Engineering bacterium for degrading herbicide 2, 4-D as well as construction method and application of engineering bacterium

Abstract

The invention belongs to the technical field of construction of genetic engineering strains, and particularly relates to an engineering bacterium for degrading herbicide 2, 4-D as well as a construction method and application of the engineering bacterium. The invention provides an engineering bacterium for degrading a herbicide 2, 4-D. According to the engineering bacterium, Halomonas cupida J9 is used as a basic bacterium, and the engineering bacterium is used for degrading the herbicide 2, 4-D. According to the engineering bacterium, Halomonas cupida J9 and Halomonas cupida J9 are used as the basic bacterium; and tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into non-functional sites of a genome of the Halomonas craganeensis J9. The engineering bacterium (J9U2, 4-D) takes H.cupida J9 as a basic bacterium, can survive and maintain activity in a high-salt-concentration environment, rapidly and efficiently degrade 2, 4-D, completely mineralizes 2, 4-D, can effectively convert harmful pollutants into harmless substances, reduces durable pollution in the environment, reduces treatment cost and reduces negative effects on the environment.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering strain construction, and specifically relates to an engineering bacterium for degrading the herbicide 2,4-D, and a construction method and application thereof. Background Art

[0002] 2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide commonly used to control broadleaf weeds in crops. This organochlorine pesticide persists in the environment and can accumulate in organisms through the food chain. There is evidence that its accumulation can have adverse effects on non-target organisms, affect the abundance and distribution of soil microorganisms, and even induce human diseases and endanger human health.

[0003] The degradation of pesticides by natural microorganisms is affected by factors such as the chemical structure, concentration and soil composition of the pesticides. The degradation of 2,4-D is relatively slow, and relying solely on naturally isolated microorganisms is not enough to achieve rapid and effective 2,4-D biodegradation. In addition, many industrial activities discharge high-salt and high-nutrient wastewater. If not properly treated, these wastewater streams may cause significant environmental pollution. However, the use of microorganisms for wastewater treatment may be hindered by high salinity. Therefore, there is an urgent need for an engineered bacterium that is salt-tolerant and can efficiently degrade the herbicide 2,4-D. Summary of the invention

[0004] The purpose of the present invention is to provide an engineered bacterium for degrading the herbicide 2,4-D and a construction method and application thereof. The engineered bacterium of the present invention can efficiently degrade the herbicide 2,4-D, can adapt to a high-salt environment and has mineralization ability, can reduce processing costs, and reduce negative impacts on the environment.

[0005] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D. The engineered bacterium is based on Halomonas cupida J9; tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into non-functional sites of the genome of Halomonas cupida J9;

[0006] The nucleotide sequence of the tfdA is shown as SEQ ID NO.1; the nucleotide sequence of the tfdB is shown as SEQ ID NO.2; the nucleotide sequence of the tfdC is shown as SEQ ID NO.3; the nucleotide sequence of the tfdD is shown as SEQ ID NO.4; the nucleotide sequence of the tfdE is shown as SEQ ID NO.5; and the nucleotide sequence of the tfdF is shown as SEQ ID NO.6.

[0007] As a preferred embodiment, the upp gene is deleted in the genome of the Halomonas aspirans J9, and the nucleotide sequence of the upp gene is shown in SEQ ID NO.11.

[0008] As a preferred embodiment, a promoter is also inserted into the 5′ end of each of the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

[0009] As a preferred embodiment, the promoter includes P15 promoter; the nucleotide sequence of the P15 promoter is shown in SEQ ID NO.9.

[0010] As a preferred embodiment, the insertion site of the tfdA gene is located at 3150569-3150961bp of the genome of Halomonas aspirans J9; the insertion site of the tfdB gene is located at 2822684-2823050bp of the genome of Halomonas aspirans J9; the insertion site of the tfdC gene is located at 2825177-2825719bp of the genome of Halomonas aspirans J9; the insertion site of the tfdD gene is located at 7980206-789550bp of the genome of Halomonas aspirans J9; the insertion site of the tfdE gene is located at 2827638-2828669bp of the genome of Halomonas aspirans J9; the insertion site of the tfdF gene is located at 2834359-2834775bp of the genome of Halomonas aspirans J9; the reference genome of Halomonas aspirans J9 is GenBank accession No.CP094345.

[0011] As a preferred embodiment, the genome of the Halomonas aspirans J9 further has a gfp gene and a vgb gene inserted therein; the nucleotide sequence of the gfp gene is shown in SEQ ID NO.7; and the nucleotide sequence of the vgb gene is shown in SEQ ID NO.8.

[0012] The present invention also provides a method for constructing the engineering bacteria described in the above scheme, comprising the following steps:

[0013] The complete reverse selection marker gene upp in Halomonas aspirans J9 was knocked out to obtain H. cupida J9U;

[0014] The complete reverse selection marker gene upp in Halomonas aspirans J9 was connected to pK18mobSacB to obtain the pKJU plasmid vector;

[0015] The tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes and the upstream and downstream homologous arms of each gene insertion site are respectively connected to the pKJU plasmid vector to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F respectively;

[0016] The gene was inserted into vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F and transformed into H. cupida J9U to obtain the engineered bacteria J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and ftdF genes.

[0017] As a preferred embodiment, the gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F and pKJU-GV are transformed into the H. cupida J9U, comprising: the gene insertion vector pKJU-A is transformed into the H. cupida J9U to obtain the strain J9U-A containing the tfdA gene; the gene insertion vector pKJU-B is transformed into the strain J9U-A to obtain the strain J9U-AB containing the tfdA and tfdB genes; the gene insertion vector pKJU-C is transformed into the strain J9U-AB to obtain the strain J9U-ABC containing the tfdA, tfdB and tfdC genes; the gene insertion vector pKJU-D is transformed into the strain J9U-ABC to obtain the strain J9U-ABC containing the tfdA, tfdB, The strain J9U-ABCD contains tfdC and tfdD genes; the genes are inserted into the vector pKJU-E and transformed into the strain J9U-ABCD to obtain the strain J9U-ABCDE containing tfdA, tfdB, tfdC, tfdD and tfdE genes; the genes are inserted into the vector pKJU-F and transformed into the strain J9U-ABCDE to obtain the strain J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

[0018] As a preferred embodiment, after obtaining the strain J9U-ABCDEF, the method further comprises: connecting the gfp and vgb genes and the upstream and downstream homologous arms of the insertion site with the pKJU plasmid vector to obtain a gene insertion vector pKJU-GV; transforming the gene insertion vector pKJU-GV into the strain J9U-ABCDEF to obtain the strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes. 2,4-D .

[0019] The present invention also provides the use of the engineered bacteria described in the above scheme or the engineered bacteria obtained by the construction method described in the above scheme in degrading the herbicide 2,4-D.

[0020] Beneficial effects:

[0021] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D. The engineered bacterium is based on Halomonas cupida J9; tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into the non-functional site of the genome of Halomonas cupida J9; the nucleotide sequence of tfdA is shown in SEQ ID NO.1; the nucleotide sequence of tfdB is shown in SEQ ID NO.2; the nucleotide sequence of tfdC is shown in SEQ ID NO.3; the nucleotide sequence of tfdD is shown in SEQ ID NO.4; the nucleotide sequence of tfdE is shown in SEQ ID NO.5; and the nucleotide sequence of tfdF is shown in SEQ ID NO.6. The engineered bacterium (J9U 2,4-D ) uses salt-tolerant cell Halomonas cupida J9 (H. cupida J9) as the basic bacteria, contains degradation genes for degrading the herbicide 2,4-D, can survive and maintain activity in a high salt concentration environment, quickly and efficiently degrade 2,4-D, and completely mineralize it, and can effectively convert harmful pollutants into harmless substances, reducing persistent pollution in the environment. The use of the engineered bacteria of the present invention improves the degradation efficiency, reduces the processing cost, and reduces the negative impact on the environment. The results of the embodiment show that J9U 2,4-D It can completely degrade 25 mg / L of 2,4-D within 6 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 is the fitting curve of the inhibition rate of H. cupida J9 cell growth by different concentrations of 2,4-D;

[0024] Figure 2 The figure is a schematic diagram of the integration of foreign genes into the H. cupida J9U genome; Kan is kanamycin; Upp is uracil phosphoribosyltransferase; X is the foreign gene; UP is the upstream homology arm; DN is the downstream homology arm;

[0025] Figure 3 For the engineered bacteria J9U 2,4-D Schematic diagram of gene metabolism;

[0026] Figure 4 It is a PCR detection diagram in Example 2; wherein, lane: M is Marker III; 1 is tfdA, 864bp; 2 is tfdB, 1797bp; 3 is tfdC, 768bp; 4 is tfdD, 1113bp; 5 is tfdE, 705bp; 6 is tfdF, 1065bp; 7 is gfp, 720bp; 8 is vgb, 411bp;

[0027] Figure 5 It is the Rt-PCR detection diagram in Example 3; wherein A is tfdA; B is tfdB; C is tfdC; D is tfdD; E is tfdE; F is tfdF; G is gfp; H is vgb; lane: M is Marker III; 1 is PCR with cDNA as template; 2 is PCR with genomic DNA as template; 3 is PCR with mRNA as template; 4 is PCR with ddH2O as template;

[0028] Figure 6 J9U in Example 4 2,4-D Degradation diagram of 2,4-D in MM60; A is 25 mg / L 2,4-D; B is 50 mg / L 2,4-D; C is 100 mg / L 2,4-D;

[0029] Figure 7 The HPLC analysis chart of 25 mg / L 2,4-D at different sampling times in Example 4;

[0030] Figure 8 The HPLC analysis chart of 50 mg / L 2,4-D at different sampling times in Example 4;

[0031] Fig. 9 The HPLC analysis chart of 100 mg / L 2,4-D at different sampling times in Example 4;

[0032] Fig.10 J9U under high concentration NaCl conditions in Example 5 2,4-D Schematic diagram of the degradation of 2,4-D; A is 80g / LNaCl; B is 100g / LNaCl; C is 120g / LNaCl;

[0033] Fig.11 J9U in Example 6 2,4-D Mineralization diagram of 2,4-D; A is J9U in MM60 with 25 mg / L 2,4-D added 2,4-D Mineralization of 2,4-D; B is J9U in MM100 with 25 mg / L 2,4-D added 2,4-D mineralization of 2,4-D;

[0034] Fig.12 The hypoxic condition J9U in Example 7 2,4-D The degradation diagram of 2,4-D is shown below;

[0035] Fig.13 J9U in Example 8 2,4-D Growth performance diagram; A is the growth curve diagram; B is the maximum specific growth rate (μ max );

[0036] Fig.14 Example 9 River water J9U 2,4-D Schematic diagram of the degradation of 2,4-D; A is J9U in river water with 25 mg / L 2,4-D and 60 g / L NaCl added 2,4-D Mineralization diagram of 2,4-D; B is J9U in river water with 100 mg / L 2,4-D and 100 g / L NaCl added 2,4-D Mineralization map of 2,4-D. DETAILED DESCRIPTION

[0037] The present invention provides an engineered bacterium for degrading the herbicide 2,4-D. The engineered bacterium is based on Halomonas cupida J9; tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into non-functional sites of the genome of Halomonas cupida J9;

[0038] The nucleotide sequence of the tfdA is shown as SEQ ID NO.1; the nucleotide sequence of the tfdB is shown as SEQ ID NO.2; the nucleotide sequence of the tfdC is shown as SEQ ID NO.3; the nucleotide sequence of the tfdD is shown as SEQ ID NO.4; the nucleotide sequence of the tfdE is shown as SEQ ID NO.5; and the nucleotide sequence of the tfdF is shown as SEQ ID NO.6.

[0039] The Halomonas cupida J9 (H. cupida J9) described in the present invention is a halophilic microorganism that can survive and maintain activity in a high salt concentration environment. Using it as a basic bacterium can overcome the problem of reduced activity of traditional microorganisms in high-salt wastewater, and has higher adaptability in high-salt wastewater treatment. The present invention does not specifically limit the source of the H. cupida J9 strain. In a specific embodiment of the present invention, the H. cupida J9 strain is derived from the Halomonas cupida J9 strain with a deposit number of CGMCC No. 24707. The tfdA of the present invention encodes 2,4-dichlorophenoxyacetic acid dioxygenase; tfdB encodes 2,4-dichlorophenol hydroxylase; tfdC encodes chlorophthalide-1,2-dioxygenase; tfdD encodes chloromuconate cycloisomerase; tfdE encodes chlorodiene lactone hydrolase; tfdF encodes maleoacetic acid reductase, and the above genes are related to the main pathways for microbial degradation of 2,4-D. The complete metabolic pathway of the engineered bacteria of the present invention is as follows Figure 3 As shown, the six degradation genes inserted into the engineered bacteria can convert 2,4-D into 3-oxoadipic acid, which is then converted into succinyl-CoA and acetyl-CoA by the endogenous genes PcaI, Pca J and FadA in H. cupida J9. These products then enter the tricarboxylic acid cycle, allowing the engineered bacteria to use them as carbon sources. The present invention does not specifically limit the site where the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into the H. cupida J9 genome, and insertion into a non-functional gene site of the genome is sufficient. In the present invention, the schematic diagram of the integration of exogenous genes into the H. cupida J9 genome is shown in Figure 2 shown.

[0040] As an embodiment, the upp gene is deleted in the genome of the Halomonas aspirans J9, and the nucleotide sequence of the upp gene is shown in SEQ ID NO. 11. When the upp gene in the genome of H. cupida J9 itself is successfully knocked out, the mutant strain constructed using the vector carrying the upp gene as a vector for inserting genes has 5-FU resistance, and this reverse marker can be used to screen the mutant strain.

[0041] As an embodiment, the genome of the Halomonas aspirans J9 is further inserted with a gfp gene and a vgb gene; the sequence of the GFP gene is shown in SEQ ID NO.7. In the present invention, the gfp gene can express green fluorescent protein (GFP). The introduction of the GFP gene into the engineered bacteria can enable the engineered bacteria to emit green fluorescence, which is convenient for real-time monitoring of its activity and position through a confocal microscope. This visualization method is of great significance in the actual environmental remediation process and helps to evaluate the effect of bioremediation. The vgb gene encodes Vitreoscilla hemoglobin, which can enhance the oxygen absorption capacity of the engineered bacteria.

[0042] As an embodiment, a promoter is also inserted into the 5′ end of each of the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes. In a specific embodiment of the present invention, the promoter includes a P15 promoter; the nucleotide sequence of the P15 promoter is shown in SEQ ID NO.9. In a specific embodiment of the present invention, an RBS sequence is also connected between the P15 promoter and the gene, and the nucleotide sequence of the RBS is shown in SEQ ID NO.10. In the present invention, the P15 promoter is directly connected to the gene through the RBS. As an embodiment, the tfdA gene is used as an example for explanation, and P15-RBS-tfdA will be inserted into the corresponding non-functional site as a whole.

[0043] As an embodiment, the insertion site of the tfdA gene is located at 3150569-3150961 bp of the genome of Halomonas aspirans J9; the insertion site of the tfdB gene is located at 2822684-2823050 bp of the genome of Halomonas aspirans J9; the insertion site of the tfdC gene is located at 2825177-2825719 bp of the genome of Halomonas aspirans J9; the insertion site of the tfdD gene is located at 798 0206~789550bp; the insertion site of the tfdE gene is located at 2827638~2828669bp of the genome of Halomonas aspirans J9; the insertion site of the tfdF gene is located at 2834359~2834775bp of the genome of Halomonas aspirans J9; the insertion sites of the gfp and vgb genes are located at 3148104~3149096bp of the genome of Halomonas aspirans J9; the reference genome of Halomonas aspirans J9 is GenBank accession no.CP094345. Taking the insertion site of the tfdA gene as an example, the insertion site of the tfdA gene described in the present invention can be any position in the 3150569-3150961bp of the genome of Halomonas aspirans J9; in a specific embodiment of the present invention, the insertion site of the tfdA gene is the 3150799bp of the genome of Halomonas aspirans J9, that is, the tfdA gene is inserted between the 3150799-3150800bp of the genome of Halomonas aspirans J9.

[0044] The present invention also provides a method for constructing the engineering bacteria described in the above scheme, comprising the following steps: knocking out the complete reverse screening marker gene upp in Halomonas aspirans J9 to obtain H. cupida J9U; connecting the complete reverse screening marker gene upp in Halomonas aspirans J9 to pK18mobSacB to obtain a pKJU plasmid vector; connecting the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes and the upstream and downstream homologous arms of each gene insertion site to the pKJU plasmid vector, respectively, to obtain gene insertion vectors pKJU-A and pKJU-B, respectively. , pKJU-C, pKJU-D, pKJU-E and pKJU-F; inserting the gene into the vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F and transforming them into the H. cupida J9U to obtain the engineered bacteria J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

[0045] The present invention knocks out the complete reverse screening marker gene upp in H. cupida J9 to obtain H. cupida J9U. The present invention has no special limitation on the knockout method of the upp gene, and the knockout can be performed by a conventional gene knockout method known to those skilled in the art. In the present invention, the knockout of the upp gene is performed with reference to Chinese Patent 202310723544.X.

[0046] The present invention connects the complete reverse screening marker gene upp in H. cupida J9 to pK18mobSacB to obtain a pKJU plasmid vector. The present invention does not specifically limit the connection method, and the connection method of genes and vectors well known to those skilled in the art can be used. In the present invention, the preparation of the pKJU plasmid vector is carried out with reference to Chinese Patent 202310723544.X. The pK18mobSacB of the present invention is a shuttle suicide plasmid with kanamycin resistance, which can be replicated freely in Escherichia coli, but when the host bacteria is Pseudomonas, it does not have the ability to replicate independently, and can only be site-integrated and inserted into the chromosome of the host bacteria, otherwise it will be cut and degraded as an exogenous fragment by the host strain. The pK18mobSacB of the present invention has kanamycin (Kan) resistance, and the pKJU plasmid vector obtained by connecting the reverse screening marker gene upp to pK18mobSacB has Kan resistance and 5-FU resistance. The mutant strain constructed by transforming the pKJU plasmid vector into H. cupida J9U has Kan resistance and 5-FU resistance, and the mutant strain can be obtained by double exchange screening.

[0047] As an embodiment, the present invention fuses the P15 promoter and RBS sequence with tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes respectively to obtain P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE and P15-tfdF genes. Taking the P15-tfdA gene as an example, the nucleotide sequence of the obtained P15-tfdA gene is P15 promoter+RBS sequence+tfdA gene. As an embodiment, the present invention also includes: fusing the P15 promoter and RBS sequence with gfp and vgb genes to obtain the P15-gfp-vgb gene. The nucleotide sequence of the P15-gfp-vgb gene of the present invention is P15 promoter+RBS sequence+gfp gene+RBS sequence+vgb gene. In the present invention, there is no particular limitation on the fusion method, and the fusion can be performed using a gene fusion method known to those skilled in the art.

[0048] The present invention connects the P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, P15-tfdF and P15-gfp-vgb genes and the upstream and downstream homologous arms of each gene insertion site with the pKJU plasmid vector respectively to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F and pKJU-GV.

[0049] The invention inserts the gene into vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F and pKJU-GV and transforms them into H. cupida J9U to obtain an engineered bacterium J9U containing tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, GFP and Vgb genes. 2,4-D .

[0050] The invention comprises the steps of inserting the gene into vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F and pKJU-GV to transform the gene into H. cupida J9U, comprising: inserting the gene into vector pKJU-A to obtain strain J9U-A containing the tfdA gene; inserting the gene into vector pKJU-B to obtain strain J9U-AB containing the tfdA and tfdB genes; inserting the gene into vector pKJU-C to obtain strain J9U-ABC containing the tfdA, tfdB and tfdC genes; and inserting the gene into vector pKJU-D to obtain strain J9U-ABC containing the tfdA, tfdB, The strain J9U-ABCD containing tfdC and tfdD genes is prepared; the gene is inserted into the vector pKJU-E and transformed into the strain J9U-ABCD to obtain the strain J9U-ABCDE containing tfdA, tfdB, tfdC, tfdD and tfdE genes; the gene is inserted into the vector pKJU-F and transformed into the strain J9U-ABCDE to obtain the strain J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes. As an embodiment, the present invention inserts the gene into the vector pKJU-GV and transforms into the strain J9U-ABCDEF to obtain the strain J9U containing tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, GFP and Vgb genes. 2,4-DIn a specific embodiment of the present invention, a single-double exchange screening method is used to screen strains. Kanamycin resistance is first used to screen strains that undergo the first single exchange. The screened single exchange strains are screened again in a medium containing 5-Fu to obtain target gene insertion strains that undergo the second exchange, and finally the target strains are obtained.

[0051] The present invention also provides the use of the engineered bacteria described in the above scheme or the engineered bacteria obtained by the construction method described in the above scheme in degrading the herbicide 2,4-D.

[0052] To further illustrate the present invention, an engineered bacterium for degrading the herbicide 2,4-D provided by the present invention, as well as its construction method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Main experimental materials:

[0054] LB60 medium (1 L): 70 g NaCl, 10 g tryptone, 5 g yeast extract.

[0055] MM60 medium (1L): 60g NaCl, 30g glucose, 0.5g yeast extract, 2g NH4Cl, 0.2g MgSO4, 9.65g Na2HPO4.12H2O, 1.5g KH2PO4, 10mL trace elements I, 1mL trace elements II.

[0056] Trace element solution I: 5 g / L ammonium ferric citrate, 2 g / L CaCl2, prepared with 1M HCl.

[0057] Trace element solution II: 100 mg zinc sulfate heptahydrate, 30 mg manganese chloride tetrahydrate, 300 mg boric acid, 200 mg cobalt chloride hexahydrate, 10 mg copper sulfate pentahydrate, 20 mg nickel chloride hexahydrate, 30 mg sodium aluminate dihydrate, dissolved in 1 M HCl.

[0058] 2,4-Dichlorophenoxyacetic acid (2,4-D) mother solution (10 g / L): Weigh 0.1 g 2,4-D, dissolve it in 10 mL methanol solution, and store it away from light.

[0059] Example 1 Toxicity Analysis

[0060] The half effective concentration (EC50) was used to evaluate the antibacterial activity of 2,4-D against H. cupida J9: H. cupida J9 was inoculated into MM60 medium and cultured at 37°C for 24 h, and the transfer was repeated 3 times; then the strain was transferred to fresh MM60 medium to increase the OD 600nm was 0.1, and after continuing to culture for 6 h to reach the logarithmic growth phase of the strain, different concentrations of 2,4-D (0 mg / L, 50 mg / L, 100 mg / L, 200 mg / L, 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L) were added respectively; the culture was incubated at 37 °C for 12 h, and the OD was measured. 600 The value of nm.

[0061] The inhibition data is fitted to obtain a fitting curve, such as Figure 1 As shown. According to the fitting curve, the EC50 value was 1554 mg / L. According to previous studies (W. Wilms, M. -Karczewska,M.Niemczak,A.Parus,R.Frankowski, Wolko,J.Czarny,A.Piotrowska-Cyplik,A. HJ Heipieper, Chrzanowski, 2,4-D versus 2,4-D based ionic liquids: Effect ofcation on herbicide biodegradation, tfdA genes abundance and microbiomechanges during soil bioaugmentation, J. Hazard. Mater. 452 (2023) 131209.), EC50 values ​​greater than 1000 mg / L are considered harmless. This indicates that H. cupida J9 is a robust chassis cell that can survive in an environment with high concentrations of 2,4-D, making it a promising candidate for treating 2,4-D contaminated wastewater.

[0062] Example 2 Construction of engineered bacteria

[0063] 1. Amplify genes: A. Entrust Genscript Biotechnology Co., Ltd. in Nanjing to synthesize genes responsible for the 2,4-D degradation pathway (tfdA, tfdB, tfdC, tfdD, tfdE and tfdF), as well as genes encoding GFP and VHB proteins (gfp and vgb), and optimize the codons according to the genus Halomonas. The sequence information is as follows: SEQ ID NO.1 to SEQ ID NO.6. B. Using the genes obtained in step A as templates, design primers, and use PCR amplification to obtain tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes.

[0064] 2. Extraction of genome: The genome of Halomonas cupida J9 strain (H. cupida J9) was extracted according to the kit instructions of Vazyme Company; the reference genome of the Halomonas cupida J9 strain is GenBank accession no. CP094345. The upstream homology arm UP of the tfdA gene is 679 bp upstream of the 3150799 bp of the genome, and the downstream homology arm DN is 679 bp downstream of the 3150799 bp of the genome; the upstream homology arm UP of the tfdB gene is 800 bp upstream of the 2823040 bp of the genome, and the downstream homology arm DN is 800 bp downstream of the 2823040 bp of the genome; the upstream homology arm UP of the tfdC gene is 666 bp upstream of the 2825294 bp of the genome, and the downstream homology arm DN is 666 bp downstream of the 2825294 bp of the genome; the upstream homology arm UP of the tfdD gene is 666 bp upstream of the 7980210 bp of the genome, and the downstream homology arm DN is 980210bp downstream 665bp; the upstream homology arm UP of the tfdE gene is the upstream 680bp of the 2828468bp of the genome, and the downstream homology arm DN is the downstream 680bp of the 2828468bp of the genome; the upstream homology arm UP of the tfdF gene is the upstream 666bp of the 2834728bp of the genome, and the downstream homology arm DN is the downstream 666bp of the 2834728bp of the genome; the upstream homology arm UP of the GFP and Vgb genes is the upstream 700bp of the 3148803bp of the genome, and the downstream homology arm DN is the downstream 700bp of the 3148803bp of the genome; taking the upstream and downstream homology arms of the tfdA gene as an example, the H. cupida The 3150798 bp of the J9 strain genome is the 679 bp of the 3' end of the upstream homology arm UP of the tfdA gene; the 3150800 bp of the genome is the 1 bp of the 5' end of the downstream homology arm DN of the tfdA gene. The upstream and downstream homology arm sequences UP and DN of the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb gene insertion sites were obtained by PCR amplification. The gene insertion sites are shown in Table 1.

[0065] 3. Connect the P15 promoter: Using the J9 genome in step 2 as a template, design primers according to the gene insertion site, amplify the promoters connected to different genes, and introduce the same RBS sequence (SEQ ID NO.10) at the end of each promoter sequence; fuse the amplified P15 promoter with the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes using Vazyme high-fidelity polymerase to obtain P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE and P15-tfdF fragments; fuse the P15 strong promoter with the gfp and vgb genes using Vazyme high-fidelity polymerase to obtain the P15-gfp-vgb fragment.

[0066] Table 1 Gene sources and their integration sites

[0067]

[0068] 4. Use Vazyme high-fidelity polymerase to fuse the upstream and downstream homologous arms amplified in step 2 with the P15-tfdA, P15-tfdB, P15-tfdC, P15-tfdD, P15-tfdE, P15-tfdF and P15-gfp-vgb genes (refer to step 3) to obtain UP-P15-tfdA-DN, UP-P15-tfdB-DN, UP-P15-tfdC-DN, UP-P15-tfdD-DN, UP-P15-tfdE-DN, UP-P15-tfdF-DN and UP-P15-gfp-vgb-DN fragments. Taking the UP-P15-tfdA-DN fragment as an example, the UP-P15-tfdA-DN is obtained by sequentially connecting the upstream homology arm UP of the tfdA gene, the P15 promoter, the RBS sequence, the tfdA gene, and the downstream homology arm DN of the tfdA gene, without a linker. The UP-P15-gfp-vgb-DN fragment is obtained by sequentially connecting the upstream homology arm UP of the gfp-vgb gene, the P15 promoter, the RBS sequence, the gfp gene, the RBS sequence, the vgb gene, and the downstream homology arm DN of the gfp-vgb gene, without a linker.

[0069] 5. Insertion vector construction: Knock out the reverse screening marker gene upp in the H. cupida J9 strain to obtain H. cupida J9U; connect the reverse screening marker gene upp in H. cupida J9 to pK18mobSacB to obtain the pKJU plasmid vector (see Chinese Patent 202310723544.X).

[0070] 6. Construction of J9U-A strain: (1) The plasmid pKJU was digested with restriction endonuclease EcoRⅠ to obtain a linearized plasmid. The UP-P15-tfdA-DN sequence in step 4 was then ligated with the linearized plasmid using Vazyme single fragment ligase to obtain pKJU-A, which was then transformed into DH5α competent cells.

[0071] (2) Plasmid extraction: The plasmid was extracted using the FastPure Plasmid Mini Kit from Vazyme. Primers were designed based on the pKJU plasmid vector, and bacterial liquid PCR was used to verify whether the inserted gene was inserted. The verified plasmid was sequenced. After sequencing, the target plasmid was transferred into E. coli S17-1λpir.

[0072] (3) Conjugation transformation: A. Activate the E. coli S17-1λpir containing the inserted vector by streaking on an LB plate, and activate the H. cupida J9 strain by streaking on an LB60 plate; B. Inoculate the E. coli S17-1λpir into LB liquid culture medium, and inoculate the H. cupida J9 strain into LB60 liquid culture medium, and culture them at 37°C, 180 rpm for 16 h; C. Take the cultured bacterial solution in step 2) and transfer it to new LB and LB60 liquid culture medium at a ratio of 1% (v / v), and culture them at 37°C, 180 rpm for 6 h; D. Take the cultured E. coli S17-1λpir and H. cupida containing the plasmid and culture them at 10% (v / v). J9 bacterial solution, 5000rpm, centrifuge for 5min, and discard the supernatant; E. Use 10mmoLMgSO4 and 10mmoLMgSO4 containing 60g / LNaCl to wash and resuspend E.coliS17-1λpir and H.cupida J9 bacteria three times respectively; F. Add 100μL LB liquid to resuspend the bacteria respectively, mix the two bacterial solutions, and drop them on the cellulose membrane of LB60 solid plate.

[0073] (4) Odd-Double Exchange Screening: A. Scrape the bacterial lawn obtained in step (3) combined with the culture after transformation, dissolve it in 1 mL of LB60 liquid culture medium, and spread it on an LB60 plate containing kanamycin sulfate resistance. Incubate it upside down at 37°C for 24 h until colonies grow on the plate. B. Pick a single colony and place it in LB60 liquid culture medium containing 100 μg / mL Kan, culture it at 37°C, 180 rpm, for about 6 hours, design primers according to the upstream and downstream homologous arms of the inserted gene, perform PCR verification on the bacterial solution, and screen for single exchange; C. Transfer the correctly verified strain to a LB60 test tube without resistance, culture it at 37°C, 180 rpm for 24 hours to complete the double exchange, and the suicide plasmid is lost; D. Take 100 μL of the cultured bacterial solution, dilute it 1000 times with LB60 liquid culture medium, and then spread it on LB60 liquid culture medium containing 5-Fu resistance for culture, design primers according to the inserted gene, verify the double exchange by bacterial solution PCR, and sequence the PCR product of the double exchange strain; F. Insert the correctly sequenced gene into the strain, that is, the strain containing the tfdA gene, and store it in a glycerol tube at -80°C, which is recorded as the J9U-A strain.

[0074] 7. Construction of J9U-AB strain: The UP-P15-tfdB-DN obtained in step 5 is used to construct the pKJU-B plasmid according to the method described in step 6(1), and then it is transferred into the J9U-A strain by conjugation transformation according to the method described in steps 6(2) to (3). The strains that have undergone exchange are screened for kanamycin resistance according to the method described in step 6(4). The verified correct strains are screened again in a medium containing 5-Fu to obtain the target gene-inserted strains that have undergone the second exchange, thereby obtaining the J9U-AB strain containing the tfdA and tfdB genes.

[0075] 8. Construction of J9U-ABC strain: UP-P15-tfdC-DN in step 5 was used to construct the pKJU-C plasmid according to the method described in step 6(1), and then transferred into J9U-AB by conjugation transformation according to the method described in steps 6(2) to (3). The strains that underwent exchange were screened for kanamycin resistance according to the method described in step 6(4). The correct strains were screened again in a medium containing 5-Fu to obtain the target gene-inserted strains that underwent the second exchange, thereby obtaining the J9U-ABC strain containing the tfdA, tfdB and tfdC genes.

[0076] 9. Construction of J9U-ABCD strain: UP-P15-tfdD-DN in step 5 was used to construct the pKJU-D plasmid according to the method described in step 6(1), and then transferred into the J9U-ABC strain by conjugation transformation according to the method described in steps 6(2) to (3). The strains that underwent exchange were screened for kanamycin resistance according to the method described in step 6(4). The correct strains were screened again in a medium containing 5-Fu to obtain the target gene-inserted strains that underwent the second exchange, thereby obtaining the J9U-ABCD strain containing the tfdA, tfdB, tfdC and tfdD genes.

[0077] 10. Construction of J9U-ABCDE strain: UP-P15-tfdE-DN in step 5 was used to construct the pKJU-E plasmid according to the method described in step 6(1), and then transferred into the J9U-ABCD strain by conjugation transformation according to the method described in steps 6(2) to (3). The strains that underwent exchange were screened for kanamycin resistance according to the method described in step 6(4). The correct strains were screened again in a medium containing 5-Fu to obtain the target gene-inserted strains that underwent the second exchange, thereby obtaining the J9U-ABCDE strain containing the tfdA, tfdB, tfdC, tfdD and tfdE genes.

[0078] 11. Construction of J9U-ABCDEF strain: UP-P15-tfdF-DN in step 5 is used to construct pKJU-F plasmid according to the method described in step 6(1), and then it is transferred into J9U-ABCDE strain by conjugation transformation according to the method described in steps 6(2) to (3). The strains that have undergone exchange are screened for kanamycin resistance according to the method described in step 6(4). The verified correct strains are screened again in a medium containing 5-Fu to obtain strains with the target gene inserted after the second exchange, thereby obtaining J9U-ABCDEF strain containing tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

[0079] 12. Construction of J9U2,4-D strain: The UP-P15-gfp-vgb-DN in step 5 was used to construct the pKJU-GV plasmid according to the method described in step 6(1), and then transferred into the J9U-ABCDEF strain by conjugation transformation according to the method described in steps 6(2) to (3). The strains that underwent exchange were screened using kanamycin resistance. The correct strains were screened again in a medium containing 5-Fu to obtain the target gene-inserted strain that had undergone the second exchange, thereby obtaining the J9U2,4-D strain containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes.

[0080] After the foreign gene was integrated into the H. cupida J9U genome, J9U 2,4-D The complete metabolic pathway is Figure 3 As shown in the figure, 2,4-D is converted into 2,4-dichlorophenol (2,4-DCP) by the action of 2,4-dichlorophenoxyacetic acid dioxygenase encoded by the tfdA gene; then, 2,4-DCP is degraded into 3,5-dichlorocatechol (3,5-DCC) by the action of 2,4-dichlorophenol hydroxylase encoded by the tfdB gene; 3,5-DCC is converted into 2,4 -dichloro-cis, cis-muconate; then it is converted into 2,4-chlorodienolide by the action of dichloromuconate cycloisomerase (encoded by tfdD gene); 2,4-chlorodienolide generates 2,4-chloromaleacetic acid by the action of dienolide hydrolase encoded by tfdE gene; then it is further converted into maleoacetic acid by the action of maleoacetate reductase encoded by tfdF, and then becomes β-ketoadipate; β-ketoadipate generates β-ketoadipate-CoA by the action of succinyl-CoA:3-oxooxalate-CoA transferase α subunit and β subunit encoded by J9 endogenous genes Pca I and Pca J; then it generates acetyl-CoA and succinyl-CoA by the action of β-ketoadipate-CoA thiolase encoded by J9 endogenous FadA gene, and finally enters the tricarboxylic acid cycle to realize the mineralization of 2,4-D. It can be seen that the six degradation genes can convert 2,4-D into 3-oxoadipate, but the subsequent conversion into succinyl-CoA and acetyl-CoA is completed by the endogenous genes PcaI, PcaJ and FadA in H. cupida J9, and these products then enter the tricarboxylic acid cycle, making J9U 2,4-D They can be used as a carbon source.

[0081] 13. Strain verification: A. Use SparkJade Bacterial RNA Rapid Extraction Kit to extract RNA, see the instructions for details; use Vazyme IIQ RT SuperMix for qPCR kit removes residual DNA in the solution more thoroughly, followed by reverse transcription. Primers are designed based on the inserted gene, and PCR is used to detect J9U 2,4-D Whether the strain is successfully constructed. Figure 4 shown.

[0082] The strain was subjected to Sanger sequencing, and the eight exogenous genes were successfully inserted into the H. cupida J9 genome, which was verified by PCR and Sanger sequencing. Finally, the engineered strain J9U was constructed. 2,4-D .

[0083] Example 3 RT-PCR detection:

[0084] J9U in Example 2 2,4-DThe activated strain was activated; the activated strain was incubated in LB60 overnight for 12 h; the cells were collected after centrifugation and the SPARKeasy improved bacterial RNA kit (Sparkjade) was used to extract RNA from J9U 2,4-D Total RNA was extracted from the culture medium; the residual genomic DNA in the RNA was removed using the Vazyme reverse transcription kit to obtain mRNA; the RNA was reverse transcribed into cDNA after the residual DNA was removed; the J9U 2,4-D The genome of the strain was extracted to obtain genomic DNA.

[0085] Subsequently, primers were designed according to the inserted genes, and PCR amplification was performed using cDNA, genomic DNA, mRNA, and ddH2O as templates to analyze the transcription of tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp, and vgb. The results are shown in Figure 5 shown.

[0086] The results showed that the cDNA and genomic DNA corresponding bands showed bands matching the expected size of the target gene, while no bands were observed in the negative control. These results confirmed that all eight exogenous genes were expressed in J9U 2,4-D Successful transcription in .

[0087] Example 4 Degradation experiment of different concentrations of 2,4-D

[0088] Degradation experiments were performed in MM60 culture medium supplemented with different concentrations of 2,4-D. 2,4-D The strain was inoculated into 100 mL LB60 medium at a 1% (v / v) inoculum and cultured for 9 to 12 h until the logarithmic growth phase. The bacteria were washed three times with 10 mM phosphate buffered saline (PBS60) containing 60 g / L NaCl and inoculated into 20 mL MM60 medium to adjust the initial OD 600 The nm value was 0.4. B. 25, 50, and 100 mg / L of 2,4-D were added to MM60 culture medium to detect J9U 2,4-DDegradation of 2,4-D. The control group was transformed with H. cupida J9U strain without any gene. C. The above culture was incubated on a shaker at 37°C and 180rpm, and samples were collected at different time points for analysis. D. The residual concentration of 2,4-D was determined by HPLC: Agilent 1260 Infinity II was used for liquid chromatography analysis, equipped with a ZORBAX StableBond C18 column and a variable wavelength detector (VWD). The detection wavelength of 2,4-D was set to 230nm, and the column temperature was maintained at 30°C. The mobile phase consisted of 70% methanol and 30% water (containing 0.1% phosphoric acid, v / v), the flow rate was 1mL / min, and the sample volume was 5μL.

[0089] In the HPLC chromatogram, the retention time of the characteristic peak of 2,4-D is 3.5min. Figure 6 to Figure 9 As shown, J9U 2,4-D It was able to degrade a large amount of herbicides within 2 hours and completely degrade 25 mg / L of 2,4-D within 6 hours. The strain also showed good degradation ability at higher concentrations of 2,4-D, with a degradation rate of 95% for 50 mg / L 2,4-D and 90% for 100 mg / L 2,4-D within 6 hours. The control group did not show significant degradation of 2,4-D.

[0090] Example 5 Degradation experiment of different concentrations of NaCl:

[0091] Degradation experiments were performed in MM medium supplemented with different concentrations of NaCl. A. Follow the steps in step A of Example 4. B. Use MM medium containing 25 mg / L 2,4-D and 80 g / L, 100 g / L or 120 g / L NaCl (MM80, MM100 and MM120) to evaluate the performance of the engineered bacteria under high salt conditions; the control group was transferred with the H. cupida J9U strain without any genes. C. Incubate the culture on a shaker at 37°C and 180 rpm, and collect samples at different time points for analysis. D. Determine the residual concentration of 2,4-D by HPLC, and the conditions are the same as step D in Example 4.

[0092] The results are as follows Fig.10 As shown, J9U 2,4-D It was able to rapidly degrade 25 mg / L of 2,4-D within 8 h in MM80 medium containing 80 g / L NaCl. The strain also maintained excellent degradation performance in MM100 and MM120 medium, indicating its potential for treating pollutants in high-salinity wastewater environments.

[0093] Example 6 Mineralization Experiment (Isotope Experiment)

[0094] By monitoring J9U 2,4-D In degradation 13 C6-2,4-D released during 13 CO2 was used to evaluate its ability to mineralize pollutants. 2,4-D Inoculate to contain 25mg / L 13 In MM60 and MM100 medium for C6-2,4-D, adjust the initial OD 600 nm value was 0.4, and the culture was incubated at 37 ° C for 3 days under sealed culture conditions. At the end of the incubation, the culture was acidified to a pH value below 2 with 2M HCl; then the culture was shaken at 180 rpm for 30 minutes, and the gas in the sealed vial was analyzed using an isotope mass spectrometer (CAAS ESI lab). The carbon isotope ratio was defined using the δ13C value (in ‰) relative to the V-PDB standard. The control group was transformed with the H. cupida J9U strain without any gene.

[0095] The results are as follows Fig.11 The results show that J9U was incubated in MM60 medium containing 25 mg / L 2,4-D. 2,4-D After 3D, J9U 2,4-D The amount of CO2 produced 13 The C value (2712‰) was much higher than that of the control group (128‰). 2,4-D In the incubation gas, 13 C / ( 12 C+ 13 C)% value increased by 319.87% compared with the control group. 2,4-D The ability to mineralize pollutants was also maintained under high salt conditions. 2,4-D The mineralization capacity of NH4Cl2 was also confirmed by stable isotope experiments, showing that it can perfectly mineralize pollutants under hypersaline conditions.

[0096] Example 7 Degradation experiment under hypoxic conditions

[0097] To evaluate the J9U 2,4-D The degradation ability of J9U under hypoxic conditions 2,4-D The strain was inoculated into MM60 medium containing 25 mg / L 2,4-D, and the initial OD 600 nm was adjusted to 0.4, and cultured in a sealed bottle at 37°C and 80 rpm to maintain hypoxic conditions. The degradation of 2,4-D was analyzed at different time points using HPLC, and the detection and analysis were the same as in Example 4.

[0098] The results are as follows Fig.12 As shown, the engineered strain J9U 2,4-DIt showed better growth ability under hypoxic conditions and was able to completely degrade 25 mg / L 2,4-D within 8 h.

[0099] Example 8 Gene stability and growth dynamics study:

[0100] J9U 2,4-D The cells were subcultured 20 times in 5 mL of MM60 and LB60 medium. PCR was used to verify the presence of eight exogenous genes in the 20th generation strains. 2,4-D The overnight cultures were inoculated into 100 mL of LB60 and MM60 medium, respectively. 600 After nm was adjusted to 0.1, the cells were cultured at 37 °C and 180 rpm for 48 h, and the OD was measured every 3 h. 600 nm to construct growth curves.

[0101] The results are as follows Fig.13 As shown in the figure, the 8 exogenous genes of the 20th generation strain were stably expressed in both MM60 and LB60. The degradation experiment also proved that J9U still had a very outstanding ability to degrade 2,4-D after multiple passages. 2,4-D The growth curves showed that there was no significant difference in the growth trend and maximum specific growth rate of the two strains, indicating that the insertion of exogenous genes did not affect the growth of J9U.

[0102] Example 9 River water degradation experiment

[0103] Water was collected from an unpolluted stream on the campus of Nankai University in Tianjin, China. 2,4-D was inoculated into river water culture medium containing 25 mg / L 2,4-D and 60 g / L NaCl or 100 mg / L 2,4-D and 100 g / L NaCl to achieve 10 6 The culture was incubated at 37°C and 180 rpm in a shaker and samples were taken regularly for HPLC analysis. 2,4-D The mineralization capacity of river water is achieved by using 13 C6-2,4-D was further evaluated using 25 mg / L 2,4-D and 100 mg / L NaCl.

[0104] The results are as follows Fig.14 As shown, J9U 2,4-D It can effectively degrade pollutants in river water containing 25mg / L 2,4-D and 60g / L NaCl, and the degradation is completed within 8 hours. Even in river water with high salt concentration (100mg / L 2,4-D and 100g / L NaCl), J9U 2,4-DIt still maintains its high degradation capacity, degrading 90% of 2,4-D within 15 hours, while showing good growth performance. 2,4-D The amount of CO2 produced 13 The C value was significantly higher than that of the control group, indicating that the herbicide was successfully mineralized in the natural water environment.

[0105] In summary, the present invention uses the halophilic microorganism H. cupida J9 as the chassis cell and successfully constructs the herbicide degrading bacteria J9U with a complete metabolic pathway containing six degradation genes (tfdA, tfdB, tfdC, tfdD, tfdE and tfdF) through synthetic biology. 2,4-D , which can convert 2.4-D into 3-oxoadipate, which is then converted into succinyl-CoA and acetyl-CoA by the endogenous genes PcaI, PcaJ and FadA in H. cupida J9 and enters the tricarboxylic acid cycle, making J9U 2,4-D They can be used as carbon sources to ultimately achieve efficient degradation of the herbicide 2,4-D. They can also effectively convert harmful pollutants into harmless substances, reducing persistent pollution in the environment. The engineered bacteria of the present invention improve the degradation efficiency of the herbicide 2,4-D, reduce processing costs, and reduce negative impacts on the environment.

[0106] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An engineered bacterium for degrading the herbicide 2,4-D, characterized in that: The engineered bacteria is based on Halomonas cupida J9; tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes are inserted into the non-functional sites of the genome of Halomonas cupida J9; The nucleotide sequence of the tfdA is shown in SEQ ID NO.1; the nucleotide sequence of the tfdB is shown in SEQ ID NO.2; the nucleotide sequence of the tfdC is shown in SEQ ID NO.3; the nucleotide sequence of the tfdD is shown in SEQ ID NO.4; the nucleotide sequence of the tfdE is shown in SEQ ID NO.5; and the nucleotide sequence of the tfdF is shown in SEQ ID NO.

6.

2. The engineered bacteria according to claim 1, characterized in that The upp gene is deleted in the genome of the Halomonas aspirans J9, and the nucleotide sequence of the upp gene is shown in SEQ ID NO.

11.

3. The engineered bacteria according to claim 1, characterized in that A promoter is also inserted into the 5′ end of each of the tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

4. The engineered bacteria according to claim 3, characterized in that The promoter includes P15 promoter; the nucleotide sequence of the P15 promoter is shown in SEQ ID NO.

9.

5. The engineered bacteria according to claim 1, characterized in that The insertion site of the tfdA gene is located at 3150569-3150961bp of the genome of Halomonas aspirans J9; the insertion site of the tfdB gene is located at 2822684-2823050bp of the genome of Halomonas aspirans J9; the insertion site of the tfdC gene is located at 2825177-2825719bp of the genome of Halomonas aspirans J9; the insertion site of the tfdD gene is located at 7980206-789550bp of the genome of Halomonas aspirans J9; the insertion site of the tfdE gene is located at 2827638-2828669bp of the genome of Halomonas aspirans J9; the insertion site of the tfdF gene is located at 2834359-2834775bp of the genome of Halomonas aspirans J9; the reference genome of Halomonas aspirans J9 is GenBank accessionno.CP094345.

6. The engineered bacteria according to claim 1, characterized in that The genome of the Halomonas aspirans J9 also has a gfp gene and a vgb gene inserted therein; the nucleotide sequence of the gfp gene is shown in SEQ ID NO.7; and the nucleotide sequence of the vgb gene is shown in SEQ ID NO.

8.

7. The method for constructing an engineered bacterium according to any one of claims 1 to 6, characterized in that: The following steps are involved: The complete reverse selection marker gene upp in Halomonas aspirans J9 was knocked out to obtain H. cupida J9U; The complete reverse selection marker gene upp in Halomonas aspirans J9 was connected to pK18mobSacB to obtain the pKJU plasmid vector; The tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes and the upstream and downstream homologous arms of each gene insertion site are respectively connected to the pKJU plasmid vector to obtain gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F respectively; The gene was inserted into vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E and pKJU-F and transformed into H. cupida J9U to obtain the engineered bacteria J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and ftdF genes.

8. The construction method according to claim 7, characterized in that: The gene insertion vectors pKJU-A, pKJU-B, pKJU-C, pKJU-D, pKJU-E, pKJU-F and pKJU-GV are transformed into the H. cupida J9U, which includes: the gene insertion vector pKJU-A is transformed into the H. cupida J9U to obtain the strain J9U-A containing the tfdA gene; the gene insertion vector pKJU-B is transformed into the strain J9U-A to obtain the strain J9U-AB containing the tfdA and tfdB genes; the gene insertion vector pKJU-C is transformed into the strain J9U-AB to obtain the strain J9U-ABC containing the tfdA, tfdB and tfdC genes; the gene insertion vector pKJU-D is transformed into the strain J9U-ABC to obtain the strain J9U- The strain J9U-ABCD contains tfdC and tfdD genes; the genes are inserted into the vector pKJU-E and transformed into the strain J9U-ABCD to obtain the strain J9U-ABCDE containing tfdA, tfdB, tfdC, tfdD and tfdE genes; the genes are inserted into the vector pKJU-F and transformed into the strain J9U-ABCDE to obtain the strain J9U-ABCDEF containing tfdA, tfdB, tfdC, tfdD, tfdE and tfdF genes.

9. The construction method according to claim 8, characterized in that: After obtaining the strain J9U-ABCDEF, the method further comprises: connecting the gfp and vgb genes and the upstream and downstream homologous arms of the insertion site with the pKJU plasmid vector to obtain a gene insertion vector pKJU-GV; transforming the gene insertion vector pKJU-GV into the strain J9U-ABCDEF to obtain the strain J9U containing the tfdA, tfdB, tfdC, tfdD, tfdE, tfdF, gfp and vgb genes. 2,4-D .

10. Use of the engineered bacteria according to any one of claims 1 to 6 or the engineered bacteria obtained by the construction method according to any one of claims 7 to 9 in degrading the herbicide 2,4-D.

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