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Method for screening single-copy T-DNA transgenic plant

A transgenic, single-copy technology, applied in the field of molecular biology, can solve the problems of amplification errors, lack of simple and reliable methods, and inability to distinguish two-fold differences in copy number, etc., achieving great application prospects, improving efficiency, and direct method. Effect

Pending Publication Date: 2021-12-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multi-step calculations greatly magnify error, making it impossible to distinguish two-fold differences in copy number
Therefore, although there are many methods for detecting single-copy transgenic plants, there is still a lack of simple and reliable methods.

Method used

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  • Method for screening single-copy T-DNA transgenic plant
  • Method for screening single-copy T-DNA transgenic plant
  • Method for screening single-copy T-DNA transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The principle and design idea of ​​embodiment 1SC PCR method

[0034] In the case of ensuring the amplification efficiency, the PCR amplification is close to 2 n Amplification was carried out (n is the number of cycles). Real-time quantitative PCR judges the copy number of the template by comparing the number of cycles (Ct value) at which the fluorescent signal of the amplified product reaches the threshold value. The number of templates amplified by a single-copy T-DNA template is reduced by half relative to that of a double-copy T-DNA, so the Ct value increases by 1. We use a pair of reference sequences (R 1 and R 2 ) to evaluate the change of Ct value of T-DNA. In our design, a pair of reference sequences R 1 and R 2 The amplification Ct values ​​of the double-copy T-DNA plants are between R 1 and R 2 between, forming a sandwich of R 1 -T-R 2 The sandwich Ct relation of . Compared with double-copy plants, the Ct value of T-DNA amplification increases by ab...

Embodiment 2

[0035] Example 2 Establishment and verification of SC PCR method for screening single-copy T-DNA transgenic plants

[0036] (1) We selected the transgenic plant seb19 inserted at a single site as the standard template (Hu,Y.,Chen, Z.,Zhuang,C.and Huang,J.(2017)Cascade of chromosome rearrangements caused by aheterogeneous T-DNA integration supports the double-stranded break repairmodel for T-DNA integration. Plant J, 90, 954-965.), to calibrate the sandwich relationship of Ct value. The seb19 transgenic plant has only one insertion site, and its progeny seb19-S is a homozygous plant with two T-DNA copies; while seb19-L is a heterozygous plant with only one T-DNA copy. The vegetative growth performances of the two offspring plants were different, and it was easy to distinguish them. The vegetative growth of seb19-L was consistent with wild type, while the vegetative growth of seb19-S was dwarf. We designed two pairs of reference sequences for the single-copy gene At1g06570 in ...

Embodiment 3

[0046] Embodiment 3 A method for obtaining single-copy T-DNA transgenic plants

[0047] (1) Obtaining Arabidopsis transgenic plants

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Abstract

The invention discloses a method for screening a single-copy T-DNA transgenic plant. The method comprises the following steps: forming a sandwich relationship (R1-T-R2) in a double-copy transgenic plant through a pair of reference amplified fragments (R1 and R2) in a plant genome and a Ct value amplified by T-DNA (T); and enabling the Ct value of the double-copy T-DNA transgenic plant to be between R1 and R2, and forming a sandwich type R1-T-R2 sandwich Ct relationship. Compared with a double-copy plant, the T-DNA amplification Ct value of the single-copy plant is increased by about 1, and the Ct value of the single-copy plant is shifted out of the sandwich relationship, so that a Ct mode of R1-R2-T is generated; and a single-copy T-DNA transgenic plant is found out by comparing different Ct value relationships. The method does not need to calculate [delta]Ct, [delta][delta]Ct and 2-[delta][delta]Ct, the change of the transgenic copy number is judged by directly displaying the relationship between the two pairs of reference sequences (R1 and R2) and the amplification Ct value of T-DNA, and the method is direct, simple and reliable.

Description

technical field [0001] The invention relates to the technical field of molecular biology, more specifically, to a method for screening single-copy T-DNA transgenic plants. Background technique [0002] Agrobacterium-mediated transformation of plants depends on the Ti plasmid (tumor inducing plasma) possessed by Agrobacterium tumefaciens. The Ti plasmid contains a T-DNA sequence (Transfer DNA) between two short sequences LB (left border) and RB (right border). The T-DNA can enter the host cell and integrate into the plant as Agrobacterium infects the plant in the genome. In addition to T-DNA, the Ti plasmid also encodes some toxic proteins (Vir region, figure 1 ). Toxin acts as a trans factor to assist Agrobacterium to infect plants and realize the transfer and integration of T-DNA. On the Ti plasmid of wild Agrobacterium tumefaciens, the protein encoded by the T-DNA gene can promote the continuous division of plant cells and cause root cancer. The discovery and transfor...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6851
CPCC12Q1/6895C12Q1/6851C12Q2600/13C12Q2531/113C12Q2537/125C12Q2561/113C12Q2563/107Y02A40/146
Inventor 胡宇飞陈晓静黄吉雷庄楚雄
Owner SOUTH CHINA AGRI UNIV
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