JP3416071B2 - Method for producing higher plant cell and higher plant into which fatty acid desaturase gene has been introduced - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene encoding a protein having the activity of desaturating the Δ9-position of a fatty acid bound to lipid (hereinafter referred to as Δ9-desaturase), a vector containing the gene, The present invention relates to a plant into which the gene is introduced and a method for producing the plant.

[0002]

2. Description of the Related Art Membrane lipids, which are lipids that compose biological membranes of living things, change (phase-separate) from a liquid crystal state to a solid state as the ambient temperature decreases. Then, the properties of the biological membrane change with the phase separation. That is, in the solid state of membrane lipids, the selectivity of substance permeation is lost, so that the biological membrane cannot perform its original function, resulting in damage to cells (cold damage).
Is believed to occur.

The phase transition temperature of the membrane lipid, which is the temperature at which the liquid crystal state changes to the solid state or vice versa, is mainly the degree of unsaturation of the fatty acid acyl group bonded to the lipid (the number of double bonds in the carbon chain). ). That is, when both fatty acid acyl groups bonded are saturated fatty acids, the phase transition temperature of this lipid molecular species is higher than room temperature,
The phase transition temperature of a lipid molecular species having at least one double bond in the bound fatty acid acyl group is approximately 0 ° C. or lower (Santaren, JF et al., Biochim. Biophys. Acta, 68).
7: 231, 1982).

Generally, the position of the double bond of fatty acid is
The number of carbon atoms from the end of the carboxyl group to the carbon having a double bond is shown following Δ (delta). In addition, the total number of double bonds is shown after the colon after the total number of carbon atoms. For example, linoleic acid is described as 18: 2Δ9,12 and its structure is CH ₃ (CH ₂ ) ₄ CH═CHCH ₂ CH═CH (CH ₂ ) ₇ C
It is OOH. The position of the double bond may be described after ω (omega), which indicates the number of carbon atoms from the terminal of the methyl group of the fatty acid to the carbon having the double bond.

Among the membrane lipids of higher plants, only phosphatidylglycerol (PG) has a relatively large number of saturated molecular species, and the cause of cold injury in plants is due to the phase transition of PG (Murata, N. et al. ., Plant Cell Physiol., 23: 1071,19
82; Roughan, PG, Plant Physiol., 77: 740, 1985), and glycerol whose molecular species composition of PG exists in chloroplasts.
Determined by the substrate selectivity of 3-phosphate acyltransferase (ATase) (Frentzen, Me
t al., Eur. J. Biochem., 129: 629,1983; Murata, N., Plant
Cell Physiol., 24:81, 1983; Frentzen, M. et al., Plant
Cell Physiol., 28: 1195,1988) was strongly suggested.

Based on these assumptions, Nishizawa et al. Reduced the saturated molecular species content of PG by introducing and expressing the ATase gene obtained from Arabidopsis thaliana, which is a plant resistant to low temperature, in the tobacco to a temperature lower than that of the wild strain. It has been shown that it can be strengthened (PCT patent application: PCT / JP92 / 0
0024, 1992). However, ATase is also present in the original plant,
Even if a large amount of exogenous ATase is expressed in plants, it is inevitable that it will compete with the endogenous ATase, and the effect of exogenous ATase may be diluted. For example, the AT of Arabidopsis thaliana among the created transformed tobacco
The content of PG saturated molecular species in the leaf of the clone expressing the highest amount of ase is about 28%, which is about 8% compared to the tobacco wild strain.
Although it was small, it was about 8% more than the wild strain of Arabidopsis thaliana (PCT patent application: PCT / JP92 / 00024, 1992).

[0007] Furthermore, acyl-ACPs generally produced by plastids are mainly 16: 0-ACP and 18: 1-ACP, and their proportions are considered to be almost equal. The ratio of 16: 0-ACP and 18: 0-ACP may be higher than that of 18: 1-ACP (Toriyama, S. et al., Plant Cell Physio
L., 29: 615, 1988). In such tissues, it may be difficult to sufficiently reduce the saturated molecular species content by exogenous ATase.

By the way, the composition of the membrane lipid of cyanobacteria (Cyanobacteria), which is a photosynthetic bacterium, is similar to the lipid composition of the membrane system that constitutes chloroplasts of higher plants (Murata, N.et).
al., in “The Biochemistry of Plants”, Academic Pres
s, 1987). In cyanobacteria, the degree of unsaturation of fatty acids bound to membrane lipids is controlled by an enzyme that desaturates fatty acids bound to lipids. And Anacystis nidula which can put only one double bond in fatty acid bound to lipid
ns (also known as Synechococcus PCC 7942) is cold-sensitive (Ono, T. et al., Plant Physiol., 67: 176,1981), but two or more Synechocystis PCC6803 are known to be cold-resistant. (Wada, H. et al., Plant Cell Phy
siol., 30: 971,1989).

All fatty acid desaturases in cyanobacteria use a lipid as a substrate and introduce a double bond into the fatty acid bound to the lipid. Therefore, cyanobacteria are membrane lipids consisting of saturated molecular species of 16: 0/16: 0- and 18: 0/16: 0-, PG and SQ.
It is possible to introduce a cis-type double bond into fatty acids of DG, MGDG and DGDG (Murata, N. et al., In
"The Biochemistry of Plants", Academic Press, 198
7). This point has an enzyme that introduces a double bond into the Δ9-position of stearoyl-ACP (18: 0-ACP) as a fatty acid desaturase enzyme, and once it was 16: 0/16: 0- (and slightly Do
18: 0/16: 0-) PG and SQDG consisting of saturated molecular species
Is different from higher plants that never introduce cis-type double bonds into their fatty acids when they are synthesized.

At present, by introducing and expressing the Δ12 desaturase gene of Synechocystis PCC6803 in Anacystis nidulans, it does not originally exist in Anacystis nidulans.
Anacystis nidu, which is capable of producing 16: 2 Δ9,12 and 18: 2 Δ9,12, is naturally cold-sensitive
It has been shown that lans can be converted to cold tolerance (Wada, H. et al., Nature, 347: 200, 1990).

Among the desalting enzymes of cyanobacteria, Δ6 position (Reddy, A Set al., Plant Mol. Biol., 27:29).
3,1993) and Δ12th position (Wada, H. et al., Nature, 347: 20).
0,1990) The desaturase gene has been obtained. However, unless a double bond is introduced at the Δ9 position, the Δ6 position and Δ12 position desaturases cannot desaturate the Δ6 position and the Δ12 position, respectively. Further, if both the Δ9-position and the Δ12-position are not desaturated, the Δ15-desaturase cannot desaturate the Δ15-position. Therefore, if a gene for an enzyme desaturating the Δ9 position of fatty acid is introduced into a higher plant and expressed, the content of the saturated molecular species in the higher plant can be reduced, and as a result, the higher plant can be made tolerant to low temperature. Should be. However, to date, no gene for an enzyme that desaturates the Δ9 position of fatty acids has been obtained.

[0012]

Accordingly, it is an object of the present invention to provide a gene for an enzyme that desaturates the Δ9 position of fatty acids and a polynucleotide containing a part thereof. Another object of the present invention is to provide a vector containing a polynucleotide containing an enzyme gene for desaturating the Δ9 position of fatty acid or a part thereof. Further, the present invention also aims to provide a plant cell and a plant into which a polynucleotide containing a gene for an enzyme desaturating the Δ9 position of fatty acid or a part thereof is introduced.

[0013]

In order to achieve the above object, the present inventor cloned a gene encoding a Δ9-desaturase from genomic DNA of cyanobacteria belonging to the genus Anacystis and integrated the gene. After obtaining a vector DNA, a plant cell was transformed with the vector DNA, and this was differentiated to regenerate the plant body, thereby successfully imparting low temperature tolerance to the plant, and thus completing the present invention. It was That is, the present invention has the following matters.

(1) A gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to lipid. (2) The gene according to 1 or a part of the gene, wherein the protein having the activity of desaturating the Δ9 position of the fatty acid bound to lipid has substantially the amino acid sequence set forth in SEQ ID NO: 4. A polynucleotide comprising. (3) The gene encoding the protein having the activity of desaturating the Δ9 position of the fatty acid bound to lipid is SEQ ID NO: 3
A gene comprising the gene according to 1, which is a DNA chain containing the nucleotide sequence according to 1., or a polynucleotide comprising a part of the gene.

(4) A vector containing the gene according to any one of 1 to 3 or a polynucleotide containing a part of the gene. (5) A plant cell into which the gene according to any one of 1 to 3 or a polynucleotide containing a part of the gene is introduced. (6) A method for producing a plant, which comprises regenerating a plant by differentiating the plant cell according to 5. (7) A plant into which the gene according to any one of 1 to 3 or a polynucleotide containing a part of the gene is introduced. In addition, the gene of the present invention includes Δ9 of fatty acid bound to lipid.
As long as it has the activity of desaturating a position, a part of the gene encoding the protein having the activity is included.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The Δ9-desaturation enzyme referred to in the present invention is an enzyme originally present in cyanobacteria as described in the above "Conventional Technology". The chemical structure of the Δ9-desaturase is shown in mouse (Kaestner, KH et al., J. Biol. Chem., 26.
4: 14755,1989), rat (Mihara, K., J. Biochem., 108: 10.
22, 1990) and yeast (Stukey, JE et al., J. Biol. Chem.,
265: 2014 4, 1990), which is locally similar to the chemical structure of the stearoyl-CoA desaturase, but differs greatly overall. In addition, desaturases at the Δ6 and Δ12 positions of fatty acids bound to known cyanobacterial lipids and desaturases at the ω3 position of fatty acids bound to lipids of higher plants (Yadav, NS
et al., Plant Physiol., 103: 467, 1993). When the gene of the present invention is produced from a natural material, cyanobacteria may be used as a raw material. The cyanobacteria used here is not particularly limited, and examples include Anacys
Examples include cyanobacteria belonging to the genus tis, the genus Synechocystis, the genus Anabaena, and the like. For the purpose of desaturating saturated molecular species of higher plants, Anacystis type (Murata, N. et al., Plant Cell Physiol., 33: 933, 1
992. The Δ9-desaturase of group 1 type cyanobacteria referred to in this document is preferable to Anabaena type and Synechocystis type enzymes.

That is, Synechocystis PCC6803 and Anab
In aena variabilis, most of its membrane lipids are sn-1 and s.
A fatty acid having 18 carbon atoms (C18) and a fatty acid having 16 carbon atoms (C16) are bonded to n-2 (Sato, N. et al., Biochi).
m.Biophys.Acta, 710: 279,1982; Wada, H.et al., Plant C
ell Physiol., 30: 971,1989) while Anacystisnid
In ulans, most of sn-1 and sn-2 bind C16 (Bishop, DG et al., Plant Cell Physiol., 27: 159).
3, 1986). Therefore, the Δ9 desaturases of Anabaena and Synechocystis mainly use the species of 18: 0/16: 0- as a substrate for sn.
-1 seems to have the activity of desaturating 18: 0 to 18: 1 Δ9. On the other hand, the Δ9 desaturase of Anacystis mainly uses 16: 0/16: 0- as a substrate and 16: 1 of sn-1 as 16: 1.
It seems to have the activity of desaturating at Δ9. further,
Since 16: 0/16: 0- is the most saturated molecular species found in higher plants, the Anacystis-type Δ9 desaturase is more suitable for desaturating saturated molecular species in higher plants.
And more suitable than Synechocystis type enzymes.

As shown in Examples described later, the gene of the present invention includes a gene encoding a Δ9-desaturase having substantially the amino acid sequence shown in SEQ ID NO: 4, and differs only in a degenerate codon. And degenerate isomers capable of encoding the same polypeptide.
The gene of the present invention has a specific form mainly as a DNA chain. In addition, "substantially the amino acid sequence described in SEQ ID NO: 4" refers to the amino acid sequence described in SEQ ID NO: 4 as long as it has Δ9-desaturase activity in addition to the amino acid sequence described in SEQ ID NO: 4. Deletion, substitution in part of the amino acid sequence,
It includes an amino acid sequence which may be added or the like. The gene of the present invention can be produced from the above-mentioned cyanobacterial cells by a generally known method.

Specifically, cyanobacterial cells are cultured and accumulated,
Genomic DNA is prepared from the cyanobacterial cells by a commonly known method such as ethanol precipitation, a gene library based on the genomic DNA is prepared, and a clone containing a desired gene is selected from the library. It is possible to manufacture by amplifying. Examples of the gene library producing vector used here include those usually used as the vector. Specifically, phages such as λDASH II (Stratagene); pWE1
5 (Stratagene) and other cosmids; pBluescript II (Stratag
ene) and the like. As a specific method for introducing a gene into the above vector, a commonly known method depending on each vector can be used.

From the gene library thus prepared, clones into which the gene of the present invention has been introduced are selected.
As the selection method, a commonly known selection method, for example, an immunological method such as a plaque hybridization method or a colony hybridization method using an antibody, or a plaque hybridization method or a colony hybridization method using a nucleotide probe can be used. As a selection criterion for the above nucleotide probe, a part of the nucleotide sequence that is presumed to be similar to the gene of the present invention (for example, a part of amino acid sequence numbers 260 to 295 of MSCD2 in FIG. 1 is read as the nucleotide sequence). Is preferably used as a probe.

The determination and confirmation of the nucleotide sequence of the gene of the present invention in the clone thus selected can be carried out by a generally known method. For example, Maxam-Gilbert, Methods Enzymol., 65: 499,
1980) or the dideoxynucleotide chain termination method using M13 phage (Messing, J. et al., Gene, 19: 269, 1982). Whether or not the Δ9-desaturase is actually expressed can be confirmed, for example, according to the method of Wada et al. (J. Bacteriol., 175: 6056, 1993).

The gene of the present invention whose nucleotide sequence has been determined as described above can also be synthesized by a commonly known means, for example, a commercially available DNA synthesizer using the phosphite method. A polynucleotide encoding the polypeptide of the present invention or a part of the gene of the present invention and having Δ9-desaturation activity is isolated from the above clone, and this is integrated into a vector for gene transfer into a plant. By introducing into a plant cell and expressing the Δ9-desaturase in a plant, low temperature tolerance can be imparted to a desired plant. There are no particular restrictions on the types of plants into which the above-mentioned genes can be introduced.

The gene transfer vector referred to here is Δ9
It is necessary that the desaturase gene is constructed so that it can be stably expressed in plants. Specifically, a promoter, a DNA chain encoding a translational regulatory region, a DNA chain encoding a chloroplast transfer peptide, a gene of the present invention or a part of the gene of the present invention and having a Δ9 position desaturation activity are included. It is necessary that the polynucleotide encoding the peptide, the DNA chain encoding the translation stop codon, and the terminator be incorporated in an appropriate positional relationship. As the constituent elements of the gene transfer vector other than the gene of the present invention, known ones can be used. As the DNA chain encoding the transfer peptide to chloroplast, for example, the small subunit gene of pea ribulose-1,5-diphosphate carboxylase can be preferably used as the DNA chain encoding the transfer peptide. . As the promoter, for example, the cauliflower mosaic virus 35S promoter can be used, and as the terminator, for example, the nopaline synthase terminator can be used.

As a method for introducing a gene into a plant cell, a generally known method such as "" Plant genetic transformati
on and gene expression; a laboratory manual ", Drape
r, J. et al. eds., Blackwell Scientific Publications, 1
988 ”. Examples thereof include a biological method using a virus, a method using Agrobacterium, a physical / chemical method such as electroporation, a polyethylene glycol method, and microinjection. Of these, the method using Agrobacterium is preferable for dicotyledons such as tobacco, because stable transformation can be surely performed. For the method using Agrobacterium, an intermediate vector method using a wild-type tumor plasmid (Nature, 287 (1980), p. 654; Cell, 32 (19
83) p. 1033; EMBO J., 3 (1984) P. 1525), T-DNA.
Intermediate vector method using a vector lacking the above tumorigenic gene region (EMBO J., 2 (1983) P. 2143; Bio / Tec
hnology, 3 (1985) p.629), binary vector method (Bio
/ Technology, 1 (1983) p.262; Nature, 303 (1983) p.1
79; Nucl. Acids Res., 12 (1984) p.8711), and any of these methods may be used. Examples of methods for infecting plants with Agrobacterium include direct inoculation method to cultured cells, protoplast co-culture method, leaf disk method, etc., but many transformed plants can be directly and easily prepared. From this point of view, it is preferable to use the leaf disk method.

Further, in order to regenerate the plant body, MS-
Culture may be performed in a known medium such as HF medium in which a selective antibiotic, plant growth hormone or the like is added. If rooted seedlings are transplanted to soil and cultivated, they can be grown to complete plants. Whether or not the transformed plant grown to a complete plant has low temperature tolerance can be examined as follows. After cultivating the test plant at a temperature that does not suffer low temperature injury (for example, 25 ° C), temporarily (for example, for one week) under low temperature (for example, 4 ° C)
It can be examined by cultivating the plant in a plant and measuring damage to the plant, for example, chlorosis of leaves and reduction of fertility, or by comparing the amount of growth at low temperature with a control plant.

[0026]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Cloning of an open reading frame DNA fragment adjacent to the upstream of the Δ12 desaturase gene (desA) of Anabaena variabilis Anabaena variabilis IAM M-3 (sold by the Institute of Molecular and Cellular Biology, University of Tokyo) ) To about 100 ml of BG-11 medium ("Plant Molecular Biology", Shaw, CHed., P.279, IR).
L PRESS, 1988). The cells were shaken 120 minutes per minute under a fluorescent lamp of 1,000 lux at 25 ° C. to grow the bacteria sufficiently. The cells were collected as a precipitate by centrifuging the culture at room temperature at 5,000 g for 10 minutes.

To prepare genomic DNA, 50 ml of bacterial cells
The solution was suspended in A solution (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), washed, and centrifuged to collect the bacterial cells as a precipitate. Next, 15 ml of solution B (50 mM Tris-HCl, 20 mM EDTA, 50
The cells were suspended in mM NaCl, 0.25 M sucrose, pH 8.0), 40 mg of lysozyme (Sigma) dissolved in solution B was added, and the mixture was shaken at 37 ° C for 1 hour. Next, 15 mg of proteinase K and SDS were added so that the final concentration was 1%, and the mixture was shaken overnight at 37 ° C. Then add NaClO4 to a final concentration of 1M, and add 20 more
After adding ml of chloroform / isoamyl alcohol (24: 1) and shaking for 10 minutes, the aqueous layer was recovered by centrifugation. After re-extraction with chloroform / isoamyl alcohol (24: 1), 50 ml of ethanol was added to the aqueous layer and the genomic DNA preparation was wound on a glass rod and collected. This DNA preparation was dissolved in 20 ml of solution A, and NaCl was added to a final concentration of 0.
The concentration was adjusted to 1 M, RNase was further added so that the final concentration was 50 mg / ml, and the mixture was incubated at 37 ° C for 1 hour. Next, after extracting twice with an equal amount of phenol saturated with solution A, the genomic DNA in the aqueous layer was recovered as a precipitate by adding ethanol, washed with 70% ethanol, and dissolved in 1 ml of solution A. It was used as a genomic DNA solution of Anabaena variabilis.

Sakamoto et al. Announced the cloning of Δ12 desaturase gene of fatty acid bound to membrane lipid derived from Anabaena variabilis (1993 Annual Meeting of the Plant Physiology Society of Japan,
Abstracts, No.3aF04), there was an open reading frame (ORF) adjacent to the upstream of the Δ12 desaturase gene, which may have some relation with the desaturase. , Its function has not been identified. The present inventors are interested in the ORF and the function, paying attention to the nucleotide sequences at three positions in the DNA chain of the ORF, and synthesizing four primers (SEQ ID NO: 5 to SEQ ID NO: 8), Anabaena variabilis PCR was carried out using the genomic DNA of E. coli as a template.

Among the above four primers, SEQ ID NO: 5
The primers having the nucleotide sequences shown in 6 and 6 encode the sense strand, and the primers having the nucleotide sequences shown in SEQ ID NOS: 7 and 8 encode the antisense strand, and the nucleotide sequences shown in SEQ ID NOS: 6 and 7 are the same. It is derived from the amino acid sequence of.
One kind of primer was arbitrarily selected from each of the sense strand and the antisense strand, and PCR was performed with a combination of a total of four kinds of primers. The reaction was carried out by using GeneAmp PCR Kit (Takara Shuzo) in which 20 μM of each primer and 1 μg of genomic DNA of Anabaena variabilis were put in 100 μl of reaction solution. The temperature control of the reaction is 95 ℃ (1 minute), 45 ℃
(1 minute) and 72 ° C. (2 minutes) were set as one cycle, and 35 cycles were performed. However, 95 ° C in the first cycle was 3 minutes.
After the reaction was completed, 10 μl of the reaction solution was electrophoresed on a 2% agarose gel to separate and analyze the synthesized DNA. As a result, a DNA fragment of the expected size (about 190 bp) was detected as a major band in the DNA synthesized with the combination of primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 8. After blunting both ends of this DNA (hereinafter, des 9 var) fragment with Klenow fragment, it was cloned into the SmaI site of plasmid pTZ18R (Pharmacia), and the nucleotide sequence was determined using a fluorescent DNA sequencer (Applied Biosystems). . The obtained nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence deduced from this nucleotide sequence (SEQ ID NO: 2) showed significant homology with mouse stearoyl-CoA desaturase [Fig. 1: amino acid sequence encoded by des 9 var fragment and mouse stearoyl]. -Co
A comparison of the amino acid sequences of A desaturase (MSCD2) is shown].

Next, using the des 9 var fragment as a probe,
Southern analysis was performed on the genomic DNA of Anacystis nidulans.
Cleavage of about 0.1 μg Anacystis nidulans genomic DNA using each of the restriction enzymes XhoI, PstI and BamHI alone, and separation of DNA fragments by 0.8% agarose gel electrophoresis, followed by nylon membrane (Hybond-N +; Amersham) Blotted. Probe DNA is Multiprime DNA
Labeling Kit (Amersham) [α-32P] dC
Labeled with TP. 6 × SSPE [1 × SSPE is 10 mM phosphate buffer (pH 7.0), 1 mM EDTA, 0.15 M NaCl], 0.2% SDS and 100 μg / ml herring sperm DNA at 55 ° C., 16
The probe DNA was allowed to react with the membrane by incubation for a period of time. After that, put the membrane on 2 x SSC [1 x
SSC is 0.15 M NaCl, 15 mM sodium citrate] at room temperature,
Autoradiography was performed by shaking twice for 15 minutes and then shaking in 0.1 × SSC at 40 ° C. for 15 minutes twice. As a result, one DNA fragment was digested with any restriction enzyme.
Only the fragment was detected (Fig. 2: Non is Genome D in the figure)
It shows that NA is not cleaved with a restriction enzyme).

[Example 2] Cloning of DNA chain in Anacystis nidulans genome highly homologous to des 9 var fragment Anacystis nidulans R2-SPc (contributed by Institute of Molecular and Cellular Biology, University of Tokyo) culture and preparation of genomic DNA Is
The procedure was the same as for Anabaena variabilis. About 100
After partial digestion of μg of genomic DNA with Sau3AI, Molecul
ar Cloning 2nd edition, pp.2.85-2.87 (Sambrook, J.et a
l.eds., Cold Spring Harbor Laboratory, 1989), by ultracentrifugation under a sucrose density gradient to about 9
A 23 kbp DNA fragment was recovered from BamHI and Hin
Lambda phage vector λDASH II (Stra
After cloning into a tagene), it was packaged in a phage particle to obtain a genomic DNA library of Anacystis nidulans. This phage library was used for Escherichia coli P2392.
The plaques of about 100,000 in total were formed on a petri dish of about 15 cm in diameter containing NZYM medium, and then blotted on a nylon membrane (Hybond-N +; Amersham). Similar to the Southern analysis above, [α- 32 P]
The des 9 var fragment labeled with dCTP was reacted with this membrane, and positive phages detected by autoradiography were screened again in the same manner to obtain about 30 phage clones having different signal intensities. Twelve clones were arbitrarily selected from this and phage DNA was obtained according to a conventional method. The obtained phage DNA was cleaved with several kinds of restriction enzymes, separated by 0.8% agarose gel electrophoresis, and then blotted on a nylon membrane. This membrane was subjected to Southern analysis under the same conditions as in the above screening, and the length of the DNA fragment hybridizing with the probe DNA and its signal intensity were compared. As a result, the two clones of λ5 and λ15 showed the strongest signal, and the lengths of the insert DNA fragments were 11 and 15 kbp, respectively, so it was judged that the clones were sufficient to contain the entire ORF of interest. Furthermore, Southern analysis was performed by cutting with several restriction enzymes.
As a result, when cleaved with XhoI and hybridized, a DNA fragment of about 5 kbp was detected in both clones.
By subcloning into the XhoI site of Bluescript SK- (Stratagene), plasmids p5X and p15X containing DNA fragments derived from λ5 and λ15 were obtained. When a detailed restriction map of p5X and p15X was created and compared, both were judged to contain the same genomic DNA fragment [Fig. 3: λ5,
The interrelation between the insert DNA fragments of λ15 and p15X is shown. The shaded rectangle represents a DNA fragment hybridized with the des 9 var fragment of the probe during the screening process. Thick arrows indicate the direction of the sense strand and the region of des 9 nid (described later). A thin arrow indicates the direction of the sequence of the region containing des 9 nid. The 5,1.25 and 0.5 kbp bars indicate size markers in each figure on the left. Abbreviations for restriction enzymes are B, BamHI; H, HindIII; N, NotI; Hp, HpaI; RI, EcoRI; R
V, EcoRV; S, SalI; P, PstI; X, XhoI].

Therefore, a deletion plasmid with a restriction enzyme or ExoIII was prepared from p15X and des 9 va
Approximately 2 kbp DN containing the region to which the r fragment hybridizes
The base sequence of the A fragment was determined using a fluorescent DNA sequencer (Fig. 3). As a result, the DNA fragment contained 834 bp.
Was present (SEQ ID NO: 3), and the amino acid of 278 residues was deduced to be encoded (SEQ ID NO: 4). Anabaena v cloned earlier
The homology with the amino acid sequence (SEQ ID NO: 2) encoded by the des 9 var fragment derived from ariabilis was about 80%.
Furthermore, analysis software for nucleic acid and amino acid sequences (GEN
ETYX; software development) and a database of nucleic acid and amino acid sequences (EMBL and DDBJ) were used to search for highly homologous amino acid sequences. As a result, the overall homology with the mouse stearoyl-CoA desaturase was found. It was about 30%, but it was extremely high locally [Fig. 4: Comparison of des9 nid and amino acid sequence of mouse stearoyl-CoA desaturase (MSCD2)]. It was strongly suggested that it encodes an oxidative enzyme.

Example 3 Activity Measurement by Expression of des9nid Gene in Escherichia coli Anacystis nidulans has only Δ9-desaturase activity as desaturase which desaturates the 9-position of saturated fatty acid bound to lipid. (Bishop, DGet al., PlantCell Phy
siol., 27: 1593, 1986), an attempt was made to express the polypeptide encoded by des9nid in E. coli and measure its activity. Since it is difficult to express directly from p15X, a vector for expression in E. coli was prepared. That is, pET3a (Novagen) was used as a vector, and des9ni was inserted between the NdeI and BamHI.
Cloning of d was carried out as follows without adding an extra amino acid to the amino terminus. des
In order to insert a BamHI site immediately after the C-terminal side of the 9nid-encoded protein, a PCR reaction was carried out using two base sequences scissoring the C-terminal. That is, sense primer; 5'-ACGTCATGGC CTGCAG T (underlined is PstI
Site) (SEQ ID NO: 9) antisense primer; 5'-CGC GGATCCT TA GTTGTTTGGAG
ACG (single line is BamHI site, double line is stop codon)
(SEQ ID NO: 10)

A PCR reaction was carried out using p15X as a template and the above two primers to obtain a product of about 140 bp, which was subcloned into the SmaI site of pUC19 to confirm that the nucleotide sequence was correct. An EcoRI site was created downstream of BamHI in the resulting plasmid.
This is cut with EcoRI and PstI in turn, and p15X is also cut with the same restriction enzyme.
BamHI site was introduced. This plasmid was cleaved with SalI, and then a Fillin reaction was carried out using the DNA polymerase Klenow fragment in the presence of four kinds of dNTPs, followed by a HindIII digestion. An NdeI site was introduced at the amino terminal side by introducing an adapter consisting of the following two types of synthetic DNA into this. That, 5'- CATATG ACCCTTGCTATCCGACCCA (underlined NdeI) (SEQ ID NO: 11) and 5'- AGCTT GGGTCGGATAGCAAGGGT C ATATG (1 double lines NdeI site, double line some HindIII) (SEQ ID NO: 12) an equimolar The amounts were mixed and used as an adapter. The plasmid (pDes9Nde) prepared as described above was used in a conventional method (Molecular cl
oning pp.250-251; 1982) E. coli strain BL
21 (DE3) (Novagen) was introduced into competent cells, and the transformant BLDES1 was selected by selection with ampicillin resistance.
Got

BL21 with only BLDES1 and pET3a
The strain (BL1) was inoculated into 100 ml of M9 medium (containing 200 μg / ml ampicillin, 4 mg / ml glucose, 10 μM FeCl3, 0.5 μg / ml vitamin B1, 1 mg / ml casamino acid), and 37
Cultured at ° C. After continuing the culture until the turbidity of the culture reached 0.5 OD at a wavelength of 600 nm, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM. After further culturing for 1 hour, the expression of the Δ9 position desaturase gene was induced. After washing the recovered E. coli pellet with 1.2% NaCl,
The lipid was extracted. For lipids, the method of Bligh and Dyer (Can J. Bi
ochem. Physiol., 37: 911, 1959) and extract 2.5
The mixture was completely sealed with 5 ml of 5% hydrochloric acid methanol and reacted at 85 ° C. for 2 hours and a half to methylate fatty acids. The resulting fatty acid methyl ester was extracted 4 times with 2.5 ml of hexane, the solvent was removed with nitrogen gas, and the mixture was concentrated. Gas chromatography was used for analysis of fatty acid methyl ester. The fatty acid was identified by comparing the retention time with standard fatty acid methyl.
Chromatopack C-R7A plus (Shimadzu) was used for quantification. The results are shown in Table 1 below.

[0036]

[Table 1]

Here, the time indicates the induction time of the protein by IPTG. It is clear that in BLDES1 there is an increase of 16: 1. That is, this gene was shown to have desaturation activity to 16: 0. Moreover, when these strains were cultured in M9 medium containing 0.1 mM stearic acid and compared in the same manner, it was 1 in BLDES1 as compared with BL1 strain.
Generates not only 6: 1 but also 18: 1 (9), des 9 ni
The polypeptide encoded by d is not only 16: 0 but also 1
It was shown that 8: 0 also produces unsaturated fatty acids as substrates.

Example 4 Introduction of des9nid Gene into Tobacco Plants Anacystis nidulans-derived des9nid gene was incorporated into tobacco as follows. (1) Construction of plant expression vector plasmid By cutting pDes9Nde with SacI and SalI, a des9nid gene fragment flanked by the cleavage sites of both enzymes can be obtained. on the other hand,
The clone pSNIP9 (Schre
icher et al., EMBO J. 4,25 (1985)) to chloroplast transit
By excising the sequence with HindIII and SphI and cloning into pUC118 cut with the same restriction enzymes, tr
A plasmid (pTRA3) having a multicloning site downstream of the ansit sequence was obtained. After cutting this HindIII site, it was filled in with Klenow enzyme and XbaI linker was added (pTRA3
X). Cut this plasmid pTRA3X with Sal I and Sac I,
Des9n obtained by cutting with the same restriction enzyme
The id gene fragment was inserted (pTRA3Xdes9). In this plasmid, the des9nid gene is translated in the same reading frame as that of the RuBisCO transit sequence. This is Sac I and X
It is cut with ba I and inserted into the plant vector described below. Plant-expressing binary plasmid pBI121 (Clonetec
Plasmid pBI obtained by cleaving h) with the restriction enzymes SacI and XbaI.
(-GUS) does not contain the β-Glucuronidase gene (GUS gene), and the 35S cauliflower mosaic virus
By inserting the above-mentioned transgene between the promoter and the nopaline synthase (NOS) terminator, a vector for introduction into plants (pBI121 (-GUS) Rbsc-des9) was obtained.

(2) Introduction of pBI121 (-GUS) Rbsc-des9 into Agrobacterium Agrobacterium tumefaciens LBA4404 (Clonetech) 50 m
l YEB medium (5g beef extract, 1 yeast extract per l)
g, peptone 1 g, sucrose 5 g, 2 mM MgSO4 (pH 7.4)) and inoculated at 28 ° C for 24 hours, and then the culture solution was 3,000 rpm, 4 ° C,
The cells were collected by centrifugation for 20 minutes. 10 ml of 1 mM Hepes-KOH
After washing 3 times with (pH 7.4), add 1 ml of 3 ml of 10% glycerol.
Wash once and finally suspend in 3 ml of 10% glycerol D
The Agrobacterium for NA introduction was used.

50 μl of the bacterial solution thus obtained and 91 μg of the above-mentioned plasmid pBI121 (-GUS) Rbsc-des91 were put in a cuvette, and an electroporation apparatus (Gene Pulser; Bio) was used.
(Rad) was used to introduce an electric pulse under the conditions of 25 μF, 2500 V, and 200 Ω to introduce the plasmid DNA into Agrobacterium. Transfer this bacterial solution to an Eppendorf tube,
800 μl SOC medium (20 g tryptone / l, yeast extract 5 g, NaCl 0.5 g, 2.5 mM KCl, 10 mM MgSO4, 1 per liter)
Add 0 mM MgCl2, 20 mM glucose, pH 7.0) and add 28
Static culture was carried out at ℃ for 1.5 hours. Add 50 μl of this culture to 100
YEB agar with ppm kanamycin (agar 1.2%)
It was sprinkled on top and incubated at 28 ° C for 2 days.

A single colony is selected from the obtained colony group, and the plasmid DN is selected from this colony by the alkaline method.
A was adjusted. After digesting this plasmid DNA with an appropriate restriction enzyme, the DNA fragment was separated by 1% agarose gel electrophoresis and subjected to Southern analysis using the 32P-labeled des9nid gene fragment as a probe to obtain plasmid pBI121 (-GU
It was confirmed that (S) Rbsc-des9 was included. This Agrobac
Terbium tumefaciens is called ALBBSDES.

(3) Tobacco transformation LB containing 50 ppm of kanamycin was prepared from the above strain ALBBSDES.
It was shake-cultured in a liquid medium at 28 ° C. for 2 days. 1.5 ml of culture solution
The cells were collected by centrifugation at 10,000 rpm for 3 minutes and washed with 1 ml of LB medium to remove kanamycin. Further 10,000 rpm, 3
After centrifugation for 1 minute to collect the cells, the cells were resuspended in 1.5 ml of LB medium to prepare a bacterial solution for infection. To infect tobacco, young leaves were collected, soaked in 0.5% sodium hypochlorite aqueous solution for 10 minutes, washed 3 times with sterilized water, and wiped with water on sterilized filter paper to make leaves for infection. . This leaf is aseptically cut with a scalpel so that one piece becomes 1 cm 2, and the leaf back is placed on the above-mentioned Agrobacterium bacterial solution, and it is gently shaken for 2 minutes, and then sterilized. The leaves were placed on filter paper to remove excess Agrobacterium. MS-B5 medium in a petri dish (benzyl adenine 1.0 ppm, naphthalene acetic acid 0.1 ppm, and agar.
8% included) (Murashige, T. and Skoog, F. Plant Ph
ysiol., 15: 473, (1962)), Whatman No. 1 filter paper (φ 7.0 cm) was placed, and leaves were placed on the filter paper with the back side facing up. The petri dish was sealed with parafilm and incubated at 25 ° C. for 2 days in a cycle of 16 hours light and 8 hours dark. Then, the cells were transferred to MS-B5 medium containing 250 ppm of Claforan and similarly cultured for 10 days to remove Agrobacterium. Furthermore, it was placed on an MS-B5 medium containing 250 ppm of Claforan and 100 ppm of kanamycin, and similarly cultured for 7 days. During this time, callus was formed around the leaf pieces, and shoot primordia were formed.
After culturing for another 10 days, the expanded shoots were
MS-HF medium containing 250 ppm and kanamycin 100 ppm (MS-HF containing no benzyladenine and naphthaleneacetic acid)
It was placed in B5 medium). After culturing for 10 days, the rooted shoots were used as kanamycin-resistant transformants and transplanted to MS-HF medium containing 250 ppm of Claforan in the plant box.

Example 5 Genomic Southern and Northern Analysis of Transformed Tobacco To confirm the introduction of the target gene, DNA was extracted from kanamycin-resistant tobacco and subjected to Southern and Northern analysis. The method for extracting genomic DNA is the CTAB method.
gers, SO & Bendich, AJ; Plant Molecular Biol
ogy Manual A6; 1 (1988)). That is, 2
g tobacco leaf was ground in liquid nitrogen and genomic DNA was obtained in CTAB extraction buffer. 10 μg of DNA was digested with restriction enzymes EcoRI and XbaI and electrophoresed on 0.7% agarose gel, and then 0.4 N was applied to a nylon membrane (Hybond N +; Amersham).
Transferred with NaOH. Using the desaturase gene with transit from pTRA3Xdes9 as a probe, this membrane was
By hybridizing for 6 hours, it was confirmed that the target gene was integrated in the tobacco genome.

In addition, in order to examine the expression of the transgene,
RNA analysis was performed from about 2 g of tobacco leaves. The method involves extraction with guanidinium thiocyanate (Nagy,
F. et al: Plant Molecular Biology Manual B4; 1 (198
8)), poly (A) + RNA was electrophoresed on an agarose gel containing formaldehyde, and then nylon membrane (Hybond N; Amersha
m) and analyzed by hybridization similar to the Southern method. Although there were individuals expressing various amounts of RNA, lipid analysis was performed on those individuals with high expression levels.

Example 6 Fatty Acid Analysis of Lipids in Transformed Tobacco From tobacco transformants confirmed to have high RNA expression in Example 5 and tobacco leaves transformed with pBI121 as a control, the following method was used. Phosphatidylglycerol (P
G), sulfoquinovosyl diacyl glycerol (SQ
A lipid such as DG) was prepared and its fatty acid composition was analyzed.
Root lipids were also analyzed from some individuals.

(1) Extraction of total lipids Lipids were extracted by Bligh-Dyer method (Can J. Biochem. Physio
l., 37: 911, 1959). Wet 2 g of leaves (1 g when using some roots as a sample) was shredded with a scalpel, added 20 ml of chloroform: methanol (1: 2, volume ratio), and crushed the leaves with a homogenizer. , Let stand for 15 minutes. Chloroform (12 ml) and distilled water (12 ml) were added thereto and mixed vigorously, and then centrifuged at 3000 rpm at 4 ° C. for 30 minutes to separate into an aqueous layer and an organic layer, and an organic layer (lower layer) was recovered. To this was added an appropriate amount of ethanol, and the solvent was removed under reduced pressure at 30 ° C. using a rotary evaporator. This was dissolved in 2 ml of chloroform: methanol (1: 4, volume ratio) to give a total lipid extract. A part of this was treated with 5% methanolic hydrochloric acid by the method described below to obtain a methylated fatty acid.

(2) Fractionation of lipids 2.5 ml of a suspension of DEAE-Toyopearl 650C (Tosoh) was mixed with 25 ml of a 1 M sodium acetate aqueous solution (pH 7.0) to give an acetic acid form. This is washed successively with distilled water and methanol, and finally suspended in methanol to a column with an inner diameter of 2 cm and a height of 1.5 cm.
And then add 50 ml of chloroform: methanol (1:
(4, volume ratio). Then the total lipid extract was applied to the column and 50 ml of chloroform: methanol (1: 4,
Volume ratio of monogalactosyldiacylglycerol (M
GDG), digalactosyldiacylglycerol (DG
DG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are eluted to give a neutral lipid (M
GDG, DGDG, PE, PC) fraction. Then 5 ml
The phosphatidylserine (PS) was eluted with acetic acid of 10%, and the acetic acid was washed with 20 ml of chloroform: methanol (1: 4, volume ratio), and then 50 ml of chloroform: methanol: 10 M ammonium acetate aqueous solution (20:80). : 0.2, volume ratio) to obtain a fraction containing PG, SQDG and phosphatidylinositol (PI). 15 ml of ethanol was added to this fraction, and the solvent was removed under reduced pressure. Dissolve this in 0.2 ml of chloroform: methanol (2: 1, volume ratio),
The acidic lipid (PG, SQDG, PI) fraction was used.

The MGDG, DGDG, PE and PC fractions are
Further fractionation was performed by silicic acid column chromatography (Iatrobeads, Yatron). That is, a sample dissolved in 1 ml of chloroform was applied to a column equilibrated with chloroform, and eluted with chloroform: acetone (4: 1), acetone, and methanol in that order to obtain glycolipids (MGDG,
DGDG) was eluted with acetone and phospholipids (PC, PE) were eluted with methanol.

(3) Isolation and Purification of PG by Thin Layer Chromatography (TLC) and Analysis of Fatty Acids The fraction obtained in (2) was purified by silica gel-TLC plate # 5721 (Me).
rck) separated. As the developing solvent, in the case of acidic lipid, chloroform: acetone: methanol: acetic acid: water (5
0: 20: 10: 15: 5, volume ratio) and chloroform: methanol: water (70: 21: 3, volume ratio) for neutral lipids. TLC
After separation with, primulin (80% acetone solution) was sprayed, and fluorescence was developed under ultraviolet light, and the lipid fraction of each class was estimated by comparing the mobility with the standard lipid,
The colored lipid was scraped off together with the silica gel and placed in a test tube with a screw cap. To estimate this fatty acid composition, 3 ml of methanolic 5% hydrochloric acid was added to this test tube, and the reaction was carried out at 85 ° C. for 2 hours and a half under complete sealing to perform fatty acid methylation. On the other hand, in order to determine the fatty acid composition for each sn-1 and sn-2, the lipid was recovered from the scraped silica gel with 5 ml of a mixture of chloroform: methanol (2: 1), dried, and then dried to 1 ml.
50 mM TrisCl (pH 7.2) and 0.05% Triton X-100 are added, the mixture is vigorously stirred to disperse the lipid, and lipase (2,500 U; Boehringer) derived from Rhizopus delemar is added and kept at 37 ° C for 30 minutes. By doing so, the fatty acid at the sn-1 position was selectively decomposed. After the reaction product was concentrated, it was separated into unreacted lipid, lyso form, and fatty acid by TLC (chloroform: acetone: methanol: acetic acid: water = 10: 4: 2: 3: 1). These were also recovered from the gel and methylated fatty acids were obtained with methanolic hydrochloric acid as described above. 3 ml of the resulting fatty acid methyl ester
It was extracted 4 times with hexane, the solvent was removed under reduced pressure, and the mixture was concentrated. Gas chromatography was used for the analysis of fatty acid methyl. The fatty acid was identified by comparing the retention time with standard fatty acid methyl. Chromatopack C-R7 for quantification
A plus (Shimadzu) was used. The results of total lipids are shown in Table 2,
Table 3 shows the PG, and Table 4 shows the analysis results of other representative lipids. The table shows the average of the analytical values of 2 individuals for the control plant and 2 or 3 independent individuals for the transformant.

[0050]

[Table 2]

[0051]

[Table 3]

[0052]

[Table 4]

From the results of analysis of fatty acids bound to PG,
In transformed tobacco expressing a fatty acid desaturase derived from acystis nidulans, 16: 0 (palmitic acid) was significantly reduced, and 16: 1 cis was increased instead, and a small amount was present 18: It became clear that 0 almost disappeared and conversely 18: 1 increased. As a result, saturated fatty acids (16: 0 + 16: 1trans + 1
8: 0 (stearic acid) content is 7 for control tobacco
While it is 0%, it is 55% in tobacco transformed with the gene of the desaturase, which is remarkably low. From the analysis results of PG sn-1 and 2-position, sn-2 is occupied by saturated fatty acids (16: 0 or 16: 1 trans) in an amount of 98% or more, and is newly generated by gene transfer 16:
It was found that all 1s were in sn-1. Therefore, s of tobacco PG transformed with this desaturase gene
It is clear that the saturated fatty acid at the n-1 position is extremely low. Therefore, the amount of so-called saturated molecular species consisting of saturated fatty acids at both the sn-1 and 2-positions is greatly reduced,
It can be seen that the composition of the lipid molecular species changed to a form that was extremely resistant to low temperatures.

On the other hand, MGDG and DGD of other lipids
Also in G, SQDG, PC, PE and PI, a decrease of 16: 0 and a corresponding increase of around 16% in 16: 1 were apparent, and desaturation at 18: 0 was also advanced. Among these, for MGDG and DGDG, 16: 1 production was mainly at the sn-1 position, but a small amount was also detected at the sn-2 position. MGDG, DGDG, SQDG and PG
Is a lipid mainly present in chloroplasts and is the cyanobacteria Anac
It is clear that the desaturation of ystis nidulans was surprisingly advanced by expressing it in the chloroplasts of higher plants. More than that, these four lipids are
It was also present in the membrane of tis nidulans and was likely to become a substrate for desaturation, but other PCs, PE and PI were lipids not present in the membrane of Anacystis nidulans, and higher plants. Since it is a lipid mainly existing outside the chloroplast, it is surprising that palmitic acid and stearic acid are also desaturated in them.

As described above, the results of lipid analysis of the transformed tobacco revealed that the fatty acid desaturase derived from Anacystis nidulans was almost all of the lipids of 16: 0 and 18 in the transformants of tobacco, which is a higher plant. This example proves that 0 can be desaturated very efficiently. Table 5 shows the results of fatty acid analysis of total root lipids.

[0056]

[Table 5]

From these results, it is clear that the fatty acid desaturase derived from Anacystis nidulans catalyzed the desaturation of 16: 0 and 18: 0 not only in the leaves but also in the roots. This not only has the possibility that the fatty acid desaturase gene of the present invention changes the low temperature tolerance of the plant, but also has the possibility of increasing the unsaturated fatty acid content, and is useful in the industry where plants are used as raw materials for oil. Is shown.

[Example 7] Cold Tolerance Test of Transformed Tobacco The next-generation seeds were collected by self-fertilization from the individuals considered to be promising in the above RNA expression analysis and lipid analysis. MS-HF containing a part of 800 kanamycin
Spread on a medium and light for 2 days at 25 ℃, 16 hours light, 8 hours dark.
Kanamycin resistant seedlings were selected after weekly cultivation. This seedling was transplanted to a plant box and further cultivated for 4 weeks. Further, as a control, the above operation was carried out for an individual transformed with pBI121. Next, 4 ℃
After low temperature treatment for 11 days under continuous light, it was cultivated at 25 ° C. for 2 days. As a result, in the control plants (plants transformed with pBI121), large atrophy symptoms and chlorosis of leaves were observed, whereas in the transformed plants, almost no damage was observed. Therefore, it was estimated that the low temperature tolerance was improved by the introduction of the desaturase gene.

[0059]

INDUSTRIAL APPLICABILITY By introducing the gene encoding the Δ9-desaturase of the present invention into plants, it became possible to impart low temperature tolerance to the plants and increase the unsaturated fatty acid content in the plants. .

[0060]

[Sequence list]                                SEQUENCE LISTING    <110> KIRIN BEER KABUSHIKI KAISHA    <120> A METHOD FOR CREATING A HIGHER PLANT CELL OR A HIGHER       PLANT HAVING IMPROVED CHILLING-RESISTANCE    <130> divisional application of P96-0198    <140> <141>    <150> JP 05-352858 <151> 1993-12-28    <160> 12    <170> PatentIn Ver. 2.0    <210> 1 <211> 196 <212> DNA <213> Anabaena variabilis    <400> 1 gctctggggt tgttgctgtt atatctaggc gggtggtctt ttgtggtctg gggagttttc 60 tttcgcatcg tttgggttta ccactgtact tggttggtaa acagcgctac ccataagttt 120 ggctaccgca cctatgatgc tggtgacaga tccactaact gttggtgggt agctgtccta 180 gtgtttggtg aaggtt 196    <210> 2 <211> 65 <212> PRT <213> Anabaena variabilis    <400> 2 Ala Leu Gly Leu Leu Leu Leu Tyr Leu Gly Gly Trp Ser Phe Val Val   1 5 10 15    Trp Gly Val Phe Phe Arg Ile Val Trp Val Tyr His Cys Thr Trp Leu              20 25 30    Val Asn Ser Ala Thr His Lys Phe Gly Tyr Arg Thr Tyr Asp Ala Gly          35 40 45    Asp Arg Ser Thr Asn Cys Trp Trp Val Ala Val Leu Val Phe Gly Glu      50 55 60    Gly  65    <210> 3 <211> 837 <212> DNA <213> Anacystis nidulans    <400> 3 atgacccttg ctatccgacc caagcttgcc ttcaactggc cgaccgccct gttcatggtc 60 gccattcaca ttggagcact gttagcgttc ctgccggcca actttaactg gcccgctgtg 120 ggcgtgatgg ttgcgctgta ttacattacc ggttgttttg gcatcaccct aggctggcac 180 cggctaattt cgcaccgtag ctttgaagtt cccaaatggc tggaatacgt gctggtgttc 240 tgtggcacct tggccatgca gcacggcccg atcgaatgga tcggtctgca ccgccaccat 300 cacctccact ctgaccaaga tgtcgatcac cacgactcca acaagggttt cctctggagt 360 cacttcctgt ggatgatcta cgaaattccg gcccgtacgg aagtagacaa gttcacgcgc 420 gatatcgctg gcgaccctgt ctatcgcttc tttaacaaat atttcttcgg tgtccaagtc 480 ctactggggg tacttttgta cgcctggggc gaggcttggg ttggcaatgg ctggtctttc 540 gtcgtttggg ggatcttcgc ccgcttggtg gtggtctacc acgtcacttg gctggtgaac 600 agtgctaccc acaagtttgg ctaccgctcc catgagtctg gcgaccagtc caccaactgc 660 tggtgggttg cccttctggc ctttggtgaa ggctggcaca acaaccacca cgcctaccag 720 tactcggcac gtcatggcct gcagtggtgg gaatttgact tgacttggtt gatcatctgc 780 ggcctgaaga aggtgggtct ggctcgcaag atcaaagtgg cgtctccaaa caactaa 837    <210> 4 <211> 278 <212> PRT <213> Anacystis nidulans    <400> 4 Met Thr Leu Ala Ile Arg Pro Lys Leu Ala Phe Asn Trp Pro Thr Ala   1 5 10 15   Leu Phe Met Val Ala Ile His Ile Gly Ala Leu Leu Ala Phe Leu Pro              20 25 30    Ala Asn Phe Asn Trp Pro Ala Val Gly Val Met Val Ala Leu Tyr Tyr          35 40 45    Ile Thr Gly Cys Phe Gly Ile Thr Leu Gly Trp His Arg Leu Ile Ser      50 55 60    His Arg Ser Phe Glu Val Pro Lys Trp Leu Glu Tyr Val Leu Val Phe  65 70 75 80    Cys Gly Thr Leu Ala Met Gln His Gly Pro Ile Glu Trp Ile Gly Leu                  85 90 95    His Arg His His His Leu His Ser Asp Gln Asp Val Asp His His Asp             100 105 110    Ser Asn Lys Gly Phe Leu Trp Ser His Phe Leu Trp Met Ile Tyr Glu         115 120 125    Ile Pro Ala Arg Thr Glu Val Asp Lys Phe Thr Arg Asp Ile Ala Gly     130 135 140    Asp Pro Val Tyr Arg Phe Phe Asn Lys Tyr Phe Phe Gly Val Gln Val 145 150 155 160    Leu Leu Gly Val Leu Leu Tyr Ala Trp Gly Glu Ala Trp Val Gly Asn                 165 170 175    Gly Trp Ser Phe Val Val Trp Gly Ile Phe Ala Arg Leu Val Val Val             180 185 190    Tyr His Val Thr Trp Leu Val Asn Ser Ala Thr His Lys Phe Gly Tyr         195 200 205    Arg Ser His Glu Ser Gly Asp Gln Ser Thr Asn Cys Trp Trp Val Ala     210 215 220    Leu Leu Ala Phe Gly Glu Gly Trp His Asn Asn His His Ala Tyr Gln 225 230 235 240    Tyr Ser Ala Arg His Gly Leu Gln Trp Trp Glu Phe Asp Leu Thr Trp                 245 250 255    Leu Ile Ile Cys Gly Leu Lys Lys Val Gly Leu Ala Arg Lys Ile Lys             260 265 270    Val Ala Ser Pro Asn Asn         275    <210> 5 <211> 18 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 5 atgacaattg ctacttca 18    <210> 6 <211> 15 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 6 gctctggggt tgttg 15    <210> 7 <211> 15 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 7 caacaacccc agagc 15    <210> 8 <211> 18 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 8 rtgrtgrttr ttrtgcca 18    <210> 9 <211> 17 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 9 acgtcatggc ctgcagt 17    <210> 10 <211> 26 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 10 cgcggatcct tagttgtttg gagacg 26    <210> 11 <211> 25 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 11 catatgaccc ttgctatccg accca 25    <210> 12 <211> 29 <212> DNA <213> Artificial Sequence    <220> <223> Description of Artificial Sequence: synthetic DNA    <400> 12 agcttgggtc ggatagcaag ggtcatatg 29

[0061]

[Sequence list free text] SEQ ID Nos. 5 to 12 are synthetic DNs
The nucleotide sequence of A is shown.

[Brief description of drawings]

FIG. 1 is an amino acid sequence encoded by the des 9 var fragment and mouse stearoyl-CoA desaturase (M
The comparison of the amino acid sequences of SCD2) is shown. In the figure, when both are the same amino acid, and when they are amino acids having similar properties, * is compared. X shows the range with high homology between them.

[Fig. 2] Fig. 2 shows that the des 9 var fragment was used as a probe.
1 is an electrophoretic photograph showing an autoradiogram obtained by Southern analysis of genomic DNA of acystis nidulans.

FIG. 3 shows the interrelationship of λ5, λ15 and p15X insert DNA fragments. Thick arrows indicate the part encoding the protein and the direction, and thin arrows indicate the site and the direction in which the sequence was determined.

FIG. 4 shows des 9 nid and mouse stearoyl-
The comparison of the amino acid sequences of CoA desaturase (MSCD2) is shown. The amino acid sequences were compared in the same manner as in FIG.

FIG. 5 is a photograph of the morphology of organisms showing the effect of cold treatment on transgenic tobacco at the plant level. The left shows the result of low temperature treatment of the tobacco into which the desaturase gene was introduced, and the right shows the result of low temperature treatment of the tobacco with pBI121 introduced as a control.

Continuation of front page (51) Int.Cl. ⁷ identification code FI C12R 1:91) (58) Fields investigated (Int.Cl. ⁷ , DB name) A01H 1/00 A01H 5/00 BIOSIS (DIALOG) JISST file (JOIS)

Claims (2)

(57) [Claims] 1. A method for producing a higher plant cell, which comprises introducing a gene encoding a Δ9 desaturase derived from Anacystis that desaturates a Δ9 position of a fatty acid bound to a lipid. 2. A method for producing a higher plant, which comprises introducing a gene encoding a Δ9 desaturase derived from Anacystis that desaturates a Δ9 position of a fatty acid bound to a lipid.