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Gossypium raimondii functional centromere sequence and molecular marker of same

A technology of functional centromere and Raymond's cotton, applied in the fields of bioinformatics and molecular cytogenetics, can solve the problems of undetermined DNA sequence CENH3 binding, the authenticity of the functional centromere sequence is questionable, etc. Real and reliable, good primer specificity, single band effect

Inactive Publication Date: 2015-08-05
FUJIAN AGRI & FORESTRY UNIV
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AI Technical Summary

Problems solved by technology

[0004] In recent years, cotton genome research has developed rapidly. However, there are few reports on the study of cotton centromere, and the only reports are basically based on random DNA sequence analysis and screening. It is impossible to determine whether the DNA sequence binds to CENH3. Therefore, Its authenticity as a functional centromere sequence is questionable

Method used

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  • Gossypium raimondii functional centromere sequence and molecular marker of same

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Embodiment 1

[0019] Example 1: Implementation of ChIP-Seq Technology

[0020] ChIP-Seq technology refers to the combination of chromatin immunoprecipitation ChIP and next-generation sequencing. This method is feasible and stable. It is not only suitable for the study of the binding relationship between a single gene and protein, but also for the genome-wide study of specific protein binding sites.

[0021] The basic process of ChIP-Seq used in the present invention is:

[0022] 1. Grind fresh and young leaves with liquid nitrogen; 2. Extract cell nuclei and enzymatically hydrolyze them with micrococcal ribozyme; 3. Add the antibody of centromere-specific histone CENH3 to combine with the target protein-DNA complex; 4. Add ProteinA binds to the antibody-target protein-DNA complex and precipitates it; 5. Washes the precipitated complex to remove some non-specific binding; 6. Elutes to obtain an enriched target protein-DNA complex; 7. 8. PCR analysis and preparation of sequencing library; 9....

Embodiment 2

[0039] Embodiment 2: Primer design and PCR amplification

[0040] Use the primer design software Primer Premier 5.0 to design primers for the functional centromere sequence. The set conditions are: first, the full-length or partial sequence of the nucleic acid sequence; second, the length of the primer is 21 bp±5 bp; third, the GC content of the primer sequence 40-60%; Fourth, the PCR product size is 200-700 bp.

[0041] The conditions of PCR amplification were: pre-denaturation at 95°C for 3 minutes, 35 cycles at 95°C for 30s, 47-53°C for 30s, 72°C for 45s, and a total extension at 72°C for 7 minutes. It should be noted that different primer sequences have different annealing temperatures: 50°C for sequence 1, 47°C for sequence 2, 53°C for sequence 3, and 51°C for sequence 4. The band amplified by PCR was single and bright. Therefore, the QIAGEN kit was directly used to purify and recover the PCR product, its product number: 28104, and its name: QIAquick PCR Purification Ki...

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Abstract

The invention provides a gossypium raimondii functional centromere sequence and a molecular marker of the same. IN the invention, a method of combination of chromatin co-immunoprecipitation and high throughput sequencing (ChiP-Seq) is employed to obtain four DNA sequences in a functional centromere zone of gossypium raimondii, and primers are respectively designed according to the sequences for fluorescence in-situ hybridization (FISH) of mitosis metaphase of the gossypium raimondii. A result proves that all the four sequences generate concentrated, clear and bright signals in the centromere zone of all chromosomes. The centromere sequence can be directly used for positioning the functional centromeres and identification of relative sequences. The molecular marker can be used in genetic location of the centromere of a genetic map of cotton, can provide more accurate information to functional gene location and linkage relationship and can provide basis for sequence splicing of whole genome sequencing of the gossypium raimondii, so that the information of a physical map can be more comprehensive.

Description

technical field [0001] The invention relates to the fields of bioinformatics and molecular cytogenetics, in particular to the Raymond cotton functional centromere sequence and molecular markers thereof. Background technique [0002] Centromeres are important functional elements on chromosomes of higher eukaryotes. It is responsible for the equal separation of chromosomes during cell mitosis and meiosis, thus playing an important role in ensuring the stable transmission of genetic material. Because the centromere region is rich in repetitive sequences, it is difficult to sequence and assemble, so its research is relatively backward. For example, although the whole genome sequencing of model organisms such as human, Drosophila and Arabidopsis has been completed long ago, their centromeres often still exist as gaps of different lengths on the genome sequencing map due to the large number of repetitive sequences, which cannot be resolved. Therefore, the study of centromere is ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/158
Inventor 王凯韩金磊张文盼俞丽英单文波林爱婷
Owner FUJIAN AGRI & FORESTRY UNIV
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