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Method for screening rice plant subjected to targeted gene editing

A technology targeting genes and plants, which is applied in the field of plant genetic engineering, can solve the problems of false positives, low sensitivity, lack of single plant identification methods, etc., achieve high accuracy, simple and fast operation, and improve the efficiency of plant screening

Active Publication Date: 2015-12-16
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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  • Method for screening rice plant subjected to targeted gene editing
  • Method for screening rice plant subjected to targeted gene editing
  • Method for screening rice plant subjected to targeted gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The population of CRISPR targeted editing of the rice LOC_Os03g55240 gene is the test plants, and the unedited rice plants are the control plants. The steps of detecting and screening the plants to be tested are as follows:

[0081] 1. Extraction of rice genomic DNA

[0082](1) Shred the rice leaves and put them in a 2.0mL centrifuge tube, put a steel ball at the same time, and grind them with a tissue mill;

[0083] (2) Add 800 μL CTAB extraction buffer (Tris-HCl, 100 mM, pH8.0; EDTA, 20 mM, pH8.0; NaCl, 500 mM; CTAB, 2%), 65 ° C water bath for 40 min, shake 3-4 times during the period;

[0084] (3) Add an equal volume of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl alcohol is 24:1), mix upside down, 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, mix gently by inversion, settling at -20°C for 30min, and centrifuge at ...

Embodiment 2

[0102] Adopt the method substantially identical with embodiment 1, detect and screen the plant to be tested, and its difference is only that the PCR amplification step is different, specifically as follows:

[0103] The PCR primer pair adopted in this embodiment is 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] Synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0107] The reaction system for PCR amplification is as follows: rTaq, 0.2 μL; 2×GCbuffer, 5 μL; 2.5mixdNTP 1.6 μL; 10 μM upstream primer B2F, 0.2 μL; 10 μM downstream primer B2R, 0.2 μL; 10×EvaGreen (fluorescent dye), 1 μL; 50ng / 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°C f...

Embodiment 3

[0151] The above-mentioned CRISPR targeted edited population of rice large spot gene LOC_Os12g16720 was the test plants, and the unedited rice plants were the control plants. The steps of detecting and screening the plants to be tested are as follows:

[0152] 1. Extraction of rice genomic DNA

[0153] (1) Shred the rice leaves and put them in a 2.0mL centrifuge tube, put a steel ball at the same time, and grind them with a tissue mill;

[0154] (2) Add 800 μL CTAB extraction buffer (Tris-HCl, 100 mM, pH8.0; EDTA, 20 mM, pH8.0; NaCl, 500 mM; CTAB, 2%), 65 ° C water bath for 40 min, shake 3-4 times during the period;

[0155] (3) Add an equal volume of chloroform-isoamyl alcohol mixture (the volume ratio of chloroform to isoamyl alcohol is 24:1), mix upside down, 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, mix gently by inversion, settling at -20°C for 30m...

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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-edited plants. Background technique [0002] In modern biological research, genome editing (Genomicediting, GE) technology is an important technical means for people to understand and improve gene function. The development of genome editing technology has successively experienced zinc finger nuclease (Zincfinger nucleases, ZFNs) technology, transcription activator like effector nuclease (Transcription activator like effector nucleases, TALENs) technology, and clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats (CRISPR) / Cass (CRISPR-assoicated) technology. These artificial nucleases can generate DNA double-strand breaks (Doublestrandbreaks, DSBs) at the DNA target site, and then use the two different repair mechanisms of non-homologous end joining or homo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2531/113C12Q2527/107C12Q2563/107
Inventor 李珊舒庆尧
Owner 无锡哈勃生物种业技术研究院有限公司
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