gene by transforming sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2. A method of increasing the low-temperature storage capacity and linolenic acid content of tubers. delete Transforming sweet potato plant cells with a recombinant vector containing a gene encoding the sweet potato IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2, thereby overexpressing the IbFAD8 gene; and
A method for producing a transgenic sweet potato plant with increased low-temperature storability and linolenic acid content of the tubers compared to the wild type, comprising the step of redifferentiating the transformed sweet potato plant from the transformed sweet potato plant cell. A transgenic sweet potato plant with increased low-temperature storage and linolenic acid content of the tubers prepared by the method of claim 3, and increased low-temperature and drying stress tolerance. delete Sweet potato tubers with increased low-temperature storage and linolenic acid content of the plant according to paragraph 4. A composition for improving low-temperature storage and linolenic acid content of sweet potato tubers, containing as an active ingredient a gene encoding the sweet potato-derived IbFAD8 ( Ipomoea batatas Fatty acid desaturase 8) protein consisting of the amino acid sequence of SEQ ID NO: 2.