Antigen polypeptide of mandulapalka functional centromere histone protein CENH3 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to antigen polypeptide of mandulapalka functional centromere histone CENH3 and application of the antigen polypeptide. The amino acid sequence of the antigen polypeptide is shown as SEQ ID No: 3. The polypeptide is used for conventionally immunizing New Zealand white rabbits to prepare antiserum and an anti-CENH3 protein antibody subjected to antigen affinity purification. The verification proves that the antibody is effective and reliable, and the functional centromere of the cyperus esculentus can be accurately identified. The method has a wide application prospect in the research fields of molecular cytogenetics, genomics, epimics and the like of cyperus esculentus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an antigen polypeptide of Cyperus esculentus functional centromere histone CENH3 and application thereof. BACKGROUND

[0002] Centromere is a chromosomal region essential for proper segregation and transmission of chromosomes during mitosis and meiosis in eukaryotes. Although the function of centromere is very conservative, the centromere DNA sequences of various species of eukaryotes are highly variable. The presence of a large number of repetitive sequences in centromere DNA makes sequence analysis and genome assembly difficult. With the development of chromatin immunoprecipitation, sequencing technology combined with molecular biology, cytogenetics, bioinformatics analysis methods, we can further analyze and locate the centromere sequence, and provide certain basis for the study of its function and evolution. At present, the centromere of Arabidopsis thaliana, rice, corn and other plants has been studied in depth, and it is shown that the function of centromere is very conservative, but the centromere DNA sequences of various species of eukaryotes are highly variable. The Cyperaceae plant has the characteristics of full centromere chromosome, and the research is less. Cyperus esculentus L. as an important economic crop of Cyperaceae, the genome sequencing has been completed, which is an ideal plant for studying full centromere chromosome. SUMMARY

[0003] In order to study the genome assembly of centromere region of new oil crop Cyperus esculentus and the formation and evolution mechanism of centromere, and further provide a basis for the research field of molecular cytogenetics and epigenetics of Cyperus esculentus, the present application first provides an antigen polypeptide of Cyperus esculentus centromere histone CENH3.

[0004] The amino acid sequence of the antigen polypeptide of Cyperus esculentus centromere histone CENH3 provided by the present application is shown in SEQ ID No: 3.

[0005] The present application also provides an anti-CENH3 protein antibody, which is a polyclonal antibody prepared by taking the polypeptide of claim 1 as an immunogen.

[0006] In the anti-CENH3 protein antibody, the polyclonal antibody is a rabbit-derived antibody.

[0007] The present application also provides a method for preparing an anti-CENH3 protein antibody, which comprises preparing a polyclonal antibody by taking the polypeptide as an immunogen, and the polyclonal antibody is an anti-CENH3 protein antibody.

[0008] In the above method, the polyclonal antibody is a rabbit-derived antibody.

[0009] The application also provides application of the polypeptide in preparation of a reagent or kit for specifically recognizing the CENH3 of Cyperus esculentus.

[0010] In the application, the reagent or kit for specifically recognizing the CENH3 of Cyperus esculentus contains the anti-CENH3 protein antibody.

[0011] The application also provides application of the anti-CENH3 protein antibody in preparation of a reagent or kit for specifically recognizing the CENH3 of Cyperus esculentus.

[0012] In the application, the reagent or kit for specifically recognizing the CENH3 of Cyperus esculentus contains the anti-CENH3 protein antibody.

[0013] The application also provides a reagent or kit for specifically recognizing the CENH3 of Cyperus esculentus, which contains the anti-CENH3 protein antibody.

[0014] The anti-CENH3 protein antibody provided by the application can specifically recognize the functional CENH3 of Cyperus esculentus.

[0015] The application uses bioinformatics alignment to analyze the CENH3 protein amino acid sequence from the Cyperus esculentus genome, selects a conserved N-terminal amino acid sequence and artificially synthesizes a polypeptide, uses the polypeptide to immunize New Zealand white rabbits, prepares an antiserum, and performs antigen affinity purification on the CENH3 protein antibody. It has been verified that the antibody is effective and reliable, and can accurately recognize the functional CENH3 of Cyperus esculentus. The application has broad application prospects in the research fields of molecular cytogenetics, genomics and epigenomics of Cyperus esculentus. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Bioinformatics analysis results of the amino acid sequences of CENH3 proteins of different species in Example 1 of the application. In the figure, AtCenh3 is the CENH3 protein sequence of Arabidopsis thaliana, OsCenh3 is the CENH3 protein sequence of Oryza sativa, KlCenh3 is the CENH3 protein sequence of Kobresia pygmaea, and CeCenh3 is the CENH3 protein sequence of Cyperus esculentus.

[0017] Figure 2 ELISA experiment results in Example 3 of the application. RB3215 and RB3216 are antisera of two rabbits, and RT3217 and RT3218 are antisera of two rats.

[0018] Figure 3 Western Blotting results of the antibody RT3216 (rabbit anti) in Example 4 of the application. In the figure, the bands from left to right are protein Maker, leaf, root, and flower.

[0019] Figure 4The ratio of IP (output value) to input (control, input value) in the Chip-squence of chromosome 1 of Cyperus oleifera in Example 5 of the present invention was plotted using igv software. The position where the peak appears indicates that the position is significantly enriched for CenH3 binding DNA sites. Cyperus oleifera has multiple centromere binding sites.

[0020] Figure 5 The results of sequence analysis of the binding DNA sites significantly enriched with CenH3 in Example 5 of the present invention show that the sequences are consistent with the centromere characteristic sequences, and a logo diagram is drawn for the sequence with the highest occurrence frequency. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0022] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0023] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0024] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0025] The Cyperus oleifera Yuyousha No. 2 in the following examples is described in the non-patent document “Li Chunxin, et al.: Analysis of Germplasm Characteristics of Three Kinds of Grain Types of Cyperus oleifera”, which is available to the public from the applicant to repeat the experiments of the present invention.

[0026] Reagent formula:

[0027] Cross-linking buffer: 0.4 M sucrose, 10 mM Tris-HCl (pH 8), 1 mM PMSF, 1 mM EDTA, 1% formaldehyde.

[0028] Nuclear isolation buffer: 0.25 M sucrose, 15 mM PIPES (pH 6.8), 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Tween X-100, 2 mg / ml aprotinin, 2 mg / ml aprotinin.

[0029] Nuclear buffer: 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1% SDS, 0.1% sodium deoxycholate, 1% Tween X-100, 1 mg / ml aprotinin, 1 mg / ml aprotinin.

[0030] Pre-equilibrate Protein A agarose beads: Centrifuge 50 μL of salmon sperm DNA / Protein A agarose beads at 3800 g for 2 minutes at 4°C to pellet the beads. Discard the supernatant and add 50 μL of nuclear lysis buffer. Mix by gentle rotation at 4°C for 2 minutes. Repeat the centrifugation at 3800 g for 2 minutes at 4°C. Resuspend the Protein A agarose beads in 50 μL of lysis buffer.

[0031] Elution buffer: 0.5% SDS and 0.1 M NaHCO3

[0032] Low Salt Wash Buffer: 150 mM NaCl, 20 mM Tris–HCl (pH 8), 0.2% SDS, 0.5% Tween X-100, and 2 mM EDTA. Critical Note: Prepare immediately before use and store at 4°C.

[0033] High Salt Wash Buffer: 500 mM NaCl, 20 mM Tris–HCl (pH 8), 0.2% SDS, 0.5% Tween X-100, and 2 mM EDTA. Critical Note: Prepare immediately before use and store at 4°C.

[0034] LiCl Wash Buffer: 0.25 M lithium bromide, 1% sodium deoxycholate, 10 mM Tris–HCl (pH 8), 1% NP-40, and 1 mM EDTA. Critical Note: Prepare immediately before use and store at 4°C.

[0035] TE buffer: 1 mM EDTA and 10 mM Tris–HCl (pH = 8.0).

[0036] Reagents: Sucrose (Sigma, cat. # 84097); Formaldehyde 37% (Sigma, cat. # F8775); Filter (Fisher, cat. # 06-665-28); Disodium salt EDTA (Sigma, cat. # E5134); SDS (Sigma, cat. # L6026); Tris base (Sigma, cat. # 08656); Glycine (Sigma, cat. # G7403); Tween X-100 (Sigma, cat. # T8787); Surfact-Amps NP-40 (Pierce, cat. # 28324); Ethanol (Sigma, cat. # 459828); Phenol / chloroform / isoamyl alcohol (25:24:1) (Pierce, cat. # 17908); Sodium acetate (Sigma, cat. # S2889); Sodium chloride (Sigma, cat. # S7653); Sodium bicarbonate (Sigma, cat. # S6297); Sodium deoxycholate (Deoxycholic acid, sodium salt) (Sigma, cat. # D6750); HEPES (N-(2-Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (Sigma, cat. # H-4034); PIPES (Piperazine-N,N’-bis(2-ethanesulfonic acid)) (Sigma, cat. # P-1851); Potassium chloride (Sigma, cat. # P9541); Calcium chloride (Sigma, cat. # C2661); Magnesium chloride (Sigma, cat. # M8266); Lithium chloride (Sigma, cat. # L4408); Proteinase K (20 mg / ml) (Fermentas, cat. # EO0491); Protease inhibitors: PMSF (Sigma, cat. # P7626); Protease inhibitors: Aprotinin A (Sigma, cat. # P5318); Protease inhibitors: Bestatin (Sigma, cat. # A3428).

[0037] Example 1 Obtaining of the antigenic polypeptide

[0038] First, a gene homologous to the gene encoding the Arabidopsis CENH3 protein was searched in the Cyperus esculentus genome database, i.e. the gene encoding the CENH3 protein of Cyperus esculentus, as shown in SEQ ID No: 1, the amino acid sequence of the CENH3 protein encoded by which is shown in SEQ ID No: 2.

[0039] SEQ ID No: 1

[0040] ATGGCCCGGACCAAGCACTTCTCCGCCAAATCCTCCAGGAAGAAGGTCGTCGCGCGCCGCTCCCTCGCTGTTCGTGGATCCTCCTCTCAGCATGAGGGAAGCGATACTGACCAGCATGAAGATGTCACTGGCACTCCTCGTACAAACAACCGACA GAAGACTCCGAAAAACACTGCTAGAAAGAGTACCAGGCCTCTTGTGCCTCCTAATTTGAGAGGAAACAACACTGATGCACGCCAGCACAGACTAAAAAGCACCGATTTCGGCCTGGGACTGTTGCACTGCGGGAAATCCGCCAGTATCAGAAAA CTGCCTTTCTGTTGATACCAGCCAGACCTTTTGCAAGATTGGCAAGGGAAATAACAGCGTTCTTCAACCCAGAAATAAATCGATGGACTCCTGCAGCACTTCTTGCTCTCCAAGAAGCAGCAGAACATTACATGGTAGAACTGTTCGAGGACACC AATCTCTGTGCCATTCATGCCAAGCGTGTCACAATTAGTAAGTTCACCTTTTCTTCCTACAATGTAGATTATATCTCATCTCTTGTTCTTTATTTTGAGTTCTGGGATAAGCTTGAGATGCTTTATGGATTAGATGGAAATTCAAGAGTTCTATGA

[0041] SEQ ID No: 2

[0042] MARTKHFSAKSSRKKVVARRSLAVRGSSSQHEGSDTDQHDDVTGTPRTNNRQKTPKNTARKSTRPLVPPNLRGNTDARQQTKKHRFRPGTVALREIR QYQKTAFLLIPARPFARLAREITAFFNPEINRWTPAALLALQEAAEHYMVELFEDTNLCAIHAKRVTIMQKDMQLARRISGRRMW

[0043] Bioinformatics analysis was performed on the amino acid sequences of CENH3 proteins from different species, including Cyperus oleifera, Arabidopsis thaliana, Rice, and Kobresia chinensis. Figure 1CENH3 protein of Cyperus esculentus L. and rice is 49%, and the similarity with Kobresia myosuroides is 57%, and the similarity with Arabidopsis thaliana is the highest, which is 46%. The amino acid sequences of CENH3 proteins of different species vary greatly at the N-terminal but are relatively conservative at the C-terminal. According to a specific sequence (positions 11-26 of SEQ ID No: 2) at the N-terminal of the amino acid sequence of CENH3 protein of Cyperus esculentus L., an antigenic polypeptide for functional centromere histone CENH3 of Cyperus esculentus L. is designed, and the specific amino acid sequence is SSRKKVVARRSLAVRG (as shown in SEQ ID No: 3).

[0044] The antigenic polypeptide as shown in SEQ ID No: 3 is artificially synthesized (Jieren Biochemistry (Shanghai) Co., Ltd.), and the enzyme-linked immunosorbent assay (ELISA) of Example 2 and the Western Blot of Example 3 are carried out.

[0045] Preparation of anti-CENH3 protein antibody

[0046] The CENH3 specific antibody is a polyclonal antibody obtained by immunizing rabbits and mice with the antigenic polypeptide (as shown in SEQ ID No: 3) prepared in Example 1 as an immunogen. The specific method is as follows:

[0047] Adult New Zealand white rabbits (two, Qingdao Kangda Biological Technology Co., Ltd.) and rats (two, Qingdao Kangda Biological Technology Co., Ltd.) are used as immunized animals.

[0048] Blood is collected from the ear margin vein before immunization and the serum is separated as negative serum. The antigenic polypeptide (as shown in SEQ ID No: 3) is used as an immunogen, and the corresponding dose of immunogen is emulsified with an equal amount of adjuvant. The immunization method and immunization procedure are shown in Table 1. The second immunization (second immunization) is carried out 30 days after the first immunization (first immunization), and then the immunization is carried out every 30 days until the eighth immunization (eighth immunization). The first ear vein boost immunization (first boost immunization) is carried out 30 days after the eighth immunization, the second ear vein boost immunization (second boost immunization) is carried out 14 days after the first boost immunization, the third ear vein boost immunization (third boost immunization) is carried out 14 days after the second boost immunization, and the serum is obtained by collecting a large amount of blood from the heart 14 days after the third boost immunization to obtain the CENH3 specific antibody. The serum is referred to as antiserum hereinafter.

[0049] Table 1 Immunization method and immunization procedure

[0050]

[0051]

[0052] Antiserum was isolated by collecting 0.5-1 ml of rabbit or mouse ear vein blood. The titer was determined by ELISA (see Example 3). After the test was qualified, whole blood was collected by cardiac puncture to isolate the antiserum. Antigen affinity purification was performed, with 20 ml of rabbit antiserum and 5 ml of rat antiserum purified per antibody. The resulting antiserum contained anti-CENH3 protein antibodies.

[0053] The two rabbit antisera obtained were numbered RB3215 and RB3216, respectively, and the two rat antisera obtained were numbered RT3217 and RT3218, respectively.

[0054] Example 3 Enzyme-linked immunosorbent assay (ELISA)

[0055] 1. The antigen polypeptide synthesized in Example 1 (free peptide, as shown in SEQ ID No: 3) was diluted to 2 μg / mL with coating buffer (Thermo Fisher Scientific, 28382) to obtain a coating stock solution. 100 μL / well of this coating stock solution was added to the ELISA plate and incubated overnight at 4°C. Unbound antigen polypeptide was washed away by swishing the contents of the ELISA plate into a sink, filling the wells with deionized water, swishing again, and then repeating twice with wash buffer (Thermo Fisher Scientific, 28360).

[0056] 2. Add 200 μl of blocking solution (Thermo Fisher Scientific, 37535) to each well. Incubate at room temperature (25°C) for 2 hours. Remove the blocking solution and wash the plate three times with wash buffer.

[0057] 3. Add 100 μl of the antiserum prepared in Example 2 (dilute the antiserum prepared in Example 2 with blocking buffer to 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000, respectively) or a blank control (i.e., blocking buffer) to each well; incubate at room temperature (25°C) for 60 minutes. Wash the plate three times with wash buffer.

[0058] 4. Add 100 μl of horseradish peroxidase (HRP)-labeled goat anti-rabbit (rat) IgG (Biyuntian, A0208) (1:5000 dilution in blocking buffer) to each well and incubate at 37°C for 45 minutes. Wash the plate three times with wash buffer.

[0059] 5. Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 10 minutes. If applicable, add stop solution (Thermo Fisher Scientific, N600). Read the results on an ELISA plate reader.

[0060] See the results Figure 2In the figure, RB3215 and RB3216 are the antisera of two rabbits, RT3217 and RT3218 are the antisera of two rats, and the horizontal axis is the dilution multiple. Figure 2 It can be seen that the specificity of the antibodies in the four antisera has been verified.

[0061] Example 4 Western Blot

[0062] Crude protein extraction from cyperus juncea: Cut tissue samples from the leaves, roots, and flowers of Yuyusha No. 2 into small pieces and quickly freeze them in liquid nitrogen. Add an appropriate amount of liquid nitrogen to a mortar and pestle, then grind the frozen tissue pieces thoroughly with a pestle until they are a fine powder. Add pre-chilled extraction buffer and continue grinding until the tissue cells are fully ruptured and the proteins are released. Transfer the homogenate to a centrifuge tube and centrifuge at 4°C, 10,000-15,000 rpm, for 10-20 minutes. Collect the supernatant to obtain crude protein extracts from the leaves, roots, and flowers, which will be used as samples for western blotting.

[0063] 1. Preparation of protein samples from different tissues: 10 μg of protein was loaded per well. All sample volumes were adjusted to the same using protein lysis buffer. Protein loading buffer (Biyuntian, P0015L) was then added proportionally. Mix thoroughly, boil in a boiling water bath for 10 minutes, and then quickly place the samples on ice for 10 minutes. After cooling, centrifuge at 13,000 rpm at 4°C for 10 minutes to obtain a protein solution ready for sample loading and electrophoresis.

[0064] 2. SDS-PAGE electrophoresis: Perform electrophoresis on a 15% precast protein gel (YaZyme, PG213). Remove the gel and place it in an electrophoresis tank. Add running buffer and slowly pipette in the protein solution prepared in step 1. Simultaneously, add a prestained protein molecular weight standard next to the sample. After adding the sample, run the gel at 60V for 30 minutes, then at 120V for 60 minutes.

[0065] 3. Semi-dry transfer: During SDS-PAGE electrophoresis, cut the PVDF membrane filter paper (Biyuntian, FFP28) to the same size as the gel. Place it in a glass dish filled with methanol and activate it for 15 seconds. Then transfer it to pre-chilled 1x transfer buffer (Biyuntian, P0572) and soak the transfer filter paper for at least 5 minutes. After electrophoresis is complete, carefully remove the gel and place it in transfer buffer. Then, arrange the gel in the order of filter paper, PVDF membrane, SDS-PAGE gel, and filter paper. Transfer the membrane at 15V for 150 minutes.

[0066] 4. Blocking: After Western Blotting transfer, cut the target protein band with a razor blade according to the molecular weight of the target protein. Place the membrane in an incubator and block with 5% skim milk powder (5% skim milk powder dissolved in 1× TBST) for 2 hours. After blocking, discard the skim milk powder and wash the membrane at least four times with 1× TBST at 80 rpm for 5 minutes each time.

[0067] 5. Primary Antibody Incubation: Dilute the primary antibody (antiserum against CENH3 protein obtained in Example 2, specifically rabbit anti-RT3216 in this example) at a ratio of 1:2000 (with 1× TBST) and incubate overnight at 4°C. After overnight, recover the primary antibody incubation solution and wash the membrane four times with 1× TBST.

[0068] 6. Secondary antibody incubation: Dilute the secondary antibody (Biyuntian, A0277), incubate at 80 rpm at room temperature for 1-2 h, and then wash the membrane four times with 1×TBST at 80 rpm.

[0069] 7. Development: Prepare ECL chemiluminescent solution at a ratio of 1:1 between solution A and solution B. Insulate the washed PVDF film with toilet paper to remove excess membrane wash solution, then place it in the ECL chemiluminescent solution and keep it at room temperature in the dark for 2-3 minutes. Then place it in a chemiluminescent imager for imaging, and adjust the exposure time according to the intensity of the band brightness. The image is processed and analyzed accordingly using imaging software.

[0070] Protein extraction buffer formula: 50mmol·L-1Tris-Cl (pH 7.5), 150mmol·L- 1 NaCl, 1mmol·L-1EDTA (pH 8.0), 1mmol·L-1DTT, 1% Triton X-100, 10% Glycerol, 1xProtease Inhibitor Cocktail

[0071] The molecular weight of Cyperus oleifera CenH3 was calculated to be approximately 18 kDa. The Western Blot results are shown in Figure 3 , showing that the band size is correct, the band specificity is high, and there are no mixed bands.

[0072] Example 5 Chromatin Immunoprecipitation Experiment (Chip-squence)

[0073] The experiment was performed with reference to “Saleh, A., Alvarez-Venegas, R., and Avramova, Z. (2008). An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc 3, 1018-1025.” The specific steps are as follows:

[0074] 1. Add 14-day-old seedling tissues of Cyperus oleifera Yuyousha No. 2 into three 50 ml centrifuge tubes: 2 g of flowers, 4 g of leaves, and 4 g of roots.

[0075] 2. Add 37 ml of cross-linking buffer to the sample and cross-link under vacuum at room temperature for 10 minutes.

[0076] 3. Add 2.5 ml of 2 M glycine solution (final concentration 100 mM) to stop the cross-linking reaction and place the tissue under vacuum infiltration for another 5 minutes at room temperature.

[0077] 4. Wash the plant tissue three times in sterile deionized water, blot dry between paper towels, and snap freeze in liquid nitrogen. Grind the tissue into a fine powder using a pre-chilled mortar and pestle, ensuring that the sample does not melt during grinding.

[0078] 5. Resuspend the sample in 25 ml of nuclear isolation buffer, swirl briefly to mix, and place the sample on ice until completely homogenized (15-30 minutes).

[0079] 6. After homogenization, filter the solution through four layers of cheesecloth. Centrifuge the filtrate at 11,000 g at 4°C for 20 minutes and set aside. A compact white pellet covered with chlorophyll will be visible at the bottom of the test tube.

[0080] 7. Discard the supernatant and resuspend the pellet (nuclei) in 2 ml of nuclear buffer.

[0081] 8. Divide the sample into four 500 μl aliquots and place them into 1.5 ml centrifuge tubes. Shear the DNA into fragments of 500 bp (200-1,000 bp) and sonicate at 6x power for 15 seconds five times. Keep the sample on ice for 1 minute each time.

[0082] 9. Centrifuge the sample at 13800 g, 4°C for 10 minutes to pellet debris. Collect the supernatants from all four tubes of the same sample into one tube.

[0083] 10. Take 100 μl of the sonicated chromatin sample (step 9) and dilute it 10-fold with lysis buffer. Pre-clear the diluted sonicated chromatin by adding 50 μl of salmon sperm DNA / protein A agarose beads (Sigma, cat. no. 16-201) (pre-equilibrated with nuclear lysis buffer) and rotate gently at 4°C for 1 hour.

[0084] 11. Add 5 μl of anti-CENH3 protein antibody (anti-CENH3 protein antiserum is obtained in Example 2, specifically rabbit anti-RT3216 is used in this example) to the supernatant and incubate at 4°C with slow rotation for at least 5 hours to overnight.

[0085] 12. Add 60-75 μl of salmon sperm DNA / Protein A agarose beads and continue incubation at 4°C for 2 hours. Centrifuge at 3800 g, 4°C for 2 minutes to collect the agarose beads and chromatin.

[0086] 13. Wash the beads with 1 ml of each of the following buffers for 5 minutes at 4°C with gentle rotation, followed by centrifugation at 3800 g for 2 minutes at 4°C: low salt buffer (1 time); high salt buffer (1 time); LiCl buffer (1 time); and TE buffer (2 times).

[0087] 14. Add 250 μl of fresh elution buffer and incubate at room temperature for 15 minutes with gentle shaking to elute the immune complex from the agarose beads. Centrifuge at 3800 g for 2 minutes at 4°C and transfer the supernatant to a fresh centrifuge tube.

[0088] 15. Add another 250 μl of elution buffer to the beads from step 14 and repeat the elution step in step 14. Incubate at room temperature for 30 minutes with gentle shaking. Centrifuge at 3800 g, 4°C for 2 minutes.

[0089] 16. Combine the two elutions (total 500 μl). At the same time, add 450 μl of elution buffer to the 50 μl sonicated chromatin in step 9 as an input control (positive control).

[0090] 17. Add 20 μl of 5 M NaCl solution to each tube and incubate at 65°C for at least 4 hours or overnight to reverse cross-linking.

[0091] 18. Add the following solution to each tube: 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris–HCl (pH 6.5), and 1 μl of proteinase K (20 mg / mL). Incubate at 45°C for 1.5 hours to digest the protein.

[0092] 19. Add equal volume (550 μl) of phenol / chloroform / isoamyl alcohol to each tube, vortex briefly. Centrifuge at 13800 g for 15 min at 4°C, transfer supernatant (about 500 μl) to 2 ml microcentrifuge tubes.

[0093] 20. Add the following solution to each tube: 2.5 volumes of 100% ethanol, 1 / 10 volume of 3 M sodium acetate solution (pH 5.2), 4 μl of glycogen (20 mg / ml), incubate at -80°C for 1 hour to precipitate DNA. Centrifuge at 13800 g for 15 min at 4°C. Discard the supernatant, wash the pellet with 500 μl of 70% ethanol (v / v), centrifuge again at 13800 g for 10 min at 4°C. Discard the supernatant, dry the pellet at room temperature.

[0094] 21. Dissolve the DNA in 50 μl of TE buffer, store at -80°C. Subsequent library sequencing is completed by Wuhan Wanmole Technology Co., Ltd.

[0095] Result analysis, use BWA software to align CleanReads to Cyperus esculentus reference genome, sort the alignment results by SAMtools software, remove redundancy, and then count the sequencing depth and genome coverage of each sample. Extract the results of Chip-sequence, find that the Cyperus esculentus chromosome has multiple CenH3 binding sites ( Figure 4 ), and the bound sequence conforms to the centromere characteristic sequence ( Figure 5 ).

[0096] The above results prove that the obtained antibody can specifically recognize the functional centromere of Cyperus esculentus, and the antibody can be used to determine the position, size and sequence information of the functional centromere of Cyperus esculentus, and provide information reference for Cyperus esculentus centromere region genome assembly and centromere formation and evolution mechanism.

[0097] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID No:

3.

2. An anti-CENH3 protein antibody, characterized in that: The anti-CENH3 protein antibody is a polyclonal antibody prepared using the polypeptide according to claim 1 as an immunogen.

3. The anti-CENH3 protein antibody according to claim 2, characterized in that: The polyclonal antibody is a rabbit-derived antibody.

4. A method for preparing an anti-CENH3 protein antibody, characterized in that: The method comprises preparing polyclonal antibodies using the polypeptide according to claim 1 as an immunogen, wherein the polyclonal antibodies are antibodies against CENH3 protein.

5. The method according to claim 4, characterized in that: The polyclonal antibody is a rabbit-derived antibody.

6. Use of the polypeptide according to claim 1 in preparing a reagent or kit for specifically recognizing centromeres of Cyperus oleifera.

7. The use according to claim 6, characterized in that: The reagent or kit for specifically recognizing the centromere of Cyperus oleifera contains the anti-CENH3 protein antibody according to claim 2 or 3.

8. Use of the anti-CENH3 protein antibody according to claim 2 or 3 in the preparation of a reagent or kit for specifically recognizing centromeres of Cyperus oleifera.

9. The use according to claim 8, characterized in that: The reagent or kit for specifically recognizing the centromere of Cyperus oleifera contains the anti-CENH3 protein antibody according to claim 2 or 3.

10. A reagent or kit for specifically identifying centromeres of Cyperus oleifera, characterized by: Contains the anti-CENH3 protein antibody according to claim 2 or 3.

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