A copia retrotransposon specific to the centromere region of p genome and its application

A centromere, specific primer pair technology, used in recombinant DNA technology, microbial assay/inspection, DNA/RNA fragments, etc.

Active Publication Date: 2017-07-21
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no research in this area has been seen in Agropyron

Method used

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  • A copia retrotransposon specific to the centromere region of p genome and its application
  • A copia retrotransposon specific to the centromere region of p genome and its application
  • A copia retrotransposon specific to the centromere region of p genome and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, the discovery of pAcpCR1 and its distribution characteristics on the chromosome of wheatgrass

[0063] 1. Discovery of pAcpCR1

[0064] The 6P short arm DNA sequence fragment was obtained by microdissection and DOP-PCR using wheat-Agropyron 6P short arm system as material. These sequences were connected to the pMD19-T vector and introduced into Escherichia coli to obtain a library of 6P short DNA sequence fragments. Differential DNA clones between wheatgrass Z559 and wheat Fukuho were screened by dot hybridization and sequenced.

[0065] According to BLAST analysis, there is a DNA fragment with a length of 1742bp and a GC content of 43.8%. The DNA fragment is named pAcpCR1, as shown in sequence 1 of the sequence listing.

[0066] Nucleotides 1041-1664 at the 5' end of pAcpCR1 have a maximum of 82% sequence similarity with a long-terminal (LTR) Copia-like retrotransposon in the wheat 3B-specific BAC library. This wheat retrotransposon The transposon is part...

Embodiment 2

[0072] Embodiment 2, the development of specific primer pair

[0073] 1. Based on pAcpCR1, a specific primer pair was designed as follows:

[0074] AcpCR1F (sequence 2 of the sequence listing): 5'-GCAAAGCTGACCAGATGACA-3';

[0075] AcpCR1R (SEQ ID NO: 3 of the Sequence Listing): 5'-TCGAAAGAGCAAAAGCACAA-3'.

[0076] 2. Extract the genomic DNA of each sample to be tested respectively.

[0077] 3. Using each genomic DNA as a template, perform PCR amplification using the specific primer pair obtained in step 1 to obtain PCR amplification products.

[0078] 4. Carry out 1% agarose gel electrophoresis for each PCR amplification product obtained in step 3, the results are shown in image 3 .

[0079] image 3 Among them, M corresponds to marker, 1 corresponds to Wheatgrass Z559, 2 corresponds to wheat Fukuho, 3 corresponds to wheat-Wheatgrass 6P disomy addition line 4844-12, 4 corresponds to wheat-Wheatgrass addition line 5113, and 5 corresponds to wheat-Wheatgrass addition line 5...

Embodiment 3

[0082] Embodiment 3, the specificity of specific primer pair

[0083] 1. Extract the genomic DNA of each sample to be tested.

[0084] 2. Taking each genomic DNA as a template and using a specific primer pair composed of AcpCR1F and AcpCR1R to carry out PCR amplification to obtain PCR amplification products.

[0085] 3. Perform 1% agarose gel electrophoresis on each PCR amplification product obtained in step 2, the results are shown in Figure 4 .

[0086] Figure 4 Among them, M corresponds to marker, 1 corresponds to wheatgrass Z1842, 2 corresponds to wheatgrass Z559, 3 corresponds to wheatgrass Z1750, 4 corresponds to wheat Chinese spring, 5 corresponds to wheat Fukuho, and 6 corresponds to cultivating einkorn wheat MO 4 , 7 corresponds to durum wheat Jing DR 3 , 8 corresponds to Timofeywheat TI 1 , 9 corresponds to Ae14, 10 corresponds to Y93, 11 corresponds to Y258, 12 corresponds to Ae49, 13 corresponds to Y39, 14 corresponds to RM2161, 15 Corresponds to Thinopyrum...

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Abstract

The present invention discloses a P genome acrocentric region specific Copia retrotransposon and applications thereof, wherein the P genome acrocentric region specific Copia retrotransposon is the following (a) or (b): (a) a DNA molecule represented by the nucleotides site 250-883 from the 5' terminal in the sequence 1, and (b) a DNA molecule represented by the sequence 1. The invention further discloses a specific primer pair comprising a single-stranded DNA molecule represented by the sequence 2 in the sequence list and the single-stranded DNA molecule represented by the sequence 3 in the sequence list. According to the present invention, the obtaining of the dispersed repetitive sequence pAcpCR1 provides the important effect for the research of the Agropyron cristatum plant evolution; a lot of wheat-Agropyron cristatum translocation lines are obtained, such that the new challenge is provided for the rapid detection of the exogenous Agropyron cristatum chromatin; and with the obtaining of the retrotransposon specific molecular marker based on the P genome acrocentric region, the efficient detection of the Agropyron cristatum acrocentric region P chromatin under the wheat background can be performed.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Copia retrotransposon specific for the near-centromere region of the P genome and an application thereof. Background technique [0002] DNA repeats are a major component of most plant genomes. It can be divided into two categories: tandem repeats and scattered repeats. Most of the dispersed repeats have the characteristics and characteristics of transposons and retrotransposons. Such scattered repetitive DNA sequences are not only an important part of plant genomes but also a major force in the evolution of plant genomes. Long-terminal retrotransposons are the focus of research in repetitive sequences. In recent years, some new scattered repeat sequences have been isolated from plants, which provides a better understanding of the structure of retrotransposons and the evolution of plant genomes. However, no research in this area has been found in the genus Agropyron....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李立会韩海明刘伟华胡赞民张锦鹏鲁玉清杨欣明李秀全
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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