Strain for specifically degrading herbicide propanil, amidase degrading gene and application

A technology of amidase and degrading bacteria, applied in the field of degrading amidase gene and application, can solve the problems of toxicity and carcinogenicity

Active Publication Date: 2021-10-08
HUAIBEI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, it is also toxic to mammal

Method used

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  • Strain for specifically degrading herbicide propanil, amidase degrading gene and application
  • Strain for specifically degrading herbicide propanil, amidase degrading gene and application
  • Strain for specifically degrading herbicide propanil, amidase degrading gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The isolation and identification of embodiment 1 bacterial strain

[0037] The invention provides a bacterial strain capable of specifically and efficiently degrading the herbicide propanil and its degrading amidase gene and application thereof. The bacterial strain used is a Gram stain-negative strain P5, which is isolated from the soil of an abandoned pesticide factory in Xinyi, Jiangsu. The specific isolation and screening methods for strains are as follows:

[0038] Take 10.0 g of the soil sample and add it to 100 mL of liquid inorganic salt medium (hereinafter referred to as MSM) containing 30 mg / L propanil, culture it on a shaker at 30°C and 180 rpm for 7 days, and transfer it to fresh In MSM, serial enrichment was subcultured four times. Use an ultraviolet spectrophotometer to scan in the range of 200-350nm to detect the degradation effect of the fifth-generation enrichment solution. Dilute and spread the effective enrichment solution on inorganic salt solid me...

Embodiment 2

[0040] Example 2 Evaluation of the effect of strain P5 on degrading propanil and analysis of metabolites

[0041] 1.1 Preparation of seed solution

[0042]Insert bacterial strain P5 (CCTCC M 2021603) containing 30mg L -1 Cultivate propannil in 100mL LB medium at 30°C and 160rpm on a shaker, collect the bacteria by centrifugation at 6,000rpm after 48 hours, wash the bacteria twice with sterilized MM medium, and finally resuspend with 10mL MM medium as seeds liquid for use.

[0043] 1.2 Degradation of the herbicide propanil by strain P5

[0044] The bacterial strain P5 seed liquid is received respectively to contain 30mg L by the inoculum amount of 5%. -1 Propannil 100mL MM culture medium, 30 ℃, 160rpm shaker culture. Every 24 hours, take 3 mL of culture solution, extract with an equal volume of dichloromethane, remove water with anhydrous sodium sulfate, take 2 mL of organic phase and blow dry, then redissolve in 0.5 mL of methanol, filter with a filter membrane (pore size ...

Embodiment 3

[0064] Example 3 High-efficiency expression and purification of the amidase gene that specifically degrades propanil in Escherichia coli BL21[pET-29a(+)]

[0065] 3.1 PCR amplification of the amidase gene that specifically degrades propanil

[0066] With forward primer F1:5'-AAA CATATG TCACATGATCAACAGGGCCCCAAAA-3' (SEQ ID NO.3) and reverse primer R1:5'-TAT AAGCTT Using TGACCGCGTGTCGCCCCC-3' (SEQ ID NO.4) as a primer, the amidase gene fragment that specifically degrades propanil is amplified from the Boseasp.P5 genome by PCR.

[0067] Amplification system:

[0068]

[0069] PCR amplification program:

[0070]

[0071]

[0072] 3.2 PCR products and vectors were digested step by step with Nde I and Hind III.

[0073] Enzyme cutting system:

[0074]

[0075] The digested product was purified and recovered with a purification recovery kit.

[0076] 3.3 Transformation and expression

[0077] The DNA fragment digested in 3.2, pET-29a(+) was enzyme-ligated (refer ...

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Abstract

The invention discloses a strain for specifically and efficiently degrading propanil and an amidase degrading gene thereof. The propanil degrading strain Bosea sp.P5, has the strain preservation number CCTCC M 2021603. The nucleotide sequence of the amidase gene capable of specifically degrading propanil is SEQ ID NO. 1. An engineering strain constructed by using the gene can efficiently express amidase capable of specifically degrading propanil. A produced enzyme preparation can be used for removing residues of amide herbicide propanil.

Description

technical field [0001] The invention belongs to the field of applied environmental microorganisms, and relates to a herbicide propanil specific degradation strain, a degradation amidase gene and application thereof. Background technique [0002] According to statistics, the area of ​​chemical pesticide control in my country has exceeded 2.8 billion hectares per time, and the annual use of pesticides in the country is about 300,000 tons (technical medicine). However, the utilization rate of pesticides in my country is only 20-30%, and the remaining residues all enter the ecological environment such as crops, soil, and water bodies, causing serious damage to the ecological environment. [0003] Propanil is one of the amide herbicides and is currently a good herbicide for controlling barnyardgrass in rice fields. Because of its high herbicidal efficacy, strong selectivity, wide range of herbicides, and convenient use, it has rapidly become one of the most widely used herbicide...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/78C12N15/55C12N15/70C12N1/21B09C1/10C02F3/34A01N25/32C12R1/01C02F101/38
CPCC12N1/20C12N9/78C12N15/70B09C1/10C02F3/34C02F3/342A01N25/32C02F2101/306C02F2101/38
Inventor 张龙宋嫚李峰刘远王光利
Owner HUAIBEI NORMAL UNIVERSITY
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