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Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application

A herbicide amide, amido hydrolase technology, applied in hydrolase, application, genetic engineering and other directions, can solve environmental problems, crops, phytotoxicity, serious problems, etc.

Active Publication Date: 2016-07-20
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Although the use of herbicides improves agricultural production efficiency, it also brings serious environmental problems and crop phytotoxicity problems

Method used

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  • Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application
  • Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application
  • Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Effect evaluation and metabolite analysis of strain LR2014-1 degrading Ligulong

[0037] 1.1 Preparation of seed solution

[0038] The strain LR2014-1 (CCTCCNO: M2015236) was inserted into 100mL LB medium containing 30mg / L Liguron, cultured on a shaker at 30°C and 150rpm, and after 48h, the bacteria were collected by centrifugation at 6000rpm, washed twice with MM, and finally Resuspend with 10mLMM and use as seed solution for later use.

[0039] 1.2 Degradation of the herbicide Liguron by strain LR2014-1

[0040] The bacterial strain LR2014-1 was inoculated into 100mLMM containing 30mg / L Liguron according to the inoculum amount of 5%, cultured on a shaker at 30°C and 150rpm, and after culturing for 12h, 3ml of the culture solution was extracted with an equal volume of dichloromethane. After removing water with sodium sulfate, take 2ml of dichloromethane and blow dry, then redissolve in 1ml of methanol, filter with a filter membrane (pore size 0.22 μm), and ...

Embodiment 2

[0042] Cloning of embodiment 2 rituron amidohydrolase gene

[0043] 2.1 Extraction of bacterial genome total DNA

[0044]Strain Diaphorobactersp.LR2014-1 (Diaphorobactersp.LR2014-1, Liguron efficient degradation strain isolated and screened from Liguron contaminated soil). After mass cultivation, the CTAB method was used to extract the total genomic DNA of Diaphorobactersp.LR2014-1 with high purity and large fragments, dissolved in TE (pH8.0), and stored at -20°C. For specific methods, refer to F. Osper et al. Edited "Refined Molecular Biology Experiment Guide".

[0045] 2.2 Draft Bacterial Genome Sequencing

[0046] The qualified Diaphorobactersp.LR2014-1 genomic DNA was sent to Shanghai Meiji Biomedical Technology Co., Ltd. for scanning the whole genome draft of the bacteria. Genome sequencing results showed that the draft genome size of the strain LR2014-1 was 3,825,957 bp, with 87 scaffolds, 87 scaffolds larger than 1,000 bp, and a total of 4,238 open reading frames (OR...

Embodiment 3

[0078] Example 3 High expression of rituron amidohydrolase gene in BL21 (pET-29a (+))

[0079] 3.1 PCR amplification of rituron amidohydrolase enzyme gene

[0080] Using forward primer F2:5'-TTAGGATCCCCAACGGACTGGAGAGTTGAA-3'(SEQ ID NO.5) and reverse primer R2:5'-AACCTCGAGTTGCGGTTCCGAACGCGGATA-3'(SEQ ID NO.6) as primers, amplified from Diaphorobactersp.LR2014-1 by PCR A gene fragment of glutaminase hydrolase was produced.

[0081] Amplification system:

[0082]

[0083] PCR amplification program:

[0084] a. Denaturation at 98°C for 2 minutes

[0085] b. Denaturation at 98°C for 15s, annealing at 63°C for 15s, extension at 68°C for 1min30s, for 30 cycles

[0086] c. Extend at 68°C for 10 minutes,

[0087] d.10°C for 5min, and cool to room temperature.

[0088] 3.2 PCR products and vectors were digested step by step with BamHI and XhoI.

[0089] Enzyme digestion and enzyme-linked system (refer to 1.6)

[0090] 3.3 Transformation and expression

[0091] The pET-29a(+)...

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Abstract

The invention discloses a broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application and provides an amidohydrolase gene of a substituted urea herbicide and hydrolase protein induced by the gene.The total length of the gene is 1377 bp, the G+C content is 63.5%, 458 amino acids are encoded, and the gene is a new amidohydrolase gene resource having a broad-spectrum hydrolysis function on the substituted urea herbicide, 30 mg / L N-methoxyl-N-methyl substituted urea herbicide (linuron) and N,N-dimethyl substituted urea herbicide (diuron, chlortoluron and fluometuron) can be completely degraded within 24 h, and produced enzymic preparations can be used for removing substituted urea herbicide residues on soil, water and crops.The gene can be used for cultivation of transgenic crops resisting substituted urea herbicide.

Description

technical field [0001] The invention belongs to the field of applied environmental microorganisms, and relates to broad-spectrum substituted urea herbicide-degrading bacteria and amidohydrolase genes and applications. Background technique [0002] Although the use of herbicides has improved the production efficiency of agriculture, it has brought serious environmental problems and crop phytotoxicity problems at the same time. Substituted urea herbicides have been produced commercially for more than 50 years and are among the most widely used important herbicides worldwide. Due to its extensive repeated use and relatively high water solubility, it is often detected in various polluted habitats and agricultural by-products, and herbicide crop phytotoxicity also occurs from time to time. Therefore, replacing urea herbicide residues has become an urgent problem to be solved in ecological environment protection and crop phytotoxicity relief. The method of microbial remediation ...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/78C12N15/70C12N1/21A62D3/02C02F3/34B09C1/10A01N25/32A01H5/00A62D101/04A62D101/28C02F101/38
CPCA01N25/32A62D3/02A62D2101/04A62D2101/28B09C1/10C02F3/34C02F2101/306C02F2101/34C02F2101/36C02F2101/38C12N9/78C12N15/8274
Inventor 蒋建东张龙陈凯
Owner NANJING AGRICULTURAL UNIVERSITY
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