WO2016039300A1 - Nouveau vecteur de transformation de diatomée, et nouvelle séquence de promoteur comprenant celui-ci - Google Patents

Nouveau vecteur de transformation de diatomée, et nouvelle séquence de promoteur comprenant celui-ci Download PDF

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WO2016039300A1
WO2016039300A1 PCT/JP2015/075372 JP2015075372W WO2016039300A1 WO 2016039300 A1 WO2016039300 A1 WO 2016039300A1 JP 2015075372 W JP2015075372 W JP 2015075372W WO 2016039300 A1 WO2016039300 A1 WO 2016039300A1
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gene
vector
promoter
polynucleotide
seq
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Japanese (ja)
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伊福　健太郎
康浩 菓子野
秀哉 福澤
昌孝 梶川
順 小川
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公立大学法人兵庫県立大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats

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  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fatty acid hydroxylase gene eg, fatty acid 2-hydroxylase gene, more specifically, oleic acid 12-hydroxylase gene
  • 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).
  • NTC Knowluseotricin
  • 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
  • TP 40 pulses for each polarity, 8 V, 50 ms duration, 50 ms interval, 40% decay rate
  • TP 40 pulses for each polarity, 8 V, 50 ms duration, 50 ms interval, 40% decay rate
  • Each ACAT promoter was placed immediately before the foreign gene cloning site.
  • 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
  • Gracilis transformation vector map-Lhcf4 fcp gene
  • promoter-Nourseotricin resistance gene nat
  • C. with restriction enzyme cleavage site marked .
  • 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 ⁇ g) 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.
  • DIG digoxigenin
  • 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 ⁇ m.
  • 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.
  • Gracilis transformation vector map-Lhcf4 (fcp gene) promoter-Zeocin resistance gene (ble) C. with restriction enzyme cleavage site marked .
  • 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.
  • 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
  • the “target gene” is a gene encoding a protein desired to be produced, and is not particularly limited.
  • Vector of 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the target gene may be inserted into the first expression cassette.
  • the second expression cassette can be used to select transformants.
  • 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.
  • a promoter sequence for example, CgLhcr14p; CgACATp; CgLhcr5p; CgACSLp; CgNRp; CgbTublinp; CgATPSp; CgLhcf4p; CgLhcf1p_A
  • an expression cassette containing a terminator sequence for example, CgLhcr14ter
  • 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.
  • a promoter sequence for example, CgLhcr14p; CgACATp; CgLhcr5p; CgACSLP; CgNRp; CgbTublinp; CgATPSp; CgLhcf4p; CgLhcf1p_A
  • an expression cassette comprising a terminator sequence (eg, CgLhcr14ter) incorporated downstream thereof, and
  • the present invention provides a method for transforming algae, which comprises introducing the vector into a host algal cell.
  • the host used in the method of the present invention is not particularly limited as long as it is an algal cell.
  • 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 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.
  • 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 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 ⁇ -9 fatty acid having 18 carbon atoms. Is synthesized by adding a hydroxyl group.
  • fatty acid hydroxylase for example, fatty acid 2-hydroxylase, more specifically oleic acid 12-hydroxylase
  • fatty acid hydroxylase gene examples 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.
  • 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.
  • 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.
  • the host cell when the host cell is a diatom (for example, hornflower), it is preferably 15 to 35 ° C, more preferably 18 to 25 ° C.
  • an example of the medium is Daigo IMK culture medium supplemented with sea salt and 0.2 mM Na 2 SiO 3 .
  • the culture conditions include shaking culture under 50 ⁇ mol photon m ⁇ 2 s ⁇ 1 .
  • the following table shows the terminator sequence of CgLhcr14: CgLhcr14ter (SEQ ID NO: 11).
  • CpFAH fatty acid 2-hydroxylase gene
  • 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 ⁇ mol photons m ⁇ 2 s ⁇ 1 under continuous light conditions.
  • 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.
  • 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).
  • 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.
  • TM In-Fusion
  • 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.
  • the resulting plasmid has a short cloning site (BamHI, XbaI, SalI, PstI), which makes it possible to insert the desired gene.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ mol 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 ⁇ g / mL).
  • 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.
  • 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). .
  • Reverse transcription (RT) -PCR was performed with PrimeScript TM 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).
  • 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), ⁇ -tubulin (bTublin), ATP synthase (ATPS), and nitrate reductase (NR).
  • ACAT acetyl -CoA acetyltransferase
  • ACSL long chain acetyl-CoA synthetase
  • bTublin ⁇ -tubulin
  • ATPS ATP synthase
  • NR nitrate reductase
  • 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.
  • 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.
  • fcp fucoxanthin chlorophyll a / c binding protein
  • NR nitrate reductase
  • 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.
  • ACAT acetyl-CoA acetyltransferase
  • FIG. 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.
  • a fluorescent protein green fluorescent protein, green coral Galaxidae-derived thistle green (AG) (Non-patent Document 3)
  • AG green coral Galaxidae-derived thistle green
  • 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.
  • 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 ⁇ mol photon m ⁇ 2 s ⁇ 1 for 8 days.
  • CpFAH fatty acid 2-hydroxylase
  • FIG. 9 shows transgenic C. elegans transformed with pCgLhcr5-CpFAH .
  • the lipid analysis result of gracilis is shown.
  • 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 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 ( ⁇ -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).
  • 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.
  • a nourseotricin resistant plasmid pCgLhcf4p and pCgNRp
  • 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.

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Abstract

L'invention fournit un nouveau promoteur qui possède une séquence de bases des SEQ ID N°1 à 10 ou une séquence de bases possédant une homologie de 80% ou plus avec cette séquence de bases, et qui est destiné à la transformation des algues (de la diatomée, par exemple). L'invention fournit également un nouveau vecteur de transformation comprenant ce nouveau promoteur, et un procédé de transformation mettant en œuvre ce vecteur.
PCT/JP2015/075372 2014-09-08 2015-09-07 Nouveau vecteur de transformation de diatomée, et nouvelle séquence de promoteur comprenant celui-ci WO2016039300A1 (fr)

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JP2016547436A JP6573400B2 (ja) 2014-09-08 2015-09-07 珪藻の新規形質転換ベクターおよびその含有する新規プロモーター配列

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JP2014-182607 2014-09-08
JP2014182607 2014-09-08

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WO2016039300A1 true WO2016039300A1 (fr) 2016-03-17

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1149695A (ja) * 1997-08-07 1999-02-23 Noevir Co Ltd 老化防止用皮膚外用剤
JPH11116431A (ja) * 1997-10-13 1999-04-27 Noevir Co Ltd 皮膚外用剤
JP2014511140A (ja) * 2011-02-02 2014-05-12 ソラザイム、インク 組み換え油産生微生物から生成される用途に応じた油
WO2014088560A1 (fr) * 2012-12-04 2014-06-12 Exxonmobil Research And Engineering Company Promoteurs de tetraselmis et terminateurs destinés à être utilisés dans des cellules eucaryotes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1149695A (ja) * 1997-08-07 1999-02-23 Noevir Co Ltd 老化防止用皮膚外用剤
JPH11116431A (ja) * 1997-10-13 1999-04-27 Noevir Co Ltd 皮膚外用剤
JP2014511140A (ja) * 2011-02-02 2014-05-12 ソラザイム、インク 組み換え油産生微生物から生成される用途に応じた油
WO2014088560A1 (fr) * 2012-12-04 2014-06-12 Exxonmobil Research And Engineering Company Promoteurs de tetraselmis et terminateurs destinés à être utilisés dans des cellules eucaryotes

Also Published As

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JP6573400B2 (ja) 2019-09-11
JPWO2016039300A1 (ja) 2017-06-22

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