JP2003102498A - Method for analyzing chromosome - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing chromosome with which the action of a chromosome can be analyzed in a state where the shape in a living body is kept. SOLUTION: A sample derived from a hybrid plant organism of an analysis object is brought into contact with a solution containing a compound capable of fixing a nucleic acid in the sample, to fix the sample and to obtain a fixed sample. The labeled probe is hybridized with the fixed sample under the following conditions: a labeled probe obtained by labeling, with a labeling material, a nucleic acid derived from an analysis species which may provide a genome to the plant organism of the hybrid can penetrate into the hybrid sample; and the labeled probe can be annealed specifically with a nucleic acid existing in the sample and having a sequence complementary to the sequence of the probe. Thus, the action of the chromosome in the sample is observed via a signal derived from each labeling material of the labeled probe.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a chromosome analysis method. More specifically, it relates to a chromosome analysis method capable of analyzing the dynamics of plant chromosomes.

[0002]

2. Description of the Related Art Currently, various attempts have been made to breed useful plants. For example, a cross between wheat and corn is performed.

When corn pollen is crossed with wheat,
The pollen tube elongates and the maize sperm nucleus fertilizes with the wheat egg.

However, the corn chromosome is shed at an early stage of embryogenesis, and a wheat haploid is produced. Therefore, it is the current situation that the corn mutation is not utilized in wheat due to this far crossing. Investigating the process of the loss of maize chromosomes is important to break this cross incompatibility.

[0005] The loss of corn chromosomes from wheat x corn hybrid embryos was reported by Laurie and Bennett (Laurie
and Bennett) [Genome, 32: 953-961 (1989)] to determine the timing of dropout. However, since the findings of Raleigh et al. Are observations by the crushing method, it is currently difficult to obtain information on the spatial arrangement of chromosomes, dynamics in interphase nuclei, and the like. Therefore, at present, the positional relationship between the chromosomes of both species is unknown.

[0006] Therefore, there is a demand for a technique capable of specifically staining and discriminating and observing a chromosome, particularly a heterologous chromosome, in a state close to that in a living body, that is, in a highly conserved state.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a chromosome analysis method capable of analyzing the dynamics of chromosomes in a state in which the morphology in a living body is maintained, that is, in a highly conserved state.

[0008]

The gist of the present invention is [1]
(A) fixing the sample by contacting a sample derived from a hybrid plant individual to be analyzed with a solution containing a compound capable of fixing the nucleic acid in the sample to obtain a fixed sample, ( b) In the fixed sample obtained in step (a), a labeled probe obtained by labeling a nucleic acid derived from an analyte suspected of providing a genome to the hybrid plant individual with a labeling substance is provided. Under permeable conditions, and under conditions suitable for specifically annealing the labeled probe and a nucleic acid present in the sample, the nucleic acid having a sequence complementary to the sequence of the probe, The step of hybridizing the 2-labeled probe with the fixed sample obtained in the step (a), and (c) the kinetics of the chromosome in the sample are derived from the respective labeling substances of the labeled probe. Comprising the step of observing through Gunaru method chromosome analysis, as well as (2) a plant from an individual sample hybrids to be analyzed, a hybrid embryo, chromosomal analysis method according to claim 1, relates.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The chromosome analysis method of the present invention comprises:
(A) fixing the sample by contacting a sample derived from a hybrid plant individual to be analyzed with a solution containing a compound capable of fixing the nucleic acid in the sample to obtain a fixed sample, ( b) In the fixed sample obtained in step (a), a labeled probe obtained by labeling a nucleic acid derived from an analyte suspected of providing a genome to the hybrid plant individual with a labeling substance is provided. Under permeable conditions, and under conditions suitable for specifically annealing the labeled probe and a nucleic acid present in the sample, the nucleic acid having a sequence complementary to the sequence of the probe, The step of hybridizing the 2-labeled probe with the fixed sample obtained in the step (a), and (c) the kinetics of the chromosome in the sample are derived from the respective labeling substances of the labeled probe. It has one feature in comprising a step of observing through Gunaru.

According to the chromosome analysis method of the present invention, the (early) hybrid embryo can be removed and the dynamics of the heterologous chromosome can be directly observed. Since the embryo of a plant generally exists inside the ovary, it has conventionally been necessary to prepare a section. It exhibits an excellent effect that it is possible to investigate the dynamics of heterologous chromosomes in the development of hybrid embryos in plants, which has been difficult to observe in the past. Further, the chromosome analysis method of the present invention is that it is not necessary to prepare a section, that spatial position information can be obtained as compared with the crushing method, that many fluorescently labeled probes can be used, etc. It is advantageous to effectively analyze the dynamics of.

According to the chromosome analysis method of the present invention, for example, the dynamics of chromosomes in hybrid embryos, the dynamics of heterologous chromosomes in tissues of hybrid plants, and the like can be investigated.

In the chromosome analysis method of the present invention, first, a sample derived from a hybrid plant individual to be analyzed is brought into contact with a solution containing a compound capable of immobilizing a nucleic acid in the sample to give the sample. Fix and obtain a fixed sample [referred to as step (a)].

The "plant individual" includes all plants capable of producing hybrids such as wheat, rice and tobacco.

In the present specification, the "hybrid" may be a hybrid produced by a distant cross or a cross between close relatives.

The sample derived from the hybrid plant individual to be analyzed includes, for example, divided tissues such as embryos, root tips, and growing points, pollen mother cells that perform meiosis, and the like.

Examples of the "solution containing a compound capable of immobilizing nucleic acid in a sample" include ethanol-acetate, methanol, formalin and paraformaldehyde-containing solutions. Among them, ethanol acetate is preferable from the viewpoint of sufficiently obtaining the permeability of the labeled probe into a sample derived from a plant individual.

The contact time between the sample and the solution containing the compound capable of immobilizing the nucleic acid in the sample, that is, the time required for immobilizing the sample is, for example, about 1 week in the case of ethanol acetate, containing paraformaldehyde. In the case of a solution, it may be overnight at 4 ° C.

In the step (a), the cell wall may be further digested if necessary.

The digestion of the cell wall can be carried out, for example, by proteinase, specifically proteinase K, cellulase, pectinase, detergent (eg, Tween 20).
And the like.

The method of the present invention is advantageous in that, for example, when it is applied to a hybrid embryo of a hybrid produced by distant crosses, the cause of the cross incompatibility and its process can be investigated.

Then, in the fixed sample obtained in the step (a), nucleic acids derived from the analyte suspected to provide the genome to the hybrid plant individuals are labeled with different labeling substances. Suitable for allowing specific annealing of the labeled probe and the nucleic acid present in the sample and having a sequence complementary to the sequence of the probe under a condition that allows the labeled probe to penetrate. Under the conditions, the labeled probe is hybridized with the fixed sample obtained in the step (a) [referred to as step (b)].

The above-mentioned "conditions suitable for specifically annealing the (labeled probe) and a nucleic acid having a complementary sequence" include, for example, a sense strand and an antisense strand of a nucleic acid encoding a specific gene. However, a strand of a nucleic acid encoding another gene means a condition that only the corresponding strand is annealed without being annealed. Such conditions can be appropriately selected with reference to, for example, the hybridization conditions described in Molecular Cloning Laboratory Manual, Second Edition [Zanbrook, et al., Cold Spring Harbor Laboratory, (1989)].

The labeled probe is a labeled probe obtained by labeling a nucleic acid derived from an analyte that is suspected to provide a genome to the hybrid plant individual with a different labeling substance. It can be isolated from an analyte suspected of providing the genome to a hybrid plant individual according to conventional nucleic acid (eg, genomic DNA) isolation methods.
In addition, the nucleic acid may be a nucleic acid having a sequence specific to an analyte that is suspected to provide the genome to a hybrid plant individual. The nucleic acid may have any length as long as it is suitable for hybridization, for example, 4
The base length is from 00 to 200. The nucleic acid may be interspersed repeats present throughout the genome.

In the present specification, the "analyzed species suspected of providing the genome to a hybrid plant individual" means an individual presumed to be a parent of the hybrid plant individual.

Examples of the labeling substance include a fluorescent substance and FITC. From the viewpoint of ease of operation in measurement and shortening of time required for measurement, a fluorescent substance is preferable. Examples of the labeling substance include FIT
C, Cy3, Texas Red, Cy5, Cy7, biotin, digoxigenin, ULYSIS oregan G
reen 488 (manufactured by Molecular Probe) and the like.

The hybridization in step (b) can be carried out in the same manner as, for example, the method in genomic in situ hybridization. For example, a support holding a test sample (such as a slide glass) and a hybridization solution [composition: deionized formaldehyde (manufactured by Roche) 5 μl, 50%
Dextran sulfide 2 μl, 20 × SSC (1 × SSC indicates 0.15M NaCl, 0.015M sodium citrate, pH 7.0) 1 μl, labeled probe
0.5 μl (0.1 μg / μl), blocking DNA
(When wheat x corn, wheat genomic DNA
) Was incubated at 70 ° C. for 5 minutes and then at 37 ° C. overnight, and the resulting slide glass was washed with 2 × SSC at room temperature in the dark for 10 minutes, and then 0. 1% Triton X
The conditions include washing with 100 × 2 × SSC at room temperature in the dark for 10 minutes, and then with 2 × SSC at room temperature in the dark for 10 minutes.

Next, the dynamics of the chromosome in the sample are observed via the signals derived from the labeling substances of the labeled probe [referred to as step (c)].

The signal can be detected by a conventional confocal laser microscope, fluorescence microscope, or the like. In addition, it is desirable that the fluorescence microscope has equipment for obtaining an image in the z-axis direction in addition to the two-dimensional direction (x-axis, y-axis),
An example of a system having such equipment is a deconvolution image system.

Further, the whole sample may be stained with DAPI, PI, Hoechst 33258, YOYO1 or the like to carry out counterstaining.

According to the chromosome analysis method of the present invention, when the chromosome is analyzed, the state close to that of the living body is maintained. It has the excellent effect that it can be analyzed with.

[0031]

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

Example 1 A wheat line as a female parent was a bread wheat variety, China.
se Spring (CS) was used. Strain 919J was used as corn as a pollen parent.

Maize pollen was crossed with the emasculated wheat pistil. Twenty-four hours after the crossing (the first division of the fertilized egg has occurred), the wheat pistil was immersed and fixed in a fixing solution [ethanol: acetic acid = 3: 1 (volume ratio)]. The fixed wheat pistil was dyed by immersing it in a 45% carmine acetate stain solution and allowing it to stand overnight at room temperature.

Then, the ovules were extracted from the pistil of the wheat under a stereoscopic microscope. Furthermore, under a stereoscopic microscope, embryos (hybrid embryos) were extracted from within the embryo species in 45% acetic acid dropped on silane-coated slide glasses. Then, the silane-coated slide glass was dried as it was, and the extracted embryo and the slide glass were attached.

Hybridization was carried out as follows using a silane-coated slide glass to which hybrid embryos were attached.

The maize nuclear DNA extracted by the nuclear isolation method was used as ULYSIS oregon Green.
n 488 (manufactured by Molecular Probe) was directly labeled and used as a probe. That is,
Saccharomyces cerevisiae nuclear DNA extracted by the nuclear isolation method was fragmented into 400 to 200 bp by a sonicator. The obtained fragmented nuclear DNA was used as ULYSIS Labelin.
Labeled with oregan Green 488 of g System (manufactured by Molecular Probe) to obtain a labeled probe DNA.

Hybridization solution [composition: deionized formaldehyde (manufactured by Roche) 5 μ per slide (when using a 22 × 22 mm cover glass)]
1, 50% dextran sulfide 2 μl, 20 × SSC 1
μl, labeled probe DNA 0.5 μl (0.1 μg /
μl), blocking DNA (for wheat x corn, genomic DNA of wheat is sonicated)]
10 μl was used. The amount of the hybridization solution is appropriately changed so as to be in the above ratio depending on the thickness of the sample and the area for hybridization,
Prepared.

The prepared hybridization solution was incubated in boiling water for 10 minutes to prepare labeled probe D.
Denature each of NA and blocking DNA,
Immediately, the mixture was poured into ice and cooled for 10 minutes to give single-stranded DNA.

The slide glass prepared above is 70%
70 in 2 × SSC with deionized formaldehyde
Incubated at 2.5 ° C for 2.5 minutes. Then, the slide glass after the incubation was treated with 70% ethanol at -20 ° C, 90% ethanol at -20 ° C, and 100% at -20 ° C.
Each was immersed in ethanol for 5 minutes, and then the slide glass was air dried at room temperature.

Then, the hybridization solution was dropped on a slide glass, and the slide sealed with a cover glass was treated at 70 ° C. for 5 minutes. 37 in 2 × SSC wet chamber
Hybridization was performed overnight at ℃.

After the hybridization, the cover glass was removed from the slide glass, and the slide glass was replaced with 2.
X SSC, wash at room temperature in the dark for 10 minutes, then 2 x SSC containing 0.1% Triton X 100
At room temperature in the dark for 10 minutes, then 2 x SS
C Wash at room temperature in the dark for 10 minutes.

Regarding the slide glass, 1 ng /
Vector shield containing μl of propidium iodide (PI) [trade name: Vectashield, Vector (Vecta)
Manufactured by the company).

Images are from BioRad confocal laser microscope MRC1024 and LaserSharp Ve.
r. Obtained by 2.1A.

As shown in FIG. 1, it was found that in the hybrid embryo 48 hours after the crossing, the corn chromosome had already dropped out as a micronucleus, and the corn chromosome did not exist in the wheat nucleus.

In the hybrid embryos 24 hours after the crossing, it was possible to clearly observe the dynamics of the maize chromosome from the first metaphase to the late division after the crossing. The maize chromosome was observed to be delayed in alignment with the equatorial plate in the middle stage as compared with the wheat chromosome. further,
The distribution of maize chromosomes to the two poles was delayed at the late stage of division, and the maize chromosome distribution was incomplete.

It was speculated that the delay in the chromosome arrangement during this division process causes the loss of the maize chromosome.

Further, FIGS. 1 to 4 show the kinetics at each division stage in detail. This figure will be submitted separately as a reference photograph.

As shown in panel A of FIG. 1, it was found that the chromosomes on the wheat side (red fluorescence) were aligned with the equatorial plate in the first metaphase after the crossing. on the other hand,
The maize chromosome (yellow fluorescence) was found not to be perfectly aligned. In addition, 10 maize chromosomes (yellow fluorescence) were observed.

As shown in panel B of FIG. 1, FIG. 2 and FIG. 3, the maize chromosome was replicated relatively late in the meiotic phase of the first post-crossing, and 20 maize chromosomes were confirmed. However, it turns out that they do not exist in parallel. On the other hand, the wheat chromosome was pulled to both poles.

As shown in FIG. 4, maize DNA in the early embryos that became 8-cell stage 48 hours after crossing
Exists outside the wheat nucleus as a micronucleus, and seems to form and disappear.

As described above, by the chromosome analysis method of the present invention, it was possible to investigate the dynamics of chromosomes, which are the developmental stages of hybrid embryos in plants, which were difficult to observe in the past.

[0052]

EFFECTS OF THE INVENTION According to the chromosome analysis method of the present invention, there is an excellent effect that it is possible to examine the dynamics of chromosomes in plants which have been difficult to observe in the past.

[Brief description of drawings]

FIG. 1 is a diagram showing a result of detecting a corn chromosome in a wheat × corn hybrid embryo. In the figure, panel A shows the first metaphase hybrid embryo after the cross, and panel B shows the first metaphase hybrid embryo after the cross. In the figure, the corn chromosome is heavily stained.
The scale bar shows 10 μm.

FIG. 2 is a diagram showing the results of examining the dynamics of chromosomes in the first post-mitotic stage after crossing of wheat × corn hybrid embryos. In the figure, the corn chromosome is heavily stained. The scale bar shows 10 μm.

[Fig. 3] Fig. 3 is a diagram showing the results of examining the dynamics of chromosomes at the first meiotic stage (later meiotic stage after a lapse of time than in Fig. 2) after the first crossing in a wheat x corn hybrid embryo. The scale bar shows 10 μm.

[Fig. 4] Fig. 4 is a diagram showing the results of examining the dynamics of chromosomes in early embryos at the 8-cell stage, 48 hours after crossing, in wheat x corn hybrid embryos. The scale bar shows 10 μm.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Minoru Murata             2-20-1 Chuo, Kurashiki City, Okayama Prefecture Okayama University             Institute of Genome Science F-term (reference) 2B030 CA01 CA24                 4B063 QA08 QA11 QQ09 QQ41 QR31                       QR56 QR78 QS03 QS34 QX02

Claims (2)

[Claims] 1. A fixed sample, which comprises: (a) contacting a sample derived from a hybrid plant individual to be analyzed with a solution containing a compound capable of fixing a nucleic acid in the sample, thereby fixing the sample. And (b) in the fixed sample obtained in step (a), a nucleic acid derived from an analyte suspected of providing a genome to the hybrid plant individual is labeled with a labeling substance. Suitable for allowing the labeled probe to permeate, and specifically annealing the labeled probe and the nucleic acid present in the sample, which has a sequence complementary to the sequence of the probe. Under the conditions described above, the step of hybridizing the labeled probe with the fixed sample obtained in the step (a), and (c) the kinetics of the chromosome in the sample A method for chromosomal analysis, which comprises the step of observing via a signal derived from. 2. The method for chromosome analysis according to claim 1, wherein the sample derived from the individual plant of the hybrid to be analyzed is a hybrid embryo.