KR100254285B1 - Process for creating the plant - Google Patents

Abstract

A plant cell into which a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to a lipid is introduced: A plant harvesting method characterized by regenerating the plant by differentiating the plant cell.

Description

Plant cropping method {PROCESS FOR CREATING THE PLANT}

The present invention provides a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to a lipid (hereinafter referred to as the Δ9 position desaturase), a vector containing the gene, a plant into which the gene is introduced, and a method of producing the same. It is about.

Membrane lipids, which are lipids constituting biological membranes of living organisms, change (phase separation) from a liquid crystal state to a solid state with a decrease in external temperature. With this phase separation, the properties of the biofilm change. In other words, it is thought that the membrane is unable to perform its original function because the selectivity of the substance permeation disappears in the solid state, and as a result, the injury (low temperature injury) is caused to the cells.

The phase transition temperature of the membrane lipid, which is the temperature that changes from the liquid crystal state to the solid state or vice versa, is mainly determined by the degree of unsaturation (the number of double bonds in the carbon chain) of the fatty acid acyl group bound to the lipid. That is, when two of the fatty acid acyl groups bound are polysaturated fatty acids, the phase transition temperature of the lipid molecular species is higher than room temperature, but the phase transition temperature of the lipid molecular species having at least one double bond to the bound fatty acid acyl group is Near 0 ° C. (Santaren, JF et al., Biochem. Biophys. Acta, 687: 231, 1982).

In general, the position of the double bond of a fatty acid indicates the number of carbons from the carboxyl terminal to the carbon having a double bond, followed by Δ (delta). The total number of double bonds is described after the colon after the total carbon number. 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 COOH

to be. In addition, although the position of a double bond may be described next to (omega), this shows the carbon number from the methyl group terminal of a fatty acid to the carbon with a double bond.

Among the membrane lipids of higher plants, only saturated phosphatidylglycerol (PG) is relatively high, and the low-temperature injury of plants is due to the phase transition of PG (Murata, N. et al., Plant Cell Physiol., 23: 1071). , 1982; Roughan, PG, Plant Physiol., 77: 740, 1985), and the molecular species composition of PG is determined by the substrate selectivity of glycerol-3-acyltransferase (ATase) present in the chloroplasts (Frentzen). , M. et 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).

Based on these assumptions, Nishizawa et al. Proposed that ATase genes obtained from Arabidopsis, a plant resistant to low temperatures, could be introduced and expressed in tobacco to lower the saturated molecular species content of PG and to make tobacco stronger than wild strains (PCT). Patent application: PCT / JP92 / 00024, 1992). However, ATase is also present in the original plants. For example, even if a large amount of foreign ATase is expressed in the plant, competition with endogenous ATase is unavoidable. Therefore, the possibility of diluting the effect of foreign ATase cannot be denied. For example, the content of saturated molecular species of PG in the leaves of clones expressing the largest amount of ATase in Arabidopsis transgenic tobacco was about 28%, which is about 8% less than tobacco wild wine. About 8% more than the state (PCT patent application: PCT / JP92 / 00024, 1992).

In general, the acyl-ACPs made of plastids are mainly 16: 0-ACP and 18: 1-ACP, and their ratio is almost equivalent, but depending on the tissue, 16: 0-ACP or 18: 0-ACP It is also conceivable that the ratio may be higher than 18: 1-ACP (Toriyama, S. et al., Plant Cell Physiol., 29: 615, 1988). In such a tissue, it may be considered that it is difficult to sufficiently reduce the saturated molecular species content by the foreign ATase.

However, the composition of membrane lipids of photosynthetic bacteria cyanobacteria (namzo) is similar to the lipid composition of membrane systems that make up chloroplasts of higher plants (Murata, N. et al., In 'The Biochemistry of Plants', Academic Press). , 1987). In South Korea, 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 nidulans (aka Synechococcus PCC 7942), which can only have one double bond to fatty acids bound to lipids, are cold-sensitive (Ono, T. et al., Plant Phy Siol., 67; 176, 1981). It is known that Synechocystis PCC6803 can be put at low temperature (Wada, H. et al., Plant Cell Physiol., 30: 971, 1989).

In addition, all fatty acid desaturating enzymes in the southern tank use lipids as substrates and introduce double bonds into fatty acids bound to lipids. Therefore, South Korea can introduce cis-type double bonds into fatty acids of PG, SQDG, MGDG and DGDG, which are membrane lipids of 16: 0/16: 0- and 18: 0/16: 0- saturated molecular species. (Murata, N. et al., In 'The Biochemistry of Plants' Academic Press, 1987). This is an fatty acid desaturase that has an enzyme that introduces a double bond at the Δ9 position of stearoyl-ACP (18: 0-ACP), once it is 16: 0/16: 0- (and slightly present 18: Synthesis of PG and SQDG with 0/16: 0-) saturated molecular species is very different from higher plants that never introduce cis-type double bonds to their fatty acids.

By introducing and expressing the Δ12 position desaturase gene of Synechocystis PCC6803 into Anacystis nidulans, it is possible to produce 16: 2Δ9,12 and 18: 2Δ9,12, which are not present in the original Anacystis nidulans, and consequently the low temperature susceptibility Anacystis nidulans It is shown that the conversion to cold resistance is possible (Wada, H. et al., Nature, 347: 200, 1990).

In addition, Δ6 position (Reddy, AS et al., Plant Mol. Biol., 27: 293, 1993) and Δ12 position (Wada, H. et al., Nature, 347: 200, 1990) ) The desaturase gene has been obtained. However, if a double bond is not introduced at the Δ9 position, the Δ6 position and the Δ12 position desaturase cannot unsaturate the Δ6 position and the Δ12 position, respectively. If the Δ9 and Δ12 positions are not desaturated together, the Δ15 position desaturase cannot desaturate the Δ15 position. Therefore, introducing and expressing genes of enzymes that desaturate the Δ9 position of fatty acids into higher plants will lower the saturated molecular species content of higher plants and consequently make the higher plants resistant to low temperatures. However, until now, no enzyme has been obtained that desaturates the Δ9 position of fatty acids.

Accordingly, an object of the present invention is to provide a polynucleotide comprising a gene of an enzyme that desaturates the Δ9 position of a fatty acid and a part thereof.

It is another object of the present invention to provide a vector containing a polynucleotide containing a gene or part of an enzyme that desaturates the Δ9 position of a fatty acid.

It is another object of the present invention to provide a plant cell and a plant into which a polynucleotide containing a gene or a part of an enzyme that desaturates the Δ9 position of a fatty acid is introduced.

Figure 1 shows a comparison of the amino acid sequence encoded by the des 9 var fragment with the amino acid sequence of stearoyl-Co A desaturase (MSCD2) in mice. In the figure, in the case of amino acids of the same amino acid: in the case of amino acids having similar properties, the comparison was made with s. X shows the range with high homology between them.

FIG. 2 is an electrophoresis photograph showing an autoradiogram of Southern analysis of genomic DNA of Anacystis nidulans using a des 9 var fragment as a probe.

Figure 3 shows the interrelationship of insert DNA fragments of λ5, λ15 and p15X. The thick arrows indicate the part and direction that encodes the protein, and the thin arrows indicate the site and direction that determined the sequence.

Figure 4 shows a comparison of the amino acid sequence of des 9 var and stearoyl-CoA desaturase (MSCD2) in mice. Comparison of amino acid sequence was performed similarly to FIG.

Figure 5 is a photograph of the form of the organism showing the effect of cold treatment on transgenic tobacco at the plant level. The left side shows the result of the low temperature treatment of the tobacco into which the desaturase gene was introduced, and the right side shows the result of the low temperature treatment of the tobacco into which pBI121 was introduced.

In order to achieve the above object, the present inventors cloned a gene encoding the Δ9 desaturase from a genome DNA of a South Korean genus belonging to the genus Anacystis, and obtained a vector DNA containing the gene, and then, plant cell was used as the vector DNA. It was successful to give low temperature resistance to plants by transforming and differentiating them to regenerate plants, thus completing the present invention. That is, this invention makes the following matter a summary.

(1) A gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to a lipid.

(2) A polynucleotide comprising the gene according to (1), or a part of the gene, wherein the protein having an activity of desaturating the Δ9 position of the fatty acid bound to the lipid has substantially the amino acid sequence of SEQ ID NO: 4.

(3) The polynucleotide containing the gene as described in (1) whose gene which codes the protein which has the activity which desaturates the Δ9 position of a fatty acid bound to a lipid is a DNA chain containing the nucleotide sequence of SEQ ID NO: 3, or a part of the gene. .

(4) The vector containing the polynucleotide containing the gene as described in any one of (1)-(3), or a part of the gene.

(5) The plant cell into which the polynucleotide containing the gene as described in any one of (1)-(3) or a part of the gene was introduce | transduced.

(6) A plant harvesting method characterized by regenerating a plant by differentiating the plant cells described in (5).

(7) Plant into which the polynucleotide containing the gene as described in any one of (1)-(3) or a part of the gene was introduce | transduced.

Best Mode for Carrying Out the Invention

The Δ9 position desaturating enzyme referred to in the present invention is an enzyme originally present in the bath as described in the above-mentioned 'conventional technique'. The chemical structure of the Δ9 position desaturase is shown in mice (Kaestner, KH et al., J. Biol., Chem., 264: 14755, 1989), rats (Mihara, KJ Biochem., 108: 1022, 1990) and yeast ( Stukey, JE et al., J. Biol. Chem., 265: 20144, 1990), which are locally similar to the chemical structure of stearoyl-CoA desaturase, but differ greatly in overall. In addition, the Δ6 and Δ12 desaturating enzymes of fatty acids bound to known Namzo lipids and the ω3 desaturating enzymes of fatty acids bound to lipids of higher plants (Yadav, NS et al., Plant Physiol., 103: 467, It is not at all similar to the chemical structure of 1993). In the case where the gene of the present invention is produced from natural materials, it may be used as a raw material. The south bath used herein is not particularly limited, and for example, a ramzo belonging to the genus Anacystis, Synechocysits, Anabaena, and the like. In addition, Δ9 of Anacystis type (Murata, N. et al., Plant Cell Physiol., 33: 933, 1992. Group 1 type algae referred to in this document) may be used to desaturate saturated species of higher plants for the following reasons. Gastric desaturating enzymes are preferred over enzymes of Anabaena and Synechocystis types.

That is, in Synechocystis PCC6803 and Anabaena variabilis, almost all of the membrane lipids bind sn-1 and sn-2 with 18 carbon atoms (C18) and 16 carbon atoms (C16), respectively (Sato, N. et al., For Biochem, Biophys.Acta, 710: 279, 1982; Wada, H. et al., Plant Cell Physiol., 30: 971, 1989), in Anacystis nidulans, almost all of sn-1 and sn-2 use C16. (Bishop, DG et al., Plant Cell Physiol., 27: 1593, 1986). Therefore, Δ9 position desaturase of Anabaena and Synechocystis is considered to have the activity of desaturating 18: 1 to 18: 1Δ9 of sn-1, mainly based on the molecular species of 18: 0/16: 0-. On the other hand, Anacystis Δ9 position desaturase is presumed to have the activity of desaturating 16: 1 of Sn-1 to 16: 1Δ9, mainly based on the molecular species of 16: 0/16: 0-. In addition, saturated molecular species found in higher plants are 16: 0/16: 0-, so the Δ9 position desaturating enzyme of Anacystis type is more suitable than the enzymes of Anabaena and Synechocystis type to desaturate saturated species.

The gene of the present invention includes encoding a Δ9 position desaturase having substantially the amino acid sequence as set forth in SEQ ID NO: 4, as shown in the examples below, and differs only in the degeneracy codon, thereby encoding the same polypeptide. It includes decondensation agents that can be used. The gene of the present invention mainly has a specific form as a DNA chain. In addition, the term "substantially the amino acid sequence shown in SEQ ID NO: 4" may have a Δ9 position desaturase activity in addition to the amino acid sequence shown in SEQ ID NO: 4, and may be deleted, substituted or added to a part of the amino acid sequence shown in SEQ ID NO: 4. The amino acid sequence which may be etc. may be included.

The gene of the present invention can be produced from the cells of the haematozoa using a conventionally known method.

In other words, culturing and accumulating cyanobacteria cells, genomic DNA is produced from the hematopoietic cells by a conventionally known method such as ethanol precipitation, a gene library based on the genomic DNA is prepared, and the desired gene is contained from this library. One clone can be selected and produced by amplifying it.

As a vector for gene library preparation used here, what is normally used as this vector is mentioned. Specifically, phage, such as lambda DASH II (Stratagene); cosmids such as pWE15 (Stratagene); and phagemids such as pBluescript II (Stratagene). As a specific gene introduction method for the vector, a conventionally known method can be used according to each vector.

The clone into which the gene of this invention was introduced is selected from the gene library thus prepared.

As the selection method, a known selection method is usually known, for example, an immunological method such as a flag hybridization method or a hybrid hybridization method using an antibody or a flag hybridization method using a nucleotide probe. Or the colonization hybridization method can be used. In addition, a part of the base sequence (for example, a part of amino acid sequence numbers 260 to 295 of MSCD2 shown in FIG. 1) changed to the base sequence was used as a probe as a selection criterion for the nucleotide probe. It is desirable to.

In this way, the determination and confirmation of the nucleotide sequence of the gene of the present invention in the selected clone can be carried out using a known method. For example, Maxam-Gilbert, Methods Enzymol., 65: 499, 1980 or dideoxy nucleotide chain termination using M13 phage (Messing, J. et al., Gene, 19: 269, 1982) Or the like.

In addition, confirmation of whether or not the Δ9 position desaturase is actually expressed can be performed, for example, by a method such as Wada et al. (J. Bacteriol., 175: 6056, 1993).

The gene of the present invention having the base sequence determined as described above can also be synthesized by a commercially available DNA synthesizer using a known means, for example, the phosphite method.

Polynucleotides encoding the gene of the present invention or a polypeptide containing a part of the gene of the present invention and having a Δ9 position unsaturated activity are isolated from the clone, incorporated into a vector for plant gene introduction, and the vector is introduced into a plant cell to place the Δ9 position. By expressing unsaturated enzymes in a plant, low temperature resistance can be imparted to a desired plant.

And there is no particular limitation on the kind of plant that can be introduced into the gene.

Here, the gene for introduction vector is necessary to be configured so that the Δ9 desaturase gene can be stably expressed in the plant. Specifically, a polynucleotide encoding a polypeptide having a Δ9 position unsaturated activity containing a promoter, a DNA chain encoding a translational regulatory region, a DNA chain encoding a transfer peptide to chloroplast, a gene of the present invention or a part of the gene of the present invention, The DNA strands and terminators encoding the translation end codons need to be incorporated in the appropriate positional relationship. Moreover, a well-known thing can be used normally as a component of the vector for gene introduction other than the gene of this invention. As the DNA chain encoding the transfer peptide to the chloroplast, for example, the bovine subunit genes of the ribolos-1, 5-2 phosphate carboxylase of endo can be suitably used as the DNA chain encoding the transfer peptide. As a promoter, for example, the 35S promoter of cauliflower mosaic virus can be used, and as a terminator, for example, a terminator of noparin synthase can be used.

As a gene introduction method into a plant cell, it is usually carried out using a known method, for example, the method described in 'Plant genetic transformation and gene expression: a laboratory manual' Draper, J. et al., Blackwell Scientific Publications, 1988. You can do it. Examples thereof include a method of using a virus, which is a biological method, a method of using Agrobacterium, an electroporation method, a polyethylene glycol method, a microinjection method, and the like that are physical and chemical methods. Among them, a method of using agrobacterium is preferable since it is possible to reliably transform the dicotyledons including tobacco. Methods using Agrobacterium include intermediate vector methods using wild-type tumor plasmids (Nature, 287 (1980), p.654; Cell, 32 (1983) p.1033; EMBO J., 3 (1984) p.1525 ), Intermediate vector method using a vector lacking a tumorigenic gene region on TDNA (EMBO J., 2 (1983) p.2143; Bio / Technology, 3 (1985) p.629), binary vector method (Bio / Technology , 1 (1983) p.262; Nature, 303 (1983) p.179; 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 of cultured cells, protoplasm co-culture, and leaf disk method. It is desirable to.

In addition, in order to regenerate a plant, it is cultured in the medium which added antibiotics for selection, a plant growth hormone, etc. to well-known medium, such as MS-HF medium. Rooted young plants can be transplanted to the soil and grown to complete plants.

Whether or not the transformed plants grown to complete plants have low temperature resistance can be examined as follows.

After cultivation of the black plants at temperatures not affected by cold injury (eg 25 ° C), they are grown temporarily (eg 1 week) at low temperatures (eg 4 ° C) and injured by plants, e.g. chlorosis or leaves. The decrease in toughness can be measured or the growth rate under low temperature can be examined by comparison with the control plant.

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

Example 1

Cloning of a DNA fragment in an open reading frame adjacent upstream of the Δ12 desaturase gene (des A) of Anabaena variabilis

Incubated with Anabaena variabilis IAM M-3 (from the Institute of Molecular and Cellular Biology, University of Tokyo, Japan) in approximately 100 ml of BG-11 medium ('Plant Molecular Biology', Shaw, CH ed., P.279, IRL PRESS, 1988) It was. The mixture was shaken 120 times per minute under a fluorescent lamp of 25 ° C. and 1,000 lux to sufficiently grow. The cells were recovered as a precipitate by centrifugation for 10 minutes at 5,000 g at room temperature.

In order to prepare genomic DNA, the cells were suspended, washed with 50 ml of A solution (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), and the cells were recovered as a precipitate by centrifugation. Next, it was suspended in 15 ml of B liquid (50 mM Tris-HCl, 20 mM EDTA, 50 mM NaCl, 0.25 M sucrose, pH8.0), 40 mg of rizzo (Sigma) dissolved in B liquid was added and shaken at 37 ° C for 1 hour. . Then 15 mg of proteinase K and SDS were added to a final concentration of 1% and shaken overnight at 37 ° C. Thereafter, NaClO ₄ was added so as to have a final concentration of 1 M, and 20 ml of chloroform / isoamyl alcohol (24: 1) was added thereto, followed by shaking for 10 minutes, and 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 to recover the genomic DNA preparations by winding them on a glass rod. This DNA preparation was dissolved in 20 ml of A solution, NaCl was added to a final concentration of 0.1 M, RNase was added to a final concentration of 50 mg / ml, and incubated at 37 ° C for 1 hour. Next, the mixture was extracted twice with an equivalent amount of phenol saturated with A solution. The genomic DNA in the aqueous layer was recovered as a precipitate by adding ethanol, washed with 70% ethanol, and then dissolved in 1 ml of A solution, followed by a genomic DNA solution of Anabaena variabilis. It was made.

Sagamodo et al. Announced the cloning of the Δ12 position desaturase gene of fatty acids bound to membrane lipids derived from Anabaena variabilis (1993 Japanese Society of Plant Physiology, Lecture Summary, No.3aF04). There was an open reading frame (ORF) upstream of the gene, and it was reported that this had some relationship with desaturase, but its function was not identified. The present inventors are interested in the ORF and function, paying attention to three base sequences in the DNA chain of the ORF, and synthesizing four primers (SEQ ID NO: 5 to SEQ ID NO: 8) to form a genome DNA of Anabaena variabilis PCR was carried out as.

Among the four primers, a primer having a nucleotide sequence shown in SEQ ID NOs: 5 and 6 has a sense chain, and a primer having a nucleotide sequence shown in SEQ ID NOs: 7 and 8 codes an antisense chain, and a base sequence shown in SEQ ID NOs: 6 and 7 It is derived from the same amino acid sequence. One kind of primer was arbitrarily selected from a sense chain and an antisense chain, respectively, and PCR was performed by combining four types of primers. The reaction was carried out using a GeneAmp PCR Kit (Takara Shuzo) with 20 μM of primers and 1 μg of genomic DNA of Anabaena variabilis in 100 μl of reaction solution. The temperature control of the reaction was carried out for 35 cycles at 95 ° C (1 minute), 45 ° C (1 minute) and 72 ° C (2 minutes) as one cycle. However, 95 degreeC of the 1st cycle was made into 3 minutes. After completion of the reaction, 10 μl of the reaction solution was electrophoresed on a 2% agarose gel to separate and synthesize the synthesized DNA. As a result, DNA fragments of the expected size (about 190 bp) were detected as major bands in the DNA synthesized by the combination of the primers having the nucleotide sequences shown in SEQ ID NOs: 6 and 8. Both ends of this DNA (hereinafter referred to as des 9 var) fragments were blunted with Klenow fragments, cloned into the sma I site of plasmid pTZ18R (Pharmacia), and nucleotide sequences were determined using fluorescent DNA sequencers (Applied Biosystems). It was. The obtained nucleotide sequence is shown in SEQ ID NO: 1. The amino acid sequence estimated 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 stearate. A comparison of the amino acid sequence of loyl-CoA desaturase (MSCD2) is shown.

Next, the genomic DNA of Anacystis nidulans was analyzed by des 9 var fragment as a probe. Each of the restriction enzymes Xho I, Pst I and BamH I alone was used to cleave about 0.1 μg of genomic DNA of Anacystis nidulans, and DNA fragments were isolated by 0.8% agarose gel electrophoresis, followed by nylon membrane (Hybond-N). ⁺ ; Amersham). Probe DNA was labeled with [α- ³² P] dCTP using Multiprime DNA labeling Kit (Amersham). Incubate probe DNA and membrane at 55 ° C for 16 hours in 6 x SSPE [1 x SSPE in 10 mM phosphate buffer (pH7.0), 1 mM EDTA, 0.15 NaCl], 0.2% SDS and 100 μg / ml herring sperm DNA. Reacted. The membrane was then washed by shaking twice at room temperature for 15 minutes in 2 × SSC [1 × SSC for 0.15M NaCl, 15 mM sodium citrate], followed by 40 ° C. for 15 minutes in 0.1 × SSC and autoradiography. . As a result, only one DNA fragment was detected even when cleaved by any restriction enzyme (Fig. 2: Non indicates that the genomic DNA was not cleaved with restriction enzyme).

Example 2

Cloning of the DNA Chain in the Anacystis nidulans Genome with High Homology to the des 9 var Fragment

Culture of Anacystis nidulans R2-SPc (prepared from the Institute of Molecular Cell Biology) and preparation of genomic DNA were performed as in the case of Anabaena variabilis. After partial digestion of about 100 μg of genomic DNA with Sau3AI, seconds under sucrose density gradient according to the method of Molecular Cloning 2nd edition, pp.2.85-2.87 (Sambrook, J. et al. Eds., Cold Spring Harbor Laboratory, 1989). The DNA fragment of about 9-23 kbp was recovered by centrifugation. This was cloned into lambda phage vector λDASH II (Stratagene) digested with BamH I and HindIII, and then packaged with phage particles to obtain a genomic DNA library of Anacystis nidulans. The phage library was infected with E. coli P2392, plated in a Petri dish of about 15 cm in diameter with NZYM medium to form a total of about 100,000 flags, and blotted onto a nylon membrane (Hybond-N ⁺ ; Amersham). Similar to the Southern analysis above, about 30 phage clones with different signal intensities were reacted with a des 9 var fragment labeled with [α- ³² P] dCTP and reacted with this membrane to again screen positive phage detected by autoradiography. Got. Among them, 12 clones were randomly selected to obtain phage DNA according to the conventional method. The obtained phage DNA was digested with several kinds of restriction enzymes, separated by 0.8% agarose gel electrophoresis, and blotted onto a nylon membrane. The membrane was Southern-analyzed under the same conditions as those of the above screening, and the length and signal intensity of the DNA fragment hybridized with the probe DNA were compared. As a result, since 2 clones of λ5 and λ15 showed the strongest signal and the length of the insert DNA fragment was 11 and 15 kbp, respectively, it was judged that it was sufficient to cover the entire ORF of interest. Again, several restriction enzymes were cut and Southern analysis was performed. As a result, DNA fragments of about 5kbp were detected when both clones were cut and hybridized with Xho I. Subcloning these into the Xho I site of pBluescript SK- (Stratagene) resulted in DNA fragments derived from λ5 and λ15, respectively. Plasmids p5X and p15X were obtained. A detailed restriction map of p5X and p15X was made and compared, and it was judged that they contained the same genomic DNA fragment. [FIG. 3: Correlation of insert DNA fragments of λ5, λ15 and p15X. The meshed rectangles represent DNA fragments hybridized with des 9 var fragments of the probe during screening. The thick arrows indicate the region of des 9 nid (described below) and the direction of the sense chain. Thin arrows indicate the direction of the sequence of the region containing des 9 nid. Each bar of 5, 1.25 and 0.5 kbp represents a size marker in each figure on the left. Abbreviations for restriction enzymes include B, BamHI; H, HindII; N, NotI; Hp, HpaI ,; RI, EcoRI; RV, Eco V; S, Sa1I; P, PstI; X, XhoI].

Thus, a restriction enzyme or ExoIII digestion plasmid was prepared from p15X, and the base sequence of the DNA fragment of about 2 kbp including the region where the des 9 var fragment hybridized was determined using a fluorescent DNA sequencer (Figure 3). As a result, an ORF (des 9 nid) of 834 bp was present in the DNA fragment (SEQ ID NO: 3), and 278 residues of amino acid were encoded (SEQ ID NO: 4). The homology with the amino acid sequence (SEQ ID NO: 2) encoded by the des 9 var fragment derived from Anabaena variabilis, which was first cloned, was about 80%. In addition, we searched homologous amino acid sequences using nucleic acid and amino acid sequence analysis software (GENETYX: software development) and nucleic acid and amino acid sequence databases (EMBL and DDBJ). The homology with the enzyme is about 30% in total, but is very high locally (Fig. 4: Comparison of amino acid sequence of des 9 nid and mouse stearoyl-CoA desaturase (MSCD2)]. 9 nid was strongly suggested to encode an enzyme that desaturates fatty acids.

Example 3

Activity measurement by expression of des 9 nid gene in Escherichia coli

Anacystis nidulans has only Δ9 position desaturase activity that desaturates the 9th position of saturated fatty acids bound to lipids as desaturase (Bishop, DG et al., Plant Cell Physiol., 27: 1593, 1986). An attempt was made to measure activity by expressing the polypeptide encoded by 9 nid in E. coli.

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 cloning was carried out so that des 9 nid was not attached to the amino terminus between the NdeI and BamHI so as not to attach an extra amino acid to the amino terminus. Immediately after the C-terminal side of the protein encoded by des 9 nid, PCR was performed using two nucleotide sequences with the C-terminal inserted into the BamHI site.

Sense primer; 5'-ACGTCATGGC CTGCAGT (underline is PstI site) (SEQ ID NO: 9)

Anthense primers; (Single line is BamHI site, double line is stop codon) (SEQ ID NO: 10).

PCR reaction using the two primers using p15X as a template yielded a product of about 140 bp, which was subcloned into the SmtI site of pUC19 to confirm that the sequence was correct. An EcoRI site was generated downstream of BamHI of the resulting plasmid. The BAMHI site was introduced immediately after the stop codon by sequentially cleaving with EcoRI and PstI, and cutting with p15X with the same restriction enzyme. This plasmid was digested with Sa1I, followed by Fi111 in reaction using DNA polymerase Klenow fragment in the presence of four dNTPs, followed by cleavage with HindIII. In addition, the NdeI site | region was introduce | transduced into the amino acid terminal side by introducing the adapter which consists of the following 2 types of synthetic DNA. In other words

5'- CATATG ACCCTTGCTATCCGACCCA (underline NdeI) (array number 11) and

(Singlet is NdeI site doublet is part of HindIII) (SEQ ID NO: 12)

Was mixed in an equimolar amount to obtain an adapter. The resulting plasmid (pDes9Nde) was introduced into the competent cell of Escherichia coli BL21 (DE3) (Novagen) adjusted according to the conventional method (Molecular cloning pp. 250 to 251; 1982) and transformed by selection by ampicillin resistance. Obtained the week BLDES1.

BL21 strain (BL1) with BLDES1 and pET3a only was inoculated in 100 ml of M9 medium (containing 200 μg / ml ampicillin, 4 mg / ml glucose, 10 μM FeCl ₃ , 0.5 μg / ml vitamin B1, 1 mg / ml gazamino acid) , And incubated at 37 ℃. After culturing was continued until the turbidity of the culture solution became 0.5 OD at a wavelength of 600 nm, isopropylthiogarakside (IPTG) was added to a final concentration of 1 mM. Incubation was again carried out for 1 hour to induce expression of the Δ9 position desaturase gene. The recovered E. coli pellets were washed with 1.2% NaCl, and then lipids were extracted. Lipids were extracted according to Bligh and Dyer's method (Can J. Biochem. Physiol., 37: 911, 1959), completely sealed with 2.5 ml of 5% hydrochloric acid methanol, and reacted at 85 ° C. for 2 hours to methylate fatty acids. It was. The resulting fatty acid methyl ester was extracted four times with 2.5 ml of hexane, and concentrated by removing the solvent with nitrogen gas. Gas chromatography was used for the analysis of fatty acid methyl esters. Identification of fatty acids was carried out by comparing the retention time with methyl standard fatty acid. Chromatography pack C-R7A plus (Shimazu Seisakusho) was used for quantification. The results are shown in the first table below.

Here, time represents the induction time of the protein by IPTG.

Obviously, 16: 1 is increasing in BLDES1. In other words, this gene has an unsaturated activity of 16: 0.

These strains were also cultured in M9 medium containing 0.1 mM stearic acid and compared in the same manner.BlDES1 produced not only 16: 1 but also 18: 1 (9) compared to BL1 strain, and the polypeptide encoded by des 9 nid was 16. Not only: 0 but also 18: 0 showed unsaturated fatty acid as substrate.

Example 4

Introduction of the des 9 nid Gene into Tobacco Plants

The des 9 nid gene derived from Anacystis nidulans was introduced into tobacco as follows.

(1) Construction of vector plasmid for plant expression

By cleaving pDes9Nde with SacI and SaII, a des 9 nid gene fragment inserted at the cleavage site of both enzymes is obtained. Meanwhile, the transit sequence from the clone pSNIP9 (Schreicher et al., EMBO J. 4,25 (1985)) containing the endo RuBisCO gene to chloroplast was cleaved with HindIII and SphI and cloned into pUC118 digested with the same restriction enzyme. Plasmids with multicloning sites (pTRA3) were obtained downstream of the transit array. After cleaving this HindIII site, it was filled in with Klenow enzyme and put into XbaI linker (pTRA3X). The plasmid pTRA3X was digested with Sal I and Sac I, and the des 9 nid gene fragment obtained by cleavage with the same restriction enzyme was inserted first (pTRA3Xdes9). In this plasmid, following the RuBisCO tranit sequence, the des 9 nid gene is translated into the same reading frame. This is cut into Sac I and Xba I and inserted into the plant vectors described below. Plasmid pBI (-GUS) obtained by cleaving the plant-expression binary plasmid pBI121 (Clonetech) with restriction enzymes Sac I and Xba I does not contain the β-Glucuronidase gene (GUS gene), which contains the cauliflower mosaic virus. A plant introduction vector (pBI121 (-GUS) Rbsc-des9) was obtained by inserting the above-mentioned transgene between the 35S promoter and the nopaline synthase (NOS) terminator.

(2) introduction into Agrobacterium of pBI121 (-GUS) Rbsc-des9

Agrobacterium tumefaciens LBA4404 (Clonetech) was inoculated in 50 ml of YEB medium (5 beef extracts per 1 liter, 1 g yeast extract, 1 g peptone, 5 g sucrose, 2 mM MgS04 (pH 7.4)), and cultured at 28 ° C. for 24 hours. The cells were collected by centrifugation at 3,000 rpm, 4 ° C. for 20 minutes. The cells were washed three times with 10 ml of 1 mM Hepes-KOH (pH 7.4), then washed once with 3 ml of 10% glycerol, and finally suspended in 3 ml of 10% glycerol to prepare Agrobacterium for DNA introduction.

10 μl of the obtained bacterial solution and 1 μg of the above-mentioned plasmid pBI121 (-GUS) Rbsc-des9 were put into a cuvette, and an electric pulse was applied under conditions of 25 μF, 2500 V, and 200 Ω using an electroporation device (Gene Pulser; BioRad). DNA was introduced into Agrobacterium. Transfer this bacteria solution to an Eppendorf tube and add 800 μl of SOC medium (20 g of tryptone per liter, 5 g of yeast extract, 0.5 g of NaCl, 2.5 mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ , 20 mM glucose, pH7.0) It was incubated for 1.5 hours at 28 degreeC. 50 µl of this culture solution was sprayed on YEB agar medium (1.2% agar) containing 100 ppm of kanamycin and incubated at 28 ° C for 2 days.

Single colonies were selected from the obtained colonies, and plasmid DNA was adjusted from the colonies by the alkali method. After digesting this plasmid DNA with an appropriate restriction enzyme, DNA fragments were isolated by 1% agarose gel electrophoresis, and plasmid pBI121 (-GUS) Rbsc by Southern analysis probed for the des 9 nid gene fragment labeled with 32P. It was confirmed that it contained -des9. This Agrobacterium tumefaciens is called ALBBSDES.

(3) transformation of tobacco

The strain ALBBSDES was shaken for 2 days at 28 ° C. in an LB liquid medium containing 50 ppm of kanamycin. 1.5 ml of the culture solution was collected by centrifugation at 10,000 rpm for 3 minutes, and then washed with 1 ml of LB medium to remove kanamycin. After centrifugation for 3 minutes at 10,000 rpm, the cells were collected and resuspended in 1.5 ml of LB medium to obtain bacterial cells for infection.

When infected with tobacco, young leaves were taken, immersed in 0.5% sodium hypochlorite aqueous solution for 10 minutes, washed three times with sterile water, and wiped with water on a sterile space to obtain leaves for infection. One leaf is cut aseptically with a scalpel so that one piece is 1 cm ² , the back side of the leaf is placed on the Agrobacterium fungus solution, shaken quietly for 2 minutes, and then the leaf is placed on the sterilized place. Agrobacterium was removed. Watts on MS-B5 medium (containing 1.0 ppm benzyladenin, 0.1 ppm naphthalene acetate, and 0.8% agar) in Petri dishes (Murashige, T. and Skoog, F. Plant Physiol., 15: 473, (1962) Bay No. 1 (φ 7.0) was placed, and the leaves were placed with the rear face up, and the Petri dishes were sealed with parafilm, and incubated for 16 hours at 25 ° C. for 2 hours with a bright 8 hour dark cycle. It was transferred to MS-B5 medium containing 250 ppm of claporan and incubated for 10 days as well to remove Agrobacterium, and 250 ppm of clapola was then placed on MS-B5 medium containing 100 ppm of kanamycin and cultured for 7 days as well. In the meantime, the periphery of the flanks was callusized, shoots emerged, and after 10 days of incubation, the elongated shoots contained MS-HF medium containing 250 ppm of claphoran and 100 ppm of kanamycin (benzyladenin and naphthalene acetate). MS-B5 medium) . 10 days, to as transformants of kanamycin for rooting the shoots resistant, were transferred to a MS-HF medium containing 250ppm Cloud sulfolane in the plant box after culture.

Example 5

Genomic Southern and Northern Analysis of Transgenic Tobacco

In order to confirm the introduction of the gene of interest, DNA was extracted from kanamycin-resistant tobacco and Southern and northern analysis was performed. Genomic DNA extraction was performed by CTAB method according to the Bible (Rogers, SO & Bendich, AJ; Plant Molecular Biology Manual A6; 1 (1988)), ie, 2 g of tobacco leaves were pulverized in liquid nitrogen and used as CTAB extraction buffer. Genomic DNA was obtained: 10 μg of DNA was digested with restriction enzymes EcoRI and XbaI and electrophoresed with 0.7% agarose gel, which was then transcribed into 0.4 N NaOH on a nylon membrane (Hybond N ⁺ ; Amersham) from pTRA3Xdes9. Using the transit desaturated gene as a probe, hybridization at 65 ° C. for 16 hours confirmed that the target gene was contained in the tobacco genome.

In addition, RNA was analyzed from about 2 g of tobacco leaves to investigate the expression of the transgene. The method was extracted with guanidium thiocyanic acid (Nagy, F. et al .: Plant Molecular Biology Manual B4; 1 (1988)), followed by electrophoresis of ploy (A) + RNA with an agarose gel containing formaldehyde. It was transferred to a nylon membrane (Hybond N; Amersham) and analyzed by hybridization similar to that of the Southern method. Although there were individuals expressing various amounts of RNA, lipid analysis was performed in Example 6 with respect to the individuals with a large amount of expression.

Example 6

Fatty Acid Analysis of Lipids in Transgenic Tobacco

From the tobacco transformants in which high expression of RNA was confirmed in Example 5, and tobacco leaves transformed with pBI121 as a control, phosphotadyl glycerol (PG), sulfokinobosildiacyl glycerol (SQDG), etc. by the following method Lipid was adjusted and the fatty acid composition was analyzed. Some individuals also analyzed lipids in roots.

(1) Extraction of Whole Lipids

Lipid extraction was performed using the Bligh-Dyer method (Can J. Biochem. Physiol., 37: 911, 1959). 2 g of wet weight leaves (1 g when using some roots as a sample) are shredded with a scalpel, and 20 ml of chloroformum: methanol (1: 2, volume ratio) is added thereto, and the leaves are shredded with a homogenizer. Let stand for a minute. 12 ml of chloroform and 12 ml of distilled water were added thereto, and the mixture was mixed vigorously, followed by centrifugation at 3000 rpm, 4 ° C for 30 minutes, and divided into two layers, an aqueous layer and an organic layer, to recover an organic layer (lower layer). An appropriate amount of ethanol was added thereto, and the solvent was removed at 30 ° C. under reduced pressure using a rotary evaporator. This was dissolved in 2 ml of chloroform: methanol (1: 4, volume ratio) to obtain a whole lipid extract. Methylated fatty acid was obtained by treating this one volume by the method mentioned later using 5% methanolic hydrochloric acid.

(2) fraction of lipids

2.5 ml of a suspension of DEAE-Toyopearl 650C (Doso) was mixed with 25 ml of an aqueous 1 M sodium acetate solution (pH 7.0) to obtain acetic acid. This was successively washed with distilled water and methanol, and finally suspended in methanol, and filled into a column of 2 cm in diameter up to 1.5 cm in height, and then washed with 50 ml of chloroform: methanol (1: 4, volume ratio).

Next, the whole lipid extract was added to a column with 50 ml of chloroform: methanol (1: 4, volume ratio), monogalactosyldiacylglycerol (MGDG), digaractosyldiacylglycerol (DGDG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) was eluted to a neutral lipid (MGDG, DGDG, PE, PC) fraction. Next, phosphatidylserine (PS) was eluted and removed with 5 ml of acetic acid, and acetic acid was washed with 20 ml of chloroform: methanol (1: 4, volume ratio), and then 50 ml of chloroform: methanol: 10M aqueous ammonium acetate solution (20: 80). : 0.2, volume ratio) to obtain a fraction containing PG, SQDG, and phosphatidylinositol (PI). 15 ml of ethanol were added to this fraction, and the solvent was removed under reduced pressure. This was dissolved in 0.2 ml of chloroform: methol (2: 1, volume ratio) to obtain an acidic lipid (PG, SQDG, PI) fraction.

MGDG, DGDG, PE, and PC fractions were re-fractionated by silicate column chromatography (Itorobis, Yatron). In other words, a sample dissolved in 1 ml of chloroform is eluted in a column equilibrated with chloroform in the order of chloroform: acetone (4: 1), acetone, and methanol, and glycolipid (MGDG, DGDG) is acetone, and phospholipid (PC, PE) is methanol. Eluted.

(3) Isolation and Fatty Acid Analysis of PG by Thin Layer Chromatography (TLC)

The fraction obtained in (2) was separated by silica gel-TLC plate # 5721 (Merck). As the developing solvent, chloroform: acetone: methanol: acetic acid: water (50: 20: 10: 15: 5, volume ratio) for acidic lipids, and chloroform: ethanol: water (70: 21: 3, volume ratio for neutral lipids) ) Was used. After separation by TLC, priming phosphorus (80% acetone solution) was sprayed to fluoresce under ultraviolet light, and the fraction of lipids of each class was estimated by comparing the mobility with standard lipids. It was removed and placed in a test tube with a screw head. In estimating this fatty acid composition, 3 ml of methanolic 5% hydrochloric acid was added to the test tube, and the mixture was completely sealed and reacted for 2 and a half hours at 85 ° C. for fatty acid methylation. Meanwhile, 5 ml of chloroform: methanol (2: 1) mixture was collected and dried from silica gel sieved to determine the fatty acid composition of every sn-1, 2, and then dried, and then 1 ml of 50 mM TrisCl (pH 7.2) and 0.05% Triton. Lipids are dispersed by vigorous stirring with X-100, and lipase (2500U; Boehringer) derived from Rhizopus delemar is added to insulate for 30 minutes at 37 ° C. to selectively decompose the fatty acid of sn-1. I was. The reaction product was concentrated and separated into unreacted lipids, lysates, and fatty acids by TLC (chloroform: acetone: methanol: acetic acid: water = 10: 4: 2: 3: 1). These were also recovered from the gel to obtain methylated fatty acids with methalen hydrochloric acid as described above. The resulting fatty acid methyl ester was extracted four times with 3 ml of hexane, and the solvent was concentrated under reduced pressure. Gas chromatography was used for the analysis of fatty acid methyl. Identification of fatty acids was carried out by comparing the retention time with methyl standard fatty acid. Chromatography pack C-R7A plus (Shimazu Seisakusho) was used for quantification. The results of all lipids are shown in Table 2, PG in Table 3, and other representative lipid analysis results in Table 4. The table shows the average value of the analysis values of two or three individuals for control plants and independent two or three for transformants.

From the results of fatty acid analysis bound to PG, the transgenic tobacco expressing fatty acid desaturase derived from Anacystis nidulans significantly reduced 16: 0 (palmitic acid), and instead increased 16: 1 cis and a small amount 18: 0, which was also almost gone, turned out to be 18: 1 increasing. As a result, the saturated fatty acid (16: 0 + 16: 1 trans + 18: 0 (stearic acid)) content was significantly lower, compared to 70% in the control tobacco, 55% in tobacco transfected with the desaturase gene. have. From the analysis results of sn-1 and 2nd place of PG, sn-2 is occupied by more than 98% unsaturated fatty acids (16: 0 or 16: 1trans), and all of the 16: 1 produced by the introduction of new genes are sn-1. It turned out to be. Therefore, it is clear that the saturated fatty acid on sn-1 of PG of the tobacco transformed with this desaturase gene is extremely small. Therefore, it can be seen that the amounts of so-called saturated molecular species, both of which are both saturated fatty acids and both of sn-1 and 2, are greatly reduced, so that the composition of the molecular species of lipids is changed to a form that is significantly resistant to low temperatures.

Meanwhile other lipids MGDG, DGDG, SQDG, PC. In PE and PI, the decrease of 16: 0 and the increase of about 10% of 16: 1 corresponding to it were obvious, and the unsaturation of 18: 0 also advanced. Among them, the production of 16: 1 was mainly found in sn-1 for MGDG and DGDG, but a small amount was detected from sn-2. MGDG, DGDG, SQDG and PG are lipids present mainly in the chloroplasts, and it can be seen that the desaturation progresses surprisingly by expressing desaturation enzymes of Anacystis nidulans, which is a male alga, into chloroplasts of higher plants. Moreover, these four lipids are also present in the membranes of Anacystis nidulans, and are likely to be unsaturated substrates, but other PCs, PEs, and PIs are lipids that do not exist in the membranes of Anacystis nidulans, and also in higher plants. It is surprising that palmitic acid and stearic acid are unsaturated in them because they are lipids which are mainly present outside chloroplasts.

As a result of the lipid analysis of transgenic tobacco, it was found that fatty acid desaturase derived from Anacystis nidulans can desaturate almost all of the lipids 16: 0 and 18: 0 with extremely good efficiency in the transformants of tobacco which is a higher plant. This example proved.

The results of fatty acid analysis on all lipids in the roots are shown in Table 5.

From these results, it was found that fatty acid desaturase derived from Anacystis nidulans catalyzed 16: 0 and 18: 0 desaturation not only in the leaves but also in the roots. This indicates that the fatty acid desaturase gene of the present invention has not only the possibility of changing the cold resistance of plants, but also the possibility of increasing the content of unsaturated fatty acids, and is also useful in industries in which plants are used as oil.

Example 7

Cold Resistance Test of Transgenic Tobacco

The next generation of seeds was collected by the offspring of individuals considered promising in the expression analysis and lipid analysis of the above RNA. Some of them were planted in MS-HF medium containing 800 ppm of kanamycin, and brightened at 25 ° C., 16 hours a day, and darkened for 8 hours, followed by cultivation of kanamycin resistance after 2 weeks of cultivation. This seedling was transplanted to the plant box and grown again for 4 weeks. In addition, the above-mentioned operation was performed also about the individual transformed with pBI121.

After 11 days of low temperature treatment under continuous light at 4 ° C, the cells were grown at 25 ° C for 2 days. As a result, injuries were hardly observed in the transgenic plants, while the atrophy and chlorosis were observed in the leaves in the control plants (plants transformed with pBI121). Therefore, it was assumed that low temperature resistance was improved by introduction of desaturase gene.

By introducing the gene encoding the Δ9 position desaturase of the present invention into the plant, it has become possible to impart low temperature resistance to the plant and to increase the unsaturated fatty acid content in the plant.

Claims (4)

It is a protein substantially having the amino acid sequence set forth in SEQ ID NO: 4, characterized in that the plant cells are regenerated by differentiating plant cells into which a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to lipids is introduced. How to harvest plants. It is a DNA chain gene comprising the nucleotide sequence of SEQ ID NO: 3, characterized in that to regenerate the plant by differentiating plant cells into which a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to lipids is introduced. How to harvest plants. It is a protein substantially having the amino acid sequence set forth in SEQ ID NO: 4, and regenerating plants by differentiating plant cells of a dicotyledonous plant into which a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to lipids is introduced. A plant harvesting method characterized by the above-mentioned. Regenerates plants by differentiating plant cells of dicotyledonous plants in which a gene is a DNA chain comprising the nucleotide sequence set forth in SEQ ID NO: 3, and a gene encoding a protein having an activity of desaturating the Δ9 position of a fatty acid bound to lipids is introduced. Plant harvesting method characterized in that.