A diuron-degrading enzyme and cloning, expression and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a diuron-degrading enzyme pPuhA-47 with diuron-degrading capacity, and further discloses application thereof. The present application obtains a strain 47 capable of growing on a culture medium containing 0.5 mM diuron by screening from a long-term diuron-sprayed soil sample, and finds that the strain can degrade diuron through preliminary detection by HPLC. After genome sequencing of the strain, a diuron-degrading gene pPuhA-47 is cloned. The present application provides a new diuron-degrading enzyme and its coding gene, and has a great application prospect in the fields of herbicide pollution bioremediation and cultivation of new herbicide-resistant plant varieties.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a diuron-degrading enzyme pPuhA-47 with diuron-degrading ability, and further disclosing its cloning, expression and application. Background Technology

[0002] Diuron (N′-3,4-dichlorophenyl N-dimethylurea), a phenylurea herbicide, is a systemic herbicide with some contact activity. Diuron can be used as a selective herbicide at low doses and as a non-selective herbicide at high doses (YueXiaoLi 2020). After application to plants, diuron is absorbed by the roots and leaves, primarily by the root system. Once absorbed by the weed roots, it spreads from the roots to the above-ground parts of the plant, mainly the leaves, and then diffuses along the veins, inhibiting the Hill reaction of photosynthesis. This leads to chlorosis, yellowing, discoloration of leaf tips and margins, and ultimately wilting and death of the plant. Diuron is an active ingredient in several plant protection products and fungicide formulations. It is widely used for pre- or post-emergence control of various types of broadleaf and grass weeds in a variety of crops (such as cotton, fruit and grain), as well as for algae control in fish production ponds (Castillo et al 2006, Stork et al 2008, Lu et al 2019, Tandon and Pant 2019).

[0003] Diuron has a half-life of up to 330 days in soil, and the amount of diuron accumulated in the soil does not decrease due to volatilization. With long-term and large-scale use, its concentration in the soil continues to accumulate, and it drifts in the environment, posing a threat to non-target organisms. Therefore, with the long-term and large-scale use of diuron, its degradation has received widespread attention.

[0004] Microbial degradation is considered the primary pathway for diuron degradation. It has been reported that the degradation of diuron in microorganisms first involves the N-demethylation of one or two urea groups, generating two metabolites, DCPMU (3(3,4-dichlorophenyl)-1-methylurea) and DCPU (3,4-dichlorophenylurea), respectively. This is followed by amide bond hydrolysis, yielding the metabolite 3,4-DCA (3,4-dichloroaniline). Other studies have shown that some strains can directly convert diuron to DCPU or 3,4-DCA without forming other intermediates (Cui et al 2014). The hydrolysis of the amide bond to generate 3,4-DCA is a key enzymatic step in the degradation of diuron (Hussain et al 2015). The first biodegradation pathway of 3,4-DCA involves direct oxidative deamination to form 1,2-dichlorobenzene, followed by ortho-cleavage of 4,5-dichlorocatechol. The benzene ring of 4,5-dichlorocatechol then breaks, yielding 3,4-dichlorohexyl-3-en-1,6-diol and 3-chloro-4-oxoadipic acid. 3-chloro-4-oxoadipic acid subsequently enters the succinic acid degradation pathway. The second biodegradation pathway of 3,4-DCA involves aromatic ring dechlorination to form 4-chloroaniline (4-CA) (Silambarasan et al 2020). 4-CA undergoes further metabolism via dechlorination and deoxygenation.

[0005] The initial degradation of diuron mainly occurs under the action of amidases or amidolytic enzymes. The first phenylurea hydrolase was purified from *Bacillus spheroidae* in 1971; it catalyzes the degradation of N-methoxy-N-methylphenylurea (OMs) but not diuron or other N,N-dimethylphenylureas (Engelhardt et al 1971, Engelhardt et al 1973). Following the genetic characterization of PuhA, the first enzyme capable of degrading diuron, PuhB was later discovered. Both are amidolytic enzymes with approximately 83% homology, catalyzing the hydrolysis of amide or ester functional groups at carbon or phosphorus centers. Both enzymes maintain their highest activity at around 40°C (Khurana et al 2009).

[0006] In summary, the genes reported in current research that have the ability to degrade diuron are mainly used to degrade diuron residues in the environment and soil. There is currently no research on transgenic diuron-resistant plants. Summary of the Invention

[0007] Therefore, the technical problem to be solved by the present invention is to provide a degrading enzyme with diuron-methyl degrading activity;

[0008] The second technical problem to be solved by the present invention is to provide the degrading enzyme and its encoding gene for the degradation of diuron in the field of cultivating new diuron-resistant crops.

[0009] To solve the above-mentioned technical problems, the present invention provides a diuron-degrading enzyme, the degrading enzyme being designated pPuhA-47, the amino acid sequence of which has at least 95% similarity to the amino acid sequence shown in SEQ ID No: 1.

[0010] Specifically, the diuron-degrading enzyme has the amino acid sequence shown in SEQ ID No: 1.

[0011] The present invention also discloses a gene pPuhA-47 encoding the diuron-degrading enzyme of claim 1 or 2, comprising the nucleotide sequence shown in SEQ ID No: 2.

[0012] Specifically, the gene pPuhA-47 encoding the diuron-degrading enzyme has the nucleotide sequence shown in SEQ ID No: 2.

[0013] The present invention also discloses an expression vector for tolerance to diuron, wherein the expression vector contains the gene pPuhA-47 encoding the diuron-degrading enzyme.

[0014] Specifically, the expression vector is the recombinant plasmid pCDFDuet-pPuhA47.

[0015] The present invention also discloses a transgenic cell line resistant to diuron, wherein the transgenic cell line contains the gene pPuhA-47 encoding the diuron-degrading enzyme.

[0016] The present invention also discloses a genetically engineered bacterium resistant to diuron, wherein the genetically engineered bacterium contains the gene pPuhA-47 encoding the diuron-degrading enzyme.

[0017] The present invention also discloses a transgenic plant resistant to diuron, wherein the transgenic plant contains the gene pPuhA-47 encoding the diuron-degrading enzyme;

[0018] Preferably, the genetically modified plant is genetically modified tobacco.

[0019] The present invention also discloses the application of the diuron-degrading enzyme or the gene pPuhA-47 encoding the diuron-degrading enzyme in the field of cultivating transgenic plants capable of degrading diuron.

[0020] The present invention also discloses a method for cultivating transgenic plants with diuron degradation ability, comprising the step of transforming the gene pPuhA-47 encoding the diuron degradation enzyme into the target plant.

[0021] This invention screened soil samples treated with diuron over a long period to obtain a strain 47 that can grow on a culture medium containing 0.5 mM diuron. Preliminary HPLC analysis revealed that this strain can degrade diuron. Genome sequencing of this strain led to the cloning of a diuron-degrading gene, pPuhA-47. Under the action of pPuhA-47, the amide bond of diuron is broken to generate non-herbicidal 3,4-DCA, thereby achieving the degradation of diuron. This invention provides a novel diuron-degrading enzyme and its encoding gene, which has significant application prospects in the fields of herbicide pollution bioremediation and the breeding of new herbicide-tolerant plant varieties.

[0022] The diuron-degrading enzyme pPuhA-47 described in this invention, after enzyme activity assay and kinetic parameter determination, has a Kc of pPuhA-47. m 0.44mM, V max 0.0040 mM.min -1 K cat 20 minutes -1 K cat / K m 45.45mM -1 min -1 Compared with previously reported genes, this gene exhibits reduced affinity and catalytic efficiency. In particular, HPLC and UPLC-MS analysis revealed that the degradation rate of diuron by this gene was approximately 30%. Under the influence of this gene, the amide bond of diuron was broken, generating 3,4-DCA, which has no herbicidal activity.

[0023] This invention constructs a plant expression vector for the pPuhA-47 gene, which is resistant to diuron, and then uses the leaf disc method to transform it into diuron-resistant transgenic tobacco. Diuron resistance experiments have shown that wild-type plants wilt and die under the influence of 0.002% diuron, while tobacco plants transgenic with the pPuhA-47 gene grow normally under the influence of 0.005% diuron. This indicates that the transgenic plant can tolerate more than twice the minimum lethal dose of diuron and has the potential for transgenic application. Attached Figure Description

[0024] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0025] Figure 1 The degradation of diuron by strain 47 in Example 1;

[0026] Figure 2This is an SDS-PAGE image of the purified pPuhA-47 protein;

[0027] Figure 3 The results show the degradation performance of pPuhA-47 on diuron.

[0028] Figure 4 The UPLC-MS results are for the degradation products of pPuhA-47.

[0029] Figure 5 This refers to the results of Western blot analysis.

[0030] Figure 6 The results of the resistance of tobacco transgenic pPuhA-47 to diuron. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0032] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, all experimental conditions are conventional conditions well known to those skilled in the art.

[0033] Example 1: Screening and Identification of Diuron-Degrading Strains

[0034] In this embodiment, soil samples from different locations sprayed with diuron were mixed together and cultured in M63 liquid medium containing 0.5 mM diuron and 1 / 5 glucose. The cultured bacterial solution was then gradually transferred to M63 liquid medium containing 1 / 10 glucose without glucose for enrichment. The bacterial solution in the glucose-free medium was then spread on solid M63 medium containing 0.5 mM diuron, and single colonies were picked and streaked to screen for a strain that could grow on M63 medium containing 0.5 mM diuron.

[0035] The selected strains were cultured for 11 days in liquid culture medium with 0.5 mM diuron, chlorpyrifos, and linuron as the sole carbon source, respectively. High-performance liquid chromatography (HPLC) was used to detect whether the herbicide content in the bacterial culture decreased significantly. The results are shown in the appendix. Figure 1 .

[0036] Liquid chromatography-mass spectrometry (LC-MS) analysis of the original strain 47 revealed that the strain could degrade diuron after 10 days of culture, and the degradation rate gradually increased with the number of culture days, reaching approximately 36% after 10 days.

[0037] In this embodiment, genomic DNA was extracted using a bacterial genomic DNA extraction kit and sent to Shanghai Panoson Biotechnology Co., Ltd. for sequencing analysis. Based on the whole-genome sequencing results and annotations, an amidase (pPuhA-47) homologous to the previously reported PuhA was identified in NCBI. The amino acid sequence of pPuhA-47 was compared with the sequence of a previously reported diuron degradation gene. Phylogenetic analysis using Mega 7.0 software was used to analyze the phylogenetic relationship between pPuhA-47 and other reported degradation genes. The highest sequence identity was 17.68%, indicating that pPuhA-47 obtained in this embodiment is a novel amidase.

[0038] Specifically, the amino acid sequence of the degrading enzyme is shown in SEQ ID No: 1, and the gene pPuhA-47 encoding the diuron degrading enzyme has the nucleotide sequence shown in SEQ ID No: 2.

[0039] SEQ ID No.1

[0040] pPuhA-47 amino acid sequence MSALHLHCSTLFDGTGLQARPLHTLIIEGGVIRHVGPTAEAPRPRPGDRETQEHFVMPGLVDVHTHLAFGNAQSEEDIDIWTSDEFRALRGLFFAQHVLAAG VTSMVCPGDSGQLSIAVRNTVNGGLFEGPRIAASSRVITNRQSLNDWFPSRVGAPEYFTAALVTSRTEAIAQIRKQAKDGVDLIKIAMDGTHRRPNGEIIAAFTADETREMVDEA HRLGCRVATHAYGREAVMYAAKAGVDLVFHAFYMDDACIEALLEAGSVLAPTMTFPQNTVDFCQPHDPAISTGYAGYCARTLEVGTPVLKRAKAAGVPFACGSDSGFAITPYGEW HARELELLVRRLGFTPAEALYAATNVGARLMPKGETLGTLEPGKQADLLVLDGSPLDDIRILQDRSRLLAVYKAGEPVRLQRTGYNSKQVSDFNSLKWTDLYTRDRVAELGKWSL*

[0041] SEQ ID No.2

[0042] pPuhA-47 nucleotide sequence:

[0043]

[0044] Example 2 Cloning and Expression of Diuron Degradation Gene

[0045] In this embodiment, the amplification of the pPuhA-47 gene was performed by PCR using the bacterial genome as a template. The specific PCR reaction procedure was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 100 sec, for a total of 30 cycles; 25℃ incubation for 5 min.

[0046] The specific PCR reaction system (50 μL) is as follows: 4 μL dNTPs (2.5 mM), 10 μL 5×FastPfu Buffer, 1 μL each of forward and reverse primers, 1 μL template, 1 μL FastPfu DNA polymerase (5 U / μL), and add H2O to 50 μL.

[0047] In this embodiment, the BamHI / SalI restriction site was introduced by PCR. After the target fragment was amplified by PCR, it was digested with BamHI / SalI and then ligated into the vector pCDFDuet-1, which was digested with the same restriction.

[0048] The constructed recombinant plasmid pCDFDuet-pPuhA47 was electroporated into E. coli DE3(BL21) expression host cells. After identifying positive clones, the plasmid was activated overnight by streaking with 25 μg / mL streptomycin on LB agar plates. The activated plasmid was then inoculated into 10 mL of LB broth containing 25 μg / mL streptomycin and cultured overnight at 37°C with shaking at 180 rpm. A 1% inoculum was then transferred to a large Erlenmeyer flask containing 500 mL of LB broth and incubated at 37°C for 2-3 hours. When the OD... 600 When the concentration reached 0.6, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 0.2 mmol / L, and the mixture was induced at 18°C ​​and 180 rpm for 16 h. After induction, the cells were collected by centrifugation at 7500 rpm for 10 min, washed with pre-cooled Hepes buffer (pH 7.0), centrifuged twice to remove the supernatant, and then fully resuspended in 100 mL of fresh Hepes buffer. The cells were then disrupted using a low-temperature high-pressure homogenizer (Guangzhou Juneng Nanobiotechnology Co., Ltd.), and the cell lysate was collected. The resulting lysate was centrifuged at 12000 rpm and 4°C for 60 min, and the supernatant was collected for purification by Ni column affinity chromatography. The protein eluted with 500 mM imidazole was purified using an AKTA protein purifier to remove the imidazole. (See attached image) Figure 2As shown in the results, the pPuhA-47 protein was successfully purified by SDS-PAGE electrophoresis, with a size of approximately 48.5 kDa, consistent with the predicted value.

[0049] Example 3: Determination of the degradation effect of pPuhA-47 on diuron.

[0050] In this embodiment, the purified enzyme was used to detect the degradation effect of pPuhA-47 on diuron. High-performance liquid chromatography (HPLC) was used to detect whether the absorption peak of diuron decreased. An Agilent C18 reverse-phase column was used, with acetonitrile:water = 40:60 as the mobile phase and a flow rate of 0.8 mL / min. After the mobile phase was prepared, it was filtered through 0.22 μm filter paper, and air bubbles were removed using an ultrasonic instrument.

[0051] In this embodiment, the reaction system for detecting the degradation effect of the enzyme on diuron was as follows: 0.5 μM pPuhA-47, 0.5 mM diuron, with the remainder made up with PBS buffer. The prepared system was reacted at 37°C for 3 h, and then inactivated by boiling in water for 10 min. The control reaction system consisted of buffer plus an equal amount of diuron.

[0052] The completed reaction sample must be ultrafiltered using a 0.5 mL ultrafiltration tube with a molecular weight cutoff of 3 kDa before it can be detected by high-performance liquid chromatography (HPLC). Results are attached. Figure 3 As shown, HPLC analysis revealed that the purified protein achieved a 30% degradation rate of 0.5 mM diuron.

[0053] Example 4: Determination of enzyme activity and kinetic parameters of pPuhA-47

[0054] In this embodiment, enzyme activity is determined by detecting the reduction in substrate content. The reduction in substrate content after the reaction is determined by HPLC when different concentrations of substrate are added under the same reaction conditions.

[0055] In this embodiment, the standard curve of diuron is prepared by using diuron standard, and diuron standard solutions with concentrations of 1mM, 2mM, 3mM, 4mM and 5mM are prepared respectively. The peak area of ​​the absorption peak at 254nm is detected by high performance liquid chromatography according to the aforementioned conditions to plot the standard curve.

[0056] In this embodiment, the reaction system for determining enzyme kinetic parameters was as follows: 0.5 μM pPuhA-47, (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6 mM) diuron, with the remainder made up with buffer. The prepared system was reacted at 37°C for 3 h, followed by boiling in water for 10 min to inactivate the enzyme (three replicates per group). The degradation activity was detected by HPLC, the reduction in diuron was quantified, and K was calculated using Graphpad Prism 6 software. m (Mi constant) and V max (Maximum reaction rate), then substitute into the protein concentration to calculate the catalytic constant k. cat .

[0057] In this embodiment, the results of the enzyme kinetic parameters measurement are shown in Table 1 below.

[0058] Table 1. Dynamic parameters of pPuhA-47

[0059] Enzyme <![CDATA[K m (mM)]]> <![CDATA[V max (mM / min)]]> <![CDATA[k cat (min -1 )]]> <![CDATA[k cat / K m (mM) -1 bad -1 )]]> pPuhA-47 0.44±0.13 0.0040±0.0006 20 45.45

[0060] Example 5: UPLC-MS analysis of degradation products of diuron by pPuhA-47

[0061] In this embodiment, after pPuhA-47 degrades diuron, the presence of diuron degradation markers such as 3,4-DCA is detected by UPLC-MS.

[0062] In this embodiment, the reaction system preparation and sample pretreatment were as follows: the blank control group contained 0.5 mM diuron (Aladdin, purity higher than 99%), DMSO, and PBS buffer; the experimental group contained 0.5 mM diuron, 0.5 μM pPuhA-47, DMSO, and PBS buffer; the samples were ultrafiltered, treated with liquid nitrogen, and then analyzed using a Thermo Scientific QExactive Ultimate 3000 UPLC ultra-high performance liquid chromatography-tandem mass spectrometry system. The results are shown in the attached figure. Figure 4 As shown.

[0063] UPLC-MS results showed that diuron was successfully detected in both the analytical standard and the reaction solution of pPuhA-47 [m / z = 231.0092]. Diuron was detected at approximately 8.8 minutes, with a peak value of 3.92 × 10⁻⁶. 9 (like Figure 4(a) Furthermore, compared with the mass spectrometry results of the pPuhA-47 reaction solution and the diuron analytical standard, in addition to diuron, 3,4-DCA, the product of diuron hydrolysis by amidase, was detected in the reactants at 8.32 minutes, with a peak value of 1.05 × 10⁻⁶. 9 The concentration detected was 3.92 × 10⁻⁶ compared to the standard variety with the same concentration. 9 Compared to a significant reduction (such as) Figure 4 (b)

[0064] It is evident that the pPuhA-47 gene encodes an amidase, under which the amide bond of diuron is broken, generating 3,4-DCA.

[0065] Example 6: Construction of diuron-resistant transgenic tobacco using leaf disc method and detection of transgenic tobacco.

[0066] In this embodiment, in order to analyze the diuron resistance of the pPuhA-47 gene in plants, a transgenic tobacco containing pPuhA-47 and the Flag tag was constructed by using pCAMBIA1300S as a vector and "Yanyan 97" as a recipient through leaf disc transformation.

[0067] First, sterile tobacco seedlings were cultivated. Mature seeds of "Yanyan 97" were selected and immersed in clean water for 5 minutes, filtered through gauze, and rinsed again with clean water. They were then soaked in 70% alcohol for 8 seconds, 2% sodium hypochlorite for 10 minutes, and rinsed 5 times with sterile water. The seeds were inoculated onto MS germination medium and cultured alternately at 25℃, light intensity of 1000-1500 lux, 16 hours of light, and 8 hours of darkness. The seeds germinated in about one week, and sterile seedlings emerged after two weeks. Once the seedlings had 5-6 leaves, they were ready for transformation.

[0068] Using a sterile punch, take leaf samples approximately 0.5 cm long (avoiding veins) and transfer them to a pre-culture medium, with the upper epidermis facing upwards. Culture for 1 day under 18 hours of light followed by 6 hours of darkness. In a liquid solution containing rifampicin, tetracycline, and kanamycin, incubate a small amount of *Agrobacterium tumefaciens* containing the target gene at 28°C for 3 days, then transfer to 50 mL of medium for further culture. Suspend *Agrobacterium* in the liquid co-culture medium, adjust the OD600 value to 0.5-1.0, and store on ice. Infect the pre-cultured leaf discs with *Agrobacterium* solution for approximately 30 minutes. Gently remove each leaf disc one by one with tweezers, remove excess solution, and place them in MS co-culture medium, epidermis facing upwards. Place a filter paper on the surface of the co-culture medium. Seal with sealing film, epidermis facing upwards, and culture at 23°C in the dark for 3 days. First, wash the co-cultured leaf discs with sterile water, then wash them with an aqueous solution containing cephalosporin, and blot dry the surface moisture with filter paper. Transfer the explants to selection medium, gently pressing the leaf disc edges into the medium. Culture at 24℃, with alternating light and dark cycles of 12 hours per day and 12 hours per day for approximately two weeks. In the selection medium, callus will grow from the explant edges, and buds will emerge from the callus. When the buds reach 3 mm in length, transfer them to rooting medium, placing 2-3 plants per bottle. Once the roots reach 3-4 cm in length, the seedlings are ready for hardening off and transplanting. Leaves from the regenerated seedlings are cut, and DNA is extracted using the CTAB method. Glyphosate resistance is then verified in transgenic positive seedlings using hygromycin resistance gene-specific primers.

[0069] In this embodiment, plant protein was extracted for Western blot analysis, as follows:

[0070] Plant protein extraction: Plant tissue was ground in a mortar with liquid nitrogen; the dry weight was weighed, and 10 mL of Western blot buffer, 1% protease inhibitor, and 1% DTT were added to every 1 g of plant tissue; the mixture was incubated on ice for 30 min, with repeated inversion during the incubation period; the mixture was centrifuged at 4 °C and 12000 rpm for 10 min, the precipitate was discarded, and the supernatant was collected. This process was repeated twice; the supernatant was filtered through a filter membrane, and the resulting liquid was the total protein from the plant leaves.

[0071] Western blot: Prepare two identical SDS-PAGE gels and load samples at the same locations. Spot pre-stained protein markers onto the gel used for transfer. Use one gel for Coomassie Brilliant Blue staining and the other for transfer. Cut out the target region. Cut filter paper and NC membrane according to the gel size (filter paper slightly larger than the gel, NC membrane slightly larger than the filter paper). Wet the NC membrane and filter paper in transfer buffer (NC membrane soaked for 10 min, filter paper wetted for 30 sec). For transfer, wet the carbon plate with transfer buffer, aligning it in the following order: anode carbon plate, filter paper, NC membrane, gel, filter paper. Remove air bubbles after each layer. Transfer at 4°C for 30 min. Coat overnight at 4°C with 5% skim milk (prepared with PBST). Wash the membrane twice (10 min each time) with 1 / 1000 Tween 20 in BS solution; incubate with primary antibody (Flag antibody purchased from Proteintech, a mouse monoclonal antibody), dilute the primary antibody to a suitable concentration with 5% skim milk, and incubate for 2 h in a decolorizing shaker; wash the membrane three times with PBST, dilute the secondary antibody labeled with horseradish oxidase (purchased from Proteintech, goat anti-mouse secondary antibody) with 5% skim milk, and incubate for 1 h; discard the secondary antibody, wash the membrane three times with PBST, and then develop the color; turn on the chemiluminescence imaging system (MF-ChemiBIS) for pre-cooling, absorb the surface liquid of the NC membrane to be tested, place it in the imaging system, mix the two components of the color development solution in equal volumes, and then evenly drop it onto the surface of the NC membrane, and expose for 30 sec-10 min for imaging.

[0072] Four transgenic tobacco plants and one negative-negative plant were selected, and the expression of the target gene in tobacco was detected by the Western blot method described above. The results are as follows: Figure 5 As shown, the pPuhA-47 gene is successfully expressed in tobacco.

[0073] Example 7: Resistance Test of Transgenic Tobacco Resistant to Dichlorvos

[0074] In this embodiment, when the transgenic tobacco reached the 5-7 leaf stage, diuron herbicide at concentrations of 0.002% and 0.005% was applied, respectively. The growth of the tobacco was observed 15 days after application, and the results are shown in the attached figure. Figure 6 As shown.

[0075] It is evident that wild-type plants wilted and died after being treated with 0.002% diuron, while tobacco plants transgenic with the pPuhA-47 gene still grew normally after being treated with 0.005% diuron, indicating that the transgenic plants can tolerate more than twice the minimum lethal dose of diuron.

[0076] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A diuron-degrading enzyme, characterized in that, The degrading enzyme is denoted pPuhA-47, and the amino acid sequence of the degrading enzyme is shown as SEQ ID No:

1.

2. A gene encoding the diuron-degrading enzyme of claim 1 pPuhA-47 characterized in that, The nucleotide sequence thereof is shown as SEQ ID No:

2.

3. A dicamba-tolerant expression vector, characterized in that, The expression vector contains the gene encoding the diuron-degrading enzyme according to claim 2 pPuhA-47 .

4. A genetically engineered bacteria which is tolerant to diuron, characterized in that, The genetically engineered bacteria contain the gene encoding the diuron-degrading enzyme according to claim 2 pPuhA-47 .

5. The diuron-degrading enzyme of claim 1 or the gene encoding the diuron-degrading enzyme of claim 2 pPuhA-47 Use in the field of breeding transgenic plants capable of degrading diuron.

6. A method for breeding a diuron-degrading transgenic plant, comprising the steps of, comprising the gene encoding the diuron-degrading enzyme of claim 2 pPuhA-47 the step of transforming into a plant of interest.

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