Method for selecting promoter that functions in organelle

Abstract

To provide a method capable of selecting a promoter that functions in an organelle and has desired activity intensity, on a large scale using a next-generation sequencer, and to provide a promoter that functions in an organelle, which is obtained by the selection.SOLUTION: The present invention provides a method for selecting a promoter that functions in an organelle, the method comprising: (1) the step of preparing sequence information obtained by RNA sequencing analysis; (2) the step of mapping the sequence information prepared in the step (1) onto a sequence of organellar DNA; (3) the step of calculating an amount of change in RNA expression in each region on the basis of the mapping information obtained in the step (2); (4) the step of selecting regions in which the amount of change obtained in the step (3) is within a range of preset reference values; and (5) the step of identifying a proper region, among the regions selected, as a promoter functioning in the organelle.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for selecting a promoter that functions in an organelle, and to a promoter DNA containing a novel sequence selected by the method. [Background technology]

[0002] By introducing foreign genes into plant cells, it is possible to give plants useful traits that cannot be obtained through cross breeding, which is extremely significant for the future improvement of agricultural crops and the development of agriculture, as it contributes to increasing food production and promoting environmental improvement amid the recent rapid changes in agricultural conditions due to rapid population growth and climate change. Furthermore, from the perspective of restrictions on the supply of fossil fuels, it is essential to improve plant genetic engineering technology in order to realize the production of substances using plants from carbon dioxide as a raw material.

[0003] As one of the gene recombination techniques for plant cells, gene recombination techniques targeting organelle DNA such as plastids (e.g., chloroplasts) and mitochondria, rather than the nucleus, are known (e.g., Non-Patent Document 1). The organelles are usually present in tens to thousands in a cell (e.g., in the case of chloroplasts, about 100 are present per cell), and each organelle usually has tens to hundreds of genomes, so high expression of genes introduced into the organelles is expected. In addition, even if a protein or ribonucleic acid is harmful in the cytoplasm, it can be accumulated in the organelle without specific cleavage or modification in each organism, and there is no silencing, so gene expression is stable even in progeny. Furthermore, since the organelles are often limited to maternal or paternal inheritance, the risk of spreading foreign genes is extremely low. In order to stably and efficiently express proteins in such organelles, it is important to select a promoter that functions in the organelle.

[0004] Meanwhile, the so-called RNA sequencing method (RNA-Seq method), which performs gene expression analysis using a next-generation sequencer, is rapidly becoming popular (e.g., Non-Patent Document 2). By using the RNA-Seq method, gene expression analysis can be performed with efficiency comparable to that of microarrays in terms of cost and labor. In addition, the expression analysis of IncRNA (long noncoding RNA) is also performed using the RNA-Seq method (e.g., Non-Patent Document 3). However, as far as the present inventors know, there is no known method that can select a promoter that functions in an organelle, and moreover, a promoter that has a desired activity strength, using the RNA-Seq method. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Day, A. and Goldschmidt-Clermont, M., Plant Biotechnol. J., 9:540 (2011). [Non-Patent Document 2] Mortazavi A., et al., Nat Methods. 5(7):621-628 (2008) [Non-Patent Document 3] Di C., et al., Plant J., 80(5):848-861 (2014) Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, an object of the present invention is to provide a method for large-scale selection of promoters that function in organelles and have desired activity strength using a next-generation sequencer, and to provide promoters that function in organelles obtained by said selection. [Means for solving the problem]

[0007] The present inventors have been searching for a promoter capable of expressing a large amount of a target protein, which is toxic when highly expressed, in an organelle as stably as possible. Until now, reverse transcription PCR has been mainly used to detect gene expression in organelles, so that the strength of promoters cannot be compared, and the screening method for promoters has been limited to qualitative classification such as high expression promoters or low expression constitutive expression promoters. However, with a high expression promoter, the target protein exerts toxicity, so there is a problem that the gene construct expressing the target protein cannot be stably maintained. On the other hand, with a low expression constitutive expression promoter, the construct can be stably maintained, but the target protein cannot be expected to be expressed in large amounts. Therefore, the inventors came up with the idea that if a promoter that can achieve an expression level intermediate between the above two types of promoters can be selected from the promoters present in the organelle DNA, a promoter that can be stably maintained in the organelle and can fully express the target protein can be obtained.

[0008] In selecting such promoters, the present inventors thought that it would be possible to comprehensively screen promoters that meet the above conditions by using an RNA sequencing method (RNA-Seq method). In the RNA-Seq method for analyzing gene expression levels, a sample prepared by isolating mRNAs with polyA sequences from mRNAs is usually used. However, since it is known that mRNAs derived from organelle DNA do not have polyA sequences added in their active form, it was decided to use data obtained by performing RNA-Seq using a sample containing mRNAs that do not have polyA sequences. Using the data, the amount of change in FPKM (fragments per kilobase of exon per million reads mapped) was used as an index, and by selecting promoters whose change was between the value of a high-expression promoter and the value of a low-expression promoter, the inventors succeeded in identifying 12 promoters. Moreover, they found that some of these promoters contained sequences that could not be predicted from the ORF information of the base sequence. Based on these findings, the present inventors further conducted research and completed the present invention.

[0009] That is, the present invention is as follows. [1] A method for selecting a promoter that functions in an organelle, comprising the following steps (1) to (5): (1) preparing sequence information obtained by RNA sequencing analysis; (2) mapping the sequence information prepared in step (1) onto the sequence of organelle DNA; (3) calculating the change in RNA expression before and after each region based on the mapping information obtained in step (2); (4) selecting a region in which the amount of change obtained in step (3) is within a range of a preset reference value; and (5) identifying the upstream region of the region selected in step (4) as a promoter that functions in an organelle; [2] The method according to [1], further comprising the step of: (6) creating a construct containing one or more promoters identified in step (5) and verifying that the promoters function in the organelle. [3] The method according to [1] or [2], wherein the change in expression before and after the region is the change in FPKM. [4] The method according to any one of [1] to [3], wherein the organelle is a plastid or a mitochondrion. [5] A DNA having promoter activity in an organelle, comprising the following sequence (a), (b) or (c): (a) a sequence shown in any one of SEQ ID NOs: 1 to 12 (b) a sequence in which one or more bases are deleted, substituted, or added in the sequence in (a); (c) a sequence having at least 90% identity with the sequence in (a). [6] A transformation vector comprising the sequence described in [5]. [7] A cell having an organelle transformed with the transformation vector described in [6]. [8] The cell described in [7], wherein the organelle is a plastid or a mitochondrion. [9] The cell described in [8], wherein the cell is a plant cell.

[10] A plant body having the plant cell described in [9]. Effect of the Invention

[0010] According to the screening method of the present invention, a promoter that functions in an organelle and has a desired activity strength can be selected on a large scale. By transforming an organelle with the promoter obtained by such screening, a cell expressing a desired protein can be produced. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows a schematic diagram of the RNA-Seq gene analysis method. Gene analysis by RNA-Seq can quantify gene expression levels and identify promoter positions. [Diagram 2] 2 shows a map of the vector used in the examples. In the figure, the promoter region to be tested was inserted into the AtCpP03 portion. [Diagram 3] 3 shows the results of the relative activity of the promoter 10 to 11 hours after the start of amplification using Escherichia coli. The horizontal axis shows the sequence number of the promoter. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 1. Method for selecting promoters that function in organelles The present invention provides a method for selecting a promoter that functions in an organelle (hereinafter, sometimes referred to as the "selection method of the present invention"). The selection method of the present invention includes the following steps (1) to (5). (1) preparing sequence information obtained by RNA sequencing (hereinafter sometimes referred to as "RNA-Seq") analysis; (2) mapping the sequence information prepared in step (1) onto the sequence of organelle DNA; (3) calculating the change in RNA expression in each region based on the mapping information obtained in step (2); (4) selecting a region in which the amount of change obtained in step (3) is within a range of a preset reference value; and (5) identifying or determining that the upstream region of the region selected in step (4) is a promoter that functions in an organelle. In this specification, the term "region" includes not only a single region, but also a region group consisting of a plurality of regions.

[0013] As shown in the Examples below, it has been demonstrated that promoters that are correlated with functions in organelles can be selected by carrying out the above steps (1) to (5). Therefore, the selection method of the present invention is sufficient to include steps (1) to (5), but may also include a step of verifying that the promoters identified in step (5) function in organelles. Thus, in one aspect, the selection method of the present invention includes a step (6) of preparing a construct containing one or more promoters identified in step (5) and verifying that the promoters function in organelles.

[0014] As used herein, the term "organelle" refers to an organelle having DNA other than nuclear DNA, and includes, for example, plastids and mitochondria. Examples of the plastids include chloroplasts, etioplasts, amyloplasts, elaioplasts, chromoplasts, and leucoplasts, among which chloroplasts are preferred, as many transformation methods are known for them.

[0015] In the above step (1), the sequence information to be prepared may be, for example, information published as a database (e.g., PRJNA213635 in Non-Patent Document 3), or information obtained by performing RNA-Seq using a sample containing RNA without a polyA sequence prepared by a method known per se. Preparation of a sample containing RNA without a polyA sequence can be performed, for example, by extracting RNA from a sample, removing nuclear rRNA sequences, fragmenting the RNA, synthesizing cDNA with random primers, etc., and adding an aptamer sequence to the cDNA.

[0016] In the above step (2), mapping onto the organelle DNA sequence can be performed using known mapping software (e.g., BWA, Bowtie, STAR, etc.). The organelle sequence can also be obtained from a database (e.g., the sequence of the Arabidopsis thaliana chloroplast genome is published under GenBank Accession No: NC_000932.1, and the sequence of the Arabidopsis thaliana mitochondrial genome is published under GenBank Accession No: NC_001284.2). Alternatively, a new DNA sequence may be identified using next-generation sequencing or the like, and the sequence information may be used.

[0017] In the above step (3), the expression of each gene can be calculated using, for example, FPKM, RPKM (calculated by the following formula [Number 1]), TPM (transcripts per kilobase million), etc. In addition, the amount of change in the expression of the RNA before and after the region can be calculated using the values ​​calculated above. FPKM (RPKM in the case of single-end reads) means a value obtained by correcting the read count data obtained from the RNA-Seq data by the total number of reads and then correcting by the transcript length. TPM means a value representing how many transcripts are present per transcript when there are 1 million total transcripts in the sample. FPKM and RPKM can be calculated by the following formula ([Number 1]).

[0018]

number

[0019] In the above formula, FPKM i is the FPKM of transcript i, N is the total number of reads that could be mapped to the reference sequence, and Y i indicates the number of reads mapped to the region of transcript i out of the total number of reads, and Li indicates the length of transcript i.

[0020] And TPM can be calculated as follows: Y tLet be the read count mapped to transcript t, and L t If x is the length of transcript t, the number of reads per 1,000 bp of transcript t can be calculated as follows ([Equation 2]):

[0021]

number

[0022] Next, the total read count after correction for transcript length is corrected to 1 million. In this case, the TPM of transcript t t can be calculated as follows ([Mathematical formula 3]):

[0023]

number

[0024] In the step (4), the range of the reference value can be set depending on the strength of the desired promoter activity. For example, when the purpose is to select a promoter having intermediate promoter activity between a promoter known to be a high-expression promoter and a promoter known to be a low-expression promoter, the change in RNA expression in the promoter region and the region under its control can be calculated as the change in FPKM, RPKM, TPM, or the like, and the range of the values ​​can be set as the range of the reference value. In one embodiment, in the case of chloroplasts, the range of the reference value is 10 1 ~10 5 , preferably 10 2 ~10 4.5 In the case of mitochondria, the range of the reference value is 10 or more, preferably 10 1.5 ~10 4 It can be set as follows.

[0025] In the above step (5), the upstream region of a gene or the like that falls within the range of the reference value in step (4) can be identified as a promoter that functions in an organelle. The above judgment or identification of the promoter region can be performed, for example, by the following steps (i) to (iii), but is not limited to this method. (i) Predicting ORFs from sequence information of the region. (ii) If an increase in FPKM value is observed near the start codon of an ORF, confirm whether there is a sequence that is likely to be an SD (Shine-Dalgarno) sequence before the start codon, and if an SD sequence is present, the region up to the vicinity of the SD sequence is considered to be a candidate for the promoter region. If no ORF is found or if no SD sequence is found, the sequence up to the vicinity of the change in FPKM value is considered to be a candidate for the promoter region. The SD sequence refers to a common sequence found upstream of the start codon in prokaryotes, and is known to bind ribosomes that translate mRNA into proteins and promote translation initiation. (iii) To prevent an ORF from including a start codon in the promoter sequence, the intergenic region is considered as a candidate for the promoter region. Furthermore, the candidates may be narrowed down based on the ease of designing PCR primers and the presence or absence of ORF.

[0026] In the above step (6), for example, a nucleic acid in which the promoter identified in step (5) and a gene (e.g., the reporter gene described in 2. below) are linked to the 3' side of the promoter sequence is introduced into a cell, and the expression level of the gene in the cell is measured, and it can be confirmed whether the promoter has the desired activity. In this case, gene expression in the organelle may be detected or measured, but cells having a prokaryotic transcription-translation mechanism (e.g., Escherichia coli, etc.) may be used, as in the organelle. In addition, when the promoter does not have an SD sequence, it is preferable to appropriately add an SD sequence (e.g., the SD sequence of the T7g10 gene, etc.). Furthermore, the promoter identified by the selection method of the present invention can be appropriately included in an expression construct, and an expression vector suitable for the promoter can be selected and identified by introducing the expression vector into a cell and verifying whether it shows the desired expression activity.

[0027] 2. Promoters that function in organelles In another embodiment, the present invention provides a DNA having promoter activity in an organelle, i.e., functioning as a promoter in an organelle, obtained by the selection method of the present invention (hereinafter, sometimes referred to as the "promoter DNA of the present invention"). Definition of Organelle The types are as described in 1. above.

[0028] As shown in the examples below, a sequence shown in any one of SEQ ID NOs: 1 to 12 was incorporated into a vector for transformation, and a gene linked to a promoter was expressed in Escherichia coli, which has a prokaryotic transcription-translation mechanism similar to that of organelles. As a result, gene expression was observed to reflect the strength of promoter activity predicted from the results of RNA-Seq analysis, and therefore all of the sequences shown in any one of SEQ ID NOs: 1 to 12 can have promoter activity in organelles. Therefore, in one aspect, the promoter DNA of the present invention includes or consists of a sequence shown in any one of SEQ ID NOs: 1 to 12. SEQ ID NO: 1 is the sequence of the region from 66967th base to 66739th base in the sequence of the Arabidopsis thaliana chloroplast genome (GenBank Accession No: NC_000932.1). SEQ ID NO: 2 is the sequence of the region from 1467th base to 1574th base in the sequence. SEQ ID NO: 3 is the sequence of the region from 54960th base to 54593rd base in the sequence. SEQ ID NO: 4 is the sequence of the region from the 34250th base to the 34084th base in the sequence. SEQ ID NO: 5 is the sequence of the region from the 13505th base to the 13608th base in the sequence. SEQ ID NO: 6 is the sequence of the region from the 64320th base to the 64558th base in the sequence. SEQ ID NO: 7 is the sequence of the region from the 41893rd base to the 42081st base in the sequence. SEQ ID NO: 8 is the sequence of the region from the 112600th base to the 112806th base in the sequence. SEQ ID NO: 9 is the sequence of the region from the 32636th base to the 32390th base in the sequence. SEQ ID NO: 10 is the sequence of the region from the 14768th base to the 14889th base in the sequence. SEQ ID NO: 11 is the sequence of the region from base 232031 to base 231696 in the sequence of the Arabidopsis thaliana mitochondrial genome (GenBank Accession No: NC_001284.2). SEQ ID NO: 12 is the sequence of the region from base 206221 to base 206059 in the sequence.

[0029] In another aspect, the promoter DNA of the present invention comprises or consists of a sequence in which one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) bases have been deleted, substituted or added in the sequence shown in any one of SEQ ID NOs: 1 to 12, and has promoter activity in an organelle.

[0030] In yet another aspect, the promoter DNA of the present invention comprises or consists of a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) (excluding 100%) identity to a sequence shown in any one of SEQ ID NOs: 1 to 12, and has promoter activity in an organelle.

[0031] The identity of base sequences can be calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) with various parameters set to default values.

[0032] The promoter DNA of the present invention can have promoter activity even if it contains other sequences as long as it has the above-mentioned sequence. Therefore, the length of the promoter DNA of the present invention is not particularly limited, but is preferably 500 bases or less, more preferably 400 bases or less.

[0033] The method for synthesizing the promoter DNA of the present invention is not particularly limited, and a conventionally known method can be used. Examples of the synthesis method include a synthesis method using a genetic engineering technique, a chemical synthesis method, and the like. Examples of the genetic engineering technique include an in vitro transcription synthesis method, a method using a vector, and a method using a PCR cassette. The vector is not particularly limited, and examples thereof include a non-viral vector such as a plasmid, a viral vector, and the like. The chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method. For example, a commercially available automatic nucleic acid synthesizer can be used for the chemical synthesis method. In the chemical synthesis method, an amidite is generally used. The amidite is not particularly limited, and examples of commercially available amidites include ACE amidite, TOM amidite, CEE amidite, CEM amidite, and TEM amidite.

[0034] Whether the above DNA has the desired promoter activity in the organelle can be confirmed by the method described in 1 above.

[0035] 3. Transformation vector containing the promoter of the present invention The present invention also provides a transformation vector (hereinafter sometimes referred to as the "vector of the present invention") comprising the promoter of the present invention described above in 2. In the vector of the present invention, the promoter of the present invention is operably linked to a gene (hereinafter sometimes referred to as the "gene of interest") that encodes a target protein or ribonucleic acid (e.g., non-coding RNA).

[0036] In the organelle, even proteins that are harmful in the cytoplasm can accumulate, so the target gene is not particularly limited, and examples thereof include the merA gene, pest resistance genes, herbicide resistance genes, genes involved in the biosynthesis of carotenoids and vitamin E having antioxidant properties, and genes involved in photosynthesis, such as fructose 1,6-bisphosphatase / sedoheptulose 1,7-bisphosphatase genes. When the vector of the present invention has a metallothionein gene such as the merA gene in the organelle, the cell into which the vector has been introduced can acquire resistance to harmful heavy metals and mercury in the environment, such as soil. When the vector of the present invention has a pest resistance gene or an antibiotic resistance gene (aadA), the cell into which the vector has been introduced can acquire resistance to pests and resistance to infectious bacteria. When the vector of the present invention has a gene involved in the biosynthesis of carotenoids and vitamin E having antioxidant properties, the cell into which the vector has been introduced can improve the content of useful components, such as carotenoids and vitamin E, in the oils and fats produced by the cell. Furthermore, when the vector of the present invention contains a cyanobacterium-derived fructose 1,6-bisphosphatase / sedoheptulose 1,7-bisphosphatase gene involved in photosynthesis, cells into which the vector has been introduced will be taller, have a larger leaf area, grow faster, and have an increased ability to synthesize sugar and starch compared to wild-type strains.

[0037] When a harmful protein (such as a CMS causative protein) is expressed in the organelle, pollen development is inhibited, resulting in cytoplasmic male sterility (CMS), but since fertilization is possible when pollen from another pollen parent is provided, this phenomenon is also utilized in the production of F1 crop seeds, etc. The above-mentioned target gene is not particularly limited, and examples thereof include CMS causative genes such as the orf79 gene, orfH79 gene, orf284 gene, orf352 gene, urf13 gene, orf355 gene, atp6-C gene, orf256 gene, orf260 gene, orf138 gene, orf125 gene, orf224 gene, orf222 gene, orf463 gene, orfH522 gene, orf107 gene, preSatp6 gene, orf129 gene, G cox2 gene, pcf gene, orf239 gene, Ψatp6-2 gene, and orf456 gene. When the vector of the present invention has a CMS causative gene, the individual into which the vector has been introduced has inhibited germ cell development and is therefore unable to self-fertilize, and can easily cross-pollinate to produce F1 seeds. In addition, by using an individual having an appropriate fertility restorer, it is possible to maintain the individual with the target gene in its organelle.

[0038] In the organelle, even ribonucleic acid that undergoes a unique host-specific metabolism in the cytoplasm can accumulate in the transcribed form, so the target gene is not particularly limited, and examples thereof include constructs that express target pest-specific conserved sequences contained in genes essential for pests, such as the acetylcholinesterase gene, chitin synthase gene, and actin gene, so that they eventually become double-stranded ribonucleic acid. When the vector of the present invention contains a construct that expresses a pest-specific conserved region to become double-stranded ribonucleic acid, a pest that has eaten a cell into which the vector has been introduced will metabolize ribonucleic acid independently in the pest's body by double-stranded ribonucleic acid that does not undergo plant-specific metabolism, causing RNAi, and the expression of essential genes will be suppressed, which may result in lethality or a repellent effect on the cell.

[0039] To the vector of the present invention, it is preferable to add a sequence that is homologous to or highly identical to the gene sequence of the organelle DNA (e.g., trnG (tRNA-Gly(GCC)), trnV (tRNA-Val(GAC)), trnfM (tRNA-fMet(CAU)), rbcL gene, accD gene, trnI (tRNA-Ile(GAU)) and trnA (tRNA-Ala(UGU)), 3'rps12 (ribosomal protein S12 exon-3) gene, trnV (tRNA-Val(GAC)), etc.) to the 5' and 3' ends of the gene to be introduced, so that the gene can be introduced into the organelle DNA by homologous recombination. The length of the homologous sequence may be, for example, about 500 to 1500 bases. The homologous sequence can be appropriately designed based on known information on the base sequence of the organelle DNA.

[0040] It is preferable that the vector of the present invention has a gene described in a codon table corresponding to the organelle in which it is desired to express the gene. For example, in the case of vertebrates, the UGA codon represents a stop codon in the nucleus, but encodes tryptophan in mitochondria. It is preferable to control the expression of an appropriate protein sequence only in an appropriate organelle in accordance with such differences in the codon table.

[0041] The vector of the present invention preferably has a marker gene for selecting a transformant. Examples of the marker gene include reporter genes (e.g., genes encoding fluorescent proteins, genes encoding luminescent proteins, genes encoding proteins that assist fluorescence, luminescence, or coloration), drug resistance genes, and the like. Only one type of marker gene may be used, or two or more types (e.g., a combination of a gene encoding a fluorescent protein and a drug resistance gene) may be used. In this specification, a protein encoded by a marker gene may be referred to as a "marker protein".

[0042] Examples of fluorescent proteins include blue fluorescent proteins such as Sirius, TagBFP, and EBFP; cyan fluorescent proteins such as mTurquoise, TagCFP, AmCyan, mTFP1, MidoriishiCyan, and CFP; green fluorescent proteins such as TurboGFP, AcGFP, TagGFP, Azami-Green (e.g., hmAG1), ZsGreen, EmGFP, EGFP, GFP2, and HyPer; yellow fluorescent proteins such as TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, and mBanana; and KusabiraOrange. (e.g., hmKO2), mOrange, and other orange fluorescent proteins; TurboRFP, DsRed-Express, DsRed2, TagRFP, DsRed-Monomer, AsRed2, mStrawberry, and other red fluorescent proteins; TurboFP602, mRFP1, JRed, KillerRed, mCherry, HcRed, KeimaRed (e.g., hdKeimaRed), mRasberry, mPlum, and other near-infrared fluorescent proteins, but are not limited to these.

[0043] Examples of luminescent proteins include, but are not limited to, aequorin, and examples of proteins that assist fluorescence, luminescence, or coloration include, but are not limited to, enzymes that decompose fluorescent, luminescent, or color precursors, such as luciferase, phosphatase, peroxidase, and β-lactamase.

[0044] Examples of drug resistance genes include drug resistance genes such as the hygromycin resistance gene (hygromycin phosphotransferase gene, hpt), spectinomycin resistance gene (aadA), kanamycin resistance gene, and neomycin resistance gene, and herbicide resistance genes such as the ALS (AHAS) gene and PPO gene.

[0045] It is preferable that the upstream or downstream of the marker gene contains an enhancer, promoter, leader and / or terminator sequence for controlling the expression of the gene. In the case of an organelle, the gene may be a plurality of genes to form an operon sequence. The enhancer may be, for example, an enhancer region containing an upstream sequence in the CaMV35S promoter. The promoter may be, for example, an rrn promoter, a cox2 promoter, a psbA promoter, an accD promoter, a clpP promoter, an atpB promoter, an rpl32 promoter, an H strand promoter, a promoter of the elongation factor 1α gene (EF1α promoter), a 35S promoter, a PPDK promoter, a PsPAL1 promoter, a PAL promoter, a UBIZM1 ubiquitin promoter, and the like. Among them, promoters derived from organelles are preferable. The leader sequence may be, for example, a cry9Aa2 leader, an atpB leader, and the like. When one promoter is used to induce the expression of multiple genes, it is preferable to contain a leader sequence. Examples of the terminator include an rbcL terminator, an rps16 terminator, a CaMV35S terminator, an ORF25polyA transcription terminator, a psbA terminator, etc. Among these, organelle-derived terminators are preferred.

[0046] The vector of the present invention may also have a nucleic acid encoding an organelle transport signal peptide. A mitochondrial transport signal peptide is usually composed of a pattern in which 2-3 hydrophobic amino acids and basic amino acids alternate, for example, MLSLRQSIRFFK (SEQ ID NO: 37). A chloroplast transport signal peptide is usually composed of a sequence of several tens to about 100 amino acid residues that is rich in serine and threonine and poor in acidic amino acids (Javis P. and Lopez-Juez E., Nat. Rev. Mol. Cell Biolo., 14, 787-802 (2013)). An example of a peroxisome transport peptide is C-terminal SKL.

[0047] The vector of the present invention can be constructed, for example, by introducing a DNA in which a gene of interest is linked to the promoter of the present invention, and, if necessary, the above-mentioned marker gene, etc., into an appropriate vector. Specific examples of the method include, but are not limited to, the method described in Svab et al., Proc. Nal. Acad. Sci. USA, 87, 8526 (1990), the method described in Sikdar et al., Plant Cell Rep., 18, 20 (1998), the method described in Sidorov et al.: Plant J., 19, 209 (1999), the method described in Ruf, S. et al., Nature biotechnol., 19, 870 (2001), and the method described in Hou et al., Transgenic Res., 12, 111 (2003). Such vectors can also be prepared based on pBI vectors, pUC vectors, pPZP vectors (Hajdukiewicz P., et al., Plant Mol Biol., 25: 989-94, (1994)), pCAMBIA vectors, pSMA vectors, etc., which can introduce a target gene into a plant via Agrobacterium. Examples of the pBI binary vectors include pBI121, pBI101, pBI101.2, and pBI101.3. Examples of the pUC vectors include pUC18, pUC19, and pUC9. Plant virus vectors such as cauliflower mosaic virus (CaMV), bean mosaic virus (BGMV), and tobacco mosaic virus (TMV) may also be used.

[0048] The method for inserting the promoter of the present invention into a vector includes, for example, DNA containing the nucleotide sequence, etc., is digested with an appropriate restriction enzyme and then inserted into the restriction enzyme site of the vector or into a multiclonal fragment. Examples of such methods include inserting the gene into a targeting site and ligating it to a vector.

[0049] 4. Cells containing organelles transformed with the vector of the present invention By introducing the transformation vector prepared as described in 3. above into a host cell, a cell having a transformed organelle (hereinafter sometimes referred to as the "cell of the present invention") can be produced.

[0050] The host cell is not particularly limited as long as it is a cell having an organelle, and examples thereof include plant cells, animal cells, microorganisms, and insect cells, and is preferably a plant cell. The plant from which the plant cell is derived is not particularly limited, and is preferably a monocotyledonous or dicotyledonous plant. Examples of monocotyledonous plants include, but are not limited to, plants of the family Poaceae and Amaryllidaceae. Examples of the Poaceae include plants belonging to Oryza, Triticum, Hordeum, Secale, Saccharum, Sorghum, or Zea, and specific examples thereof include, but are not limited to, corn, sorghum, wheat, rice, oats, barley, rye, and foxtail millet. Preferred Poaceae plants are corn, wheat, and rice. Examples of the Amaryllidaceae plants include, but are not limited to, onion, leek, and garlic. Preferred Amaryllidaceae plants are onion and leek.

[0051] Examples of dicotyledonous plants include, but are not limited to, cruciferous plants, legumes, solanaceae, cucurbits, convolvulaceae, and Asteraceae. Examples of cruciferous plants include plants belonging to the genus Raphanus, Brassica, Arabidopsis, Wasabia, or Capsella, and specifically include, but are not limited to, Chinese cabbage, rapeseed, cabbage, cauliflower, radish, rapeseed, Arabidopsis, wasabi, and shepherd's purse. Preferred cruciferous plants are Arabidopsis, Chinese cabbage, and rapeseed. Examples of legumes include, but are not limited to, soybean, adzuki bean, kidney bean, and cowpea. Preferred legumes are soybean and kidney bean. Examples of solanaceous plants include, but are not limited to, tobacco, tomato, petunia, eggplant, and potato. Preferred solanaceous plants are tobacco, tomato, and petunia. Examples of Cucurbitaceae include, but are not limited to, Japanese cantaloupe, cucumber, melon, and watermelon. A preferred Cucurbitaceae plant is Japanese cantaloupe. Examples of Asteraceae include, but are not limited to, sunflower, chrysanthemum, and dandelion. A preferred Asteraceae plant is sunflower. Examples of Convolvulaceae include, but are not limited to, morning glory, sweet potato, and bindweed. A preferred Convolvulaceae plant is sweet potato.

[0052] Further examples of plants other than those mentioned above include plants of the Rosaceae, Lamiaceae, Liliaceae, Chenopodiaceae, Polygonaceae, Amaranthaceae, Umbelliferae, and Araceae families, as well as any tree species, any fruit species, plants of the Mulberry family (e.g., rubber), and plants of the Malvaceae family (e.g., cotton).

[0053] Examples of the animal cells include cell lines such as monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary (CHO) cells, dhfr gene-deficient CHO cells, mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FL cells, pluripotent stem cells such as human and other mammalian iPS cells and ES cells, and primary culture cells prepared from various tissues. Examples of the microorganisms include yeasts (e.g., Saccharomyces cerevisiae AH22, AH22R-, NA87-11A, DKD-5D, and 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, and Pichia pastoris KM71, etc.), and Chlamydomonas (e.g., Chlamydomonas reinhardtii). The insect cells include a cell line derived from the larvae of the night thief moth (Spodoptera frugiperda cell; Sf cell), MG1 cells derived from the midgut of Trichoplusiani, and High Five TM cells, cells derived from Mamestrabrassicae, cells derived from Estigmena acrea, and a cell line derived from silkworm (Bombyxmori N cells; BmN cells).

[0054] As a method for introducing the vector of the present invention into a host cell to transform it, known methods such as the particle gun method (Svab Z., et al., Proc Natl acad Sci USA, 87:8526-8530 (1990)), the PEG method (Golds T., et al., Bio Technol., 11:95-97 (1992)), and electroporation can be preferably used. For example, in the particle gun method, the vector is coated on extremely fine particles of gold or tungsten, and the particles to which the vector is attached are shot into the host cell with gunpowder or high-pressure gas, thereby introducing the vector into the host cell. The definition and types of organelles are as described in 1 above, but plastids and mitochondria are preferred, and among them, chloroplasts and mitochondria are more preferred. It is also preferable to introduce the vector of the present invention into an organelle using a complex of an organelle localization signal peptide and a cell membrane permeable peptide, as described in Chuah JA, et al., Scientific Reports, 5:7751 (2015).

[0055] When the organelles in the cells are in a heteroplasmic state, homoplasmic individuals may be produced by a method known per se. Examples of the above-mentioned known method include a method of obtaining homoplasmic individuals by repeating selective culture two or three times. In addition, as described in WO 2013 / 077420 A1, homoplasmicization can be promoted by introducing a nucleic acid encoding an organelle-translocating lethal protein (e.g., barnase) into the nucleus and introducing a nucleic acid encoding an inhibitor of the protein into the organelle, and selectively erasing only organelles that do not have the nucleic acid encoding the inhibitor.

[0056] After successful transformation of the organelle and, if necessary, homoplasmicization, the exogenous nucleic acid incorporated into the organelle by the introduction of the vector of the present invention may be removed. Methods for removing the nucleic acid incorporated into the organelle include a method using the Cre-loxP system or the FLP-FRT system, a method using a transposon, and a natural removal method via endogenous homologous recombination activity.

[0057] The target protein can be produced by culturing the cells of the present invention or a plant body having the cells. In this specification, the term "plant body" includes any of an individual plant, a plant organ, a plant tissue, a plant cell, and a seed. Examples of plant organs include roots, leaves, stems, and flowers. In addition, plant cells include cultured cells as well as cells in a plant body. In addition, plant cells of various forms, such as suspension culture cells, protoplasts, leaf slices, root slices, callus, immature embryos, pollen, etc. are included.

[0058] The cells of the present invention can be cultured according to a known method depending on the type of the cells. The medium used for the culture is preferably a solid medium (e.g., agar medium, agarose medium, gellan gum medium, etc.). The medium preferably contains a carbon source, a nitrogen source, inorganic substances, etc. necessary for the growth of the transformant. When the cells of the present invention are plant cells, for example, N6 medium, MS medium, LS medium, B5 medium, etc. are used as the basal medium. Plant growth substances (e.g., auxins, cytokinins, etc.) may be added to the medium as appropriate. The pH of the medium is preferably about 5 to about 8. The culture temperature can be appropriately selected within the range of usually about 20°C to about 35°C depending on the type of plant cells. For example, rice callus can be cultured usually at 28 to 33°C, preferably 30 to 33°C.

[0059] Examples of media for culturing animal cells include Minimum Essential Medium (MEM) containing about 5 to about 20% fetal bovine serum [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], and 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)]. The pH of the medium is preferably about 6 to about 8. The culture is usually performed at about 30°C to about 40°C. Aeration and stirring may be performed as necessary.

[0060] Examples of media for culturing microorganisms include Burkholder's minimal medium [Proc. Natl. Acad. Sci. USA, 77, 4505 (1980)] and SD medium containing 0.5% casamino acids [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably about 5 to about 8. The culture is usually carried out at about 20°C to about 35°C. Aeration and stirring may be performed as necessary.

[0061] As a medium for culturing insect cells or insects, for example, Grace's Insect Medium [Nature, 195, 788 (1962)] with additives such as 10% inactivated bovine serum, etc., is used. The pH of the medium is preferably about 6.2 to about 6.4. The culture is usually carried out at about 27°C. Aeration and stirring may be performed as necessary.

[0062] The medium may also contain sugars as a carbon source, vitamins, a support for solidifying the medium, and the like. Examples of sugars include glucose and sucrose. The amount of sugar added is about 1 to 10% by weight, preferably about 2 to 5% by weight. Examples of vitamins include thiamine hydrochloride, pyridoxine hydrochloride, nicotinic acid, inositol, and the like. Examples of supports include agar, gellan gum, and paper bridge. The medium may also contain amino acids (e.g., glycine, etc.), adenine, coconut water, and the like.

[0063] When the cells of the present invention are callus, they can be redifferentiated into a plant by a redifferentiation method known per se, such as the method described in Toki S. et al., Plant Physiol. 100(3):1503-1507 (1992), Christou P. et al., Bio / Technology 9:957-962 (1991), or Hiei Y. et al., Plant J., 6:271-282, (1994), in the case of rice.

[0064] Once a plant containing cells whose organelles have been transformed is obtained in this manner, it is possible to obtain progeny from the plant through sexual or asexual reproduction. It is also possible to obtain propagation materials (e.g., seeds, fruits, cuttings, stumps, calli, protoplasts, etc.) from the plant or its progeny or clones, and to mass-produce the plant based on these.

[0065] The target protein can be isolated by extracting and purifying useful components from the cells cultured as described above by a method known per se, or by secreting the target protein into the culture solution and recovering the protein. Alternatively, the cells may be frozen and dried and used as is. Similarly, the target protein can be isolated from the plant body by extracting and purifying useful components from the target protein from the plant body by a method known per se. Alternatively, the plant body may be used as is. As an example of the above-mentioned known method, there is a method in which the plant body is crushed in a mixer or a mortar, then immersed in water or physiological saline, and the crushed residue is removed by centrifugation or filtration.

[0066] The present invention will be described below with reference to examples, although the present invention is not limited to these examples. EXAMPLES

[0067] Example 1: Identification of organelle promoters <Analysis of next-generation sequencing data> For the analysis of next-generation sequencing data, RNA extracted from Arabidopsis was pretreated using Ribo minus RNA-seq and sequenced using Illumina's HiSeq 2000 (PRJNA213635), as described in Non-Patent Document 3. FastQC was used for sequence quality check. We also mapped the sequences to the Arabidopsis chloroplast genome (NC_000932.1) and the Arabidopsis mitochondrial genome (NC_001284.2) using BWA. From this mapping information, we calculated the FPKM (reads per kilobase of exon per million mapped) in each region using igv (integrative genomics viewer), and estimated that the first half of the region with the largest change in FPKM was the promoter sequence of the organelle.

[0068] <Plasmid construction> To test promoter activity in E. coli and plant chloroplasts, we used pKKP23 (Figure 2), which expresses the spectinomycin resistance gene (aadA) and red fluorescent protein (TagRFP) in the known strong organelle promoter rrn, and green fluorescent protein (GFP) in the promoter sequence to be tested. pKKP23 was created by amplifying each component by PCR and analyzing them with the NEBuilder iFi Using the DNA Assembly Master Kit (NEB), a promoter was constructed for each promoter to be tested. The primers used for PCR are shown in Table 1.

[0069] [Table 1]

[0070] result Based on the data of Non-Patent Document 3, which is an analysis of Arabidopsis RNA using Ribo minus RNA-seq, the transcription start position and expression level were estimated from FPKM. In addition, the upstream region of the point where FPKM increases was predicted to be a promoter region in the organelle. Specifically, (i) an orf was predicted from the sequence information of the region, and (ii) if an increase in FPKM value was observed at a position close to the start codon of the orf, it was confirmed whether a sequence that is likely to be an SD (Shine-Dalgarno) sequence exists before the start codon, and if an SD sequence exists, the vicinity of the SD sequence was set as a candidate for the promoter region. If an orf was not found, or if an SD sequence was not found, the sequence up to the vicinity of the change in FPKM value was set as a candidate for the promoter region. (iii) In order to prevent the orf from being included in the promoter sequence in a form that includes the start codon, the intergenic region was set as a candidate for the promoter region. Furthermore, the candidates were narrowed down using the ease of designing the promoter and the presence or absence of an orf as indicators. As a result, 12 promoter sequences listed in Table 2 were selected as those predicted to have different expression levels. In the table, "Predicted Intensity" indicates the change in FPKM value before and after the region.

[0071] [Table 2]

[0072] Example 2: Activity analysis of organelle promoters <Measurement of promoter activity in E. coli> To measure the activity of each promoter in E. coli, pKKP23 was prepared for each promoter to be tested and transformed into E. coli JM109 strain. The seed culture was made by shaking overnight in the presence of antibiotics. This culture was inoculated into LB medium containing antibiotics dispensed into a 96-well plate and rotated at 37°C. The growth curve of each E. coli strain under these conditions was measured using EPOCH2 (BioTek). In addition, the fluorescence values ​​of GFP and RFP were measured using a plate reader at each time point after inoculation. SectraMax Paradigm (Moleculu Devises) or GloMax (Promega) were used as the plate reader. GFP / RFP was calculated as the activity value of each promoter, and the relative value to the promoter sequence that showed the highest activity was calculated. The results are shown in Figure 3.

[0073] <Measurement of promoter activity in plants> To measure the activity of each promoter in plant chloroplasts, the plasmids used to measure promoter activity in E. coli are used. Young leaves of tobacco (Nicotiana tabacum cv. Petit Havana) are used as plants, and DNA-coated gold particles are introduced by particle gun method using a PDS-1000 / He (Bio-Rad) equipped with a Hepta adapter (Bio-Rad). The protocol of Bio-Rad is used for the use of the device, and the method described by Pal Maliga and Tarinee Tungsuchat-Hung (Methods Mol Biol. 1132:205-220, (2014)) is followed for the selection of transformed plants. Individuals with transformed chloroplasts are selected with 500 mg / L spectinomycin, and the amount of GFP per total protein in the young leaves is calculated as the activity value of each promoter.

[0074] result Since E. coli has a prokaryotic transcription-translation mechanism, just like organelles, the promoter activity of these regions was confirmed from the expression level of each protein in E. coli. When GFP was inserted downstream of the predicted promoter sequence, GFP expression was observed in correlation with the promoter strength. This demonstrated that the promoter functions within the organelle. Surprisingly, P04, P11, and P12 were found to have promoter activity, even though the promoter regions were set based only on RNA-seq data, not on the predicted ORF results. [Industrial Applicability]

[0075] According to the present invention, it is possible to select promoters that function in organelles and have the desired activity strength on a large scale. By transforming organelles with promoters obtained by such selection, it is possible to produce cells that stably express a sufficient amount of a target protein, even if the maximum expression level of the target protein would adversely affect the host.

[0076] This application is based on patent application No. 2019-042535 filed in Japan (filing date: March 8, 2019), the contents of which are incorporated in their entirety by reference in this specification.

Claims

1. A method for selecting a promoter that functions in an organelle, comprising the following steps (1) to (5): (1) preparing sequence information obtained by RNA sequencing analysis using a sample containing mRNA that does not have a polyA sequence; (2) mapping the sequence information prepared in step (1) onto the sequence of organelle DNA; (3) calculating the change in RNA expression before and after each region based on the mapping information obtained in step (2); (4) selecting a region in which the amount of change obtained in step (3) is within a range of a preset reference value; and (5) identifying the upstream region of the region selected in step (4) as a promoter that functions in an organelle;

2. 2. The method of claim 1, further comprising the step of: (6) generating a construct comprising one or more promoters identified in step (5) and verifying that the promoters function in the organelle.

3. The method according to claim 1 or 2, wherein the change in expression before and after the region is the change in FPKM.

4. The method according to any one of claims 1 to 3, wherein the organelle is a plastid or a mitochondrion.

5. A DNA having promoter activity in an organelle, comprising the following sequence (a), (b) or (c): (a) a sequence shown in any one of SEQ ID NOs: 1 to 12 (b) A sequence in which one or more bases are deleted, substituted, or added in the sequence of (a). (c) a sequence having at least 90% identity with the sequence of (a).

6. A transformation vector comprising the sequence of claim 5.

7. A cell having an organelle transformed with the transformation vector according to claim 6.

8. The cell of claim 7 , wherein the organelle is a plastid or a mitochondrion.

9. The cell of claim 8 , wherein the cell is a plant cell.

10. A plant body having the plant cell according to claim 9.

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