WO2016039300A1 - Novel diatom transformation vector and novel promoter sequence including same - Google Patents

Abstract

　The invention provides a novel promoter for transforming algae (e.g., diatoms) having a base sequence of SEQ ID NOS: 1-10 or a base sequence having 80% or higher homology with base this sequence, a novel transformation vector including this promoter, and a method for transforming algae using this vector.

Description

A novel diatom transformation vector and a novel promoter sequence contained therein

The present invention relates to a novel promoter for transforming algae, a vector containing the promoter, and a method for transforming algae using the vector.

Marine diatoms, one of the algae, are unicellular eukaryotes belonging to a group called so-called yellow plants. Diatoms are estimated to be responsible for about 20-40% of the world's CO ₂ fixation, and are extremely important organisms for the global environment as primary producers. Diatoms are organisms that synthesize fats and oils useful for humans, such as EPA (eicosapentaenoic acid). In general, transformation techniques are used for industrial use of living organisms, but it is known that the transformation efficiency of algae is low. Although various studies have been made to increase the transformation efficiency, the transformation method is still limited. In particular, for the horned diatom, which is one of the marine diatoms, its gene introduction method has been studied, but its transformation efficiency is low. Chaetoceros sp. Using a Tpfcp / nat plasmid containing a promoter derived from Thalassiosira pseudona . In this transformation, the transformation efficiency was as low as 1.5 to 6 transformants / 10 ⁸ cells, and no transgene expression was detected (Non-patent Document 1).

An object of the present invention is to provide a novel promoter for transforming algae (for example, diatom), a novel transformation vector containing the promoter, and a method for transforming algae using the vector.

As a result of investigations to solve the above problems, the present inventors have found that a promoter of a gene highly expressed in Chaetoceros gracilis can transform algae (for example, diatoms) extremely efficiently, and completed the present invention. did.

The present invention includes the following:
(1) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 10;
(2) a polynucleotide having a homology of 80% or more with respect to any one of the nucleotide sequences of SEQ ID NOS: 1 to 10 and functioning as a promoter that causes algae to express a target gene;
(3) a promoter having a base sequence in which one or several bases are added, deleted, and / or substituted to any one of SEQ ID NOS: 1 to 10 and causing algae to express a target gene A polynucleotide that functions as:
I will provide a. In the present specification, the polynucleotides (1) to (3) may be referred to as the promoter sequence according to the present invention or the promoter sequence according to the present invention.

The present invention provides a vector containing the polynucleotide.

The present invention
Provided is a vector comprising a first expression cassette comprising the polynucleotide and terminator sequences; or a second expression cassette comprising the polynucleotide, drug resistance gene and terminator sequences; or comprising both of these expression cassettes.

The present invention provides a method for transforming algae, characterized by introducing the above vector into a host algal cell.

Furthermore, the present invention provides a transformant by introducing a vector containing the above-mentioned polynucleotide and a fatty acid hydroxylase gene (eg, fatty acid 2-hydroxylase gene, more specifically, oleic acid 12-hydroxylase gene) into a host algal cell. The present invention provides a method for producing ricinoleic acid, characterized in that ricinoleic acid is obtained by culturing the transformant.

The promoter sequence according to the present invention can efficiently transform algae (for example, diatom).

C. Effect of Knowluseotricin (NTC) on Gracilis Growth Cells were subcultured in IMK medium containing various concentrations of antibiotics at OD700 = 0.07 and continuously irradiated (30 μmol photon m ⁻² s ^{− 1} ) The cells were grown at 20 ° C. for 28 days. Multiple pulse electroporation pulse plan explanation Multiple high-voltage poreing pulses (PP: 9 pulses, 300 V, 5 ms duration, 25 ms interval, 10% decay rate) promote the formation of temporary pores in the cell membrane The subsequent multiple low voltage transfer pulse (TP: 40 pulses for each polarity, 8 V, 50 ms duration, 50 ms interval, 40% decay rate) facilitates the introduction of DNA into the cell. C. using pUC vectors containing various promoters that drive the expression of the nourseotricin resistance gene (nat) . Transformation efficiency of gracilis cells Transformed cells were selected on IMK agar plates containing 400 μg / mL nourseotricin. Error bars indicate ± SE (n = 3-7). Compared with the conventional vector (left end), the promoter isolated this time showed a transformation efficiency of 100 to 200 times. Colonies of nourseotiricin resistant transformants on selective media C. with restriction enzyme cleavage site marked . Gracilis transformation vector map-Lhcr5 (fcp gene) promoter-Nourseotricin resistance gene (nat) Lhcr5 (fcp gene), Lhcf4 (fcp gene), nitrate reductase gene (NR), and acetyl-CoA acetyltransferase gene ( Each ACAT promoter was placed immediately before the foreign gene cloning site. In the vector, the constitutive ACAT promoter drives expression of the nourseotricin resistance gene (nat). A terminator for the Lhcr14 gene was placed at the end of both expression cassettes. A HindIII site or EcoRI site can be used to linearize the vector for transformation. The construct also contains an ampicillin resistance gene (AmpR) and an Escherichia coli origin of replication. C. with restriction enzyme cleavage site marked . Gracilis Transformation Vector Map-Nitrate Reductase Gene (NR) Promoter-Nourseotricin Resistance Gene (nat) C. with restriction enzyme cleavage site marked . Gracilis transformation vector map-Lhcf4 (fcp gene) promoter-Nourseotricin resistance gene (nat) C. with restriction enzyme cleavage site marked . Gracilis transformation vector map-acetyl-CoA acetyltransferase gene (ACAT) promoter-nourseotricin resistance gene (nat) Transgenic C.I. Transgene integration and mRNA expression in Gracilis cells (A) Transgenic C. elegans transformed with pCgLhcr5p-luc . PCR amplification of nat, luc and psb31 genes in Gracilis strains Genomic DNA isolated from wild type (WT) and four nourseotricin resistant clones was used as template DNA. M: 100 bp size ladder marker (B) RT-PCR analysis showing mRNA expression of each gene (C) Genomic Southern blot analysis Total genomic DNA (5 μg) was cleaved with EcoRI (left) or BamHI (right) and 1% agarose The gel was separated and blotted onto a nylon membrane. A DNA fragment containing the transgene was detected using a digoxigenin (DIG) labeled DNA probe for the luciferase coding region. Stability of luciferase activity after repeated subcultures Transgenic Lhcr5p-luc cells (strain 2) are subcultured in a one-week cycle in the presence (black) or absence (white) of nourseotricin Cultured. The activity of the first subculture in the presence of nourseotricine was defined as 100%. Transgenic C. expressing monomeric thistle green protein (mAG) under the control of the Lhcr5 promoter . Light micrograph (A) and fluorescence micrograph (B) of gracilis cells The presence of mAG protein was indicated by green fluorescence. The scale bar is 10 μm. Induction of luciferase (luc) activity under the control of the nitrate reductase gene (NR) promoter C. cerevisiae transformed with pCgNRp-luc . Gracilis transgenic cell lines were cultured in ammonium medium and transferred to nitrate medium (NO ₃ ⁻ ) or fresh ammonium medium (NH 4 ⁺ ). The x-axis shows the time after medium change. a. u. : Arbitrary unit error bar indicates ± SE (n = 3). Transgenic C. transformed with pCgLhcr5-CpFAH . Gracilis lipid analysis MS profile of CpFAH-4 strain MS profile of CpFAH-4 strain MS profile of CpFAH-4 strain MS profile of CpFAH-4 strain RT-PCR results Amount of ricinoleic acid in CpFAH-3 strain and CpFAH-4 strain Ricinoleic acid accumulated to 0.2 to 0.3% (w / w) of dry cell weight. C. Major fatty acid composition of Lacr5p-CpFAH strain of gracilis ricinoleic acid (18: 1-OH): 4.8-6.7% of total fatty acids in CpFAH strain 16: 1-OH: 1 of total fatty acids in CpFAH strain .0% C. with restriction enzyme cleavage site marked . Gracilis transformation vector map-Lhcf4 (fcp gene) promoter-Zeocin resistance gene (ble) C. with restriction enzyme cleavage site marked . Gracilis transformation vector map-nitrate reductase gene (NR) promoter-zeocin resistance gene (ble)

Promoter sequence of the present invention In the present invention, the promoter sequence means a sequence capable of controlling the expression of any target gene located downstream in an algal cell, for example, a diatom, particularly a hornwort cell.

The promoter sequence of the present invention is obtained by subjecting a polynucleotide constituting a non-coding region located upstream of a gene highly expressed in Chaetoceros gracilis to a method generally used in the art, such as draft analysis and RNA sequencing analysis. You can search by doing it.

Examples of the polynucleotide constituting the non-coding region located upstream of the gene highly expressed in Chaetoceros gracilis include the following sequences:
CgLhcr14p (SEQ ID NO: 1);
CgACATp (SEQ ID NO: 2);
CgLhcr5p (SEQ ID NO: 3);
CgACSLp (SEQ ID NO: 4);
CgNRp (SEQ ID NO: 5);
CgbTublinp (SEQ ID NO: 6);
CgATPSp (SEQ ID NO: 7);
CgLhcf4p (SEQ ID NO: 8);
CgLhcf1p_A (SEQ ID NO: 9);
CgLhcf1p_B (SEQ ID NO: 10).

The promoter sequence of the present invention includes (1) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 10.

The promoter sequence of the present invention has (2) 80% or more, 90% or more, 95% or more, 97%, or 99% or more homology to any one of SEQ ID NOS: 1 to 10, Furthermore, a polynucleotide that functions as a promoter that brings about expression of a target gene in algae can be mentioned.

The promoter sequence of the present invention includes (3) one or several of the nucleotide sequences of SEQ ID NOS: 1 to 10 (preferably 1 to 100, preferably 1 to 50, more preferably 1 to 30). More preferably, 1 to 10 or less is more preferable, and 1 to 5 or less is particularly preferable. As a promoter having an added, deleted and / or substituted base sequence and causing algae to express the target gene Examples include functional polynucleotides.

The promoter sequence of the present invention can be obtained by a method generally used in the art, for example, by separating from the upstream of a gene highly expressed in Chaetoceros gracilis , and can be amplified and used by PCR method. it can. Or you may synthesize | combine chemically.

The “target gene” is a gene encoding a protein desired to be produced, and is not particularly limited.

Vector of the present invention The present invention provides a vector comprising the above promoter sequence.
The vector of the present invention may contain one or more promoter sequences (for example, 1 to 5, 1 to 3, 1 or 2) according to the present invention.
The type of vector is not particularly limited as long as it can be introduced into algal cells. For example, vectors such as a plasmid vector, a phage vector, and a cosmid vector are preferable, and a plasmid vector is preferable.

The vector of the present invention can be used to introduce any desired gene. Examples of the target gene include ricinoleic acid synthase.

The vector of the present invention may contain any drug resistance gene whose expression can be controlled by the promoter sequence of the present invention. For example, drug resistance genes for selecting transformants (for example, nourseotricin resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, zeocin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene) Can be mentioned. Selection using multiple drug resistances can be used for multiple introductions of genes into host cells.

The vector of the present invention may contain other sequences included in general vectors. For example, it may contain a so-called regulatory sequence such as an operator, terminator, enhancer or the like that regulates the expression of the target gene. Furthermore, a promoter different from the present invention and a gene controlled by the promoter (for example, drug resistance gene) may be included.

The vector of the present invention can be prepared by methods well known to those skilled in the art. For example, it can be obtained by amplifying the promoter sequence of the present invention using a primer provided with an appropriate restriction enzyme site and inserting it into a donor vector cut with a restriction enzyme. Alternatively, it can be prepared by a method using an In-Fusion ^(trademark) reaction.

The promoter sequence of the present invention may be introduced into a donor vector as an expression cassette having a terminator sequence incorporated downstream thereof. The target gene may be incorporated between the promoter sequence and the terminator sequence.
In order to insert the target gene, it is desirable that a multiple cloning site having a plurality of restriction enzyme cleavage sites exists between the promoter sequence and the terminator sequence.
The terminator sequence in the present invention is not particularly limited as long as it functions in algal cells, and examples thereof include CgLhcr14 terminator sequence: CgLhcr14ter (SEQ ID NO: 11).

In the present invention, the expression cassette is:
A nucleic acid construct comprising at least a promoter sequence and a terminator sequence;
A nucleic acid construct comprising at least a promoter sequence and a gene of interest; or a nucleic acid construct comprising at least a promoter sequence, a gene of interest and a terminator sequence;
And may contain introns, spacer sequences, enhancer sequences and the like.

The vectors of the present invention are:
A first expression cassette comprising a promoter sequence and a terminator sequence; or a second expression cassette comprising a promoter sequence, a drug resistance gene and a terminator sequence, or both.
The first expression cassette can be used to insert a gene of interest. Alternatively, the target gene may be inserted into the first expression cassette.
The second expression cassette can be used to select transformants.

In the present invention, the first expression cassette is a promoter sequence according to the present invention (for example, CgLhcr14p; CgACATp; CgLhcr5p; CgACSLp; CgNRp; CgbTublinp; CgATPSp; CgLhcf4p; CgLhcf1p_A; And an expression cassette containing a terminator sequence (for example, CgLhcr14ter) incorporated downstream thereof.

In the present invention, the second expression cassette includes a promoter sequence according to the present invention (for example, CgLhcr14p; CgACATp; CgLhcr5p; CgACSLP; CgNRp; CgbTublinp; CgATPSp; CgLhcf4p; CgLhcf1p_A; And an expression cassette comprising a terminator sequence (eg, CgLhcr14ter) incorporated downstream thereof, and a drug resistance gene (eg, nat, ble) incorporated between the promoter sequence and the terminator sequence.

Transformation Method The present invention provides a method for transforming algae, which comprises introducing the vector into a host algal cell.

In order to introduce the vector of the present invention into host algae cells, methods well known to those skilled in the art can be used. For example, particle gun method, glass bead stirring method, microinjection method, Agrobacterium method, lithium acetate method, Examples of the method include a calcium phosphate method, a protoplast method, and a multiple pulse electroporation method.

The host used in the method of the present invention is not particularly limited as long as it is an algal cell. Examples of preferred host algae cells include diatoms (e.g., central diatoms or pterygium diatoms) and variants thereof, preferably hornflower and variants thereof, more preferably Chaetoceros gracilis and variants thereof.

In the method of the present invention, the host cell can be transformed by being retained in the host cell in a state in which the target gene is incorporated into the chromosome, plasmid, plastid or mitochondrial DNA in the host cell.

Culture The culture conditions of the transformant can be appropriately selected based on the host algal cell, the promoter sequence, and the target gene.
The transformant can be cultured using a normal culture method such as shaking culture or continuous culture. The culture conditions may be appropriately selected depending on the culture method and the like.

A method for producing ricinoleic acid Further, the present invention provides a transformant by introducing a vector containing the promoter sequence and fatty acid hydroxylase gene according to the present invention into a host algal cell, and cultivating the transformant to obtain ricinoleic acid. A method for producing ricinoleic acid is provided.
Ricinoleic acid is a monounsaturated fatty acid having 18 carbon atoms and a hydroxyl group at the C12 position. Ricinoleic acid is a fatty acid hydroxylase (for example, fatty acid 2-hydroxylase, more specifically oleic acid 12-hydroxylase), which is the 12th position of oleic acid, which is a monounsaturated fatty acid of ω-9 fatty acid having 18 carbon atoms. Is synthesized by adding a hydroxyl group.

Examples of the fatty acid hydroxylase gene include a fatty acid 2-hydroxylase (oleic acid 12-hydroxylase) gene (CpFAH (SEQ ID NO: 12)) derived from Claviceps purpurea NBRC6263 strain. The fatty acid hydroxylase gene of the present invention is also a gene encoding a protein having a nucleotide sequence having homology of 80% or more, 90% or more, 95% or more, or 97% or more with the CpFAH and having fatty acid hydroxylase activity. include.
Algae are generally known to have the ability to biosynthesize oleic acid. In the production of ricinoleic acid, the host is not particularly limited as long as it is an algal cell. Examples of preferred host algae cells include diatoms (e.g., central diatoms or pterygium diatoms) and variants thereof, preferably hornflower and variants thereof, more preferably Chaetoceros gracilis and variants thereof.
The medium for synthesizing ricinoleic acid is not particularly limited as long as it is a medium in which ricinoleic acid is synthesized by culturing the transformant.
When the host cell is diatom, it may be a general medium in which diatom can grow.
The culture temperature is not particularly limited as long as it is a temperature at which ricinoleic acid is produced. For example, when the host cell is a diatom (for example, hornflower), it is preferably 15 to 35 ° C, more preferably 18 to 25 ° C.
When the host is marine diatom, an example of the medium is Daigo IMK culture medium supplemented with sea salt and 0.2 mM Na ₂ SiO ₃ .
When the host is a marine diatom, the culture conditions include shaking culture under 50 μmol photon m ⁻² s ⁻¹ .

The following table shows examples of promoter sequences of the present invention: SEQ ID NOS: 1 to 10.

The following table shows the terminator sequence of CgLhcr14: CgLhcr14ter (SEQ ID NO: 11).

The table below shows the fatty acid 2-hydroxylase gene (CpFAH) (SEQ ID NO: 12) derived from ergot fungus ( Claviceps purpurea NBRC6263).

Hereinafter, the present invention will be described in more detail with reference to production examples, but the present invention is not limited to these examples.

Example 1
Materials and methods <Diatom culture>
C. Gracilis obtained from the University of Texas Culture Collection, Daigo IMK culture medium (Nippon Pharmaceutical Co., Ltd., Osaka, Japan) supplemented with sea salt (Sigma, St. Louis, MO, USA) and 0.2 mM Na ₂ SiO ₃ Cultured. Grow at 20 ° C. in an artificial weather incubator. A white fluorescent lamp provided an irradiance of 30 μmol photons m ⁻² s ⁻¹ under continuous light conditions. In particular, the diatom cell is an f / 2 medium containing 0.88 mM NH ₄ Cl (ammonium medium) or 0.88 mM NaNO ₃ (nitrate medium) as the only nitrogen source (Non-patent Document 6). In culture.

Construction of Nourseotricin Resistance Plasmid The nourseotricin resistance gene nat (SEQ ID NO: 13) was excised from the pYL16 plasmid (WERNER BioAgents, Jena, Germany) and subcloned into the BamHI-PstI site of pUC118 (Takara Bio). Then, using the primers described in Table A, fucoxanthin chlorophyll a / c-binding proteins (fcp) gene CgLhcr14 terminator region: CgLhcr14ter (110bp, SEQ ID NO: 11) were grown in genomic PCR, an In-Fusion ^{( TM)} reaction (Clontech, Palo Alto, CA, USA) was inserted into the downstream HindIII site. Finally, C.I. The Gracilis promoter region was amplified by genomic PCR and subcloned into the EcoRI-BamHI site upstream of pUC118. Table B below shows the length of the amplified promoter sequences and the accession numbers in the GenBank / EMBL / DDBJ database. The primer sets used in genomic PCR are shown in Table A below.

<Construction of pCgLhcr5p vector (registration number AB981621) and pCgNRp vector (registration number AB981622)>
(Registration number is DNA DATA BANK of JAPAN (DDBJ) registration number)
A constitutive gene expression plasmid pCgLhcr5p was constructed in several steps. The PCR primers used in the construction process below are listed in Table A above. To create the first expression cassette, the promoter region (811 bp) of fcp CgLhcr5 was inserted into the EcoRI-BamHI site of pUC118 in which the CgLhcr14 terminator sequence was inserted into the HindIII site. The resulting plasmid has a short cloning site (BamHI, XbaI, SalI, PstI), which makes it possible to insert the desired gene.
To construct a second expression cassette for antibiotic selection, a pUC118 vector containing the promoter region (626 bp) of the acetyl-CoA acetyltransferase (ACAT) gene and the CgLhcr14 terminator sequence was similarly constructed. The nat gene fragment with BglII and NsiI sites at the 5′-end and 3′-end was then amplified from pYL16 by PCR and inserted into the BamHI-PstI site of the second expression cassette. This destroys the BamHI-PstI site in the second expression cassette (BamHI / BglII and PstI / NsiI pairs create a common complementary end). Finally, the two expression cassettes are combined (the complete promoter-terminator cassette of the first expression cassette is amplified by PCR and the In-Fusion ^™ reaction in the EcoRI site of the pUC118 vector containing the second expression cassette. PCgLhcr5p vector was obtained. An MfeI site was introduced between the two cassettes to facilitate replacement of either expression cassette.
In order to construct a pCgNRp vector for inducible gene expression, the promoter region of CgLhcr5 in the pCgLhcr5p vector was excised by EcoRI / BamHI digestion and nitrate excised by the same restriction enzyme treatment from the nourseotricin resistant plasmid constructed above. It was replaced with the promoter region (631 bp) of the reductase CgNR gene.
The luciferase gene (SEQ ID NO: 44) and thistle green gene (SEQ ID NO: 45) were amplified by PCR using the primers in Table A above and inserted into the BamHI-PstI site of pCgLhcr5p or pCgNRp.

Sequence of pCgLhcr5p vector (registration number AB981621) (SEQ ID NO: 53)

Sequence of pCgNRp vector (registration number AB981622) (SEQ ID NO: 55)

< C. Gracilis transformation>
Multiple pulse electroporation was performed using a NEPA21 apparatus (NEPAGENE, Chiba, Japan) as described in Non-Patent Document 2, with some modifications. Logarithmic growth phase (OD ₇₀₀ = 0.2-0.4) diatom cells are collected by centrifugation (700 × g, 4 min), washed and 0.77 M with 10% (v / v) IMK medium. Resuspended with mannitol. Suspension (0.15 mL) containing 5.9 × 10 ⁶ cells was mixed with HindIII linearized plasmid (5 μg) and then transferred to a 0.2 cm wide electroporation cuvette. A 300V rectangular poreing pulse (pulse duration, 5 ms; 9 pulses; interval 50 ms; 10% decay rate) was applied, followed by an 8 V transfer pulse (pulse duration, 50 ms; 40 pulses for each polarity; interval 50 ms; 40% attenuation). After electroporation, the cells were transferred to 4 mL of IMK medium and then incubated at 20 ° C. for 16-20 hours under a light intensity of 30 μmol photons m ⁻² s ⁻¹ to recover with non-selective medium. Cells were collected by centrifugation (700 xg, over 4 minutes) and resuspended in IMK medium (0.2 mL). Transformed cells were selected on IMK agar plates containing 1% agar and nourseotricin (clonNat, WERNER BioAgents) (400 μg / mL).

<Analysis of insertion of introduced DNA into genome>
Genomic DNA was isolated from wild type and transformed cells subcultured 2-3 times on selective media using a DNeasy Plant Mini Kit (Qiagen, Venlo, The Netherlands). The primer pairs listed in Table A above were also used for this analysis for PCR detection of integrated genes in isolated genomic DNA. For Southern blotting, isolated genomic DNA was digested with EcoRI or BamHI, separated on a 1% agarose gel, and transferred onto Hybond NX membrane (GE Healthcare, Piscataway, NJ, USA) by capillary transfer. . UV cross-linking of the DNA transferred onto the Hybond NX membrane was performed using UVP CL-1000 crosslinker (UVP Inc., Upland, CA, USA). The digoxigenin labeling of the nat and lu cDNA probes, the hybridization between the probe and membrane-bound DNA, and the detection of the hybridization were performed according to the instructions using the DIG DNA Labeling and Detection Kit (Roche, Indianapolis, IN, USA). .

<Analysis of introduced DNA expression in transformed cells>
Total RNA was isolated from wild type and transformed cells using the RNeasy Plant Mini Kit (Qiagen). Reverse transcription (RT) -PCR was performed with PrimeScript ^™ RT reagent with gDNA Eraser (Takara Bio, Otsu, Japan) using the same primers used for genomic PCR analysis. Green fluorescence of monomeric thistle green protein (mAG) in transformed diatom cells was analyzed with a BZ-9000 fluorescence microscope (Keyence, Osaka, Japan). The area containing cells was activated at 480 nm and fluorescence emission was detected at 510 nm. Luciferase assays were performed using a luciferase assay kit (Promega, Madison, WI, USA) and a Lumat LB 9507 luminometer (Berthold, Oak Ridge, TN, USA). Luciferase activity was normalized by the protein concentration of the cell extract quantified using an Rc-Dc protein assay kit and a bovine serum albumin standard (Bio-Rad, Hercules, CA, USA).

Results <Selection of transformants Determination of concentration of nourseotricin>
C. Transformation that acquired resistance to nourseotiricin To screen for gracilis , C. a. We tested for the concentration of nourseotricine required to inhibit the growth of gracilis . As shown in FIG. 1, during the culture period of 28 days, 400MyugmL ^-1 Knowle Theo tricine (liquid IMK medium) is C. Gracilis growth was completely inhibited. Cell growth was inhibited with 300 μgmL- ¹ nourseotricin but began to recover 7 days after inoculation. Similar results were obtained with IMK agar plates regardless of cell density differences. Therefore, C.I. IMK medium containing 400 μgmL- ¹ nourseotricin was used for selection of gracilis transformants and subsequent maintenance culture.

< C. Using a nourseotricin resistant plasmid containing various promoters . Gracilis transformation>
Ten promoters with high expression of downstream genes (estimated by RNA sequencing analysis) were selected. Each was isolated using the primers in Table A above. The respective sequences are shown in Tables 1-10. The genes downstream of the isolated promoter encoded the following proteins (Table B above); four FCP proteins (Lhcr5, Lhcr14, Lhcf1 (CgLhcf1p_A and CgLhcf1p_B gene products), and Lhcf4), acetyl -CoA acetyltransferase (ACAT), long chain acetyl-CoA synthetase (ACSL), β-tubulin (bTublin), ATP synthase (ATPS), and nitrate reductase (NR). In pUC118, an isolated promoter containing a 5′-UTR was inserted upstream of the nat gene. After the nat gene, the 3′-UTR sequence of the Lhcr14 gene was inserted as a transcription terminator. The vector was linearized by restriction enzyme digestion and used for the transformation reaction.
C. of nourseotricin resistant plasmid Introduction into gracilis cells was performed by multiple pulse electroporation (Non-patent Document 2). Short multiple high voltage pulses (pored pulses) promote the formation of temporary holes in the cell membrane, followed by multiple multiple low voltage pulses (transfer pulses) that facilitate the delivery of DNA into the cell. .

FIG. 3A shows antibiotic-resistant colony count / 10 ⁸ transformed cells obtained using pUC vectors containing different promoters. Although multiple pulse electroporation was used, consistent with previous reports (Non-Patent Document 1), the pTpfcp / nat vector produced only a few antibiotic-resistant colonies. The remaining vector containing the promoter provided 100-400 antibiotic resistant colonies / 10 ⁸ transformed cells.

Differences in transformation efficiency with promoters may reflect differences in promoter activity during selection with selective media. In this experiment, IMK medium containing NaNO ₃ as the nitrogen source was used, so that the inducible NR promoter was active and could produce a significant number of antibiotic resistant colonies.
As shown in FIG. 3B, clear colonies were observed 14 days after transformation, and the presence of the nat gene was confirmed in all antibiotic-resistant colonies by PCR analysis.

<Construction of expression plasmids pCgLhcr5p and pCgNRp>
The vector pCgLhcr5p / CgACATp-nat contains a constitutive promoter for the fucoxanthin chlorophyll a / c binding protein (fcp) gene, and the vector pCgNRp / CgACATp-nat is a nitrate reductase (NR) gene to drive transgene expression. Of inducible promoters (FIG. 4). Both vectors had a second expression cassette, and the acetyl-CoA acetyltransferase (ACAT) promoter in the second expression cassette driven nat gene expression for antibiotic selection. For simplicity we will call these vectors pCgLhcr5p and pCgNRp. BamHI, XbaI, SalI, and PstI sites can be used to insert foreign genes, and EcoRI or HindIII sites can be used to linearize the vector for transformation reactions.

<Transformed C.I. Constitutive expression of transgene in Gracilis cells>
C. To test the expression of foreign genes in Gracilis , the firefly luciferase gene (luc) was inserted into pCgLhcr5p and pCgNRp, resulting in pCgLhcr5p-luc and pCgNRp-luc. Using these vectors, C.I. Gracilis cells were transformed and the transformation efficiency was> 100 transformants / 10 ⁸ cells. On average, about 50% of antibiotic resistant strains contained both nat and luc genes. FIGS. 5 (A) and 5 (B) show the results of genomic PCR and reverse transcription (RT) PCR analysis of four Lhcr5p-luc transformants (strains 1-4), in which the transgene Both (nat and luc) genomic insertion and mRNA expression were confirmed. The psb31 gene, which encodes the photosystem II membrane superficial subunit, was analyzed as a control. The psb31 fragment size is different in genomic PCR (765 bp, including intron) and RT-PCR (566 bp in mature mRNA), suggesting that genomic DNA contamination in RT-PCR analysis is negligible, and the luc gene It was confirmed that mRNA and nat gene mRNA were certainly expressed in these strains. The transgene copy number in the transgenic genome was analyzed by Southern blot analysis using the luc gene as a probe. Genomic DNA was digested with HindIII or BamHI. These have zero and one recognition site, respectively, in the linearized pCgLhcr5p-luc vector. The result shows that the introduced foreign DNA has incorporated at most only one or two copies into the chromosomal DNA (FIG. 5C), which helps to reduce the risk of unwanted insertional mutations. I suggested it would be.

To test the stability of the heterologously expressed gene, the strain 2 Lhcr5p-luc (which showed high luc activity) was repeatedly subcultured in the presence or absence of nourseotricin, Luciferase activity was assessed at the end of the subculture period. As shown in FIG. 6, there is no difference between the four subcultures regardless of the presence or absence of antibiotics, which makes the transgene stable even in the absence of antibiotic selection pressure. It was shown to be inherited and expressed.

Using the pCgLhcr5p vector and the pCgNRp vector to visualize the expression, the expression of a fluorescent protein (green fluorescent protein, green coral Galaxidae-derived thistle green (AG) (Non-patent Document 3)) targeting intracellular protein products is performed. Experimented. AG has a high extinction coefficient, fluorescence quantum yield, and acid stability and produces a brighter green fluorescence in HeLa cells than eGFP. A monomeric version of modified AG (mAG) is well suited for visualization of the intracellular localization of the fusion protein. As shown in FIG. 7, about 50% of the transformants having the transgene showed green fluorescence of mAG.

<Inducible expression of luciferase gene by NR promoter>
By simply changing the nitrogen source in the culture medium, the promoter of the nitrate reductase gene is transferred to Cylindrotheca fusiformis and T. et al . It has been shown that it can be used to control the expression of transgenes in pseudona (Non-patent Documents 4 and 5). C. Gracilis NR gene (CgNR) expression may be controlled by the same mechanism: induction is turned off in ammonium media and induced when growing in nitrate media.
C. To test the effectiveness of PCgNRp vectors for inducible expression of the transgene in gracilis, incorporate luc gene into the multiple cloning site to give the pCgNRp-luc. After selection with nourseotricin, about 50% (14/29) of antibiotic resistant colonies carry the luc gene, and about 60% (9/14) of transformants carrying the luc gene. Luciferase activity was shown in IMK medium containing only nitrate as the nitrogen source.

To analyze the kinetics of inducible luc expression driven by the CgNR promoter, one of the CgNRp-luc transformant strains was grown in f / 2 medium containing NH ₄ Cl as a single nitrogen source and then Transferred to f / 2 medium containing NaNO ₃ (FIG. 8). The luc activity was induced after 60 minutes and increased more than 20 times the pre-induction value within 8 hours.

Example 2
Synthesis of ricinoleic acid A fatty acid 2-hydroxylase (CpFAH) gene derived from ergot fungus ( Claviceps purpurea NBRC6263 ) was incorporated into the multiple cloning site of the pCgLhcr5p vector to obtain pCgLhcr5p-CpFAH by multiple pulse electroporation . Introduced into Gracilis . The obtained transformants CpFAH-3 and CpFAH-4 were transformed into Daigo IMK culture medium (Nippon Pharmaceutical Co., Ltd.) supplemented with sea salt (Sigma, St. Louis, MO, USA) and 0.2 mM Na ₂ SiO ₃ . (Osaka, Japan) (50 mL) was cultured with shaking (100 rpm) at 20 ° C. under 50 μmol photon m ⁻² s ⁻¹ for 8 days.

FIG. 9 shows transgenic C. elegans transformed with pCgLhcr5-CpFAH . The lipid analysis result of gracilis is shown. In the CpFAH-4 strain, ricinoleic acid was detected as the fourth largest peak.
FIG. 10 shows the MS profile of the CpFAH-4 strain.

FIG. 11 shows the RT-PCR results.
PCR conditions

PCR enzyme, KOD FX NEO (TOYOBO JAPAN)
25 cycles (10 seconds, 98 ° C .; 30 seconds, 55 ° C .; 20 seconds, 68 ° C.)
Expected product length; 152 bp (CpFAH) and 168 bp (α-tubulin)
Size marker: 1 kb + ladder (Life Technologies Carlsbad, CA, USA)

FIG. 12 shows the amount of ricinoleic acid in the CpFAH-3 and CpFAH-4 strains. Ricinoleic acid accumulated to 0.2-0.3% (w / w) of dry cell weight.
FIG. 13 shows the main fatty acid composition of the CpFAH-3 and CpFAH-4 strains.
Ricinoleic acid (18: 1-OH) was 4.8-6.7% of the total fatty acids in both CpFAH strains. 16: 1-OH was 1.0% of the total fatty acids in the CpFAH strain.

Example 3
An expression vector conferring zeocin resistance was constructed in the same manner as described above in <Construction of pCgLhcr5p vector (Registration number AB981621) and pCgNRp vector (Registration number AB981622)>.
Specifically, in order to construct a second expression cassette for zeocin selection, a ble gene having BglII and NsiI sites at the 5′-end and 3′-end from the pPha-T1 vector (GenBank: AF219942.1) ( The gene) fragment conferring zeocin resistance was amplified by PCR. This fragment was inserted into the BamHI-PstI site of the pUC118 vector containing the promoter region (626 bp) of the acetyl-CoA acetyltransferase (ACAT) gene and the terminator sequence of CgLhcr14 to create a second expression cassette. This insertion destroys the BamHI-PstI site in the second expression cassette (BamHI / BglII and PstI / NsiI pairs create a common complementary end). Next, this second expression cassette was excised with SacI and HindIII and inserted into a nourseotricin resistant plasmid (pCgLhcf4p and pCgNRp) cleaved with the same restriction enzyme, thereby replacing the second expression cassette for zeocin selection.
Using the zeocin resistant plasmids (pCgLhcf4p-ble and pCgNRp-ble) constructed in this manner, the C.I. When Gracilis was transformed, it was found to have a transformation efficiency (> 100 transformants / 10 ⁸ cells) equivalent to the nourseotricin resistant plasmid.
A map of a zeocin resistant plasmid ( C. gracilis transformation vector) in which restriction enzyme cleavage sites are described is shown in FIG.

The sequence of pCgLhcf4p-ble (SEQ ID NO: 57)

pCgNRp-ble sequence (SEQ ID NO: 60)

Claims (5)

1. (1) a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1 to 10;
   (2) a polynucleotide having a homology of 80% or more with respect to any one of SEQ ID NOS: 1 to 10 and functioning as a promoter that brings about expression of a target gene in algae; or (3) SEQ ID NOs: 1 to A polynucleotide having a base sequence in which one or several bases are added, deleted, and / or substituted with respect to any one of the base sequences, and functioning as a promoter that causes algae to express a target gene.
2. A vector comprising the polynucleotide according to claim 1.
3. A first expression cassette comprising the polynucleotide of claim 1 and a terminator sequence; and a second expression cassette comprising the polynucleotide of claim 1, a drug resistance gene, and a terminator sequence;
   The vector according to claim 2, comprising:
4. An algal transformation method comprising introducing the vector of claim 2 or 3 into a host algal cell.
5. A ricinol acid is obtained by introducing a vector comprising the polynucleotide of claim 1 and a fatty acid hydroxylase gene into a host algae cell to obtain a transformant, and culturing the transformant to obtain ricinoleic acid. Acid production method.

Images