JPH1014579A - Monogalactosyl diacylglycerol synthase and gene coding the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject enzyme having a specific amino acid sequence, a monogalactosyl diacylglycerol(MGDG) synthase found abundantly in plants, etc., useful for improving the lipid composition of organisms, esp. controlling the quantity of MGDG. SOLUTION: This enzyme is a new monogalactosyl diacylglycerol(MGDG) synthase as a galactolipid found abundantly in plants, etc., having an amino acid sequence of the formula or that virtually identical with the above-mentioned amino acrid sequence. This enzyme is useful for improving the lipid composition of animals, plants or microorganisms owing to its gene, esp. for controlling the quantity of MGDG. This MGDG synthase is obtained by a process wherein cucumber seeds are allowed to germinate, a mRNA is separated from the resultant greened cotyledons; using the mRNA, a cDNA library is constructed and then retrieved using a synthetic DNA probe coding a partial peptide sequence estimated from the purified enzyme, and the resultant gene is integrated into an expression vector and expressed in host cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an enzyme for synthesizing monogalactosyldiacylglycerol (MGDG), which is a major lipid of plants and some microorganisms, and a gene encoding the same.

[0002]

2. Description of the Related Art Basic components of biological membranes are lipids and proteins. Among lipids, there are polar lipids such as phospholipids and glycolipids and neutral lipids such as pigments and sterols. In the cell membrane of chloroplasts and some photosynthetic microorganisms (cyanobacteria) in plants,
There are several types of lipids that are thought to be associated with photosynthesis. Most of the lipids in these membranes are monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), and the lower ones are phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol. (SQDG). The synthesis of the main lipid, MGDG, occurs in plants in the chloroplast envelope. That is, 1,2-
The transfer of galactose from UDP-galactose to the 3-position of diacyl-sn-glycerol gives 1,2-diacyl-
Produces 3-O- (β-D-galactopyranosyl) -sn-glycerol (MGDG) (EC 2.4.1.
46). Galactolipids (MGDG, DGDG) and SQD
Since G uses diacylglycerol as a common substrate, it is believed that the ratio of galactolipid to SQDG is regulated by MGDG synthase. In fact, galactolipids are estimated to be present in 5-20 times the amount of SQDG molecules. MGDG is abundant in photosynthetic tissues,
There is 0.6 to 15 μmol / g fresh leaf weight. Considering that the chloroplast membrane is the most abundant in the cell membrane system, it is speculated that MGDG is the most abundant lipid in the cell. Galactolipids contain a large amount of polyunsaturated fatty acids, of which about 95% is linolenic acid. Therefore, as the molecular species, sn-1, 2
Both are often occupied by 18: 3, but in a so-called "16: 3 plant" such as spinach, sn-1
Is 18: 3, while sn-2 is occupied by 16: 3. On the other hand, the molecular species occupied by both positions at 18: 3 is a so-called “eukaryotic” lipid which is synthesized in the cytoplasmic endoplasmic reticulum and then transferred to the chloroplast.
What replaces n-2 at 16: 3 is a so-called "prokaryotic" lipid that is totally synthesized in chloroplasts.

[0003] Since MGDG synthase is a trace enzyme present in the chloroplast envelope, it has been extremely difficult to purify the MGDG synthase despite its extremely large amount. First, the envelope is a small component in the total membrane components of cells, and its protein is also 1 to 2% of the total chloroplast protein.
Just occupy. Second, the envelope has a much larger amount of lipids than proteins, and it is difficult that the enzyme is not easily solubilized. As the content of MGDG synthase,
It is said to be 0.1 to 0.5% of the envelope protein. Two groups have reported partial purification of this enzyme, all of which have successfully concentrated enzymatic activity several hundred times from the chloroplast envelope of spinach, but have not yet achieved complete purification ( CR Acad. Sci. Paris, 313: 521,
1991; Planta, 184,319, 1991). The molecular weight is about 20
kDa.

[0004]

A large number of mutants involved in plant lipid synthesis have been isolated from Arabidopsis thaliana. Particularly, enzymes that perform desaturation reactions have been analyzed in detail. However, as for MGDG synthase, the amount of MGDG is extremely large as described above, and no mutation has been reported, though it seems that the mutation is easily detected. Although MGDG is a lipid that exists widely in the chloroplast membrane system, it is known that it forms a complex strongly bound to the reaction center of photosystem II, in particular, as well as the thylakoid membrane. (Biochim. Biophys. Acta, 101
9: 261, 1990). In addition, it is said that MGDG in particular plays a role in stabilizing the curved portion of the thylakoid membrane and folding hydrophobic proteins (FEBS LETT., 150: 19,198).
2). From these facts, it is assumed that the physiological role of MGDG in photosynthesis and the like is extremely large. However, techniques to manipulate membrane stabilization by adjusting its amount, including the use of mutants, were not possible.

[0005] An object of the present invention is to isolate an enzyme involved in the synthesis of MGDG, which plays an important role in plant organisms, and a gene encoding the same.
An object of the present invention is to produce a plant or the like in which the MGDG-producing ability is artificially modified by introducing the gene.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventor succeeded in isolating a gene encoding MGDG synthase, and further introduced the gene into Escherichia coli. And succeeded in imparting MGDG production ability, and completed the present invention.

That is, the present invention provides a monogalactosyldiacylglycerol synthase having the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3; A fusion protein consisting of another protein.

[0008] The present invention also relates to a monogalactosyldiacylglycerol synthase gene encoding the amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. Furthermore, the present invention is a recombinant vector containing the above gene and a cell into which the above gene has been introduced.

Hereinafter, the present invention will be described in detail. The MGDG synthase of the present invention has an amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. Also, the MGDG of the present invention
The synthase gene encodes the amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. Here, "SEQ ID NO: 3
"An amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3" is a sequence in which some amino acid residues of the amino acid sequence represented by SEQ ID NO: 3 have undergone a change such as substitution, deletion, or addition. And an amino acid sequence having an activity of synthesizing MGDG, similar to the amino acid sequence represented by SEQ ID NO: 3. Inducing changes such as substitutions, deletions, and additions for some amino acid residues can be achieved by techniques commonly used at the time of filing the present application, such as site-directed mutagenesis (Nucl
eic Acid Research, Vol. 10, No. 20, p6487-6500). In general, when a DNA chain encodes a polypeptide having an amino acid sequence, there are a plurality of genetic codes (codons) corresponding to one amino acid (degenerate isomers), but the MGDG synthase gene of the present invention Of course, any genetic code corresponding to the amino acid sequence represented by SEQ ID NO: 3 can be used.

[0010] The MGDG synthase of the present invention is an enzyme originally present in plants and some microorganisms (cyanobacteria), as described in the above “Conventional Technology” and the like. The amino acid sequence of MGDG synthase is locally similar to the protein encoded by the MurG gene of Bacillus subtilis and Escherichia coli, but largely different overall (FIG. 6). In addition, it does not resemble the chemical structure of other enzymes involved in the synthesis of fatty acids and lipids.

[0011] MGDG synthase is contained in plants and cyanobacteria and can be isolated therefrom. Specifically, it can be performed by the following method.
First, cells of a plant or cyanobacteria are disrupted, and a microsomal fraction is obtained by centrifugation. Next, a surfactant is added to the microsomal fraction to solubilize the MGDG synthase bound to the membrane. The surfactant used at this time is not particularly limited, but LDAO is preferably used. Solubilized M
GDG synthase is purified by various types of chromatography using its activity as an index. The activity of MGDG synthase was determined by the method of Teucher and Heinz (Planta 184,319,1991).
Can be measured. In addition to using the above-mentioned natural materials as raw materials, MGDG synthase can be isolated from transformed cells into which the MGDG synthase gene has been introduced.

The MGDG synthase of the present invention can be fused with another protein to form a fusion protein. M
The protein to be fused with the GDG synthase is not particularly limited, for example, glutathione S-transferase,
β-galactosidase, maltose binding protein and the like can be used. Such a fusion protein
It can be prepared by inserting a base sequence encoding the other protein between the MGDG synthase gene and its promoter, and expressing this fusion gene. MGD
The use of the G synthase as a fusion protein has advantages such as improvement of expression level and easy purification of the protein.

[0013] The MGDG synthase gene can be obtained, for example, as follows. First, MGDG synthase is isolated from a plant or cyanobacterium, fragmented with peptidase, and the amino acid sequence of the fragment is determined. Next, an oligonucleotide corresponding to the fragmented peptide whose amino acid sequence has been determined is synthesized. Separately, total RNA is extracted from plants or cyanobacteria and poly-A is extracted from them.
Is isolated and a DNA complementary to this RNA
(CDNA) is synthesized. This cDNA was converted to phage λgt
Connect to an appropriate vector such as 11 to create a cDNA library. Here, a cDNA library is screened using the oligonucleotide synthesized above. Screening can be performed not only by a method using a nucleotide probe but also by an immunological method using an antibody. Further, as another frequently used method, based on a known base sequence, a short DNA located at both ends of a target sequence is used.
A primer corresponding to the NA sequence is designed, and DNA obtained from the material used to determine the entire sequence is used as a template,
By performing PCR, a target sequence can also be obtained.

The determination and confirmation of the nucleotide sequence of the gene of the present invention in the clones selected as described above can be performed by a generally known method. For example, the dideoxynucleotide chain termination method using M13 phage (Gene,
19: 269, 1982). In addition, even without using the above method, the gene of the present invention can be obtained from commercially available DN using the phosphite method based on SEQ ID NO: 2.
It can also be synthesized with an A synthesizer.

In order to express a DNA chain or a fragment thereof to produce a protein or polypeptide encoded thereby, an expression control region is required in addition to the DNA sequence (coding region) corresponding to the amino acid sequence. Such a region is necessary for expressing the MGDG synthase gene of the present invention, but they need not be special ones, and the original expression control region of the MGDG synthase gene may be used as it is, Expression control regions derived from other organisms may be used.

The “recombinant vector containing the MGDG synthase gene” of the present invention can be prepared by connecting the MGDG synthase gene alone or together with the expression control region to a vector. Known vectors such as plasmids and phages can be used as the vector.

The “cells into which the MGDG synthase gene has been introduced” of the present invention can be prepared by introducing a vector containing the MGDG synthase gene into the cells.
The vector used here is preferably one that can express the MGDG synthase gene in the introduced cell. The cells may be any of microbial cells, plant cells, and animal cells, regardless of the type of organism. The method of introduction into cells
It can be performed according to a conventional method, and when introducing into plant cells, for example, refer to “Plant Molecular Biology Manual, Second
Edition; SGGelvin, and RA Schilperoort eds, K
luwer Academic Publishers, 1995 ". Examples include physicochemical methods such as a method using a virus which is a biological method, a method using Agrobacterium, an electroporation method, a polyethylene glycol method, and a particle gun method.

Since the MGDG synthase is a protein present in the chloroplast envelope in plants, a DNA chain encoding a chloroplast transit peptide should be added upstream of the MGDG synthase. Is preferred. For example, the small subunit gene of pea ribulose-1, 5-2 phosphate carboxylase can be used as the gene encoding the translocation peptide.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION

[0020]

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

[Example 1] MGD from cucumber cotyledons
Purification of G synthase Cucumber seeds (Cucumis sativus L. cv. Aonagajibai /
(Obtained from seeds of Sakata) was germinated with moist vermiculite in the dark at 27 ° C. Four days later, light irradiation was performed with a fluorescent lamp (5 W / m2) for 29 hours, and the green cotyledons excluding the hypocotyl were harvested and stored in a freezer at -80 ° C until use. To about 3.2 kg of cotyledons collected, add an equal volume of TSPB buffer (50
mM Tris-HCl, pH 7.8, 10 mM dithiothreitol, 0.8 M
sodium acetate, 1 mM phenylmethylsulfonyl fluorid
e, 20% glycerol, 0.02% sodium azide, 1 μM leup
eptin), and the mixture was crushed with a Waring blender, followed by filtration with gauze. The filtrate was centrifuged at 15,000 × g at 4 ° C. for 15 minutes, and the supernatant was further centrifuged at 165,000 × g for 1.5 minutes.
After centrifugation for hours, a microsomal fraction was obtained as a precipitate, which was stored frozen at -30 ° C. Although MGDG synthase is an enzyme present in the chloroplast envelope, it is difficult to prepare a large amount of the envelope, so a microsomal fraction containing a large amount of the envelope was used as a starting material.

Regarding the solubilization of enzymes by surfactants
sodium cholate, Brij-35, TritonX-100, CHAPS,
LDAO (lauryldimethylamine oxide), octylglucosid
A comparative study was conducted using several drugs such as e. Among them, the solubilization rate by CHAPS, LDAO and octylglucoside was high. Among these three, LDAO was selected as a solubilizing agent, which is advantageous in that ion exchange chromatography can be used for purification because of its non-ionicity and that it is suitable for mass purification at low cost. After adding 12 mM LDAO to the previously prepared microsomal fraction, 16
After centrifugation at 5,000 × g for 1.5 hours, the supernatant was used as a solubilized enzyme. The solubilized enzyme was subjected to metal chelate affinity chromatography using zinc (Pharmacia), hydroxyapatite chromatography (Wako Pure Chemical Industries), gel filtration with Toyopearl HW-55 (Toyo Soda), cation exchange chromatography (2 types: CM-Toyopearl, manufactured by Toyo Soda Co .; MonoS, manufactured by Pharmacia) and affinity chromatography using UDP-hexanolamine agarose (manufactured by Sigma) were sequentially performed for purification. As a result, the MGDG synthase was finally purified 11,000 times or more as compared with the crude fraction immediately after solubilization. One of the steps having a high purification effect is cation exchange chromatography. By repeating this step twice with different kinds of ligands, the concentration can be more than 10 times as much as the final specific activity. Is 6
It became 00 nmol / min mg protein. Also, SDS-P
From the results of AGE, the molecular weight of MGDG synthase was 45 k
Da. This is due to the reported molecular weight of the enzyme of spinach chloroplast envelope of about 20 kDa (Marechal et al.).
(1991), and Teucher and Heinz (1991)).

Next, the purified enzyme was fragmented into peptides by digestion with protease, and the fragmentation was performed according to the method of Iwamatsu (Biochemistry, 63: 139,
1991; Electrophoresis 13: 142, 1992) to determine the amino acid sequence of the fragmented peptide. To the solution of the protein purified by the above-described method, double the amount of acetone was added to precipitate the protein with acetone.
After dissolving in μl of an electrophoresis buffer, SDS electrophoresis was performed at an acrylamide concentration of 12%, and the protein was transferred to a PVDF membrane (manufactured by Applied Biosystems) using an electroblotting apparatus (manufactured by Atto). The PVDF membrane onto which the protein was transferred
% Ponceau S (manufactured by Sigma) / 1% acetic acid, and a band having a molecular weight of about 45 kDa was cut out.
Decolorized with OH. This is reduced S-carboxymethylated and lysyl endopeptidase (Achromobacter protea)
se 1) was added at an enzyme: substrate (mol: mol) ratio of 1: 100, and reacted at 30 ° C. for 16 hours. The resulting fragmented peptide was equilibrated with solvent A (98%) and solvent B (2%) and μ-Bondashere 5μC8-300A (2.1 × 150 mm, Wate
rs) (2-50% for solvent B)
With a linear gradient of 30 minutes and a flow rate of 0.25 ml /
(Solvent A: 0.05% TFA aqueous solution, solvent B: 0.02% TF in 2-propanol: acetonitrile = 7: 3 (v / v)
A). The eluted peptide was detected by absorption at 214 nm, and each peak fraction was manually collected.
Each peak fraction obtained was analyzed using a gas phase protein sequencer (Applied Biosystems, Model 470A). As a result of analyzing all the obtained peak fractions, the amino acid sequence of the fragmented peptide was clearly determined. The following shows some of them.

# 1: GVSDE (N-terminal; AP-10) # 2: LVTRCYCPSTEVAK (AP-5) # 3: GFVTK (AP-1) # 4: IVFTTVVTD (AP-13) # 5: RVLILMSDTGG (AP- 9) The activity of MGDG synthase will be described in detail later. However, UDP-galactose labeled with ³ H and 1,2-dioleoyl-sn-gl
Using ycerol as a substrate, incorporation of [ ³ H] -galactose into the lipid fraction was measured as an index.

Example 2 Preparation of cDNA Library Derived from Cucumber Cotyledons Cucumber seeds were germinated with moist vermiculite at 30 ° C. in the dark for 5 days, and the green cotyledons were kept under fluorescent light for 6 hours. Used for material. Chirgwin from 10g cotyledons
Total RNA was obtained according to the method (Biochemistry, 18: 5294-5299, 1979). Based on this total RNA, RNA having poly A was isolated according to the method of Aviv et al. (Proc. Natl. Acad. Sci. USA., 69: 1408, 1972). Poly A above
The DNA complementary to the RNA having was synthesized according to the method of Gubler et al. (Gene, 25: 263, 1983). At this time, oligo (dT) and random oligonucleotide were used as primers. D consisting of a synthesized double strand
After attaching an EcoRI / NotI adapter (Pharmacia) to both ends of NA, the ends were phosphorylated. Next, the excess adapter was removed from the cDNA fraction by gel filtration, and the cD
The arm of the phage λgt11 which had been preliminarily cut and dephosphorylated with NA and EcoRI was ligated. This DNA was subjected to in vitro packaging (Gigapack Gold II; Stratagene) to obtain a λ
Phage particles were packaged to obtain a λgt11 library.

[Example 3] Screening of MGDG synthase gene-carrying strain A part of the partial peptide sequence # 2 deduced from the purified enzyme (C
DNA probe was synthesized based on YCPSTEVAK) (Applied Bio
syste; model 394). 5'-TGYTAYTGYCCIWSIACIGARGTIGCIAAR (IUB code)
(SEQ ID NO: 1)

After labeling the 5 'end with ³² P-dCTP, Sambrook et al. (Molecular Cloning; S)
econd edition, Cold Spring Harbor Laboratory Pres
s, 1989). The conditions were 5 × SSC (1 × SSC was 0.15M NaCl, 15m
M sodium citrate), 10 mM EDTA, 10 × De
nhardt solution (50 × Denhardt solution is Ficoll (Type400, Pha
rmacia), polyvinylpyrrolidone, bovine serum albu
min (fraction V, Sigma) at 10 g / l each) and 2
60 ° C. in a solution consisting of 50 μg / ml salmon sperm DNA,
16 hours. After that, the membrane was changed to 5 × SSC,
After washing twice in a 0.1% SDS solution at 45 ° C. for 15 minutes, autoradiography was performed. After purifying positive clones,
NA was subcloned into pBluescript II SK +, and the nucleotide sequence was determined according to a conventional method. The DNA sequence (1647 bp) and the predicted amino acid sequence are shown in FIGS. 1 to 3 (each represented by SEQ ID NO: 2 and
NA sequence and the amino acid sequence represented by SEQ ID NO: 3). The arrows in the figure indicate the expected breakpoints of the transit sequence into the chloroplast, the thin underlined part indicates the part corresponding to the sequence deduced from the sequence of the limited digested peptide of the purified enzyme, and the thick underlined part indicates the expression in E. coli. Shows the positions of the primers used for PCR. This amino acid sequence was compared with the amino acid sequences of other proteins in the database (analysis software GENETYX). FIG. 6 shows the results. The mark * in FIG. 6 is 3.
The position of the corresponding amino acid in the species gene is indicated. As shown in FIG. 6, this amino acid sequence is identical to Bacillus subtilis.
It showed about 20% significant homology with the MurG gene of E. coli (a gene involved in the synthesis of peptidoglycan) (clone name: pBSMGDG1).

Example 4 Activity Measurement of MGDG Synthase Candidate Gene (pBSMGDG1) by Expression in Escherichia coli To confirm what kind of enzyme activity the MGDG synthase candidate gene has in Escherichia coli, this gene was expressed in Escherichia coli. And tried to measure the activity. Using the MGDG synthase candidate gene and the expression vector pGEX-3X (Pharmacia) in Escherichia coli, Ba was used to express Glutathione S-transferase (GST) as a fusion protein at the C-terminus.
mHI and EcoRI sites were added to both ends of the MGDG synthase candidate gene by PCR.

Sense primer; 5'-GG GGATCC CTGGTGTT
TCAGATGAAACCAATG (underlined BamHI site) (SEQ ID NO: 4) Antisense primer; 5'-CC GAATTC CCGCCGGAATATTG
TGGTACAAAAC (EcoRI site underlined) (SEQ ID NO: 5)

Using the above two kinds of primers, pBSMGDG1
When a PCR was carried out using as a template, a product of about 1.3 kbp was obtained. The reaction conditions were 94 ° C. (1.5 minutes) and 60 ° C. (2 minutes) according to the manual of PerkinElmer.
) And 72 ° C (3 minutes) as one cycle were repeated 30 times. After the reaction product was cut with BamHI and EcoRI, it was cloned into pGEX-3X previously cut with the same set of restriction enzymes (referred to as pGEX-GT). This plasmid was introduced into competent cells of Escherichia coli XL1-Blue strain prepared according to a conventional method (Molecular cloning pp. 1.76-1.84; 1989), and a transformant was obtained by selection with ampicillin resistance (1b-2). ). The original plasmid pGEX-3X
The E. coli strain into which was introduced was named GEX-3X.

GEX-3X and 1b-2 were combined with 50 μg / ampicillin /
The cells were cultured at 37 ° C. for 3 hours in 50 ml of an LB medium containing the same, and a final concentration of 0.1 mM isopropylthiogalactoside (IPTG) was added, followed by further 2 hours of culture. The cells are collected by centrifugation at 3,000 rpm for 5 minutes, and the cells are MOD of 5 ml.
Buffer solution (50 mM MOPS-NaOH (pH7.9), 10 mM dithiothreito
l) and crushed by ultrasonic waves (output 2, duty 60, twice for 30 seconds; manufactured by Tommy).

Using this crushed liquid, the activity of MGDG synthase was measured according to the method of Teucher and Heinz (Planta 184, 319, 1991). At this time, 400 μM UDP was used as a substrate.
Added -galactose. 50 μl of 1, 1 in 30 μl of enzyme solution
2-dioleoyl-sn-glycerol (This is a 0.01% Tween 20
And adjusted to a concentration of 4 mg / ml in a TSPB buffer solution) and 110 μl of MOD buffer solution, and pre-incubated at 30 ° C. for 5 minutes. Reaction is 10 μl UDP
Starting with the addition of-[4,5- ³ H] galactose (92.5 Bq nmol-1), the reaction was performed at 30 ° C for 10 minutes. Next, 1 ml of ethyl acetate was added to stop the reaction. After vortexing well, 0.5 ml of 0.45% (W / V) aqueous NaCl solution was added, and the mixture was centrifuged at 500 × g for 5 minutes. Discard the lower layer,
A 0.45% NaCl aqueous solution was added again, the mixture was stirred, and the radioactivity of the upper layer after centrifugation was measured with a liquid scintillation counter (LS6500CE, manufactured by Beckman-RIIC). At this time, 4 ml of scintillation cocktail (ACS
-II) and 0.5 ml of methanol were added.

[0033]

[Table 1]

Here, the amount of protein is determined by Bensadoun and Weinstein.
(Anal. Biochem., 70, 241, 1976).
Only in the Escherichia coli strain (1b-2) expressing the MGDG synthase candidate gene, galactose was incorporated, and the amount was significantly induced by IPTG.

Next, it was examined by TLC whether the lipid produced by the incorporation of galactose in the transformed Escherichia coli was MGDG. First, a crushed product of Escherichia coli induced by IPTG was prepared as described above, and 50 μM of UDP- [ ¹⁴ C] galactose (493 Bq n
mol-1) was added. The reason is that with ³ H-labeled galactose, it is difficult to detect the reaction product after fractionation by TLC described below with an image analyzer (BAS2000, manufactured by Fujifilm Corporation). After performing the reaction at 30 ° C. for 10 minutes, the reaction was stopped by adding 1 ml of ethyl acetate in the same manner as described above, and after washing twice with 0 and 45% NaCl, 500 μl of the obtained ethyl acetate layer was removed. Dried and again 10
It was dissolved in 0 μl of a chloroform: methanol (1: 1) mixed solution, and the whole amount was subjected to TLC to fractionate with a developing solvent 1 (chloroform: methanol: water = 65: 15: 2, V / V). At this time, as a lipid control, spinach leaf extract was simultaneously developed, and glycolipids were colored by spraying an anthrone reagent to compare the mobilities. The result is shown in FIG. In FIG. 5, lane 1 is a lipid extracted from spinach leaves, and lanes 2 to 5 are lipids synthesized by an E. coli extract. Lanes 2 and 3
GEX-3X and lanes 4 and 5 were derived from 1b-2. Among them, lanes 2 and 4 used extracts from cells before induction of IPTG, and lanes 3 and 5 used extracts from cells after induction. Lane 6 shows a product obtained by a reaction using an enzyme purified from cucumber cotyledons. As shown in FIG. 5, radioactive MGDG was not detected in GEX-3X regardless of the presence or absence of induction by IPTG, but in 1b-2, incorporation into the lipid fractions and the As a result, a band due to apparent radioactivity was detected at the position of MGDG, and there was induction by IPTG. From the above, it became clear that this gene encodes the MGDG synthase gene.

Example 5 MGDG synthase gene (p
Lipid Composition of Escherichia coli Expressing BSUDGT1) Lipid composition was examined using Escherichia coli strains GEX-3X and 1b-2 used for the above activity measurement. Lipids are prepared by the method of Bligh and Dyer (Ca
n. J. Biochem. Physiol., 37: 911, 1959).
Extracted from the cells induced by IPTG, MGDG
Is developing solvent 1 and phospholipid is developing solvent 2
(Chloroform: methanol: acetic acid = 65: 20: 1
0, V / V). Phosphatidylglycerol (PG),
cardiolipin (CL), Phosphatidylethanolamine (PE) is Dit
Reacted with tmer reagent (J. Lipid Res., 5: 126, 1964), and MGDG was reacted with anthrone reagent and detected by comparison with each standard lipid. In the determination of lipids, phospholipids were determined by the method of Fiske-Subbarow (J. Biol. Chem., 66: 375, 1925).
Using lactose as a standard, the method of Radin et al. (J. Biol. Che
m., 217: 789, 1955).

[0037]

[Table 2]

From the result of analyzing the lipid composition, it was found that MGDG, which is not originally present in Escherichia coli, surprisingly produced 17% or more. Also, other lipids generally decreased relatively.

[0039]

The gene encoding the MGDG synthase of the present invention is useful for improving the lipid composition of animals, plants, and microorganisms by transformation, particularly for controlling the amount of MGDG.

[0040]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 30 Sequence type: Nucleic acid (DNA) Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: TGYTAYTGYC CIWSIACIGA RGTIGCIAAR

SEQ ID NO: 2 Sequence length: 1266 Sequence type: nucleic acid (DNA) Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin: Organism: Cucumis sativus Stock name: Aochi Chiku sequence: GGT GTT TCA GAT GAA ACC AAT GGG ATT AGA GAC GAT GGA TTT GGT GTT TCG CAA GAT GGG GCA CTG CCA TTG AAT AAA ATC GAG GCT GAG AAC CCC AAA CGG GTT CTT ATT TTA ATG AGT GAC ACT GGT GGA GGT CAT CGG GCT TCT GCT GAG GCA ATC AAG GCA GCC TTT AAT GAA GAA TTT GGG AAC AAT TAT CAG GTG TTT ATA ACT GAT TTG TGG ACG GAC CAC ACT CCT TGG CCT TTC AAT CAA TTA CCA AGA TGC TAC ATC GTG AAA CAT GGC ACA TTG TGG AAG ATG ACT TAC TAT GTG ACT GCT CCA AAA GTG ATT CAT CAG TCA AAT TTT GCT GCA ACT TCA ACA TTC ATA GCT CGA GAA GTA GCA AAA GGA CTG ATG AAA TAT AGG CCA GAT ATT ATT ATC AGT GTT CAT CCT CTG ATG CAG CAT GTT CCC ATT CGT ATT TTG AGG TCG AAG GGC CTC TTG AAT AAG ATT GTT TTC ACC ACA GTA GTC ACA GAT TTG AGC ACC TGC CAC CCA ACA T GG TTT CAC AAG CTT GTT ACA AGA TGC TAC TGC CCA TCT ACG GAG GTA GCA AAG AGG GCT TTG AAA GCT GGA CTC CAG CCT TCC AAA CTA AAG GTT TTT GGC CTT CCT GTG CGG CCT TCC TTT GTT AAG CCT ATT CGT CCG AAG ATT GAG TTA AGA AAA GAA TTG GGC ATG GAT GAA AAT CTT CCT GCC GTG TTG CTT ATG GGA GGG GGG GAA GGC ATG GGT CCC ATT GAG GCT ACT GCA AAG GCG CTA AGT AAG GCA TTG TAT GAT GAA AAT CAT GGA GAG CCA ATA GGC CATT GTG ATC TGT GGC CAC AAC AAA AAA CTA GCT GGC AGG TTG CGT TCA ATT GAT TGG AAG GTT CCT GTC CAG GTG AAG GGG TTT GTC ACA AAA ATG GAG GAA TGC ATG GGA GCT TGT GAT TGC ATT ATA ACA AAG AGG GCG GGC CCT GGA ACGATT GCT GAA GCC ATG ATA AGA GGT CTT CCT ATA ATT CTG AAT GAC TAC ATT GCT GGA CAG GAA GCT GGA AAT GTG CCA TAT GTC GTC GAA AAC GGA TGT GGG AAG TTT TCA AAA TCT CCT AAA GAG ATA GCG AAC ATT GTA GCA AAA TGG GGG CCA AAA GCA GAT GAG TTG CTG ATC ATG TCA CAG AAT GCC TTG AGG CTT GCT AGA CCT GAT GCT GTA TTT AAG ATT GTT CAT GAT CTC CAT GAG CTT GTT AAA CAA AGA AGT TTT GTA CCA CAA TAT TCC GGC

SEQ ID NO: 3 Sequence length: 422 Sequence type: Protein Topology: Linear Sequence type: Peptide Origin: Organism: Spinach (Cucumis sativus) Strain: Blue Nagachizoku Sequence features: Features Symbol indicating: CDS Location: 130.1395 Method for determining characteristics: P sequence: Gly Val Ser Asp Glu Thr Asn Gly Ile Arg Asp Asp Gly Phe Gly Val 16 Ser Gln Asp Gly Ala Leu Pro Leu Asn Lys Ile Glu Ala Glu Asn Pro 32 Lys Arg Val Leu Ile Leu Met Ser Asp Thr Gly Gly Gly His Arg Ala 48 Ser Ala Glu Ala Ile Lys Ala Ala Phe Asn Glu Glu Phe Gly Asn Asn 64 Tyr Gln Val Phe Ile Thr Asp Leu Trp Thr Asp His Thr Pro Trp Pro 80 Phe Asn Gln Leu Pro Arg Ser Tyr Asn Phe Leu Val Lys His Gly Thr 96 Leu Trp Lys Met Thr Tyr Tyr Val Thr Ala Pro Lys Val Ile His Gln 112 Ser Asn Phe Ala Ala Thr Ser Thr Phe Ile Ala Arg Glu Val Ala Lys 128 Gly Leu Met Lys Tyr Arg Pro Asp Ile Ile Ile Ser Val His Pro Leu 144 Met Gln His Val Pro Ile Arg Ile Leu Arg Se r Lys Gly Leu Leu Asn 160 Lys Ile Val Phe Thr Thr Val Val Thr Asp Leu Ser Thr Cys His Pro 176 Thr Trp Phe His Lys Leu Val Thr Arg Cys Tyr Cys Pro Ser Thr Glu 192 Val Ala Lys Arg Ala Leu Lys Ala Gly Leu Gln Pro Ser Lys Leu Lys 208 Val Phe Gly Leu Pro Val Arg Pro Ser Phe Val Lys Pro Ile Arg Pro 224 Lys Ile Glu Leu Arg Lys Glu Leu Gly Met Asp Glu Asn Leu Pro Ala 240 Val Leu Leu Met Gly Gly Gly Glu Gly Met Gly Pro Ile Glu Ala Thr 256 Ala Lys Ala Leu Ser Lys Ala Leu Tyr Asp Glu Asn His Gly Glu Pro 272 Ile Gly Gln Val Leu Val Ile Cys Gly His Asn Lys Lys Leu Ala Gly 288 Arg Leu Arg Ser Ile Asp Trp Lys Val Pro Val Gln Val Lys Gly Phe 304 Val Thr Lys Met Glu Glu Cys Met Gly Ala Cys Asp Cys Ile Ile Thr 320 Lys Ala Gly Pro Gly Thr Ile Ala Glu Ala Met Ile Arg Gly Leu Pro 336 Ile Ile Leu Asn Asp Tyr Ile Ala Gly Gln Glu Ala Gly Asn Val Pro 352 Tyr Val Val Glu Asn Gly Cys Gly Lys Phe Ser Lys Ser Pro Lys Glu 368 Ile Ala Asn Ile Val Ala Lys Trp Phe Gly Pro Lys Ala Asp Glu Leu 384 Leu Ile Met Ser Gln As n Ala Leu Arg Leu Ala Arg Pro Asp Ala Val 400 Phe Lys Ile Val His Asp Leu His Glu Leu Val Lys Gln Arg Ser Phe 416 Val Pro Gln Tyr Ser Gly 422

SEQ ID NO: 4: Sequence length: 32 Sequence type: Nucleic acid (DNA) Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: GGGGATCCCTGGTGTTTCAGATGAAACCAATG

SEQ ID NO: 5 Sequence length: 33 Sequence type: nucleic acid (DNA) Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence: CCGAATTCCCGCCGGAATATTGTGGTACAAAAC

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the cucumber MGDG synthase gene and the corresponding amino acid sequence (following FIG. 2).

FIG. 2 shows the nucleotide sequence of the MGDG synthase gene of cucumber and its corresponding amino acid sequence (continued from FIG. 3).

FIG. 3 shows the nucleotide sequence of the MGDG synthase gene of cucumber and the corresponding amino acid sequence.

FIG. 4 is a schematic diagram showing how to prepare a plasmid for expressing the MGDG synthase gene in Escherichia coli.

FIG. 5 is a photograph showing a chromatogram of a lipid synthesis product in an Escherichia coli extract expressing MGDG synthase.

FIG. 6: MGDG synthase and Bacillus subtilis, E. coli MurG
FIG. 2 is a view showing the homology of amino acid sequences to genes.

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[Procedure amendment]

[Submission date] July 10, 1996

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 5

[Correction method] Change

[Correction contents]

FIG. 5

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical display location C12N 9/10 C12N 9/10 // (C12N 1/21 C12R 1:19) (C12N 9/10 (C12R 1:19) (72) Inventor Kenichiro Takamiya 4259 Nagatsuda, Midori-ku, Yokohama-shi, Kanagawa Prefecture Tokyo Institute of Technology Life Science and Engineering

Claims (5)

[Claims] 1. A monogalactosyldiacylglycerol synthase having an amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. 2. A fusion protein comprising the monogalactosyldiacylglycerol synthase according to claim 1 and another protein. 3. A monogalactosyldiacylglycerol synthase gene encoding an amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3. 4. A recombinant vector containing the monogalactosyldiacylglycerol synthase gene according to claim 3. 5. A cell into which the monogalactosyldiacylglycerol synthase gene according to claim 3 has been introduced.