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A method for screening rice targeted gene edited plants

A technology for targeting genes and plants, applied in the field of plant genetic engineering, can solve problems such as poor sensitivity, lack of single-plant identification methods, false positives, etc., and achieve the effects of simple and fast operation, improved plant screening efficiency, and high accuracy.

Active Publication Date: 2018-06-05
无锡哈勃生物种业技术研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is faster and less expensive than the clone sequencing method, but the sensitivity is not as good as the clone sequencing method, and there are false positives
Therefore, for the mutations in the CRISPR targeted editing population, there is still a lack of a high-accuracy, low-cost, fast, and high-throughput single-plant identification method

Method used

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  • A method for screening rice targeted gene edited plants
  • A method for screening rice targeted gene edited plants
  • A method for screening rice targeted gene edited plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The above-mentioned population of CRISPR-targeted editing of the rice LOC_Os03g55240 gene is the test plant, and the unedited rice plant is the control plant. The steps to detect and screen the plants to be tested are as follows:

[0081] 1. Extraction of rice genomic DNA

[0082] (1) The rice leaves are cut into pieces and placed in a 2.0mL centrifuge tube, and a steel ball is placed at the same time, and ground with a tissue mill;

[0083] (2) Add 800 μL CTAB extraction buffer (Tris-HCl, 100 mM, pH 8.0; EDTA, 20 mM, pH 8.0; NaCl, 500 mM; CTAB, 2%), water bath at 65°C for 40 min, and shake 3-4 times during this period;

[0084] (3) add an equal volume of chloroform-isoamyl alcohol mixed solution (wherein the volume ratio of chloroform and isoamyl alcohol is 24:1), invert up and down and mix, and centrifuge at 10000r / min for 10min;

[0085] (4) Transfer the supernatant to a new 1.5mL centrifuge tube, add an equal volume of isopropanol pre-cooled at -20°C, gently invert ...

Embodiment 2

[0102] The plants to be tested are detected and screened using the same method as in Example 1, the difference is only that the PCR amplification steps are different, and the details are as follows:

[0103] The PCR primer pairs adopted in this example are as follows:

[0104] Upstream primer B2F: 5'-TCACCGAGCACGACGTGACCTT-3'; (SEQ ID NO:6)

[0105] Downstream primer B2R: 5'-CACGCTGAGGGAGACCTCGAACA-3'; (SEQ ID NO:7)

[0106] It was synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0107] The PCR amplification reaction system is as follows: rTaq, 0.2 μL; 2×GC buffer, 5 μL; 2.5mix dNTPs, 1.6 μL; 10 μM upstream primer B2F, 0.2 μL; 10 μM downstream primer B2R, 0.2 μL; 10×Eva Green (fluorescence dye), 1 μL; 50 ng / μL DNA, 1 μL; sterile water 1.3 μL; mineral oil 10-20 μL.

[0108] The PCR reaction program was: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, a total of 40 cycles; extension at 72 ...

Embodiment 3

[0151] The population of the above-mentioned large spot gene LOC_Os12g16720 CRISPR-targeted editing is the test plant, and the unedited rice plant is the control plant. The steps to detect and screen the plants to be tested are as follows:

[0152] 1. Extraction of rice genomic DNA

[0153] (1) The rice leaves are cut into pieces and placed in a 2.0mL centrifuge tube, and a steel ball is placed at the same time, and ground with a tissue mill;

[0154] (2) Add 800 μL CTAB extraction buffer (Tris-HCl, 100 mM, pH 8.0; EDTA, 20 mM, pH 8.0; NaCl, 500 mM; CTAB, 2%), water bath at 65°C for 40 min, and shake 3-4 times during this period;

[0155] (3) add an equal volume of chloroform-isoamyl alcohol mixed solution (wherein the volume ratio of chloroform and isoamyl alcohol is 24:1), invert up and down and mix, and centrifuge at 10000r / min for 10min;

[0156] (4) Transfer the supernatant to a new 1.5mL centrifuge tube, add an equal volume of isopropanol pre-cooled at -20°C, gently in...

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Abstract

The invention discloses a method for screening a rice plant subjected to targeted gene editing. The method comprises the following steps: extracting genomic DNA of a rice plant to be detected and genomic DNA of a contrast rice plant, adding specific primers and a fluorescent dye, performing PCR amplification on DNA segments containing editing sites in the extracted genomic DNA, then, performing HRM (High Resolution Melting) analysis, and contrasting corresponding high resolution melting curves of the plant to be detected and the contrast plant by taking the high resolution melting curve corresponding to the contrast plant as a reference line to obtain the maximum fluorescence difference delta F; if the absolute value of the delta F is smaller than 0.05, determining that the plant to be detected is not successfully edited; if the absolute value of the delta F is not smaller than 0.05, determining that the plant to be detected is successfully edited. The method provided by the invention is simple, convenient, quick, effectively, accurate in result, high in flux, and low in use cost; when the method is used for assisting seed breeding, the plant screening efficiency can be greatly improved.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a method for screening rice targeted gene editing plants. Background technique [0002] In modern biological research, genome editing (GE) technology is an important technical means for people to understand and improve gene function. The development of genome editing technology has experienced successively the technology of zinc finger nucleases (Zinc finger nucleases, ZFNs), transcription activator like effector nucleases (Transcription activator like effector nucleases, TALENs) technology, and clustered regularly interspaced short palindromic repeats (clustered regularly interspaced repeats). short palindromic repeat, CRISPR) / Cas (CRISPR-assoicated) technology. All of these artificial nucleases can generate DNA double-strand breaks (DSBs) at DNA target sites, and then use two different repair mechanisms of non-homologous end joining or homologous recombinat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 李珊舒庆尧张渭章
Owner 无锡哈勃生物种业技术研究院有限公司
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