Chinese cabbage eIF (iso) 4E.c site transposon degenerated hull

A Chinese cabbage, transposon technology, applied in the field of transposon insertion mutants of eukaryotic translation initiation factor eIF4E.c in Chinese cabbage

Inactive Publication Date: 2014-07-02
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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  • Chinese cabbage eIF (iso) 4E.c site transposon degenerated hull
  • Chinese cabbage eIF (iso) 4E.c site transposon degenerated hull
  • Chinese cabbage eIF (iso) 4E.c site transposon degenerated hull

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Cloning of eIF(iso)4E.c in different Chinese cabbage inbred line materials

[0033] 1.1 Chinese cabbage genomic DNA extraction

[0034] (1) Put the leaves of Chinese cabbage seedlings into a liquid nitrogen pre-cooled mortar, and grind them into powder in liquid nitrogen;

[0035] (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;

[0036] (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;

[0037] (4) Centrifuge at 12000 rpm for 15 minutes at room temperature;

[0038] (5) Use a pipette to carefully suck out the upper aqueous phase, add it ...

Embodiment 2

[0057] Example 2 Development and verification of co-dominant ASM markers

[0058] Since there are more than 3,000 base insertion mutations in the genome sequences of BrA.eIF(iso)4E.c and BrA.eIF(iso)4e.c2, the pair of primers used to amplify the gene are highly conserved and can be Used for developing tags. We used the primers to amplify this gene in the backcross population of (DaQinBai×06-247)×DaQinBai for verification. The specific implementation rules are as follows:

[0059] (1) The genomic DNA of each individual plant in the backcross population was extracted as described in 1.1.

[0060] (2) PCR amplification: The preparation and amplification conditions of the PCR reaction solution are as described in item (2) of 1.2.

[0061] (3) Detection of PCR products is as described in Example 2. Test results such as Figure 4 As shown, M is the molecular weight standard DL5000, and 1-10 is 10 individual plants of the backcross population. From the amplification results, it...

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Abstract

The invention discloses site specific dominant ASM marks directly relevant to Chinese cabbage eIF (iso) 4E.c wild type and transposon degenerated hull identification. The mark relevant to the wide site detection is named as ASM-4E.c-W, the size of segments is 1216bp, which is shown as SEQ ID No.1; the corresponding mark relevent to the mutation site detection is named as ASM-4e.c-I, and the size of segments is 4419bp, which is shown as SEQ ID No.2. The invention utilizes a homologous cloning technology for finding one transposon degenerated hull of the eIF (iso) 4E.c site and developing the molecular marks for itentifying the site. People can utilize the mark for accurately selecting the gene types of backcross-transformation later generations so as to seak and create hull materials with more abundant genetic backgrounds.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68A01H1/04
Inventor 刘栓桃赵智中张志刚李巧云卢金东
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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