JPWO2019163601A1 - Transformed plants and their use - Google Patents

Abstract

An object of the present invention is to provide a transformed plant in which the production amount of 7-dehydrocholesterol, which is a precursor of active VD3, is increased, and a technique for utilizing the same. In plants having two different types of genes encoding the first and second 7-dehydrocholesterol reductases, the expression of the genes encoding the first and second two 7-dehydrocholesterol reductases is suppressed. The above problem is solved by providing.

Description

The present invention relates to a transformed plant and a technique for utilizing the transformed plant, for example, a method for producing 7-dehydrocholesterol using the transformed plant.

Vitamin D3 (hereinafter, also referred to as "VD3") and active VD3 are components that regulate calcium and phosphorus metabolism in vivo, and are effective for prevention / treatment of osteoporosis and motor function decline, deficiency treatment, etc. It has been known. Therefore, it is required to provide VD3 or active VD3, or a precursor thereof, in a large amount and at a low price.

As a technique for mass-producing VD3 or active VD3, or a precursor thereof, production using plants is being studied. For example, in Solanaceae plants, in addition to the biosynthetic system of plant sterols, there is a biosynthetic system of cholesterol that synthesizes 7-dehydrocholesterol (hereinafter, also referred to as “7-DHC”) which is a precursor of VD3. It is known that VD3 or active VD3, or a precursor thereof, is mass-produced by utilizing this (Patent Document 1).

Japanese Unexamined Patent Publication No. 2012-239412

However, the above-mentioned technique is not sufficient as an efficient production technique for active VD3 and the like.

Therefore, one aspect of the present invention is to provide a transformed plant in which the production amount of 7-DHC, which is a precursor of active VD3, is increased, and a technique for utilizing the same.

As a result of diligent studies to solve the above problems, the present inventors have obtained by simultaneously suppressing the expression of these two types of enzyme genes in Solanaceae plants having two different types of 7-dehydrocholesterol reductase. , 7-DHC production amount can be increased, and the present invention has been completed. That is, one aspect of the present invention includes the following inventions:
In plants having two different types of genes encoding the first and second 7-dehydrocholesterol reductases, the expression of the genes encoding the first and second two 7-dehydrocholesterol reductases is suppressed. A plant characterized by.

According to one aspect of the present invention, it is possible to provide a transformed plant in which the production amount of 7-DHC, which is a precursor of VD3, is increased.

FIG. 1 is a diagram showing a presumed plant sterol synthesis pathway and a cholesterol synthesis pathway. FIG. 2 is a diagram showing the route of conversion from 7-DHC to cholesterol or VD3. FIG. 3 is a diagram showing a sequence design of a guide RNA for knocking out LeDWF5H. FIG. 4 is a diagram showing a sequence design of a guide RNA for knocking out LeDWF5. FIG. 5 is a diagram showing a guide RNA expression vector (pmgP237-2A-gfp). FIG. 6 is a diagram showing a guide RNA expression vector (pmgP237). FIG. 7 is a diagram showing a vector for knocking out both LeDWF5H and LeDWF5. FIG. 8 is a diagram showing the results of analysis by LC-MS of tomato hairy roots in which LeDWF5H was knocked out. FIG. 9 is a diagram showing the results of analysis by GC-MS of tomato hairy roots in which both LeDWF5H and LeDWF5 were knocked out. FIG. 10 is a diagram showing the results of analyzing the 7-DHC and cholesterol contents of tomato hairy roots in which both LeDWF5H and LeDWF5 were knocked out. FIG. 11 is a diagram showing the results of LC-MS analysis of tomato hairy roots in which both LeDWF5H and LeDWF5 were knocked out. FIG. 12 is a diagram showing the results of LC-MS analysis of tomato hairy roots in which both LeDWF5H and LeDWF5 were knocked out. FIG. 13 is a diagram showing the results of analyzing the content of 7-DHC by GC-MS in tomato hairy roots in which both LeDWF5H and LeDWF5 were knocked out.

An embodiment of the present invention will be described in detail below. All academic and patent documents described in the present specification are incorporated herein by reference. Unless otherwise specified in the present specification, "A to B" representing a numerical range means "A or more (including A and larger than A) and B or less (including B and smaller than B)".

As used herein, the term "gene" is used interchangeably with "polynucleotide", "nucleic acid" or "nucleic acid molecule" and is intended to be a polymer of nucleotides. Here, the gene can be present in the form of DNA (eg, cDNA or genomic DNA) or RNA (eg, mRNA). The DNA or RNA may be double-stranded or single-stranded. The single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). The gene may also be chemically synthesized and the codon usage may be modified to improve the expression of the encoding protein. It is also possible to replace codons that encode the same amino acid. Also, the term "protein" is used interchangeably with "peptide" or "polypeptide". As used herein, the notation of bases and amino acids uses the one-letter or three-letter notation defined by IUPAC and IUB as appropriate.

<1. Outline of the present invention>
In one embodiment of the invention, in a plant having two different genes encoding the first and second 7-dehydrocholesterol reductases, the genes encoding the first and second two 7-dehydrocholesterol reductases. A plant in which the expression of is suppressed (hereinafter, may be referred to as “plant of the present invention”) is provided. Hereinafter, "two different types of first and second 7-dehydrocholesterol reductase" will be referred to as "DWF5" and "DWF5H" (* H means homologue), respectively, and these genes will be referred to as "DWF5 gene", respectively. And sometimes referred to as the "DWF5H gene". In addition, "7-dehydrocholesterol reductase" as a general term may be referred to as "C7R".

The production of VD3 occurs when the double bonds present at the 5- and 7-positions of 7-DHC become pre-VD3 by a ring-opening reaction with ultraviolet rays or the like, and then isomerize to VD3. Therefore, the presence of two double bonds at positions 5 and 7 is important.

Here, Solanaceae plants including tomato have a cholesterol synthesis pathway in addition to the plant sterol synthesis pathway normally possessed by many plants (Fig. 1). In such plants, the reduction of the double bond at position 7 of 7-DHC proceeds rapidly, and as a result, 7-DHC is converted to cholesterol and the amount of 7-DHC accumulated is small (Fig. 2). ).

Examples of plants having a cholesterol synthesis pathway in addition to the plant sterol synthesis pathway include tomato (Solanum Lycopersicum). In tomato, it has LeDWF5 and LeDWF5H as DWF5 and DWF5H, respectively. Here, in tomatoes, it has been conventionally considered that LeDWF5 is involved in the plant sterol synthesis pathway and LeDWF5H is involved in the cholesterol synthesis pathway (Prashant et al., “Plant cholesterol biosynthetic pathway overlaps with phytosterol metabolism,” Nature Plant. , (2016)). Therefore, the present inventors conducted experiments on the assumption that suppression of LeDWF5H in tomato would prevent the conversion of 7-DHC to cholesterol in the cholesterol synthesis pathway, resulting in the accumulation of a large amount of 7-DHC. It was. However, unexpectedly, there was no increase in the accumulation of 7-DHC.

Therefore, as a result of further diligent studies, the present inventors surprisingly, as shown in Examples described later, produce 7-DHC by simultaneously suppressing two genes, LeDWF5 and LeDWF5H. Was found to increase for the first time.

Therefore, the plant of the present invention exerts a remarkable effect that the amount of 7-DHC, which is a precursor of VD3, is increased.

<2. Plants in which the expression of genes encoding two different types of 7-dehydrocholesterol reductase is suppressed>
In the plant of the present invention, the expression of the DWF5 gene and the DWF5H gene may be suppressed, and other specific configurations are not particularly limited. Hereinafter, aspects of the present invention will be described in detail.

<Gene encoding 21-1.7-dehydrocholesterol reductase>
As used herein, "7-dehydrocholesterol reductase (C7R)" means a protein having enzymatic activity that reduces the double bond at position 7 of the steroid skeleton of cholesterol and / or plant sterols. For example, a protein having an enzymatic activity that reduces the double bond between the 7-position and the 8-position of the steroid skeleton of 7-DHC can be exemplified. By the action of C7R, 7-DHC is converted to cholesterol. Cholesterol is further converted to steroid saponins, steroid glycoalkaloids (hereinafter, also referred to as "SGA") and the like.

In one embodiment of the present invention, C7R is not particularly limited as long as it is a protein having the above-mentioned reducing activity. The DWF5 gene is, for example, any gene selected from the group consisting of the following (a) to (e).

(A) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) In the amino acid sequence set forth in SEQ ID NO: 1, one or several amino acid residues consist of an amino acid sequence substituted, deleted, inserted and / or added, and encode a protein having C7R activity. gene;
(C) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having C7R activity;
(D) A gene consisting of the nucleotide sequence shown in SEQ ID NO: 2;
(E) A gene encoding a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (a) to (d) and has C7R activity.

The DWF5H gene is, for example, any gene selected from the group consisting of the following (f) to (j).

(F) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3;
(G) In the amino acid sequence set forth in SEQ ID NO: 3, a protein comprising an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added and having C7R activity is encoded. gene;
(H) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3 and having C7R activity;
(I) A gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4;
(J) A gene encoding a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (f) to (i) and has C7R activity.

The gene of (a) above will be described. SEQ ID NO: 1 is a type of tomato-derived C7R, which is a protein composed of 435 amino acid residues in total length.

Subsequently, the gene of (f) above will be described. SEQ ID NO: 3 is a type of tomato-derived C7R, which is a protein composed of 435 amino acid residues in total length. The gene (f) is a homologue of the gene (a).

The genes (b) and (g) are functionally equivalent variants, derivatives, variants, alleles, homologs, orthologs, partial peptides, or proteins having the amino acid sequences set forth in SEQ ID NOs: 1 and 3, respectively. As long as it encodes a protein having C7R activity, such as a fusion protein with another protein / peptide, the specific sequence thereof is not limited. Here, the number of amino acids that may be deleted, substituted or added is not limited as long as the above function is not lost, but is deleted or substituted by a known introduction method such as a site-specific mutagenesis method. Or the number that can be added. The number of such amino acids is usually 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, still more preferably 7 amino acids or less, and particularly preferably 5 amino acids or less (eg,). 5, 4, 3, 2 or 1 amino acid). Further, in the present specification, the term "mutation" mainly means a displacement artificially introduced by a site-specific mutagenesis method or the like, but it may be a naturally occurring similar mutation.

The mutated amino acid residue is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, the properties of the amino acid side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G). , H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side Amino acids with chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side chains Amino acids having (H, F, Y, W) can be mentioned (all in parentheses represent one-letter notation of amino acids). It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution with another amino acid of one or more amino acid residues to an amino acid sequence maintains its biological activity. There is. Furthermore, it is more preferable that the target amino acid residue is mutated to an amino acid residue having as many common properties as possible.

As used herein, "functionally equivalent" means that the protein of interest has the same (identical and / or similar) biological function as the protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1 and 3. Or intended to have a biochemical function. In the present specification, the biological and biochemical functions of the protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 1 and 3 include, for example, the 7th and 8th positions of the steroid skeleton of 7-DHC. The function of reducing the double bond between the two can be mentioned. The biological function may also include the specificity of the site of expression, the amount of expression, and the like. Whether or not the mutated protein is functionally equivalent is determined by obtaining a transformant in which a gene encoding the protein is introduced and expressed, and this transformant uses 7-DHC as a substrate and the 7- It can be determined by examining whether the double bond between the 7th and 8th positions of the steroid skeleton of DHC can be reduced.

The genes (c) and (h) are also functionally equivalent variants, derivatives, variants, alleles, homologs, orthologs, partial peptides, or proteins having the amino acid sequences set forth in SEQ ID NOs: 1 and 3, respectively. It is intended as a fusion protein with other proteins / peptides, and has an enzymatic activity that reduces the double bond between the 7th and 8th positions of the steroid skeleton of 7-DHC (that is, an activity that reduces 7-DHC). As long as it encodes a protein having), its specific sequence is not limited.

Amino acid sequence identity is at least 90% or more, more preferably 95% or more (eg, 95%, 96%, 97%, 98%, 99%) of the entire amino acid sequence (or region required for functional expression). It means that they have the same sequence of (above). Amino acid sequence identity can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). There is. When analyzing the base sequence by BLASTN, the parameters are, for example, score = 100 and wordlength = 12. When analyzing the amino acid sequence by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. In addition, when analyzing the amino acid sequence using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known. Additions or deletions (eg, gaps, etc.) may be tolerated in order to optimally align the base sequence or amino acid sequence to be compared.

As used herein, "identity" refers to the proportion of the same number of amino acid residues. The properties of amino acids are as described above.

Regarding the genes (d) and (i) above, SEQ ID NOs: 2 and 4 have the base sequences (Open Reading Frame: ORF) of the genes encoding the polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 3, respectively. Shown.

The gene (e) is intended to be a gene that hybridizes to a polynucleotide having a base sequence complementary to any of the genes (a) to (d) under stringent conditions. Further, the gene (j) is intended to be a gene that hybridizes to a polynucleotide having a base sequence complementary to any of the genes (f) to (i) under stringent conditions. Here, the stringent condition refers to a condition in which a double-stranded polynucleotide specific to a so-called base sequence is formed and a non-specific double-stranded polynucleotide is not formed. In other words, conditions for hybridizing highly homologous nucleic acids, for example, from the melting temperature (Tm value) of a perfectly matched hybrid to 15 ° C, preferably 10 ° C, more preferably 5 ° C lower. It can be said that. For example, to give an example, the temperature is 60 to 68 ° C., preferably 65 ° C., more preferably 65 ° C. in a buffer solution consisting of 0.25M Na ₂ HPO ₄ , pH 7.2, 7% SDS, 1 mM EDTA, and 1 × Denhardt solution. causes the 16 to 24 hours hybridized under the conditions of 68 ° C., further _{20mM Na 2 HPO 4, pH7.2,1%} SDS, temperature in a buffer consisting of 1 mM EDTA is 60 to 68 ° C., preferably 65 ° C., More preferably, the condition of performing the washing for 15 minutes twice under the condition of 68 ° C. can be mentioned. Other examples include 25% formamide, under more severe conditions 50% formamide, 4 × SSC (sodium chloride / sodium citrate), 50 mM Hepes pH 7.0, 10 × Denhardt solution, 20 μg / mL denatured salmon sperm DNA. After prehybridization in the hybridization solution at 42 ° C. overnight, a labeled probe is added and the mixture is kept warm at 42 ° C. overnight for hybridization. The cleaning liquid and temperature conditions in the subsequent cleaning are about "1 x SSC, 0.1% SDS, 37 ° C.", and the stricter conditions are about "0.5 x SSC, 0.1% SDS, 42 ° C.". As a stricter condition, it can be carried out at about "0.2 × SSC, 0.1% SDS, 65 ° C.". As the conditions for washing the hybridization become stricter, the hybridization becomes more specific. However, the combination of SSC, SDS and temperature conditions is exemplary and one of ordinary skill in the art will determine the stringency of hybridization as described above or other factors (eg, probe concentration, probe length, hybridization reaction). It is possible to realize the same stringency as described above by appropriately combining (time, etc.). This is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory (2001) and the like.

In addition, 1 to 50 base sequences are substituted, deleted, inserted and / or added to the genes (e) and (j) above in the DNA consisting of the base sequences shown in SEQ ID NOs: 2 and 4, respectively. It also includes a gene consisting of the same DNA and a gene consisting of DNA having 90% or more homology with the DNA consisting of the nucleotide sequences shown in SEQ ID NOs: 2 and 4.

As a method for obtaining the above gene / protein, a commonly used method for modifying a polynucleotide may be used. That is, a polynucleotide having the genetic information of a desired recombinant protein can be prepared by substituting, deleting, inserting and / or adding a specific base of the polynucleotide having the genetic information of the protein. Specific methods for converting the base of a polynucleotide include, for example, a commercially available kit (KOD-Plus Site-Directed Mutagenesis Kit; Toyobo, Transformer Site-Directed Mutagenesis Kit; Clontech, QuickChange Site Directed Mutagenesis Kit; Stratagene, etc. ), Or the use of the polymerase chain reaction (PCR). These methods are known to those skilled in the art.

Further, the gene may consist only of the polynucleotide encoding the protein, but may have other base sequences added. The base sequence to be added is not particularly limited, but is limited to a label (for example, histidine tag, Myc tag or FLAG tag, etc.), a fusion protein (for example, streptavidin, cytochrome, GST, GFP or MBP, etc.), a promoter sequence, and Examples include a base sequence encoding a signal sequence (for example, an endoplasmic reticulum translocation signal sequence and a secretory sequence). The site to which such a base sequence is added is not particularly limited, and may be, for example, the N-terminal or the C-terminal of the protein to be translated.

In the plant of the present invention, as described above, the expression of the DWF5 gene and the DWF5H gene is suppressed.

The method for suppressing the expression of the above gene is not particularly limited, and any method used in the art is used. For example, ZFN (Zinc Finger Nucleases), TALEN (Transcription Activator Like Effector Nucleases), CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR Associated Proteins 9), PPR (Pentatrico Peptide Repeat) motif, ion beam irradiation, ultraviolet irradiation, Examples thereof include methods such as siRNA, miRNA, shRNA, antisense RNA, and homologous recombination. Each method is described, for example, in the following literature: ZFN (Kim YG et al., Proc. Natl. Acad. Sci. USA 93: 1156-1160, 1996), TALEN (Christian et al., Genetics, 186: 757-761, 2010), CRISPR / Cas9 (Gilbert et al., Cell, 154: 442-451, 2013), PPR motif (International Publication No. 2014/175284), ion beam irradiation (A. Tanaka et) a1., Int. J. Radiat. Bio1. 72: 121-127, 1997), UV irradiation (Yasuo Ukai, Plant Breeding Science, University of Tokyo Publishing Center (2003)), siRNA (Elbashir SM et al., Nature. 2001) May 24; 411 (6836): 494-8), miRNA (Caudy AA et al, Genes & Devel 16: 2491-96, 2002), shRNA (Bernstein E, Caudy AA et al, Nature; 409 (6818): 363 -6, 2001), Antisense RNA (Ching et al., Proc. Natl. Acad. Sci. USA 86: 10006-10010 (1989).

It should be added that the method for producing a plant of the present invention by the above-mentioned gene expression suppression method is also included in the scope of the present invention.

In one embodiment of the present invention, examples of the plant material for which the above gene expression is suppressed include plant tissues such as roots, stems, leaves, seeds, embryos, ovules, ovules, shoot apex, anthers, and pollen. Examples thereof include, but are not limited to, the sections, cells, callus, and plant cells such as protoplasts obtained by subjecting them to enzyme treatment to remove the cell wall.

In one embodiment of the present invention, an appropriate vector capable of causing modification (disruption) to the above genes (a) to (j) is constructed, introduced into a plant cell, and the transformed plant is regenerated from the cell. Therefore, the plant of the present invention can be produced.

The plant cells transformed as described above can be regenerated into individual plants by a method known to those skilled in the art. For example, a method in which callus-like transformed cells are transferred to a medium having different hormone types and concentrations and cultured to form adventitious embryos to obtain a complete plant body can be mentioned.

In addition, when plant tissues such as leaf discs are used as plant materials to be transformed, these are used as inorganic salts, vitamins, carbon sources (sugars as an energy source, etc.), and plants after Agrobacterium infection. It is possible to form foliage by culturing under appropriate light and temperature conditions on a redifferentiated solid medium sterilized by adding growth regulators (plant hormones such as auxin and cytokinin) and selective agents such as canamycin. it can. Next, adventitious roots can be induced by culturing the foliage on a medium (rooting medium) from which the plant growth regulator has been removed from the solid medium, and the plant can be regenerated into a complete plant. Examples of the medium to be used include general media such as LS medium and MS medium.

In the confirmation of whether or not the expression of the gene is suppressed in the transformant, it is determined whether or not any of the genes (a) to (j) above is present or whether or not the expression of the gene is suppressed. It is done by doing.

Since the genes (a) to (j) above encode proteins having an activity of reducing 7-DHC, the presence or absence of these genes in plants or whether or not the expression of the genes is suppressed is determined. By the determination, it can be easily determined whether or not the plant can produce a large amount of 7-DHC.

As a specific determination method, a conventionally known method can be used. For example, (i) a DNA sample is obtained from a target plant, and the presence or absence of a gene or a mutation is introduced into the gene to suppress expression. Examples include a method for examining whether or not the gene is produced, (ii) a method for examining the presence or absence or amount of mRNA which is a transcript of the above gene, and (iii) a method for examining the presence or absence or amount of a protein which is a transcript of the above gene. Can be done.

As a method for examining the above DNA, RNA or protein, a conventionally known method can be used, and the method is not particularly limited, and for example, a method using a probe, a PCR method, an RT-PCR method, various immunoassay methods using an antibody, and a microarray can be used. The method of using it can be mentioned.

<2-2. Plants>
In one embodiment of the present invention, the plant to be targeted for suppressing the expression of genes encoding two different types of C7R is not particularly limited as long as it is a plant having two different types of C7R.

In one embodiment of the invention, the plant can be, for example, a plant that produces large amounts of cholesterol-derived steroid saponins. It is presumed that cholesterol is produced in large quantities in plants that produce large amounts of cholesterol-derived steroid saponins. Therefore, in such a plant, by suppressing the conversion of 7-DHC to cholesterol, it is possible to realize a plant in which the amount of 7-DHC produced is increased. Such plants include, for example, Solanaceae, Liliaceae, Scrophulariaceae, Yam or Labiatae. More specifically, such plants include Solanaceae potatoes (Solanum tuberosum), tomatoes (Solanum lycopersicum), lurianagi (Solanum glaucophyllum), yams (Cestrum nocturnum), tobacco tobacco (Nicotiana glauca), figworts (Lamiaceae). Digitalis purpurea), Dioscorea sp. Of the family Dioscorea, and Ajuga reptans of the Labiatae family. Preferably, the plant can be tomato or potato. Since the metabolic pathways of these plants have been clarified, they are suitable as targets for suppressing the expression of genes encoding two different types of C7R.

The plant of the present invention includes the whole plant body, plant organs (for example, roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (for example, epidermis, sieve, parenchyma, xylem, canal bundle, etc.), plants. Includes both cells, xylem, etc. It also includes protoplasts, shoot primordia, multi-buds and hairy roots.

Further, once a transformed plant in which the genes (a) to (j) in the chromosome are disrupted or whose expression is suppressed can be obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. Is. It is also possible to obtain breeding materials (for example, seeds, fruits, cut ears, tubers, tubers, roots, strains, callus, protoplasts, etc.) from the above plants, their offspring, or clones, and mass-produce the above plants based on them. In addition, the plant of the present invention includes progeny plants such as the redifferentiated current generation "T0 generation" that has undergone transformation treatment and the "T1 generation" that is a self-fertilized seed of the T0 generation plant, and mating with them as one parent. Includes hybrid plants and progeny plants.

<Method for producing 3.7-dehydrocholesterol>
In one embodiment of the present invention, a method for producing 7-DHC (hereinafter, also referred to as “the production method of the present invention”) using the plant of the present invention described in <2> above is provided.

The production method of the present invention is not particularly limited as long as it uses the plant of the present invention described in <2> above.

As described above, the amount of 7-DHC produced is increased in plants in which the expression of two different C7R-encoding genes is suppressed. Therefore, according to the production method of the present invention, 7-DHC, which is a precursor of vitamin D3, can be efficiently produced.

In the production method of the present invention, the plant of the present invention can be cultivated by using any method in the art (for example, Katsuji Osawa, Basic knowledge of pictorial book / plant biotech, Rural Culture Association Japan (2005). ), Katsuji Osawa, Actual Plant Biotech, Rural Culture Association (2003), etc.).

The 7-DHC produced by the production method of the present invention can be separated from the plant and purified as a substantially pure and uniform compound. For the separation and purification of 7-DHC, the separation and purification methods used in the purification of ordinary compounds may be used. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, sulfur precipitation or ethanol precipitation, acid extraction, dialysis and re-precipitation. 7-DHC can be separated and purified by appropriately selecting or combining crystals and the like. Furthermore, a plurality of these methods can be combined.

Chromatography includes affinity chromatography, anion or cation ion exchange chromatography, hydrophobic chromatography, phosphocellulose chromatography, hydroxyapatite chromatography, lectin chromatography, gel filtration, reverse phase chromatography and adsorption chromatography. And so on.

One embodiment of the present invention includes the following configurations.

(1) In plants having two different types of genes encoding the first and second 7-dehydrocholesterol reductases, the expression of the genes encoding the first and second two 7-dehydrocholesterol reductases is suppressed. A plant characterized by being.

(2) The gene encoding the first 7-dehydrocholesterol reductase is one of the genes selected from the group consisting of the following (a) to (e), according to (1). plant:
(A) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) In the amino acid sequence set forth in SEQ ID NO: 1, one or several amino acid residues consist of an amino acid sequence substituted, deleted, inserted and / or added, and have 7-dehydrocholesterol reductase activity. Genes encoding proteins;
(C) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having 7-dehydrocholesterol reductase activity;
(D) A gene consisting of the nucleotide sequence shown in SEQ ID NO: 2;
(E) Encodes a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (a) to (d) and has 7-dehydrocholesterol reductase activity. gene.

(3) The gene encoding the second 7-dehydrocholesterol reductase is any gene selected from the group consisting of the following (f) to (j), according to (1). plant:
(F) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3;
(G) In the amino acid sequence set forth in SEQ ID NO: 3, one or several amino acid residues consist of an amino acid sequence substituted, deleted, inserted and / or added, and have 7-dehydrocholesterol reductase activity. Genes encoding proteins;
(H) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3 and having 7-dehydrocholesterol reductase activity;
(I) A gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4;
(J) Encodes a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (f) to (i) and has 7-dehydrocholesterol reductase activity. gene.

(4) The plant according to any one of (1) to (3), wherein the plant is a plant that produces a large amount of cholesterol-derived steroid saponin.

(5) The plant according to any one of (1) to (4), wherein the plant is a plant of the Solanaceae, Liliaceae, Scrophulariaceae, Yams or Labiatae families.

(6) The plant according to any one of (1) to (5), wherein the plant is a tomato, a potato, a yakoboku, a luryanagi or a tobacco tobacco.

(7) Suppression of the expression of the gene encoding 7-dehydrocholesterol reductase is ZFN, TALEN, CRISPR / Cas9, PPR motif, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA or homologous recombination. The plant according to any one of (1) to (6), which is characterized by being performed by.

(8) A method for producing 7-dehydrocholesterol using any of the plants (1) to (6).

The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims, and the embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. Is also included in the technical scope of the present invention.

Examples of the present invention will be described below. In this example, tomatoes of Solanaceae plants whose metabolic pathways are almost clear were used. Further, in this example, CRISPR / Cas9 is used as a method for suppressing gene expression, but the method is not particularly limited to this method.

<1. Construction of plant transformation vector pMgP237 for CRISPR / Cas9>
As the CRISPR / Cas9 plant transformation backbone vector, pMgP237-2A-gfp (distributed by Takashi Tokushima University) was used (Fig. 5). This vector is a vector in which the guide RNA is driven by the Arabidopsis thaliana U6-26 (AtU6-26) promoter and Cas9 is driven by the 2 × 35S promoter. The feature of this vector is that guide RNA and tRNA are connected in tandem, and a plurality of guide RNAs are expressed directly under one promoter. Since tRNA is processed in the plant body, a plurality of guide RNAs transcribed as one mRNA are cleaved in the plant body, and each guide RNA appears. The vector creation method is shown below.

We designed a long oligo from the selected guide RNA sequence and asked Hokkaido System Science to synthesize it. PCR was performed using the purchased long oligo as a primer and pMD_gtRNA as a template. The composition for PCR was adjusted as follows. The primer sequences used are shown in Table 1.
-Template DNA (20 pg) 1.0 μL
・ 10 μM F primer 0.75 μL
・ 10 μM R primer 0.75 μL
-DNTP Mixure 0.75 μL
・ 5 × PS buffer 5 μL
-PrimeSTRA HS 0.25 μL
・ MilliQ 15.25mL
PCR was performed under the following reaction conditions.
95 ° C., 30 seconds → (98 ° C., 10 seconds / 55 ° C., 20 seconds / 72 ° C., 10 seconds) for 35 cycles → 72 ° C., 5 minutes The PCR reaction product was subjected to 2.0% agarose gel electrophoresis. Extraction and purification from the gel was performed using the WizardR SV Gel and PCR Clean-UP System (Promega). The purified PCR fragment and pMgP237-2A-gfp, which is a backbone vector, were mixed and golden gate cloning was performed with the following reaction composition.
-Vector (100 ng / μL) 1.0 μL
-PCR product (10 ng / μL) 3.0 μL
・ 10 × T4 DNA ligase buffer 2.0 μL
・ BsaI 1.0 μL
T4DNA ligase 1.0 μL
Using MilliQ, the total was 25 μL.

One PCR product was used to express two guide RNAs, and three PCR products were used to express four guide RNAs. The reaction conditions are as shown below.
・ (37 ℃, 5 minutes / 16 ℃, 10 minutes) for 10 cycles → 12 ℃
In order to digest the undigested vector in the reaction solution, 0.5 μL was added to 20 μL of the golden gate cloning solution, and the reaction was carried out under the conditions of 50 ° C., 60 minutes → 80 ° C., 15 minutes. Escherichia coli DH5α was transformed with the obtained digested Golden Gates Loning Solution and seeded on LB agar medium (Km: 50 mg / L). Then, the colonies of Escherichia coli were scraped off, and the liquid culture was carried out in an LB liquid medium (Km: 50 mg / L) to extract a plasmid. The obtained plasmid was sequenced and confirmed to be constructed without sequence errors. This was used as a transformation vector (Fig. 6).

＜２．毛状根の形質転換＞
〔２−１．形質転換ベクターのアグロバクテリウム・リゾゲネスＡｇｒｏｂａｃｔｅｒｉｕｍ． ｒｈｉｚｏｇｅｎｅｓへの導入〕
上記＜１＞で構築した形質転換ベクターを、以下の方法で、Ａ． ｒｈｉｚｏｇｅｎｅｓ １５８３４株へ導入した。まず、構築した形質転換ベクター２μＬとＡ． ｒｈｉｚｏｇｅｎｅｓ １５８３４株のコンピテントセル５０μＬとを十分に混合し、ＭｉｃｒｏＰｕｌｓｅｒエレクトロポレーションキュベット／０．１ｃｍギャップ（ＢＩＯ−ＲＡＤ製）に注入した。続いて、キュベットをＭｉｃｒｏＰｕｌｓｅｒエレクトロポレーター（ＢＩＯ−ＲＡＤ製）にセットした後、パルス送出を行った。得られたＡ． ｒｈｉｚｏｇｅｎｅｓ １５８３４株を含む溶液を、ＹＥＢ液体培地１ｍＬに懸濁し、ロータリーシェイカーを用いて、暗所２８℃、２００ｒｐｍの条件下で４時間振とう培養した。振とう培養後の溶液を、ＹＥＢ寒天培地（カナマイシン：５０ｍｇ／Ｌ）に播種し、暗所２８℃で７２時間培養した。得られたシングルコロニーをＹＥＢ液体培地（カナマイシン：５０ｍｇ／Ｌ）３ｍＬに接種し、ロータリーシェイカーを用いて暗所２８℃、２００ｒｐｍで７２時間振とう培養した。得られた培養液３ｍＬのうち２ｍＬはアルカリＳＤＳによるプラスミド抽出に用いた。得られたプラスミドを鋳型として適当なプライマーでＰＣＲを行い、目的遺伝子の挿入を確認した。ＰＣＲはＥｘ Ｔａｑ ＨＳ（ＴａＫａＲａ製）を用いて行った。ＰＣＲ反応液の組成は、Ｅｘ Ｔａｑ ＨＳ ０．１μＬ、プラスミド０．５μＬ、ｄＮＴＰ ｍｉｘｔｕｒｅ ０．８μＬ、１０×Ｅｘ Ｔａｑ Ｂｕｆｆｅｒ １μＬ、各プライマー（１００μＭ）０．５μＬ、ＭｉｌｌｉＱ水 ６．６μＬを加え、全量１０μＬとした。反応条件は、９５℃で５分間の初期変性後に、９４℃で３０秒間の変性、５６℃で３０秒間のアニーリング、７２℃で１分間の伸長反応を３５サイクル繰り返し、その後、７２℃で５分間の最終伸長を行った。ＰＣＲによる増幅が見られ、目的遺伝子の挿入が確認できたものは、培養液の残りの１ｍＬでグリセロールストックを作製し、−８０℃で保存した。

<2. Hairy root transformation>
[2-1. Transformation vector Agrobacterium rhizogenes Agrovactium. Introduction to rhizogenes]
The transformation vector constructed in <1> above was prepared by the following method. It was introduced into rhizogenes 15834 strain. First, 2 μL of the constructed transformation vector and A. 50 μL of competent cells of the rhizogenes 15834 strain were thoroughly mixed and injected into a MicroPulser electroporation cuvette / 0.1 cm gap (manufactured by BIO-RAD). Subsequently, the cuvette was set in a MicroPulser electropolator (manufactured by BIO-RAD), and then pulse transmission was performed. The obtained A. A solution containing rhizogenes 15834 strain was suspended in 1 mL of YEB liquid medium and cultured with a rotary shaker in a dark place at 28 ° C. and 200 rpm for 4 hours with shaking. The solution after shaking culture was seeded on YEB agar medium (kanamycin: 50 mg / L) and cultured at 28 ° C. in the dark for 72 hours. The obtained single colony was inoculated into 3 mL of YEB liquid medium (kanamycin: 50 mg / L) and cultured in a dark place at 28 ° C. at 200 rpm for 72 hours using a rotary shaker. Of the 3 mL of the obtained culture solution, 2 mL was used for plasmid extraction with alkaline SDS. Using the obtained plasmid as a template, PCR was performed with an appropriate primer to confirm the insertion of the target gene. PCR was performed using Ex Taq HS (manufactured by TakaRa). The composition of the PCR reaction solution is as follows: Ex Taq HS 0.1 μL, plasmid 0.5 μL, dNTP mixture 0.8 μL, 10 × Ex Taq Buffer 1 μL, each primer (100 μM) 0.5 μL, MilliQ water 6.6 μL, and the total amount. It was set to 10 μL. The reaction conditions were as follows: initial denaturation at 95 ° C. for 5 minutes, denaturation at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, extension reaction at 72 ° C. for 1 minute, repeated for 35 cycles, and then at 72 ° C. for 5 minutes. Was finally stretched. For those in which amplification by PCR was observed and insertion of the target gene was confirmed, a glycerol stock was prepared from the remaining 1 mL of the culture medium and stored at -80 ° C.

[2-2. Preparation of tomatoes]
Twenty-five seeds of Micro-Tom (Scott and Harbaugh, 1989) were placed in an Eppendorf tube, 1 mL of 70% ethanol was added thereto, and the mixture was inverted and mixed for 1 minute, and 70% ethanol was removed by Pipetman. Next, 200 μL of household kitchen highter and 800 μL of MilliQ were added and mixed by inversion for 15 minutes, and the mixed solution was removed using a pipetman in a clean bench. The seeds were washed by adding 1 mL of sterile MilliQ water and discarding the sterile MilliQ water three times. Sterile seeds were placed in plastic pots containing 50 mL of MS (1.5% Sucrose) agar medium and grown in the dark at 25 ° C. for about 10 days. When the stem grew about 10 cm, it was left at 25 ° C. for 16 hours under bright conditions (40 to 50 μmol of photons / m ² / s) for about 2 to 3 days to color the leaves.

[2-3. Hairy root transformation]
A. in which each gene was introduced in the above [2-1]. A glycerol stock of rhizogenes was spread on YEB agar medium (kanamycin: 50 mg / L) and cultured in the dark at 28 ° C. for 72 hours. The tomato stem obtained in the above [2-2] was cut into 1 to 1.5 cm, and the tip of the stem on the root side was A. It was attached to rhizogenes colonies, and a section of this stem was erected in a plastic pot containing 50 mL of B5 agar medium with the root side facing up. Leave this in a dark place at 25 ° C for 20 days, cut only the upper part of the stem where hairy roots can be confirmed, transfer it to MS agar medium (2% Sucrose, CEF: 250 mg / L), and leave it in a dark place at 25 ° C for 7 days. There was. About 1 cm of the root tip of the elongated hairy root was subcultured on B5 agar medium (2% sucrose, CEF: 250 mg / L) so as to be distinguishable for each individual, and left in a dark place at 25 ° C. for 7 days. The work of substituting the tip of the root with B5 agar medium (2% Sucrose, CEF: 250 mg / L) was repeated until hairy roots were formed.

<3. Genome extraction of hairy roots and confirmation of transformation by PCR>
It was confirmed by genomic PCR whether or not the target gene was inserted into the hairy root. One hairy root planted on B5 agar medium (2% Sucrose, CEF: 250 mg / L) was placed for each gene line, and the tip 1 cm was placed in an Eppendorf tube, and BufferA (100 mM Tris-HCl (pH 9.). 5) 150 μL of 1 M KCl, 10 mM EDTA) was added and ground with a toothpick. Centrifuge at 10,000 rpm for 3 minutes and transfer the supernatant to a new tube. 150 μL of isopropanol was added thereto, and the mixture was sufficiently pipetted, and then centrifuged at 15,000 rpm for 10 minutes to remove the supernatant. 300 μL of 70% ethanol was added, and the mixture was centrifuged at 15,000 rpm for 1 minute at 4 ° C., and the supernatant was removed by decantation. Centrifugation was performed again at 15,000 rpm for 1 minute at 4 ° C., and the supernatant was removed by Pipetman. It was dried under light irradiation, dissolved in 20 μL of TE buffer, and sufficiently pipetted.

PCR of the extracted genome was performed using Ex Taq HS (manufactured by TakaRa). The primers used are shown in Table 1. The composition of the PCR reaction solution was 10 μL by adding 0.05 μL of Ex Taq, 1 μL of template, 0.8 μL of dNTP mixture, 1 μL of 10 × Ex Taq Buffer, 0.6 μL of each primer (5 μM), and 5.95 μL of MilliQ water. .. The reaction conditions were as follows: initial denaturation at 95 ° C. for 5 minutes, denaturation at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, extension reaction at 72 ° C. for 1 minute, repeated for 35 cycles, and then at 72 ° C. for 5 minutes. Was finally stretched. Hairy roots in which the insertion of the target gene was confirmed by agarose gel electrophoresis were used as transformed hairy roots for later analysis.

<4. Mutation analysis using electrophoresis of transformed hairy roots>
Mutation analysis of the target region was performed using the extracted genomic DNA. The Heteroduplex mobility assay (HMA) method was used for mutation analysis. The specific method is shown below. The extracted genomic DNA was quantified using a Qubit 2.0 Fluorometer (manufactured by Invitrogen) and diluted with TE buffer to 1 ng / μL. PCR was performed on the diluted genomic DNA solution using primers (Table 1) designed to have a fragment size of about 500 bp so as to sandwich the target sites in the templates. PrimeSTAR HS DNA Polymerase (manufactured by TakaRa) was used for PCR. The reaction composition and reaction conditions are shown below.

PrimeSTAR HS DNA Polymerase 0.1 μL, 5 × PrimeSTAR Buffer 2 μL, dNTP Mixture (2.5 mM each) 0.8 μL, Template genomic DNA (1 ng / μL) 1 μL, Primer (10 μM) 0.25 μL, Primer (10 μM) 0.25 μL, A total of 10 μL of reaction solution was prepared. The reaction conditions were as follows: initial denaturation at 98 ° C. for 3 minutes, denaturation at 98 ° C. for 10 seconds, annealing at 58 ° C. for 5 seconds, extension reaction at 72 ° C. for 30 seconds, repeated 35 cycles, and then at 72 ° C. for 5 minutes. Was finally stretched. The obtained PCR product was subjected to migration at a constant voltage of 100 V using 5% acrylamide and a migration buffer of 1xTBE buffer (Tris 89 mM, Bolic acid 89 mM, EDTA 2 mM). The composition of the gel is as follows. 40% acrylamide 1.25 mL, 5xTBE buffer 2 mL, MilliQ 6.75 mL, 10% ammonium persulfate 100 μL, and TEMED 10 μL were mixed. The ratio of the mutation introduction lines was calculated by using the line in which the extra band was generated as a result of electrophoresis as the mutation introduction line.

<5. Liquid culture and transformation of hairy roots>
Hairy roots 10 to 15 sections grown on B5 agar medium (cefotaxim: 250 μg / mL) for 7 days were placed in 50 mL of B5 liquid medium (2% Sucrose, CEF: 250 mg / L) in a 200 mL flask and placed in a dark place. The cells were shake-cultured at 25 ° C. and 100 rpm for about 14 days. The recovered roots were frozen in liquid nitrogen and stored at −80 ° C. [2-3. Hairy root transformation] was performed in the same manner as for hairy root transformation.

<6. Genome extraction of hairy roots and confirmation of transformation by PCR>
It was confirmed by genomic PCR whether or not the target gene was inserted into the hairy root. For extraction of genomic DNA and confirmation of insertion by PCR, <3. The procedure was the same as the method of Genome extraction of hairy roots and confirmation of transformation by PCR>.

<7. Mutation analysis using electrophoresis of transformed hairy roots>
Similar to <4> above, mutation analysis of the target region was performed using a primer designed to sandwich the target region on the DWF5H. The primers used are shown in Table 2 below. The following two primers were used as one set.
・ SLDWF5H_ex1 Fw and SLDWF5H_ex1 Rv
・ SLDWF5H_ex5 Fw and SLDWF5H_ex5 Rv
・ SLDWF5H_ex6 Fw and SLDWF5H_ex6 Rv
・ SLDWF5H_ex9 Fw and SLDWF5H_ex9 Rv

＜８．形質転換毛状根の内生ＳＧＡおよび蓄積産物のＬＣ−ＭＳ解析＞
ＬＣ−ＭＳを用いて形質転換毛状根の内生ＳＧＡの分析を行った。具体的には、液体培養を行った毛状根を液体窒素で凍結し破砕した。粉末約３０ｍｇを１．５ｍＬチューブに量り取り、３００μＬの１００％ ＭｅＯＨを加えた。ミルミキサー（Retch社製）を用いて、２７Ｈｚ、１０分間試料を破砕した。その後、４℃、１５０００ｒｐｍ、１０分間遠心分離した。上清を新しい１．５ｍＬチューブに移し、残渣に再び抽出溶液を３００μＬ加え、同様の操作を繰り返した。抽出溶液を４℃、１５０００ｒｐｍ、５分間遠心分離し、上清を１００％ ＭｅＯＨで適宜希釈し、ＬＣ−ＭＳ分析用の試料とした。各試料は０．２μｍ ＰＴＦＥフィルター（Waters社製）を用いてフィルター濾過し、ＬＣ−ＭＳおよび分析に供した。ＬＣ−ＭＳ分析の条件を以下に示す。

<8. LC-MS analysis of endogenous SGA and accumulated products of transformed hairy roots>
Endogenous SGA of transformed hairy roots was analyzed using LC-MS. Specifically, the hairy roots that had undergone liquid culture were frozen in liquid nitrogen and crushed. About 30 mg of powder was weighed into a 1.5 mL tube and 300 μL of 100% MeOH was added. The sample was crushed at 27 Hz for 10 minutes using a mill mixer (manufactured by Retch). Then, it was centrifuged at 4 ° C., 15000 rpm, and 10 minutes. The supernatant was transferred to a new 1.5 mL tube, 300 μL of the extract solution was added to the residue again, and the same operation was repeated. The extract solution was centrifuged at 4 ° C., 15000 rpm for 5 minutes, and the supernatant was appropriately diluted with 100% MeOH to prepare a sample for LC-MS analysis. Each sample was filtered using a 0.2 μm PTFE filter (manufactured by Waters) and subjected to LC-MS and analysis. The conditions for LC-MS analysis are shown below.

(LC-MS analysis conditions)
-UPLC-ESI-MS ACQUITYTM (manufactured by Waters), column: ACQUITY UPLC HSS T3 Solvent 1.8 μm (2.1 x 100 mm) (manufactured by Waters), column oven temperature: 40 ° C., flow velocity: 0.2 mL / min , Solvent condition: Dient elution with two solvents, Solvent A: H ₂ O (0.1% formic acid), Solvent B: 100% MeCN, Gradient condition: 0-25 minutes: 90% A / 10% B-52% A / 48% B, 25-30 minutes: 0% A / 100% B
(Mass spectrometer)
-Positive ESI mode, capillary voltage: 3 kV, cone voltage: 60 V, source temperature: 120 ° C, desolation gas temperature: 350 ° C, nebulizer / N2 Law2 , Measurement mode: Positive ion mode, MS scan mode (m / z 350-1400)
The analysis result is shown in FIG. In FIG. 1, DWF5ko1, DWF5Hko2, DWF5Hko3, and DWF5Hko4 are transformed vectors pMgP237_DWF5Hko1, pMgP237_DWF5Hko2, pMgP237_DWF5Hko3, and pMgP2, respectively.

As shown in FIG. 1, it was found that the amount of SGA produced was not significantly reduced only by knockout of LeDWF5H. From this, it was considered that the function of LeDWF5 and the function of LeDWF5H overlapped, and it is possible that LeDWF5 complemented the production of SGA. Therefore, the present inventors created a plant in which both LeDWF5 and LeDWF5H were knocked out, as shown below.

<9. In vitro enzyme function analysis of DWF5H and DWF5>
[9-1. Total RNA extraction and cDNA synthesis]
The tomato leaves were placed in a mortar pre-cooled with liquid nitrogen and mashed until powdered. Total RNA was extracted from the powdered sample using RNeasy Plant Mini Kit (manufactured by QIAGEN). The operation was carried out at room temperature according to the following procedure.

To 100 mg of the powdered sample, 500 μL of RLT buffer and 5 μL of mercaptoethanol were added. It was mixed well with vortex, transferred to RNeasy Mini Spin Volume (purple volume), and centrifuged at 15,000 rpm for 3 minutes. The supernatant of the passing solution was transferred to a 1.5 mL Eppendorf tube and centrifuged at 15,000 rpm for 10 minutes. The supernatant was transferred to a new 1.5 mL Eppendorf tube, 225 μL of 100% ethanol was added and mixed. The mixed solution was placed in RNeasy Mini Spin Color (pink color) and centrifuged at 15,000 rpm for 30 seconds. The passing liquid was discarded, 225 μL of 100% ethanol was added to the pink volume, the direction of the volume was reversed by 180 ° C., and the mixture was centrifuged at 15,000 rpm for 30 seconds. I did this again. The passing solution was discarded, and then 350 μL of RW1 buffer was placed in a pink volume and centrifuged at 15,000 rpm for 15 seconds to remove the eluate. 70 μL of RDD buffer was added to 10 μL of DNase I stock solution, and the mixture was gently mixed. This was placed in a pink volume and allowed to stand for 15 minutes. 350 μL of RW1 buffer was placed in a pink volume and centrifuged at 15,000 rpm for 15 seconds to remove the eluate. 500 μL of RPE buffer was placed in a pink volume and centrifuged at 15,000 rpm for 15 seconds to remove the eluate. Further, 500 μL of RPE buffer was placed in a pink volume, the direction of the volume was reversed by 180 ° C., and the mixture was centrifuged at 15,000 rpm for 2 minutes to remove the eluate. Centrifuge at 15,000 rpm for 1 minute. The pink volume was transferred to a 1.5 mL Eppendorf tube, 15 μL of RNase free water was added, and the mixture was allowed to stand at 4 ° C. for 1 minute and centrifuged at 15,000 rpm for 1 minute. This operation was performed once again to obtain total RNA. From the obtained total RNA, cDNA was synthesized using ReverseTra Ace qPCR RT Master Mix with gDNA Remover (manufactured by TOYOBO).

[9-2. Primer design]
Primers for acquiring ORFs were designed based on MiBASE (Table 1). Restriction enzyme recognition sites for inserting the obtained clone into the TA cloning vector and the yeast expression vector were added to the N-terminal and C-terminal of the primer.

[9-3. Amplification of DNA fragments by PCR]
For SlDWF5H and SlDWF5, a PCR reaction was carried out using the cDNA obtained in [9-1] above as a template. PCR was performed based on the protocol of TakaRa Ex Taq Hot Start Version (manufactured by TakaRa). The composition is Ex Taq HS 0.1 μL, 10 × Ex Taq Buffer 2 μL, dNTP mixer (2.5 mM each) 1.6 μL, cDNA 2 μL, 10 μM Forward Primer 1 μL, 10 μM Reverse Primer 1 μL, and MilliQ in total. Was 20 μL.

The reaction conditions were as follows: initial denaturation at 95 ° C. for 2 minutes, denaturation at 95 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, extension reaction at 72 ° C. for 1 minute and 30 seconds repeated for 30 cycles, and then 5 at 72 ° C. Stretched for minutes. All PCR products were subjected to electrophoresis on a 1% (w / v) agarose gel prepared with 1x TAE buffer stained with GelRed Nucleic Acid Gel Stein, 10,000x in DMSO.

After electrophoresis, a single band near the target size was excised from the agarose gel with a scalpel, and cDNA was extracted using Wizard SV Gel and PCR Clean-UP System (manufactured by Promega). The operation was performed according to the attached instruction manual.

[9-4. TA cloning]
4 μL of the DNA fragment purified in [9-3] above, 1 μL of T-vector pMD19 (manufactured by TakaRa), and 5 μL of DNA ligation Kit Mighty Mix (manufactured by TakaRa) were mixed, and a ligation reaction was carried out at 16 ° C. for 30 minutes. In addition, the ligation solution was added to Escherichia. It was transformed into E. coli (DH5α strain).

[9-5. Confirmation of insert DNA by restriction enzyme treatment]
Restriction enzyme treatment was performed to confirm that the desired DNA fragment was inserted into the vector. 2 μL of plasmid solution, 2 μL of 10 × H buffer, terminal restriction enzyme sites and internal restriction enzyme sites were selected, and 10 μL of the reaction solution containing 0.5 μL of each was incubated at 37 ° C. for 1 hour. The treated sample was subjected to agarose gel electrophoresis to confirm digestion and the size of the insert DNA fragment. The reagent used was manufactured by Toyobo Co., Ltd. unless otherwise specified.

[9-6. Decoding the base sequence of insert DNA]
In order to obtain the nucleotide sequence of the inserted DNA fragment, a sequencing reaction was performed using the obtained plasmid of about 100 ng with a BigDye Terminator v3.1 / 1 Cycle Sequence Sequencing Kit (manufactured by Applied Biosystems).

[9-7. Construction of expression vector]
SlDWF5H and SlDWF5 gene full-length sequences subcloned into plasmid pMD19 were treated with each restriction enzyme. After confirming digestion by electrophoresis, the target fragment was cut out and purified. The DNA fragment of each clone was ligated to a yeast expression vector pYES2 (manufactured by Thermo Fisher) that had undergone the same restriction enzyme treatment as above, and transformation was performed using the ligation product. After heat shocking the competent cells, 300 μL of SOC medium at room temperature was added, 100 μL of the solution was sprinkled on LB solid medium (ampicillin: 100 μg / mL), and the mixture was incubated at 37 ° C. for about 18 hours. The next day, the formed colonies were grown in 2 mL of LB liquid medium containing 100 μg / mL ampicillin, plasmid extraction and restriction enzyme treatment were performed to prepare an expression vector containing an accurate sequence.

[9-8. Recombinant enzyme expression using yeast (INVSc1)]
The yeast expression vector constructed in [9-7] above was introduced into yeast (INVsc1). EZ-Yeast Transformation Kit (manufactured by MP) was used for the introduction, and the attached protocol was followed for the operation. Using the obtained transformed yeast, the recombinant protein was expressed by the following procedure.

(1.) Single colonies of transformed yeast were scraped off and cultured overnight at 30 ° C. in 15 mL of SC-U medium (2% raffinose).

(2.) The OD600 value of the culture solution was measured, and a bacterial suspension was added so that the OD600 value was 0.4 in 50 mL of the expression-inducing medium (SC-U, 2% galactose, 1% raffinose). ..

(3.) The bacterial solution obtained in (2.) was cultured at 30 ° C. for 24 hours to induce the expression of recombinant protein.

(4.) The obtained culture solution was centrifuged at 1,500 × g, 5 min, 4 ° C, and the cells were collected.

[9-9. Preparation of enzyme solution]
The cells obtained in [9-8] above were washed 3 times with Buffer 1 (20 mM Tris-HCL-pH 7.5, 50 mM NaCl). 500 μL of Buffer 1 was added to the washed cells, 250 μL of acid-washed glass beads (manufactured by SIGMA) was added thereto, and vortexing was performed at 4 ° C. for 60 sec. This vortex was performed 5 times to crush the recombinant yeast. The obtained yeast crushed solution was transferred to another 1.5 mL microtube without sucking up the beads, and this homogenate was used as an enzyme solution.

[9-10. Recombinant enzyme assay]
Using the obtained recombinant enzyme solution, an enzyme assay was performed with the reaction compositions shown in Table 3 below. The prepared reaction solution was reacted at 30 ° C. for 2 hours.

〔９−１１．酵素反応物のＧＣ−ＭＳ解析〕
反応後の酵素溶液から、以下のようにステロールの抽出を行った。具体的には、反応後の酵素溶液（２００μＬ）に等量の酢酸エチルとヘキサンを加え、ボルテックスミキサーで懸濁した。その後、室温、１５０００ｒｐｍで１分間遠心分離を行い、上層の有機層を回収した。下層には再び等量の酢酸エチルとヘキサンを加えて、同様の操作を行った。この混合溶媒を用いた抽出を合計３回行った。回収した有機層は、ガラス製褐色ミクロチューブに集めて減圧乾固操作を施した。乾固物サンプルに４２μＬのＮ−メチル−Ｎ−（トリメチルシリル）トリフルオロアセトアミド（ＭＳＴＦＡ、Thermo scientific1社製）を加え、ヒートブロックで、８０℃、３０分の熱処理を施して試料のトリメチルシリル化（ＴＭＳ化）を行った。ＴＭＳ化を行ったサンプルをＧＣ−ＭＳ−ＱＰ２０１０ Ｕｌｔｒａ（SHIMADZU社製）に供し、分析を行った。分析条件を以下に示す。

[9-11. GC-MS analysis of enzyme reactants]
Sterols were extracted from the enzyme solution after the reaction as follows. Specifically, equal amounts of ethyl acetate and hexane were added to the enzyme solution (200 μL) after the reaction, and the mixture was suspended by a vortex mixer. Then, centrifugation was performed at room temperature at 15,000 rpm for 1 minute, and the upper organic layer was recovered. Equal amounts of ethyl acetate and hexane were added to the lower layer again, and the same operation was performed. Extraction using this mixed solvent was performed a total of 3 times. The recovered organic layer was collected in a glass brown microtube and subjected to a vacuum drying operation. 42 μL of N-methyl-N- (trimethylsilyl) trifluoroacetamide (MSTFA, manufactured by Thermo scientific 1) was added to the dry sample, and the sample was heat-treated at 80 ° C. for 30 minutes in a heat block to trimethylsilylate the sample (TMS). ) Was performed. The TMS-ized sample was subjected to GC-MS-QP2010 Ultra (manufactured by SHIMADZU) for analysis. The analysis conditions are shown below.

(GC-MS analysis conditions)
-Column: DB-1 (J & W scientific, length 30 m, film thickness 0.25 μm, inner diameter 0.25 mm)
(GC condition)
-Column oven temperature: 180 ° C. (0 to 1 minute), 180 to 280 ° C. (1 to 6 minutes), 280 to 300 ° C. (6 to 16 minutes), 300 ° C. (16 to 32 minutes), vaporization chamber temperature: 250 ° C., injection mode: splitless, sampling time: 1 minute, carrier gas: He, pressure (103.8 kPa), total flow rate (107.0 mL / min), column flow rate (1.00 mL / min), linear velocity (38) .4 cm / sec), purge flow rate (6.0 mL / min), split ratio (100)
(MS condition)
-Ion source temperature: 250 ° C., interface temperature: 300 ° C., solvent elution time: 1.5 minutes, scan range: m / z 50 to 700, 15 to 30 minutes The results of GC-MS analysis are shown in FIG. In addition, in FIG. 9, 7-DHC std. Refers to a sample for qualitative 7-DHC. Empty shows the results of using hairy roots transformed with an empty transformation vector as a sample. DWF5dko3_ # 1 and # 9 show the results of using hairy roots transformed with the transformation vector pMgP237 dko3 as a sample.

No 7-DHC was detected in empty_ # 1 acid hydrolysis and empty_ # 1 alkaline hydrolysis in which an empty transformation vector was introduced as a control, regardless of acid or alkali treatment. In contrast, 7-DHC was detected in all of DWF5dkо3_ # 1 acid hydrolysis, DWF5dkо3_ # 1 alkaline hydrolysis, DWF5dkо3_ # 9 acid hydrolysis, and DWF5dkо3_ # 9 alkaline hydrolysis in which both LeDWF5H and LeDWF5 were knocked out. (That is, 7-DHC was detected regardless of acid treatment or alkali treatment). From this, it was found that the accumulation of 7-DHC requires knockout of both LeDWF5H and LeDWF5.

The result of analyzing the content of cholesterol and 7-DHC from the result of GC-MS analysis is shown in FIG. As shown in FIG. 10, it was found that knocking out both LeDWF5H and LeDWF5 reduced the cholesterol (CHR) content and increased the 7-DHC content. From this, it was clarified that by knocking out both LeDWF5H and LeDWF5, the reduction reaction by C7R to CHR shown in FIG. 2 was inhibited, and as a result, 7-DHC was increased.

[9-12. LC-MS analysis of SGA in hairy roots]
(A) In addition, the content of SGA in the hairy roots of DWF5dko2 and DWF5dko3 was analyzed by LC-MS. To 100 mg of hairy root sample, 300 μL of methanol was added in 3 portions and sonicated for 10 minutes. Then, centrifugation was performed at 15,000 rpm for 10 minutes. Methanol was recovered and subjected to LC-MS analysis.

The analysis result of DWF5dko3 is shown in FIG. In FIG. 11, the peak corresponding to 7-dehydrotomatine (hereinafter, also referred to as 7-DHT) is marked with a circle.

The analysis result of DWF5dko2 was the same as that of DWF5dko3 (data is not shown).

In addition, the results of FIG. 11 are further analyzed, and the results of calculating the contents of α-tomatine and 7-DHT in each hairy root are shown in FIG. As shown in FIG. 12, it was found that knocking out DWF5 and DWF5H reduces the content of α-tomatine, which is a kind of SGA. On the other hand, it was suggested that part of 7-DHC may have been converted to 7-DHT.

(B) Further, the content of 7-DHC contained in the hairy root of DWF5 dko3_ # 9 is further reduced to unhydrolyzed free 7-DHC and alkaline hydrolyzed ester type 7-DHC. Separately, it was analyzed by GC-MS. The GC-MS analysis is performed in the above [9-11. GC-MS analysis of the enzyme reaction product] was performed in the same manner. The results are shown in FIG. In the wild strain, 7-DHC is present in an amount below the detection limit, but as shown in FIG. 13, by knocking out DWF5 and DWF5H, free 7-DHC is accumulated in an amount of 10 μg / gFW. It turned out.

The present invention can be used for food additives, feed additives, drug substances and intermediates, health foods and the like.

Claims (8)

In plants having two different types of genes encoding the first and second 7-dehydrocholesterol reductases, the expression of the genes encoding the first and second two 7-dehydrocholesterol reductases is suppressed. A plant characterized by. The plant according to claim 1, wherein the gene encoding the first 7-dehydrocholesterol reductase is any gene selected from the group consisting of the following (a) to (e). :
(A) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1;
(B) In the amino acid sequence set forth in SEQ ID NO: 1, one or several amino acid residues consist of an amino acid sequence substituted, deleted, inserted and / or added, and have 7-dehydrocholesterol reductase activity. Genes encoding proteins;
(C) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 1 and having 7-dehydrocholesterol reductase activity;
(D) A gene consisting of the nucleotide sequence shown in SEQ ID NO: 2;
(E) Encodes a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (a) to (d) and has 7-dehydrocholesterol reductase activity. gene. The plant according to claim 1, wherein the gene encoding the second 7-dehydrocholesterol reductase is any gene selected from the group consisting of the following (f) to (j). :
(F) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 3;
(G) In the amino acid sequence set forth in SEQ ID NO: 3, one or several amino acid residues consist of an amino acid sequence substituted, deleted, inserted and / or added, and have 7-dehydrocholesterol reductase activity. Genes encoding proteins;
(H) A gene encoding a protein having an amino acid sequence having 90% or more identity with the amino acid sequence set forth in SEQ ID NO: 3 and having 7-dehydrocholesterol reductase activity;
(I) A gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4;
(J) Encodes a protein that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes (f) to (i) and has 7-dehydrocholesterol reductase activity. gene. The plant according to any one of claims 1 to 3, wherein the plant is a plant that produces a large amount of cholesterol-derived steroid saponin. The plant according to any one of claims 1 to 4, wherein the plant is a plant of the Solanaceae, Liliaceae, Figwortaceae, Yamidae or Labiatae family. The plant according to any one of claims 1 to 5, wherein the plant is a tomato, a potato, a yakoboku, a luryanagi or a tobacco tobacco. Suppression of the expression of the gene encoding the 7-dehydrocholesterol reductase is performed by ZFN, TALEN, CRISPR / Cas9, PPR motif, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA or homologous recombination. The plant according to any one of claims 1 to 6, characterized in that. A method for producing 7-dehydrocholesterol using the plant according to any one of claims 1 to 6.

Images