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Resistance gene capable of degrading herbicide glyphosate and encoded protein of resistance gene

A technology for encoding protein and glyphosate, applied in the field of resistance gene isolation

Inactive Publication Date: 2014-12-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although my country has made some progress in herbicide-resistant transgenic crops, there is still a huge gap compared with developed countries. Therefore, it is very necessary to screen and improve new glyphosate degradation genes.

Method used

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  • Resistance gene capable of degrading herbicide glyphosate and encoded protein of resistance gene
  • Resistance gene capable of degrading herbicide glyphosate and encoded protein of resistance gene
  • Resistance gene capable of degrading herbicide glyphosate and encoded protein of resistance gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Cloning of embodiment 1 bacillus cereus glycine oxidase BceGO original gene

[0029] According to the published GenBank glycine oxidase gene thiO of Bacillus cereus (GenBank sequence number KC203486), specific primers were designed for PCR amplification of the original gene of BceGO glycine oxidase of Bacillus cereus.

[0030] The primer names and DNA sequences are as follows:

[0031] BceGO-F:5'-CGC GGATCC ATGTGTAAGAAGTATGATGTAGCGAT-3'

[0032] BceGO-R:5'-CCG CTCGAG CTAAACTCTCCTAGAAAAGCAATGAAT-3'

[0033] Restriction sites are underlined: BamHI and XhoI.

[0034] The PCR reaction system is shown in Table 1.

[0035] Table 1 PCR reaction system

[0036]

[0037] The PCR reaction procedure is:

[0038] Pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 90 sec, a total of 30 cycles; extension at 72°C for 10 min; incubation at 15°C for 5 min. After the reaction, the amplified products were d...

Embodiment 2

[0040] Example 2: Construction of a random mutant library using error-prone PCR

[0041] The error-prone PCR reaction system is shown in Table 2

[0042] Table 2 Error-prone PCR reaction system

[0043]

[0044]

[0045] The error-prone PCR reaction procedure is:

[0046] Pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 90 sec, a total of 30 cycles; extension at 72°C for 10 min; incubation at 15°C for 5 min. After the reaction, the amplified products were detected by 0.8% agarose gel electrophoresis.

[0047] After recovering the error-prone PCR products, they were digested with BamHI-XhoI, enzyme-linked to the pGEX-6p-1-BamHI-XhoI double-cut vector, and then transformed into E. coli DH5α to construct a random mutation library. Ten clones were randomly selected for sequencing. After sequence comparison, it was found that the number of nucleotide substitutions in each gene was 1 to 3 bases, and the...

Embodiment 3

[0048] Example 3 Using site-directed mutagenesis to superimpose mutation sites

[0049] Two single-point mutants 22D11 (mutation site: G51R) and 23B1 (mutation site: D60G) were obtained from the first round of random mutation library screening, and the two points were merged into one by site-directed mutagenesis (Cadwell and Joyce1992). On the mutant, a double-point mutant B1R (G51R / D60G) was obtained. The coded nucleotides (Weiner and Costa1994) of the mutation site were designed using the codon preference frequency of Escherichia coli, and the site-directed mutagenesis primer name and sequence are:

[0050] G51R-F:5'-GCTGCTGGTTTACTT CGT GTTCAGGC-3'

[0051] G51R-R:5'- ACG AAGTAAACCAGCAGCTGCTTTTG-3'

[0052] The restriction site is underlined: G51R (GGT→CGT)

[0053] See Table 3 for site-directed mutagenesis PCR reaction system

[0054] Table 3 site-directed mutagenesis PCR reaction system

[0055]

[0056]

[0057] The PCR reaction procedure is:

[0058] Pre-...

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Abstract

The invention belongs to the field of genetic engineering, relates to separation, determinate evolution in vitro and functional identification of a resistance gene capable of degrading herbicide glyphosate efficiently, and further relates to the resistance gene capable of degrading herbicide glyphosate in plants. The nucleotide sequence of the gene B3S1 is as shown in the figure SEQ ID NO: 1; the amino acid sequence of encoded protein of the resistance gene is as shown in the figure SEQ ID NO: 2. According to the invention, the glycine oxidase (thiO) from marine bacteria bacillus cereus (Bacillus cereus) is amplified, and two rounds of random mutagenesis and a round of DNA shuffling test are performed continuously on the glycine oxidase (thiO), and combined with an activity screening technology on the basis of T7 phage pyrolysis-horseradish peroxidase / dianisidine enzyme coupling, the final glyphosate oxidase mutant B3S1 is obtained. The function of the gene and the application approach of the gene in degrading the resistance of herbicide glyphosate are proved in the invention.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the separation of a resistance gene for efficiently degrading the herbicide glyphosate, and also relates to the application of the resistance gene for efficiently degrading the herbicide glyphosate in the breeding of glyphosate-resistant transgenic plants Applications. Background technique [0002] Glyphosate (glyphosate) is an organophosphorus herbicide developed by Monsanto Company (St.Louis, MO) in 1970. Since it was registered in the United States in 1974 (trade name Roundup, Chinese name Nongda), it has been widely used to control weeds in agriculture, towns, gardens, forests and other environments, and has become the most widely used herbicide in the world. The products also include Aquamaster (Monsanto Company, St.Louis MO) and Rodeo (Dow Agrosciences, Indianapolis, IN), which are mainly used in the control of weeds in aquatic environments. [0003] Glyphos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02A01H5/00
Inventor 刘子铎詹滔林拥军吴高兵张利莉
Owner HUAZHONG AGRI UNIV
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