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Resistance gene for degrading herbicide glyphosate and coded protein of resistance gene

A technology for encoding protein and glyphosate, applied to the resistance gene and its encoded protein for degrading herbicide glyphosate, application of resistance gene in plant herbicide glyphosate, glyphosate oxidase B4S7 gene field , can solve the problem that glyphosate degradation gene has not been found yet

Inactive Publication Date: 2015-03-25
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the 1980s, microorganisms and genes have been reported to be able to degrade the herbicide glyphosate, and the reported genes are all derived from microorganisms. So far, no plant-derived glyphosate degradation genes have been found (Duke 2011)

Method used

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  • Resistance gene for degrading herbicide glyphosate and coded protein of resistance gene
  • Resistance gene for degrading herbicide glyphosate and coded protein of resistance gene
  • Resistance gene for degrading herbicide glyphosate and coded protein of resistance gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of mutant library using DNA shuffling technology

[0027] The present invention uses the mutants B3S1, B3S4, B3S6 and B3S7 from Bacillus cereus glycine oxidase (BceGO) as templates (Zhan et al.2013), referring to the DNA shuffling method reported by Stemmer and using ultrasonication to process DNA fragments, DNA shuffling (Stemmer 1994; Miller et al.2002), recombination of beneficial mutation sites to achieve further improvement of glycine oxidase from Bacillus cereus, so as to obtain glyphosate oxidase with better glyphosate degradation activity , lay a solid foundation for the application of glyphosate-resistant crops, and reduce the residual amount of glyphosate in plants, so that plants can reduce the threat to human health through the food chain while removing glyphosate's growth inhibition.

[0028] The specific steps are as follows:

[0029] (1) Amplification of the template gene

[0030] Extract pGEX-6p-B3S1 ( figure 2 ), pGEX-6p-B3S4 ...

Embodiment 2

[0054] Example 2 Screening of mutants with high glyphosate oxidation activity (high-throughput screening based on 96-well plate)

[0055] This method refers to the screening method reported by (Zhan et al.2013). Inoculate the control bacteria (Escherichia coli DH5α containing the recombinant plasmid pGEX-6p-B3S1) and the clones in the mutant library into 96 deep wells containing 600 μL of liquid LB medium (adding ampicillin to a concentration of 100 μg / ml) At the same time, the bacteria in each well were backed up on the ampicillin-resistant plate. Seal the 96-deep-well plate, shake and culture at 200 rpm at 37°C for 16 hours, add 200 μL of LB liquid medium containing ampicillin (100 μg / ml), IPTG (final concentration 0.1 mM) and T7 phage (Tarahovsky et al.1994) to each well , 28 ℃ 200rpm shaking culture for 6h to induce protein expression and be lysed by phage to release. Pipette 159 μL of crude enzyme solution (lysed supernatant, containing target protein) into a 96-microwe...

Embodiment 3

[0056] Example 3 Protein expression and purification of mutant B4S7

[0057] The recombinant plasmid pGEX-6p-B4S7 ( Figure 7 ) mixed with E.coli BL21(DE3) competent cells, transformed by electric shock, spread on an ampicillin-resistant plate (ampicillin concentration 100 μg / mL), and cultured overnight at 37°C until a single colony visible to the naked eye grew out. Pick a single clone and inoculate it into 20 mL liquid LB medium (containing 100 μg / mL ampicillin), and culture overnight at 37°C with shaking at 200 rpm. Transfer 1% of the bacterial solution to 2L of fresh liquid LB medium (containing 100 μg / mL ampicillin), shake culture at 200 rpm at 37 °C until the OD600 is 0.6, add the inducer IPTG with a final concentration of 0.1 mM, and induce at 160 rpm at 22 °C Cultured for 12h to express the protein. The cells were collected by centrifugation, washed twice with 50 mM sodium pyrophosphate buffer (pH 7.0), and then resuspended in 40 mL of pre-cooled 50 mM sodium pyropho...

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Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a resistance gene for degrading herbicide glyphosate and coded protein of the resistance gene. The gene and the protein are screened from a resistance gene which is capable of effectively degrading herbicide glyphosate; the gene is named glyphosate oxidase B4S7 gene, a nucleotide sequence is as shown in SEQ ID NO: 1, and the protein of the protein coded by virtue of the gene is as shown in SEQ ID NO: 2. Through DNA reshuffling on the coding gene of glycine oxidase mutant B3S1 of bacillus cereus as well as the enzyme-coupling activity screening based on T7 bacteriophage lytic-horseradish peroxidase / o-dianisidine, the glyphosate oxidase mutant B4S7 is finally obtained. The resistance gene disclosed by the invention preliminarily verifies the functions of the glyphosate oxidase B4S7 gene as well as application potential of the glyphosate oxidase B4S7 gene in glyphosate-resistance crops.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a resistance gene for degrading the herbicide glyphosate and its encoded protein. The gene is named glyphosate oxidase B4S7 gene. Glyphosate is obtained from the resistance gene with high efficient degrading activity. The invention also relates to the use of a high-efficiency degrading herbicide glyphosate resistance gene in the plant herbicide glyphosate. Background technique [0002] Glyphosate is a non-selective broad-spectrum herbicide developed in 1970 by Monsanto Company (St.Louis, MO). Its trade name is Roundup, and its Chinese name is Nongda. Since its commercialization, glyphosate has been widely used all over the world, and its usage is increasing year by year. [0003] The molecular formula for glyphosate is C 3 h 8 NO 5 P, whose chemical name is N-(phosphonomethyl)glycine (GLP), is a derivative of glycine with a relative molecular weight of 169.1. P...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06A01H5/00
Inventor 刘子铎姚佩张利莉吴高兵林拥军洪玉枝
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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