JP3002735B2 - Soybean phosphoenolpyruvate carboxylase gene - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a phosphoenolpyruvate carboxylase gene contained in plant seeds, particularly soybeans. Since the gene is related to increased protein production, expression of this gene can produce soybeans and other plants with high protein content, while suppression of gene expression can lead to soybean and other plants. By increasing the production of fat and utilizing the present invention, soybeans and other various plants having desired properties can be bred very 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 a reaction for producing oxaloacetic acid and orthophosphate from phosphoenolpyruvate and carbonate ions. .

[0003] Sugimoto et al. (Agric. Bio)
l. Chem. 53 , 885, 1989) report that there is a positive and negative correlation between PEPC contained in soybean seed and protein content and fat content, respectively. That is, it is shown that by controlling the expression level of PEPC, the production amount of these can be controlled. In a previous study, the PEPC gene of maize, a C4 plant, was isolated (Izui et al.,
Nucl. Acids Res. 14 , 1615, 19
86), isolation of genes from C3 plant seeds has not been reported.

[0004]

[Problems to be solved by the invention] As the population increases,
Increasing food production is an urgent need worldwide, but increasing the acreage is unfavorable because it leads to the destruction of forests and other nature. In order to increase food production under such conditions, there can be considered a method of increasing or enlarging individual plants themselves, and a method of particularly increasing the concentration of specific components in plants.

[0005] The present invention is directed to the latter idea, which is intended to be solved by using a genetic engineering technique. In particular, from the viewpoint of increasing the production of proteins and fats, the present inventors clone the genes involved in the latter. Therefore, it is an ultimate object to breed a desired plant.

[0006]

DISCLOSURE OF THE INVENTION The present invention has been made to achieve the above object, and the present inventors have proposed to increase the protein content or the fat production in plant seeds, leaves and cultured cells. As a means to achieve
The cloning of the C gene was planned, and two cDNAs were successfully cloned from the seeds of the soybean ripening stage. Further, as a result of research from various fields, the structure of the soybean PEPC gene was successfully determined. Was completed. Hereinafter, the present invention will be described in detail.

[0007] In order to carry out the present invention, it is necessary to first separate and purify PEPC from soybean seeds. For this purpose, a conventional method used for enzyme purification is appropriately used. For example, after soybean seeds are crushed, The suspension is suspended in a buffer, and the ammonium sulfate fraction of the supernatant is purified according to a known method such as column chromatography.

[0008] After purifying PEPC from soybean seeds to homogeneity, an antibody against rabbits was prepared using the purified PEPC. Next, poly (A) RNA was prepared from soybean ripening seeds, and cDNA was synthesized using the RNA as a template. This cDNA was transformed into a lambda phage vector λg.
At t11, an integrated cDNA library was obtained. This library was further amplified using Escherichia coli strain Y1088 before screening. Screening was performed using the previously prepared rabbit antibody. As a result, six λ phages producing proteins that specifically bind to this antibody were obtained. To confirm that they have the PEPC gene, antibodies that bind to the protein produced by these λ phages were purified, and Western analysis was performed using them. As a result, it was confirmed that all the antibodies specifically contained only PEPC from the soybean seed proteins separated by SDS electrophoresis, and thus contained the PEPC gene. Therefore, λ DNA was prepared from these λ phages, cleaved with the restriction enzyme EcoRI, and the cDNA portion was recovered.
It was subcloned into S +. When these restriction enzyme maps were prepared, four were obtained with insert cD of 1 kbp or less.
Only NA was contained, but two contained 3 kbp of insert DNA. These two clones were replaced with SPC1
And SPC16. Both of them differed in the position of the restriction enzyme, and were found to be PEPC genes derived from different genes. By determining these nucleotide sequences, the nucleotide sequences of the cDNAs of the two PEPC genes were successfully obtained (SEQ ID NO: 1 and SEQ ID NO: 2).

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

Hereinafter, the present invention will be described in detail with reference to examples.

[0011]

Embodiment 1

(1) Purification of soybean PEPC Soybean seeds (500 g) were pulverized and 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. From this supernatant, a 30% to 50% ammonium sulfate fraction was obtained. This ammonium sulfate precipitate was added to a 0.1 M Tris buffer (p
H7.5, containing 30% ammonium sulfate), and then using the same buffer solution, a butyl toyopearl column (5 × 50 cm).
To elute the PEPC activity. After dialyzing this fraction, it was applied to a DEAE Toyopearl column (5 × 50 cm). From this column, the PEPC activity was eluted with a concentration gradient of sodium chloride. This fraction was precipitated with ammonium sulfate and then subjected to gel filtration using a Sepharose CL6B column (2 × 90 cm). This fraction is applied to a hydroxyapatite column (1 × 20 cm), and the
Elution was performed with M phosphate buffer (pH 6.8). Thus, PEPC uniformly purified was obtained.

(2) Preparation of antibody against soybean PEPC 1 mg of soybean PEPC 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 soybean PEPC in the same manner to increase the antibody titer. After 60 days, blood was collected from the rabbit and an equal volume of 50
mM sodium citrate was added and the supernatant was used as an antibody.

(3) Poly (A) R from soybean ripening seeds
Preparation of NA The soybean seeds in the ripening stage were immediately poured into liquid nitrogen, frozen, and stored at -135 ° C until use. This soybean seed (50 g) was crushed using a mortar in the presence of liquid nitrogen until it became powdery. To this was added 200 ml of extraction buffer (20 mM vanadyl complex, 2% SDS, Tris-HCl, pH 8.0) and immediately 500 × g
And the supernatant was collected. An equal amount of phenol / tris (pH 8.0) was added to the supernatant, and the mixture was stirred for about 10 minutes and centrifuged at 2000 × g to collect the supernatant. One-tenth volume of 3M sodium acetate (p
H5.2) and 2 volumes of ethanol were added to give -20.
C. 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 five more times. The obtained supernatant was precipitated with ethanol, and the RNA was dissolved in distilled water so that the RNA concentration was 1 mg / ml and the lithium chloride concentration was 2 M, followed by standing on ice for 8 hours. This was centrifuged at 2000 × g, and the precipitate was recovered. This RNA precipitate was dissolved in distilled water. Preparation of poly (A) RNA is performed using oligo dT
This was performed using a spin column (manufactured by Pharmacia) according to the instruction manual for this column. 100 μg of poly (A) RNA could be obtained.

(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, both ends of the synthesized cDNA are
Using a ligation kit (manufactured by Takara Shuzo Co., Ltd.), a ligation reaction was performed using the coRI adapter according to the instructions. The thus obtained λDNA was reconstituted with phage particles using a packaging kit (manufactured by Stratagene) in accordance with the instructions of the kit to prepare a cDNA library. This library contained approximately one million recombinants. Before screening this library, all the phages contained in this library were infected into E. coli strain Y1088, which was cultured on an agar medium at 42 ° C. Collected using the solution. The recovered solution was stored at 4 ° C. with a small amount of chloroform. In the following screening, this phage solution was prepared using cDN
Used as A library.

(5) Screening of cDNA A part of the phage solution contained in the cDNA library obtained as described above is mixed with E. coli (Y1090 strain) and cultured at 37 ° C. for 15 minutes, whereby the phage is E. coli was infected. This was melted at 50 ° C and 10m
LB agar medium (diameter 15c)
m a petri dish) and the soft agar was solidified. At this time, the amounts of Escherichia coli and the phage solution were adjusted so that 100,000 plaques were formed per petri dish. After culturing the 10 Petri dishes thus prepared at 42 ° C. for 3 hours, infiltrating a 10 mM IPTG solution, covering the dried nitrocellulose filter on the agar medium, and further heating at 37 ° C. for 3 hours. Was performed. afterwards,
Carefully remove these filters from the agar medium and add 1%
For 16 hours. After adding and mixing PEPC rabbit antibody (0.1 ml) to 100 ml of BSA solution, the filter was immersed in this solution, and allowed to react with the antibody for 1 hour at room temperature with gentle shaking. The color reaction was performed using a secondary antibody method kit (Promega, Pico Blue) using alkaline phosphatase according to the instructions. The positions of the six 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 these six kinds of phage solutions contain a large number of phages other than the target phage, this time about 1 petri dish was used.
Phage plaques giving purple spots were collected from each phage solution in the same manner (secondary screening). Further, a tertiary screening was performed in the same manner, and finally, six kinds of phage solutions were obtained.

(6) Determination of base sequence Gross was obtained from the six kinds of phage thus obtained.
Berger's method (Nuc. Acids Res. 1)
5 , 6737, 1987). This λDNA was digested with the restriction enzyme EcoRI, and then ligated to the plasmid vector pKS + (Stratagene), which had been digested with EcoRI and treated with CIP, according to the instructions 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 cDNAs considered to be full length were found. These are SPC1
And SPC16. These show similar restriction maps, but differed 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 inserted into another plasmid vector pUC.
It was subcloned into 118. Using these, a deletion clone was prepared using Promega's erase-a-base kit 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 both of the two PEPC clones had a full-length cDNA insert including the translation initiation site.

That is, the present invention relates to the soybean PEPC shown in SEQ ID NOs: 1 and 2 in the sequence listings shown in Tables 1 to 16 below.
It relates to the gene encoding

[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]

EFFECT OF THE INVENTION From the viewpoint that the amount of protein produced in plant cells having high PEPC activity is increased as compared with the synthesis of fat, it is possible to increase the production of protein by using PEPC or, conversely, to suppress the expression of PEPC. 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, using genetic recombination technology to produce plant seeds, plants with increased protein content in leaves and cultured cells, Conversely, the production of plants with an increased fat content can be expected.

────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Robert F. Whittier 31-14 Sakuragaoka, Tsuchiura City, Ibaraki Prefecture (56) References JP-A-3-172187 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 GenBank / EMBL / DDBJ

Claims (1)

(57) [Claims] 1. A cDNA of a phosphoenolpyruvate carboxylase gene represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing.