Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application

A super-engineering and construction method technology, applied in the direction of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of pesticide residue pollution, people's health hazards, etc., and achieve the effect of broadening the degradation spectrum

Inactive Publication Date: 2005-08-10
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem that my country has to use a large amount of pesticides as a large agricultural production country, and there is a serious pollution problem of pesticide residues in the environment. In order to eliminate the losses caused by pesticide residue pollution to the national economy and the harm to people's health , so as to provide a super engineered bacteria that can effectively degrade organophosphorus, carbamate and pyrethroid pesticides at the same time

Method used

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  • Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application
  • Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application
  • Super engineering bactrerin, its expressed detoxicating enzyme, construction method and application

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Embodiment 1

[0048] Embodiment 1, construction super engineering bacteria and detoxification enzyme expressed by the recombinant plasmid pETDuet-b1-opd of the present invention

[0049] The cloning of detoxification enzyme and hydrolase gene super engineering bacterium provided by the present invention is as follows:

[0050] 1.1 Plasmid and recipient bacteria

[0051] Escherichia coli strain BL-21 (DE3) was used as expression bacteria, and plasmid pETDuet was purchased from Novagen as a carrier for expressing opd gene and b1 gene. The plasmid was propagated in E. coli E.coli.DH5α, and all host strains BL-21( DE3), E.coli.DH5α were preserved in our laboratory, and the plasmid pPNCO33 carrying the hydrolase opd gene was donated by Wilfred Chen (University of California, Riverside, California.) as a template for the opd gene; the plasmid carrying the detoxification esterase b1 gene As the source of b1 gene, pET28a-b1 was provided by the Resistance Molecular Genetics Group of Institute of Zo...

Embodiment 2

[0079] Embodiment 2, super engineering bacteria of the present invention are to the degradative action of malathion

[0080]Add 20ml water, 1ml 2% TritonX-100, 5μl (165μg) malathion into a 100ml Erlenmeyer flask, mix it well, add the immobilized super engineered bacteria gene cell pellets, shake at 30°C The bed shakes. Take out 1ml of the treatment solution every once in a while and place it in a 5ml test tube, add 1ml of redistilled n-hexane, shake it on the mixer for 3min to fully mix and extract, then centrifuge at 6500rpm for 15min, take the supernatant, and repeat the extraction once. After the supernatants taken out were combined, a gas chromatograph and a nitrogen and phosphorus detector were used to measure the residual content of malathion in the n-hexane extract at different times, so as to detect the degradation rate of malathion by super engineering bacteria. The specific test The conditions are as follows: use HP 5890 II gas chromatograph, BPX-50 capillary column...

Embodiment 3

[0102] Embodiment 3, the detoxification experiment of the dichlorvos poisoning of experimental chicken

[0103] Adult aircraft carrier chickens (Gallus gallus) (12 months old, chicken weight about 1.5kg) were purchased from the experimental chicken farm of Beijing Agricultural University. After the chickens were purchased, they were fed individually, and the experiment began after at least one week of adaptation to the environment. Free access to food and water, during the experiment, the temperature of the chicken house was controlled at about 20°C, and the light was about 10h per day. The experimental chickens were divided into 4 groups:

[0104] Experimental group: give dichlorvos (loaded into empty capsules, the dose is 170mg / kg, orally) and genetically engineered bacteria expressing detoxification esterase (0.5g wet bacterium is loaded into empty capsules, orally) at the same time;

[0105] Experiment two groups, first give dichlorvos (into the empty capsule, dosage is 1...

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Abstract

A super engineered bacterium able to degradate the residual agricultural chemical contains the gene to code detoxicating lipase and hydrolase, so it can induce the expression of detoxicating lipase and hydrolase. Its configuring process includes cloning the genes of detoxicating lipase and hydrolase, respectively cloning them to expression carrier pETDuet-1, configuring fusion expression carrier pETDuet-b1-opd, transferring to obtain recombinant strain, and inductive culture. It can be used to eliminate residual agricultural chemical.

Description

technical field [0001] The present invention relates to a super engineering bacterium and its construction and application, as well as the detoxification enzyme expressed by the super engineering bacterium and its application, in particular to a super engineering bacterium capable of degrading pesticide residues and the detoxification enzyme expressed by it, and Their construction methods and applications. technical background [0002] Modern agricultural production has played a huge role in satisfying people's desire for high food production, and was once regarded as a panacea for third world countries to get rid of poverty and hunger. However, in the face of the decline in the quality of agricultural by-products and the reduction of biodiversity in modern agriculture, the increasing incidence of crop diseases and insect pests, the waste of agricultural resources, the degradation of the agricultural environment, and the increasing food crisis in Africa, people are delighted...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/00C12N15/52C12N15/55C12N15/70C12P21/02
Inventor 乔传令兰文生黄菁
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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