JP3094485B2 - Oligonucleotide for detecting plant transformant and method for detecting the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a probe for efficiently detecting or confirming a transformant when a new variety having a new trait is developed by introducing a gene into a plant by genetic engineering techniques. As an oligonucleotide. More specifically, when a transformed tissue is induced from a plant tissue using a recombinant microorganism or the like, a probe that is extremely effective in determining whether the obtained tissue is a parent plant tissue or a transformed tissue As an oligonucleotide.

[0002]

2. Description of the Related Art In recent years, in a plant breeding technology, a new trait not existing in existing species has been introduced by introducing a certain foreign gene into a plant body by genetic engineering techniques and expressing a trait peculiar to the gene. There have been many attempts to add. Suitable microorganisms to be used as recombinant microorganisms are those originally capable of actively infecting plants, and these are so-called phytopathogenic microorganisms. In order to use these microorganisms for plant transformation, it is desired to eliminate the expression of pathogenicity inherent in plants while maintaining at least the ability to infect plants, and to express desired useful genes.

[0003] Here, in order to confirm whether or not a newly obtained plant tissue is truly a transformant, it is necessary to analyze at the gene level for both confirmation of introduction and expression.

[0004]

However, at the present stage, efforts are being made to obtain useful genetic resources for use in recombinant DNA, and it has not yet reached practical use.

[0005] Therefore, an object of the present invention is to detect, at the genetic level, whether or not a plant tissue is truly a transformant.

[0006]

Means for Solving the Problems The present inventor has made intensive studies to achieve the above object, and as a result, a specific gene present in the vicinity of a plant cell was identified by a gene amplification method using an oligonucleotide as a primer. The inventors have found that detection is efficient, and have conducted further studies to complete the present invention. Here, the gene amplification method is based on the Polymerase Chain Reaction method developed by Saiki et al.
R method; Science, 230, 1350 (1985)).

According to this method, when detecting a specific nucleotide sequence region, one end of the region is hybridized by recognizing a + strand and the other end is recognized by a − strand. Prepared as a primer for a template-dependent nucleotide polymerization reaction of a sample nucleic acid which has been converted to a single-stranded state by thermal denaturation, and the generated double-stranded nucleic acid is separated into single-stranded nucleic acids. Wake up. By repeating this series of operations, the number of copies in the region between the two primers increases until the region can be reliably detected.

[0008] Materials include all tissues and organs of a plant, such as dedifferentiated cells, tumor tissues, etc.
Leaves and petals may be used. In order to use these materials as samples for a PCR reaction, pretreatment for releasing nucleic acid components from plant materials is required. However, nucleic acids to which primers can hybridize are
Since the R reaction progresses, a simple operation of treating the material with oxygen, a surfactant, alkali, or mechanical crushing for a short time can prepare a sample solution with a sufficient amount of nucleic acid to advance the PCR reaction. .

The oligonucleotide used as a primer in the present invention is a gene encoded by Pseudomonas syringae DNA, which is a gene relating to the synthesis of an ice nucleation active protein, ina Z (ice nucleation protein gene).
(5 ′) d-CTGCACTGAAGGTTTTCACTG (3 ′)... (A) (5 ′) d-GAATAGCATCATGACGCCAG The following oligonucleotide (a) or (b) complementary to the nucleotide sequence of the gene: (3 ′)... (B) Alternatively, a gene encoded by the DNA of Erwinia herbicolaA, which targets a gene iceE (ice nucleation protein gene) related to the synthesis of an ice nucleation active protein, and has a nucleotide sequence of Oligonucleotides having the following sequence (c) or (d) are complementary: (5 ') d-GAACCAGAGGAGTAGACTG (3') (c) (5 ') d-GTTAGTCCGTTCTCAAAGCC (3') (d) Used.

[0010] The oligonucleotide used as a primer in the present invention may be a nucleotide fragment of at least about 15 to about 30 bases in view of specificity, detection sensitivity and reproducibility. May be.

[0011] The two primers in the present invention are, for example, an oligonucleotide containing the sequence a if one of the primers is an oligonucleotide containing the sequence a.

[0012] The primer may not be particularly labeled for detection. DN defined by primer
The amplification region in A may be from 50 bases to 2000 bases, preferably from 200 bases to 300 bases.

In the polymerization reaction of template-dependent nucleotides,
Uses thermostable DNA polymerase, which is 90-95
As long as the activity is maintained at a temperature of ° C., it may be derived from any species. This enzyme is a known substance, for example, Perkin E
A product manufactured by lmer Cetus may be used. The heat denaturation temperature is usually 90 to 95 ° C, the temperature of the annealing operation for hybridizing primers is usually 37 to 65 ° C, and the nucleotide polymerization reaction is usually 50 to 75 ° C. Depending on the DNA weight of the target nucleotide, the copy number of the target nucleotide fragment is amplified usually by 20 cycles or more, preferably 30 to 45 cycles. Upon detection, if the enzyme reaction solution is directly subjected to agarose gel electrophoresis, the presence and length of the amplified nucleotide fragment can be confirmed.

From the results, it can be determined whether or not a nucleic acid having a sequence to be recognized by the primer exists in the material. This determination directly determines the presence or absence of DNA.

For detection of the amplified nucleotide fragment, various kinds of electrophoresis such as acrylamide electrophoresis and chromatography are also effective. In addition, in electrophoresis, detection can be performed without labeling a primer or the like by determining a molecular weight by inserting a marker and / or using a nucleic acid staining method using ethidium bromide or the like.

[0016]

According to the present invention, a specific gene is amplified by the PCR method and the gene is detected, so that it is possible to confirm at the gene level whether or not the transformant is a transformant.

[0017]

[Experimental example 1: Pseudomonas syringae ice nu]
Detection of cleavage protein gene ) Preparation of sample Pseudomonas syring, which was shake-cultured overnight in LB medium, on the surface of developed leaves of tobacco (Nicotiana tabacum cv. Samsun)
2 μl of the ae strain Ni23 culture suspension was applied.

A radius of 2.5 m from the center point where the cell suspension was applied
m into a 1.5 ml Eppendorf tube, and add 40 μl of buffer solution (0.1 M Potassium Phosph
atepH7.0) and crushed in a 0.5 ml Eppendorf tube.

Then, 2 μl of Lysozyme (10 mg / ml) was added.
Incubate at 25 ° C. for 15 minutes. Subsequently, 50 μl of an Alk-SDS solution (0.2N NaOH, 1% SDS) was added, and the mixture was allowed to stand at 25 ° C. for 10 minutes. Next, add 37.5 μl of 3M Potassium acetate
It was left still at 25 ° C. for 10 minutes.

The supernatant was recovered by centrifugation at 4 ° C. at 15,000 rpm for 20 minutes, and 0.5 volume of 2-propanol was added.
Centrifugation was performed at rpm for 2 minutes. 75% e of the resulting precipitate
After washing with tha-nol and drying, 1 ml of distilled water was added to obtain a sample solution.

The nucleotide sequence of the gene inaZ for the synthesis of ice nucleation active protein of DNA synthesis Pseudomonas syringae primer's, (5 ') d-CTGCACTGAAGGTTTCACTG (3') ...... (a sequence; SEQ ID NO: 1) and, (5 ′) d-GAATAGCATCATGACGCCAG (3 ′)... (B sequence; SEQ ID NO: 2) was selected, and oligonucleotides having the same sequence were chemically synthesized to obtain primers (a) and (b), respectively. MILLIPORE's Cyclon plu for chemical synthesis
This was performed by the phosphonate method using s DNA synthesizer. For purification of the synthesized oligonucleotide, a C18 reverse phase column was used.

PCR Using 3 μl of the above sample solution, add 14.55 μl of sterile distilled water
l, 10 × reaction buffer 3 μl, dNTP solution 4.8 μl, NP40
3 µl of solution, 0.75 µl of primer (a), 0.75 µl of primer (b), and 0.15 µ of thermostable DNA polymerase
was added to prepare 30 μl of the reaction solution. 50 μl of mineral oil (SIGMA) was overlaid on the container containing the reaction solution.

The details of the additive liquid are as follows. 10 × reaction buffer; 500 mM KCl, 100 mM Tris-HCI (pH 8.
3), 15 mM MgC12, 0.1% (w / v) gelatin NP40 solution; mixture of NONIDET P-40 and Tween 20 (final concentration: 0.05%) dNTP solution; mixture of dATP, dCTP, dGTP, dTTP (Final concentration: 1.25 mM) Primers (a) and (b); aqueous solutions of the above-mentioned chemically synthesized purified products (5 ODU / ml) Thermostable DNA polymerase; Taq DNA polymerase (5 un
it / ml; Perkin Elmer Cetus).

The reaction conditions are as follows. Thermal denaturation; 94 ° C for 1 minute Annealing; 55 ° C for 1 minute Polymerization reaction; 72 ° C for 1 minute.

The process from the thermal denaturation to the polymerization reaction through annealing is defined as one cycle (the required time is 5.7 minutes).
42 cycles (about 4 hours total time) were repeated. These operations were performed by programming the above reaction conditions in a DNA Thermal Cycler manufactured by Perkin Elmer Cetus.

To detect the amplified nucleotide fragment from the detection reaction solution, agarose gel electrophoresis was performed under the following conditions.

Agarose gel has a gel concentration of 2% (w / v)
A solution containing ethidium bromide (0.5 μg / ml) was used. The electrophoresis was performed at a constant voltage of 150 V for 35 minutes. The operation method and other conditions are as described in Molecula Cloni using maniatis.
ng (1982). In addition to the reaction solution, migration of a molecular weight marker was also performed at the same time, and the length of nucleotide fragments detected in the gel by ultraviolet light or the like was calculated by comparing the relative mobilities.

Results As described above, in the DNA of Pseudomonas syringae,
The nucleotide sequence of the inaZ gene has already been determined, and the size of the oligonucleotide of the present invention, that is, the nucleotide to be amplified by the primer by PCR can be estimated. According to this, when the primer (a) and the primer (b) were used, an estimated 155 base pairs of nucleotides would have been amplified. This estimate was in good agreement with the results of agarose gel electrophoresis.

FIG. 1 shows the results of agarose gel electrophoresis of the amplified nucleotide fragment. In the figure, (a) shows the marker (φ × 174 / HinC II) and (b) shows the Pseudomonas
syrin-gae strain Ni23, (c) tobacco leaves (control)
(D) shows tobacco-infected leaves.

(Experimental Example 2: ice nu of Erwinia herbicola)
Detection of cleavage protein gene) Erwinia herbicola strain M42 culture suspension was used as a sample, and (5 ′) d-GAACCAGAGGAGGTAAGACTG was used as a primer.
(3 ′)... (C sequence, SEQ ID NO: 3) (5 ′) d-GTTAGTCGTTCTCAAAGCC
Sample preparation, primer synthesis, PCR, and detection were performed in the same manner as in Experimental Example 1, except that the base sequence of (3 ′)... (D sequence, SEQ ID NO: 4) was synthesized.

As described above, Erwinia herbicola D
The nucleotide sequence of the iceE gene in NA has already been determined, and the oligonucleotide of the present invention, ie, P
The size of the nucleotide to be amplified by the primer by CR can be estimated. According to this, when the primer (c) and the primer (d) were used, an estimated 189 base pairs of nucleotides would have been amplified. This estimate was in good agreement with the results of agarose gel electrophoresis.

FIG. 2 shows the results of agarose gel electrophoresis of the amplified nucleotide fragment. In the figure, (a) shows the marker (φ × 174 / HinC II) and (b) shows the Pseudomonas
syrin-gae strain Ni23, (c) tobacco leaves (control)
(D) shows tobacco-infected leaves.

[0033]

According to the present invention, primers amplify the copy number of a target nucleotide in a plant by employing PCR.
Moreover, since the nucleotide sequence to be amplified can be defined by the reaction of two or more primers, according to the present invention, a specific foreign gene can be easily detected from a transformant with high sensitivity, and high selectivity can be obtained upon detection. Can be

In the present invention, a large amount of material is not required for detection despite high detection sensitivity, and material pretreatment is simple. Moreover, the reaction time is short, the detection is simple, and the operation is easy, so that the time until the result is obtained can be greatly reduced. Also, in the present invention, by using agarose gel electrophoresis and nucleic acid staining with ethidium bromide for detection, detection can be performed without labeling primers and the like, and the length of nucleotide fragments can be confirmed, so that the reliability of the results can be confirmed. The nature becomes high.

Furthermore, the detection of a microorganism having a target nucleotide has a high selectivity for the target nucleotide. Therefore, for example, in a plant transformation experiment using a recombinant microorganism, if the target nucleotide is incorporated into the microorganism, it can be detected in a plant. It can be used as an indicator of the presence of microorganisms.

[0036]

[Sequence List] SEQ ID NO (SEQ ID NO): 1 sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Pseudomonas syringae sequence The method of determining the characteristics S sequence CTGCACTGAAGGTTTCACTG

Sequence number (SEQ ID NO): 2 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear type of sequence Genomic DNA Hypothetical sequence NO Antisense NO Origin Pseudomonas syringae Sequence Features Method of determining features S array GAATAGCATCATGACGCCAG

Sequence number (SEQ ID NO): 3 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Erwinia herbicola Sequence Features Method for determining features S Sequence GAACCAGAGGAGTAAGACTG

SEQ ID NO: 3 Sequence length 20 bases Sequence type Number of nucleic acid strands Single strand Topology Linear Sequence type Genomic DNA Hypothetical sequence NO Antisense NO Origin Erwinia herbicola Sequence Features Method of determining features S Sequence GTTAGTCGTTCTCAAAGCC

[Brief description of the drawings]

FIG. 1 is an agarose gel electrophoresis diagram of the amplified nucleotide fragment of Experimental Example 1.

FIG. 2 is an agarose gel electrophoresis diagram of the amplified nucleotide fragment of Experimental Example 2.

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:18) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12Q 1 / 68 BIOSIS (DIALOG) GenBank / EMBL / DDBJ

Claims (2)

(57) [Claims] 1. A gene encoded by the DNA of Pseudomonas syringae, which targets the gene inaZ (ice nucleation protein gene) relating to the synthesis of an ice nucleation protein, and is complementary to the nucleotide sequence of the gene described below. Or b) an oligonucleotide having the sequence. (5 ′) d-CTGCACTGAAGGTTTCACTG (3 ′) (a) (5 ′) d-GAATAGCATCATGACGCCAG (3 ′) (b) 2. A gene encoded by the DNA of Erwinia herbicola A, which targets a gene iceE (ice nucleation protein gene) relating to the synthesis of an ice nucleation protein, and is complementary to the nucleotide sequence of the gene as follows: An oligonucleotide having the sequence of c) or (d). (5 ') d-GAACCAGAGGAGGTAAGACTG (3') (c) (5 ') d-GTTAGTCCGTTCTCAAAGCC (3') (d)