JP7220604B2 - Promoter derived from yew and use thereof - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law The 139th Annual Meeting of the Pharmaceutical Society of Japan PROGRAM, Pharmacia Vol.

Application of Article 30, Paragraph 2 of the Patent Law Homare Tabata et al., Abstracts of the 139th Annual Meeting of the Pharmaceutical Society of Japan, published on February 1, 2019, 22PO-am065, website <http://nenkai. pharm. or. jp/139/pc/ipdfview. asp? i=2972>

The present invention relates to a promoter useful for expressing a gene of interest in plant cells, an expression vector and DNA fragment having the promoter, and a transformant containing any of these.

As useful plants that produce various physiologically active substances including anticancer agents, Taxus genus plants, Solanaceae plants belonging to the genus Nicotiana, Brassicaceae Arabidopsis thaliana plants and the like are known.
Plants belonging to the genus Taxus, for example, Taxus brevifolia NUTT, contain paclitaxel, which is a taxane anticancer agent used as a therapeutic agent for ovarian cancer, breast cancer, lung cancer, and the like. At present, the production of taxane compounds as active ingredients of anticancer agents involves the production of raw material compounds using Taxus plantations or Taxus plant cell culture methods, and the taxane compounds are obtained using these raw material compounds as precursors. is done by the method. However, Taxus plants are slow-growing plants, taking more than 10 years to grow to a height of 20 cm above the ground. In addition, the growth rate in cell culture of Taxus plants is extremely slow, 5-10 times in 3-4 weeks. Therefore, the method for producing the taxane compound described above is insufficient for industrial production, and it may be difficult to obtain a large amount of the raw material compound.
Therefore, in considering the introduction of genes that contribute to productivity improvement, promoters for genes that are consistently highly expressed in Taxus are desired.
Patent Document 1 discloses a method for producing a transgenic plant containing yew whose growth is increased compared to the wild type by changing the expression level of genes involved in growth using a strong promoter. there is

Japanese Patent Publication No. 2010-511405

Accordingly, the present invention provides a promoter useful for expressing a desired gene, an expression vector and a DNA fragment having the promoter, and any of these, using a plant such as Taxus genus or a cultured cell of a plant such as Taxus genus as a host. The object is to provide a transformant containing

As a result of intensive studies by the present inventors in order to achieve the above-mentioned purpose, they discovered an elongation factor 1α gene (hereinafter referred to as "EF1α gene") that is highly expressed in taxus, and a new gene was found at 5.1 kb upstream of the gene. The present inventors have discovered a region containing such a promoter and completed the present invention.
The promoter according to the present invention is characterized by comprising one kind of DNA selected from the group consisting of (a) to (e) below.
(a) a DNA consisting of a nucleotide sequence having promoter activity selected from SEQ ID NO: 1;
(b) a DNA consisting of 1 to 3 base sequences selected from SEQ ID NOs: 2 to 5 and having activity as the promoter;
(c) a DNA comprising a base sequence having at least 70% identity with a base sequence of one DNA selected from (a) and (b) and having activity as the promoter;
(d) in the base sequence of one DNA selected from (a) and (b), one or more bases are deleted, substituted or added within the range of maintaining the activity as the promoter; DNA consisting of a base sequence,
(e) hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of one DNA selected from the group consisting of (a) to (d), and as the promoter DNA with activity.
An expression vector according to the present invention is characterized by containing the promoter described above. The expression vector can contain a promoter and an expression gene operably linked downstream of the promoter.
A DNA fragment according to the present invention is characterized by comprising the promoter described above and an expression gene functionally linked downstream of the promoter.
One form of the transformant according to the present invention is characterized by containing the above expression vector or DNA fragment.
Another form of the transformant according to the present invention is characterized by having an expression gene inserted downstream of the above-mentioned promoter present in the genome of the host and functionally linked to the promoter.
The above-mentioned transformants include plant transformants obtained by transforming plants or plant cells as hosts.
The promoter according to the present invention can also be used in the following inventions.
(1) The use or method of using one DNA selected from the group consisting of (a) to (e) above as a promoter for expressing a gene for expression in a host.
(2) Forming in a host an expression unit having a promoter consisting of one kind of DNA selected from the group consisting of (a) to (e) and an expression gene operably linked to the promoter. A method for transforming a host, comprising steps.
(3) A method for producing a target product by transforming a host, wherein the host comprises a promoter consisting of one DNA selected from the group consisting of (a) to (e) above, and forming a transformant by transforming the host with an expression unit having an expression unit having an expression gene encoding the desired product linked to the expression gene using the promoter activity in the transformant and expressing the target product to produce the target product.
In each of the above inventions, a heterologous gene different from the original EF1α gene involved in the promoter activity obtained by DNA consisting of SEQ ID NO: 1 can be used as an expression gene.
Formation of an expression unit in a host includes a method of introducing an expression vector or a DNA fragment having the promoter and the gene for expression into the host, and a method of recombination downstream of the above-mentioned promoter possessed by the chromosome of the host for expression. It is possible to use at least one method selected from a method of functionally introducing and binding a gene, and a method of functionally introducing and binding the above-mentioned promoter upstream of the expression gene of the host chromosome by recombination. can.

According to the present invention, a promoter useful for expressing a desired gene in a plant such as Taxus genus or a cultured cell of a plant such as Taxus genus as a host, an expression vector and a DNA fragment having the promoter, and any of these A transformant containing either can be provided.

It is a figure plotting the expression level of each gene measured by cDNA sequence analysis. FIG. 10 is a diagram showing the state of cultured Taxus cells into which a plasmid has been introduced, observed under a fluorescence microscope.

The present invention will be described in detail below.
The EF1α gene of the media yew (Taxus x media), which is a cross between Taxus cuspidata and European yew (Taxus baccata), has a promoter region in its upstream that constitutively expresses the EF1α gene. The present inventors have newly found that this promoter region has promoter activity useful for improving the expression level of a target gene in plants such as Taxus or cultured plant cells.
The promoter according to the present invention consists of DNA derived from a region having promoter activity existing upstream of the EF1α gene. This promoter allows the gene for expression located downstream thereof to be constantly expressed in plants and plant cells.
That is, the promoter according to the present invention is characterized by comprising one kind of DNA selected from the group consisting of (a) to (e) below.
(a) a DNA consisting of a nucleotide sequence having promoter activity selected from SEQ ID NO: 1;
(b) a DNA consisting of 1 to 3 base sequences selected from SEQ ID NOs: 2 to 5 and having activity as the promoter;
(c) a DNA comprising a base sequence having at least 70% identity with a base sequence of one DNA selected from (a) and (b) and having activity as the promoter;
(d) in the base sequence of one DNA selected from (a) and (b), one or more bases are deleted, substituted or added within the range of maintaining the activity as the promoter; DNA consisting of a base sequence,
(e) hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of one DNA selected from the group consisting of (a) to (d), and as the promoter DNA with activity.
SEQ ID NO: 1 shows the nucleotide sequence of the region consisting of 5.1 kb upstream of the EF1α gene.
The nucleotide sequence of each SEQ ID NO is described from the 5' end to the 3' end, and the EF1α gene is located on the 3' end side of the nucleotide sequence of SEQ ID NO: 1 in the taxus media chromosome.
The DNA of (a) above consists of a nucleotide sequence having promoter activity selected from SEQ ID NO:1.
SEQ ID NO: 1 is the entire nucleotide sequence (also referred to as nucleotide sequence) of the 5.1 kb promoter-containing region of the EF1α gene.
A base sequence or nucleotide sequence is hereinafter simply referred to as "sequence".
This SEQ ID NO: 1 consists of SEQ ID NO: 2: promoter region, SEQ ID NO: 3: 5' untranslated region 1, SEQ ID NO: 4: intron, SEQ ID NO: 5: 5' untranslated region 2.
The DNA contained in (a) above consists of a base sequence having promoter activity selected from SEQ ID NO:1. This DNA includes DNA consisting of SEQ ID NO: 1 and DNA consisting of a partial sequence selected from SEQ ID NO: 1 and having promoter activity. This partial sequence can be composed of one partial sequence of SEQ ID NO: 1 or a combination of two or more partial sequences.
Examples of the DNA consisting of the partial sequence of SEQ ID NO: 1 include the DNA contained in (b) above. The DNA contained in (b) above consists of one sequence selected from SEQ ID NOs: 2 to 5, or a sequence consisting of a combination of two or three, so as to obtain the desired promoter activity.
A preferable DNA contained in the above (b) is a 2.4 kb region obtained by connecting SEQ ID NOS: 2, 3 and 5 in the 5′ to 3′ direction, that is, SEQ ID NO: 1 excluding SEQ ID NO: 4 as an intron. DNA consisting of

The DNA constituting the promoter according to the present invention is not limited to the DNA defined in (a) and (b) above, and the DNA defined in (c) to (e) above is used to construct the promoter according to the present invention. be able to.
The DNA defined in (c) above has a sequence possessed by the DNA defined in (a) and (b) above and at least 70% (but less than 100%) within the range where the desired promoter activity can be obtained. It is DNA consisting of a sequence having identity (also referred to as degree of identity or homology).
The lower limit of sequence identity is preferably 80% or more, preferably 90% or more, and more preferably 95% or more.
The identity value is obtained by aligning the reference sequence and the comparison target sequence using a well-known homology search program, and the comparison target sequence matches the reference sequence. It is a value calculated as the ratio of bases that In this method for calculating the identity value, the DNA sequences (a) and (b) above are reference sequences, and the DNA sequence (c) above is a comparison sequence.

The DNA defined in the above (d) has one or more bases substituted, deleted, or substituted in the sequence of the DNA defined in the above (a) and (b) within the range where the desired promoter activity can be obtained. A DNA consisting of an added or inserted sequence.
The multiple bases (nucleotides) to be substituted, deleted, added or inserted in the DNA defined in (d) above are, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50, Most preferably, it means 2 to 25 bases. Based on the nucleotide sequence of one DNA having promoter activity selected from (a) and (b) above, substitution, deletion, addition or substitution with the number of bases within these ranges so as to obtain promoter activity Insertion may be performed.
In addition, substitution, deletion, addition, or insertion of multiple bases is such that the identity described in (c) above is 70% or more, preferably 80% or more, and more preferably 90%, within the range in which promoter activity can be obtained. Above, most preferably 95% or more may be carried out. Also in this case, based on the base sequence of DNA having promoter activity selected from (a) and (b) above, substitutions, deletions, and additions are made within these ranges so as to obtain promoter activity. Or an insertion may be made.
The DNA defined in (e) above hybridizes under stringent conditions to a DNA comprising a sequence complementary to the sequence possessed by the DNAs (a) to (d) above, and the desired DNA having promoter activity.
Here, to hybridize under stringent conditions means to maintain binding under 2×SSC washing conditions at 60°C. Hybridization can be performed by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory (1989).
Promoter activity can be verified by a reporter assay using an appropriate host. Suitable hosts include plant cells of yew plants, tobacco plants, Arabidopsis plants, and the like. In particular, it is preferred to use cells of Taxus plants. The reporter gene used in the reporter assay is not limited as long as it has the intended reporter function. Examples of reporter genes that can be used include green fluorescent protein (GFP) gene, luciferase (LUC) gene, β-glucuronidase (GUS) gene, and the like. Assays using these reporter genes can be performed according to known protocols or by appropriately modifying known protocols within the scope obvious to those skilled in the art.
The promoter according to the present invention can be used in the form of single-stranded DNA, or in the form of double-stranded DNA consisting of a single-stranded DNA consisting of a sequence having promoter activity and its complementary strand, depending on its use.

By using the promoter according to the present invention, it is possible to provide a nucleic acid construct such as a DNA fragment having an expression region with excellent transcriptional activity. In addition to the above promoter, the nucleic acid construct may contain a cis-element that enhances the transcriptional activity of the promoter. This nucleic acid construct can also be constructed to have restriction enzyme recognition sequences at both ends.
A promoter according to the present invention can be used as a promoter region of an expression vector.
The expression vector may be in the form of having an expression gene or in the form of not having an expression gene (having an insertion site for an expression gene).
The promoter of the present invention is incorporated into an expression vector that enables the expression of a desired gene for expression, and the gene for expression is functionally linked downstream of the promoter, thereby improving the expression of the gene at the transcription level. An expression vector can be provided that can

Portions other than the promoter of the expression vector may be configured for expression of the gene for expression of interest. The construction of this expression vector other than the promoter is not particularly limited as long as the gene for expression of interest can be expressed, and can be appropriately selected according to the expression system to be used.
The expression vector may be either in the form of being introduced into the chromosome (genome) in the host cell or in the form of being independently retained extrachromosomally. In the form of introduction into the chromosome (genome) in the host cell, a method of inserting a gene for expression downstream of the promoter of the present invention in the genome of the host and functionally binding to the promoter, or Methods are available for recombinantly introducing such a promoter and a set of genes for expression operably linked to the promoter into the genome of the host.
The term "downstream of the promoter" refers to the downstream side in the 5' to 3' direction of the promoter sequence.
An expression vector can comprise at least one of a terminator, an enhancer, a selectable marker, a replication origin, a multiple cloning site, etc. as a structural unit, as a portion other than the promoter. These structural units can be selected from known materials and used.
Expression vectors can also be provided in the form of single-stranded DNA or in the form of double-stranded DNA.
In addition, like an expression vector, the promoter of the present invention can be used to construct a DNA fragment for expression of a gene of interest in a host. The structure of this DNA fragment can be the same as that of the above expression vector.
Here, the selection marker is a gene to be introduced as a marker for the introduction of the expression vector, and fluorescent proteins and drug resistance genes are usually used. In the present invention, these selectable marker genes may also be used to construct an expression vector, and cells can be selected using the selectable marker, or only transfected cells can be obtained by applying selective pressure with a drug.

The host in which the promoter, the expression vector containing the promoter, and the DNA fragment of the present invention are allowed to act is not limited as long as the desired action can be obtained.
A transformant that expresses the desired gene can be obtained by transforming the host by introducing the expression vector or DNA fragment described above.
One form of transformant formation method is a method of transforming a host with an expression unit. This expression unit has a promoter according to the invention and a gene for expression operably linked to this promoter.
For forming a transformant of a host having an expression unit, a method of forming an expression unit in the host and transforming the host can be used. At least one of a method of introducing an expression unit prepared outside the host into the host and a method of forming the expression unit in the chromosome of the host can be used to form the expression unit in the host. As a method of forming an expression unit in the chromosome of the host, a method of introducing one or both of the promoter of the present invention and the gene for expression into the chromosome of the host by recombination can be used.
Expression units can be used in the form of expression vectors or DNA fragments as described above.

As the gene for expression, a gene encoding a protein to be produced in the host can be used. Examples of this protein include enzymes involved in the synthesis system of the target product in the host.
A conventionally known transformation method can be used to transform the host. Transformation methods include Agrobacterium method, particle bombardment method, electroporation method, protoplast method, PEG method, whisker method and the like. The transformation method is not limited to these transformation methods as long as the expression vector and DNA fragment are introduced into the cell.
Plants and plant cells that can be used as hosts are not particularly limited, but plants belonging to the genus Taxus such as Taxus baccata, Taxus cuspidata, and Taxus x media, the genus Nicotiana such as Nicotiana tabacum, and the genus Arabidopsis such as Arabidopsis thaliana, and their cells (e.g. cultured cells).
The target gene for expression that is operably linked to the promoter of the present invention may be any gene that can be expressed by the action of the promoter of the present invention in a host for transformation.
Taxadiene synthase (TXS), taxadiene 5α hydroxylase (T5α H), taxadiene 5α-ol when using a plant of the genus Taxus or a plant cell such as a cultured cell of the genus Taxus as a host and aiming to improve the productivity of paclitaxel O-acetyltransferase (TDAT), taxan 10β-hydroxylase (T10β H), taxadiene 13α-hydroxylase (Tα H), taxan 2α-O-benzoyltransferase (DBBT), 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylalanine Transforming enzymes involved in paclitaxel synthesis selected from aminomutase (PAM), Baccatin III:3-amino, 3 phenylpropanoyltransferase (BAPT), 3'-N-debenzoyl-2-dexytaxol-N-benzoyltransferase (DBTNBT), etc. Genes for production in the body can be mentioned.

EXAMPLES The present invention will be described in detail with reference to examples below, but the technical scope of the present invention is not limited to the examples described below. "%" is based on mass unless otherwise specified.
[Example 1]
1. Extraction of Total RNA from Taxus Cultured Cells As Taxus cultured cells, callus was obtained from seed embryos of T. x media using WP agar medium containing 10 −5 M NAA and 3% sucrose, and the agar was removed from the medium. A TxN16-1 strain was obtained with repeated culture passages in WP medium and repeated stable growth. TxN16-1 strain was transplanted to WP medium at 8.0 g FW, added with 100 μM methyl jasmonate, and cultured at 25° C. in the dark for 14 days. In addition, the same strains were also cultured at the same time without the addition of methyl jasmonate. After culturing, the cultured cells were frozen with liquid nitrogen, pulverized in a mortar, and 0.1 g was transferred to a 1.5 ml tube. Add 1 ml of CATB solution (2% hexadecyltrimethylammonium bromide, 0.1 M Tris-HCl (pH 9.5), 20 mM EDTA, 20 mM EDTA, 1.4 M NaCl, 1% β-mercaptoethanol) and °C for 10 minutes. An equal amount of chloroform/isoamyl alcohol was added, and after stirring, the mixture was centrifuged at 15,000 rpm for 10 minutes. After centrifugation, the aqueous phase was recovered, 1/4 volume of 10 M lithium chloride solution was added, allowed to stand at -20°C for 2 hours, and then centrifuged at 15,000 rpm for 10 minutes. After centrifugation, the supernatant was removed, 500 μl of TE buffer was added, and the mixture was centrifuged at 15,000 rpm for 10 minutes to collect the supernatant. The supernatant is treated with phenol and phenol/chloroform, centrifuged, the aqueous phase is recovered, 1/4 volume of 10 M lithium chloride solution is added, left at -20°C for 2 hours, then 15,000 rpm for 10 minutes. Centrifuged. The supernatant was removed, the precipitate was washed with 70% ethanol, and dissolved in an appropriate amount of sterilized water to obtain a total RNA extraction solution.

2. cDNA sequence analysis (de novo RNA-seq)
Based on the extracted total RNA (methyl jasmonate-added cell RNA: TxN16-1 MJ, methyl jasmonate-free cell extract: TxN16-1 cont.), a next-generation sequencer was used to perform comprehensive gene sequencing. Acquisition and expression quantification analysis were performed. Preparation of the sequence library and sequence analysis were outsourced to Takara Bio Inc.
2.1 Preparation of sequence library
A sequence library was prepared using TruSeq Stranded mRNA Sample Prep Kit (Illumina) and Agilent XT-Auto System (Agilent Technologies). PolyA+ RNA was isolated from the obtained total RNA. Next, PolyA+ RNA was fragmented, and single-stranded cDNA was synthesized by reverse transcription reaction using this RNA fragment as a template. Using this single-stranded cDNA as a template, a double-stranded cDNA incorporating dUTP was synthesized. Both ends of the obtained double-stranded cDNA were blunted and phosphorylated, followed by 3'-dA protrusion treatment and ligation with indexed adapters. Using the adapter-ligated double-stranded cDNA as a template, PCR amplification was performed using a polymerase that does not selectively amplify the strand having dUTP, and a sequence library was obtained.
Sequence library preparation conditions were carried out as shown in Table 1.

2.2 Sequence analysis Tables 2 to 4 show the analysis conditions, equipment used for cluster formation and sequence analysis, manual names and version numbers used for each task.

2.3 Results of Sequence Analysis Table 5 shows the results of sequence analysis.

2.4 Data Analysis 2.4.1 Outline of Analysis After trimming the adapter sequences and bases with low quality values from the obtained sequence data, assembly was performed using Trinity software. Then, using the obtained contig sequence as a reference, the gene expression level was determined for each sample using RSEM software. For the annotation of the contig sequences, blastn search by NCBI nt database, ORF prediction, blastp search by UniPortKB database of amino acid sequences, and Gene Ontology assignment were performed.
2.4.2 Pretreatment of read sequences As pretreatment of read sequences, adapter sequences and bases with low quality values were trimmed. The cutadapt software (v1.3) was used for recognition and removal of adapter sequences. If the length of the overlap with the adapter sequence was 12 bases or more, the region was trimmed to the 3, terminal side of the read sequence. Trimming of low-quality bases at the 3′ end of the read sequence was performed using sickle software (v 1.200) on the read sequence after processing of the adapter sequence. In the process, a 10 base-length window is set on the 3' end side of the read sequence, and if the average value is less than 30, it is regarded as a low quality region, and the window is slid to the 5' end side. When the average value exceeded 30, the 3'-terminal side was trimmed from that position. If the number of bases remaining after trimming was 40 bases or less, the read was removed from the analysis. As a result, when either the paired-end R1 side or R2 side was excluded from the analysis, the remaining one read was also excluded from the analysis.

2.4.3 Assembly by Trinity The adapter sequences and the read sequences of all samples from which low-quality bases were removed were mixed and assembled using Trinity software (v2.0.6). Create a k-mer fragment sequence from the entire read sequence, lexiconize the sequence and the number of occurrences (k = 25), then low-complexity k-mer sequences and occurrences from the dictionary A k-mer sequence with a count of 1 was deleted. The k-mer sequence with the highest number of occurrences in the created dictionary was used as the seed sequence, and those that matched this seed sequence under the k-1 mer overlap condition were searched in order of the number of occurrences. The search was performed in both directions of the seed sequence, and both ends were ligated so as to extend to create a contig. The k-mer sequence once selected was deleted from the dictionary. These operations were repeated until all k-mer sequences were deleted from the dictionary.
The contigs obtained above were clustered to construct a de Bruijn graph. The conditions for clustering were that the contigs of each other had a perfect match at k-1 mer or longer, the read sequences were mapped to the contigs, and even if a sequence connecting the two contigs existed under certain conditions, they were classified into the same group. From the obtained de Bruijn graphs, transcripts containing alternatively spliced isoforms were reconstructed.
2.4.4 Gene expression analysis by RSEM
Gene expression levels (FPKM) were determined for each sample using the RSEM software (v1.2.19) using the sequences obtained by Trinity as a reference. Default values were used for all analysis parameters.

2.4.5 Annotation of Contig Sequences
Contig sequences obtained by Trinity were subjected to homology searches with NCBI's nt database using blast+ software. (blast + version 2.2, retrieved from 28 database nt (ftp://ftp.ncbi.nim.nih.gov/blast/db/) on 2016/11/23, blast search conditions are blast program: blastn, E-value : le-3 or less, maximum number of hits: up to 20).
In addition, ORF estimation was performed on the contig sequence obtained by Trinity using TransDecoder software (r2014-07-04). The amino acid sequences obtained by TransDecoder were searched for homology with the UniProtKB database of UniProt using blast+ software. (blast + version 2.2.28, database UniProtKB (got uniprot_sprot_fasta.gz from ftp://ftp.uniprot.org/pub/databases/uniprot/current release/knowledgebase/complete te/ on 2016/11/30), blast Search conditions are blast program: blastp, E-value: 1e-6 or less, maximum number of hits: up to 5).
Gene Ontology Terms were assigned based on UniProt IDs matched by homology searches between amino acid sequences and the UniProtKB database. Blast2G0 software (v2.5) was used to link UniProt ID to Gene Ontology.
2.5 Analysis Results Table 6 shows the analysis results.

3. Analysis of highly expressed genes Based on the gene expression level values obtained from data analysis, the contig sequences are rearranged and shown in a diagram (Fig. 1). indicates After picking up contigs showing high expression in both treatment plots, a contig encoding the EF1α gene was found by referring to the annotation information attached to the contig number indicated by the arrow in FIG. (SEQ ID NO: 6)

・ Sequence number 6
ATGGGAAAAG AAAAGGTTCA TATCAACATT GTTGTTATTG GCCATGTTGA CTCTGGAAAG
TCAACTACTA CTGGGCATCT GATTTATAAG TTGGGTGGTA TTGACAAGCG TGTGATTGAG
CGTTTTGAGA AGGAAGCTGC TGAAATGAAC AAAAGGTCAT TCAAGTATGC TTGGGTGCTT
GATAAGCTAA AGGCTGAGCG TGAACGTGGT ATTACCATTG ATATTGCACT ATGGAAGTTT
GAGACCACTA GATACTACTG CACTGTCATT GATGCTCCAG GACATCGTGA TTTCATTAAG
AACATGATTA CAGGAACTTC TCAGGCTGAT TGTGCAGTGC TGATCATTGA TTCAACGACT
GGAGGTTTTG AGGCTGGTAT TTCCAAAGAC GGTCAGACCCC GAGAGCATGC TCTTCTAGCA
TTTACACTGG GAGTGAAGCA AATGATTTGC TGCTGTAACA AGATGGATGC TACTACCCCA
AAATACTCAA AGGCTCGGTA TGAAGAAATT GTCAAGGAAG TGTCTTCTTA TTTGAAGAAG
GTTGGATACA ATCCAGACAA GATCCCTTTT GTGCCAATCT CTGGTTTTGA GGGCGATAAC
ATGATTGAGA GATCAAGCAA CCTTGATTGG TACAAGGGTC CAACACTGTT GGATGCCCTT
GACCAGGTTT CAGAGCCCAA ACGTCCATCA GACAAGCCAC TCAGGTTGCC ACTTCAGGAT
GTTTACAAGA TTGGTGGTAT TGGAACTGTA CCTGTGGGGC GAGTTGAGAC TGGAGTGATT
AAGCCAGGTA TGGTTGTAAC CTTTGGTCCA TCTGGGTTGA CAACTGAAGT TAAGTCTGTT
GAAATGCATC ATGAAGCTTT GTTAGAGGCA TTGCCTGGTG ACAATGTCGG ATTCAATGTC
AAGAATGTTG CTGTGAAAGA TCTCAAGCGA GGGTATGTAG CCTCTGATTC AAAGAATGAC
CCAGCGAAGG AGGCTGCAAA CTTCACTGCC CAGGTCATCA TCATGAACCA TCCAGGACAA
ATTGGAAATG GTTATGCACC TGTGCTGGAT TGCCACACAT GCCACATTGC TGTTAAGTTT
GCTGAGATCT TGACTAAGGT TGACAGACGA TCTGGTAAGG AACTTGAGAA GGAACCAAAG
TTCATTAAGA ATGGGGATGC TGCATTTGTC AAGATGATTC CAACCAAGCC CATGGTTGTG
GAAACATTCT CGGAGTACCC TCCATTGGGT CGTTTTGCTG TGAGGGATAT GCGTCAGACT
GTTGCTGTTG GAGTCATCAA GGCTGTTGAG AAGAAGGAAC CTTCTGGTGC TAAGGTTACC
AAGGCTGCTG CTAAGAAGAA GTGA

4. Determination of EF1α promoter sequence 4.1 Extraction of genomic DNA
Cultured cells of the TxN16-1 strain were frozen in liquid nitrogen and crushed in a mortar. Transfer 3.0 g of crushed cells to a 50 ml tube, add 6 ml of 2x CTAB solution (2% CTAB, 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA), and add 3 ml of 1x CTAB solution. It was added and mixed while incubating at 70° C. until there were no lumps. After that, the mixture thus obtained was reciprocally shaken at 55° C. and 40 rpm for 40 minutes, added with an equal amount of chloroform/isoamyl alcohol, and centrifuged at 2,800 rpm for 20 minutes after stirring. After centrifugation, the upper layer was collected. 3 ml of 1×CTAB solution was added to the remaining lower layer, shaken for 10 minutes, centrifuged at 2,800 rpm for 20 minutes, and added to the previously obtained upper layer to obtain a mixture. A 1/10 amount of a 10% CTAB solution (10% CTAB, 0.7 M NaCl) was added to this mixed solution, and the mixture was allowed to stand at 70° C. for 1 minute, and then an equal amount of chloroform/isoamyl alcohol was added. The mixed liquid thus obtained was stirred and then centrifuged at 2,800 rpm for 20 minutes. Collect the upper layer after centrifugation, add an equal amount of CTAB precipitation buffer (1% CTAB, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA) and mix, add 5 ml of the same buffer and mix, 5,000 rpm, 5 Centrifuge for 1 minute. After centrifugation, remove the supernatant, add 3 ml of NaCl-TE (1M NaCl, 10mM Tris-HCl (pH8.0), 1mM EDTA) to the precipitate and dissolve at 55°C. After ethanol precipitation, dissolve in TE buffer. to obtain a genomic DNA solution.

4.2 Determination of promoter sequences by inverse PCR
When the nucleotide sequence (SEQ ID NO: 6) of the contig encoding the EF1α gene was examined for restriction enzyme sites, it was found to have a restriction enzyme SphI site. The genomic DNA obtained above was treated with Sph I according to a standard method, purified by phenol/chloroform treatment and ethanol precipitation, and then ligated using T4 DNA ligase. Using this as a template, inverse PCR was carried out using the primers of SEQ ID NO: 7 and SEQ ID NO: 8. When this amplified product was subjected to agarose gel electrophoresis, a non-specific amplified product was confirmed, so the inverse PCR amplified product was used as a template, and primers were designed so that the annealing position would be closer to the inside (SEQ ID NO: 9, SEQ ID NO: 10). A single amplified product was obtained by nested PCR. In order to determine whether this amplification product contains a sequence located upstream of the EF1α gene, primers (SEQ ID NO: 11 and SEQ ID NO: 12) were designed, and PCR was performed using these primers using genomic DNA as a template. A single amplicon was observed. Using this amplified product as a template, primers of SEQ ID NOs: 13 to 17 were designed and sequence analysis was performed using a 3130xl Genetic Analyzer (Life Technologies) according to a standard method. In addition, in order to clarify whether the region upstream of the start codon contains an intron, the region encoding the 5'UTR portion was amplified from genomic DNA using the primers of SEQ ID NO: 18 and SEQ ID NO: 19, and sequenced in the same manner. Analysis was performed. As a result, the sequence shown in SEQ ID NO: 1 was successfully decoded, and this sequence includes a promoter region (SEQ ID NO: 2), a 5' untranslated region of about 0.3 kb (5' untranslated region 1: SEQ ID NOS: 3 and 5 'untranslated region 2: SEQ ID NO: 5) and an intron of about 2.4 kb (SEQ ID NO: 4).
The decoded sequences of SEQ ID NOs: 1 to 5 and the sequences of each primer are shown below.

・ Sequence number 1
GCATGCATCA ACAAACATTA GAGATTAGTA TTTCAAAAAA ATTGCTGGAT CTAAAATTAT
ATCCATTCAA GAATGGTTTC CGTCTCTGAT TGGATATCAT AGATTAAATT ATGATGGAGC
TTCAAAATAA TAAAAACCAG GTATATTTGT CACAAAAATC GCCCATCATA GACGCAACAG
ACAAAACATG AAAGCTTTAA TCAACCATAT ATTATTCAATC AAGATCTACA GAAAAACCAA
AACACAAAAT GATACACAGT GAAAGCTCTC GGTACTAGAT GACGAGAAAA CACCCACAGA
GCAAGGCAGA AAATATGATC AAGCTTTGAA TCTTTATTTC CACATTGCAG AGAAGGCTCC
AAAAATAGGT GTATGCCGCT TTTGGTGGCC CGCACACAGT ATTATTTAGT AAAAAGCGAA
AGAATAGTTC AGTATGTGTT GACCACGTGG CTCCGTGAAC TTTGTAAGGT TATTGACATT
ACTACTTATA ACACATCTTG GTAGGGAGTA CTTGTTTCGC CATCCATGTG GGATTAAAAT
TAATGATAAT ATCTTGGTGT GTTAGTTTAC ATTCAAAAAG GAAGCAACTT GATAACACAC
ATCATCCTAC GAGGATTTAA CTTACAGTTA CATATAATCT ACGTATTCAT CCAAGGTATA
ATGTCATCGG CCAATGAGCA GTATGATATT TATTAATGGT GGTTTGATAT TCATCTACTC
AAGGGATACT TTCATTATTG TCAAATGTAC ACATTTCTAC CACACTTCTA ATAATTAAGA
TGATGATAAA TCAATTTCTC TCATACGAAA ACACCATAAA ACAATTATAT TTTGCTTCTT
GCCCATTCTC ACTTTTCCTT TTTATGATCC TTGTTTCATG GATTACATAA TGCGAGGTGC
ATTAGAGACC TGACACTCGA CAACTATTTT GTTTCTAGAC CATTCTAACT TTTTTCCTTC
ATTTTTCCAGG GCTTTTATAA TGTGAAGTGC ATTGGAGACC TTACTCTTGA AGAATATATT
TCGTTAGGAT TTCAAGCTGA TGGCCTAGAG GTGAGCTTTT TTGAGATTTT TCTAGATGGG
TTGGGGTTTG GTAGTGCGAC ATTCTGATGA TTGTTAGTCT CTCTGTCTTT CTTTTATTCA
ATATTCTCAT TTTCAAAGCT CTTTTTTTTT CACAGTGGTG ATGCAATCAT TTTTAATGGT
AATTGATTGT TCTGACCAAA ACCATCTTGG TTAAAGTACT CTTGGGTGTT TGTAATTGGG
AAACATTTCT TACTTTTCTT TCGCTGACTA CACCTAGGTC ATCCATGGGG TATAACTATC
TACCCCACGG GTCTATGTTA TTGTATTTTT CTTTTGATTC AAAATGTTAA CCTTACAACA
TCTATGGTTA CATTGTGCCA ATTTTTTGGCA TTTTTCTCAA TTACGAGTTG ATGAACTTAA
ACTAACGTGT TTGGATTTTC AAGGTTGCAT TTATAATTTT GGAAAAACTC TACAACGAAG
TTAGAAGAGT GATGGAAAAC GCTGATCCCT AGCCTAGAAC AACACATATC TCTCATCCAA
TGGTCGATAC TCCCTTTGAG GCCAATCTTC ACCGTATGTT GGTAAGGATA TTCACAAAAT
GAGGTTATTG AATACACCTT GGGAACATTT AAATGCCAAA ATAGGAGATC TAGTATATAA
AAGCCAAGTG TTCGAATAAA TGACGAAACG TGATGTATTT TTGGTAAATA TCATAATTAC
AACTTAAAGA AAACATGGTC ATCTTCAAGA GCCAGTGTGA TTGTTCTATC AAATGAAACA
AACAGATATT CACCATGATT AGATCACTTT TGCGAACATA CTCATAGAAA TAAGAACTTT
GCAATAACGT ATGAACATTT GTCAAGGCAT AATAGGTAGT GGCAACTCAC TACATTGTTC
TCTCCTTCCC ATCACACAAA CATCAAACAT TTTAAGATAA CATACATTTC ATCGCAAAGT
ACAACTCCAA CAAACTAAAA ATTACTATAC GGTAAGGGCG GAAATTGCAA CCCATGTGCA
ATTGCTTGAA TTTTTTTTAT TTTTTTTTGG GAAGGTTCCT TCTTTCACAA AAGGTCACAA
GATTCTTTCT CGGAGATTAC CCACAGACAA TCTCACCGTT CGATATAAAA TTCGACGGTG
CGAAATAGAA ACAAATCATA ACCGTTGAAT GGTACATCGG ACGGCGAGGA AAATACCCTA
ATGCGCGAGG AAGGGTTTAG AGGCAAAGAG GCATATATGT TGGTTTGAAA GCATTTGTTA
GGTCTCTATT TTTGAGTATT CGTTTCTCCT TCATTTTTCT GTTCCGCCGC ACCGAGGATA
ACCTTCTTCG ATCTGCCAGG CGCTTCTGCA ACAAGGTGCT CTTTCTCATC CTAATCATCT
TCTTTGCTTA TTTATCTCCT GTCTTTCGGA AGAAAAAATG CTTATTTTCA ATTGCTGTCT
GTGCTGTTGA TGTATTTGCC TATGGATTGT CGAAGATCTG AGGAGATCTT GTCTTTATGC
TTTTTTTGTG TGATGGCGCA CGGTTATGGT GTTTGTTTTG AAGGATTGCA AGGTTATGGT
GTTTGTTTTG AAGGATTCCA AGGTTCGATG TCAAGTGTTT TATCGGGGGT GTTAGAGTTA
ATTCTATAGA ACATTAGCCG ATCTATCTGT GCAGTCGTCC TCTATTTCAG TTGTGTTTTA
GATCTGGCTG TCTATTTCAA TATCGTGTTC GTTTACCGCT TGTCGTTTTT GGCGTGAGTA
TTTTTTTTGC TTTTTTCCGT TTCTCGGATA AACTCCATAG ATCTGTTGGT TTCAGTTCGA
CGGATTGGTT GTTTATTTGT TCTTGGTTGT ATTCCACTTG GTCTCGAAAA GGCCGATCTT
TTGCAGGGTT TATCTGCTTA TACACATTGT CCTGTGTTTT TCGATTACAA CGTGATTTTA
TTCCCGCAAA AGTTTCCGTC AGAGCTTACA TTATTTTTTTT CGTTTCTCAG TACCTTACTT
TTCGTGTATT TAGATGCACT GAATTTGTGT CATAACGTGT ATGAATCTTA AAAGTTCTAT
AAGCTGCATA TGGTTGCAAT GTTAAAAATT ACCAGAATCC TCACTCTTTA ATTTTAACGT
CTTATACACT GTTGTTCTAT TGCACTACAT GAGAATTTGT TCTTGCAATA ATTTCCACCC
GATTTCTCTG AAAGTTTTCTT CTCGATAGGC AATTAAAGTG AATATATA CACACACACA
CACACACACA CTGTTGCACT GAATTTGTGC CATTTTCTGT ATATTAGGAA TTTTATAAAC
TGCATACGGT CGCTAGGTAA AATCTTCGCGC GAACTATTCA GTTTTATTTT TTAGCATATG
GAAATATTCT TTTCAGAAGA ATTTTTGACT GTAAGATAAT ATTTATATTT TCCCATGTAC
AGTTATAACC TTGTAGTTCT TTTGTTGCTT ATACCGTTTT ACCTGAACTT GCCCCCTTAA
ATATGAACTA TTATATTGAT ATTAAGTGCC CATTTTTGCT GATATCTTGA AGCCTTATAA
TATAATCAAT TTTTGATGTA TAGGAATTGT GTGGTCTAGC AAAAGGGGGA CTATAATGAG
AGATTTCTAT TCTAGTTTTA TTTTGTAGAT GGTACACTCA ATTTTTTTGTT GCAACTCTAC
ATAATATAAT CCTTTGCTTA ACGTGTGGAG GAGTGACCCA AGCAATTCTA ATTAGCCCCA
CAGCTGCATA GGGATTGAAG GGATTCTTTT GACAAAATGT TACCTGCAGA TTAGTTCAGG
TCAAAATTTT ACTTTTATTT TGAAATCTAC TTCTTTATAA TTCCTATAAT TTAGAAGCCA
TCCATTTTTT ACAAAACTTTA GCACCACTTA CCTGCAAAAA GTCTCCTCAA ATTTAGTTGG
ACACAAGTTG GTTGTTTGCC CCCTGCATGG GATCACAAGT AGCACGTGTT TTATATCGG
ACTGAATTCT CTAGGCTCAT TGTATTACTG GTTGTTAAAT AACCTATACT TCTTAATTCTT
TATCTCTGAA GTTAAAATAGC GTATGAATTT TTATATTTGA AGTCCAGTCA GAAACGTTTA
ATAATTTAAC TGTGATTATA GAGTTTCATG TATTGCTTCA ATGATCTAAA TATTCCATAA
TAGTATAT CTACTGTTAT TAATTGGCCA GCCATTTTTG TATGTTTAAT AATTTAAGTG
TGATTATAGA GTTTCATGTA TTGCTTCAAT GATCCAAATA TTCTTTAATA GTATATATCT
ATCGTTATCA ATTGGCCATC CATTTTTTAC AAACAACAGG ACCACTTGCC TGCAAAAAAGT
CTCCTCAAAT TTAGTTGGAC ACAAATTAGT TGTTTGCCCT CTCCACAAGT AGCCCGTGTT
TTATATCGG ACTGAATCTT CTAGGCTCAT TGTACTAGTG GTTGTTAAAT AACCTTTACC
TTTGAATCTT TATCTTTGAA GTAAAATAAC CTATACTTAT GAATATTTAA GTTTGAAGTC
CAGTCAGAAA TGTTAATAAT TAACTGATTA TAGAGTTTCA TGTATGCTTC AATGATCTAA
ATATTCAAAG CAATTGATAA TGGTAGATGT ATAATGGCAA AAGCAATTGA TAACGGCAGA
TATATACTAT AGGAATTTCT TGTTTCATGT CAAAGTGGTT TGGGTTGGGC TTTGAATTTT
TACACGATAA TAACTTTGAT GGGCATCTAT ATAGATTTTA GTGCTGTTTT TTATGTTCAT
CTGGTTGTTT AACATTTATG CTAAAATATG TATTATAATG TGCCCTAATA CTTTGGTCTG
TTCATACTGC AGTTTTTAAT CTCAAGCAAC CATG

・ Sequence number 2
GCATGCATCA ACAAACATTA GAGATTAGTA TTTCAAAAAA ATTGCTGGAT CTAAAATTAT
ATCCATTCAA GAATGGTTTC CGTCTCTGAT TGGATATCAT AGATTAAATT ATGATGGAGC
TTCAAAATAA TAAAAACCAG GTATATTTGT CACAAAAATC GCCCATCATA GACGCAACAG
ACAAAACATG AAAGCTTTAA TCAACCATAT ATTATTCAATC AAGATCTACA GAAAAACCAA
AACACAAAAT GATACACAGT GAAAGCTCTC GGTACTAGAT GACGAGAAAA CACCCACAGA
GCAAGGCAGA AAATATGATC AAGCTTTGAA TCTTTATTTC CACATTGCAG AGAAGGCTCC
AAAAATAGGT GTATGCCGCT TTTGGTGGCC CGCACACAGT ATTATTTAGT AAAAAGCGAA
AGAATAGTTC AGTATGTGTT GACCACGTGG CTCCGTGAAC TTTGTAAGGT TATTGACATT
ACTACTTATA ACACATCTTG GTAGGGAGTA CTTGTTTCGC CATCCATGTG GGATTAAAAT
TAATGATAAT ATCTTGGTGT GTTAGTTTAC ATTCAAAAAG GAAGCAACTT GATAACACAC
ATCATCCTAC GAGGATTTAA CTTACAGTTA CATATAATCT ACGTATTCAT CCAAGGTATA
ATGTCATCGG CCAATGAGCA GTATGATATT TATTAATGGT GGTTTGATAT TCATCTACTC
AAGGGATACT TTCATTATTG TCAAATGTAC ACATTTCTAC CACACTTCTA ATAATTAAGA
TGATGATAAA TCAATTTCTC TCATACGAAA ACACCATAAA ACAATTATAT TTTGCTTCTT
GCCCATTCTC ACTTTTCCTT TTTATGATCC TTGTTTCATG GATTACATAA TGCGAGGTGC
ATTAGAGACC TGACACTCGA CAACTATTTT GTTTCTAGAC CATTCTAACT TTTTTCCTTC
ATTTTTCCAGG GCTTTTATAA TGTGAAGTGC ATTGGAGACC TTACTCTTGA AGAATATATT
TCGTTAGGAT TTCAAGCTGA TGGCCTAGAG GTGAGCTTTT TTGAGATTTT TCTAGATGGG
TTGGGGTTTG GTAGTGCGAC ATTCTGATGA TTGTTAGTCT CTCTGTCTTT CTTTTATTCA
ATATTCTCAT TTTCAAAGCT CTTTTTTTTT CACAGTGGTG ATGCAATCAT TTTTAATGGT
AATTGATTGT TCTGACCAAA ACCATCTTGG TTAAAGTACT CTTGGGTGTT TGTAATTGGG
AAACATTTCT TACTTTTCTT TCGCTGACTA CACCTAGGTC ATCCATGGGG TATAACTATC
TACCCCACGG GTCTATGTTA TTGTATTTTT CTTTTGATTC AAAATGTTAA CCTTACAACA
TCTATGGTTA CATTGTGCCA ATTTTTTGGCA TTTTTCTCAA TTACGAGTTG ATGAACTTAA
ACTAACGTGT TTGGATTTTC AAGGTTGCAT TTATAATTTT GGAAAAACTC TACAACGAAG
TTAGAAGAGT GATGGAAAAC GCTGATCCCT AGCCTAGAAC AACACATATC TCTCATCCAA
TGGTCGATAC TCCCTTTGAG GCCAATCTTC ACCGTATGTT GGTAAGGATA TTCACAAAAT
GAGGTTATTG AATACACCTT GGGAACATTT AAATGCCAAA ATAGGAGATC TAGTATATAA
AAGCCAAGTG TTCGAATAAA TGACGAAACG TGATGTATTT TTGGTAAATA TCATAATTAC
AACTTAAAGA AAACATGGTC ATCTTCAAGA GCCAGTGTGA TTGTTCTATC AAATGAAACA
AACAGATATT CACCATGATT AGATCACTTT TGCGAACATA CTCATAGAAA TAAGAACTTT
GCAATAACGT ATGAACATTT GTCAAGGCAT AATAGGTAGT GGCAACTCAC TACATTGTTC
TCTCCTTCCC ATCACACAAA CATCAAACAT TTTAAGATAA CATACATTTC ATCGCAAAGT
ACAACTCCAA CAAACTAAAA ATTACTATAC GGTAAGGGCG GAAATTGCAA CCCATGTGCA
ATTGCTTGAA TTTTTTTTAT TTTTTTTTGG GAA

・ Sequence number 3
GGTTCCTTCT TTCACAAAAG GTCACAAGAT TCTTTCTCGG AGATTACCCA CAGACAATCT
CACCGTTCGA TATAAAATTC GACGGTGCGA AATAGAAACA AATCATAACC GTTGAATGGT
ACATCGGACG GCGAGGAAAA TACCCTAATG CGCGAGGAAG GGTTTAGAGG CAAAGAGGCA
TATATGTTGG TTTTGAAAGCA TTTGTTAGGT CTCTATTTTT GAGTATTCGT TTCTCCTTCA
TTTTTCTGTT CCGCCGCACC GAGGATAACC TTCTTCGATC TGCCAGGCGC TTCTGCAACA
AG

・ Sequence number 4
GTGCTCTTTC TCATCCTAAT CATCTTCTTT GCTTATTTAT CTCCTGTCTT TCGGAAGAAA
AAATGCTTAT TTTCAATTGC TGTCTGTGCT GTTGATGTAT TTGCCTATGG ATTGTCGAAG
ATCTGAGGAG ATCTTGTCTT TATGCTTTTT TTGTGTGATG GCGCACGGTT ATGGTGTTTG
TTTTGAAGGA TTGCAAGGTT ATGGTGTTTG TTTTGAAGGA TTCCAAGGTT CGATGTCAAG
TGTTTTATCG GGGGTGTTAG AGTTAATTCT ATAGAACATT AGCCGATCTA TCTGTGCAGT
CGTCCTCTAT TTCAGTTGTG TTTTAGATCT GGCTGTCTAT TTCAATATCG TGTTCGTTTA
CCGCTTGTCG TTTTTGGCGT GAGTATTTTT TTTGCTTTTT TCCGTTTCTC GGATAAACTC
CATAGATCTG TTGGTTTCAG TTCGACGGAT TGGTTGTTTA TTTGTTCTTG GTTGTATTCC
ACTTGGTCTC GAAAAGGCCG ATCTTTTGCA GGGTTTATCT GCTTATACAC ATTGTCCTGT
GTTTTTCGAT TACAACGTGA TTTTATTCCC GCAAAAGTTT CCGTCAGAGC TTACATTATT
TTTTTCGTTT CTCAGTACCT TACTTTTCGT GTATTTAGAT GCACTGAATT TGTGTCATAA
CGTGTATGAA TCTTAAAAGT TCTATAAGCT GCATATGGTT GCAATGTTAA AAATTACCAG
AATCCTCACT CTTTAATTTT AACGTTCTTAT ACACTGTTGT TCTATTGCAC TACATGAGAA
TTTGTTCTTG CAATAATTTC CACCCGATTT CTCTGAAAGT TTCTTCTCGA TAGGCAATTA
AAGTGAATAT ATATACACAC ACACACACAC ACACACTGTT GCACTGAATT TGTGCCATTT
TCTGTATATT AGGAATTTTA TAAACTGCAT ACGGTCGCTA GGTAAAATCT TCCGCGAACT
ATTCAGTTTT ATTTTTTAGC ATATGGAAAT ATTCTTTTCA GAAGAATTTT TGACTGTAAG
ATAATATTTA TATTTTCCCA TGTACAGTTA TAACCTTGTA GTTCTTTTGT TGCTTATACC
GTTTTACCTG AACTTGCCCC CTTAAATATG AACTATTATA TTGATATTAA GTGCCCATTT
TTGCTGATAT CTTGAAGCT TATAATATAA TCAATTTTTG ATGTATAGGA ATTGTGTGGT
CTAGCAAAAG GGGGACTATA ATGAGAGATT TCTATTCTAG TTTTATTTTG TAGATGGTAC
ACTCAATTTT TTGTTGCAAC TCTACATAAT ATAATCCTT GCTTAACGTG TGGAGGAGTG
ACCCAAGCAA TTCTAATTAG CCCCACAGCT GCATAGGGAT TGAAGGGATT CTTTTGACAA
AATGTTACCT GCAGATTAGT TCAGGTCAAA ATTTTACTTT TATTTTGAAA TCTACTTCTT
TATAATTCCT ATAATTTAGA AGCCATCCAT TTTTTACAAA CTTTAGCACC ACTTACCTGC
AAAAAGTCTC CTCAAATTTA GTTGGACACA AGTTGGTTGT TTGCCCCCTG CATGGGATCA
CAAGTAGCAC GTGTTTTATA TCCGGACTGA ATTCTCTAGG CTCATTGTAT TACTGGTTGT
TAAATAACCT ATACTTCTTA ATCTTTATCT CTGAAGTTAA ATAGCGTATG AATTTTTATA
TTTGAAGTCC AGTCAGAAAC GTTTAATAAT TTAACTGTGA TTATAGAGTT TCATGTATTG
CTTCAATGAT CTAAATATTC CATAATAGTA TATATCTACT GTTATTAATT GGCCAGCCAT
TTTTGTATGT TTAATAATTT AAGTGTGATT ATAGAGTTTC ATGTATTGCT TCAATGATCC
AAATATTCTT TAATAGTATA TATCTATCGT TATCAATTGG CCATCCATTT TTTACAAACA
ACAGGACCAC TTGCCTGCAA AAAGTCTCCT CAAATTTAGT TGGACACAAA TTAGTTGTTT
GCCCTCTCCA CAAGTAGCCC GTGTTTTATA TCCGGACTGA ATCTTCTAGG CTCATTGTAC
TAGTGGTTGT TAAATAACCT TTACCTTTGA ATCTTTATCT TTGAAGTAAA ATAACCTATA
CTTATGAATA TTTAAGTTTG AAGTCCAGTC AGAAATGTTA ATAATTAACT GATTATAGAG
TTTCATGTAT GCTTCAATGA TCTAAATATT CAAAGCAATT GATAATGGTA GATGTATAAT
GGCAAAAGCA ATTGATAACG GCAGATATAT ACTATAGGAA TTTCTTGTTT CATGTCAAAG
TGGTTTGGGT TGGGCTTTGA ATTTTTACAC GATAATAACT TTGATGGGCA TCTATATAGA
TTTTAGTGCT GTTTTTTATG TTCATCTGGT TGTTTAACAT TTATGCTAAA ATATGTATTA
TAATGTGCCC TATATACTTTG GTCTGTTCAT ACTGCAG
・ Sequence number 5
TTTTTAATCT CAAGCAACC

・ Sequence number 7
CACCTCGAGA GATTGTCTGT GGGTAATCT
・ Sequence number 8
CCCGGATCCT CTCAGGCTGA TTGTGCAGT
・ Sequence number 9
CACCTCGAGC CGAGAAAGAA TCTTGTGAC
・ Sequence number 10
CCCGGATCCA GAACATGATT ACAGGAACT
・ Sequence number 11
GCTCTCGGTA CTAGATGACG AGA
・ Sequence number 12
GAGCCTAGGA TGGACAGGAG CAC
・ Sequence number 13
TTAGCCGATC TATCTGTGCA
・ Sequence number 14
AACTAATTTGTGTCCAACTA
・ Sequence number 15
CACATCATCCTACGAGGATT
・ Sequence number 16
CTCTTCTAAC TTCGTTGTAG
・ Sequence number 17
GCACTGAATTTGTGCCATT
・ Sequence number 18
CACCTCGAGT GACTTTCCAG AGTCAACAT
・ Sequence number 19
AGATTACCCA CAGACAATCT

5. 5. Analysis of Promoter Activity 5.1 Construction of Plasmid To verify whether the obtained sequence has promoter activity, promoter activity was analyzed by incorporating it into a vector and introducing it into a plant. The activity was verified by replacing the cauliflower mosaic virus 35S promoter of the pGWB506 vector with the sequence of SEQ ID NO:1. The pGWB506 vector was treated with Hind III and Xba I to remove the cauliflower mosaic virus 35S promoter, and then ligated with the annealed oligo DNAs of SEQ ID NO: 20 and SEQ ID NO: 21 having the Apa I site, resulting in plasmid pGWB506-EF1a-. Built delCaMV.
・ Sequence number 20
AGCTTGTGG GCCCAAT
・ Sequence number 21
CTAGATTGGG CCCACA
First, the primers of SEQ ID NO: 22 and SEQ ID NO: 23 and yew genomic DNA were used as a template to amplify by PCR, the amplified product was treated with Apa I and Nhe I, and the plasmid pGWB506-EF1a-delCaMV was treated with Apa I and Xba I. The treated product was ligated according to a standard method to construct a plasmid pGWB506-EF1a-F. In addition, the primers of SEQ ID NO: 22 and SEQ ID NO: 24 and yew genomic DNA were amplified as a template, and the plasmid pGWB506-EF1a-delCaMV treated with Apa I and Xba I was ligated, and the intron (SEQ ID NO: 4) was extracted from pGWB506-EF1aF. Removed plasmid pGWB506-EF1a-delI, further amplified and ligated in the same manner using SEQ ID NO: 22 and SEQ ID NO: 25, and a plasmid in which intron (SEQ ID NO: 4) and 5' untranslated region 1 (SEQ ID NO: 3) were removed pGWB506-EF1a-delUI was constructed and used for activity analysis. In both constructed plasmids, the GFP gene was arranged downstream of the introduced sequence, which was used as an indicator of promoter activity.
・ Sequence number 22
GATGGGCCCT AGCATGCATC AACAAACATT
・ Sequence number 23
GGGGCTAGCG GTTGCTTGAG ATTAA
・ Sequence number 24
GGGGCTAGCG GTTGCTTGAG ATTAAAAACT TGTTGCAGA
・ Sequence number 25
GTGACCTTTT GTGAAAGAAG CTAGCTTCCCAA

5.2 Introduction and Observation by Particle Bombardment Method The constructed plasmid was introduced into yew cultured cells on the 12th day of passage according to a standard method using PDS-1000/He (Bio-Rad). 0.6 mg of gold particles (0.6 μm) was placed in a tube, 300 μl of 70% ethanol was added, and the mixture was stirred with a vortex mixer and centrifuged to precipitate the gold particles. The supernatant was removed, 100 µl of ethanol was added, the mixture was stirred and centrifuged in the same manner, and the supernatant was removed. Plasmid DNA, 50 μl of 2.5 M CaCl2 solution, 20 μl of 0.1 M spermidine, and 100 μl of ethanol were added to the solution with stirring so as to make 10 μg, and the supernatant was removed after centrifugation. 60 µl of ethanol was added to the solution to prepare a plasmid preparation solution. Add 12 μl of the plasmid preparation solution to the center of the microcarrier and leave it to dry at room temperature. Spread 1 g of yew cultured cells around the center of the WP agar medium and set them together in PDS-1000/He, and apply a pressure of 1100 PSI according to the standard method. typed in. After introduction, the cells were allowed to stand at 25°C for 24 hours, and the introduced cells were observed under a fluorescence microscope. As a result, GFP fluorescence was observed in all cells introduced with pGWB506-EF1a-F, pGWB506-EF1a-delI and pGWB506-EF1a-delUI (Fig. 2).
Compared to pGWB506-EF1a-F-introduced cells (Fig. 2(a)), pGWB506-EF1a-delUI-introduced cells (Fig. 2(b)) show weaker fluorescence, demonstrating that the 5' untranslated region enhances promoter activity. became. In addition, the pGWB506-EF1a-delI-introduced cells in which the intron was removed (Fig. 2(c)) had a higher fluorescence intensity and were equivalent to the pGWB506 (cauliflower mosaic virus 35S promoter)-introduced cells (Fig. 2(d)). was observed, and it was clarified that the promoter without the intron contributed to the high activity.

Claims (13)

A promoter consisting of one kind of DNA selected from the group consisting of the following (a) to ( d ) ;
(a) a DNA consisting of a nucleotide sequence having promoter activity selected from SEQ ID NO: 1;
(b) a DNA comprising the base sequence represented by SEQ ID NO: 2 and having promoter activity;
(c) a DNA having promoter activity , comprising a nucleotide sequence having at least 90% identity with a nucleotide sequence of one DNA selected from (a) and (b);
(d) in the nucleotide sequence of one DNA selected from (a) and (b) above, 1 or 2 to 25 bases are deleted, substituted or added within the range of having the promoter activity; DNA consisting of a base sequence. 2. The promoter according to claim 1, which is for plants. 3. The promoter according to claim 2, wherein the plant belongs to the genus Taxus, Nicotiana or Arabidopsis. 4. The promoter of claim 3, wherein the plant belongs to the genus Taxus. An expression vector comprising the promoter according to any one of claims 1 to 4. 6. The expression vector according to claim 5, comprising said promoter and an expression gene operably linked downstream of said promoter. A DNA fragment comprising the promoter according to any one of claims 1 to 4 and an expression gene operably linked downstream of the promoter. A transformant comprising the expression vector according to claim 6 or the DNA fragment according to claim 7. 9. The transformant according to claim 8, wherein a host transformed by said expression vector or said DNA fragment is a plant. 5. A transformant having an expression gene inserted downstream of the promoter according to any one of claims 1 to 4 existing in the genome of the host and functionally linked to the promoter. 11. The transformant according to claim 10, wherein said host is a plant. 12. The transformant according to claim 9 or 11, wherein the plant belongs to the genus Taxus, Nicotiana or Arabidopsis. 13. The transformant according to claim 12, wherein the plant belongs to the genus Taxus.

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