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Transgenic method for automatically deleting selectable marker

A selectable marker and automatic deletion technology, applied in the field of molecular biology, can solve the problems of limited application and low frequency of homologous recombination, and achieve reliable and effective results

Pending Publication Date: 2021-07-27
WUHAN BIORUN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low frequency of homologous recombination limits the application of this technique

Method used

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  • Transgenic method for automatically deleting selectable marker
  • Transgenic method for automatically deleting selectable marker
  • Transgenic method for automatically deleting selectable marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Optimization of Arr5 promoter sequence

[0051] In Arabidopsis, the expression of the Arr5 promoter can be induced by cytokinin, but its activity in other species is unknown. This embodiment obtains the Arr5 promoter gene sequence (Arr5 gene) from the Arabidopsis genome, and then the gene The sequence was incorporated into the promoter activity analysis vector to study its activity in rice transformed.

[0052] In order to verify the relationship between the activity and length of the Arr5 promoter, and finally find a promoter sequence that can effectively function and have the shortest sequence, in this example, three Arr5 gene sequences with different lengths were amplified, each with a length of 2k+bp. (2196bp, see SEQ ID NO:1 for the specific sequence), 1k+bp (997bp, the sequence from the 607th position to the 1603rd position in SEQ ID NO:1), 0.5k+bp (517bp, is the sequence of SEQ ID NO:1 NO: the sequence from the 1st site to the 517th site in NO:1), and ...

Embodiment 2

[0087] Embodiment 2 A kind of transgene carrier and its construction of automatic deletion selection marker

[0088] The DNA sequence of a transgenic vector that automatically deletes the selectable marker is arranged according to "carrier backbone---loxP---Arr5---Cre---selectable marker---loxP---carrier backbone", and its specific sequence is shown in SEQ ID NO: 2. In this example, the hygromycin resistance gene was used as the selection marker gene.

[0089] see figure 1 , the method for constructing a transgenic vector for automatically deleting a selectable marker, comprising the following steps:

[0090] S1. Construct a vector system including accepting vector A (pBWA), supplying vector B1 (pBWD(a)), and supplying vector B2 (pBWD(b)). For the construction method of the vector system, refer to Chinese patent CN103215296B, the patent name is: A multi-segment DNA molecular assembly method and its application; the present invention will not repeat them.

[0091] S2. Cloni...

Embodiment 3

[0169] Example 3 The transgenic vector that automatically deletes the selection marker is applied to the rice transgenic experiment

[0170] The pBWARE (I) vector constructed in Example 2 was transformed into rice M294 through Agrobacterium-mediated transformation, and the specific experimental process was as follows: Figure 14 Shown:

[0171] After mature rice embryos are dehulled, after a series of treatments, they are added to callus culture medium (N6macro+B5micro+B5vit+2,4-D 2mg / L+CH 300mg / L+L-proline 500mg / L+L- Glutamine 500mg / L+sucrose 30g / L+agar 7.5g / L pH5.8) to form callus, then add Agrobacterium containing pBWARE(I) vector to infect the callus, transfer to screening culture Base (N6macro+B5micro+B5vit+2,4-D2mg / L+CH 300mg / L+L-proline 500mg / L+L-glutamine 500mg / L+sucrose30g / L+agar7.5g / L+Hm 50mg / L+ ceph 250mg / L pH5.8), carry out resistance selection twice, obtain the transgenic callus tissue of resistance to hygromycin and kana, transfer the above-mentioned transgenic...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a transgenic method for automatically deleting a selectable marker. The method comprises the steps that a transgenic vector containing the selectable marker and capable of automatically deleting the selectable marker is constructed, and the transgenic vector is transformed into a plant to obtain a callus; the selectable marker gene can be normally expressed, and after cytokinin is added, the selectable marker gene is induced to be automatically deleted; the DNA sequence of the transgenic vector capable of automatically deleting the selectable marker is arranged according to a 'vector skeleton-loxP-Arr5-Cre-selectable marker-loxP-vector skeleton'. The transgenic vector comprises a Cre / loxP recombination system, the selectable marker, an Arr5 promoter gene and a Cre gene are located between two loxP sites, an Arr5 promoter can promote expression of Cre recombinase, and then loxP recombination reaction is induced to delete a DNA sequence between the two loxP sites, so that deletion of the selectable marker gene is achieved, and a possible technical approach is provided for the safety of transgenic plants.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a transgenic method for automatically deleting selection markers. Background technique [0002] Transgenic plants refer to crops whose genomes have been modified using genetic engineering techniques to improve existing traits or introduce exogenous target genes to produce new traits. When using genetic engineering technology to introduce target genes into plant cells, antibiotic resistance marker genes or herbicide resistance marker genes are usually used as selection marker genes in the transformation system to screen transformed cells and tissues. Under selection pressure, non-transformed cells that do not contain the selectable marker gene and its products will die; transformed cells and tissues have integrated the selectable marker gene and are resistant, so that they can continue to survive and differentiate into plants. Although the selection marker gene used to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H5/00A01H6/46
CPCC12N15/8209C12N15/66C12N2800/30
Inventor 张越李杨李阳
Owner WUHAN BIORUN BIO TECH