Specific molecular markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers

A mutation site, molecular marker technology, applied in DNA/RNA fragment, microbial determination/inspection, recombinant DNA technology, etc., to achieve the effect of improving screening efficiency, stable results, and easy operation

Inactive Publication Date: 2012-10-03
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 markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers
  • Specific molecular markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers
  • Specific molecular markers of eIF4E.a mutation site of Chinese cabbage and application of Specific molecular markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Cloning of eIF4E.a in different Chinese cabbage inbred lines

[0035] 1.1 Chinese cabbage genomic DNA extraction

[0036] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0037] (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;

[0038] (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;

[0039] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0040] (5) Use a pipette to carefully suck out the upper aqueous phase, add it to a new 1.5ml...

Embodiment 2

[0061] Example 2 Development of dominant ASM markers

[0062] 2.1 Primer design

[0063] Carefully compare the genome sequences of BrA.eIF4E.a1, BrA.eIF4E.a2, BrA.eIF4e.a1, and BrA.eIF4e.a2. The regions with large differences are between 150-600bp, and the key is located in the exon region Therefore, for the difference between the wild type and the mutant, site-specific primers were designed with Primer Premier 5.0 software. The forward primer sequence related to mutant detection is Fm: 5'-GGAAGCTCCTTACGATCCGTCTTCT-3', the forward primer related to wild type detection is FW: 5'-GGAAGCTCCTTACGATCCGTCTTCAC-3', and the universal reverse primer is RWm: 5 '-TGGCACAGATAGGATCCTCCCAC-3'. The primers were synthesized by Beijing Huada Gene Company.

[0064] 2.2 Acquisition of ASM mark:

[0065] (1) Amplification using (FW+RWm) and (Fm+RWm) primer combinations in anti- / sensitivity materials respectively: the preparation of PCR reaction solution and amplification conditions are as des...

Embodiment 3

[0068] Example 3 Identification of different resources of Chinese cabbage by ASM markers

[0069] (1) Genomic DNA extraction of different Chinese cabbage resources: 8 different Chinese cabbage materials were randomly selected, and the genomic DNA of these materials was extracted as described in 1.1.

[0070] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0071] (3) Detection of PCR products is as described in item (3) of 1.2. Test results such as Figure 6 As shown, A: FW+RWm combination amplification result; B: Fm+RWm combination amplification result; M molecular weight standard DL2000, 1-8 are eight Chinese cabbage materials. It can be seen from the figure that 1-4 are wild type and 5-8 are mutants. The identification efficiency of the two sets of primers for homozygous wild type and mutants was 100%.

[0072] (4) Sequencing verification of the eIF4E site was as described in 1.2. Th...

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Abstract

The invention discloses two site-specific dominant ASM markers which are directly related with the identification of a wild type and a mutant of eIF4E.a of Chinese cabbage. One marker is that: a marker which is related with the detection of a wild type site is named ASM-4E.a-s and is shown as SEQ ID No.1; and a marker which is related with the detection of the corresponding mutation site is namedASM-4e.a-s and is shown as SEQ ID No.3. The other marker is that: a marker which is related with the detection of a wild type site is named ASM-4E.a-lc and is shown as SEQ ID No.2; and a marker whichis related with the detection of the corresponding mutation site is named ASM-4e.a-1c and is shown as SEQ ID No.4. The invention also discloses a primer for identifying the two specific dominant molecular markers. The primer is shown as SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7. The markers and the primer can be taken as molecular markers and applied to the identification of germplasm resources ofthe Chinese cabbage and the aided selection of breeding; and a germplasm material or a transformation offspring containing an eIF4E.a mutation type in 06-247 or Guan291 is selected.

Description

technical field [0001] The present invention relates to the development of a gene mutant and its related site-specific molecular markers, in particular to the development and application of a mutation of Chinese cabbage eukaryotic translation initiation factor eIF4E.a and its site-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 perennial suppl...

Claims

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Application Information

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