Recombinant vector and construction method thereof, engineering bacteria for degrading fenothrin esterase est3385, preparation method and application thereof
A technology of fenpropathrin esterase and fenpropathrin, which is applied in the field of recombinant vector and its construction, engineering bacteria for degrading fenpropathrin esterase est3385 and preparation, to achieve simple process, meet industrial production, and environment-friendly Effect
Active Publication Date: 2019-11-22
HUNAN PLANT PROTECTION INST
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Problems solved by technology
In biodegradation, microbial degradation is an important part. Many degrading bacteria resources with excellent performance and their corresponding degrading genes and degrading enzymes have been isolated and cloned and identified (Yang CR, Jian JZ (2010) Recent advance in biodegradation in China: New microorganism and pathway, biodegradation engineering, and bioenergy from pollutant biodegradation.), however, in the actual application process, due to the influence of natural environmental conditions, the effect of removing pyrethroid pesticide residues is indeed far lower than that of the laboratory; in addition, the degradation The large-scale industrial production of bacterial resources and degrading enzymes is still one of the key technical bottlenecks restricting the application of microorganisms and related enzymes in the treatment and restoration of pyrethroid pesticide residues
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Embodiment 1
[0043] A kind of engineering bacterium degrading fenpropathrin esterase est3385 of the present invention, its construction method comprises the following steps:
[0044] 1. Extraction of Rhodopseudomonas palustris genomic DNA: The liquid-cultured Rhodopseudomonas palustris was collected by centrifugation and then extracted by CTAB method, and the concentration and integrity of DNA were detected by agarose. The specific operation is as follows:
[0045] 1.1. Inoculate the PSB-S strain of Rhodopseudomonas palustris into the liquid medium according to the inoculation amount of 0.5% (v / v) (the medium is filled with the culture bottle and then sealed), at 30 ℃, the light intensity is 2000 Lx Cultured for 7 d under light conditions. The composition of the liquid medium is: 0.2 g / L K 2 HPO 4 , 0.8 g / L KH 2 PO 4 , 0.2 g / L MgSO 4 , 0.1 g / L CaSO 4 2H 2 O, 0.0033 g / L NaMoO 4 2H 2 O, 0.005 g / L FeSO 4 ·7H 2 O, 1.5 g / L yeast extract; pH 7.2.
[0046] 1.2. Centrifuge 2 mL of Rho...
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The invention discloses a recombinant vector, a construction method of the recombinant vector, engineering bacteria of fenpropathrin degrading esterase est3385, a preparing method and application.The recombinant vector comprises a pGEX-3x expression vector and a fenpropathrin degrading esterase gene est3385, wherein the sequence of the fenpropathrin degrading esterase gene est3385 is shown in SEQ ID NO.1, and the fenpropathrin degrading esterase gene est3385 is located between two restriction enzyme sites of Bam H I and Eco RI of the pGEX-3x expression vector.The construction method of the recombinant vector comprises the steps of genomic DNA extraction, primer design, PCR amplification, enzyme digestion, connection and the like.The engineering bacteria are obtained by converting the recombinant vector into competence cells through heat shock.The engineering bacteria are high in yield, simple in process, environmentally friendly and capable of meeting the requirement for industrial production and being applied to degrading fenpropathrin.
Description
technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant vector and a construction method thereof, an engineering bacterium for degrading fenpropathrin esterase est3385, a preparation method and application thereof. Background technique [0002] Fenpropathrin is one of the pyrethroid pesticide varieties, widely used in my country to control the pests of major crops such as grain, potato, tobacco, cotton and fruit. Since commercial production in the 1980s, pyrethroid pesticides have become an ideal substitute for some highly toxic pesticides (organochlorine and organophosphorus pesticides) due to their advantages of high efficiency, low dosage, and low toxicity to mammals products; the demand for the use of pyrethroid pesticides such as fenpropathrin continues to grow. However, as a large amount of pyrethrin pesticides such as fenpropathrin are put into agricultural production, the residues of pyrethrin pestic...
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IPC IPC(8): C12N15/70C12N15/66C12N1/21C12N9/18A62D3/02A62D101/04A62D101/26A62D101/28
CPCA62D3/02A62D2101/04A62D2101/26A62D2101/28C12N9/18C12N15/66C12N15/70
Inventor 张松柏刘勇张德咏吴龙飞罗香文郑立敏彭静张卓杜娇
Owner HUNAN PLANT PROTECTION INST