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A kind of repeated dna sequence of black mustard genome and its application

A genome and DNA molecule technology, applied to the repeated DNA sequence of the black mustard genome and its application fields, can solve the problems of small chromosomes and difficulty in morphological distinction, and achieve the effect of easy identification

Inactive Publication Date: 2019-03-26
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the small chromosome size of Brassica crops and the difficulty in morphological distinction, it is necessary to establish a simple and reliable detection method

Method used

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  • A kind of repeated dna sequence of black mustard genome and its application
  • A kind of repeated dna sequence of black mustard genome and its application
  • A kind of repeated dna sequence of black mustard genome and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1, the discovery of DNA repeat sequence CL5

[0020] Using black mustard 'G1 / 1' as material, using BrCENH3 protein antibody (a centromere functional protein of Brassica, constructed by myself, the article has been published, Wang et al., 2011, Chromosoma, 120:353-365 ) for Chromatin Immunoprecipitation (ChIP-Cloning) and sequencing.

[0021] The main steps:

[0022] 1) The newly extracted nuclei of the black mustard were digested with micrococcal nuclease (micrococcal nuclease, Sigma, St.Louis, MO) to obtain a DNA fragment of 300-700 bp, mainly containing 2-3 nucleosomes in size.

[0023] 2) The enzymatically digested chromatin was incubated with BrCENH3 protein antibody and original serum (control), and the immune complexes were collected and eluted with protein A sepharose (GE Healthcare) to obtain immunoprecipitated DNA.

[0024] 3) The DNA sequence obtained by immunoprecipitation was cloned, sequenced, and sequenced, and a centromere functional DNA repeat ...

Embodiment 2

[0026] Embodiment 2, the application of DNA repeat sequence CL5 as detection probe

[0027] 1. Fluorescence in situ hybridization (FISH) test method using CL5 as a detection probe

[0028] 1. Chromosome production

[0029] 1) Put the plant seeds to be tested in a petri dish with moist filter paper, and take root at room temperature;

[0030] 2) When the root tip length is 0.5-1cm, cut off the root tip and put it into the d 2 h 2 O in a test tube, and put the test tube into an ice box filled with ice-water mixture and place it in the refrigerator for 22-24h for pretreatment;

[0031] 3) Put the pretreated root tip into fixative solution (absolute ethanol:glacial acetic acid=3:1 (v / v)) and fix it for 1-7 days;

[0032] Note: In order to obtain better results in in situ hybridization, the fixation time should not be too long. Of course, if the fixation time is less than two days, it is relatively difficult to make slices.

[0033] 4) Excise part of the root tip meristem and ...

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Abstract

The invention discloses repeated DNA sequences of the black mustard genome and applications of the repeated DNA sequences. The repeated DNA sequences of the black mustard genome are DNA molecules shown by the sequence 1 of the sequence table, and can be applied for identifying that whether the genome of the to-be-detected plant contains the genetic material derived from the B genome of black mustard or not. The experiment proves that (1) the acquiring of CL5 enables the chromosome of the brassica crop to be easily and accurately identified, and therefore, the blank that no ideal probe is available for identifying the chromosome of the brassica crop is filled up; (2) and CL5 can be used for identifying that whether the chromosome of the brassica crop contains that derived from the B genome of the black mustard or not.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a repetitive DNA sequence of the black mustard genome and its application. Background technique [0002] Cruciferous Brassica vegetables include the A genome Brassica.rapa (AA, n = 10) represented by Chinese cabbage, the B genome B of black mustard, B. nigra (BB, n = 8), and the C genome represented by cabbage B. oleracea (CC, n = 9), and three allotropic tetraploid compound species Brassica napus (AACC, n = 19), Brassica napus B. juncea (AABB, n =18), and Ethiopian mustard B.carinata (BBCC, n=17), forming the famous U-triangle. In molecular biology studies such as species evolution, gene function, and cytological analysis, it is often necessary to distinguish the diploid ancestral chromosomes from their respective sources in tetraploid materials. However, due to the small chromosome size of Brassica crops and the difficulty in morphological distinction, it is necessary to establis...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841C12N15/11
CPCC12Q1/6895C12Q2600/158
Inventor 王桂香刘凡何群燕蔡泽希李静郭宁韩硕宗梅
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES