Specific molecular marker of deletion mutation of base at site of eIF(iso) 4E.c of Chinese cabbage and application thereof

A deletion mutation and molecular marker technology, applied in DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effect of simple operation, stable results, and improved screening efficiency

Inactive Publication Date: 2014-02-26
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, there have been no reports at home and abroad on the mutation of Chinese cabbage at the above two gene loci and the development of markers for the detection of related mutants.

Method used

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  • Specific molecular marker of deletion mutation of base at site of eIF(iso) 4E.c of Chinese cabbage and application thereof
  • Specific molecular marker of deletion mutation of base at site of eIF(iso) 4E.c of Chinese cabbage and application thereof
  • Specific molecular marker of deletion mutation of base at site of eIF(iso) 4E.c of Chinese cabbage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Cloning of eIF(iso)4E.c in different Chinese cabbage inbred line materials

[0034] 1.1 Chinese cabbage genomic DNA extraction

[0035] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0036] (2) After the liquid nitrogen evaporates to dryness, transfer it to a 2ml centrifuge tube immediately, add about 0.6ml of CTAB extract preheated to 65°C for every 100mg of material, after melting, vigorously shake and mix the sample, place it in a 65°C water bath for 40- 60 minutes to lyse the cells;

[0037] (3) After the lysis is complete, take out the sample and let it cool down to room temperature completely. Add an equal volume of chloroform (chloroform), gently invert to mix, and place at room temperature for 10 minutes;

[0038] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0039] (5) Use a pipette to carefully suck out the upper aqueous phase, add it ...

Embodiment 2

[0059] Example 2 Development of co-dominant ASM markers

[0060] Since the genomic sequences of BrA.eIF(iso)4E.c and BrA.eIF(iso)4e.c have four base insertion / deletion mutations, and the nucleic acid sequences on both sides of the mutation site are not well conserved, according to Therefore, we used a nested PCR strategy to amplify the mutation site. First, a pair of primers (PF1 and PR1) for amplifying the genome sequence were used for the first round of PCR amplification, and then inner nested primers PF2 and PR1 were designed on both sides of the mutation site. For PR2 (as shown in SEQ ID NO.4, 6), the second round of PCR amplification is performed using the first PCR amplification product as a template, and the PCR amplification conditions are as shown in item (2) of 1.2.

[0061] The amplified products were separated by electrophoresis on 8% polyacrylamide gel and stained with silver nitrate. The staining procedure was referred to Zhang Chunlei et al. Comparison. Journal...

Embodiment 3

[0062] Example 3 Identification of F2 population of Chinese cabbage combination He102×06-247 by ASM markers

[0063] (1) The genomic DNA of each individual plant of the F2 population was extracted as described in 1.1.

[0064] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0065] (3) Detection of PCR products is as described in Example 2. Test results such as Figure 5 As shown, P1 is parent 1, that is, 06-247, P2 is parent 2, that is, He102A, and 1-21 are 21 F2 generation single plants. It can be seen from the figure that 6, 11, 12, 15, 17 and 20 are the same as parent 1 and are wild type, while 10 and 16 are the same as parent 2 and are mutants, and the rest are heterozygous. It can be seen that the nested PCR strategy is adopted, and the accuracy rate of identifying the wild type, base deletion mutant type and heterozygous type of the eIF(iso)4E.c site is 100% through two PCR amplifi...

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Abstract

The invention discloses a specific codominant acid sphingomyelinase (ASM) marker at a site of a Chinese cabbage, and the specific codominant ASM marker at the site of the Chinese cabbage is directly related to identification of a wild type and a base deletion mutant at the eIF(iso) 4E.c of the Chinese cabbage. A marker related to detection of the site of the wild type is named as ASM-W, the size of a segment is 128bp, as shown in SEQ ID No.1. A marker related to detection of the corresponding site of the base deletion mutant is named as ASM-m, and the size of a segment is 124bp, as shown in SEQ ID No.2. According to a specific molecular marker of the deletion mutation of a base at the site of eIF(iso) 4E.c of the Chinese cabbage and application of the specific molecular marker, the homologous cloning technology is utilized to find a mutant at the side of eIF(iso) 4E.c, and the specific molecular marker used for indentifying the site is developed. The specific molecular marker can be utilized for accurate selection of genotypes of backcrossing descendants. At the same time, the specific molecular marker can also be used for sorting out Chinese cabbage germplasm resources to seek for mutant materials which are more abundant in genetic background.

Description

technical field [0001] The present invention relates to the development of a gene mutant and related site-specific molecular markers, in particular to a base deletion mutation of Chinese cabbage eukaryotic translation initiation factor eIF(iso)4E.c and its site Development and application of specific molecular markers. The mutant can provide a source of variation for the selection of virus-resistant Chinese cabbage germplasm containing the mutation site, and the site-specific molecular markers can be directly applied to molecular marker-assisted breeding to select Chinese cabbage materials containing the mutation site and improve eIF4E mutation The invention relates to the selection efficiency of a body, and belongs to the field of biotechnology. Background technique [0002] Chinese cabbage (Brassica rapa L.ssp pekinensis) is an important vegetable crop of the Brassicaceae family, which originated in China and is currently grown all over the world. Especially in the peren...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 刘栓桃赵智中卢金东张志刚李巧云
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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