Fluorescent labeled probes for identifying centromeric region of chromosome of pennisetum sinense and application thereof

Abstract

The present application relates to the fluorescence marker for identifying the centromere region of the chromosome of P. wallichii and its application, belongs to the technical field of molecular cytogenetics, the fluorescence marker probe is named HS-CHTZ5, the probe belongs to the centromere region repeat sequence of P. wallichii, can be combined with the centromere of P. wallichii chromosome and show fluorescence signal, and the centromere segment of P. wallichii chromosome is identified.The fluorescence marker provided by the present application can be used as a beneficial supplement to the prior art, and through in situ hybridization, the efficient identification of the centromere region of the chromosome of P. wallichii is realized, and the present application has important significance for cytogenetic map construction, chromosome karyotype analysis and different genus and species chromosome relationship analysis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular cytogenetics, and particularly relates to a fluorescently labeled probe for identifying the centromere region of Aegilops sharonensis and application thereof. BACKGROUND

[0002] The centromere is a specialized structure on the chromosome, and is an important element of all eukaryotic chromosomes. Its main components are centromere proteins and centromere DNA. In various division processes, the centromere plays a crucial role in the equal division of cells and the accurate distribution of genetic material. In different plant chromosomes, the evolution speed of the centromere is also different. However, the organization mode and DNA sequence type are basically similar, and are all composed of retrotransposons and multiple tandem repeats interspersed therein. The genetic material in the centromere region generally has a low recombination exchange rate, and the DNA sequence is relatively complete, so the research on this region is conducive to the analysis of the phylogenetic relationship and evolution of species.

[0003] The fluorescence in situ hybridization (FISH) technology is a non-radioactive molecular hybridization technology that began to rise in the 1980s. Based on the principle of base complementary pairing, a specific sequence is labeled with fluorescence and hybridized with single-stranded DNA. After combination, the local double-stranded fluorescence is emitted. Through the excitation light of different wavelengths of the fluorescence microscope, the position and distribution density of the target sequence on the chromosome are studied. During cell division, the chromosomes will be in different morphological characteristics. The most commonly used stage of FISH technology is the mitotic metaphase chromosome. The chromosome in this morphology is short and thick, and is separated two by two, which is convenient for the observation and comparative analysis of the fluorescence signal. The FISH technology can also be used for the analysis of the centromere position of the mitotic metaphase chromosome, such as the development of specific markers for detecting the centromere region of wheat's close relative species barley, rye, and hordeum bogdanii.

[0004] Achnatherum sibiricum is a Chinese endemic perennial diploid grass from the Huashan region of Shaanxi Province, China. Its self-incompatibility results in a small population size and narrow distribution range, and it has been listed as the first batch of national class one rare and protected plants (priority protection species) and urgently needed protection of wild relatives of crops, with the grade of critically endangered, and is known as the "panda" of the Poaceae family. Achnatherum sibiricum is a close relative of wheat, and has many excellent traits such as cold and drought tolerance, salt and alkali tolerance, and resistance to rust, powdery mildew, take-all disease, scab, and yellow dwarf disease. Through distant hybridization between Achnatherum sibiricum and wheat, the excellent traits of Achnatherum sibiricum can be transferred to cultivated wheat. Therefore, Achnatherum sibiricum is also a rare germplasm resource for enriching the genetic diversity of wheat and achieving wheat disease and stress resistance improvement. Scholars have carried out research on the agronomic traits of the derived offspring of wheat-Achnatherum sibiricum. However, due to the relatively high difficulty of chromosome preparation of Achnatherum sibiricum, the centromere region of Achnatherum sibiricum has not been reported, and the centromere region of Achnatherum sibiricum is unknown. SUMMARY

[0005] In order to solve the problem of the unknown centromere region of Achnatherum sibiricum in the prior art, the present application provides a fluorescently labeled probe for identifying the centromere region of Achnatherum sibiricum chromosome, which is developed and designed based on the centromere repeat sequence of Achnatherum sibiricum. The fluorescently labeled probe can accurately locate the centromere region of Achnatherum sibiricum, and can lay a molecular foundation for karyotype analysis and related cytological experiments of Achnatherum sibiricum.

[0006] The first aspect of the present application is to provide a fluorescently labeled probe for identifying the centromere region of Achnatherum sibiricum chromosome. The 5' end of the fluorescently labeled probe carries a 6-FAM fluorescent group, and emits fluorescence under the excitation light of the GFP channel after specific binding to the centromere region of Achnatherum sibiricum chromosome. The nucleotide sequence of the fluorescently labeled probe is shown in SEQ ID NO: 01.

[0007] The second aspect of the present application is to provide a composition comprising the above fluorescently labeled probe. Preferably, the composition is a fluorescently labeled hybridization solution, which comprises 1xTE and the fluorescently labeled probe.

[0008] The third aspect of the present application is to provide the use of the above fluorescently labeled probe or the composition comprising the fluorescently labeled probe in the identification of the centromere region of Achnatherum sibiricum chromosome.

[0009] The fourth aspect of the present application is to provide a method for identifying the centromere region of Achnatherum sibiricum chromosome, comprising the following steps:

[0010] Step one, chromosome preparation of Achnatherum sibiricum root tip in metaphase;

[0011] Step two, preparation of fluorescently labeled hybridization solution: dissolve the fluorescently labeled probe dry powder in 1xTE to form fluorescently labeled hybridization solution;

[0012] Step three, drop the fluorescently labeled hybridization solution prepared in step two on the preparation in step one for in situ hybridization treatment;

[0013] Step four, fluorescence microscopy: under GFP excitation light, the bright green fluorescent signal area on each chromosome is the centromere region.

[0014] Preferably, the fluorescently labeled hybridization solution in step two contains 7 μL of 1xTE and 3 μL of fluorescently labeled probe with a concentration of 20 ng / μL in 10 μL.

[0015] Preferably, the in situ hybridization treatment in step three is as follows: drop the fluorescently labeled hybridization solution on the preparation, incubate at 42°C for 4 hours, then wash off the fluorescently labeled hybridization solution with pure water, air dry, then add 10 ul of anti-fluorescence quenching mounting agent H1300, cover with a cover glass, and place in the dark for natural staining for 20 minutes.

[0016] Compared with the prior art, the beneficial effects of the present application are as follows: the fluorescently labeled centromere repeat sequence of A. formosana provided by the present application can accurately identify the centromere region of the A. formosana chromosome set, which is helpful for in-depth cytological research on the national endangered species A. formosana, including karyotype analysis, genetic relationship, and evolutionary analysis, and promotes the study of chromosome centromere recombination of the A. formosana-wheat distant hybrid offspring. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the fluorescence in situ hybridization result of the fluorescently labeled centromere repeat sequence of A. formosana and the A. formosana chromosome set; in the figure, the arrow indicates the centromere region. DETAILED DESCRIPTION

[0018] The fluorescently labeled centromere repeat sequence of A. formosana is named HS-CHTZ5, and the marker sequence is shown in SEQ ID. 1, and a 6-FAM fluorescent group is added to the 5' end of the marker.

[0019] A method for applying the fluorescently labeled centromere repeat sequence of A. formosana, which comprises performing fluorescence in situ hybridization experiment of the marker and A. formosana chromosomes, and the specific content is as follows:

[0020] 1) A. formosana root tip metaphase chromosome preparation;

[0021] 2) Preparation of fluorescently labeled hybridization solution;

[0022] 3) In situ hybridization treatment;

[0023] 4) Fluorescence microscopy.

[0024] The components of the fluorescently labeled hybridization solution in step 2) are 10 ul, including 1xTE (7 ul) and 20 ng / uL of fluorescently labeled HS-CHTZ5 (3 ul).

[0025] The in situ hybridization process in step 3) is as follows: drop the hybridization solution on the slide, incubate at 42°C for 4 hours, then wash off the hybridization solution with pure water, and after air drying, add 10 ul of anti-fluorescence quenching mounting agent H1300 to the in situ position, cover the coverslip and place it in the dark for natural staining for 20 minutes.

[0026] The microscope used in step 4) has a DAPI and GFP excitation light module.

[0027] The centromere region of the A. sinensis chromosome emits a bright green fluorescent signal under GFP excitation light.

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application.

[0029] Example 1

[0030] This example provides a method for preparing mitotic slides of A. sinensis root tip cells, and the specific steps are as follows:

[0031] 1) Soak mature seeds of A. sinensis in water overnight, take out the seeds and place them on a wet filter paper to germinate, and after the main root emerges, place them in a 4°C cold treatment for 24 hours. Then take them out and wait for the tender root tips to grow to 1-2 cm in a 25°C incubator, cut off the root tips and place them in an ice water bath for 24 hours, and then take them out and store them in Carnoy's fixative I (anhydrous ethanol: glacial acetic acid = 3:1, v / v);

[0032] 2) Take the A. sinensis root tips and place them in a 0.5 mL centrifuge tube, rinse them with water, then add a mixture of 2% cellulase and 1% pectinase and incubate at 37°C for 50 minutes;

[0033] 3) Gently aspirate the enzyme solution, wash the root tips with 75% ethanol 3 times, then add glacial acetic acid at a ratio of 20 uL per root tip, and grind the root tips;

[0034] 4) Use a pipette to take 8 uL of cell suspension and drop it onto the center of the slide, place it in an incompletely sealed humidified black box, and after 10 minutes of standing, observe and select the good chromosome slides in the division phase.

[0035] Example 2

[0036] This example provides a method for preparing A. sinensis Ns chromosome marker hybridization solution, which requires light protection, and the specific steps are as follows:

[0037] 1) The dry powder state fluorescently labeled HS-CHTZ5 synthesized by Shengwo Bioengineering (Shanghai) Co., Ltd. was diluted to 20 ng / uL using 1xTE as a probe solution, and stored at -20℃ in the dark.

[0038] 2) 7 uL of 1xTE and 3 uL of the above-mentioned HS-CHTZ5 probe solution were sequentially added to a 0.5 mL centrifuge tube, and mixed uniformly as a hybridization solution.

[0039] Example 3

[0040] This example provides a fluorescence in situ hybridization process, which needs to be carried out in the dark, and the steps are as follows:

[0041] The hybridization solution was dropped onto the appropriate area of the chromosome preparation, a cover glass was placed on top, and incubation was carried out at 42℃ for 4 hours. At the time, the slide was removed, the cover glass was knocked off, and the hybridization solution was washed off by soaking in pure water. The slide was taken out and air-dried on the desktop. 10 ul of anti-fluorescence quenching mounting medium H1300 was added at the position where the original hybridization solution was added, a cover glass was placed on top, and natural staining was carried out in the dark for 20 minutes.

[0042] Example 4

[0043] This example provides a fluorescence microscope examination method, and the steps are as follows:

[0044] Under the DAPI light channel of the fluorescence microscope, the chromosomes were found, the wave wheel was switched to the GFP light channel, and the focus was adjusted to the clearest image. The observed field was photographed.

[0045] The results of the fluorescence in situ hybridization of the centromere repeat sequence marker HS-CHTZ5 and the A. sinuatus chromosomes are shown in Figure 1 The fluorescent signal region on each chromosome is the centromere region of the chromosome, indicating that the marker specifically pairs and binds with the centromere of the A. sinuatus chromosome.

[0046] The probe described in the present application belongs to the centromere region repeat sequence of A. sinuatus, and can specifically bind with the centromere of the A. sinuatus chromosome to show a fluorescent signal, and identify the centromere segment of the A. sinuatus chromosome. The fluorescent marker provided by the present application can be used as a beneficial supplement to the prior art, and through in situ hybridization, efficient identification of the centromere region of the A. sinuatus chromosome is achieved, which has important significance for cell genetics map construction, chromosome karyotype analysis, and analysis of the genetic relationship of chromosomes of different genera and species.

[0047] The above embodiments are only for helping the reader to understand the implementation method of the present application, and are not intended to limit the protection scope of the present application. The protection scope of the present application is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to the present application without departing from the spirit and scope of the present application shall still fall within the protection scope of the present application.

Claims

1. A fluorescently labeled probe for identifying the centromere region of chromosomes in *Leymus chinensis*, characterized in that: The fluorescently labeled probe carries a 6-FAM fluorescent group at its 5' end, which specifically binds to the centromere region of the Huashan New Wheatgrass chromosome and emits fluorescence under the excitation light of the GFP channel; the nucleotide sequence of the fluorescently labeled probe is shown in SEQ ID NO:

01.

2. A composition comprising the fluorescently labeled probe of claim 1.

3. The composition according to claim 2, characterized in that: The composition is a fluorescently labeled hybridization solution, which contains 1xTE and the fluorescently labeled probe.

4. The application of the fluorescently labeled probe of claim 1 or the composition of claim 2 in the identification of the centromere region of chromosomes in *Leymus chinensis*.

5. A method for identifying the centromere region of chromosomes in *Leymus chinensis* using the composition of claim 3, characterized in that: Includes the following steps: Step 1: Preparation of metaphase chromosomes from the root tips of *Huashan New Wheatgrass*; Step 2: Prepare fluorescently labeled hybridization solution: Dissolve the fluorescently labeled probe powder in 1xTE to form a fluorescently labeled hybridization solution; Step 3: Perform in situ hybridization on the slide prepared in Step 1 using the fluorescently labeled hybridization droplets prepared in Step 2. Step 4: Fluorescence microscopy examination: Under GFP excitation light, the bright green fluorescent signal area on each chromosome is the centromere region.

6. The method according to claim 5, characterized in that: The fluorescently labeled hybridization solution described in step two, in 10 μL, contains 7 μL of 1xTE and 3 μL of fluorescently labeled probe with a concentration of 20 ng / μL.

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