WO1995014094A1 - Chaine d'adn codant pour la glycerol-3-phosphate acyltransferase et son utilisation - Google Patents

Chaine d'adn codant pour la glycerol-3-phosphate acyltransferase et son utilisation Download PDF

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WO1995014094A1
WO1995014094A1 PCT/JP1994/001956 JP9401956W WO9514094A1 WO 1995014094 A1 WO1995014094 A1 WO 1995014094A1 JP 9401956 W JP9401956 W JP 9401956W WO 9514094 A1 WO9514094 A1 WO 9514094A1
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plant
dna
atase
amino acid
fatty acids
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PCT/JP1994/001956
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English (en)
Japanese (ja)
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Osamu Nishizawa
Toshihiro Toguri
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Kirin Beer Kabushiki Kaisha
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Publication of WO1995014094A1 publication Critical patent/WO1995014094A1/fr

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition

Definitions

  • the present invention relates to a glyceronol-3-transphosphate acyltransferase produced by spinacia (Spinacia. 1 erace L.). (Hereinafter referred to as ATase) and its use in the use of DNA strands capable of biotechnological production o
  • phosphatidylglycerol (hereinafter referred to as PG) has a high temperature at which it changes from a liquid crystal state to a solid state, and hardens at high temperatures. It is a fat. Therefore, the temperature sensitivity of the biological membrane changes greatly depending on the nature of PG.
  • the stiffness of PG is determined by the type of fatty acids that make it up.
  • the transfer of fatty acids to glycerol-3-phosphoric acid (hereinafter referred to as G-3-P) is performed by ATase. That is, ATase catalyzes the transfer of the fatty acid moiety to G-3-P from the complex of fatty acid and acyl carrier-protein (hereinafter referred to as ACP). .
  • the complex of fatty acid and ACP, which are substrates of ATase, is palmitoyl-ACP (hereafter 16: 0-ACP) and oleoyl-ACP (hereafter 18: 1) -ACP).
  • which substrate A AT se selects depends on the nature of AT A se itself, that is, the substrate selectivity of AT A se.
  • the substrate selectivity of A Tase has been investigated in various plants. For example, the cold-tolerant plants spinach and end ATase have high substrate selectivity for 18: 1-ACP, and the PGs of these plants are comparable. Liquid crystal state even at extremely low temperatures [European Jiannore, Obno "" Euro Chemistry (Eur. J. 5 iocii em.
  • proteins that are synthesized in the cytoplasm and translocate to chloroplasts require a translocation peptide on the amino-terminal side of the protein [Naturia -(Nature 313 (1985) 358-363).
  • the content of saturated molecular species in PG of spinach is about one-third of endogenous species and about one-fourth of Arabidopsis thaliana.
  • the substrate selectivity of urnose ATase for 18: 1-ACP is about twice that of endogenase and about three times that of Arabidopsis thaliana.
  • the gene for spinach ATase is conferring cold tolerance on low temperature sensitive plants, and is far superior to those known so far. It seems to be that .
  • the amino acid sequence of spinach ATase is similar to that of Arabidopsis thaliana and Endo as a whole, but differs locally. It is conceivable that the difference in substrate selectivity of ATase is due to these regions.
  • the present invention provides a DNA chain capable of biotechnologically producing ATAse useful for changing the properties of chloroplast membrane lipid PG, and a use thereof.
  • the present inventor has found it difficult to obtain cDNA for spinach ATase by a method in which cDNAs of ATase derived from four kinds of plants are used as probes. Derived from these plants Focusing on the amino acid sequence of ATase, a region with high homology is selected to synthesize the corresponding DNA, and these are combined as a primer to obtain cDNAZ mRNA. Include between primers by PCR method with Neubrid as type
  • the DNA chain capable of producing glycerol-3-transphosphate from the present invention in a biotechnological manner is characterized by that of glycerol-3-3.
  • the present invention was the first to elucidate the amino acid sequence of A Tase derived from spinach and the nucleotide sequence of its gene.
  • the present invention also relates to the use of the above-described DNA strand, specifically, a plant obtained by incorporating the DNA into a plant cell to change its properties, and a method for imparting such a property change.
  • the present invention relates to the present invention, wherein the above-mentioned DNA is incorporated, and the expression of this DNA allows the glycerol 13-phosphate to be produced. This is because the production of acyltransferase has resulted in higher levels of unsaturated fatty acids in the fatty acids bound to lipids than in the original composition.
  • the above-mentioned DNA is incorporated into plant cells, and the DNA is expressed in a cultured plant.
  • acyltransferase which reduces the amount of unsaturated fatty acids in the fatty acids bound to the lipids of this plant, It also relates to a method for changing the fatty acid composition in plant lipids, characterized in that the composition is higher than the composition.
  • the present invention relates to a plant which is naturally suffering from a cold injury, wherein the above-mentioned DNA is incorporated, and the expression of this DNA causes the glycerolysis.
  • 1-phosphoryl acyltransylase phenylase is produced, and the phosphazigrellicellose contained in the biological membrane of the cells of this plant. Because the content of unsaturated fatty acids in the fatty acids bound to the plant is higher than its original composition, it can cause injury even at temperatures lower than the temperature at which the plant is naturally injured.
  • the present invention relates to a plant which is characterized as not to receive the above DNA, and incorporates the above DNA into a plant cell, and integrates this DNA in a cultured plant.
  • the present invention also relates to a method for changing the sensitivity of a plant to temperature.
  • FIG. 1 is an explanatory diagram showing the results of Northern analysis of mRNAs of five types of plants using the cDNA of Cabochina ATase as a probe.
  • RNA derived from pumpkin, spinach, rice, rice, and end, respectively are electrically Transcribed after electrophoresis.
  • FIG. 2 is an explanatory diagram showing a comparison of known amino acid sequences of ATase from four kinds of plants.
  • the underlined portions of 1 to 6 indicate the regions used to synthesize the primers of PCR.
  • the number to the left of the amino acid sequence indicates the number of the amino acid when the reported N-terminal amino acid is number 1.
  • FIG. 3 is an explanatory diagram following FIG. 2 and showing a comparison of amino acid sequences.
  • FIG. 4 is an explanatory diagram showing the positional relationship between cDNA clones SpAT # 1 and SpAT # 4 of spinach ATase and a simple restriction map.
  • Bold lines indicate open reading frames.
  • FIG. 5 shows the nucleotide sequence of cDNA of spinach ATase.
  • FIG. 3 is an explanatory diagram showing a column and an amino acid sequence.
  • FIG. 6 is an explanatory diagram showing an array following FIG.
  • FIG. 5 is an explanatory diagram showing the results of Northern analysis using a cDNA of spinach ATable as a probe.
  • the DNA strand capable of producing ATase according to the present invention which is capable of producing ATase in a biotechnological manner, that is, the ATase gene has ATase activity, and the amino acid sequence is substantially the same as SEQ ID NO: 1.
  • the amino acid sequence shown in Figure 10 from position 103 to position 472 or the amino acid sequence shown in Figures 5 and 6) It encodes the polypeptide that is the constituent of the acid sequence (from A to B).
  • the term “DNA strand” refers to a polynucleotide chain having a certain length.
  • the “DNA strand” includes the amino acid sequence of the polypeptide encoded by the amino acid sequence (including the modified or modified amino acid sequence described below).
  • the DNA strand (degenerate as described below) is used. Isomers) and finite ones.
  • the DNA strand contains a gene encoding the ATase and is useful for biotechnological production of the polypeptide. In most cases, only this finite length of DNA strand can be used.
  • the 5 '-upstream and 3'-downstream of this, the appropriate length of DNA strands can be linked to this polypeptide to produce the polypeptide in a biotechnological manner. That is,
  • a transfer peptide is required at the amino-terminal side of mature ATase in order for ATase to be synthesized in the cytoplasm and transferred to chloroplasts. That's it. Therefore, when it is necessary to transfer the ATase synthesized in the cytoplasm to chloroplasts, the gene encoding ATase should be located at the 5 'upstream of this transfer peptide. It is necessary to have a DNA strand that encodes the code.
  • DNA strand refers to the specific length (from amino acid at position 103 to amino acid 47 at position 2 in the sequence of SEQ ID NO: 1).
  • Length to amino acid The length of A to B in the case of the corresponding amino acid in Figs. 5 and 6), as well as the length of this specific length. Includes those in the form of strands or circular DNA strands, of which the DNA strands are members
  • DNA chains according to the present invention may be present in plasmids or phage DNAs as part of members.
  • Microorganisms in the form in which the DNA strands have been inserted, as well as inserted into plasmid or phage or genomic DNA Or a form that exists in phage particles or plants.
  • the bacteria here do not even contain Escherichia coli bacterium.
  • the preferred form of the DNA strand according to the present invention is
  • the promoter, the DNA strand encoding the translation regulatory region, and the transfer peptide to the chloroplast are encoded.
  • Known DNA strands and terminators can be used by appropriately combining known ones.
  • the DNA strand encoding the transfer peptide to chloroplast is the nucleotide sequence of the ATase gene obtained by the present invention (SEQ ID NO: 1 (or FIG. 5), base number 238). — 360, or 55 — 360), or a well-known, end-to-end rib mouth-1,5-diphosphate
  • SEQ ID NO: 1 or FIG. 5
  • base number 238 or a well-known, end-to-end rib mouth-1,5-diphosphate
  • the small subunit gene of bboxylase can also be used.
  • the DNA strand according to the present invention is specified by the amino acid sequence that it encodes.
  • This polypeptide has ATase activity, and the amino acid sequence is substantially 103 in the amino acid sequence represented by SEQ ID NO: 1.
  • the amino acid sequence shown in Fig. 6 is from A to B (Fig. 6 is a continuation of Fig. 5). That's not true).
  • the amino acid sequence is substantially the amino acid sequence from position 103 to position 472 of the amino acid sequence shown in SEQ ID NO: 1.” This means that some of the amino acids may have deletions, substitutions, additions, etc., as long as the polypeptide has ATase activity. It is an indication of this.
  • SEQ ID NO: 1 (or FIGS. 5 and 6) Amino acid sequences (from 61 to 102 (from J to A)) shown in FIG.
  • the position from position 1 to position 102 (C 'to A) is considered to be at least a part of the transfer peptide from the structural comparison with Cabochya ATase. This portion also includes any alterations or modifications to the amino acid sequence described above.
  • a typical polypeptide having ATase activity in the present invention is 103 of the amino acid sequences of SEQ ID NO: 1 (or FIGS. 5 and 6). From position 42 to 72 (A to B) [or from position 61 to position 47 to 72 (C to B) or from position 1 to position 47 (C 'to B) )] And consists of 370 (or 411 or 472) amino acids, which are conventionally known as amino acids. The array is unknown.
  • the ATase targeted by the present invention is EC 2.3.1. It is the enzyme specified in 15.
  • the DNA chain encoding A TAS e is SEQ ID NO: 1.
  • Degenerate isomer means a DNA strand that differs only in a degenerate coden and is capable of encoding the same polypeptide.
  • SEQ ID NO: 1 (or FIGS. 5 and 6) from position 361 to position 1470 (from A to B)
  • the code corresponding to any of the amino acids for example, the code corresponding to A sn (AAC) is in a degenerate relationship with this.
  • a substance converted into AAT is referred to as a degenerate isomer in the present invention.
  • Preferred embodiments of the DNA strand according to the present invention are those having at least one termination code (for example, TAG) at the 3'-end. .
  • TAG termination code
  • the 5′-upstream and (or) 3′-downstream of the DNA strand of the present invention Good. Acquisition of DNA strand
  • One method for obtaining a DNA strand having a base sequence that encodes the amino acid sequence of ATase described above is to reduce the amount of the DNA strand according to the method of nucleic acid synthesis. Is partly chemically synthesized.
  • the ATase gene has been isolated from four types of plants (Cabochia, Arabidopsis thaliana, Endo and Kiuri), and its DNA structure has been isolated. Has been clarified.
  • the present inventors have made DNA synthesized from several conserved sequences into a primer, Further, a cDNA fragment of spinach ATase was obtained by performing PCR using spinach mRNA as a type. Using this fragment as a probe, a spinach-based cDNA library was screened to obtain the DNA strand of the present invention (for details, see Examples below).
  • the present invention has a higher substrate selectivity for 18: 1 ACP than ATase derived from cold-tolerant plants (Arabidopsis thaliana and Endo) with known DNA structures. (Eur. J. Biochem, 129 (1983) 629-636: PCT / J P92 for the Arabidopsis thaliana and the window) / 0002), the DNA strands of the present invention can be transformed into cold-sensitive plants (for example, rice, corn, potato, sweet potato, cucumber, pee man).
  • cold-sensitive plants for example, rice, corn, potato, sweet potato, cucumber, pee man.
  • an appropriate peptide in addition to the DNA according to the present invention is required.
  • the expression control sequences, such as the encoding DNA strand and terminator be introduced into the plant cells in an integrated form.
  • Promoters include, for example, a calorimeter promoter, a 35 s promoter, and a promoter for nonolin synthase. , Such as promoters for ribose nitric acid carboxylase / oxygenase subunits, etc.
  • the DNA of the present invention can be obtained by a known method using an appropriate combination of a terminator of nopaline synthase, a terminator of octabin synthase, and the like.
  • the chains can be introduced (transformed) into a plant cell, and the transformed plant can be obtained by culturing.
  • the DNA strand encoding the chloroplast transfer peptide is the nucleotide sequence of the ATase gene obtained by the present invention (SEQ ID NO: 1 (or FIG. 5)). 3 8 — 36 0, or 5 5 — 360 0), or a known one, for example, end rebose-1, 5-nilinic acid
  • SEQ ID NO: 1 or FIG. 5
  • the small subunit gene of Norreboxylase can also be used.
  • Methods for introducing foreign genes into plants include various methods that have already been reported and established, for example, the Ti Plus of the agglom- ber liposome.
  • the method of using the gene as a vector and the method of introducing the gene into the plant protoplasts by using an electoral opening section, etc. Can be used as appropriate depending on the plant in which the plant is to be introduced (for example, 'P 1 ant ge netictr an sfo rma ti on and en e exp ressi on;
  • Suitable plant materials for introducing genes include leaf pieces, stem pieces, tuber pieces, protoplasts, callus, pollen, pollen tubes, etc. You can choose.
  • a saturated molecular species of phosphatidylglycerol that is, a lipid molecular species that causes phase separation of a biological membrane and causes cold damage to a plant , which results in plants that are resistant to low temperatures.
  • RNA containing polyA [po1y (A) + RNA] was isolated.
  • DNA (cDNA) complementary to the above-mentioned poly (A) + RNA was obtained by the method of Gubler (Gub 1er) et al. [Gene, 25 (1983) 263-269]. And synthesized. In this case, Oligo is used as the primer.
  • the synthesized double-stranded cDNA is treated with EcoRl methylase to obtain a restriction enzyme.
  • EcoRI linker (dGGAATTCC; manufactured by Takara Shuzo) was added to both ends.
  • the excess linkers are cleaved and removed with the restriction enzyme EcoRI, and the free linkers are removed from the cDNA fraction by the gel filtration method. And were concatenated.
  • DNA a scan full A over sheet over the children; to (G i gap a ck Go ld S trata made gene) and Nono 0 Tsu cage in g, host of vinegar gtll U-les-down Soviet ⁇ cDNA La I got one library.
  • the pre-digestion fraction is 6 X SSPE [1 XSS PE is mM phosphate buffer (pH 7.0), 1 mM EDTA, 0.15 NaCl], 0.23 ⁇ 4 SDS and 100
  • the test was carried out at 60 ° C for 16 hours in a solution consisting of ug / m1 dicine sperm DNA.
  • the membrane was then placed in 2X SSC (1X SSC 0.15M Na, 15mM sodium citrate) solution at room temperature for 15 minutes twice, then 4 times.
  • the plate was washed by shaking twice at 2 ° C for 15 minutes.
  • Fig. 1 shows an autoradiogram (replicated) of a probe of the cDNA of Cabochya ATase.
  • 1 to 3 correspond to the sense chain
  • 4 to 6 correspond to the antisense chain.
  • primer sets (9 sets) selected one by one from each group were used.
  • Genomic DfiA, cDNA library DNA, etc. may also be considered as the type II used in the PCR reaction, but in this case, the mRNA is converted into type II and reverse transcriptase is used.
  • cDNA was synthesized and an eDNA / mRNA hybrid was formed, and this was used as type III.
  • This reaction was based on the Gene Amp TM RNA PCR Kit (Takara Shuzo). Synthesis reaction solution of single-stranded first c DNA is, 50 mM T ⁇ i s-HC l (H 8.
  • the cDNA library was screened.
  • a recombinant phage containing spinach cDNA was infected with E. coli Y1090 (r-) strain, and about 30,000 plaques were formed on a plate about cm in diameter. Forty plates thus formed were prepared, and the phage was transferred to a Ni-membrane (Hyb. Nd-N +; manufactured by Amersham).
  • the probe DNA was labeled with 32 P-d CTP using a Multitip DNA DNA Label Kit (Amersham).
  • the hybridization solution was a hybridization solution having the same composition as described above (Example 2), and was carried out at 65 ° C for 16 hours.
  • SpATtl ⁇ 3 SpAT # l is about 1.5 kb in length, and the amino acid sequence of its open reading frame is replaced with that of other plants. Comparison with the amino acid sequence of ATase revealed that the N-terminal amino acid was missing. Therefore,
  • SpAT # 4 is a clone that extends about 120 bp to the N-terminal side from SpAT # l, and the remaining base sequence of about bp is completely identical to SpAT # 1. did. Also, a translation start code that matches the reading frame with the open reading frame of SpAT # l
  • the homologue of the amino acid sequence of ATase from other plants with the amino acid sequence is not N-terminal, which is presumed to be a chloroplast transit signal (transit peptide). Excluding the sequence consisting of about 1QQ amino acids, it was 6Q3 ⁇ 4; strong, and more than 50% overall including the transition signal (GENETYX data Analysis software; software developed by software).
  • Example 2 The nylon membrane subjected to the Northern analysis of Example 2 was 100 in Q.015i SDS solution. C, heating for 15 seconds As a result, the probe DNA that had been hybridized was removed.
  • Northern analysis was performed using the cDNA clone SpAT # 1 of Horenso ATase as a probe and using this probe. The drying and cleaning temperatures were 65 ° C and 50 ° C, respectively. [Hybridization solution and The composition of the cleaning solution is the same as that described above (Example 2)]. As a result, a clear band of about 2 kb was detected in the lane that transcribed the spinach mRNA. This position is for the mRNAs of Kapotiya and Arabidopsis thaliana, respectively.
  • the band was the same as that detected when the ATase cDNA was analyzed as a probe.
  • Fig. 7 shows this autogram (replica).
  • CDNA encoding spinach ATase was introduced into tabaco cells as follows.
  • Binary plasmid pBI121 (C10ntech) for plant expression was cut with restriction enzymes Sacl and BamHl, and the cut end was blunt-ended with Klenow fragment. Thereafter, both ends were ligated using a T4 DNA ligation.
  • the plasmid pBI121 (-GUS) thus obtained does not contain the / S-Glucuronidase gene (G gene) and is a 35S plasmid of the molybdenum strain.
  • the plasmid containing cDNA (SpAT # 14) obtained in Example 3 was digested with the restriction enzyme EcoRI, and the vector was analyzed by low-melting point agarose gel electrophoresis. Plus pTZ R and spinning
  • the cDNA of ATase was separated, and cDNA was cut out from the gel and prepared. In addition, the cut end was made blunt by Klenow fragment.
  • the plasmid pBI12i (-GL'S) obtained above was cleaved with the restriction enzyme Xbal, and treated in the same manner as cDNA to make both ends blunt.
  • the thus obtained ATase cDNA and the plasmid pBil (-GUS) were ligated using a T4 DNA ligase to obtain a 35S promoter.
  • a plasmid pBil 21-35S SPT for expression containing DNA of the target and the spinach ATase cDNA and the NOS terminator was obtained.
  • a gr 0 bacteriumtume ⁇ aciens LBA4404 (Cl on tech) was added to 50 ml of YEB medium (beef ex 5g, yeast ex lg, peptone lg, sucrose 5 g per one) It was inoculated into 2 mM MgS0 4, P H7. 4), after 24 hours at 28 ° C, and the culture cells were collected by 3000 r pm, 4 ° C, 20 minutes of centrifugation. Bacteria cells in ml l mM
  • This culture solution 501 was spread on YEB agar medium (1.2% agar) containing lQQppm canamaisin and cultured at 28 ° C for 2 days.
  • a colony colony was selected, and a plasmid DNA was prepared from the colony by the anorecali method.
  • This flop La scan Mi-de-DNA was the separation of the by Ri DNA fragment in 1% ⁇ gas and Russia over the scan gel electrophoresis after digestion with an appropriate restriction enzyme, 3 ho was La Belle at 2 P U-les-down Soviet ⁇ AT Southern analysis using ase cDNA as a probe confirmed that it contained the plasmid pBU21-35SSPT. This acronym is called ALBSSPT.
  • the ALBSSPT obtained in (2) above was shake-cultured in an LB liquid medium containing 50 ppm of canamycin at 28 ° C for 12 hours. , Centrifuge for 3 minutes and collect cells. Washing was performed with 1 ml of LB medium to remove namosine. The cells were collected by centrifugation at 10,000 rpm for 3 minutes, and resuspended in 1.5 ml of LB liquid medium to obtain a bacterial solution for infection.
  • MS-B5 including Clarholan 250 ppm and Canamycin l QO ppm It was transferred to a medium and grown, and used as a material for the following tests.
  • tobacco transformant tobacco cell SPI
  • tobacco cell SPI tobacco cell SPI
  • Example 6 Fatty acid analysis of phosphatidylglycerol in transformed tobacco cells
  • phosphatidylglycerol was obtained by the following method. (PG) was prepared and its fatty acid composition was analyzed.
  • DEAE-Toy opear 1 A mixture of 25 ml of 650 C (Tohoku Souichi) suspension with 25 ml of 1 M aqueous sodium peracid solution (pH 7.0) was converted to acetic acid type. Wash sequentially with ethanol, and finally suspend in methanol, pack in a 2 cm inner diameter column to a height of 1.5, and further add 50 ml of chloroform. Washed with honolem: metanol (1: 4, volume ratio).
  • methanol solution (1: 4, volume ratio) Glycerol, Jacquard Sill Jacyl Glycerol, phosphatidylsolenol, and phosphatidylethanol Eluted and removed.
  • phosphatidylserine is eluted with 50 ml of acetic acid to remove it, and 15 ml of chlorozozolem: methanol is obtained.
  • the fraction obtained in (2) was separated by silica gel-TLC plate # 5721 (Merck).
  • the developing solvent used was porcine porphyrin: acetone: methanol: drunkic acid: water (50: 20: 10: 15: 5, volume ratio).
  • Serin is sprayed and fluorescence is generated under ultraviolet light, and the fraction of PG showing the same mobility as the standard PG is taken with silica gel.
  • a test tube with a screw stopper 2.5 ml of 5% methanolic hydrochloric acid was added thereto, and the mixture was allowed to react at 85 ° C for 2 and a half hours in a completely closed atmosphere to methylate the fatty acid.
  • the resulting fatty acid methyl ester was extracted four times with 2.5 ml of hexane, and the solvent was removed under reduced pressure to concentrate.
  • Gas chromatograph was used for fatty acid methyl analysis.
  • Gas chromatograph GC-17AAWFW (Shimadzu Corporation) was used for the fractionation of fatty acid methyls, and the identification of fatty acids was performed by comparing the retention time with standard fatty acid methyls. .
  • Chromatographic pack C-R7A Plus (Shimadzu Corporation) was used for quantification. Table 1 shows the results.
  • the content of saturated fatty acids ( : Q + 16: lt + stearate (: 0)) in the fatty acids bound to PG is 72 ⁇ 1% in the control untransformed tapaco.
  • the expression was as low as 65 ⁇ 1%.
  • 16: 0 and 16: 1 PG are strongly bound to the second position of PG, from the result of fatty acid analysis, The content of saturated species in PG is calculated to be 44% 2% in non-transformed tobacco> 30 ⁇ 2% in transformed tobacco cells.
  • Plant 16 0 + 16: 1 t + 18: 0 Saturated »Seed Non-transformed tobacco 72 soil 1 44 ⁇ 2 TO-transformed tobacco 65 soil 1 30 ⁇ 2 As described above, according to the present invention, From Urenzo
  • the ATase derived from spinach is more unsaturated fatty acid ( ⁇ ) than the ATase derived from cold-tolerant plants whose DNA structure is known ( ⁇ -alphanucleus and endogenous).
  • the present inventors have confirmed that the substrate selectivity for ACP is high, and the DNA strand of the present invention was introduced into a cold-sensitive plant according to a known method and expressed. By doing so, the plant can be given even more cold-tolerance than using the known gene.
  • the spinach-derived ATase had significantly higher substrate selectivity for unsaturated fatty acids than other low-temperature-resistant ATases.
  • the A ⁇ ase-like characteristics 3 ⁇ 4r and O It is understood that.
  • the DNA obtained by the present invention is used in a probe, it is present in a cold-tolerant plant and is a substrate. It seems that the cloning can be done more easily by the known method.
  • the composition of unsaturated fatty acids of PG of the plant is greatly enhanced, and thus the DNA strand of the present invention is improved.
  • the method and the method using the same are useful as a gene and a method capable of conferring strong low temperature tolerance to a low temperature sensitive plant.
  • Sequence type nucleic acid
  • Organism name Spinach
  • I3S J3S isy and 3jy jqi 3 ⁇ aqj ⁇ Siy 3 ⁇ m ⁇ H IU MV ias
  • n na na de A N3 ⁇ n I0 " ⁇ ⁇ 3 ⁇ ” S 3l V m usy B

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Abstract

L'invention concerne une chaîne d'ADN présentant une séquence de base codant pour un polypeptide présentant une activité de glycérol-3-phosphate acyltransférase (ATase) et une séquence d'acides aminés spécifiée provenant essentiellement de l'épinard. On peut, en introduisant la chaîne d'ADN dans une plante sensible au froid, induire une expression conférant à la plante une résistance au froid accrue par rapport à celle conférée en utilisant un gène d'ATase connu provenant d'une plante résistant au froid.
PCT/JP1994/001956 1993-11-19 1994-11-18 Chaine d'adn codant pour la glycerol-3-phosphate acyltransferase et son utilisation WO1995014094A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU10345/95A AU1034595A (en) 1993-11-19 1994-11-18 Dna chain coding for glycero-3-phosphate acyltransferase and use thereof

Applications Claiming Priority (2)

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JP31421293 1993-11-19
JP5/314212 1993-11-19

Publications (1)

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WO1995014094A1 true WO1995014094A1 (fr) 1995-05-26

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997005246A1 (fr) * 1995-07-27 1997-02-13 Kirin Beer Kabushiki Kaisha Brins d'adn codant la glycerol-3-phosphate acyltransferase
WO2000078974A2 (fr) * 1999-06-21 2000-12-28 National Research Council Of Canada Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01235594A (ja) * 1988-03-14 1989-09-20 Norio Murata グリセロール−３−リン酸アシルトランスフェラーゼをコードするｄｎａ鎖

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01235594A (ja) * 1988-03-14 1989-09-20 Norio Murata グリセロール−３−リン酸アシルトランスフェラーゼをコードするｄｎａ鎖

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EUR. J. BIOCHEM., Vol. 129 (1983), p. 629-636. *
NATURE, Vol. 324, No. 13 (1986), RANDALLK, SAIKI et al., "Analysis of Enzymatically Amplified beta-Globlin and HLA-DQalpha DNA with Allele-specific Oligonucleotide Probes", p. 163-166. *
SCIENCE, Vol. 240 (1988), CAROL A. RHODES et al., "Genetically Transformed Maize Plants from Protoplasts", p. 204-207. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997005246A1 (fr) * 1995-07-27 1997-02-13 Kirin Beer Kabushiki Kaisha Brins d'adn codant la glycerol-3-phosphate acyltransferase
US6160203A (en) * 1995-07-27 2000-12-12 Kirin Beer Kabushiki Kaisha DNA strands coding for glycerol-e-phosphate acyltransferase
CN100419077C (zh) * 1995-07-27 2008-09-17 麒麟控股株式会社 编码甘油-3-磷酸酰基转移酶的dna链
WO2000078974A2 (fr) * 1999-06-21 2000-12-28 National Research Council Of Canada Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase
WO2000078974A3 (fr) * 1999-06-21 2001-07-05 Ca Nat Research Council Surexpression dans des levures ou des plantes d'un gene codant pour la glycerol 3-phosphate acyltransferase

Also Published As

Publication number Publication date
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