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Engineering bacterium capable of degrading organophosphorus pesticides, and construction method and application thereof

A technology of organophosphorus pesticides and construction methods, which is applied in the field of degradable organophosphorus pesticide engineering bacteria, can solve problems such as high content of organophosphorus components, decreased soil productivity, and ecological environment damage, and achieve the effect of high degradation ability

Inactive Publication Date: 2014-10-08
XUZHOU INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] First, while organophosphorus pesticides kill target organisms, they also more or less kill non-target organisms, causing serious damage to the surrounding environment and even the entire ecological environment.
[0008] Second, the content of organic phosphorus in the soil is too high, which indirectly affects the health of humans and animals through food, vegetables, fruits, etc.
[0009] The third is that organophosphorus pesticides have a serious impact on the number, population structure and biological activity of soil microorganisms, resulting in a decline in soil productivity
However, according to estimates by relevant experts in the Newsletter of the International Union of Microbiological Societies, there are about 500,000 to 600,000 species of microorganisms in the world, and less than 5% have been studied and recorded. In such a rich microbial resource bank, due to the natural environment and other differences in physical and chemical conditions, especially in environments under long-term pesticide stress, there are still many microorganisms that can degrade pesticides that have not been discovered, and what has been isolated is only a drop in the ocean.

Method used

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  • Engineering bacterium capable of degrading organophosphorus pesticides, and construction method and application thereof
  • Engineering bacterium capable of degrading organophosphorus pesticides, and construction method and application thereof
  • Engineering bacterium capable of degrading organophosphorus pesticides, and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0031] The construction of embodiment 1 recombinant plasmid pHT43-opd

[0032] 1. Using the plasmid pDL2 carried by Flavobacterium as a template to amplify the opd gene

[0033]Extract the Flavorbacterium.spA TCC27551 plasmid pDL2, use the Flavorbacterium plasmid pDL2 as a template, synthesize primers, and amplify the opd gene sequence. The primer sequence is as follows:

[0034] P1:opda:5'-CGGGATCCCATGCAAACGAGAAGGGTTGTGCTC-3'; (SEQ ID NO.2)

[0035] P2:opds:5'-CGCCCGGGCATGACGCCCGCATCTTGACGGGGA-3'(SEQ ID NO.3)

[0036] The upstream primer (P1) introduces the BamH I site, and the downstream primer (P2) introduces the restriction site of Sma I. Two restriction sites were introduced at the 5' end to protect the bases.

[0037] The PCR product was analyzed by gel electrophoresis, and the size of the opd gene was about 1100bp ( figure 1 ), which is the expected size. After the PCR products were purified, they were sent to Nanjing GenScript Biotechnology Co., Ltd. for sequencin...

Embodiment 2

[0065] Induced expression of embodiment 2 transformants

[0066] Pick the monoclonal colony of the recombinant strain from the freshly transformed plate, add 5ml LB medium containing 5μg / mL chloramphenicol at 37°C, 200rpm, shake and culture for 10-12 hours, then inject 5ml culture solution into 50ml containing chloramphenicol culture medium to continue culturing. When the strain grows to the end of the logarithmic phase, the OD 600 =0.80, add IPTG to make the final concentration of IPTG 0.8mM, induce at 15-25°C for 15-20 hours, and obtain the engineering bacteria of the present invention. After screening, a strain CGMCC No.9272 with the highest degradation rate of organophosphorus pesticides was obtained, which was called Bacillus subtilis sp.LTG1 in this application. The degradation rate is respectively 4.4 times, 3.5 times and 4.1 times of the lowest strain (assay method with reference to Example 3).

Embodiment 3

[0067] Embodiment 3: engineering bacterium of the present invention is to the degradation of methyl parathion, malathion and acephate

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Abstract

The invention relates to an engineering bacterium capable of degrading organophosphorus pesticides and a construction method and application thereof. The engineering bacterium contains an opd gene coding wide-spectrum organophosphorus pesticide hydrolase (OPH) and can express OPH. The bacterium is the strain Bacillus subtilis sp. LTG1 which is preserved in China General Microbiological Culture Collection Center (CGMCC), with an accession number of GCMCC No. 9272. The engineering bacterium can simultaneously degrade organophosphorus pesticides like DDVP, malathion, rogor, omethoate, parathion, methyl parathion and acephate and is used for biodegradation and biological purification of organophosphorus pesticide pollution in a water source and soil.

Description

technical field [0001] The invention relates to a degradable organophosphorus pesticide engineering bacterium, and its construction method and application belong to the technical field of biodegradation. It can be used for in-situ remediation engineering of soil pesticide pollution. Background technique [0002] Pesticides are an effective means of preventing and controlling crop diseases and insect pests and aquaculture diseases all over the world. A large number of chemical pesticides, especially the use of highly toxic and high residue pesticides, have become the main "killer" of food safety and endangering people's health. [0003] At present, the main chemical pesticides used are mainly divided into three categories: insecticides, herbicides and fungicides according to their different functions. According to different chemical structures, they can be divided into organochlorines, organophosphorus, pyrethroids, carbamates, and inorganic pesticides. The common characte...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/75A62D3/02C12R1/125A62D101/04A62D101/28
Inventor 李同祥孙会刚黄天姿候进慧董玉玮汤薇刘全德刘宾宾
Owner XUZHOU INSTITUTE OF TECHNOLOGY
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