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Separation of satellite DNA pBv sequence of centromere of halogeton glomeratus and application thereof

A halophyte and centromere technology, applied in the field of plant science, can solve the problems of lack of and limited species research depth and utilization value, and achieve highly targeted and specific effects.

Inactive Publication Date: 2018-11-27
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for this species, the current lack of basic research on its genome has greatly limited the research depth and utilization value of this species. In order to further accelerate the research progress of halophytic grass and broaden the utilization of halophytic grass The Structure and Distribution Pattern of the Repeated Sequence in the Centromeric Region of Grass
[0003] Problems in the prior art: There are no reports on the isolation and application of the centromere satellite DNA pBv sequence of halophytes in the prior art, especially the research on the separation of chromosome centromere satellite DNA pBv sequence based on PCR specific amplification

Method used

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  • Separation of satellite DNA pBv sequence of centromere of halogeton glomeratus and application thereof
  • Separation of satellite DNA pBv sequence of centromere of halogeton glomeratus and application thereof

Examples

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Embodiment 1

[0029] This embodiment provides a method for isolating the centromere satellite DNA pBv sequence of Grass saline chromosome, which specifically includes the following steps:

[0030] Primers were designed according to the conserved motifs of the chromosome centromere satellite DNA pBv sequence in plants, and the genomic DNA of Halophylla saline was extracted, and the genomic DNA was used as a template for PCR amplification to obtain the target fragment ( figure 1 ), and then recover the target fragment, and connect it to the pMD19-T cloning vector, transform Escherichia coli DH-5a competent cells, screen the positive clones, and extract the plasmid of the appropriate bacterial liquid through PCR bacterial liquid detection and send it to BGI for Sequencing analysis obtained the centromere satellite DNA pBv sequence of Grass saline, specifically as shown in SEQ ID No: 1, with a size of 327 bp ( figure 1 ):

[0031] The primer sequences used in this step are: upstream primer F...

Embodiment 2

[0034] This embodiment provides the application of the centromere satellite DNA pBv sequence of the Chromosome DNA pBv as a molecular marker in the analysis of species genetic variation and evolution analysis, specifically including the following steps:

[0035] (1) Preparation of digoxigenin-labeled probes

[0036] According to Example 1, the plasmid DNA containing the centromere satellite DNA pBv sequence element of Grass saline was obtained as the probe DNA, and the DIG-Nick Translation Mix Digoxigenin Labeling Kit was used for labeling. The reaction system was 1 μL of the probe DNA, Nick Translation Mix 4 μL. Mark in a constant temperature water bath at 15°C for 1.5 h-2.0 h, then freeze at -20°C in the dark.

[0037] (2) Preparation of metaphase chromosomes in Grass saline

[0038] Place the halophytic grass seeds treated with swollenin in a petri dish and cultivate them in the dark at 28°C. When the roots grow to about 1-1.5cm, remove the root tips and treat them in a m...

Embodiment 3

[0042] This embodiment provides the application of the centromere satellite DNA pBv sequence in the species identification of Grass saline chromosome, which specifically includes the following steps:

[0043] (1) Cloning and sequencing of the pBv sequence of Grass salina

[0044] Primers were designed according to the conserved motifs of the chromosome centromere satellite DNA pBv sequence in plants, and the genomic DNA of Halophylla saline was extracted, and the genomic DNA was used as a template for PCR amplification to obtain the target fragment ( figure 1 ), and then recover the target fragment, and connect it to the pMD19-T cloning vector, transform Escherichia coli DH-5a competent cells, screen the positive clones, and extract the plasmid of the appropriate bacterial liquid through PCR bacterial liquid detection and send it to BGI for Sequencing analysis obtained the centromere satellite DNA pBv sequence of Grass saline, specifically as shown in SEQ ID No: 1, with a siz...

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Abstract

The invention relates to separation of satellite DNA pBv sequence of centromere of halogeton glomeratus. The sequence has a nucleotide sequence as shown by SEQ ID No: 1, the size of which is 327 bp. The invention further discloses an application of the satellite DNA pBv sequence for manufacturing a probe in fluorescence in situ hybridization, analyzing a repeated sequence structure of a centromereregion of chromosome of halogeton glomeratus and a distributing mode thereof. Moreover, the satellite DNA pBv sequence of centromere of halogeton glomeratus, provided by the invention, provides a ground for molecular marker development and species identification based on the satellite DNA pBv sequence of centromere of halogeton glomeratus.

Description

technical field [0001] The invention belongs to the field of plant science; in particular, it relates to a method for isolating the centromere satellite DNA pBv sequence of the halophytic grass chromosome, and its application in molecular marker development and species identification. Background technique [0002] Eukaryotic centromere sequences consist of highly repetitive satellite DNA sequences and centromere retrotransposons. The highly repetitive satellite DNA sequence of centromere is one of the sequence components of centromere in higher plants. In some species such as barley, the centromere sequence composition is dominated by satellite DNA sequences, which are the main structural factors, arranged in tandem, and have been found in many species. The common feature of centromere satellite DNA sequences is high heterogeneity, and they can expand or shrink their sequences through a series of mutation processes, such as replication jumping of mobile elements, unequal ex...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6806C12Q1/6841C12N15/11C12N15/10
CPCC12N15/1003C12Q1/6806C12Q1/6841C12Q1/6895C12Q2563/107
Inventor 李葆春王化俊汪军成姚立蓉王刚司二静杨轲孟亚雄马小乐胡娜侯静静张燕陈春舟
Owner GANSU AGRI UNIV
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