Catechol 1, 2-dioxygenase gene catD and application thereof

Abstract

The invention discloses a catechol 1, 2-dioxygenase gene catD, the nucleotide sequence of the catechol 1, 2-dioxygenase gene catD is shown as SEQ ID NO: 1, the gene codes catechol 1, 2-dioxygenase shown as SEQ ID NO: 2, and the catechol 1, 2-dioxygenase gene catD contains 304 amino acids and is about 33.5 kDa; the catechol 1, 2-dioxygenase gene catD and catechol 1, 2-dioxygenase are applied to degradation of difenoconazole, pesticide residues in crops can be removed, and pollution-free green agricultural products can be produced; meanwhile, natural environments such as pesticide-polluted soil and water can be restored, the problem of environmental pollution is solved, and ecology is protected.

Description

Technical Field

[0001] The present invention belongs to the field of gene technology, and specifically relates to a catechol 1,2-dioxygenase gene cat D and its application in the degradation of difenoconazole fungicide. Background Art

[0002] Difenoconazole (DIF) is a triazole systemic fungicide. Due to its high efficiency and broad spectrum, it is widely used to effectively control fungal diseases caused by basidiomycetes and ascomycetes. It has been widely studied and applied in the past 40 years. It is widely used in the control of diseases of various crops, providing long-lasting protection, extending the survival period of crops and increasing yields, such as rice, wheat, corn, vegetables, etc. It can inhibit many common crop pathogenic fungi, such as wheat fusarium head blight, wheat powdery mildew, vegetable blight, rice sheath blight, rust, leaf spot, brown spot, anthracnose, etc. In addition, difenoconazole is also used to control fruit tree diseases and fungal infections of turfgrass.

[0003] Due to the high stability and persistence of fenpropimorph, fenpropimorph in the soil migrates to the environmental medium through rainwater erosion and surface runoff, posing a long-term risk to aquatic ecology and soil ecology. It is difficult to degrade in the soil of various places in a short period of time. It remains in the environment for a long time, not only causing serious pollution to the environment, but also entering the food chain, causing harm to humans and other non-target organisms. At present, various toxicological effects of fenpropimorph on different organisms and adverse effects of the entire life cycle of organisms have been reported. For example, fenpropimorph can induce developmental toxicity, thyroid endocrine disorders, immune system damage, metabolic disorders and reproductive toxicity, DNA damage and mitochondria-mediated apoptosis, hepatotoxicity in zebrafish larvae and adults, and severe tissue lesions in spleen tissue. In addition, some studies have also pointed out that fenpropimorph residues can enter the water body through runoff and erosion, which may have toxic effects on aquatic organisms and non-target species, such as inhibiting the growth and development of fish and algae, and having certain toxic effects on bees. Not only that, it will also reduce the diversity of soil bacterial communities and destroy the ecological balance of the soil. This shows that difenoconazole can pose a serious threat to the ecosystem and human health.

[0004] Degradation enzymes have obvious advantages over degradation bacteria. First, degradation enzymes are usually more tolerant of abnormal environmental conditions than the microorganisms that produce such enzymes. Second, the degradation effect of degradation enzymes is far better than that of microorganisms themselves, especially for low-concentration pesticides. Therefore, degradation enzymes have high degradation efficiency, can effectively utilize low-concentration pesticides, have high environmental suitability, and low environmental risks, which are the forefront and hot spots of international research on residual pesticide treatment technology. However, there are currently no biodegradable enzyme preparations for difenoconazole fungicide residues. Summary of the invention

[0005] The present invention provides a catechol 1,2-dioxygenase gene cat D, whose nucleotide sequence is shown in SEQ ID NO:1, G+C content is 65%, and the gene encodes catechol 1,2-dioxygenase as shown in SEQ ID NO:2, containing 304 amino acids and about 33.5 kDa; the catechol 1,2-dioxygenase gene cat The application of D and catechol 1,2-dioxygenase in the degradation of fenpropimorph can remove pesticide residues in crops and produce pollution-free green agricultural products; at the same time, it can repair the natural environment such as soil and water polluted by pesticides.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions: 1. Through whole genome sequencing and transcriptome analysis, it was found that Pseudomonas ( Pseudomonas sp.) A-3 catechol 1,2-dioxygenase gene cat D expression increased, according to the catechol 1,2-dioxygenase gene obtained in the whole genome cat D sequence, design specific primers cat DF: 5'-GGGATCCGAATTCGAGCTCCATGACCGTGAAAATTTCCCACAC-3'; cat DR: 5'-GCGGCCGCAAGCTTGTCGACTCAGGCCTCTTGCAAAGCC-3'; obtained from Pseudomonas sp. Pseudomonas sp.) A-3 total DNA and the catechol 1,2-dioxygenase gene was amplified cat The D fragment is 915 bp in length and encodes 304 amino acids. cat D; Among them, Pseudomonas Pseudomonas sp.) A-3 was disclosed in patent application 202311598278.9.

[0007] 2. Linearize the vector pET-28 by reverse PCR and insert the catechol 1,2-dioxygenase gene cat The D fragment was seamlessly cloned with the linearized pET-28b to obtain the recombinant cloning vector pET-28b- cat D. After successful verification, the recombinant vector was transferred into E. coli BL21, and positive single clones were screened by colony PCR. The positive clones were used to degrade difenoconazole, and the results showed that they contained catechol-1,2-dioxygenase gene. cat Escherichia coli BL21 from D could effectively degrade difenoconazole; 3. Gene containing catechol-1,2-dioxygenase cat coli BL21 with D was cultured, protein expression was induced, and catechol 1,2-dioxygenase protein was isolated and purified. Catechol 1,2-dioxygenase was used to degrade difenoconazole. The results showed that under the action of reduced coenzyme I (NADP), catechol 1,2-dioxygenase CatD could effectively degrade difenoconazole.

[0008] The present invention has the following beneficial effects: The present invention clones the catechol 1,2-dioxygenase gene obtained from Pseudomonas A-3 cat D, which can catalyze the degradation of difenoconazole fungicide, and the enzyme preparation produced by it can be used to remove pesticide residues in agricultural products such as Chinese herbal medicine, vegetables, fruits, tea, cotton, tobacco, etc., to improve the quality of agricultural products and increase added value, and can also be used to repair the natural environment such as soil and water bodies polluted by pesticides; the catechol 1,2-dioxygenase gene of the present invention cat D can be further used to construct transgenic crops to completely solve the problem of excessive pesticide residues in agricultural production and produce pollution-free green agricultural products; the present invention can solve environmental pollution problems and protect the ecological environment and human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 For the recombinant cloning vector pET-28b- cat D. Verification gel image of colony PCR after transformation; Figure 2 For the recombinant cloning vector pET-28b- cat Plasmid map of D. Figure 3 For the transformant E.coli BL21 / pET-28b- cat D. Degradation results of difenoconazole; Figure 4 The figure is the SDS-PAGE electrophoresis result after the purification of the recombinant protein, wherein lane 1 is the impurity protein, and lanes 2-7 are the pure proteins eluted 3-8 times respectively; Figure 5 These are the results of the degradation experiment of recombinant protein on fenpropimorph. DETAILED DESCRIPTION

[0010] The technical scheme of the present invention is further described in detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following technical scheme. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Ordinary technicians in this field can refer to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of the present invention to implement; In the examples, E. coli BL21 (DE3) was purchased from Sangon Biotech (Shanghai) Co., Ltd., and the E. coli high expression vector pET-28b was donated by the Guangdong Microbial Signaling and Crop Disease Control Laboratory of South China Agricultural University and preserved in the Yunnan Key Laboratory of Sustainable Utilization of Panax Notoginseng Resources of Kunming University of Science and Technology.

[0011] Lysis Buffer (1 L) in the example: 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O), 300 mM NaCl (17.54 g), 10 mM imidazole (0.68 g), adjusted to pH 8.0 with NaOH solution, filtered with a 0.22 or 0.45 µm filter membrane; Wash Buffer (1 L): 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O), 300 mM NaCl (17.54 g), 20 mM imidazole (1.36 g), adjust pH to 8.0 with NaOH solution, and filter with 0.22 or 0.45 μm filter membrane; Elution Buffer (1 L): 50 mM NaH2PO4 (7.80 g NaH2PO4·2H2O), 300 mM NaCl (17.54 g), 250 mM imidazole (17.0 g), adjust pH to 80 with NaOH solution, and filter with 0.22 or 0.45 μm filter membrane; Example 1: Catechol-1,2-dioxygenase gene cat Acquisition of D and construction of recombinant vector 1. Catechol-1,2-dioxygenase gene cat Acquisition of D Pseudomonas was extracted using a bacterial genomic DNA rapid extraction kit (Adlai) Pseudomonas sp.) A-3 whole genome DNA, using DNA as template and forward primer cat DF: 5′-GGGATCCGAATTCGAGCTCCATGACCGTGAAAATTTCCCACAC-3′ and reverse primer cat DR: 5'-GCGGCCGCAAGCTTGTCGACTCAGGCCTCTTGCAAAGCC-3', to amplify the catechol 1,2-dioxygenase gene catD fragment, amplification system is as shown in the following table, amplification reaction conditions are 98℃ 2min; 98℃ 10s, 62℃ 30s, 72℃ 1min, 30 cycles; 72℃ 5min; obtain 915bp gene fragment, PCR product is recovered by 1.0% agarose gel electrophoresis; ; 2. Linearization of vector pET-28 by reverse PCR Use forward primer pET-28-F: 5'-GTCGACAAGCTTGCGGC-3' and reverse primer pET-28-R: 5'-GGAGCTCGAATTCGGATCCC-3' to linearize the vector by reverse PCR. The amplification system is as shown in the table below. The amplification reaction conditions are 98℃ for 2min; 98℃ for 10s, 62℃ for 30s, 72℃ for 5min30s, 29 cycles; 72℃ for 5min; PCR products are recovered by 1.0% agarose gel electrophoresis;

[0012] 3. Use the seamless cloning kit (Biyuntian) to seamlessly clone the target fragment recovered in step 1 and the linearized vector pET-28b in step 2. The cloning system is as follows: ; 4. Recombinant cloning vector pET-28b- cat Transformation of D (1) Take 10 µL of the reaction product from step 3 and add it to 100 µL of BL21 competent cells. Mix gently and place the mixture on ice for 30 min. (2) Heat shock in a 42°C water bath for 90 seconds, then quickly return to an ice bath and let stand for 3-5 minutes; (3) Add 1 mL of SOC medium without antibiotics, mix gently, and culture at 37°C with shaking for 1 h. (4) Centrifuge the bacterial solution at 5000 rpm for 1 min to precipitate the bacteria, aspirate most of the culture medium, and spread the remaining 100 µL onto LB solid medium containing 50 µg / mL kanamycin and culture in a 37°C incubator overnight; (5) The next day, clones on the plate were picked for colony PCR identification. The PCR system and procedure were based on the colony PCR kit (Biyuntian). The results are shown in Figure 1 From the figure, we can see that the band size is between 750-1000bp, indicating that the recombinant cloning vector pET-28b- cat D was successfully constructed, and the recombinant cloning vector pET-28b- cat The plasmid map of D is shown in Figure 2, and also explained the recombinant cloning vector pET-28b- cat D was successfully transformed into E. coli BL21, and positive transformants E.coli BL21 / pET-28b- cat D. The successfully constructed clones were sequenced and identified, and the results showed that the sequence had no mutations and could be used for the next degradation experiment.

[0013] Example 2: Positive transformant E. coli BL21 / pET-28b- cat D Degradation experiment of difenoconazole The positive transformants were inoculated into LB liquid medium and cultured at 37°C until OD 600 =0.5, add IPTG to a concentration of 0.5 mM, induce overnight at 18°C, centrifuge at 4000 rpm for 20 min, collect the bacteria, wash the bacteria three times with inorganic salt medium (MSM), resuspend in MSM liquid medium, and the OD of the bacterial suspension is 600 The concentration of difenoconazole was 2.7, and a final concentration of 50 mg / L difenoconazole was added to 1 mL of bacterial suspension. The suspension was cultured at 30 °C with shaking for 24 h, and samples were taken at regular intervals. At the same time, Escherichia coli BL21 transformed with the empty vector pET-28b was used as a control, and the control without bacteria was set as a CK control. Use 1 mL of ethyl acetate to extract the difenoconazole in the culture medium, repeat the extraction 3 times, evaporate the ethyl acetate to dryness and resuspend it with 1 mL of acetonitrile, and use HPLC to detect the residual amount of difenoconazole in the acetonitrile resuspension; difenoconazole residual rate (%) = An / A0 × 100%, An is the concentration of difenoconazole in the culture medium after NH, A0 is the initial concentration of difenoconazole in the culture medium; difenoconazole degradation rate (%) = 100% - difenoconazole residual rate (%).

[0014] Chromatographic conditions: Agilent 1260 Infinity II high performance liquid chromatograph was used; the chromatographic column was Elite C 18 Reversed phase column (SupersilAQ-C18, 250 nm × 4.60 mm, 5 μm), injection temperature 24 °C, injection volume 20 μL, flow rate 1.000 mL / min, mobile phase: V (acetonitrile): V (water, acetic acid adjusted to pH 3.0) = 90: 10, detection wavelength 230 nm; Results Figure 3 , transformant E.coli BL21 / pET-28b- cat After D degraded 50 mg / L difenoconazole for 36 h, the residual amount of difenoconazole was 26.37%, that is, the transformant E. coli BL21 / pET-28b- catThe degradation rate of D on fenpropimorph reached 73.63% within 36 hours, while the degradation rate of Escherichia coli BL21 and CK containing the empty vector pET-28b on fenpropimorph was only about 10%; the results showed that the recombinant Escherichia coli BL21 containing the catechol-1,2-dioxygenase gene catD could effectively degrade fenpropimorph.

[0015] Example 3: Isolation and purification of catechol-1,2-dioxygenase protein and degradation experiment of difenoconazole 1. Engineering bacteria E.coli BL21 / pET-28b- cat Protein expression of D (1) Inoculation of engineered bacteria E. coli BL21 / pET-28b- cat D to 20 mL LB liquid medium (final kanamycin concentration 50 µg / mL), culture at 37°C, 200 r / min until OD 600 =0.6; (2) Add IPTG to a final concentration of 0.5 mM to the culture medium, and induce expression for 24 h at 18°C ​​and 160 r / min; (3) Transfer 20 mL of bacterial solution to a 50 mL centrifuge tube, centrifuge at 8000 g, 4 °C for 10 min, and discard the supernatant. (4) Wash the bacteria three times with 50 mM Tris-HCl buffer (pH 7.4), resuspend the bacteria in 1 mL of Lysis buffer (Tian Di Ren He purification column buffer solution containing imidazole) for every 100 mg of wet bacteria, and disrupt the bacteria with an ultrasonic disruptor under ice bath conditions (temperature protection set to 30 °C and power 200 W), with a total disruption time of 30 min (working 5 s, interval 5 s), and disrupt twice in total; (5) Centrifuge the cell lysate at 12,000 rpm and 4°C for 30 min and collect the supernatant; 2. Protein purification (1) Equilibrate the packed Ni NTA Beads 6FF gravity column with 5 column volumes of Lysis Buffer to keep the filler in the same buffer system as the target protein. Repeat the equilibration twice. (2) Add the supernatant to the balanced gravity column and retain it for at least 2 minutes to ensure that the sample and the medium are in full contact. Collect the effluent and repeat the loading to increase the binding efficiency. (3) Wash with 10-15 column volumes of Wash Buffer to remove non-specifically adsorbed impurities, collect the wash solution, and adjust the imidazole concentration for washing if necessary; (4) Use 5-10 column volumes of Elution Buffer for elution, collect in sections, collect one tube for each column volume, and detect them separately. This can ensure that all bound target proteins are eluted and obtain high-purity and high-concentration proteins. 3. SDS-PAGE analysis (1) Evenly mix 10 μL protein loading buffer and 40 μL protein, and denature at 100°C for 8 min; (2) Load 20 μL of sample, set the separation gel voltage to 80 V, and the compression gel voltage to 120 V. Stop when the sample band reaches the bottom of the green horizontal strip on the glass plate. (3) Wash the gel with RO water. Add 150 mL of RO water to each gel (enough to cover the gel), heat in a microwave oven for 30 seconds (medium-low heat), shake on a horizontal shaker at 80-95 rpm for 5 minutes, and repeat three times to remove residual SDS. (4) Stain the gel with Coomassie Brilliant Blue protein staining solution. Shake the bottle evenly before use. Add 25 mL of staining solution to each gel and shake on a horizontal shaker at 80-95 rpm for 1 h. The results of SDS-PAGE electrophoresis are as follows Figure 4 ,From the figure, we can see that the molecular weight of the fusion protein is about 34 kDa, which is basically consistent with the theoretical value, and the band is single, indicating that the purification effect is good.

[0016] 4. Degradation experiment of recombinant protein on difenoconazole Using difenoconazole as substrate, the residual rate of catechol 1,2-dioxygenase CatD after degradation of difenoconazole for 7 hours was determined by HPLC to characterize the degradation ability of catechol 1,2-dioxygenase CatD on difenoconazole. 200µL Na2EDTA, 60µL 50mg / L difenoconazole, 60µL crude enzyme (supernatant of step 1) or purified enzyme (3rd eluate of step 2), 1.68mL phosphate buffer (pH7.5), and 60µL NADP were mixed and treated for 7h. A control group without NADP was set up, and CK was the experimental group without enzyme addition. The residual rate of difenoconazole was determined by HPLC. The results are as follows Figure 5 As shown in the figure, the residual rate of fenpropimorph after adding NADP to the crude enzyme reached 30.23%, that is, the degradation rate reached 69.77%; the residual rate of the purified enzyme after adding NADP was 63.78%, that is, the degradation rate reached 36.22%. In addition, after adding NADP, its degradation efficiency was significantly higher than that without adding NADP, which proved that NADP can effectively enhance the degradation efficiency of catechol 1,2-dioxygenase CatD on fenpropimorph.

Claims

1. A catechol 1,2-dioxygenase gene cat D, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The catechol 1,2-dioxygenase gene according to claim 1 cat Application of D in the degradation of difenoconazole.

3. A catechol 1,2-dioxygenase, whose amino acid sequence is shown in SEQ ID NO:

2.

4. Use of the catechol 1,2-dioxygenase according to claim 3 in the degradation of difenoconazole.

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