CN106636320B

CN106636320B - Method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage

Info

Publication number: CN106636320B
Application number: CN201610135140.9A
Authority: CN
Inventors: 轩淑欣; 申书兴; 冯大领; 王彦华; 陈雪平
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2016-03-10
Filing date: 2016-03-10
Publication date: 2020-09-18
Anticipated expiration: 2036-03-10
Also published as: CN106636320A

Abstract

The invention belongs to the technical field of crop molecular cytogenetics, and particularly relates to a method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage. The method comprises the steps of determination of flower buds in the mature pollen period of the Chinese cabbage, section making of mature pollen cells, probe marking, in-situ hybridization, signal detection and the like. The invention is characterized in that: taking flower buds of Chinese cabbage 1-2 days before flowering, separating fresh pollen grains in 50% glacial acetic acid solution, and breaking pollen walls by adopting a vertical tabletting method to release complete vegetative cells and germ cells; after being pretreated, the cell sample is subjected to covariation with a denaturation probe for 3min at 80 ℃, and in-situ hybridization is directly carried out, so that a fluorescent hybridization picture with clear signals is obtained, and the signal expression difference of target DNA in vegetative cells and germ cells is revealed. The invention fills the blank of the mature pollen cell fluorescence in-situ hybridization technology of brassica plants in brassicaceae, and is applied to the molecular cytogenetic research of cabbage pollen development.

Description

Method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage

Technical Field

The invention relates to a cabbage mature pollen cell fluorescence in situ hybridization method, belonging to the technical field of molecular cytogenetics.

Background

Pollen (polen) is a performer for sexual reproduction of flowering plants, is very important for controlling crop fertility and utilizing heterosis, and is an excellent material for researching a cell differentiation and development mechanism. The development of pollen comprises microsporogenesis (microsporogenesis) and male gametophyte genesis (male gametophenesis), wherein the microsporogenesis is that sporogenous cells in plant anthers are differentiated and developed into pollen mother cells, and then the pollen mother cells are subjected to meiosis to form haploid microspores; male gametophytosis is the differentiation of microspores through 1 asymmetric mitosis into 1 large loose vegetative cell and 1 small compact germ cell, with the vegetative cell then not dividing, and the germ cell undergoing 1 normal mitosis to form two sperm cells (male gametes), i.e., mature pollen grains (male gametophytes). During pollen development, meiosis recombines the parental 2 individual cell genomes to generate 1 new gamete genome and undergoes programmed and spontaneous changes during later mitosis, resulting in possible differences in vegetative and reproductive nuclear genetic material. In recent years, the genetic mechanism of the male gametophyte has become a research hotspot, and by utilizing different molecular genetic technologies and high-throughput omics technologies, important genes for regulating the formation of the male gametophyte and the growth of the pollen tube are discovered, so that a new opportunity is provided for the research of the functional genome characteristics of the vegetative cells and the germ cells. However, there has been little molecular cytogenetic study of differences in expression of specific sequences or genes in vegetative and germ cells that are completely different in function and fate.

Fluorescence in situ hybridization technique (fluoroscent)in situhybridization, FISH) is a non-radioactive molecular cytogenetic technology developed on the basis of radioactive in situ hybridization technology at the end of the 80 th 20 th century, is widely applied to the research of chromosomes or specific DNA sequences at each stage of interphase cycles of animals and plants, and relates to many aspects such as genetic mapping, gene positioning, chromosome aberration detection, chromosome rearrangement, genome structure, species evolution analysis and the like. In plants, pollen grains are the only accessible cell type and are haploid, at stage G1, without endogenous polyploidization. FISH hybridization on pollen cells helps us to directly observe whether some specific repeats or gene sequences or chromosomes pass through the pollen development stage and determine whether differences exist between vegetative cells and germ cells. At present, the method is a new research field, and no relevant research report exists. In addition, a very challenging task exists in developing fluorescence in situ hybridization of pollen cells, namely, the pollen grains are coated with a hard and completely dry pollen wall, and how to remove the obstacle of the pollen wall and make the pollen cells completely flat on a glass slide so that a probe can penetrate through cytoplasm to penetrate into nuclear chromosomes is critical to influencing the success of the fluorescence in situ hybridization of the pollen cells.

Disclosure of Invention

The invention provides a method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage, and aims to solve the problems that pollen grains of the Chinese cabbage contain hard pollen walls, the pollen cells are difficult to well spread on a glass slide, probes are difficult to permeate into pollen nuclei, the fluorescence in situ hybridization of the pollen cells is still blank and the like. The invention lays a foundation for the molecular cytogenetics research of the development of the Chinese cabbage pollen.

The basic principle of the invention is as follows: the pollen mother cells form mononuclear microspores after meiosis, and further form mature pollen (male gametophyte) containing 1 large and loose vegetative cell and 2 small and compact germ cells after twice mitosis. During this process, meiosis recombines the parental 2 individual cell genomes to produce 1 new haploid gamete genome and undergoes programmed and spontaneous changes during mitosis. By means of fluorescence in situ hybridization of pollen, it can be revealed whether the target repetitive sequence or specific gene has difference between vegetative cell and germ cell and can be transferred to descendant via germ cell.

The invention is characterized in that: taking flower buds of Chinese cabbage 1-2 days before flowering, separating fresh pollen grains in 50% glacial acetic acid solution, and breaking pollen walls by adopting a vertical tabletting method to release complete vegetative cells and germ cells; after being pretreated, the cell sample and the denaturation probe are subjected to covariation for 3min at 80 ℃, and in-situ hybridization is directly carried out, so that a fluorescent hybridization picture with clear signals is obtained, and the tissue difference of target DNA in vegetative cells and germ cells is revealed. The invention is completed by the following steps:

(1) determination of flower buds at mature pollen stage

Taking buds with different sizes, stripping 1 anther, placing the anther on a clean glass slide, dripping a drop of DAPI staining solution, slightly squeezing to release pollen grains by using forceps, removing anther walls, covering a cover glass, absorbing excess staining solution by using filter paper, slightly pressing with a thumb, and observing the development period of the pollen under a fluorescence microscope.

(2) Preparation of mature pollen cell specimen

Tabletting: taking flower buds 1-2 days before blooming, stripping 1 fresh anther, placing on a clean glass slide, dripping a drop of 50% glacial acetic acid solution, slightly squeezing with forceps to release pollen grains, removing anther wall, covering with a cover glass, and slightly pressing the cover glass vertically with a thumb to break the pollen wall and release vegetative cells and reproductive cells.

Removing the cover plate: and (5) placing the specimen slide in liquid nitrogen for 30s, taking out, and prying off the cover glass by using a blade.

Degradation of cytoplasm: and (3) dropwise adding a drop of 50-60% glacial acetic acid solution to the target area of the specimen slide, and placing on a constant temperature table at 45 ℃ for heat preservation for 3min to fully degrade cytoplasm.

Fixing: the specimen slides were tilted, and fresh Cannon fixative (alcohol: glacial acetic acid =3: 1) was added dropwise to wash out 50% glacial acetic acid and fix for 3min, and air dried naturally.

Microscopic examination: and (4) loading the slide into a phase contrast microscope for observation, and placing the slide with complete structure and clear germ cells and vegetative cells into a refrigerator at 4 ℃ for short-term storage for later use.

(3) Preparation and labeling of probes

The 45S rDNA and 5S rDNA probes for in situ hybridization were obtained by PCR amplification using genomic DNA as template and labeled with Cy3.5-dCTP and Cy 3-dUTP by nick translation, respectively.

(4) Fluorescence in situ hybridization

Pretreatment of a pollen cell specimen: making the specimen preserved in the step (1) into slices at 65 ℃ for 30 min; then respectively carrying out RNase A enzyme and pepsin incubation treatment and formaldehyde solution fixation, carrying out alcohol dehydration after 2 XSSC rinsing, and naturally drying.

Preparing and denaturing a probe hybridization solution: mixing the labeled fluorescent probe with the hybridization solution according to a proper proportion to prepare 20 mu L of hybridization solution, denaturing in a boiling water bath for 10min, and immediately placing on ice for more than 5min in a cold bath.

Co-denaturation and hybridization: mu.L of denatured probe hybridization solution was dropped onto the target area of the specimen slide, covered with a cover slip, and allowed to covariate on a 80 ℃ incubator for 3min, then placed in a moisture-retaining hybridization box, and incubated overnight in a 37 ℃ incubator.

And (3) eluting after hybridization: removing a cover glass of the hybridized specimen in a dark condition, immersing the specimen into a 2 XSSC solution for rinsing, and dehydrating in alcohol; naturally drying under dark condition.

Counterdyeing: dripping DAPI staining solution containing anti-quencher into each hybridization specimen slide, covering with cover glass, and staining for more than 5min at room temperature in dark place.

Microscopic examination: the in situ hybridized specimen slides were mounted on a Zeiss fluorescence microscope in Germany for hybridization signal observation, images were captured and photographed using a Cooled CCD device, and the images were synthesized using Adobe Photoshop CS software.

The invention has the following technical effects and advantages:

1) the mature pollen cells of the Chinese cabbage are used as target phases for fluorescence in situ hybridization, and the target probe obtains clear and different hybridization signals on the vegetative cells and the germ cells, thereby providing technical support for molecular cytogenetic research on development of the pollen of the Chinese cabbage.

2) The pollen wall of the fresh pollen grains is broken by using a mechanical method of vertically and lightly pressing a cover plate by a thumb by taking a 50% glacial acetic acid solution as a medium, the released pollen cells are complete, the vegetative cells and 2 germ cells are dispersed, the technology is simple, and the operability is strong.

3) In the operation of the existing fluorescence in situ hybridization technique, the denaturation of a target phase specimen is generally carried out by immersing a pretreated specimen slide into a 70% formamide solution (prepared by 2 XSSC) for denaturation at 70 ℃ for 2.5-3 min, then dehydrating by frozen alcohol, naturally drying, and then hybridizing with a denatured probe. In the invention, the pollen cell sample is directly added with the denaturation probe for covariation for 3min on a constant temperature table at 80 ℃ after being pretreated, and then hybridization is carried out, so that the test process is simplified, and the test process is accelerated.

4) Fresh flower buds 1-2 days before blooming are used as materials, so that sufficient supply and convenient material taking are realized; the material does not need fixing liquid for fixing, pollen grains are fresh, and pollen walls are easy to break.

Drawings

FIG. 1 shows DAPI-stained mature pollen grains of Chinese cabbage (containing 1 large vegetative cell and 2 small germ cells).

FIG. 2 is a trinuclear pollen cell released from fresh flower powder particles.

FIG. 3 shows FISH results of the ` B153 ` pollen cells of Chinese cabbage, with red being a 45S rDNA signal and yellow being a 5S rDNA signal.

FIG. 4 shows FISH results of pollen cells of cabbage oil "yellow sarson" with 45S rDNA signal in red and 5S rDNA signal in yellow.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 1 the invention is illustrated by fluorescence in situ hybridization of 45S rDNA and 5S rDNA probes to mature pollen cells of Chinese cabbage 'B153'.

(1) Determination of flower buds at mature pollen stage

In the 'B153' full-bloom stage of Chinese cabbage, 1-5 days of flower buds with different sizes capable of blooming are taken, 1 anther of each flower bud is peeled off and placed on a clean glass slide, a drop of DAPI staining solution is dripped, pollen grains are released by slight squeezing of forceps, pollen walls are removed, a cover plate is covered, slight pressing is carried out by a thumb, the development period of the pollen is detected under a fluorescence microscope, and the fact that the pollen in the flower buds 1-2 days before blooming is developed into mature trinuclear pollen grains is determined (figure 1).

(2) Preparation of mature pollen cell specimen

Tabletting: the large bud 1-2 days before blooming is taken, 1 fresh anther is peeled off and placed on a clean glass slide, a drop of 50% glacial acetic acid solution is dropped on the clean glass slide, the pollen grains are released by gently squeezing the large bud with tweezers, the anther wall is removed, a cover glass is covered, the cover glass is lightly pressed vertically with a thumb to break the pollen wall, and the vegetative cells and the germ cells are released (figure 2).

Degradation of cytoplasm: dropping a drop of 50% glacial acetic acid solution, and keeping the temperature on a constant temperature table at 45 ℃ for 3min to fully degrade cytoplasm.

Fixing: fresh Cannon fixative (alcohol: glacial acetic acid =3: 1) was added dropwise, 50% glacial acetic acid was rinsed off and fixed for 3min, and air dried naturally.

(3) Preparation and labeling of probes

Extracting genome DNA of the Chinese cabbage 'B153' by adopting a CTAB method, taking the genome DNA as a template, amplifying by a PCR method to obtain 45SrDNA and a 5S rDNA fragment, purifying an amplification product, and respectively carrying out fluorescein labeling of Cy3.5-dCTP and Cy 3-dUTP by adopting an incision translation method.

(4) Fluorescence in situ hybridization

Preparing and denaturing a probe hybridization solution: the hybridization liquid line of each glass slide is 20 mu L, and the hybridization liquid line comprises 2 XSSC, 10 percent dextran sulfate, 50 percent deionized formamide and a fluorescent probe with proper concentration, is uniformly mixed and denatured in a boiling water bath for 10min, and then is immediately placed on ice for cold bath for more than 5 min.

And (3) eluting after hybridization: removing cover glass from the hybridized specimen, immersing in 2 XSSC solution, rinsing for 5min for 3 times, and dehydrating in 70%, 95% and 100% alcohol for 3 min; and (5) naturally drying.

Counterdyeing: 14 mu L of DAPI staining solution containing an anti-quencher is dripped into each hybridization specimen slide, and the hybridization specimen slides are covered with cover slips and stained for more than 5min at room temperature in a dark place.

Microscopic examination: the specimen slides after in situ hybridization were mounted on a Zeiss fluorescence microscope in Germany for hybridization signal observation, images were captured and photographed with a Cooled CCD device, and finally images were synthesized with Adobe Photoshop CS software to obtain images as shown in FIG. 3.

Example 2 the invention is illustrated by fluorescent in situ hybridization of ribosomal 45S rDNA and 5S rDNA probes on mature pollen cells of chinese cabbage "yellow sarson" for oil.

(1) Preparation of mature pollen cell slide specimen

In the full bloom period of the Chinese cabbage for oil, 1-2 days before blooming is taken, 1 fresh anther is stripped and placed on a clean glass slide, and the subsequent flaking method has the same steps as the example 1.

(2) Preparation and labeling of probes

The CTAB method is adopted to extract the genomic DNA of the Chinese cabbage yellow sarson' for oil, the DNA is used as a template, 45S rDNA and 5S rDNA fragments are obtained by PCR amplification, and the composition of a PCR reaction system, the reaction conditions, the purification of an amplification product and the labeling of a probe are the same as those in the embodiment 1.

(3) Fluorescence in situ hybridization

Pretreatment of specimens was the same as in example 1.

Preparation and denaturation of the probe hybridization solution were the same as in example 1.

③ Co-denaturation and hybridization the same as in example 1.

After hybridization, the elution was performed in the same manner as in example 1.

DAPI counterdyeing is the same as example 1.

Sixthly, microscopic examination is carried out in the same way as in the example 1, and an image shown in the figure 4 is obtained.

Claims

1. A method for performing fluorescence in situ hybridization on mature pollen cells of Chinese cabbage is characterized by comprising the following steps:

the prepared mature pollen cells of the Chinese cabbage are taken as a target phase, and different FISH signal patterns can be observed in vegetative cells and germ cells through Fluorescence In Situ Hybridization (FISH);

fluorescence in situ hybridization was accomplished by the following steps:

1) making pollen cell specimen into sheets, carrying out pretreatment such as copying at 65 ℃, incubating RNaseA enzyme and pepsin, fixing formaldehyde solution and the like, rinsing, dehydrating and drying;

2) preparing probe hybridization solution, denaturing in boiling water bath for 10min, and immediately placing on ice for cold bath for more than 5 min;

3) dripping the denatured probe hybridization solution into a target area of a specimen slide, covering a cover glass, placing on a constant temperature table at 80 ℃ for covariation for 3min, then placing in a moisture-preserving hybridization box, and hybridizing and incubating overnight at 37 ℃;

4) and (3) making a hybridized specimen into a sheet, removing a cover glass, rinsing, dehydrating, drying, counterstaining and performing microscopic examination to obtain a FISH hybridization signal image.

2. The method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage according to claim 1, wherein the preparation method of the mature pollen cell specimen of Chinese cabbage comprises the following steps:

1) stripping 1 anther from the flower bud of the Chinese cabbage, placing the anther on a clean glass slide, dripping a drop of 50% glacial acetic acid solution, slightly squeezing the anther with forceps to release pollen grains, removing the anther wall, covering a cover glass, and slightly pressing the cover glass vertically with a thumb to break the pollen wall and release vegetative cells and germ cells;

2) placing the specimen slide in liquid nitrogen for 30s, taking out, and prying off the cover glass by using a blade;

3) dripping a drop of 50-60% glacial acetic acid solution into a target area of the specimen slide, and placing the specimen slide on a constant temperature table at 45 ℃ for heat preservation for 3min to fully degrade cytoplasm;

4) inclining the specimen slide, dripping fresh Carnoy fixing solution to wash away 50% glacial acetic acid, fixing for 3min, and naturally drying;

5) loading into a phase contrast microscope for observation, and placing the glass slide with complete structure and clear germ cells and vegetative cells in a refrigerator at 4 ℃ for short-term storage for later use;

the Carnoy stationary liquid is alcohol: glacial acetic acid is 3:1, the ratio is volume ratio;

the bud of Chinese cabbage is large bud 1-2d before blooming, and the pollen in anther has developed to mature stage by DAPI staining microscopy; the flower buds do not need to be fixed by a fixing solution during preparation of the specimen, the separated fresh pollen is easy to break the pollen wall through vertical tabletting, and the released pollen cells are complete.