Acetochlor reductive dechlorination enzyme ArdA as well as coding gene and application thereof

Abstract

The invention discloses acetochlor reductive dechlorination enzyme ArdA as well as a coding gene and application thereof. The nucleotide sequence of the arcA is SEQ ID NO.1, the full length of the arcA is 681 bp, the amino acid sequence of the arcA is SEQ ID NO.2, and 226 amino acids are encoded. ArdA is the acetochlor reductive dechlorination enzyme found for the first time, can be used for degrading acetochlor residues in soil and water, and has very important theoretical and application values.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology and relates to the dechlorination enzyme ArdA, its gene, and its application in degrading the residues of the herbicide acetochlor in the environment through a reductive dechlorination reaction. Background Technology

[0002] Acetochlor is a highly effective, selective, contact herbicide used as a pre-emergence soil treatment. It belongs to the chloroacetamide class of herbicides and is widely used in farmland to control annual grasses and some small-seeded broadleaf weeds. Statistics from 2014 show that acetochlor ranked third in usage among herbicides; in 2019, among the top ten pesticides by production volume, acetochlor ranked fourth, after glyphosate, atrazine, and paraquat. With the modernization and intensification of agriculture in my country, its demand and usage will continue to increase.

[0003] Acetochlor is chemically stable, not easily volatilized or chemically degraded, and has a long residual period in soil (up to 45 days). Its long residual period in the environment poses serious threats to human health and the environment, as its residues enter the environment and affect agricultural products. Acetochlor residues have been detected in soils and water bodies in the Northeast Plain, the Yangtze River Basin, and the Pearl River Basin in southern my country. Studies have shown that acetochlor has certain teratogenic and mutagenic properties, and it has been classified as a Group B-2 carcinogen by the U.S. Environmental Protection Agency. Furthermore, acetochlor residues can significantly reduce soil microbial diversity, inhibiting the number and activity of soil microorganisms. Excessive residues cause severe phytotoxicity to crops. In recent years, crop damage caused by improper use of acetochlor has occurred frequently in my country, causing serious agricultural losses. Therefore, given the increasing severity of acetochlor pollution in soil and water bodies, pollution remediation is receiving increasing attention.

[0004] Compared to traditional physicochemical methods, bioremediation technology offers advantages such as high efficiency, speed, no residue, and no secondary pollution. It is suitable for the degradation and remediation of large-scale non-point source pollution of toxic and harmful organic pollutants, with lower remediation costs (only about 10% of the cost of traditional chemical and physical remediation). Domestically and internationally, bioremediation technology has been widely applied to the removal of residual organic pollution from petroleum, chemical, and pesticide industries. Depending on the oxygen content of the actual pollution remediation environment, bioremediation is divided into aerobic and anaerobic bioremediation. Aerobic bioremediation relies primarily on aerobic microorganisms to degrade pollutants, while anaerobic bioremediation relies primarily on anaerobic microorganisms. In practical applications, anaerobic bioremediation offers advantages such as low energy consumption, low nutrient requirements, low volatile matter production, high treatment efficiency, and high economic benefits, and is widely used in the removal of pollutants from urban and pesticide factory wastewater treatment plants, groundwater, and deep soil.

[0005] Currently, domestic reports on the aerobic microbial degradation of acetochlor are relatively detailed, with numerous aerobic degrading bacteria isolated, the aerobic degradation pathway elucidated, and several key degradation genes cloned. For example, in the aerobic bacterium *Sphingomonas* sp. DC-6, the Riseke non-heme ferrooxidase *CndABC* is responsible for the dealkylation of acetochlor, while in the aerobic bacterium *Rhodococcus* sp. T3-1, the cytochrome P450 monooxygenase *EthBAD* is responsible for the dealkylation of acetochlor.

[0006] Anaerobic environments are widely present in farmland soils (especially under flooded conditions), wetlands, lake and river sediments, and anaerobic reactors for domestic / industrial wastewater. Studies have shown that chloroacetamide herbicides are mainly degraded by anaerobic microorganisms in anaerobic environments, but the microbial communities and molecular mechanisms involved in anaerobic degradation are still unclear. Our research group has cultivated anaerobic activated sludge that can efficiently degrade acetochlor under anaerobic conditions by simulating an industrial anaerobic reactor, and revealed the metabolic pathway of acetochlor anaerobic degradation: acetochlor first undergoes reductive dechlorination to generate 2-ethyl-6-methyl-N-(ethoxymethyl)acetanilide (EMEMA), EMEMA undergoes deethoxymethylation to generate N-(2-methyl-6-ethylphenyl)acetamide (MEPA), and then MEPA undergoes two consecutive demethylation steps to generate N-2-ethylphenylacetamide (EPA) and N-2-ethylphenyl formamide (EPF). EPF undergoes carboxylation to generate 2-ethyl-N-carboxyl aniline (ECA). To date, the key genes and enzymes involved in the anaerobic degradation of acetochlor remain unknown, which greatly restricts research on the anaerobic degradation process and mechanism of acetochlor and the development of microbial remediation technologies for acetochlor residues. Therefore, cloning the genes encoding key enzymes in the anaerobic degradation process of acetochlor has significant theoretical and practical application value. Summary of the Invention

[0007] The purpose of this invention is to provide a novel protein, its encoding gene, and its uses, said protein being capable of catalyzing the reductive dechlorination of the herbicide acetochlor.

[0008] The purpose of the invention is achieved through the following technical solution: an acetochlor reductive dechlorination enzyme ArdA, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] The encoding gene ardA of the acetochlor reductase ArdA described in this invention has a preferred nucleotide sequence as shown in SEQ ID NO.1.

[0010] The recombinant expression vector pET29a-ardA contains the encoding gene ardA described in this invention. Preferably, the recombinant expression vector pET29a-ardA is obtained by inserting the encoding gene ardA between the Nde I and Xho I sites of pET-29a(+).

[0011] The genetically engineered bacterium *E. coli* BL21(DE3)-ardA contains the encoding gene *ardA*. As a preferred embodiment of the present invention, the expression strain of the genetically engineered bacterium is *Escherichia coli* BL21(DE3)-ardA.

[0012] The application of the reductive dechlorination enzyme ardA described in this invention in the degradation and removal of acetochlor residues in soil and water.

[0013] Beneficial effects

[0014] This invention provides a reductive dechlorination enzyme ArdA and its encoding. ArdA can catalyze the reductive dechlorination reaction of the herbicide acetochlor, and has great application prospects in the treatment of acetochlor residue pollution in soil and water bodies. Attached Figure Description

[0015] Figure 1 Anaerobic degradation effect of strain BAD-20 on butachlor

[0016] A: Liquid phase diagram of butachlor degradation by strain BAD-20; B: Time-progress curve of butachlor degradation by strain BAD-20

[0017] Figure 2 Phylogenetic tree of 16S rRNA gene sequence of strain BAD-20

[0018] The number in parentheses represents the sequence number in GenBank; the number on the node represents the bootstrap value; the scale 0.02 represents the sequence deviation value.

[0019] Figure 3 LC-TOF-MS was used to detect the metabolites of butachlor degradation by strain BAD-20.

[0020] Figure 4 LC-TOF-MS was used to detect the metabolites of butachlor degradation by strain BAD-20.

[0021] Figure 5 SDS-PAGE electrophoresis image of purified ArdA reductive dechlorination enzyme. 1: Protein marker; 2: Crude enzyme from BAD-20 strain cells; 3: Ammonium sulfate fractionation purification product; 4: Ion exchange column purification product; 5: Gel filtration chromatography purification product;

[0022] Figure 6 SDS-PAGE electrophoresis image of recombinant protein ArdA. 1: Protein marker; 2: Crude enzyme solution of recombinant expression strain E. coli BL21 (DE3)-ardA; 3: 150 mM imidazole elution buffer;

[0023] Figure 7 HPLC chromatogram of acetochlor degradation by the reductive dechlorination enzyme ArdA.

[0024] A: HPLC chromatogram of acetochlor standard; B: HPLC chromatogram of EMEMA standard; C: HPLC chromatogram of ArdA degradation products of acetochlor. Detailed Implementation

[0025] Example 1: Isolation and Identification of Chloroacetamide Herbicide

[0026] 1.1 Enrichment and Isolation of Anaerobic Degrading Bacteria for Chloroacetamide Herbicides

[0027] Soil samples used to acclimatize the anaerobic degrading microbial community of chloroacetamide herbicide were collected from the rice experimental field of Jiangsu Academy of Agricultural Sciences, Nanjing City, Jiangsu Province. 3 g of the collected soil sample was weighed and added to a 30 mL serum bottle containing 20 mg / L butachlor (a chloroacetamide herbicide) in anaerobic basal salt medium (MSM). The bottle was sealed with a rubber stopper and then placed in a 30℃ constant temperature shaker at 150 r / min for incubation. During incubation, the color of the culture medium in the serum bottle should remain colorless; if the medium turns red, it indicates the introduction of oxygen. The degradation effect of butachlor was measured periodically. When approximately 60-70% of the butachlor in the serum bottle was degraded, 3 mL of the enriched solution was transferred to another serum bottle containing fresh butachlor MSM medium. This transfer process was repeated until the enriched solution could degrade 90% of the 20 mg / L butachlor within 2 weeks, at which point the acclimatization was complete.

[0028] The isolation of butachlor-degrading bacteria from the enrichment solution was carried out in an anaerobic chamber. The enrichment solution was serially diluted and spread onto PYT solid agar plates, which were then placed in an anaerobic jar and incubated at 30 °C for 7 days. During incubation, the color of the medium was carefully observed; it should remain colorless. Colonies from the plates were picked and further purified in the anaerobic chamber. The purified colonies were inoculated into MSM medium containing 20 mg / L butachlor, and samples were taken periodically to determine their butachlor-degrading ability.

[0029] Anaerobic basic salt medium (MSM, g / L): K2HPO4·3H2O (0.35 g), KH2PO4 (0.27 g), FeSO4·7H2O (0.2 g), MgCl2·6H2O (0.10 g), NH4Cl (0.53 g), CaCl2·2H2O (0.073 g), sodium citrate (0.10 g), sodium pyruvate (0.10 g), resazurite (1.0 mg), trace element complex solution (1 mL), vitamin complex solution (1 mL), pH adjusted to 7.2 with phosphate buffer.

[0030] Anaerobic isolation and culture medium (PYT, g / L): peptone (0.5 g), yeast extract (1.0 g), tryptone (0.5 g), acid-hydrolyzed casein (1.0 g), soluble starch (1.0 g), glucose (1.0 g), K₂HPO₄·3H₂O (0.6 g), MgSO₄ (0.2 g), sodium pyruvate (0.6 g), trace element complex solution (1 mL), vitamin complex solution (1 mL, filtered and sterilized before addition), pH adjusted to 7.2 with phosphate buffer. Solid medium was supplemented with 1.7% agar.

[0031] Butachlor (97% purity), metolachlor (98% purity), acetochlor (96% purity), and pretilachlor (96% purity) were purchased from Bailingwei Company (Shanghai, China). Herbicide stock solution (10000 mg / L): Weigh 0.5155 g of butachlor, 0.5102 g of metolachlor, 0.5208 g of acetochlor, or 0.5208 g of pretilachlor, dissolve in 50 ml of methanol, filter sterilize, and store at 4 °C for later use.

[0032] An anaerobic butachlor-degrading bacterium, named BAD-20, was isolated from paddy field soil. HPLC results showed that the butachlor uptake peak was significantly reduced in the culture medium inoculated with strain BAD-20. Figure 1 A), and the degradation rate increases with the extension of cultivation time, achieving a degradation rate of over 90% of butachlor (20 mg / L) within 10 days. Figure 1 B). In addition to degrading butachlor, strain BAD-20 can also degrade major chloracetamide herbicides such as metolachlor, acetochlor, and pretilachlor. The degradation half-life (Tg) of strain BAD-20 for metolachlor, acetochlor, pretilachlor, and butachlor is shown in the figure. 1 / 2 The degradation times were 1.34 d, 2.05 d, 2.78 d, and 3.20 d, respectively. Significant difference analysis showed that the degradation efficiency of the strains for chloroacetamide herbicides, from highest to lowest, was: acetochlor > acetochlor > pretilachlor > butachlor.

[0033] 1.2 Classification and identification of anaerobic bacteria that degrade chloroacetamide herbicides

[0034] Morphological identification: BAD-20 strain was streaked onto PYT solid plates and incubated in an anaerobic jar at 30 °C for 3 days. Colony morphology was observed. The bacterial cells on the plates were resuspended in phosphate buffer, fixed with 1% osmium tetroxide, and negatively stained with 2% sodium phosphotungstenate. Observation was performed using a transmission electron microscope (Hitachi H-7650, Hitachi, Japan). After 3 days of incubation on PYT solid medium at 30 °C, the colonies of BAD-20 were white, opaque, raised, and with smooth edges. The BAD-20 cells were rod-shaped, measuring 0.5-0.8 μm × 1.5-1.8 μm, without flagella, non-motile, and Gram-negative.

[0035] The 16S rRNA of this bacterium was amplified using universal primers for the bacterial 16S rRNA gene. Sequencing results showed that the 16S rRNA of BAD-20 was 1492 bp in length (NCBI GenBank accession number MW057853). Alignment with the EzTaxon-e database (www.ezbiocloud.net) for bacterial model strains showed that the 16S rRNA of strain BAD-20 was similar to that of *Proteiniphilum acetatigenes* TB 107. T It exhibits 99% homology. In a phylogenetic tree constructed using 16S rRNA from type strains closely related to strain BAD-20, strain BAD-20 clusters with type strains of the genus *Proteiniphilum*, and with *Proteiniphilum acetatigenes* TB 107. T A branch is formed with a confidence level of 100% ( Figure 2 Based on the above morphological, physiological, biochemical, and 16S rRNA phylogenetic analyses, strain BAD-20 was preliminarily identified as belonging to the genus *Proteiniphilum* sp.

[0036] 1.3 Identification of degradation metabolites of chloroacetamide herbicide

[0037] Intermediate metabolites of butachlor and acetochlor from strain BAD-20 were detected using LC-TOF-MS. The results are as follows: Figure 6 As shown, three substances were detected in the supernatant of the butachlor degradation culture medium after high-speed centrifugation. Among them, Compound I had a mass-to-charge ratio (m / z) of 312, which is the same as the molecular weight of butachlor. The mass-to-charge ratios of several fragment peaks also corresponded to the molecular structure of butachlor. Figure 3Therefore, Compound I was identified as the substrate butachlor. Compound II has a mass-to-charge ratio of 278, which is exactly the same molecular weight as N-(2,6-diethylphenyl)-N-(butoxymethyl)acetamide (DEPBMA), the product of butachlor removal. The mass-to-charge ratios of its fragment peaks also theoretically conform to the molecular structure of this product. Figure 3 Therefore, Compound II was identified as N-(2,6-diethylphenyl)-N-(butoxymethyl)acetamide (DEBMA). Compound III has a mass-to-charge ratio of 192, which is exactly the same molecular weight as the product of DEBMA after the removal of the N-butoxymethyl group. Several of its fragment peaks also show the same molecular structure as the product after the removal of the N-butoxymethyl group. Figure 3 C), therefore Compound III identified it as N-(2,6-diethylphenyl)acetamide (DEPA). In addition, N-(2-methyl-6-ethylphenyl)-N-(ethoxymethyl)acetamide (EMEMA), a dechlorination product of acetochlor degradation by this bacterium, was also detected. Figure 4 This indicates that the bacterium also degrades acetochlor via a reductive dehalogenation pathway. The above results show that the initial step of strain BAD-20 in degrading chloroacetamide herbicide is reductive dechlorination.

[0038] Example 2: Purification of the reducing dechlorinase ArdA

[0039] Cell suspensions of the acetochlor-degrading bacterium strain BAD-20 were disrupted using an ultrasonic homogenizer (Auto Science, UH-650 Ultrasonic processor, 40% intensity) for 20 min, followed by centrifugation at 12,000 rpm for 30 min. The supernatant was collected as the crude enzyme solution. Under anaerobic conditions, the crude enzyme solution was fractionated and precipitated using ammonium sulfate saturation gradients of 0-30%, 30-45%, 45-60%, 60-80%, and 80-100%. The enzyme activity of the protein precipitates at each ammonium sulfate gradient was measured. The results showed that the 60-80% ammonium sulfate precipitate exhibited acetochlor reductive dehalogenase activity. The obtained crude protein was further purified using a Q-Sepharose HP (5×7 cm, Bio-Rad) ion exchange column and a Sephades G-75 (GE Healthcare) gel filtration chromatography column. After three-step purification, the specific activity of acetochlor-reducing dehalogenase increased from the initial 0.19 U / mg protein to 13.7 U / mg protein, representing a 71.6-fold increase in acetochlor-reducing dehalogenase activity compared to the initial crude enzyme solution. Figure 5 ).

[0040] After pretreatment of the component, the digested peptides were collected by centrifugation and quantified using a NanoDrop spectrophotometer. Online separation was performed using an Ultimate 3000 RSLC nanosystem (Thermo Fisher Scientific), followed by data-dependent MS / MS analysis using an LTQ Orbitrap XL mass spectrometer equipped with a nanoelectrospray ionization source (Thermo Fisher Scientific). Raw data for protein identification were analyzed using MaxQuant (version 1.6.17.0) with the default set, yielding 23 peptide sequences: ATEINLFETDR, FPNGLFDVSPR, KFPNGLFDVSPR, NTLVINEK, NTLVINEKYGNR, SVVVFAK, SVVVFAKK, SVVVFAKKFPNGLFDVSPR, YGNRIAFGAVLTDALVPSDPLSKR, EYTHGTNAR, EYTHGTNARGFSVTQCNTCR, FDDAPE GFHPCDLYPQCK, GFSVTQCNTCR, GILSMR, GILSMRHAAMLAGLGNMGK, GILSMRHAAMLAGLGNMGKNTLVINEK, HAMLAGLGNMGK, HAMLAGLGNMGKNTLVINEK, HAMLAGLGNMGKNTLVINEKYGNR, IAFGAVLTDALVPSDPLSKR, KCIESCPVGALNGITVDQK, KCIESCPVGALNGITVDQKK, and LCPENCR. Matching analysis of the obtained peptide sequences with the protein amino acid sequences annotated in the genomic framework diagram of the degrading strains revealed an ORF annotated as a reductive dechlorination enzyme.

[0041] The acetochlor reductase dehalogenase gene was named ardA. ardA is 681 bp in length, and its sequence is shown in SEQ ID NO.1. It encodes the acetochlor reductase dehalogenase ArdA, which consists of 226 amino acids, and its amino acid sequence is shown in SEQ ID NO.2. The protein has a molecular weight of approximately 24.8 kDa. ardA forms a separate operon in the genome, and no other genes were predicted in its upstream or downstream vicinity, indicating that ardA is not located on a gene cluster. RT-PCR results showed that transcription of the ardA gene is induced by acetochlor. Comparison analysis of the protein amino acid sequence using NCBI showed that ArdA has very low similarity to some bacterial reductase proteins. Among dehalogenases whose catalytic functions have been identified and publicly reported, ArdA shares only 33.3% amino acid sequence similarity with Tetrachloroethene reductive dehalogenase derived from Desulfitobacterium hafniense, and 25.9% sequence similarity with Epoxyqueuosine reductase from Bacillus cereus ATCC 14579.

[0042] Example 3: Heterologous expression and functional verification of the acetochlor reductase gene ardA

[0043] With forward primer ardA-F:

[0044] 5'-gaaggagatatacat ATGACGGATCAAACCGAAGCGATC-3'(SeQ ID NO.3);

[0045] Reverse primer ardA-R:

[0046] The decarboxylase ardA gene was amplified by PCR using total DNA from strain BAD-20 as a template, denoted as 5'-TCACATACTCCCTTTTCCCAGCGGTTTTCGGCGAGGCGT-3' (SeQ ID NO. 4). Alternatively, the ardA gene shown in SeQ ID NO. 1 can be chemically synthesized.

[0047] Specific amplification system (50 µL):

[0048]

[0049] The reaction procedure is as follows:

[0050]

[0051] 2.1 Construction and validation of recombinant expression strains

[0052] 2.2.1 Plasmid linearization

[0053] Using the forward primer: 5'-Catatgtatatctccttc-3' (SeQ ID NO.5);

[0054] Reverse primer: 5'-caccaccaccaccaccactgagatccggctgctaacaaagcc-3' (SeQ ID NO. 6) Linearized plasmid pET-29a (+) and eliminated template contamination with Dpn I. PCR products were purified and recovered using a gel electrophoresis kit; refer to the kit instructions for specific methods. PCR products were detected by 0.75% agarose gel electrophoresis.

[0055] 2.2 Construction of recombinant expression strains

[0056] Prepare the following reaction system (10 µL) in an ice-cold water environment:

[0057]

[0058] After mixing, the mixture was incubated in a 37 ℃ water bath for 30 min. Immediately after the reaction was complete, it was cooled in an ice-water bath for 5 min. The homologous recombination product was then transformed into the *E. coli* expression strain *E. coli* BL21 (DE3). Transformants were picked and placed into 3 mL LB tubes containing 50 mg / L kanamycin and cultured at 37 ℃ and 180 rpm in a shaker. Plasmids were extracted to obtain positive transformants, which were then verified by PCR and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to verify the correctness of the DNA fragment sequence of the inserted plasmid pET-29a (+). The obtained positive clone containing pET29a-ardA, *E. coli* BL21 (DE3) expression strain, was named *E. coli* BL21 (DE3)-ardA.

[0059] 2.3 Induction and purification of ArdA

[0060] The recombinant expression strain E. coli BL21(DE3)-ardA was cultured in 100 mL LB liquid medium at 37 °C and 180 rpm until OD200 was reached. 600When the molecular weight was 0.4-0.6, 0.10 mM IPTG was added, and the cells were induced and cultured at 16 ℃ for 8 h. The cells were then collected by centrifugation at 12000 rpm for 5 min at 4 ℃, resuspended in 15 mL of 50 mM PBS (pH 7.0) buffer, sonicated for 5-10 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected. The cells and fragments were removed by filtration through a 0.22 µm aqueous filter, yielding the crude enzyme solution of the recombinant expression strain E. coli BL21 (DE3)-ardA. ArdA was then purified using a nickel affinity chromatography column. The eluent was collected and dialyzed overnight in 50 mM PBS (pH 7.0) buffer at 4 ℃ using a dialysis bag (molecular weight cutoff of 10 kDa). SDS-PAGE protein electrophoresis was used to detect the purification effect; the band size was consistent with the theoretically predicted size (24.8 kDa). Figure 6 .

[0061] 2.4 ArdA Activity Assay

[0062] A 2 mL enzyme reaction system contained 2 mM titanium (III) citrate, 2 mM methyl viologen, 0.2 mL of prepared ArdA purified enzyme, 20 mM Tris-HCl (pH 7.5), and 200 µM acetochlor. The reaction was carried out at room temperature for 24 h in an anaerobic chamber. Samples were then taken, and the degradation rate of acetochlor was determined by ultra-high performance liquid chromatography (UHPLC) under the following conditions: Thermo Fisher Syncronis C 18 (5 μm, 4.6 × 250 mm); Mobile phase: acetonitrile:water = 90:10 (V / V); Detection wavelength: 222 nm; Flow rate: 1.0 mL / min; Column temperature: 30 °C; Injection volume: 20 μL. Degradation results are shown in […]. Figure 7 Before degradation, acetochlor has a characteristic absorption peak at 16 minutes ( Figure 7 A), after degradation, the peak height and area of ​​this characteristic absorption peak decrease significantly, and correspondingly, a new peak appears at 6.5 minutes ( Figure 7 C), this peak elution time is consistent with that of the EMEMA peak of the acetochlor dechlorination product (C). Figure 7 (B) indicates that ArdA can catalyze the reductive dechlorination of acetochlor to EMEMA. Enzymatic experiments show that the specific enzyme activity of purified ArdA in catalyzing the reductive dechlorination of acetochlor is 0.43 U / mg.

Claims

1. A reduced dechlorinase gene ardA, the nucleotide sequence of which is SEQ ID NO.

1. 2.A protein ArdA encoded by the reduced dechlorinase gene ardA of claim 1, the amino acid sequence of which is SEQ ID NO.

2. 3.A recombinant expression vector pET29a-ardA containing the reduced dechlorinase gene ardA of claim 1.

4. The recombinant expression vector pET29a-ardA according to claim 3, characterized in that, 4.The recombinant expression vector pET29a-ardA of claim 3 is obtained by homologous recombination of the reduced dechlorinase gene ardA of claim 1 and a linearized pET-29a(+) plasmid. 5.A genetically engineered strain E. coli BL21 (DE3)-ardA containing the reduced dechlorinase gene ardA of claim 1.

6. The genetically engineered bacterial strain of claim 5, characterized in that, 6.The genetically engineered strain of claim 5 is E. coli BL21 (DE3)-ardA into which the recombinant expression vector pET29a-ardA is introduced. 7.Use of the decarboxylase gene ardA of claim 1, the recombinant expression vector of the reduced dechlorinase gene ardA of claim 3, or the genetically engineered strain of claim 5 in degrading and removing acetochlor residues in soil and water. 8.Use of the protein ArdA of claim 2 in removing acetochlor residues in soil or water.

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