Method for counting centromere of chromosome based on Nuf2 antibody and application

Abstract

The invention relates to the technical field of bioinformatics and molecular cytogenetics, in particular to a method for counting centromeres of chromosomes based on a Nuf2 antibody and application. The method comprises the following steps: performing chromosome centromere counting on a Nuf2 antibody and a centromere specific protein Nuf2 through an immunofluorescence reaction, so that the number of chromosome centromeres can be directly observed, and the number of chromosomes can be efficiently and quickly determined. Besides, due to the fact that the specific protein Nuf2 of the centromere is highly conservative in eukaryotes, the invention further provides application of the method to identification of the plant ploidy, the method is easy and convenient to operate and low in cost, the ploidy identification efficiency is remarkably improved, and a universal technical platform is provided for genetic breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of bioinformatics and molecular cytogenetics, and in particular to a method and application of counting chromosome centromeres based on Nuf2 antibodies. Background Art

[0002] Nuf2 (nuclear filament-containing protein 2) is part of the Ndc-80 complex, which is located at the outer kinetochore and acts as a link between the spindle microtubules and the inner kinetochore, playing a key role in the recognition and maintenance of centromeric chromatin. Nuf2 is a highly conserved gene expressed in almost all eukaryotic species from yeast to humans. Nuf2 plays various roles in cell cycle events.

[0003] Potato is a polyploid plant. The basic chromosome number of haploid potato is 12. In nature, there are a large number of diploid (2n=24), triploid (2n=36), tetraploid (2n=48), pentaploid (2n=60), hexaploid (2n=72) and octoploid (2n=84) germplasm resources. Potato has both sexual and asexual reproduction capabilities. During the potato ploidy manipulation process, more offspring with different ploidy distributions will be produced. Therefore, quickly screening out the target ploidy and then conducting genetic improvement can speed up the breeding process.

[0004] At present, there are many methods for identifying potato ploidy, such as direct identification by chromosome pressing counting method, indirect identification by guard cell chloroplast counting method, flow cytometry, etc. Among them, flow cytometry is widely used because the material is not restricted when detecting ploidy, but when there are many materials, this method is time-consuming and labor-intensive. Therefore, there is an urgent need for a method that uses centromere-specific protein Nuf2 antibody to perform immunofluorescence reaction to observe the number of centromeres of chromosomes and determine ploidy. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art. The method described in this patent obtains and uses the clear sequence of potato centromere-specific protein Nuf2 (see SEQ ID NO.1) to design antibodies, combines immunofluorescence technology to achieve the positioning and counting of chromosome centromeres, and determines the plant ploidy based on the signal statistical results. Because Nuf2 is a highly conserved gene, the scope of application of this method is also very wide.

[0006] In one aspect, the present invention provides a method for counting chromosome centromeres based on Nuf2 antibodies, the method comprising the following steps: counting chromosome centromeres by reacting the Nuf2 antibody with the centromere-specific protein Nuf2 through immunofluorescence.

[0007] On the one hand, in order to clarify the number of chromosomes, the present invention also provides the use of a method for counting chromosome centromeres based on Nuf2 antibodies in chromosome counting.

[0008] On the other hand, the present invention also provides the use of a method for counting chromosome centromeres based on Nuf2 antibodies in identifying plant ploidy.

[0009] In the above application, the plant is potato.

[0010] The above application includes the following steps: Step 1, obtaining the potato centromere-specific protein Nuf2 sequence, the protein sequence of which is shown in SEQ ID NO.1; SEQ ID NO.1: MSRFDYPTLPRQDIIAVLAEAQIASVSDEDLIKPTPDFVTKLYSSILLHIDTLQDDHDQVDFSALEHLENPDLHVDSFRTINLFHKIRDMLAALDCPEIFTLRDLIKPDPDRT RFFVGAILNFCLHRDTKLNAIRPIVEHLTLIDEQRLALEARISQLNEEIAVHNESREREMPLVQEIDSNVKELRQTISGLNNHQMSLKASIRKLKERAKEIDEKISNADFALV QAVHDNANLRSKIVQSPDKLQRALEEKKSFQAEIRNAERAAMQSFQNKTAILEVYTKAYKKMSKNFNQMQAIQEQVNSTKSIEKDVKVLKLKLSDEEVQEKSLEAKLVERQGK ADQLEELRKQLEKERNLSFEEAAKELKNVKLEVESKRHGLEARQNDLEGVLAEADAITEKINSVRESGASKCQELDRNCEEVIAEFYRHSNSIKDLLPDIEVDETSVEKRS*; Step 2, pre-treating the potato leaf sample to prepare a cell suspension, fixing the cell suspension on a chromosome slide, and performing a centromere-specific protein Nuf2 immunofluorescence reaction to obtain a fluorescent marker; Step 3, using the fluorescent marker obtained in step 2 to locate the chromosome centromere position through the fluorescent marker signal, counting the number of signals, and obtaining the number of chromosome centromeres, that is, obtaining the number of potato chromosomes; Step 4: According to the potato chromosome number obtained in step 3, determine the potato ploidy.

[0011] In the above application, the specific steps of the immunofluorescence reaction of the centromere-specific protein Nuf2 in step 2 include: Step 201, washing the chromosome slide after fixing the cell suspension with PBS and sealing it to obtain a primary treatment sample; Step 202, washing the primary treated sample obtained in step 201 with PBS, adding a primary antibody solution, and incubating to obtain a secondary treated sample; Step 203, washing the secondary treated sample obtained in step 202 with PBS, adding a secondary antibody solution, and incubating to obtain a tertiary treated sample; Step 204: wash the three-treatment samples obtained in step 203 with PBS and alcohol, and dry them at room temperature to obtain fluorescent markers.

[0012] In the above application, the primary antibody solution in step 202 is composed of BSA serum, 1×PBS buffer and primary antibody, and the volume ratio of BSA serum, 1×PBS buffer and primary antibody is 100:100:1; The secondary antibody solution in step 203 is composed of BSA serum, 1×PBS buffer and secondary antibody, and the volume ratio of BSA serum, 1×PBS buffer and secondary antibody is 100:100:1.

[0013] In the above application, the primary antibody in the primary antibody solution is Nuf2 antibody, and the secondary antibody in the secondary antibody solution is GoatAnti Rabbit IgG(H&L)-Alexa FLUOR 488.

[0014] In the above application, the step of locating the position of the chromosome centromere by the fluorescent marker signal in step 3 includes: adding a dye solution to the fluorescent marker, sealing the slide and observing the number of chromosome centromeres.

[0015] In the above application, the dye solution is DAPI dye solution.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: 1. The present invention can achieve efficient and accurate chromosome centromere positioning and counting. The present invention is based on the core function of the centromere-specific protein Nuf2 in the kinetochore structure. Through its specific antibody, namely, Nuf2 antibody, it binds to the centromere-specific protein Nuf2 and utilizes immunofluorescence reaction to achieve rapid positioning and counting of chromosome centromeres. Through high-resolution imaging of fluorescent signals, quantitative statistics of multiple centromeres can be synchronously completed at the single-cell level, greatly improving the efficiency of ploidy identification.

[0017] 2. The present invention has universal applicability and can expand the application scenarios of polyploid plants; since Nuf2 protein is highly conserved in eukaryotes, the antibodies and immunofluorescence system designed by the present invention can be adapted to the chromosome ploidy identification of various plants. Taking potato as an example, the chromosome number span of its polyploids (2n=24 to 2n=84) is large, and traditional methods are difficult to quickly distinguish. This method can be directly extended to other polyploid crops through the compatibility of Nuf2 antibodies with the kinetochore structures of different species, providing a general technical platform for genetic breeding.

[0018] 3. The present invention can avoid the error interference of traditional methods; the present invention uses the targeted recognition mechanism of Nuf2 antibody to accurately bind to the centromere-specific protein Nuf2, effectively distinguishing the counting interference caused by chromosome overlap or morphological similarity. The traditional pressing method relies on the degree of chromosome dispersion and manual interpretation, and is easily affected by the quality of sample preparation; while this method is based on the biological function of the centromere-specific protein Nuf2, and utilizes its highly conserved antigenic epitope to ensure the specificity of antibody labeling, thereby reducing the risk of cross-reaction and significantly improving the accuracy of counting results.

[0019] 4. The present invention is simple to operate and cost-controlled, and is suitable for large-scale detection needs; the present invention only requires a microscope to complete signal analysis. Compared with flow cytometry, which relies on expensive equipment and complex sample pretreatment, this method uses low-cost reagents, such as DAPI dye and general experimental equipment, which significantly reduces the technical threshold and detection costs, and is particularly suitable for large-scale germplasm resource screening and grassroots laboratory applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The graph shows the immunofluorescence staining and chromosome centromere number statistics of tetraploid potato Desiree and diploid potato CIP 703312; the vertical axis is the chromosome count.

[0022] Figure 2 The figure shows the result of flow cytometry ploidy identification of tetraploid potato Desiree and diploid potato CIP 703312; wherein the horizontal axis is the fluorescence channel value, the vertical axis is the relative value of the measured cell number, 2n refers to the number of 2 sets of chromosomes in the cell, and 4n refers to the number of 4 sets of chromosomes in the cell. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials can be purchased on the market unless otherwise specified. The % in the following embodiments, unless otherwise specified, are all mass percentages. The proportions in the following embodiments, unless otherwise specified, are all volume ratios.

[0024] Experimental instruments: 1. Upright fluorescence microscope: Zeiss; model: Axio Image M2; 2. Cell analyzer: Luminex; model: Guava Muse.

[0025] Experimental reagents: 1. Bovine serum albumin: purchased from solarbio, Cat. No. A8020; 2. Potato Nuf2 antibody (i.e. primary antibody): commissioned by Kangtuo Antibody Technology (Wuhan) Co., Ltd. to prepare; 3. Goat Anti Rabbit IgG (H&L)-Alexa FLUOR 488 (secondary antibody): purchased from ImmunoWay Biotechnology Company, Cat. No. RS3211; 4. Anti-fluorescence attenuation mounting medium (containing DAPI): purchased from Coolaber, Cat. No. SL1841-5mL; 5. PI dye: purchased from Beyotime Biotechnology, Cat. No. ST1569-10mg; 6. DAPI staining solution: purchased from solarbio, Cat. No. C0065.

[0026] 7. Potato leaf samples: The leaf samples used were from tetraploid potato Desiree and diploid potato CIP703312. Three-week-old potato tissue culture seedlings were cultured in a plant growth room at 22±2°C, 16h light / 8h dark for 30 days. Tetraploid potato Desiree and diploid potato CIP 703312 were from Northwest Agriculture and Forestry University. 8.LB01 buffer, the components of each 100mL LB01 buffer are shown in Table 1: Table 1: LB01 buffer component details

[0027] 9. Sucrose buffer, the components of each 200 mL sucrose buffer are shown in Table 2: Table 2: Sucrose buffer component details

[0028] Example 1 In this example, a potato leaf sample is blindly selected from potato leaf samples, named as potato leaf sample I to be tested, and the potato ploidy of the potato leaf sample I to be tested is determined by immunofluorescence reaction using an antibody against potato centromere-specific protein Nuf2. The specific experimental steps are as follows: Step 1, cut 10 mg of healthy potato leaf sample I to be tested and place it in pre-cooled 4% paraformaldehyde solution, fix it on ice for 20 minutes, and then wash it twice with pre-cooled Tris-buffer, each time for 10 minutes; Step 2: Place the cleaned leaves in a petri dish, add pre-cooled LB01 buffer to immerse them, and chop them with a blade. Filter out the residue with a 300-mesh filter membrane, and transfer the filtrate to a new 2 mL centrifuge tube; Step 3: Take an appropriate amount of the filtrate and mix it with sucrose buffer at a ratio of 1:1, then drop it onto an adhesion-grade glass slide and spread it evenly with a pipette tip, and dry it at room temperature to obtain a dry glass slide; Step 4, place the dried slides described in step 3 in 1× PBS buffer and wash twice on a decolorizing shaker, each time for 5 min; Step 5: Add BSA serum (4% BSA and 0.1% Tritonex-100 mixture) on the slide to block non-specific antigens on the cell surface, cover the surface with sealing film and place at 4°C for 30 minutes; Step 6. Repeat step 4; Step 7: Add the primary antibody solution (BSA serum: 1× PBS buffer: primary antibody = 100:100:1) and incubate at 4°C overnight; Step 8. Repeat step 4; Step 9: Add the secondary antibody solution (BSA serum: 1× PBS buffer: secondary antibody = 100:100:1) and incubate at 37°C for 1 h. Step 10. Repeat step 4. Step 11: Wash with 70% alcohol, 90% alcohol, and 100% alcohol in sequence, 2 minutes each time, and place the slide at room temperature to dry after washing; Step 12: Add 15 μL of DAPI staining solution to each slide after drying at room temperature to counterstain the cell nucleus, seal the slide and observe the signal; Step 13, placing the slide under a fluorescence microscope for observation; Step 14: Repeat the above steps three times, count and statistically analyze the centromeres of potato chromosomes in the three experiments by fluorescent labeling, and determine the ploidy of potato according to the number of centromeres.

[0029] The results are as follows Figure 1 As shown, the average centromere signal count of three replicates of potato leaf sample I to be tested was 42.

[0030] Example 2 In this example, a potato leaf sample is blindly selected from the potato leaf samples, named as the potato leaf sample II to be tested, and the potato ploidy of the potato leaf sample II to be tested is determined by immunofluorescence reaction using an antibody against potato centromere-specific protein Nuf2. The specific experimental steps are as follows: Step 1, cut 10 mg of healthy potato leaf sample II to be tested, place it in pre-cooled 4% paraformaldehyde solution and fix it on ice for 20 minutes, then wash it twice with pre-cooled Tris-buffer, each time for 10 minutes; Step 2: Place the cleaned leaves in a petri dish, add pre-cooled LB01 buffer to immerse them, and chop them with a blade. Filter out the residue with a 300-mesh filter membrane, and transfer the filtrate to a new 2 mL centrifuge tube; Step 3: Take an appropriate amount of the filtrate and mix it with sucrose buffer at a ratio of 1:1, then drop it onto an adhesion-grade glass slide and spread it evenly with a pipette tip, and dry it at room temperature to obtain a dry glass slide; Step 4, place the dried slides described in step 3 in 1× PBS buffer and wash twice on a decolorizing shaker, each time for 5 min; Step 5: Add BSA serum (4% BSA and 0.1% Tritonex-100 mixture) on the slide to block non-specific antigens on the cell surface, cover the surface with sealing film and place at 4°C for 60 minutes; Step 6. Repeat step 4; Step 7: Add the primary antibody solution (BSA serum: 1× PBS buffer: primary antibody = 100:100:1) and incubate at 4°C overnight; Step 8. Repeat step 4; Step 9: Add the secondary antibody solution (BSA serum: 1× PBS buffer: secondary antibody = 100:100:1) and incubate at 37°C for 1 h. Step 10. Repeat step 4. Step 11: Wash with 70% alcohol, 90% alcohol, and 100% alcohol in sequence, 2 minutes each time, and place the slide at room temperature to dry after washing; Step 12: Add 15 μL of DAPI staining solution to each slide after drying at room temperature to counterstain the cell nucleus, seal the slide and observe the signal; Step 13, placing the slide under a fluorescence microscope for observation; Step 14: Repeat the above steps three times, count and statistically analyze the centromeres of potato chromosomes in the three experiments by fluorescent labeling, and determine the ploidy of potato according to the number of centromeres.

[0031] The results are as follows Figure 1 As shown, the average centromere signal count of three repeated experiments using potato leaf sample II to be tested is 23. According to the chromosome composition of tetraploid potato 2n=2X=48 and the chromosome composition of diploid potato 2n=2X=24, the known number of centromeres of tetraploid potato Desiree is 48 and the known number of centromeres of diploid potato CIP703312 is 24. The number of centromeres observed in the experiment is basically consistent with the number of centromeres corresponding to the known ploidy, confirming that potato leaf sample II to be tested is the leaf sample of diploid potato CIP703312, and potato leaf sample I to be tested is the leaf sample of tetraploid potato Desiree.

[0032] Example 4 In this example, flow cytometry was used to verify the ploidy of the potato leaf sample I and the potato leaf sample II to be tested in Example 1.

[0033] 1. Verify the ploidy of the potato leaf sample to be tested: Step 1. Cut about 20 mg of potato leaf sample I to be tested, wash and remove the midrib, place it in a plastic culture dish, add fresh pre-cooled cell nuclear lysis solution to immerse it, and chop the sample with a double-sided blade, transfer it to a 300-mesh nylon mesh for filtration, and collect the filtrate in a 1.5 mL centrifuge tube; Step 2, add PI dye to the filtrate, mix thoroughly and incubate at low temperature and away from light for 20 minutes; Step 3. After incubation, vortex the sample to mix well and detect it using a cytometer. Select OpenModule Yellow as the detection category and adjust the GATING PROFILE according to the target area.

[0034] 2. Verify the Ⅱ ploidy of the potato leaf sample to be tested: Step 1. Cut about 20 mg of potato leaf sample II to be tested, wash and remove the midrib, place it in a plastic culture dish, add fresh pre-cooled cell nuclear lysis solution to immerse it, and chop the sample with a double-sided blade, transfer it to a 300-mesh nylon mesh for filtration, and collect the filtrate in a 1.5 mL centrifuge tube; Step 2, add PI dye to the filtrate, mix thoroughly and incubate at low temperature and away from light for 20 minutes; Step 3. After incubation, vortex the sample to mix well and detect it using a cytometer. Select OpenModule Yellow as the detection category and adjust the GATING PROFILE according to the target area.

[0035] Save and export the above data, use FlowJo software for processing and analysis, and determine the potato ploidy based on the peak position. Figure 2 As shown, the main peak of the potato leaf sample II to be tested is 2n, indicating that it is diploid, verifying that it is the leaf sample of potato CIP703312; the main peak of the ploidy of the potato leaf sample I to be tested is 4n, indicating that it is tetraploid, verifying that it is potato Desiree.

[0036] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of preferred implementations of the present invention, and the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A method for counting chromosome centromeres based on Nuf2 antibody, characterized in that: The method comprises the following steps: the Nuf2 antibody and the centromere-specific protein Nuf2 are used to count chromosome centromeres by immunofluorescence technology.

2. Use of the method according to claim 1 in chromosome counting.

3. Application of the method according to claim 1 in identifying plant ploidy.

4. The use according to claim 3, characterized in that: The plant is potato.

5. The use according to claim 4, characterized in that: The following steps are involved: Step 1, obtaining the potato centromere-specific protein Nuf2 sequence, the protein sequence of which is shown in SEQ ID NO.1; Step 2, preparing a cell suspension after pretreatment of the potato leaf sample, fixing the cell suspension on a chromosome slide, and performing a centromere-specific protein Nuf2 immunofluorescence reaction to obtain a fluorescent labeling signal; Step 3, locating the position of the chromosome centromere according to the fluorescent labeling signal obtained in step 2, counting the number of signals, and obtaining the number of chromosome centromeres, that is, obtaining the number of potato chromosomes; Step 4: Determine the potato ploidy according to the potato chromosome number obtained in step 3.

6. The use according to claim 5, characterized in that: The specific steps of the immunofluorescence reaction of the centromere-specific protein Nuf2 in step 2 include: Step 201, washing the chromosome slide after fixing the cell suspension with PBS and sealing it to obtain a primary treatment sample; Step 202, washing the primary treated sample obtained in step 201 with PBS, adding a primary antibody solution, and incubating to obtain a secondary treated sample; Step 203, washing the secondary treated sample obtained in step 202 with PBS, adding a secondary antibody solution, and incubating to obtain a tertiary treated sample; Step 204: wash the three-treatment samples obtained in step 203 with PBS and alcohol, dry at room temperature, and obtain fluorescent labeling signals.

7. The use according to claim 6, characterized in that: The primary antibody solution in step 202 is composed of BSA serum, 1×PBS buffer and primary antibody, and the volume ratio of BSA serum, 1×PBS buffer and primary antibody is 100:100:1; The secondary antibody solution in step 203 is composed of BSA serum, 1×PBS buffer and secondary antibody, and the volume ratio of BSA serum, 1×PBS buffer and secondary antibody is 100:100:

1.

8. The use according to claim 7, characterized in that: The primary antibody in the primary antibody solution is Nuf2 antibody, and the secondary antibody in the secondary antibody solution is Goat Anti Rabbit IgG (H&L)-Alexa FLUOR 488.

9. The use according to claim 5, characterized in that: The step of locating the position of the chromosome centromere by the fluorescent marker signal in step 3 includes: adding a dye solution to the fluorescent marker, sealing the slide and observing the number of chromosome centromeres.

10. The use according to claim 9, characterized in that: The staining solution is DAPI staining solution.

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