Antigenic polypeptide of poplar functional centromere histone CENH3 and application thereof

A technology for functional centromere and antigenic polypeptides, applied in the fields of bioinformatics and molecular cytogenetics, can solve problems such as increasing the difficulty of centromere sequence assembly and achieve broad application prospects

Active Publication Date: 2020-07-14
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, poplar centromere contains a large number of highly repetitive sequences, which increases the difficulty of ce

Method used

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  • Antigenic polypeptide of poplar functional centromere histone CENH3 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Obtaining the amino acid sequence of the centromeric histone CENH3 of Populus trichocarpa

[0018] First, a gene Potri.014G096400.1 homologous to the gene encoding Arabidopsis CENH3 protein (At1g01370HTR12) was searched in the Populus trichocarpa genome database, that is, the CENH3 protein gene of Populus trichocarpa, thereby obtaining the amino acid sequence of the gene. The gene is located on chromosome 14 of P. trichocarpa and is a single copy in P. trichocarpa. Through the bioinformatics analysis of the amino acid sequence of CENH3 protein in different species, including Populus trichocarpa, Arabidopsis, rice and maize. The results showed that the amino acid sequence of Populus trichocarpa CENH3 protein was 54% similar to rice, 53% similar to maize, and 60% similar to Arabidopsis. The amino acid sequence of CENH3 proteins in different species varies greatly at the N-terminus but is relatively conserved at the C-terminus.

Embodiment 2

[0019] Example 2: Preparation of poplar centromere histone CENH3 antibody

[0020] The 20 specific amino acid sequences of the N-terminal of the centromere histone CENH3 protein of Populus trichocarpa were selected, the specific sequence was MARTKHPVARKRARSPKRSD, and the polypeptide was artificially synthesized according to the sequence as the antigen. The synthesized polypeptide was coupled to a protein carrier, and then routinely immunized New Zealand rabbits. After 4 immunizations, blood was collected from the rabbit carotid artery to collect serum. After the serum was passed through the antigen affinity column, rabbit-derived polyclonal antibodies were collected. The purity of the antibody was 90%, and the concentration of the antibody was 0.4mg / ml, the antibody was aliquoted and stored at -20°C.

Embodiment 3

[0021] Example 3: Antibody Immunofluorescence Detection

[0022] Immunofluorescence detection is a cytological test for the specificity of the prepared antibody. Because the reaction between antigen and antibody is highly specific, the quality of the prepared CENH3 antibody can be judged by immunofluorescence detection. The experimental procedure is as follows:

[0023] (1) Root tip fixation: select vigorously growing root tips of poplar trees, fix with 4% (w / v) paraformaldehyde for 15 min, and then rinse with 1×PBS buffer solution for 3 times, 5 min each time.

[0024] (2) Chromosome preparation: 1) Transfer the root tip to a clean slide; 2) Cut off the root tip meristem with a double-sided blade; 3) Add 10 μL 1×PBS to the slide; 4) lightly Lightly cover the cover glass, tap the cover glass with a dissecting needle to fully disperse the apical meristem; 5) Soak the slide in a staining jar filled with 1×PBS, soak off the cover glass, take out the prepared The slices are air-...

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Abstract

The invention discloses an antigenic polypeptide of poplar functional centromere histone CENH3 and application thereof, belonging to the fields of bioinformatics and molecular cytogenetics. The invention mainly comprises the following steps: screening a specific amino acid sequence of populus tomentosa CENH3 protein and artificially synthesizing a polypeptide; immunizing New Zealand rabbits with the polypeptide to prepare antiserum; and carrying out affinity purification to obtain a CENH3 protein antibody. Immunofluorescence detection and ChIP-FISH are carried out on the antibody to verify thespecificity, thereby proving that the antibody can accurately identify the functional centromere. The antibody can be universally used in poplar species, can accurately mark the position of the poplar functional centromere, and lays a foundation for developing the research on the structure, function and evolution of the poplar functional centromere. The invention has wide application prospects inthe research fields of poplar molecular cytogenetics, genomics, epigenomics and the like.

Description

technical field [0001] The invention belongs to the fields of bioinformatics and molecular cytogenetics, and specifically relates to an antigenic polypeptide of poplar functional centromere histone CENH3 and an application thereof. Background technique [0002] The centromere is an important functional element to ensure the stability and correct segregation of chromosomes during mitosis and meiosis in eukaryotic cells. Centromere DNA contains a large number of highly repetitive sequences, which increases the difficulty of sequence determination and analysis. The comprehensive application of rapidly developing molecular biology techniques, bioinformatics analysis methods and molecular cytogenetics techniques in centromere research, especially accompanied by Chromatin Immunoprecipitation (ChIP) and high-throughput sequencing techniques The application of has accelerated people's understanding of centromere structure, function and evolutionary relationship (Fan Qifu et al., 20...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K16/16C07K16/06G01N33/68G01N33/53
CPCC07K14/415C07K16/16G01N33/53G01N33/68
Inventor 席梦利宁仪杭辛昊阳刘光欣赵乙琏尚大鑫
Owner NANJING FORESTRY UNIV
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