阅读说明：本技术 杨树功能着丝粒组蛋白cenh3的抗原多肽及其应用 (Antigenic polypeptide of poplar functional centromere histone CENH3 and application thereof ) 是由 席梦利 宁仪杭 辛昊阳 刘光欣 赵乙琏 尚大鑫 于 2020-03-10 设计创作，主要内容包括：本发明公开杨树功能着丝粒组蛋白CENH3的抗原多肽及其应用,属于生物信息学和分子细胞遗传学领域。该发明主要包括：筛选毛果杨CENH3蛋白特异的一段氨基酸序列并人工合成多肽,用该多肽免疫新西兰兔,制备抗血清,亲和纯化后得到CENH3蛋白抗体；抗体通过免疫荧光检测、ChIP-FISH进行特异性验证,证明了该抗体可以准确识别功能着丝粒。该抗体可以在杨属树种中通用,不仅可以准确标记杨树功能着丝粒的位置,也为开展杨树着丝粒的结构、功能及进化研究奠定基础。本发明在杨树分子细胞遗传学、基因组学及表观组学等研究领域具有广阔的应用前景。(The invention discloses an antigen 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 section of amino acid sequence of the populus trichocarpa CENH3 protein, artificially synthesizing polypeptide, immunizing a New Zealand rabbit with the polypeptide, preparing antiserum, and obtaining a CENH3 protein antibody after affinity purification; the antibody is subjected to specificity verification through immunofluorescence detection and ChIP-FISH, and the fact that the antibody can accurately identify functional centromeres is proved. The antibody can be universal in populus species, can accurately mark the position of functional centromere of populus, and lays a foundation for developing the research on the structure, function and evolution of the centromere of populus. The invention has wide application prospect in the research fields of molecular cytogenetics, genomics, epigenomics and the like of poplar.)

1. The antigenic polypeptide of poplar functional centromere histone CENH3 is characterized in that the amino acid sequence is MARTKHPVARKRARSPKRSD.

2. The antigenic polypeptide of poplar functional centromere histone CENH3 of claim 1, wherein the antigenic polypeptide is a polypeptide synthesized from a specific sequence at the N-terminal of the amino acid sequence of populus trichocarpa functional centromere histone CENH 3.

3. The use of the antigenic polypeptide of poplar functional centromere histone CENH3 of claim 1 in poplar species.

4. The antigenic polypeptide of claim 1 for use in the production of antibodies.

5. A method of producing the antibody of claim 4, comprising the steps of: coupling antigen polypeptide to protein carrier, immunizing New Zealand rabbit conventionally, collecting serum from the blood of rabbit neck artery after 4-immunization, and collecting rabbit polyclonal antibody after passing through antigen affinity column.

6. A method of validating the antibody of claim 4 or 5, comprising immunofluorescence detection or ChIP-FISH.

7. The method of validating an antibody of claim 6, wherein if the method is immunofluorescence detection, the method comprises the steps of: fixing root tips, preparing chromosome sheets, incubating primary antibodies, incubating secondary antibodies, sealing sheets and performing microscopic examination.

8. The method of validating an antibody of claim 6, wherein if the method is ChIP-FISH, the method comprises the steps of: the method comprises the following steps of poplar nucleus extraction and purification, chromatin digestion and inspection, chromatin pretreatment, antibody incubation, cleaning and incubation of rProteinA agarose beads, elution of an antibody chromatin complex, ChIP-DNA extraction, probe preparation, slide denaturation and hybridization, cleaning and slide making, antibody incubation, cleaning and slide making, DAPI counter staining and microscopic examination.

Technical Field

The invention belongs to the fields of bioinformatics and molecular cytogenetics, and particularly relates to an antigen polypeptide of poplar functional centromere histone CENH3 and application thereof.

Background

The centromere is an important functional element for ensuring stable and correct chromosome separation during mitosis and meiosis of eukaryotic cells. Centromeric DNA, because of its high number of highly repetitive sequences, increases the difficulty of sequencing and analysis. The comprehensive application of rapidly developed molecular biology techniques, bioinformatics analysis methods, and molecular cytogenetics techniques in centromere research, especially with the application of chromatin co-immunoprecipitation (ChIP) and high throughput sequencing techniques, has accelerated the understanding of centromere structure, function, and evolutionary relationship (fan et al, 2017).

One notable feature of centromere structure is that histone H3 in this region is mutated into CENH3 (called CENP- ｃA in mammals) (vafｃA et al, 1997) CENH3 protein is ｃA marker protein of functional centromere the core experimental procedure of the current mainstream study of plant centromere is to first prepare ｃA specific antibody capable of identifying functional centromere histone CENH3, then enrich centromere dnｃA by ChIP technology, then sequence analysis is performed on the enriched dnｃA by high throughput sequencing (szalowskiet al, 2011), and finally verification is performed by combining Fluorescence In Situ Hybridization (FISH) technology.

However, the plant species which have been studied about centromere are mostly concentrated on herbaceous plants, and no reports are found about perennial forest trees. Poplar is the model tree species for forest research due to its own biological characteristics and importance. In 2006, the American journal of science reported the results of whole genome sequencing of Populus tomentosa (Tuskan et al, 2006), and the results of genome sequencing of Populus diversifolia and Populus xinjiang were also published one after another (Ma et al, 2013; Ma et al, 2019). However, the centromere of poplar contains a large amount of highly repetitive sequences, which increases the difficulty of centromere sequence assembly, and the sequence information of centromere of poplar genome still needs to be perfected. Therefore, the preparation of specific antibodies capable of recognizing poplar functional centromere histones CENH3 is urgently needed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide the antigenic polypeptide of the poplar functional centromere histone CENH3, and the invention aims to solve another technical problem of application of the antigenic polypeptide of the poplar functional centromere histone CENH3 in poplar species, thereby laying a foundation for developing the research on the structure, function and evolution of poplar centromere.

In order to solve the problems, the technical scheme adopted by the invention is as follows: the amino acid sequence of the antigenic polypeptide of the poplar functional centromere histone CENH3 is MARTKHPVARKRARSPKRSD.

The antigen polypeptide is a polypeptide synthesized according to a specific sequence at the N end of the functional centromere histone CENH3 amino acid sequence of populus trichocarpa.

Furthermore, the populus functional centromere histone CENH3 antigen polypeptide is applied to populus species.

Further, the antigen polypeptide is used for preparing antibodies.

A method for producing the above antibody: coupling antigen polypeptide to protein carrier, immunizing New Zealand rabbit conventionally, collecting serum from the blood of rabbit neck artery after 4-immunization, and collecting rabbit polyclonal antibody after passing through antigen affinity column.

A method for verifying the antibody comprises immunofluorescence detection or ChIP-FISH.

If the method is immunofluorescence detection, the method for verifying the antibody comprises the following steps: fixing root tips, preparing chromosome sheets, incubating primary antibodies, incubating secondary antibodies, sealing sheets and performing microscopic examination.

If the method is ChIP-FISH, the method comprises the following steps: the method comprises the following steps of poplar nucleus extraction and purification, chromatin digestion and inspection, chromatin pretreatment, antibody incubation, cleaning and incubation of rProteinA agarose beads, elution of an antibody chromatin complex, ChIP-DNA extraction, probe preparation, slide denaturation and hybridization, cleaning and slide making, antibody incubation, cleaning and slide making, DAPI counter staining and microscopic examination.

The invention has the following beneficial effects: the antigen polypeptide is artificially synthesized by using the 20 specific amino acid sequences disclosed by the invention, and the polypeptide is used for immunizing a New Zealand rabbit to obtain a CENH3 protein antibody. The prepared antibody is subjected to specificity verification through immunofluorescence detection and ChIP-FISH, and the fact that the antibody can accurately identify functional centromeres is proved. The antibody can be universal in populus species, can accurately mark the position of functional centromere of populus, and lays a foundation for developing the research on the structure, function and evolution of the centromere of populus. The invention has wide application prospect in the research fields of molecular cytogenetics, genomics, epigenomics and the like of poplar.

Drawings

FIG. 1 is the result chart of the immunodetection and ChIP-FISH of poplar CENH 3. a is the metaphase chromosome of poplar; red in b is the signal of CENH3, and CENH3 signal can be seen to be concentrated at the centromere position of metaphase chromosome; green in c is the signal of ChIP DNA; d is the result after a, b and c are synthesized, the signal of CENH3 is coincident with the main signal of ChIP DNA, which indicates that the ChIP DNA is from the DNA of centromere and the DNA can be used for next sequencing analysis. Scale bar 10 μm.

Detailed Description

The invention is further described with reference to specific examples. The temperatures not indicated in the examples are all room temperature and the reagents not indicated are all conventional chemical reagents.