Method for producing sphingomyelin

Abstract

To provide a method for producing sphingomyelin inexpensively and by a large amount by using a plant.SOLUTION: A vector in which a human-derived DNA described in any one of the following (a) to (e) is functionally connected to downstream of a promoter region which can be expressed in a plant cell. (a) a DNA encoding a protein made up of a specific amino acid sequence; (b) a DNA including an encoding region of a specific base sequence; (c) a DNA encoding a protein made up of an amino acid sequence in which one or a plurality of amino acids are substituted, deleted, added and / or inserted in the specific amino acid sequence; (d) a DNA encoding a protein which has 90% or more identity with the specific amino acid sequence, and functions equal to the protein made up of a specific amino acid sequence; and (e) a DNA in which a codon of any DNA sequence of (a), (c) or (d) is optimized to a plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing sphingomyelin, and more particularly to a method for producing sphingomyelin in a plant. [Background technology]

[0002] Conventionally, sphingomyelin has been extracted inexpensively from bovine brains, chicken skin, etc. (For example, Patent Document 1). However, because of the risk of mad cow disease in the case of bovine brains, the development of other production methods has been sought, but there have been no reports of biological production from other species using genetic engineering techniques or the like. Therefore, there is a need to develop a method for producing sphingomyelin in large quantities at lower cost. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-179588 Summary of the Invention [Problem to be solved by the invention]

[0004] To provide a method for inexpensively and mass-producing sphingomyelin using plants. [Means for solving the problem]

[0005] (1) A vector in which any one of the following DNAs (a) to (e) is operably linked downstream of a promoter region that can be expressed in plant cells: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (b) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 (c) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, added, and / or inserted. (d) DNA encoding a protein that has 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 and has a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. (e) DNA in which the codons of any one of the DNA sequences (a) to (d) have been optimized for plants. Here, "functionally linked" means that the human-derived DNA is linked in such a way that it is transcribed from the promoter, translated, and the target protein is expressed. (2) A transformed plant cell into which the vector according to (1) has been introduced. (3) A plant capable of producing sphingomyelin, regenerated from the transformed plant cell according to (2). (4) A plant capable of producing sphingomyelin, which is a descendant or clone of the plant according to (3). (5) A propagation material of a plant having the ability to produce sphingomyelin according to (3) or (4). (6) A method for producing a plant capable of producing sphingomyelin, comprising: (i) introducing the vector according to (1) into a plant cell; and (ii) regenerating a plant from the transformed plant cell into which the vector has been introduced in step (i); A method comprising: (7) A method for producing sphingomyelin, characterized by using the transformed plant cell according to (2) or the plant according to (3) or (4). (8) Plant cells into which an animal-derived or synthetic sphingomyelin synthase gene has been introduced and expressed. Here, "synthetic" means both partial and complete synthesis. [Effects of the Invention]

[0006] According to the present invention, a method for inexpensively synthesizing large amounts of lactic acid bacteria from safe plants that are free from the risk of animal-derived infectious diseases is provided. [Brief explanation of the drawings]

[0007] [Figure 1]FIG. 1 shows the DNA sequence of the hSMS2 gene (Example 1). [Figure 2] FIG. 2 shows the amino acid sequence of the hSMS protein. [Figure 3] FIG. 3 is a diagram of the p35S-Ω-hSMS2-tHSP-pBCKK vector (Example 2). [Figure 4] FIG. 4 is a photograph showing the results of genomic PCR of hSMS2-transformed tobacco BY-2 cells (Example 4). [Figure 5] FIG. 5 is a photograph showing the results of RT-PCR of hSMS2-transformed tobacco BY-2 cells (Example 5). [Figure 6] FIG. 6 is a graph showing the results of quantification using a sphingomyelin quantification kit (Example 7). [Figure 7] FIG. 7 shows a table showing comprehensive lipid analysis data by LC-MS / MS and a graph showing the quantification of ceramide, glucosylceramide, and sphingomyelin using the comprehensive lipid analysis data (Example 8). [Figure 8] FIG. 8 shows the DNA sequence of the hSMS1 gene (Example 9). [Figure 9] FIG. 9 shows the DNA sequence of the hSMS1 Art gene (Example 9). [Figure 10] FIG. 10 shows the DNA sequence of the hSMS2 Art gene (Example 9). [Figure 11] FIG. 11 shows a rice transformation vector containing the hSMS1 Art gene (Example 13). [Figure 12] FIG. 12 shows the rice EF1α-1 promoter sequence (Example 12). [Figure 13] FIG. 13 shows the structure of a transformation vector containing EF1α-1 or EF1α-4 promoter-hSMS2 (Example 12). [Figure 14] FIG. 14 shows the rice seed-specific Globulin promoter sequence (Example 13). [Figure 15]FIG. 15 shows the structure of an expression vector containing globulin promoter-hSMS1 Art-DT2 (Example 13). [Figure 16] FIG. 16 is a photograph showing the results of genomic PCR of transformed rice (Example 14). [Figure 17] FIG. 17 is a photograph showing the results of RT-PCR of transformed rice grains (Example 14). [Figure 18] FIG. 18 is a graph showing the results of comprehensive lipid analysis of rice transformants (Example 15). [Figure 19] FIG. 19 shows the construction of a transformation vector in which a 35S promoter is linked to a codon-optimized SMS2 gene (Example 15). [Figure 20] FIG. 20 is a graph showing the results of measuring the expression level (A) and sphingomyelin level (B) in tobacco BY-2 cells into which codon-optimized hSMS2 was introduced (Example 10). [Figure 21] FIG. 21 shows the nucleotide sequence of the ELF1-α4 promoter (Example 12). [Figure 22] FIG. 22 is a photograph showing RT-PCR of rice transformants into which the codon-optimized SMS2 gene was introduced (Example 14). [Figure 23] FIG. 23 shows the results of quantifying sphingomyelin in transformed rice plants (Example 16). DETAILED DESCRIPTION OF THE INVENTION

[0008] The present invention is characterized by the production of sphingomyelin in plant cells by introducing and expressing a sphingomyelin synthase (SMS) gene into the plant cells. Because sphingomyelin does not naturally exist in plants, it was unclear whether sphingomyelin could be produced by introducing a foreign SMS gene. Furthermore, there have been no reports of sphingomyelin production in other organisms, such as Escherichia coli.

[0009] In the present invention, the SMS gene is preferably the SMS2 gene of human origin, but the SMS1 gene may also be used. The DNA and amino acid sequences of SMS2 and SMS1 are shown in Figures 1, 2, and 8. They are also shown in SEQ ID NOS: 1 to 4 (see sequence listing). DNA encoding a protein that has 90% or more identity with the SMS2 protein and has SMS functions equivalent to those of the SMS2 protein may also be used as the SMS gene. The SMS gene may be derived from, but is not limited to, humans; SMS genes from mammals, birds, reptiles, amphibians, fish, crustaceans, insects, and the like may also be used.

[0010] Furthermore, DNA encoding a protein having an amino acid sequence in which one to several (or multiple) amino acids have been substituted from the amino acid sequence of the hSMS2 protein and which has a function equivalent to that of the hSMS2 protein can also be used. As used herein, "having a function equivalent" means exhibiting the same level of enzymatic activity.

[0011] Furthermore, because SMS2 is originally a human gene, its codon usage is not optimized for expression in plant cells. Therefore, it is preferable to use DNA with amino acid codons optimized for plant cells, such as tobacco and rice, to enable efficient protein synthesis in plant cells. To optimize codons, simply use the most frequently occurring codon for each amino acid. More precisely, it is thought that amino acid synthesis would theoretically proceed most quickly if the codon used was the one with the highest amount of tRNA corresponding to each amino acid.

[0012] In this case, it is preferable to optimize the codons by total gene synthesis. Alternatively, if necessary, it is possible to optimize only an appropriate length of the 5'-upstream portion of the gene. This is because optimizing the initial synthesis may lead to smoother subsequent protein synthesis.

[0013] To introduce and express the SMS gene in plants, it is necessary to construct a vector containing an SMS expression cassette (a cassette in which a promoter that functions in plants, the SMS gene (coding region), and a poly(A) addition signal are operably linked). As used herein, "operably linked" means operably linked. For example, operably linking a promoter to a gene means that at least transcription of the gene occurs.

[0014] To achieve this, we constructed a vector containing a promoter that functions in plants, upstream of the SMS gene, so that the SMS gene is expressed, a poly(A) addition signal downstream of the SMS gene, and a selectable marker gene for selecting transformants. The portion of this vector to be transferred to plants was inserted between the T regions of the Ti plasmid vector by double crossover recombination (Figure 3).

[0015] Promoters functional in plants can be selected from constitutive promoters, inducible promoters, and tissue- and / or stage-specific promoters, depending on the purpose. Constitutive promoters include, but are not limited to, the 35S promoter, 35SΩ promoter, and 19S promoter derived from cauliflower mosaic virus (CMV), the nopaline synthase gene (nos) promoter of the Ti plasmid, the octopine synthase gene (ocs) promoter (ocs), and the actin promoter, ubiquitin promoter, and PEPC promoter derived from plant genomes. Tissue- and stage-specific promoters include, but are not limited to, the Rubisco small subunit (rbcss) promoter, the chlorophyll a / b-binding protein gene (cab) promoter, the globulin promoter, the phaseolin promoter, the napin promoter, and the zein promoter. Inducible promoters include, but are not limited to, heat shock protein promoters, the Adh promoter, hormone-inducible promoters, stress-inducible promoters, and drug-inducible promoters (e.g., tetracycline).

[0016] The poly A addition signal may be a sequence containing a poly A addition signal present on the 3'-side of the promoter (usually downstream of the coding region). For example, sequences containing the poly A addition signal of the novaline synthase gene, the poly A addition signal of octopine synthase, or the poly A addition signal contained in the cauliflower mosaic virus are preferably used, but are not limited to these. Poly A addition signal sequences from genes expressed in plants, such as rbcss, may also be used, and poly A addition signal sequences derived from animals or yeast may also be used as long as they function in plants. In short, any sequence capable of adding poly A may be used.

[0017] Methods for introducing vectors into plant cells include direct introduction and Agrobacterium-based methods. Direct introduction methods include, but are not limited to, injecting a plasmid (either intact or after cleavage at appropriate sites) into plant cells with a gene gun, or introducing the plasmid into protoplasted plant cells using PEG or liposomes, or electroporation. When using Agrobacterium, it is preferable to insert an SMS gene expression cassette into the T-region (enclosed by T-DNA) of the vector to be introduced into the plant, and use a vector that contains an origin of replication for Agrobacterium and a selectable marker for selecting Agrobacterium. The T-region may be derived from an Ri plasmid. Infecting plant cells with Agrobacterium carrying a T-DNA-containing plasmid allows integration of the T-DNA into the plant genome, resulting in expression of the gene contained in the expression cassette in the T-DNA. Plant transformation typically involves using Agrobacterium carrying another plasmid capable of transferring the T-DNA region into the plant; this transformation system is called a binary vector system.

[0018] The vectors used for the transformation of the present invention are shown in FIGS. By introducing these vectors into plant cells and expressing SMS, sphingomyelin was produced in tobacco and rice (Figures 6, 7, 18, 20, and 23). Transformation was confirmed by genomic PCR (Figures 4 and 16). Expression of the SMS gene was confirmed by RT-PCR or real-time PCR (Figures 5, 17, and 22). In wild-type tobacco and rice, ceramide was present, but no sphingomyelin was detected (Figures 7B and 18B). In tobacco cells into which the SMS of the present invention was introduced and expressed, sphingomyelin was produced at approximately 60% of the ceramide originally contained (Figure 7B). In the present invention, the content of sphingomyelin in plant cells may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, or 80% or more of the total amount of ceramide originally contained.

[0019] Plants that can produce the sphingomyelin of the present invention include, but are not limited to, monocotyledons and dicotyledons. Examples include Solanaceae plants (tobacco, tomato, potato, etc.), Gramineae plants (rice, wheat, barley, corn, etc.), Brassicaceae plants (Arabidopsis thaliana, Brassica napus, etc.), Asteraceae plants (lettuce, etc.), and mosses (Marchantia polymorpha, Physcomitrella patens, etc.), with tobacco or rice being preferred. Examples of Nicotiana plants include, but are not limited to, Nicotiana benthamiana, N. tabacum, and N. excelsior. When the plant is a Nicotiana plant, the plant body, a part thereof, or a plant cell includes a Nicotiana plant or a part thereof, or a cell (including cultured cells) derived from a Nicotiana plant. The same applies to other plants. The grass family includes a wide range of species, such as wheat (Triticum spp.), rice (Oryza spp.), barley (Hordeum spp.), oats (Avena spp.), rye (Secale spp.), maize (Zea spp.), and millet (Pennisettum spp.). In one embodiment of the present invention, the preferred family members are rice, wheat, and barley.

[0020] In addition, in terms of the types of sphingomyelins, SM(d18:0 / 16:0), SM(d18:1 / 24:0), and SM(d18:1 / 16:0), which are the major sphingomyelins produced by humans, were detected in SMS-transformed tobacco culture cells. Ten types of phytosphingomyelins were detected in the skin and small intestine (SM[phyto](18:1 / 24:0), SM[phyto](18:0 / 24:0), SM[phyto](18:1 / 22:0), SM[phyto](18:0 / 22:0), SM[phyto](18:1 / 26:0), SM[phyto](18:0 / 26:0), SM[phyto](18:0 / 23:0), SM[phyto](18:1 / 23:0), SM[phyto](18:0 / 25:0), SM[phyto](19:0 / 24:0)), and 11 types of phytosphingomyelins with one hydroxyl group introduced into the fatty acid of phytosphingomyelin. The following compounds were detected: SM[phyto_aOH](18:1 / 24:0), SM[phyto_aOH](18:0 / 24:0), SM[phyto_aOH](18:1 / 22:0), SM[phyto_aOH](18:1 / 26:0), SM[phyto_aOH](18:0 / 22:0), SM[phyto_aOH](18:1 / 25:0), SM[phyto_aOH](19:1 / 24:0), SM[phyto_aOH](18:1 / 23:0), SM[phyto_aOH](18:0 / 25:0), SM[phyto_aOH](18:0 / 26:0), SM[phyto_aOH](18:0 / 23:0) (Figure 7A). Six types of sphingomyelins were detected in the grains of SMS-transformed rice: SM(d18:0 / 24:0), SM[phyto](18:0 / 24:0), SM[phyto](18:0 / 26:0), SM[phyto](18:0 / 22:0), SM[2O](t42:0), and SM[2O](t42:1) (Figure 18A).

[0021] LC-MS / MS analysis revealed that approximately 15% of ceramide was converted to sphingomyelin in the SMS-transformed rice (Figure 18B). Because this is still a heterozygous state, it is believed that even greater amounts of sphingomyelin could be produced by extracting progeny with homozygous SMS alleles.

[0022] A plant-optimized SMS2 gene was synthesized and inserted into a vector (Figure 19). This vector was used to transform tobacco BY-2 cells. Measurement of sphingomyelin levels in the resulting transformants revealed a detection level of 2.5 mg / mL (Figure 20B). In contrast, in tobacco BY-2 cells transfected with human SMS (before codon optimization for plants), the sphingomyelin level was approximately 0.1 mg / mL before codon optimization, but increased to approximately 2.5 mg / mL after optimization (Figure 20B). Therefore, optimizing codon usage is believed to produce approximately 25-fold more sphingomyelin. Therefore, optimizing codon usage for plants is useful for increasing sphingomyelin production.

[0023] Similarly, a rice vector was constructed incorporating the SMS2 gene with codon usage optimized for plants (Figure 13). Rice was transformed using this vector. Expression of the codon-optimized SMS2 gene was detected in the transformants (Figure 22). When the amount of sphingomyelin in these transformants was measured, a significant increase was observed compared to the vector control, as shown in Figure 23. [Example]

[0024] (Example 1) Method for isolating human sphingomyelin synthase gene (hSMS) Total RNA was prepared from human dermal fibroblasts (Takara Bio) using the RNeasy Mini Kit (Qiagen). First-strand cDNA was reverse transcribed using the PrimeScript RT Reagent kit with gDNA Eraser (Takara Bio). RT-PCR was performed using this cDNA as a template to amplify the full-length ORF (1,098 bp) of hSMS2 (NCBI Reference Sequence: NM_001375908.1). PCR was performed using SMS2 primer 1 (SEQ ID NO: 10: 5'-CCCGGGATGGATATCATAGAGA-3') and SMS2 primer 2 (SEQ ID NO: 11: 5'-GACCTCTCAGGTCGATTTCTCAT-3'), which contain the SmaI or SacI restriction enzyme sites. PCR was performed using Takara ExTaq, with 30 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 2 minutes. The amplified DNA fragment was inserted into the pGEM T-easy vector (Promega) to create hSMS2-pGEM. Sequencing using Big Dye Terminator Ver. 3.1 (Thermo Fisher Scientific) confirmed that the hSMS2 gene contained no mutations. The DNA and amino acid sequences of hSMS2 are shown in Figure 1 and SEQ ID NO: 1, and Figure 2 and SEQ ID NO: 2, respectively. The hSMS gene can also be obtained by DNA synthesis, or a vector carrying the hSMS gene can be used.

[0025] (Example 2) Construction of tobacco plant expression vector for hSMS2 gene p35S-hSMS2-tHSPG was generated by digesting the hSMS2-pGEM and p35S-SRDX-tHSPG vectors (Oshima et al., Plant Biotechnol. 28, 201-210, 2011) with the restriction enzymes SmaI and SacI. p35S-hSMS2-tHSPG was then introduced into the plant transformation vector pBCKK (Oshima et al., Plant Biotechnol. 28, 201-210, 2011) using a Gateway LR reaction (Thermo Fisher Scientific). hSMS2 gene expression is controlled by a strong promoter, the cauliflower mosaic virus 35S promoter (p35S), a translation enhancer, the 5'-untranslated region (Ω) of the tobacco mosaic virus, and a strong terminator (poly(A) addition signal) from the Arabidopsis heat shock protein 18.2 (HSP) gene. Compared to commonly used gene expression systems, this is expected to increase hSMS2 gene expression several hundred times (Nagaya et al., Plant Cell Physiol. 51, 328-332, 2010). It also contains a kanamycin resistance gene as a selectable marker for transformants. The plant expression vector for the hSMS2 gene is shown in Figure 3.

[0026] Example 3: Preparation of transformed tobacco BY-2 cells producing sphingomyelin Genes can be introduced into plant cells by known methods depending on the plant species. For example, in the case of tobacco cultured cells BY-2, transformed tobacco cultured cells BY-2 can be obtained by infecting the cells with Agrobacterium containing the above-mentioned vector, washing the cells with a medium containing carbenicillin, an antibiotic for eliminating Agrobacterium, and kanamycin, which selects for genetically modified organisms, and then culturing the cells in a medium containing these antibiotics.

[0027] (Example 4) Genomic PCR of transformed tobacco BY-2 cells into which the hSMS2 gene was introduced To confirm that the hSMS2 gene had been integrated into the chromosomal genome of tobacco BY-2 cells, genomic PCR was performed using genomic DNA as a template. Genomic DNA was extracted from approximately 0.1 g of kanamycin-resistant transformed BY-2 cells using the DNeasy plant mini kit (Qiagen), and 1 μl of the DNA was used for genomic PCR. The primers used were hSMS2F (SEQ ID NO: 12: 5'-ACCTCGTCTTGACAACCGTC-3') and hSMS2R (SEQ ID NO: 13: 5'-CCAGAAGTGACGAGGCGAAT-3'). PCR was performed using Takara ExTaq, with 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 2 minutes. A band derived from the hSMS2 gene was amplified in all lines, confirming that the hSMS2 gene had been integrated into the chromosomal genome of tobacco BY-2 cells. Figure 4 shows the 1% agarose gel electrophoresis images of hSMS2-transformed tobacco BY-2 cell lines 3 and 8. A 482-bp amplified fragment was observed.

[0028] (Example 5) RNA analysis of transformed tobacco BY-2 cells transfected with hSMS2 gene Total RNA was prepared from approximately 0.1 g of a tobacco BY-2 cell line containing the hSMS2 gene integrated into the chromosomal genome using the RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was reverse transcribed using the PrimeScript RT Reagent kit with gDNA Eraser (Takara Bio). RT-PCR was performed using this cDNA as a template. The primers used were the same as for the genomic PCR: hSMS2F (5'-ACCTCGTCTTGACAACCGTC-3') and hSMS2R (5'-CCAGAAGTGACGAGGCGAAT-3'). RT-PCR was performed using Takara ExTaq, with 35 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 2 minutes. A band derived from the hSMS2 gene was amplified, confirming the transcription of hSMS2 mRNA (Figure 5). As a control, RT-PCR was performed using the tobacco actin gene (NCBI Reference Sequence: XM_016618073.1). The primers used were NtACTF (SEQ ID NO: 14: 5'-CCTCTTAACCCGAAGGCTAA-3') and NtACTR (SEQ ID NO: 15: 5'-GAAGGTTGGAAAAGGACTTC-3'), and the amplified fragment was 470 bp in size.

[0029] (Example 6) Lipid extraction from transformed tobacco BY-2 cells transfected with hSMS2 gene Total lipids were extracted from tobacco BY-2 cells using the Bligh & Dyer method (Bligh and Dyer, Can. J. Biochem. Physiol. 37, 911-917, 1959). Water was removed from tobacco BY-2 cells by vacuum filtration and centrifugation at 3000 rpm for 5 minutes. Approximately 1 g of cells was immersed in 10 ml of chloroform:methanol (1:2 by volume), homogenized using a Polytron homogenizer for approximately 2 minutes, and then allowed to stand at room temperature for 10 minutes. Chloroform and water were added to a chloroform:methanol:water ratio of 1:1:0.9 by volume, transferred to a glass centrifuge tube, and centrifuged at 3000 rpm for 10 minutes to separate the two layers. The lower layer was collected and concentrated using a rotary evaporator to prepare the sample.

[0030] (Example 7) Enzymatic measurement of sphingomyelin The above samples were subjected to sphingomyelin quantification using a sphingomyelin quantification kit (Cayman Chemical Company). A signal of 0.2 mg / ml was detected in both the wild-type (WT) and vector control (pBCKK-1) tobacco plants. Because the sphingomyelin quantification kit detects choline as background, it is presumed that endogenous choline is being detected. Transgenic BY-2 tobacco plants, hSMS2-3 and hSMS2-8, which contained the hSMS2 gene, showed values ​​of 0.9 mg / ml and 0.5 mg / ml, respectively, which were higher than those of WT and pBCKK-1 (Figure 6).

[0031] (Example 8) LC-MS / MS analysis Lipid extraction was performed as follows. Tobacco BY-2 cells were added with liquid nitrogen and ground to a powder using a mortar and pestle. Lyophilization was then performed. 1–5 mg of the dry weight was added with 600 μL of chloroform:methanol (1:2 by volume), sonicated for 30 seconds, and then vortexed at 750 rpm for 20 minutes at 20°C. 40 μL of ultrapure water was added, and the mixture was stirred again in the same manner. The mixture was then centrifuged at 1670 x g for 10 minutes at 20°C. The supernatant was collected and used as a sample for LC-MS / MS analysis (Tsugawa et al. J Cheminform (2017) 9:19. DOI 10.1186 / s13321-017-0205-3). UPLC (Waters) TripleTOF 6600 (SCIEX) Separation column: Acquity UPLC Peptide BEH C18 (Waters), temperature 45°C Mobile phase A: 5 mM ammonium nitrate / 10 nM EDTA-containing methanol / acetonitrile / water = 1:1:3 (v / v) Mobile phase B: 5 mM ammonium nitrate / 10 nM EDTA in 2-propanol (v / v) Gradient: Mobile phase B 0% to 64% (0-6.5 min) → 64% to 76.5% (6.5-13.5 min) → 76.5% to 98% (13.5-18 min) → 98% to 98% (19-20 min) Mobile phase flow rate: 0.3mL / min Injection volume: 3μL Ionization method: Electrospray ionization (ESI) Measurement mode: Negative-ion mode measurement, Positive-ion mode measurement

[0032] In wild-type tobacco BY-2 cells, the major phytoceramides detected were Cer[phyto](18:0 / 22:0), Cer[phyto](18:0 / 24:0), and Cer[phyto](18:1 / 24:0), and sphingomyelin was not detected. In hSMS2-transformed tobacco BY-2 cell line 3, the major sphingomyelins detected were SM[phyto](18:0 / 22:0), SM[phyto](18:0 / 24:0), and SM[phyto](18:1 / 22:0). Phosphocholine was conjugated to ceramides in the transfected tobacco BY-2 cells, resulting in the synthesis of sphingomyelin (Figure 7A; *indistinguishable from SM[phyto](19:0 / 24:0), **indistinguishable from SM[phyto_aOH](19:1 / 24:0)). The signals for each molecular species were summed and compared with those for ceramide and glucosyl (Figure 7B). Approximately 60% of the wild-type ceramide was synthesized as sphingomyelin. Glucosylceramide, synthesized by glucosylceramide synthase using ceramide as a substrate, was detected at the same level as in the wild-type. The amount of glucosylceramide produced by plants is estimated to be 0.2–0.9 mg / g dry matter (https: / / www.naro.go.jp / project / results / laboratory / harc / 2013 / harc13_s13.html). By introducing the hSMS2 gene, approximately 60% of glucosylceramide becomes sphingomyelin, and a yield of approximately 0.12–0.54 mg / g dry matter can be expected.

[0033] (Example 9) Construction of an expression vector for human sphingomyelin synthase gene optimized for plant-type codons To increase sphingomyelin production, two human sphingomyelin synthase genes, hSMS1 (Fig. 8, SEQ ID NO: 3, 1242 bp, NCBI Reference Sequence: NM_147156.4) and hSMS2 (Fig. 1, 1098 bp, NCBI Reference Sequence: NM_001375908.1), were codon-optimized for plant use and synthesized as DNA (hSMS1 Art) (Fig. 9, SEQ ID NO: 6) and hSMS2 Art (Fig. 10, SEQ ID NO: 5) (Eurofins). DNA consists of four bases (A, T, C, and G), and the type of amino acid to be translated is determined by the combination of three of these bases. These codons are called codons. Different species have different codon usage patterns, and it has been reported that changing codons from less frequently used to more frequently used ones (codon optimization) can improve expression levels (Zhou et al., J. Virol. 73, 4972-4982, 1999). p35S-Ω-hSMS2-tHSPG (Figure 3) was digested with the restriction enzymes SmaI and SacI, and a codon-optimized hSMS2 Art gene with a SmaI site at the 5' end and a SacI site at the 3' end was inserted to create p35S-Ω-hSMS2 Art-tHSPG. This vector was then introduced into the plant transformation vector pBCKK (Oshima et al., Plant Biotechnol. 28, 201-210, 2011) using a Gateway LR reaction (Thermo Fisher Scientific) (Figure 19).

[0034] (Example 10) Sphingomyelin production in transformed tobacco BY-2 cells transfected with hSMS2 Art gene p35S-Ω-hSMS2 Art-tHSP-pBCKK was introduced into tobacco BY-2 cells by Agrobacterium infection as in Example 3. Total RNA was prepared as in Example 5, and real-time PCR was performed using reverse-transcribed cDNA as a template. The primers used were hSMS2ArtF (SEQ ID NO: 16: 5'-TTCCGGGGATGCACTTTCAA-3') and hSMS2ArtR (SEQ ID NO: 17: 5'-CAAACGCAGAATGCGCTGTA-3'). Real-time PCR was performed using TB Green Premix Ex Taq II (Takara Bio, registered trademark), with 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The polypeptide chain elongation factor gene (EF1α gene) was used as a reference gene (NCBI Reference Sequence: NM_001326165.1). The primers used were NtEF1αF (SEQ ID NO: 18: 5'-GTATCCGCTCCCAGAGTTGG-3') and NtEF1αR (SEQ ID NO: 19: 5'-TGGAAAACCAAGGCACCTCA-3'). Real-time PCR was performed using the Thermal Cycler Dice Real Time System III (Takara Bio). The relative expression level of each line was calculated using the ΔΔCT method, with the expression level of the hSMS2 Art gene in line 1 set at 1 (Figure 20A). Approximately 30 lines were analyzed, and several highly expressing lines were obtained. Lines 25, 27, and 35, which showed good growth, were subjected to sphingomyelin quantification using a sphingomyelin quantification kit. Analysis was performed as in Examples 6 and 7. hSMS2-3, which uses human codons, had a concentration of 0.12 mg / mL, while hSMS2 Art-25, -27, and -35, which have codons optimized for plant cells, had concentrations of 2.5 mg / mL, 2.3 mg / mL, and 1.3 mg / mL, respectively. The amount of sphingomyelin in hSMS2 Art-25 was approximately 20 times that of hSMS2-3, and the amount of sphingomyelin produced was significantly increased by optimizing the codons to the plant type (Fig. 20B).

[0035] Example 11: Construction of rice plant expression vector for hSMS gene Rice is a plant used as a source of ceramide in cosmetics. To produce sphingomyelin in rice plants, two human sphingomyelin synthase genes, hSMS1 (Fig. 8, 1242 bp, NCBI Reference Sequence: NM_147156.4) and hSMS2 (Fig. 1, 1098 bp, NCBI Reference Sequence: NM_001375908.1), were codon-optimized for plant use and synthesized as DNA (hSMS1 Art) (Fig. 9) and hSMS2 Art (Fig. 10) (Eurofins). DNA consists of four bases (A, T, C, and G), and the combination of three of these bases determines the type of amino acid that will be translated. These are called codons, and there is a bias in the codons used by different species. It has been reported that changing codons from less frequently used to more frequently used ones (codon optimization) can improve expression levels (Zhou et al., J. Virol. 73, 4972-4982, 1999). We constructed various expression vectors using the plant-specific codon-optimized hSMS1 Art and hSMS2 Art, the plant-specific 35S promoter, the polypeptide chain elongation factor gene promoter (EF1α promoter), and the seed-specific globulin promoter (Qu and Takaiwa, Plant Biotechnol. 2, 113-125, 2004). The virus-derived 35S promoter is a strong, plant-specific constitutive promoter, but its expression level has been reported to decrease due to silencing during development (Matzke and Matzke, Curr Opin Plant Biol. 1, 142-148, 1998; Allen et al. Plant Mol Biol. 43, 361-376, 2000; Nagaya et al. Plant and Cell Physiology, 46, 438-444, 2006). On the other hand, the EF1α promoter derived from an endogenous gene is known to be a promoter that is resistant to silencing and maintains expression over a long period of time (e.g., Takara Bio website: https: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100006656).

[0036] (Example 12) Isolation of rice EF1α promoter and construction of expression vector The EF1α promoter has been isolated and used in tobacco plants (Nagaya et al., J. Biosci. Bioeng. 89 (3), 231-235, 2000; Aida et al. JARQ, 39 (4), 269-274, 2005), but has not been isolated in rice. Therefore, we isolated the rice EF1α promoter. Genomic DNA was isolated from rice leaves using the DNeasy plant mini kit (Qiagen), and PCR was performed. The rice EF1α gene region was searched using Phytozome (https: / / phytozome-next.jgi.doe.gov / ). Primers HEF1α1SF (SEQ ID NO: 20: 5'-AAGCTT-TGGTCACCCAGATTGTCTGC-3') and HEF1α1SR (SEQ ID NO: 21: 5'-CCCG-GGTTGAATAAGGAGGAAGCTAA-3') were prepared (Eurofin) to contain approximately 1.6 kb of the EF1α-1 promoter region. Primers HEF1α4SF (SEQ ID NO: 22: 5'-AAGCTT-GGACGAGGGCATGACTCTAG-3') and HEF1α4SR (SEQ ID NO: 23: 5'-CCCG-GGTTGAATAAGGAGGAAGCTAA-3') were prepared to contain approximately 2.7 kb of the EF1α-4 promoter region. GGTTGATTAAGCAGGTAGCT-3' was prepared (Eurofin). PCR was performed using Takara ExTaq, with 30 cycles of 94°C for 30 seconds, 65°C for 30 seconds, and 72°C for 2 minutes. The amplified DNA fragment was inserted into the pGEM T-easy vector (Promega) and sequenced using Big Dye Terminator Ver. 3.1 (Thermo Fisher Scientific). It was confirmed to be the EF1α-1 and EF1α-4 promoter regions. The sequences are shown in Figures 12 and 21. The EF1α-1 and EF1α-4 promoters were excised with restriction enzymes HindIII and SmaI and inserted into the p35S portion of p35S-hSMS2 Art-tHSPG to generate pEF1α-1-hSMS2 Art-tHSPG and pEF1α-4-hSMS2 Art-tHSPG.Next, the vector was introduced into the plant transformation vector pBCKH (Oshima et al., Plant Biotechnol. 28, 201-210, 2011) using a Gateway LR reaction (Thermo Fisher Scientific) (Figure 13). The vector contains a hygromycin resistance gene as a selectable marker for transformants.

[0037] (Example 13) Construction of rice expression vector using seed-specific globulin promoter p35S-DT2-pUC19 (Kuroda et al., Biosci Biotechnol Biochem. 74, 2348-2351, 2010) was digested with restriction enzymes XbaI and BamHI, and codon-optimized hSMS1 Art and hSMS2 Art genes with an XbaI site at the 5' end and a BamHI site at the 3' end were introduced to create p35S-hSMS1 Art-DT2-pUC19 and p35S-hSMS2 Art-DT2-pUC19. Systemic expression of the sphingomyelin synthase genes driven by the constitutively high-expressing 35S promoter is expected to alter the composition of sphingolipid substrates, such as ceramide. Analysis of Arabidopsis mutants has shown that reduced glucosylceramide levels result in morphological abnormalities (Msanne et al., Plant J. 84(1), 188-201, 2015). Therefore, we attempted to express a sphingomyelin synthase gene specifically in rice seeds using a seed-specific promoter. PCR was performed using primers Globulin F (SEQ ID NO: 24: 5'-GGCGCGCCGGCGCCTGGAGGGAGGAGAG-3') and Globulin R (SEQ ID NO: 25: 5'-TCTAGATGATGATCAATCAGACAATC-3'), which add an AscI site to the 5' end and an XbaI site to the 3' end of the rice seed-specific Globulin promoter (Kuroda et al., Biosci Biotechnol Biochem. 74, 2348-2351, 2010). The sequence of the rice Globulin promoter is shown in Figure 14 (NCBI Reference: AY427575.1). The rice Globulin promoter was replaced with the p35S promoter using the restriction enzymes AscI and XbaI to create pGlobulin-hSMS1 Art-DT2-pUC19 and pGlobulin-hSMS2 Art-DT2-pUC19.Next, the plant transformation vector pZH2B (Kuroda et al., Biosci Biotechnol Biochem. 74, 2348-2351, 2010; Hajdukiwqicz et al., Plant Mo Biol. 25, 989-994, 1994) and pGlobulin-hSMS2 Art-DT2-pUC19 were digested with restriction enzymes AscI and PacI to generate pGlobulin-hSMS2 Art-DT2-pZH2B. pGlobulin-hSMS1 Art-DT2-pUC19 was digested with restriction enzymes MluI and PacI and inserted into the AscI and PacI sites of pGlobulin-hSMS2 Art-DT2-pZH2B to construct pGlobulin-hSMS1 Art-DT2-pGlobulin-hSMS2 Art-DT2-pZH2B (Figure 15). The hygromycin resistance gene was used as a selectable marker for transformants.

[0038] (Example 14) Production of rice transformants pGlobulin-hSMS1 Art-DT2-pGlobulin-hSMS2 Art-DT2-pZH2B was introduced into the rice cultivar Tsukinohikari to produce transgenic rice plants. Tsukinohikari is a forage rice cultivar that is easy to cultivate and has high yields, making it suitable for sphingomyelin production. Genomic DNA was prepared from leaves of transgenic rice plants from lines 1 to 12 using the DNeasy plant mini kit (Qiagen). Genomic PCR confirmed that the hSMS1 Art and hSMS2 Art genes had been integrated into the rice chromosome genome (Figure 16). The primers used were hSMS1 Art F (SEQ ID NO: 26: 5'-GCTTCGTCCTGACCACTGTC-3') and hSMS1 Art R (SEQ ID NO: 27: 5'-ACAGTCTCGTAGTGATGTAG-3'), hSMS2 Art F2 (SEQ ID NO: 28: 5'-GTCCACCCCTACCAGACAAG-3') and hSMS2 Art R2 (SEQ ID NO: 29: 5'-TCGGAAACCACCAAGCCCTG-3'). The rice actin gene (NCBI Reference Sequence: NM_001418593.1) was used as a control. A 169-bp DNA fragment was amplified, confirming that the genomic PCR was successful. The primers used were OsAct F (SEQ ID NO: 30: 5'-ACACCGGTGTCATGGTCGG-3') and OsAct R (SEQ ID NO: 31: 5'-ACACGGAGCTCGTTGTAGAA-3'). Genomic PCR was performed in the same manner as for the analysis of tobacco BY-2 cells.

[0039] Next, RT-PCR was used to confirm the expression of the hSMS1 Art and hSMS2 Art genes (Figure 17). Total RNA was prepared from rice seeds using the RNeasy Mini Kit (Qiagen). A faint amplification band was observed for the rice actin gene used as a control. Since no clear band was amplified compared to genomic PCR, it is inferred that there may be a problem with the quality of the RNA prepared from rice seeds. Since bands for the hSMS1 Art and hSMS2 Art genes were observed in several lines with approximately the same signal intensity, it was determined that the introduced hSMS1 Art and hSMS2 Art genes were being expressed. The primers used were hSMS1 Art F (SEQ ID NO: 26: 5'-GCTTCGTCCTGACCACTGTC-3') and hSMS1 Art R (SEQ ID NO: 27: 5'-ACAGTCTCGTAGTGATGTAG-3'), hSMS2 Art F2 (SEQ ID NO: 28: 5'-GTCCACCCCTACCAGACAAG-3') and hSMS2 Art R2 (SEQ ID NO: 29: 5'-TCGGAAACCACCAAGCCCTG-3'). RT-PCR was performed in the same manner as for the analysis of tobacco BY-2 cells. Next, lipid analysis was performed on line 8.

[0040] Example 15: LC-MS / MS analysis of rice transformants LC-MS / MS analysis was performed on T1 seeds of line 8 transfected with pGlobulin-hSMS1 Art-DT2-pGlobulin-hSMS2 Art-DT2-pZH2B. The analysis was performed in the same manner as for tobacco BY-2 cells. Sphingomyelin, a conjugated form of Cer[phyto](18:0 / 24:0) and Cer[phyto](18:0 / 26:0) phosphocholines, the major phytoceramides in rice, was detected (Figure 18A), demonstrating the feasibility of sphingomyelin production not only in tobacco cultured cells but also in industrially important rice plants. The signals of each molecular species were summed and compared with those of ceramide and glucosylceramide. Approximately 15% of the wild-type ceramide and glucosylceramide were sphingomyelin (Figure 19B). Homozygous lines carrying both the paternal and maternal hSMS genes are expected to produce increased sphingomyelin levels.

[0041] Example 16 Sphingomyelin production in leaves of transgenic rice plants transfected with pEF1α-1-hSMS2 Art1-tHSPG and pEF1α-4-hSMS2 Art-tHSPG Rice transformants were prepared as in Example 11. RT-PCR was performed using leaves of the transformant rice as in Example 0035, confirming expression of the hSMS2 Art gene (Figure 22). The primers used were those described in Example 11. Of these, pEF1α-1-hSMS2 Art-tHSP-2 and pEF1α-4-hSMS2 Art-tHSP-1 were analyzed using a sphingomyelin quantification kit as in Examples 6 and 7. A high background was detected in the vector control, likely due to pigments derived from the leaves. The signal was increased in the transformed rice plants into which pEF1α-hSMS2 Art had been introduced compared to the vector control (Figure 23). Since the EF1α promoter is expressed throughout the plant, this demonstrated the feasibility of sphingomyelin production throughout the plant.

[0042] (Example 17) Preparation of transgenic rice plants that produce sphingomyelin Rice transformation was performed using known methods. Rice seeds were cultured on N6 medium containing 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mM proline, 300 mg / L casein hydrolysate, 30 g / L sucrose, and 4 g / L gellan gum (CHU et al., Sci. Sin. 18, 659-668, 1975). Calli were induced in the seeds. Agrobacterium harboring the vector was then infected and co-cultured on N6 medium containing 2 mg / L 2,4-D, 2 mg / L acetosyringone, 1 g / L casein hydrolysate, 10 g / L glucose, 30 g / L sucrose, and 2 g / L gellan gum. Vector-transfected callus cells were selected on N6 medium containing 20 mg / L meropenem, 50 mg / L hygromycin, 2 mg / L 2,4-D, 1 g / L casein hydrolysate, 30 g / L sucrose, and 2 g / L gellan gum. Rice plants were regenerated on MS medium containing 1 mg / L kinetin, 2 mg / L NAA, 250 mg / L claforan, 50 mg / L hygromycin, and 4 g / L gellan gum (Murashige and Skoog, Physiol. Plant, 15, 473–497, 1962). The regenerated rice plants were transferred to MS medium containing 250 mg / L claforan, 50 mg / L hygromycin, and 8 g / L agar, and roots were developed. The plants were then potted to obtain rice seeds. [Industrial Applicability]

[0043] The present invention can be used in the cosmetics industry and the like.

Claims

1. A vector in which any one of the following DNAs (a) to (e) is operably linked downstream of a promoter region that can be expressed in plant cells: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (b) DNA containing the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 (c) DNA encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, added, and / or inserted. (d) DNA encoding a protein that has 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 2 and has a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO:

2. (e) DNA in which the codons of any one of the DNA sequences (a) to (d) have been optimized for plants.

2. A transformed plant cell into which the vector of claim 1 has been introduced.

3. A plant capable of producing sphingomyelin, regenerated from the transformed plant cell of claim 2.

4. A plant having the ability to produce sphingomyelin, which is a descendant or clone of the plant according to claim 3.

5. A propagation material of a plant capable of producing the sphingomyelin according to claim 3 or 4.

6. A method for producing a plant capable of producing sphingomyelin, comprising: (i) introducing the vector of claim 1 into a plant cell; and (ii) regenerating a plant from the transformed plant cell into which the vector has been introduced in step (i); A method comprising:

7. A method for producing sphingomyelin, comprising using the transformed plant cell according to claim 2 or the plant according to claim 3 or 4.

8. Plant cells in which an animal-derived or synthetic sphingomyelin synthase gene has been introduced and expressed.

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