JPH0690766A - Phosphoenol pyruvic acid carboxylase gene of brassics napus - Google Patents

Abstract

PURPOSE:To obtain a new gene useful for breeding a plant having desired properties, e.g. a plant having high protein content. CONSTITUTION:A gene coding 1-964th amino acid sequence of phosphoenolpyruvic acid carboxylase of the formula. This gene is obtained from a library assembled from a genome DNA extracted from a Brassics napus plant body according to the method described in Carrent Protcols in Molecular Biology (edited by F. M. Ausubcls and published by John wileydsons, Inc., 1987).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a gene of phosphoenolpyruvate carboxylase (hereinafter abbreviated as PEPCase), which is one of the factors controlling the biosynthesis of plant storage substances. Since there are positive and negative correlations between the enzyme and the protein content and the fat content, respectively, expression of this gene makes it possible to produce rape and other plants having a high protein content. If the expression of this gene is suppressed by an antisense gene or the like, fat production in rape and other plants can be increased. Moreover, since the secretory expression of not only PEPCase but also other substances can be promoted by using the promoter sequence according to the present invention, the present invention can be used to greatly improve the breeding of rape and other various plants having desired properties. It can be done efficiently.

[0002]

2. Description of the Related Art PEPCase has been confirmed to exist in various organisms and is known to play various roles. This enzyme requires Mg ² + ion and catalyzes the reaction of adding carbon chain derived from carbonate ion to phosphoenolpyruvate to produce oxaloacetate and phosphate. This enzyme is found in higher plants, yeast and bacteria, but not in animals. Pyruvate carboxylase exists as an enzyme that performs a reaction similar to this, and it is known that this plays an important role in animals. Both of these enzymes are T
It is thought to work to supplement the oxaloacetate required for the CA cycle. In higher plants, pH stats in roots and functions as ion balancers in nitrogen metabolism have also been proposed (CS An.
dreo et al. , FEBSLett. , 21
3, 1-8 (1987)).

Among higher plants, C4 type or CAM
In photosynthetic plants, which are called type, CO _{2 in} the atmosphere
Is fixed by PEPCase localized in mesophyll cells, and the oxaloacetate produced is reduced to malic acid. Therefore, there are many studies on PEPCase in C4 type plants such as corn. C3
It is known that a series of enzymes related to photosynthesis also exist in type C plants, but C3 type plants and C
Little research has been conducted on the difference in PEPCase between type 4 plants.

So far, C4 type PEPCase has been
Gene cloning and analysis have been advanced in maize, sorghum, etc. (K. Izui et al.,
Nucl. Acids Res. , 14, 1615-1
628 (1986); Matsuoka et a
l. , Eur. J. Biochem. , 181,593
-598 (1989); L. Hudspeth &
J. W. Grula, Plant Mol. Bio
l. , 12, 579-589 (1989); C.I. Cre
tin et al. , Gene, 99, 87-94.
(1991)). In addition, in C3 type plants, gene analysis is underway in rice and tobacco (N. Koizum).
i et al. , Plant Mol. Biol. ，
17, 535-540 (1991)). Further, some studies are known for CAM type plants which are intermediate types of both (J. C. Cushman & H.
J. Bohnert, Nucl, Acids Re
s. , 17,6743-6746 (1989)). The goals of these studies have been limited to biochemical studies of enzymes due to differences in photosynthetic types.

[0005] Glycolytic system from glucose to some bilpinic acid or phosphoenolpyruvylic acid through several enzymatic reactions, and until citric acid produced by condensation of acetyl CoA and oxaloacetic acid returns to oxaloacetic acid by a series of enzymatic reactions. The TCA cycle of Escherichia coli is linked via bilpate dehydrogenase or PEPCase.
The former reaction produces citric acid from bilpic acid via acetyl CoA, and the latter produces oxaloacetic acid directly from phosphoenolpyrbipic acid. The metabolites that enter the TCA cycle are eventually consumed as energy or linked to the amino acid synthesis system, and further become proteins. On the other hand, acetyl CoA is also the starting point of fatty acid synthesis system. That is, the transition from the glycolytic system to the TCA cycle is also the branch point between fatty acid synthesis and protein synthesis.

When the PEPCase activity is relatively strong,
It is predicted that the amount of acetyl-CoA produced is relatively reduced, and fatty acid synthesis is suppressed and protein synthesis is promoted accordingly. In fact, the correlation between the storage protein content in soybean and PEPCase activity was investigated, and the results supporting this prediction have been obtained (T. Sugim.
oto et al. , Agri. Biol. Che
m. , 53, 885-887 (1989)). Therefore,
It is of interest whether the ratio of fatty acid synthesis to protein synthesis (rate of distribution) can be changed by artificially manipulating the PEPCase activity of plants.

Oilseed rape and soybean are very useful as crops. In the case of soybean, an increase in its protein content, and conversely, in oilseed rape, an increase in oil content is an important issue for breeding. However, in the conventional breeding method, it takes a long time and a lot of labor to improve such a trait. Therefore, improvement of traits by a molecular breeding method has been awaited.

[0008]

[Problems to be Solved by the Invention] With the increase in population, it is an urgent need to increase the production of foodstuffs in the world. In order to increase the production of food under such conditions, a method of increasing or enlarging individual plants themselves, and a method of particularly increasing the concentration of specific components in the plants are considered.

The present invention is based on the latter idea and intends to solve the problem by utilizing a genetic engineering technique. In particular, from the viewpoint of increasing the production of proteins and fats, the purpose is to clone the genes related thereto, and the ultimate purpose is to breed desired plants.

[0010] The inventors of the present invention aimed at the molecular breeding of rape, purifying PEPCase which is an enzyme present at a branch point when a carbohydrate produced by photosynthesis is converted into a fatty acid or a protein as a storage substance, and its gene. Of rapeseed and PEPCase
The gene was isolated, further research was conducted, and the present invention was completed.

[0011]

The present invention has been made to achieve the above object, and the present inventors have
As a means for increasing the protein content or fat production in plant seeds, leaves, and cultured cells, we planned to clone the PEPCase gene from rape and succeeded in cloning cDNA from rape seeds during ripening. Research results from various fields Brassica PEPCa
The structure of the se gene was successfully determined, and the present invention was finally completed. The present invention will be described in detail below.

In order to carry out the present invention, it is first necessary to separate and purify PEPCase from oilseed rape seeds. Information used for the purification of the enzyme is appropriately used for that purpose. For example, after crushing rapeseed seeds, they are suspended in a buffer solution, and the ammonium sulfate fraction fraction of the supernatant is purified according to a known method such as column chromatography.

In this way, oilseed rape seeds to PEPCa
After purified to a uniform level, se was used to prepare an antibody against rabbit. Next, poly A RNA was prepared from seeds of the rapeseed ripening stage, and using this as a template, cDNA was prepared.
A was synthesized. This cDNA is λ phage vector λg
An integrated cDNA library was obtained at t11. This library was further amplified using Escherichia coli Y1088 strain and then screened.

The screening was carried out using the rabbit antibody prepared above, and 6 λ phages producing a protein which specifically binds to this antibody were obtained. In order to confirm that these have the PEPCase gene, antibodies that bind to the proteins produced by these λ phages were purified and used for Western analysis. As a result, all the antibodies specifically selected only PEPCase from the rapeseed protein separated by SDS polyacrylamide gel electrophoresis.
It was confirmed to contain the Case gene.

Therefore, λDNA was prepared from these λ phages, cleaved with the restriction enzyme EcoRI, and then cDNA was prepared.
The portion was recovered and used as a plasmid vector Bluescri.
Subcloned into ptpKS +. When these physical maps were created, 4 contained only short inserts of 1 kb or less, but 2 contained inserts of about 3 kb. The longest of these
The one that was A was selected and named pRPC1. The base sequence was determined, and the base sequence of the PEPCase cDNA was successfully obtained.

Next, using this cDNA as a probe, λE
Clones that hybridize with this cDNA were screened from a canola genomic library using MBL3 as a vector. As a result, 15 clones were obtained. The λDNA of these clones was purified and a physical map of the insert was made, confirming that they were derived from 9 sites. Of these clones, four lambda phage clones, λPE3, λPE15, λPE1
05 and λPE19 are rapeseed PE consisting of sequences slightly different from each other as a result of Southern analysis and nucleotide sequence analysis.
It was revealed to contain the PCase gene.

The present invention was finally completed as a result of further research on these genomic DNA fragments. That is, the present invention relates to rape PEPCas
It relates to the e gene. The gene is incorporated in a lambda phage vector and is derived from a library constructed from genomic DNA extracted from a canola plant. The gene includes a DNA sequence encoding an enzyme protein of rape PEPCase, and a promoter sequence existing in the upstream region thereof and involved in controlling the expression of the gene in the plant body. As described above, since the gene of the present invention is incorporated into lambda phage, an application for deposit of this phage was submitted to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology. The deposit was not accepted due to the provisions of the Rules for Implementing Regulations on the Deposit of Microorganisms, etc.

The sequence of the rape PEPCase gene of the present invention was found to be homologous to the previously reported PEPCase gene isolated from corn, pinewood, cyanobacteria, and Escherichia coli, but it was not the same.

The present invention will be described in detail below based on examples. Unless otherwise specified, the procedure of operation is Current
Protocols in Molecular B
iology (edited by FM Ausubel et al., Joh
n Wiley & Sons, Inc. , 1987).
Followed the method described in.

[0020]

【Example】

[0021]

[(1) Purification of oilseed rape PEPCase] Grained oilseed rape seeds (500 g) were pulverized and added with 2 liters of 0.1 M phosphate buffer (pH 6.8, containing 25 mM magnesium chloride) and mixed well. The supernatant was collected by centrifugation. A 30% to 50% ammonium sulfate (ammonium sulfate) fraction was obtained from this supernatant. 0.1 M of this ammonium sulfate precipitate
After being dissolved in Tris buffer (pH 7.5, containing 30% ammonium sulfate), PEPCase activity was eluted by using the same buffer for a Butyl Toyopearl column (5 × 50 cm). After dialyzing this fraction, it was applied to a DEAE Toyopearl column (5 × 50 cm). PEPCa from this column
The se activity was eluted with a sodium chloride gradient. After ammonium sulfate precipitation of this fraction, Sepharose CL6B
Gel filtration was performed using (2 × 90 cm). This fraction was applied to a hydroxyapatite column (1 × 20 cm), and a 5 mM to 100 mM phosphate buffer solution (pH 6.
It eluted in 8). PE uniformly purified in this way
PCase was obtained.

[0022]

[(2) Preparation of antibody against rape PEPCase]
1 mg of rape PEPCase obtained by the above method
Was mixed well with 2.5 ml of adjuvant and then injected into the epidermis of rabbits. After 60 days, the same rabbit was again injected with rape PEPCase to increase the antibody titer. Further, after 60 days, blood was collected from the rabbit, an equal amount of 50 mM sodium citrate was added, and the supernatant was used as an antibody.

[0023]

[(3) Preparation of poly A RNA from rapeseed ripening seeds] The rapeseed rapeseed seeds were immediately put into liquid nitrogen and frozen, and stored at -135 ° C until use. The rapeseed seeds (50 g) were crushed in the presence of liquid nitrogen using a mortar until powdered. Add 200 ml of extraction buffer (20 mM vanadyl complex, 2% SDS,
Add Tris-HCl (pH 8.0)) and immediately add 500
The supernatant was recovered by centrifugation at × g. An equal amount of phenol-tris (pH 8.0) was added to this supernatant, and the mixture was stirred for about 10 minutes and then centrifuged at 200 × g to collect the supernatant. This supernatant was mixed with 1/10 volume of 3M sodium acetate (p
H5.5) and twice the amount of ethanol were added and the mixture was allowed to stand at -20 ° C for 30 minutes. This was centrifuged at 2000 × g to collect the nucleic acid precipitate. After dissolving this in 5 ml of Tris buffer, an equal amount of phenol-Tris was added and stirred for 10 minutes. The supernatant was collected by centrifugation, and the phenol extraction operation was repeated 5 times. The obtained supernatant was precipitated with ethanol, and this RNA was dissolved in distilled water.
After adjusting the mg / ml and the lithium chloride concentration to 2M, the mixture was left standing on ice for 8 hours. This was centrifuged at 2000 xg to recover the precipitate. This RNA precipitate was dissolved in distilled water. The preparation of poly ARNA was performed using an oligo dT spin column (Pharmacia) according to the instruction manual of this column. 100 μg of poly A RNA could be obtained.

[0024]

[(4) Preparation of cDNA Library] Using 5 μg of the poly A RNA obtained by the above method, a cDNA synthesis kit (Pharmacia) was used, and cDNA was synthesized according to the instruction of the kit. CDN by this kit
In the synthesis of A, EcoRI adapters were used at both ends of the synthesized cDNA using a ligation kit (manufactured by Takara Shuzo Co., Ltd.) to perform a ligation reaction according to the instruction. The λDNA thus obtained was used as a cDNA library by reconstituting phage particles using a packaging kit (manufactured by Stratagene) according to the instructions of the kit. Before screening this library,
All phages contained in this library were transformed into E. coli Y
The strain 1088 was infected, and this was cultured on an agar medium at 42 ° C., and phages were recovered from the phage plaques that appeared 6 hours later using an SM solution. A small amount of chloroform was added to this recovered solution and the solution was stored at 4 ° C. This phage solution was used as a cDNA library in the following screening.

[0025]

[(5) Screening of cDNA] A part of the phage solution contained in the cDNA library obtained as described above was mixed with Escherichia coli (Y1090 strain) and incubated at 37 ° C for 15 minutes.
The phage was infected with E. coli by culturing for a minute. This was mixed with 10 ml of soft agar melted at 50 ° C. and then overlaid on an LB agar medium (a petri dish having a diameter of 15 cm) to solidify the soft agar. At this time, 10 per dish
The amount of E. coli and phage was adjusted so that 10,000 plaques were produced. 3 pieces of 10 petri dishes prepared in this way
After culturing at 42 ° C. for 10 hours, a 10 mM IPTG solution was impregnated and the dried nitrocellulose filter was covered on an agar medium, and further incubated at 37 ° C. for 3 hours. Then, these filters were carefully peeled from the agar medium and immersed in a 1% BSA solution for 16 hours. PEP
Case of rabbit antibody (0.1 ml) with 100 ml of B
After being added to the SA solution and mixed, the above filter was dipped in this solution and allowed to react with the antibody for 1 hour at room temperature with gentle shaking. Use alkaline phosphatase for color reaction 2
The following antibody method kit (Promega, Picoblue) was performed according to the instructions. 6 appeared on the filter
The position of each purple spot is compared with the agar medium from which the filter is derived, and the phage at the corresponding position is identified as 1
It was collected in ml of SM solution. Since a large number of phages other than the target were mixed in these 6 kinds of phage liquids, this time, the phage liquids were purple-colored on each agar medium so that the plaques of about 100 phages per dish were generated. Plaques of phage that gave spots were collected (secondary screening). Third screening was performed in the same manner to finally obtain 6 phages.

[0026]

[(6) Determination of nucleotide sequence] From the thus obtained 6 kinds of phages, the method of Grossberger (Nucl
eic Acids Res. , 15, 6737 (19
87)), and each λDNA was prepared.
This λDNA was digested with the restriction enzyme EcoRI, then digested with EcoRI, and the ends were treated with alkaline phosphatase.
The pKS + (manufactured by Stratagene) and the ligation kit (manufactured by Takara Shuzo) were ligated according to the instructions. E. coli (XL1-Blue strain) was transformed with this. A plasmid was prepared from the obtained colony and digested with various restriction enzymes to prepare a physical map for each.
Comparing these, the two cDNAs that seem to be almost full length
I found it. These were named pRPC1 and pRPC16. These show similar physical maps, but with pR
PC1 was about 0.1 kb longer. So pR
To determine the nucleotide sequence of PC1, the insert fragment was inserted into pUC
Subcloned into 118. A deletion clone was prepared from these using an erase base kit of Promega Co., Ltd. according to the instructions. Single-stranded DNA was prepared from these clones and the nucleotide sequence was determined by the dideoxy method. As a result, it was revealed that pRPC1 contained the full-length cDNA insert because the translation initiation site was contained therein (sequence listing).

[0027]

[(7) Preparation of Rape Genomic DNA and Construction of Genomic Library] Brassica napus ( Brassics napu
s ) genomic DNA is available from Rogers and Bendich.
(Plant Mol. Biol., 5, 69)
(1985)). The obtained DNA was subjected to limited digestion with a restriction enzyme Sau3AI and further subjected to sucrose density gradient centrifugation to obtain a DNA fragment of 10 kb or more. This was ligated to an arm of a λEMBL3 phage vector cut with BamHI, and then in vitro packaging kit (GIGAPACK, manufactured by Stratagene).
(Gold) was used for packaging and infection of E. coli LE392 strain to obtain a large number of recombinant λ phages. This was used as a canola genomic DNA library. The size of the library is 1.9 x 1
It was 0 ⁶ .

[0028]

[(8) Isolation of genomic clone of rape PEPCase gene] 1 of the above rapeseed genomic library
For the 0.00000 plaques, the aforementioned oilseed rape P
The rape PEPCase gene was screened using the EPCase cDNA (pRPC1) as a probe. As a result, 15 positive genomic clones were obtained. The DNA of these genomic clones was prepared and the size of the insert was investigated. In addition, for the DNA of each genomic clone, rape PEPC
Southern analysis was carried out using the asase cDNA as a probe to test whether or not the PEPCase gene was contained. 5'end portion of rape PEPCase cDNA (SEQ ID NO: 2 in the sequence listing shown in Tables 15 to 23 below)
(Corresponding to the 161st to 870th base sequences of the cDNA base sequence shown in) or the 3'end portion (103 of the base sequence of the cDNA shown in SEQ ID NO: 2 in the same sequence listing).
Southern analysis was carried out by using (corresponding to the 7th to 1648th base sequences) as a probe. As a result, λP
The four genomic clones E3, λPE15, λPE105, λPE19 hybridized strongly with both probes. From this, it was suggested that the four genomic clones contained the rape PEPCase gene. In addition, these probes are
Prepared by R.

[0029]

[Table 15]

[0030]

[Table 16]

[0031]

[Table 17]

[0032]

[Table 18]

[0033]

[Table 19]

[0034]

[Table 20]

[0035]

[Table 21]

[0036]

[Table 22]

[0037]

[Table 23]

[0038]

[(9) Physical mapping and subcloning of rapeseed genome clone] λPE3, λPE15, λPE1
For each of the four rapeseed genomic clones of 05 and λPE19, the restriction enzymes BamHI, EcoRI, S
A physical map was created using alI and XhoI (FIG. 1). Furthermore, Southern analysis was performed using a cDNA of rape PEPCase as a probe to identify a DNA fragment considered to contain the PEPCase gene of rape in each clone.
Subcloned into uescript pSK +.

[0039]

[(10) Determination of nucleotide sequence of rapeseed genome clone] λPE3, λPE15, λPE105, λPE19
Attempts were made to determine the nucleotide sequence of the insert fragment from subclones derived from the four canola genomic clones of.

First, a delation kit (Takara Shuzo)
Was used to create a series of deletion clones in which the insert fragment was deleted stepwise. The nucleotide sequence was determined using the deletion clone obtained by this operation.

When the obtained base sequence was compared with the above-mentioned cDNA, the base sequence of λPE3 corresponded. However, homology was found in all other clones. Therefore,
It was confirmed that these were all rapeseed PEPCase genes. However, since the nucleotide sequences of the 4 clones were not identical, they were different from each other in PEPCa.
It was considered to be a gene encoding the se enzyme protein.

Of these, the nucleotide sequences of the entire region of the PEPCase gene of rape contained in the λPE3 clone were determined (the results are shown in SEQ ID NO: 1 in the sequence listings shown in Tables 1 to 14 below).

[0043]

[Table 1]

[0044]

[Table 2]

[0045]

[Table 3]

[0046]

[Table 4]

[0047]

[Table 5]

[0048]

[Table 6]

[0049]

[Table 7]

[0050]

[Table 8]

[0051]

[Table 9]

[0052]

[Table 10]

[0053]

[Table 11]

[0054]

[Table 12]

[0055]

[Table 13]

[0056]

[Table 14]

As a result of comparison with the above-mentioned PEPCase cDNA, the PEPCase gene of the λPE3 clone was found to be PEPCase by several introns.
It was clarified that the coding region of the enzyme protein has a divided structure. However, exons 7 and 8 were fused in this gene.

Further, other λPE15, λPE105, λ
Oilseed rape P contained in three genomic clones of PE19
Regarding the EPCase gene, most of the nucleotide sequences were similarly determined and compared. As a result, each rape PEPCase gene showed high homology at the nucleotide sequence level. The exon and intron structures differ depending on the clone as shown in the figure.
In 105, exons 3 and 4 fuse together, and exon 7
There was an intron between and 8. On the other hand, λPE1
Introns 3 and 7 were present in 9 and had an exon and intron structure similar to the maize gene. The length of each exon was not different between all clones (Fig. 2). Note that λPE15 is N
-The sequence of the terminal region was found to be missing.

Further, at the amino acid sequence level, a homology of more than 80% was observed, so it was considered that they encode substantially the same enzyme protein. As an example, FIGS. 3 to 8 show the nucleotide sequence of exon 2 or less of λPE105 and the amino acid sequence encoded thereby. Therefore, the PEPCase gene carried in these clones is
Although slightly different from the λPE3 clone, it was revealed that they are the PEPCase gene equally.

In addition, FIG. 9, FIG. 10 and FIG. 11 (three-letter representation of the one-letter representation in FIG. 9) compare the amino acid sequences of the portions corresponding to exon 10 encoded by the respective clones. Indicates. Exon 10 corresponds to the carboxyl terminus of the protein. In general, the sequences at the carboxyl terminus are often not conserved among proteins having the same function, but in the case of this PEPCase, it was also very well conserved in this part.

[0061]

[(11) Isolation of promoter sequence] The rape PEPCase gene of the λPE3 genomic clone is PEPCa.
A promoter sequence that controls the expression of the gene in the plant is provided upstream of the region encoding the se enzyme protein. This promoter sequence contains restriction enzymes BglII and Nc
It is isolated as a DNA fragment of about 900 bases by cleaving the DNA of the λPE3 clone with oI. Only the DNA fragment of about 900 bases is plasmid Blues
Subcloning was performed on the cript pSK + to obtain a clone of the promoter sequence.

[0062]

[(12) Control of PEPCase expression] Brassica PE
CDNA encoding the enzyme protein of PCase gene
The A sequence was used to construct the antisense PEPCase gene. This DNA sequence was converted into the binary vector pB1121 (R. Jefferson et al.
l. , EMBO J .; , 6,3901-3907 (19
87)), which is inserted into the SmaI site and is complementary to the mRNA encoding the PEPCase enzyme protein.
Antisense PEPC that can synthesize NA
It was designated as ase gene (pBPA3) (FIG. 12).

Furthermore, this antisense PEPCase
The transgenic tobacco having the gene introduced thereinto was subjected to a gene transfer method mediated by Agrobacterium (RB Horsch et al.
al. , Science, 227, 129 (198
5)). Transformed tobacco is PEPC
It was shown that the activity of ase was decreased, and the expression of PEPCase gene was suppressed by the antisense PEPCase gene.

[0064]

The rape PE obtained according to the present invention
The PCase gene encodes the entire region of the rapeseed enzyme protein. Therefore, this gene is used as it is, or by using an artificial gene expression system created by combining it with a promoter derived from cauliflower mosaic virus, by a method using electroporation or Agrobacterium. , Can be introduced into plants such as tobacco. In the resulting transformed plant body, it is possible to increase the enzymatic activity of PEPCase.

Further, the DNA of all or part of the PEPCase gene obtained from oilseed rape shown in the sequence listing.
The sequence can be used to construct an antisense gene for the PEPCase gene. It is possible to suppress the PEPCase activity by introducing this into plant cells.

As described above, PEPCase is a glycolytic system from glucose to phosphoenorubirpic acid,
It catalyzes the reaction that connects the TCA cycle in which living organisms receive a large amount of energy. Since the metabolites that entered the TCA cycle also lead to the synthesis of storage proteins, PE
When PCase activity is enhanced, protein synthesis is eventually promoted. On the contrary, by suppressing this PEPCase activity, it is considered that the fatty acid synthesis can be relatively promoted and the production amount of the fat and oil component can be increased. That is, by artificially manipulating the PEPCase activity of plants, it became possible to change the efficiency of protein synthesis or fat and oil synthesis.

In particular, rape is very useful as a crop for producing fats and oils. PEPCa derived from rape of the present invention
A very high economic effect is expected to be obtained by increasing the amount of fats and oils components by a molecular breeding method using the se gene.

Further, based on the base sequence of the PEPCase gene of the present invention, a part of this base sequence is specifically mutated using a technique such as a site-specific substitution method or a PCR method to artificially produce PEPCase. Can be modified to. It can be used in studies seeking improved functionality of PEPCase. At the same time, it can be used for a wide range of studies on functional expression of enzyme proteins.

[Brief description of drawings]

FIG. 1 Genomic clones of the canola PEPCase gene, λPE3, λPE15, λPE105, and λP.
A physical map of E19 is shown. In each genomic clone, the line shown at the top shows the entire region of the lambda phage vector insert. The physical map is the restriction enzyme Bam
It was created using HI, EcoRI, SalI, and XhoI. In addition, the coding region of the PEPCase enzyme protein is shown by a square at the bottom of each physical map.

FIG. 2 is a comparison diagram of the intron and exon structures of the PEPCase gene. Genomic clone of rape PEPCase gene, λPE3, λPE15, λPE10
5 shows the relationship between exons and introns of the region encoding PEPCase on 5 and λPE19. Exons are indicated by squares. The numbers are exon numbers. The length of each exon corresponds to the size of the square. Introns are indicated by bars.

FIG. 3 shows a sequence consisting of a base sequence of exon 2 or less of the genomic clone λPE105 of the canola PEPCase gene and an amino acid sequence encoded thereby.

FIG. 4 shows a sequence following the same sequence.

FIG. 5 shows a sequence following the same sequence.

FIG. 6 shows a sequence subsequent to the same sequence.

FIG. 7 shows a sequence following the same sequence.

FIG. 8 shows a sequence following the same sequence. In these figures, undetermined nucleotide sequences are indicated by N, and amino acid sequences that could not be determined? ? ? Indicated by.

FIG. 9 is a structural comparison diagram of the exon 10 region. Genomic clone of rape PEPCase gene, λPE3,
PEP of λPE15, λPE105, and λPE19
The amino acid sequences of exon 10 of Case were aligned. The amino acid sequence is represented by one letter. Gaps were introduced to maximize homology. λPE3 PEPCas
Amino acid residues homologous to e are underlined.

FIG. 10 is a drawing in which the amino acid sequence in FIG. 9 is described in three letters instead of one letter. In addition, what is shown by "-" is a gap introduced to enhance homology.

11 is a continuation of FIG.

FIG. 12 shows an antisense PEPCase gene vector. The upper line shows a physical map of PEPCase cDNA. An antisense PEPCase gene vector (pBPA3) was constructed using this cDNA sequence.

Claims (4)

[Claims] 1. A gene encoding the 1-964th amino acid sequence of phosphoenolpyruvate carboxylase shown in SEQ ID NOS: 1 and 2 of the Sequence Listing. 2. A promoter site contained in the nucleotide sequence at positions 1-892 of phosphoenolpyruvate carboxylase shown in SEQ ID NO: 1 of the Sequence Listing. 3. A gene comprising a nucleotide sequence encoding the amino acid sequence of rapeseed phosphoenolpyruvate carboxylase obtained by using all or part of the gene of claim 1 as a probe. 4. The expression level of the phosphoenolpyruvate carboxylase gene defined in any one of claims 1 to 3 is suppressed in the plant, and the amount of phosphoenolpyruvate carboxylase synthesized in the plant is increased. An antisense phosphoenolpyruvate carboxylase gene constructed to effect a change.