JPH06319567A - Phosphoenol pyruvic acid carboxylase gene of soybean - Google Patents

Abstract

PURPOSE:To provide a novel gene useful for the growth of soybean or other kinds of plants having desirable properties. CONSTITUTION:A gene coding a phosphoenol pyruvic acid carboxylase having an amino acid sequence of the formula. The gene is obtained by cloning a cDNA from a ripened soybean seed and subsequently determining the basic sequence of the clone.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a phosphoenolpyruvate carboxylase gene contained in plant seeds, especially soybeans. Since the gene is associated with increased protein production, expression of this gene can produce soybeans and other plants having a high protein content, while suppression of gene expression can improve soybean and other plants. By increasing the production of fat, and utilizing the present invention, the breeding of soybeans and various plants having desired properties can be carried out extremely efficiently.

[0002]

2. Description of the Related Art Phosphoenolpyruvate carboxylase (hereinafter abbreviated as PEPC) is an enzyme found in various tissues of plants and catalyzes the reaction of producing oxaloacetate and orthophosphate from phosphoenolpyruvate and carbonate ions. .

Sugimoto et al. (Agric. Bio
l. Chem. 53 , 885, 1989), it has been reported that there is a positive and negative correlation between PEPC and protein content and fat content contained in soybean seeds, respectively. That is, it is shown that the production amount of these can be controlled by controlling the expression amount of PEPC. Previous studies have isolated the PEPC gene of the C4 plant maize (Izui et al.
Nucl. Acids Res. 14 , 1615, 19
86), the isolation of genes from the seeds of C3 plants has not been reported.

[0004]

[Problems to be Solved by the Invention] As the population grows,
Although increasing the production of food is an urgent global task, expanding the acreage is not preferable because it leads to the destruction of nature such as forests. In order to increase food production under such conditions, a method of increasing or enlarging individual plants themselves, and a method of particularly increasing the concentration of a specific component in a plant are considered.

The present invention is based on the latter idea and intends to solve this by utilizing a genetic engineering technique. In particular, from the viewpoint of increasing the production of protein and fat, the gene involved in it is cloned. Therefore, the ultimate purpose is to breed desired plants.

[0006]

The present invention was carried out in order to achieve the above object, and the present inventors have found that the protein content or fat production in plant seeds, leaves and cultured cells is increased. As a means to achieve, soybeans to PEP
We planned to clone C gene, succeeded in cloning two kinds of cDNA from soybean ripening stage seeds, and succeeded in determining the structure of soybean PEPC gene as a result of researches from various directions. Has been completed. Hereinafter, the present invention will be described in detail.

In order to carry out the present invention, it is first necessary to separate and purify PEPC from soybean seeds. For this purpose, a conventional method used for the purification of enzymes is appropriately used. For example, after crushing soybean seeds, The suspension is suspended in a buffer solution, and the ammonium sulfate fraction in the supernatant is purified according to a known method such as column chromatography.

In this manner, PEPC was purified to homogeneity from soybean seeds and then used to prepare an antibody against rabbits. Next, poly (A) RNA was prepared from seeds of soybean during the ripening period, and cDNA was synthesized using the poly (A) RNA as a template. This cDNA is lambda phage vector λg
An integrated cDNA library was obtained at t11. This library was further amplified using Escherichia coli Y1088 strain and then screened. When the screening was carried out using the rabbit antibody prepared above, 6 λ phages producing a protein that specifically binds to this antibody were obtained. In order to confirm that these have the PEPC gene, antibodies that bind to the proteins produced by these λ phages were purified and used for Western analysis. As a result, it was confirmed that all the antibodies contained the PEPC gene because only PEPC was specifically selected from the soybean seed proteins separated by SDS electrophoresis. Therefore, λDNA was prepared from these λ phages, cleaved with the restriction enzyme EcoRI, and the cDNA portion was recovered to obtain the plasmid vector pK.
Subcloned into S +. When these restriction enzyme maps were created, four of them were insert cDs of 1 kbp or less.
Two contained only NA but two contained 3 kbp insert DNA. These two clones are SPC1
And SPC16. It was revealed that the two have different restriction enzyme positions and are PEPC genes derived from different genes. These base sequences were determined, and the base sequences of the cDNAs of the two PEPC genes were successfully obtained (sequence listing, SEQ ID NO: 1 and SEQ ID NO: 2).

The sequences of these two types of PEPC genes were found to be homologous with, but not identical to, the PEPC genes already isolated from maize, sycamore, cyanobacteria, and Escherichia coli.

The present invention will be described in detail below based on examples.

[0011]

[Example 1]

(1) Purification of Soybean PEPC Soybean seeds (500 g) were crushed to obtain 2 liters of 0.1.
M phosphate buffer (pH 6.8, containing 25 mM magnesium chloride) was added and mixed well, and the supernatant was collected by centrifugation. A 30% to 50% ammonium sulfate fraction was obtained from this supernatant. This ammonium sulfate precipitate was mixed with 0.1 M Tris buffer (p
Butyl Toyopearl column (5 x 50 cm) after dissolving in H7.5, containing 30% ammonium sulfate)
Then, the PEPC activity was eluted. After dialyzing this fraction, it was applied to a DEAE Toyopearl column (5 × 50 cm). PEPC activity was eluted from this column by a concentration gradient of sodium chloride. After ammonium sulfate precipitation of this fraction, gel filtration was performed using a Sepharose CL6B column (2 × 90 cm). This fraction was applied to a hydroxyapatite column (1 × 20 cm) and 5 mM to 100 m
Elution was performed with M phosphate buffer (pH 6.8). In this way, uniformly purified PEPC was obtained.

(2) Preparation of antibody against soybean PEPC 1 mg of soybean PEPC obtained by the above method was well mixed with 2.5 ml of adjuvant and then injected into the epidermis of rabbits. After 60 days, the same rabbit was again injected with soybean PEPC to increase the antibody titer. After this, 60 days later, blood was collected from the rabbit and an equal volume of 50
mM sodium citrate was added and the supernatant was used as the antibody.

(3) Poly (A) R from soybean ripening seeds
Preparation of NA Soybean seeds at the ripening stage were immediately put into liquid nitrogen, frozen, and stored at -135 ° C until use. This soybean seed (50 g) was crushed in the presence of liquid nitrogen using a mortar until it became powdery. To this, 200 ml of extraction buffer (20 mM vanadyl complex, 2% SDS, Tris-HCl, pH 8.0) was added, and immediately 500 × g.
The supernatant was recovered by centrifugation at. 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 2000 × g to collect the supernatant. This supernatant was mixed with 1/10 volume of 3M sodium acetate (p
H5.2) and 2 volumes of ethanol were added to give -20
Let stand at ℃ for 30 minutes. This was centrifuged at 2000 × g to obtain a 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 was repeated 5 more times. The obtained supernatant was precipitated with ethanol, the RNA was dissolved in distilled water so that the RNA concentration was 1 mg / ml and the lithium chloride concentration was 2 M, and then the mixture was allowed to stand on ice for 8 hours. This was centrifuged at 2000 xg and the precipitate was collected. This RNA precipitate was dissolved in distilled water. Preparation of poly (A) RNA was performed using oligo dT
A spin column (manufactured by Pharmacia) was used according to the instruction manual for this column. It was possible to obtain 100 μg of poly (A) RNA.

(4) Preparation of cDNA library Using 5 μg of poly (A) RNA obtained by the above method, cD
Using an NA synthesis kit (Pharmacia), cDNA was synthesized according to the instructions of the kit. In the cDNA synthesis using this kit, E was added to both ends of the synthesized cDNA.
Ligation kit (manufactured by Takara Shuzo Co., Ltd.) was used for the coRI adapter, and the ligation reaction was performed 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. This library contained approximately 1 million recombinants. Before screening this library, all the phages contained in this library were infected with Escherichia coli Y1088 strain, which was cultivated on agar medium at 42 ° C., and phages were smeared from phage plaques that appeared after 6 hours. Recovered using the solution. A small amount of chloroform was added to this recovered solution and the mixture was stored at 4 ° C. In the following screening, this phage solution was used for cDNA
Used as A library.

(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 to screen the phage. E. coli was infected. 10m which melted this at 50 degrees Celsius
l soft agar and then mixed with LB agar (diameter 15c
m petri dish) to solidify the soft agar. At this time, the amounts of Escherichia coli and the phage solution were adjusted so that 100,000 plaques were produced per dish. The 10 dishes thus prepared were incubated at 42 ° C. for 3 hours, and then the dried nitrocellulose filter was soaked in 10 mM IPTG solution, and the dried nitrocellulose filter was covered on the agar medium for another 3 hours at 37 ° C. Culture was carried out in. afterwards,
Carefully remove these filters from the agar and remove 1%
It was soaked in the BSA solution for 16 hours. After adding PEPC rabbit antibody (0.1 ml) to 100 ml of BSA solution and mixing, the above filter was dipped in this solution and reacted with the antibody for 1 hour at room temperature while gently shaking. The color reaction was carried out using a secondary antibody method kit (Promega, Picoblue) using alkaline phosphatase according to the instructions. The positions of the 6 purple spots appearing on the filter were compared with the agar medium from which the filter was derived, and the phage at the corresponding position was recovered in 1 ml of SM solution. Since a large number of non-target phages are mixed in these 6 types of phage liquids, this time, about 1 phage per plate was used.
The plaques of phages that gave purple spots were collected from each phage solution in the same manner as above, and the plaques of phages that gave purple spots were collected (secondary screening). Further, the third screening was carried out in the same manner to finally obtain 6 kinds of phage solutions.

(6) Determination of nucleotide sequence From the 6 kinds of phages thus obtained, Gross
Berger's method (Nuc. Acids Res. 1
5 , 6737, 1987), respectively, to prepare λDNA. This λDNA was digested with the restriction enzyme EcoRI, and then ligated with the plasmid vector pKS + (Stratagene), which had been digested with EcoRI and subjected to CIP treatment, according to the instruction of the ligation kit (Takara Shuzo). Using this, E. coli (XL1-Blue
Strain). Plasmids were prepared from the obtained colonies and digested with various restriction enzymes to prepare respective restriction enzyme maps. By comparing these, two types of cDNA considered to be full length were found. These are SPC1
And SPC16. They show similar restriction maps but differ in some respects and were found to be derived from different PEPC genes. Therefore, in order to determine these nucleotide sequences, for SPC1, the insert cDNA was replaced with another plasmid vector pUC.
Subcloned into 118. Using these Erase A Base Kit from Promega, a deletion clone was prepared according to the instruction. 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 each of the two PEPC clones had a full-length cDNA insert containing a translation initiation site.

That is, the present invention relates to soybean PEPCs shown in SEQ ID NOS: 1 and 2 of the sequence listings shown in Tables 1 to 16 below.
It relates to the gene that encodes.

[0019]

[Table 1]

[0020]

[Table 2]

[0021]

[Table 3]

[0022]

[Table 4]

[0023]

[Table 5]

[0024]

[Table 6]

[0025]

[Table 7]

[0026]

[Table 8]

[0027]

[Table 9]

[0028]

[Table 10]

[0029]

[Table 11]

[0030]

[Table 12]

[0031]

[Table 13]

[0032]

[Table 14]

[0033]

[Table 15]

[0034]

[Table 16]

[0035]

EFFECTS OF THE INVENTION From the viewpoint that plant cells with high PEPC activity produce more protein than fat synthesis, PEPC is used to increase protein production, or conversely to suppress PEPC expression. It can be used to increase fat production. By isolating the PEPC gene involved in increasing protein production in soybean seeds according to the present invention, the production of plants having increased protein content in plant seeds, leaves, cultured cells, etc. using gene recombination technology, On the contrary, it can be expected to produce plants with increased fat content.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Daisuke Shibata 1153-7 Nagakuni, Tsuchiura-shi, Ibaraki Prefecture (72) Inventor Robert F Whittier 31-14 Sakuragaoka, Tsuchiura-shi, Ibaraki Prefecture

Claims (1)

[Claims] 1. A gene encoding phosphoenolpyruvate carboxylase having the amino acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.