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Method for efficiently and rapidly separating T-DNA insertion site flanking sequence, and uses thereof

An insertion site and flanking sequence technology, which is applied in the field of efficient and rapid separation of T-DNA insertion site flanking sequences, can solve the problems of low efficiency, low throughput, low success rate, etc., and achieves the effect of economy and simple analysis.

Inactive Publication Date: 2017-09-22
武汉天问生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

That is, the purpose of the present invention is to overcome the shortcomings of low throughput, low efficiency and low success rate of the existing flanking sequence separation technology, combine DNA capture and second-generation sequencing technology, specifically sequence the T-DNA border sequence and through simple analysis The flanking sequence of the T-DNA insertion site can be obtained, which can be applied to the verification of gene function in plants, transgenic breeding and variety improvement

Method used

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  • Method for efficiently and rapidly separating T-DNA insertion site flanking sequence, and uses thereof
  • Method for efficiently and rapidly separating T-DNA insertion site flanking sequence, and uses thereof
  • Method for efficiently and rapidly separating T-DNA insertion site flanking sequence, and uses thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Agrobacterium-mediated genetic transformation

[0045] The Agrobacterium-mediated genetic transformation method mainly refers to the method shown in the "Agrobacterium-mediated Genetic Transformation Operation Manual" published by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (Lin Yongjun et al., Agrobacterium-mediated Mudanjiang No. 8 high-efficiency Establishment of transgenic system, Acta Crops Sinica, 2002, 28(3): 294-300).

[0046] As shown in Figure 1, the transformation vector is pC1300, the exogenous gene is the hygromycin selection marker gene, pCAMBIA1300 (GenBank No. AF234296.1), and pC1300 is used to electrotransform into Agrobacterium strain EHA105 (purchased from Huazhong Agricultural University) for standby, transformation The recipient is the embryogenic callus induced by mature seeds of rice variety Zhonghua 11. The embryogenic calli were pre-cultured for 3 days, infected with Agrobacterium containing...

Embodiment 2

[0047] Example 2: Extraction of Genomic DNA

[0048] Take embodiment 1 and randomly select 4 strains of T 0 Generation of transgenic rice, 2g of young leaves per plant, after grinding with liquid nitrogen, add 2ml 80°C 1.5×CTAB (15g CTAB; 75ml 1M Tris-HCl, pH 8.0; 30ml 0.5M EDTA, pH 8.0; 61.4g NaCl; Set volume to 1L, stir and dissolve with a stirrer for 2h); 65°C water bath for 30min; add 2ml of chloroform / isoamyl alcohol (24:1) and slowly invert up and down for 15min until the lower liquid phase turns dark green; centrifuge at 3000r / min for 15min at room temperature; take Put the supernatant in a new 10ml centrifuge tube, add 200μl 10% CTAB and 2ml chloroform / isoamyl alcohol (24:1) slowly upside down for about 15min; centrifuge at 3000r / min for 15min at room temperature; take 1.5ml supernatant, add 4ml 1% CTAB, after mixing, pick out the precipitate with a pipette tip and dissolve it in 1ml 1M NaCl (plus 1μl RNase). Add 3ml of frozen absolute ethanol to precipitate the DNA,...

Embodiment 3

[0049] Example 3: DNA library construction

[0050] DNA purification: take 1 μg DNA (prepared in Example 2) and dissolve it in 50 μl ddH 2 After being neutralized in O, it was crushed to a size of 300-500 bp by an ultrasonic crusher. Add 0.5X magnetic beads (Beckman, http: / / www.beckmancoulter.cn / ), mix well and place at room temperature for 15 minutes; use a magnetic stand (invitrogen, http: / / www.thermofisher.com / cn / zh / home. html) Adsorb the magnetic beads, transfer the supernatant to a new centrifuge tube, add 0.5X magnetic beads again, mix well and place at room temperature for 15 minutes; absorb the magnetic beads with a magnetic stand, discard the supernatant, add 200 μl 80% ethanol, and wash for 2 all over. After drying, add 10 μl ddH 2 O dissolved DNA, set aside.

[0051] DNA repair, addition of A and ligation: DNA repair and addition of A use the kit (E7546S) from NEB (http: / / www.neb-china.com / ) company, see the instructions for the operation steps. After the react...

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Abstract

The invention provides a method for efficiently and rapidly separating a T-DNA insertion site flanking sequence. The method comprises: extracting the genomic DNA of an Agrobacterium-mediated transgenic plant; fragmenting the genomic DNA, carrying out DNA repairing, adding A, linking, carrying out fragment selection by using magnetic beads, carrying out library construction, carrying out hybridization capture on the constructed library and a T-DNA boundary sequence, and carrying out PCR enrichment; and carrying out high-throughput sequencing on the library, and carrying out bioinformatics analysis on the sequenced data so as to obtain the T-DNA insertion site. According to the present invention, the capture is performed with the target DNA, and the low-throughput sequencing is specially performed on the T-DNA boundary sequence; the disadvantages of low throughput, low efficiency and low success rate of the existing flanking sequence separation technology are overcome with the method of the present invention; and with the method of the present invention, the DNA capture and the second-generation sequencing technology are combined, the T-DNA boundary sequence is specially sequenced and the simple analysis is performed to obtain the T-DNA insertion site flanking sequence, and the method has characteristics of high efficiency, economy, and simple analysis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently and rapidly isolating the flanking sequence of a T-DNA insertion site and its application. Background technique [0002] With the rapid development of plant genetic engineering, Agrobacterium-mediated plant transformation technology has become an important method and technology in the process of plant transgenic breeding and plant gene function research. The isolation and identification of the flanking sequences of the T-DNA insertion site plays a key role in the genetic analysis of transgenic plants. At present, there are mainly the following methods for separating the flanks: [0003] 1. TAIL-PCR, whose main principle is based on specific nested primers at one end and random degenerate primers at the other end, after multiple rounds of amplification, flanking sequences can be isolated; 2. Inverse PCR, this method is mainly for genomic DNA Carr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 凌飞崔莹
Owner 武汉天问生物科技有限公司
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