Agropyron cristatum centromere specific Gypsy retrotransposon sequence and application thereof

A species-specific, chromosomal centromere technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc.

Active Publication Date: 2016-03-30
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

In addition to the centromeric retrotransposons specific to the Triticum family, some species-specific cen

Method used

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  • Agropyron cristatum centromere specific Gypsy retrotransposon sequence and application thereof
  • Agropyron cristatum centromere specific Gypsy retrotransposon sequence and application thereof
  • Agropyron cristatum centromere specific Gypsy retrotransposon sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Discovery of pAcCR1 and its distribution characteristics on the chromosome of Agropyron chinensis

[0066] 1. Discovery of pAcCR1

[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] After BLAST analysis, there is a DNA fragment with a length of 871bp and a GC content of 46.3%. The DNA fragment is named pAcCR1. As shown in sequence 1 of the sequence table, pAcCR1 has a long terminal (LTR) Gypsy type retrotransposition characteristics of the child.

[0069] pAcCR1 shares 70% sequence similarity with scaffold23384 in the Uraltu wheat genome.

[0070] pAcCR1 is a new centromere sequence of Agro...

Embodiment 2

[0077] Embodiment 2, the development of specific primer pair

[0078] 1. Based on pAcCR1, a specific primer pair was designed as follows:

[0079] AcCR1F (upstream primer, sequence 2 of the sequence listing): 5'-GGCAAGGGAGTAACACAAGC-3';

[0080] AcCR1R (downstream primer, sequence 3 of the sequence listing): 5'-CCCCTCCAATTGTGAAAAGA-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 line 5113, and 5 corresponds to...

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. Taking each genomic DNA as a template and using a specific primer pair composed of AcCR1F and AcCR1R to carry out PCR amplification 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 cultivat...

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Abstract

The present invention discloses an agropyron cristatum centromere specific Gypsy retrotransposon sequence and application thereof, and an agropyron cristatum centromere specific Gypsy retrotransposon is (a) or (b): (a) is a DNA molecule shown as 37th-803th site nucleotides from 5 'end of a sequence 1, or (b) is a DNA molecule shown as the sequence 1. The present invention also provides a specific primer pair comprising a single-stranded DNA molecule shown as a sequence 2 of a sequence table and a single-stranded DNA molecule shown as a sequence 3 of the sequence table. Significances are as follows: (1) acquisition of centromeric retrotransposon pAcCR1 has an important role for the study of agropyron plant evolution; (2) due to acquisition of a large number of wheat-agropyron cristatum translocation lines, new challenges for rapid detection of exogenous chromatin in agropyron cristatum are put forward, and P chromatin of a centromeric region of the agropyron cristatum under wheat background can be efficiently detected on the basis of acquisition of a retrotransposon specific molecular marker of a P genome centromeric region.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a centromere-specific Gypsy retrotransposon sequence and application thereof. Background technique [0002] Retrotransposons can be divided into long-terminal retrotransposons and non-long-terminal retrotransposons, which are common in cereal crops. In plant centromeres, retrotransposons and microsatellite DNA are the most abundant DNA elements and associate with the centromere-specific histone CENH3. The absence of microsatellite repeats in wheat predominates centromeric retrotransposons. There are two large groups of long-terminal retrotransposons, Ty1-copia and Ty3-gypsy. CCS1 and pSau3A9 are part of the Ty3-gypsy class of retrotransposons that are well conserved at the centromere of cereal crops. In addition to the centromeric retrotransposons specific to the Triticum family, some species-specific centromeric retrotransposons were also isolated, but no research in ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李立会刘伟华韩海明宋利强胡赞民张锦鹏鲁玉清高爱农
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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