Method and application for counting chromosome centromeres based on Nuf2 antibody

Abstract

The present invention relates to the technical fields of bioinformatics and molecular cytogenetics, and particularly relates to a method and application for chromosome centromere counting based on Nuf2 antibody. The present invention comprises the following steps: performing chromosome centromere counting on the Nuf2 antibody and the centromere-specific protein Nuf2 through an immunofluorescence reaction, so as to directly observe the number of chromosome centromeres, and thus efficiently and rapidly determine the number of chromosomes. In addition, in view of the high conservation of the centromere-specific protein Nuf2 in eukaryotes, the present invention also provides the application of this method in identifying the ploidy of plants. The operation is simple, the cost is low, the efficiency of ploidy identification is significantly improved, and a general technical platform is provided for genetic breeding.

Description

Technical Field

[0001] The present invention relates to the technical fields of bioinformatics and molecular cytogenetics, and particularly relates to a method and application for chromosome centromere counting based on Nuf2 antibody. Background Art

[0002] Nuf2 (nuclear filament-containing protein 2) is a part of the Ndc-80 complex. It is located in the outer kinetochore of the centromere and acts as a link between spindle microtubules and the inner kinetochore, playing a key role in the recognition and maintenance of centromeric chromatin. Nuf2 is a highly conserved gene and is 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 germplasm resources such as diploid (2n = 24), triploid (2n = 36), tetraploid (2n = 48), pentaploid (2n = 60), hexaploid (2n = 72) and octaploid (2n = 84). Potato has both sexual and asexual reproduction abilities. During the ploidy operation of potato, offspring with more ploidy distributions will be produced. Therefore, quickly screening out the target ploidy and then carrying out genetic improvement can accelerate the breeding process.

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

[0005] The purpose of the present invention is to solve the deficiencies in the prior art. The method described in this patent designs an antibody by obtaining and using the clear sequence of potato centromere-specific protein Nuf2 (see SEQ ID NO.1), combines immunofluorescence technology to achieve the localization and counting of chromosome centromeres, and determines the plant ploidy based on the signal statistical results. Moreover, because Nuf2 is a highly conserved gene, the applicable scope of this method is very wide.

[0006] On the one hand, the present invention provides a method for chromosome centromere counting based on Nuf2 antibody, and the method includes the following steps: performing chromosome centromere counting on the Nuf2 antibody and the centromere-specific protein Nuf2 through immunofluorescence reaction.

[0007] On the one hand, to clarify the chromosome number, the present invention also provides the application of the method for counting chromosome centromeres based on Nuf2 antibody in chromosome counting.

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

[0009] In the above applications, the plant is Solanum tuberosum.

[0010] In the above applications, the following steps are included:

[0011] Step 1: Obtain the Solanum tuberosum centromere-specific protein Nuf2 sequence, and its protein sequence is as shown in SEQ ID NO.1;

[0012] SEQ ID NO.1:

[0013] MSRFDYPTLPRQDIIAVLAEAQIASVSDEDLIKPTPDFVTKLYSSILLHIDTLQDDHDQVDFSALEHLENPDLHVDSFRTINLFHKIRDMLAALDCPEIFTLRDLIKPDPDRTRFFVGAILNFCLHRDTKLNAIRPIVEHLTLIDEQRLALEARISQLNEEIAVHNESREREMPLVQEIDSNVKELRQTISGLNNHQMSLKASIRKLKERAKEIDEKISNADFALVQAVHDNANLRSKIVQSPDKLQRALEEKKSFQAEIRNAERAAMQSFQNKTAILEVYTKAYKKMSKNFNQMQAIQEQVNSTKSIEKDVKVLKLKLSDEEVQEKSLEAKLVERQGKADQLEELRKQLEKERNLSFEEAAKELKNVKLEVESKRHGLEARQNDLEGVLAEADAITEKINSVRESGASKCQELDRNCEEVIAEFYRHSNSIKDLLPDIEVDETSVEKRS*;

[0014] Step 2: Prepare a cell suspension after pretreating the Solanum tuberosum leaf sample, fix the cell suspension on a chromosome slide, and then perform a centromere-specific protein Nuf2 immunofluorescence reaction to obtain a fluorescent label;

[0015] Step 3: Locate the position of the chromosome centromere through the fluorescent label signal of the fluorescent label obtained in Step 2, count the number of signals, and obtain the number of chromosome centromeres, that is, obtain the Solanum tuberosum chromosome number;

[0016] Step 4: Determine the ploidy of the potato according to the number of potato chromosomes obtained in Step 3.

[0017] In the above application, the specific steps of the centromere-specific protein Nuf2 immunofluorescence reaction in Step 2 include:

[0018] Step 201: Wash the chromosome slide after fixing the cell suspension with PBS and then block it to obtain a first-treated sample.

[0019] Step 202: Wash the first-treated sample obtained in Step 201 with PBS, add a primary antibody solution, and incubate it to obtain a second-treated sample.

[0020] Step 203: Wash the second-treated sample obtained in Step 202 with PBS, add a secondary antibody solution, and incubate it to obtain a third-treated sample.

[0021] Step 204: Wash the third-treated sample obtained in Step 203 with PBS and alcohol, and dry it at room temperature to obtain a fluorescent label.

[0022] In the above application, the primary antibody solution in Step 202 is composed of BSA serum, 1×PBS buffer, and a primary antibody, and the volume ratio of the BSA serum, 1×PBS buffer, and the primary antibody is 100:100:1.

[0023] The secondary antibody solution in Step 203 is composed of BSA serum, 1×PBS buffer, and a secondary antibody, and the volume ratio of the BSA serum, 1×PBS buffer, and the secondary antibody is 100:100:1.

[0024] In the above application, the primary antibody in the primary antibody solution is a Nuf2 antibody, and the secondary antibody in the secondary antibody solution is GoatAnti Rabbit IgG(H&L)-Alexa FLUOR 488.

[0025] In the above application, the steps of locating the centromere position of the chromosome through the fluorescent labeling signal in Step 3 include: adding a staining solution to the fluorescent label, covering the slide, and observing the number of centromeres of the chromosome.

[0026] In the above application, the staining solution is a DAPI staining solution.

[0027] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0028] 1. The present invention can achieve efficient and accurate chromosomal centromere localization and counting. Based on the core function of the centromere-specific protein Nuf2 in the kinetochore structure, through its specific antibody, i.e., the Nuf2 antibody binds to the centromere-specific protein Nuf2, and the rapid localization and counting of chromosomal centromeres are realized by using immunofluorescence reaction. Through high-resolution imaging of fluorescence signals, quantitative statistics of multiple centromeres can be synchronously completed at the single-cell level, greatly improving the efficiency of ploidy identification.

[0029] 2. The present invention has universality and can expand the application scenarios of polyploid plants; due to the high conservation of Nuf2 protein in eukaryotes, the antibody and immunofluorescence system designed in the present invention can be adapted to the chromosomal ploidy identification of various plants. Taking potato as an example, its polyploid (2n = 24 to 2n = 84) has a large span of chromosome numbers, and it is difficult to quickly distinguish by traditional methods. This method can be directly extended to other polyploid crops through the compatibility of the Nuf2 antibody with the kinetochore structures of different species, providing a general technical platform for genetic breeding.

[0030] 3. The present invention can avoid the error interference of traditional methods; through the targeted recognition mechanism of the Nuf2 antibody, it precisely binds to the centromere-specific protein Nuf2, effectively distinguishing the counting interference caused by chromosome overlap or similar morphology. The traditional squash method depends 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, using 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.

[0031] 4. The present invention is simple to operate and has controllable costs, adapting to the needs of large-scale detection; 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 staining solution and general experimental equipment, significantly reducing the technical threshold and detection costs, and is especially suitable for large-scale germplasm resource screening and application in grass-roots laboratories. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 It is a diagram of the immunofluorescence staining and chromosomal centromere number statistics results of tetraploid potato Desiree and diploid potato CIP 703312; wherein, the vertical axis is chromosome counting.

[0034] Figure 2 It is a result diagram of flow cytometry ploidy identification for tetraploid potato Desiree and diploid potato CIP 703312; wherein, the abscissa is the fluorescence channel value, the ordinate is the relative value of the measured cell number, 2n indicates that there are 2 sets of chromosome numbers in the cell, and 4n indicates that there are 4 sets of chromosome numbers in the cell. Specific implementation manners

[0035] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. The % in the following embodiments is the mass percentage content unless otherwise specified. The ratios in the following embodiments are volume ratios unless otherwise specified.

[0036] Experimental instruments:

[0037] 1. Upright fluorescence microscope: Zeiss; Model: Axio Image M2;

[0038] 2. Cell analyzer: Luminex; Model: Guava Muse.

[0039] Experimental reagents:

[0040] 1. Bovine serum albumin: Purchased from solarbio company, Cat.No.A8020;

[0041] 2. Potato Nuf2 antibody (i.e., primary antibody): Prepared by entrusting Kangtuo Antibody Technology (Wuhan) Co., Ltd.;

[0042] 3. Goat Anti Rabbit IgG(H&L)-Alexa FLUOR 488 (i.e., secondary antibody): Purchased from ImmunoWay Biotechnology Company, Cat.No.RS3211;

[0043] 4. Anti-fluorescence quenching mounting medium (containing DAPI): Purchased from coolaber company, Cat.No.SL1841-5mL;

[0044] 5. PI dye: Purchased from Beyotime Biotechnology company, Cat.No.ST1569-10mg;

[0045] 6. DAPI staining solution: Purchased from solarbio company, Cat.No. C0065.

[0046] 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.

[0047] 8.LB01 buffer, the components of each 100mL LB01 buffer are shown in Table 1:

[0048] Table 1: LB01 buffer component details

[0049]

[0050] 9. Sucrose buffer, the components of each 200 mL sucrose buffer are shown in Table 2:

[0051] Table 2: Sucrose buffer component details

[0052]

[0053] Example 1

[0054] 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:

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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;

[0059] Step 5: Drop BSA serum (a mixture of 4% BSA and 0.1% Tritonex-100) on the glass slide to block non-specific antigens on the cell surface. Cover the surface with a sealing film and place it at 4°C for 30 min.

[0060] Step 6: Repeat Step 4.

[0061] Step 7: Drop the primary antibody solution (BSA serum: 1×PBS buffer: primary antibody = 100:100:1), and incubate overnight at 4°C.

[0062] Step 8: Repeat Step 4.

[0063] Step 9: Drop the secondary antibody solution (BSA serum: 1×PBS buffer: secondary antibody = 100:100:1), and incubate at 37°C for 1 h.

[0064] Step 10: Repeat Step 4.

[0065] Step 11: Wash with 70% alcohol, 90% alcohol, and 100% alcohol in sequence, 2 min each time. After washing, place the glass slide at room temperature to dry.

[0066] Step 12: Add 15 μL of DAPI staining solution to each glass slide dried at room temperature to counterstain the cell nuclei, and cover the slide to observe the signals.

[0067] Step 13: Place the glass slide under a fluorescence microscope for observation.

[0068] Step 14: Repeat the above steps three times. Count and statistically analyze the centromeres of potato chromosomes in the three experiments through fluorescence labeling, and determine the ploidy of the potato according to the number of centromeres.

[0069] The results are as Figure 1 shown. The average centromere signal count in the three repeated experiments of the potato leaf sample Ⅰ to be detected is 42.

[0070] Example 2

[0071] In this example, one potato leaf sample was blindly selected from the potato leaf samples and named the potato leaf sample Ⅱ to be detected. The ploidy of the potato leaf sample Ⅱ to be detected was determined by immunofluorescence reaction using the antibody of the potato centromere-specific protein Nuf2. The specific experimental steps are as follows:

[0072] Step 1: Cut 10 mg of the healthy potato leaf sample Ⅱ to be detected and place it in a pre-cooled 4% paraformaldehyde solution for fixation on ice for 20 min, then wash twice with pre-cooled Tris-buffer, 10 min each time.

[0073] 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;

[0074] 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;

[0075] 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;

[0076] 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;

[0077] Step 6. Repeat step 4;

[0078] Step 7: Add the primary antibody solution (BSA serum: 1× PBS buffer: primary antibody = 100:100:1) and incubate at 4°C overnight;

[0079] Step 8. Repeat step 4;

[0080] 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.

[0081] Step 10. Repeat step 4.

[0082] 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;

[0083] 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;

[0084] Step 13, placing the slide under a fluorescence microscope for observation;

[0085] 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.

[0086] The results are as follows Figure 1As shown, the average centromere signal count from three repeated experiments on the potato leaf sample II to be detected was 23. According to the chromosome composition of tetraploid potatoes, 2n = 2X = 48, and that of diploid potatoes, 2n = 2X = 24. Among them, the known number of centromeres in the tetraploid potato Desiree is 48, and the known number of centromeres in the 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 the potato leaf sample II to be detected is the leaf sample of the diploid potato CIP703312, and the potato leaf sample I to be detected is the leaf sample of the tetraploid potato Desiree.

[0087] Example 4

[0088] 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 detected in Example 1.

[0089] 1. Verify the ploidy of the potato leaf sample I to be detected:

[0090] Step 1: Cut about 20 mg of the potato leaf sample I to be detected, which has been washed and had its midrib removed, and place it in a plastic petri dish. Add freshly pre-cooled nuclear lysis buffer to submerge it, and use a double-sided blade to chop the sample. Then transfer it to a 300-mesh nylon sieve for filtration, and collect the filtrate in a 1.5 mL centrifuge tube.

[0091] Step 2: Add PI dye to the filtrate, mix well, and incubate at low temperature in the dark for 20 min.

[0092] Step 3: After the incubation, vortex the sample to mix well, and detect it with a cell analyzer. Select the detection category OpenModule Yellow and adjust the GATING PROFILE according to the target area.

[0093] 2. Verify the ploidy of the potato leaf sample II to be detected:

[0094] Step 1: Cut about 20 mg of the potato leaf sample II to be detected, which has been washed and had its midrib removed, and place it in a plastic petri dish. Add freshly pre-cooled nuclear lysis buffer to submerge it, and use a double-sided blade to chop the sample. Then transfer it to a 300-mesh nylon sieve for filtration, and collect the filtrate in a 1.5 mL centrifuge tube.

[0095] Step 2: Add PI dye to the filtrate, mix well, and incubate at low temperature in the dark for 20 min.

[0096] Step 3: After the incubation, vortex the sample to mix well, and detect it with a cell analyzer. Select the detection category OpenModule Yellow and adjust the GATING PROFILE according to the target area.

[0097] Save and export the above data, and use FlowJo software for processing and analysis. Determine the ploidy of potatoes based on the peak positions. The results are as follows Figure 2 shown. The main peak of the potato leaf sample II to be detected is 2n, indicating that it is diploid, and verifying that it is the leaf sample of potato CIP703312; the main peak of the ploidy of the potato leaf sample I to be detected is 4n, indicating that it is tetraploid, and verifying that it is potato Desiree.

[0098] As described above, the basic principles, main features and advantages of the present invention are preferably described. The above embodiments and the description are only descriptions of the preferred embodiments of the present invention. The present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the present invention.

Claims

1. A method for counting chromosomal centromeres based on Nuf2 antibody, characterized in that, The method includes the following steps: Step 1: Obtain the sequence of the potato centromere-specific protein Nuf2, and its protein sequence is shown in SEQ ID NO.1; Step 2: Prepare a cell suspension after pre-treating the potato leaf sample, fix the cell suspension on a chromosome slide, and then perform a centromere-specific protein Nuf2 immunofluorescence reaction to obtain a fluorescence labeling signal; Step 3: Locate the position of the chromosome centromere according to the fluorescence labeling signal obtained in Step 2, count the number of signals, and obtain the number of chromosome centromeres.

2. The method according to claim 1, characterized in that, The specific steps of the centromere-specific protein Nuf2 immunofluorescence reaction described in Step 2 include: Step 201: Wash the chromosome slide with the fixed cell suspension with PBS and then block it to obtain a first-treated sample; Step 202: Wash the first-treated sample obtained in Step 201 with PBS, add a primary antibody solution, and incubate to obtain a second-treated sample; Step 203: Wash the second-treated sample obtained in Step 202 with PBS, add a secondary antibody solution, and incubate to obtain a third-treated sample; Step 204: Wash the third-treated sample obtained in Step 203 with PBS and alcohol, dry it at room temperature to obtain a fluorescence labeling signal.

3. The method according to claim 2, characterized in that, The primary antibody solution described in Step 202 is composed of BSA serum, 1×PBS buffer, and a primary antibody, and the volume ratio of the BSA serum, 1×PBS buffer, and the primary antibody is 100:100:1; The secondary antibody solution described in Step 203 is composed of BSA serum, 1×PBS buffer, and a secondary antibody, and the volume ratio of the BSA serum, 1×PBS buffer, and the secondary antibody is 100:100:

1.

4. The method according to claim 2, wherein The primary antibody in the primary antibody solution is a Nuf2 antibody, and the secondary antibody in the secondary antibody solution is Goat Anti Rabbit IgG-Alexa FLUOR 488.

5. The method according to claim 1, wherein The step of locating the chromosome centromere position through the fluorescence labeling signal described in Step 3 includes: adding a staining solution to the fluorescence label, covering the slide and observing the number of chromosome centromeres.

6. The method according to claim 5, characterized in that, The staining solution is a DAPI staining solution.

7. Application of the method according to claim 1 in potato chromosome counting.

8. Application of the method according to claim 1 in identifying the ploidy of potatoes.

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