P genome specific Gypsy retrotransposon and application thereof

A kind of specific, specific primer pair technology, applied in the direction of recombinant DNA technology, microbial assay/inspection, DNA/RNA fragment, etc.

Active Publication Date: 2016-02-17
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • P genome specific Gypsy retrotransposon and application thereof
  • P genome specific Gypsy retrotransposon and application thereof
  • P genome specific Gypsy retrotransposon and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0065] Example 1, the discovery of pAcPR1 and its distribution characteristics on the chromosome of wheatgrass

[0066] 1. Discovery of pAcPR1

[0067] 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 DNA sequence fragment library of the 6P short arm. Differential DNA clones between wheatgrass Z559 and wheat Fukuho were screened by dot hybridization and sequenced.

[0068] According to BLAST analysis, there is a DNA fragment with a length of 1793bp and a GC content of 37.9%. The DNA fragment is named pAcPR1, as shown in sequence 1 in the sequence listing.

[0069] pAcPR1 has the most sequence similarity (73%) to a long terminal (LTR) Gypsy-like retrotransposon in the wheat 3B-specific BAC library, which is part of cgt0464b.

[0070] pAcPR1 is a new sequence.

[0071] 2. The distributio...

Embodiment 2

[0077] Embodiment 2, development and application of specific primer pair

[0078] 1. Based on the pAcPR1 sequence, a specific primer pair was designed as follows:

[0079] AcPR1F (upstream primer, sequence 2 in the sequence listing): 5'-CCCCTTCATTAAGGTATTGTTCC-3';

[0080] AcPR1R (downstream primer, sequence 3 in the sequence listing): 5'-CTTGTCCACATGTTGTGTGCTAT-3'.

[0081] 2. Extract the genomic DNA of each sample to be tested respectively.

[0082] 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.

[0083] 4. Carry out 1% agarose gel electrophoresis for each PCR amplification product obtained in step 3, the results are shown in Figure 4 .

[0084] Figure 4 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 l...

Embodiment 3

[0087] Embodiment 3, the specificity of specific primer pair

[0088] 1. Extract the genomic DNA of each sample to be tested.

[0089] 2. Using each genomic DNA as a template, PCR amplification is carried out with specific primer pairs composed of AcPR1F and AcPR1R to obtain PCR amplification products.

[0090] 3. Perform 1% agarose gel electrophoresis on each PCR amplification product obtained in step 2, the results are shown in Figure 5 .

[0091] Figure 5 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 the cultivated b...

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Abstract

The invention discloses a P genome specific Gypsy retrotransposon and application thereof. P genome specific Gypsy retrotransposon provided by the invention is as shown in either (a) or (b), wherein (a) refers to a DNA molecule shown as nucleotides on the 531st-1575th sites from 5' end of a sequence 1; and (b) refers to a DNA molecule as shown in the sequence 1. The invention also protects a specific primer pair composed of a single-stranded DNA molecule as shown in a sequence 2 in a sequence list and a single-stranded DNA molecule as shown in a sequence 3 in the sequence list. The Gypsy retrotransposon and the application thereof disclosed by the invention have the following significances: (1) the acquisition of an interspersed repetitive sequence pAcPR1 is important for researching the evolution of agropyron plants; and (2) the acquisition of a great amount of wheat-agropyron translocation lines brings a new challenge for the rapid detection of exogenous agropyron chromatins; and the acquisition of a specific molecular marker based on the P genome retrotransposon is conductive to the efficient detection of the agropyron chromatins on the background of wheat.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a P genome-specific Gypsy retrotransposon and its application. 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. Therefore, it is of great evolutionary sign...

Claims

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Application Information

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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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