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Method for screening glyphosate-resistant gene, EPSPS mutant gene, defect strain and application thereof

A screening method and anti-glyphosate technology, applied in the biological field, can solve the problems of long plant growth cycle, large land area, and long time spent on mutant plants, and achieve short cycle, small size, and fast reproduction speed. Effect

Active Publication Date: 2016-09-28
SICHUAN GEVOTO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the long growth cycle of plants, planting a large number of mutant plants not only takes a long time but also requires a large area of ​​land

Method used

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  • Method for screening glyphosate-resistant gene, EPSPS mutant gene, defect strain and application thereof
  • Method for screening glyphosate-resistant gene, EPSPS mutant gene, defect strain and application thereof
  • Method for screening glyphosate-resistant gene, EPSPS mutant gene, defect strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0049] In this embodiment, the target plant is rice (Oryza sativa), the exogenous EPSPS gene is the rice EPSPS gene (its nucleotide sequence is shown in SEQ ID NO.1), and the wild type is knocked out by direct knockout method using homologous PCR fragments The EPSPS and C-P Lyase-deficient strains obtained from the EPSPS gene and C-P Lyase gene in Escherichia coli DH5α are taken as an example to describe the screening method of the present invention in more detail. The names of primers used in this embodiment and their nucleotides See Table 1 for the sequence.

[0050] In the first step, the EPSPS gene and C-P Lyase gene in Escherichia coli DH5α were directly knocked out using homologous PCR fragments.

[0051] 1. Knockout of the C-P Lyase gene of Escherichia coli DH5α

[0052] (1) Homologous PCR fragment amplification

[0053] Use the forward primer CPF2 and the reverse primer CP5HA3 (see Table 1), and use the wild-type Escherichia coli DH5α as a template to carry out PCR, ...

Embodiment 2

[0096] In this embodiment, the target plant is soybean (Glycine max), the exogenous gene is soybean EPSPS gene (its nucleotide sequence is shown in SEQ ID NO.5), and the wild-type gene is knocked out by direct knockout method using the homologous FRT method. The EPSPS and C-P Lyase-deficient strain obtained from the EPSPS gene and C-PLyase gene in Escherichia coli DH5α are taken as an example to illustrate the screening method of the present invention. The names of primers used in this embodiment and their nucleotide sequences are shown in the table 3.

[0097] In the first step, the C-P Lyase gene of Escherichia coli DH5α was knocked out.

[0098] The EPSPS gene and C-PLyase gene in the Escherichia coli DH5α strain were knocked out by using the FRT method in two steps, first knocking out the C-P Lyase gene, and then knocking out the EPSPS gene.

[0099] 1. Preparation of Escherichia coli DH5α competent cells containing pKD46 plasmid

[0100] Take 0.5 μL of pKD46 plasmid (it...

Embodiment 3

[0148] This embodiment provides a deficient strain, specifically, the deficient strain is an EPSPS and C-P Lyase deficient strain. The EPSPS and C-P Lyase-deficient strain is obtained by knocking out the EPSPS gene and C-P Lyase gene of any one of Escherichia coli DH5α, TOP10 and BL21 by gene knockout technology. Specifically, the gene knockout method used in this example is the same as the gene knockout method used in Example 1 or Example 2.

[0149] The EPSPS and C-P Lyase-deficient strains provided in this example can be used in testing the function of EPSPS genes from target plants. Specifically, the EPSPS and C-P Lyase-deficient strains of this example are used as host bacteria, the EPSPS gene of the target plant is introduced into the host bacteria, and then the host bacteria are placed in a basal medium that does not contain amino acids or proteins, that is, restricted If it is observed that there is normal bacterium colony production on the limiting medium, it shows t...

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Abstract

The invention discloses a method for screening a glyphosate-resistant gene, an EPSPS mutant gene, a defect strain and application thereof. The method can be used for cloning glyphosate feeling related genes, including plant genes for multidirectional accelerated mutation and high-throughput glyphosate-resistant performance screening, so that a high glyphosate-resistant gene can be quickly evolved. By adopting the screening method, plants and other sourced genes can be quickly evolved to become mutant genes with glyphosate resistance by utilizing the characteristics of quick growth and reproduction and easy culture of bacteria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a glyphosate-resistant gene screening method, EPSPS mutant gene and defective strain and application. Background technique [0002] Glyphosate, developed by Monsanto, is a foliar spray, broad-spectrum, non-selective systemic glyphosate. Its main component, glyphosate, works by inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway in plants, so that the damaged plants cannot continuously synthesize essential amino acids and affect the normal growth of plants. even death. [0003] Glyphosate is a broad-spectrum glyphosate herbicide that is lethal to almost all plants. The most commonly used glyphosate-resistant gene on the market is the CP4 gene, which is isolated from Agrobacterium by Monsanto and has strong resistance to glyphosate. gene. Plants can be genetically modified for this resistance. Because glyphosate-resistant crops br...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/10C12N1/21C12Q1/68C12Q1/02C12R1/19
CPCC12N9/1092C12N9/88C12Q1/02C12Y205/01019
Inventor 王博雅卢远根胥南飞
Owner SICHUAN GEVOTO BIOTECH CO LTD
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