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A line-type retrotransposon sequence specific to the centromere of P. trichocarpa and its application

A retrotransposon and centromere technology, applied in the fields of bioinformatics and molecular cytogenetics, can solve the problems of poor knowledge of sequence information and increase the difficulty of sequence assembly of centromere regions, and achieve broad The effect of applying the foreground

Active Publication Date: 2020-11-10
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although as early as 2006, the American Journal of Science reported the whole genome sequencing results of Populus trichocarpa (Tuskan et al., 2006), the genome sequencing results of Populus euphratica and Populus Xinjiang have also been published (Ma et al., 2013; Ma et al. al., 2019), because the DNA of the centromere region contains a large number of highly repetitive sequences, which increases the difficulty of the assembly of the centromere region sequence, and the sequence information of the centromere region of poplar is still very little known

Method used

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  • A line-type retrotransposon sequence specific to the centromere of P. trichocarpa and its application
  • A line-type retrotransposon sequence specific to the centromere of P. trichocarpa and its application

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Discovery of centromeric LINE-type retrotransposons

[0030] (1) Chromatin immunoprecipitation (ChIP) was carried out using poplar centromere-specific antibodies to enrich the centromere DNA of Populus trichocarpa. Libraries of ChIP DNA and Input DNA were constructed separately.

[0031] (2) Based on the Illumina Hiseq 2000 sequencing platform, 125bp paired-end sequencing technology was used to sequence the library DNA, and ChIP-seq data and Input-seq data of the centromere of Populus trichocarpa were obtained.

[0032](3) Use the RepeatExplorer analysis platform to excavate the repeated sequences of Populus trichocarpa centromeres on the data after quality control. First, 5 million reads are randomly selected from the Input reads, uploaded to the analysis platform, and aggregated by sequence similarity. Class analysis yields genomic repeat clusters. Then use the Blast comparison tool to compare the ChIP-seq data and Input data to the clusters respectively,...

Embodiment 2

[0034] Example 2: Centromere-specific LINE-type retrotransposon sequence amplification and probe labeling

[0035] (1) Collect the young leaves of Populus trichocarpa, grind them into powder respectively in liquid nitrogen, extract the DNA of each sample according to the instructions in the Total Plant DNA Extraction Kit (TIANGEN DP320), use Nanodrop 1000 (USA) to extract DNA Quantity and concentration were measured.

[0036] (2) Use Primer Premier5 software to design primers for centromere-specific LINE-type retrotransposon sequences. According to the primer scores, we selected 2 pairs of primers with higher scores and entrusted Nanjing Qingke Biotechnology Co., Ltd. to synthesize them , using the total genomic DNA of Populus trichocarpa as a template, optimized the PCR system, observed the target band after gel electrophoresis, and found that the primer pair F: 5′-TTAGGGAAACCAATACAACCAG-3′ and R: 5′-ACGAGGCAGTCAAATCAGAAGT-3′ could The target band ( figure 1 ).

[0037] Th...

Embodiment 3

[0040] Example 3: Fluorescence in situ hybridization (FISH) verification

[0041] (1) Chromosome production

[0042] 1) Material collection: collect the young root tips of Populus trichocarpa with a length of about 5 mm in the morning on a sunny day;

[0043] 2) Pretreatment: put the root tip into 0.7mM cyclohexanamide and treat it at 25°C for 3h;

[0044] 3) Fixation: transfer the root tip to Carnot's fixative solution (3:1 volume ratio of absolute ethanol to glacial acetic acid) and fix at room temperature for more than 24 hours;

[0045] 4) Enzymolysis: Rinse the fixed root tip thoroughly in pure water, then cut out the root tip meristem, and put it in a mixed enzyme solution of 4% cellulase and 2% pectinase, at 37°C Enzymolysis in the incubator for 1h;

[0046] 5) Smear preparation: first transfer the root apical meristem to pure water, then transfer from the pure water to the glass slide, then gently squeeze out the root apical meristem cells with tweezers, drop 20 μL ...

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Abstract

The invention discloses a populus trichocarpa centromere-specific LINE type retrotransposon sequence and application thereof, belonging to the fields of bioinformatics and molecular cytogenetics. The invention mainly includes: screening out a centromere-specific LINE-type retrotransposon sequence through bioinformatics analysis of high-throughput sequencing data of Populus trichocarpa centromere DNA. Primers were designed according to the sequence, and the genomic DNA of Populus trichocarpa was used as a template to obtain the target fragment through PCR amplification and gel cutting recovery. It was verified that the sequence was located in the centromere region by probe labeling, chromosome preparation, and fluorescence in situ hybridization. The centromere-specific LINE-type retrotransposon sequence provided by the present invention can not only accurately mark the position of the centromere of poplar to construct a high-quality karyotype map, but also contribute to the development of the structure and function of the centromere of poplar It has a broad application prospect in the fields of poplar molecular cytogenetics and genomics research.

Description

technical field [0001] The invention belongs to the fields of bioinformatics and molecular cytogenetics, and specifically relates to a centromere-specific LINE-type retrotransposon sequence of Populus trichocarpa and its application. Background technique [0002] The centromere is an important functional element to ensure the stability and correct separation of chromosomes during mitosis and meiosis of eukaryotic cells. It is composed of chromosomal DNA and various protein molecules in the region. Centromere DNA usually contains two types of high-abundance repetitive sequences: centromere-specific satellite DNA repeats and retrotransposon sequences (Gong et al., 2012; Zhang et al., 2014; Zhu et al. ., 2016; Li et al., 2018). Due to the mode of transposition and replication in the genome, retrotransposons have become the most widely distributed type of DNA in eukaryotic genomes during evolution, usually accounting for more than 50% of plant nuclear genomes (Lee et al., 2005...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11
CPCC07K14/415
Inventor 席梦利辛昊阳吴玉峰张文利宁仪杭刘光欣
Owner NANJING FORESTRY UNIV
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