Mesotrione nitroreductase MnrA as well as coding gene and application thereof

Abstract

The invention belongs to the field of environmental microorganisms and agriculture, and discloses mesotrione nitroreductase as well as a coding gene and application thereof. The amino acid sequence of the mesotrione nitroreductase MnrA is as shown in SEQ ID NO. 2; the nucleotide sequence of the gene for coding mesotrione nitroreductase MnrA is as shown in SEQ ID NO. 1; the invention also provides application of the mesotrione nitroreductase and the gene as well as an expression box, a recombinant vector and a genetically engineered bacterium capable of degrading mesotrione. The homology of the nitroreductase MnrA provided by the invention and reported nitroreductase is lower than 20%, and the nitroreductase MnrA is a novel nitroreductase. The nitroreductase MnrA disclosed by the invention can catalyze nitro reduction of mesotrione, and the mesotrione is converted into a product AMBA without herbicidal activity, so that the nitroreductase MnrA and the coding gene thereof have huge application prospects in mesotrione-resistant transgenic engineering and biodegradation detoxification and repair of mesotrione residual pollution.

Description

Technical Field

[0001] The invention belongs to the fields of environmental microorganisms and agriculture, and relates to mesotrione nitroreductase MnrA and a coding gene and application thereof. Background Art

[0002] Mesotrione is a triketone herbicide developed by Syngenta. It was launched in 2001 and is widely used in corn fields to control annual broadleaf weeds and some grass weeds. Mesotrione can be used before and after seedlings. It has systemic and contact effects and a long lasting effect. It is absorbed by the leaves, stems, and roots of plants and transmitted upward and downward through the xylem and phloem. Mesotrione inhibits the activity of p-hydroxyphenylpyruvate dioxidase (HPD) in the tyrosine metabolic pathway, preventing the conversion of p-hydroxyphenylpyruvate (4-HPP) to homogentisate (HGA), resulting in the inhibition of plastoquinone and tocopherol synthesis, which in turn affects the synthesis of carotenoids and the normal function of the antioxidant system, ultimately leading to plant death. Mesotrione has the advantages of broad spectrum, high efficiency, low toxicity, and slow development of resistance, and is considered to be an ideal herbicide for transgenic targets.

[0003] However, the long-term and large-scale use of mesotrione has caused pollution to the soil, water and other environments. Mesotrione is moderately persistent in the soil, with a half-life of up to 2-3 months in the soil. Mesotrione residues in the soil affect soil enzyme activity and microbial diversity, and cause phytotoxicity to rotation crops such as beans and vegetables. Mesotrione is easily soluble in water and has strong leaching properties. It easily migrates into surface and groundwater, causing water pollution and affecting aquatic ecosystems. In addition, mesotrione has potential hazards to non-target organisms. It is reported that low concentrations of mesotrione can cause oxidative stress and DNA damage in earthworms and fish; it can also inhibit the growth of algae and reduce their photosynthetic pigment production. Therefore, it is urgent to study the migration and transformation of mesotrione in the environment and develop mesotrione residue removal technology.

[0004] Mesotrione residues are mainly removed through microbial degradation. Microbial degradation has the advantages of being cheap and free of secondary pollution, and has become the mainstream of soil organic pollutant remediation technology and the direction of future development. Obtaining mesotrione-degrading microorganisms, cloning degradation genes and enzyme resources have the following roles in the management of pesticide residues and the elimination of its pesticide damage: (i) Introducing degradation genes into crops through modern biotechnology to construct corresponding herbicide-resistant transgenic crops; (ii) Using modern microbial fermentation technology, mesotrione-degrading strains and genes are made into degradation bacteria or enzyme preparations to achieve in-situ soil remediation. In summary, mesotrione-degrading genes and enzyme resources have very important theoretical and application value in the field of eliminating the pesticide damage and biotransformation of this type of herbicide. Summary of the invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a mesotrione nitroreductase MnrA and a gene encoding the same and an application thereof.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a mesotrione nitroreductase MnrA, wherein the amino acid sequence of the mesotrione nitroreductase MnrA is shown in SEQ ID NO.2.

[0007] In a second aspect, the present invention provides a gene encoding the mesotrione nitroreductase MnrA according to claim 1, and the nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] In a third aspect, the present invention provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence.

[0009] In a fourth aspect, the present invention provides a recombinant vector comprising the gene or the expression cassette.

[0010] Optionally, the recombinant vector is obtained by inserting the gene or the expression cassette between the NdeI and XhoI sites of pET-29a(+).

[0011] In a fifth aspect, the present invention provides a genetically engineered bacterium capable of degrading mesotrione.

[0012] Optionally, the genetically engineered bacteria is obtained by transferring the above-mentioned recombinant vector into a host strain, so that mesotrione nitroreductase MnrA is expressed in the host strain.

[0013] Optionally, the host strain is Escherichia coli BL21 (DE3).

[0014] In a sixth aspect, the present invention provides the use of the genetically engineered bacteria in the degradation and transformation of mesotrione.

[0015] Optionally, the genetically engineered bacteria is used to degrade mesotrione.

[0016] Optionally, the genetically engineered bacteria is used to remove residual mesotrione in soil and water.

[0017] In a seventh aspect, the present invention provides the use of the mesotrione nitroreductase MnrA in the degradation and conversion of mesotrione.

[0018] Optionally, the nitroreductase MnrA is used in the degradation of mesotrione.

[0019] Optionally, the nitroreductase MnrA is used to remove mesotrione from soil and water.

[0020] In an eighth aspect, the present invention provides the use of the gene in constructing mesotrione-resistant transgenic crops.

[0021] The present invention has at least one of the following beneficial effects: The present invention provides a mesotrione nitroreductase MnrA, which can catalyze the conversion of mesotrione herbicide to generate a product without herbicidal activity, 2-amino-4-methylsulfonylbenzoic acid (AMBA), under the condition that NADH and NADPH are used as cofactors, and the specific enzyme activities are 92.6 and 89.8 nmol / min / mg, respectively. The nitroreductase gene mnrA has great application potential in constructing transgenic crops resistant to mesotrione, and the nitroreductase MnrA has a good application prospect in degrading mesotrione. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 To dynamically detect the degradation of mesotrione by strain JX24.

[0023] Figure 2 The HPLC detection spectrum of mesotrione degradation by strain JX24 is shown in Figure A, AMBA standard; Figure B, control with only mesotrione added; Figure C, sample after mesotrione treatment by strain JX24.

[0024] Figure 3 The SDS-PAGE detection results of exogenously expressed MnrA, where M is a protein marker and lane 1 is the purified MnrA.

[0025] Figure 4 The HPLC detection spectrum of mesotrione degradation by MnrA, wherein Figure A, AMBA standard; Figure B, mesotrione control without enzyme treatment; Figure C, mesotrione sample treated with MnrA.

[0026] Figure 5 The UPLC / MS / MS detection spectrum of mesotrione catalyzed by nitroreductase MnrA. In which, Figure A is metabolite A; Figure B is metabolite B; Figure C is metabolite C, namely AMBA. DETAILED DESCRIPTION

[0027] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] Example 1 Screening and identification of mesotrione-degrading strain JX24 and cloning of nitroreductase gene 1.1 Screening and purification of mesotrione-degrading strains Soil samples were collected from a corn field where mesotrione was used for a long time. 5.0 g of soil was added to 90 ml of basal salt medium (2.0 g glucose, 1.0 g NH 4 Cl, 1.0 g NaCl, 1.5 g K 2 HPO 4 , 0.5 g KH 2 PO 4 , 0.2 g MgSO 4 7H 2 O, pH 7.0), add 100µM mesotrione, culture at 30℃, 180r / min for 7 days, transfer to the same culture medium with 5% inoculation, and transfer 5 times continuously. Use high performance liquid chromatography to detect the residual amount of mesotrione in the enriched culture fluid of the fifth transfer and whether there is any new metabolite. The enriched fluid with degradation effect is gradient diluted and 10 -4 Up to 10 -7 0.1 ml of each gradient dilution enrichment solution was spread on LB solid medium and cultured at 30°C for 4-5 days. Single colonies on the plate were picked and further purified by streaking. The purified single bacteria were inoculated into a basal salt liquid medium with 100 µM mesotrione. After culturing at 30°C and 180 r / min for 5 days, it was verified whether each single colony had the function of mesotrione degradation. A mesotrione-degrading bacterium was obtained through enrichment, acclimation, separation, screening and purification, and was named JX24. The degradation rate of this bacterium to 100 µM mesotrione within 12 hours was about 98% ( Figure 1 ). The degradation of mesotrione by strain JX24 was analyzed by HPLC, and the results showed that three metabolites were generated, namely metabolite A, metabolite B and metabolite C. Figure 2 Figure 1 is the HPLC detection spectrum of mesotrione degradation by strain JX24. Figure A is the AMBA standard; Figure B is the control with only mesotrione added; Figure C is the sample after mesotrione was treated by strain JX24; Figure 2 It can be seen that the retention time of metabolite C is consistent with that of the AMBA (2-amino-4-methylsulfonylbenzoic acid) standard, so metabolite C is identified as AMBA. The HPLC analysis results showed that strain JX24 degraded mesotrione through the nitro reduction pathway.

[0029] 1.2 Analysis of 16S rRNA gene sequences of mesotrione-degrading strains The 16S rRNA gene fragment of strain JX24 was amplified by PCR using the universal primers 27F (5´-GAGTTTGATCCTGGCTCAG-3´) and 1492R (5´-ACG GCTACCTTGTTACGACTT-3´). The amplified fragment was submitted to the online website Ezbiocloud (www.ezbiocloud.net) for sequence comparison. The results showed that the 16S rRNA gene sequence of the bacteria was Metabacillus indicus LMG 22858 T The similarity reached 99%, and the bacteria were preliminarily identified as Metabacillus sp.

[0030] 1.3 Cloning of mesotrione nitroreductase gene The draft genome of strain JX24 was sequenced and annotated using the Rast website (https: / / rast.nmpdr.org / ). A nitroreductase gene was found in the genome and named mxD The nucleotide sequence is SEQ ID NO.1, with a size of 624 bp and encoding 207 amino acids. The amino acid sequences of MnrA and reported mesotrione nitroreductase were compared, and the results showed that the similarity between MnrA and reported mesotrione nitroreductase was very low, not exceeding 20%.

[0031] Example 2 Nitroreductase gene mxD Highly efficient expression in BL21 (pET-29a(+)) 2.1 mxD Gene synthesis and amplification Entrust a biological company to synthesize the mxD The nucleotide sequence of the gene (624 bp) is shown in SEQ ID NO.1 in the sequence list, and the MnrA protein (207 amino acids) encoded by it is shown in SEQ ID NO.2 in the sequence list. mxD The recombinant vector was cloned into pUC57 and named pUC- mxD , and then transformed into Escherichia coli DH5α.

[0032] The forward primer: 5'-TAAGGAAGGAGATATACATATGTCAGCAGCAACAACTGAAAGCA-3' and the reverse primer: 5'-GTGGTGGTGGTGGTGCTCGAGTAAAAATGTGCTTAGTTCATCAATG -3' were used to amplify the pUC- mxD As template, PCR amplification mxDGene, PCR amplification system is: PCR amplification procedure: Pre-denaturation at 95°C for 30 s → denaturation at 95°C for 15 s, annealing at 65°C for 15 s, extension at 72°C for 30 s, perform 30 cycles → extension at 72°C for 10 min, and cool to room temperature.

[0033] After PCR amplification, the amplified product was detected by agarose nucleic acid electrophoresis, and then the corresponding DNA fragments were purified and recovered using a gel purification recovery kit.

[0034] 2.2 Double restriction enzyme digestion of plasmid pET-29a(+) Using Takara's QuickCut™ N I and QuickCut™ XOt I performed double restriction digestion on plasmid pET-29a(+), and the 50 μL restriction digestion system was as follows: The enzyme digestion system was placed in a 37°C water bath and terminated with 10× Loading Buffer after 1 h. The enzyme digestion effect was detected by agarose electrophoresis, and the linearized vector was purified and recovered using a gel purification recovery kit.

[0035] 2.3 mxD Recombinant transformation of pET-29a(+) vector and screening of positive transformants The amplified mxD The fragment and the linearized vector pET-29a(+) were recombined, and the recombinant system was as follows: The recombination reaction was placed at 37°C for 30 min and immediately placed on ice. The recombination product was converted to E. coli The recombinant expression strain BL21(DE3) was obtained from BL21( mxD ).

[0036] Example 3 Expression, purification and functional identification of nitroreductase MnrA 3.1 Expression and purification of nitroreductase MnrA The recombinant expression strain BL21 ( mxD ) was inoculated into LB medium and cultured at 37°C with a shaker at 200 rpm until OD 600nm was 0.6, IPTG was added to a concentration of 0.4 mM, and the cells were induced at 16°C for 12 h. 100 ml of bacterial solution was centrifuged to collect the cells, and the cells were washed twice with PBS (50 mM, pH 7.4), resuspended with 10 ml of PBS buffer, ultrasonically disrupted (Auto Science, UH-650Bultrasonic processor, 30% intensity) for 10 min, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and MnrA was purified using a nickel ion affinity chromatography column. The purified enzyme was subjected to SDS-PAGE electrophoresis to detect purity and size. Figure 3 The SDS-PAGE detection results of exogenously expressed MnrA, where M is a protein marker and lane 1 is the purified MnrA. Figure 3 The results showed that the purified protein presented a single band on the SDS-PAGE electrophoresis pattern, with a size of approximately 23KDa, which was consistent with the predicted theory.

[0037] 3.2 Functional identification of nitroreductase MnrA 3.2.1 Determination of MnrA-catalyzed reduction of mesotrione Enzymatic reaction system (1mL): In PBS buffer (pH 7.4), 100µM mesotrione, 1mM NADH / NADPH, 100μL purified MnrA enzyme solution (purified in 3.1), 37℃ for 30min. Each reaction starts with the addition of enzyme, and an equal volume of acetonitrile is added 30min later to terminate the reaction. The activity of MnrA is determined by high performance liquid chromatography (HPLC) to detect the reduction of mesotrione, with no enzyme added as a control. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the reduction of 1.0 nmol of substrate per minute at pH 7.4 and temperature 37℃. The enzyme activities of MnrA for mesotrione with NADH and NADPH as cofactors are 92.6 and 89.8 nmol / min / mg, respectively.

[0038] 3.2.2 Identification of products of mesotrione degradation catalyzed by MnrA The degradation products of mesotrione after the enzymatic reaction were detected by HPLC technology. HPLC detection conditions: mobile phase acetonitrile: water = 40:60 (v / v), water was adjusted to pH 3.0 with phosphoric acid, flow rate 0.5mL / min, AglientTM HPLC reverse phase C18 column (150 mm×4.6 mm, 5μm), column temperature 30℃, UV detector, detection wavelength 260 nm and 355nm, injection volume 10μL. The external standard method was quantified by peak area. The HPLC analysis results of mesotrione degradation by MnrA are shown in Figure 4 As shown, Figure 4The HPLC detection spectrum of mesotrione degradation by MnrA, where Figure A is the AMBA standard; Figure B is the mesotrione control without enzyme treatment; Figure C is the sample after mesotrione treatment with MnrA. The HPLC analysis results of mesotrione degradation by MnrA are consistent with the degradation of mesotrione by strain JX24, indicating that MnrA can catalyze the conversion of mesotrione to metabolites A, B and C, among which metabolite C is AMBA.

[0039] Liquid chromatography-mass spectrometry (LC-MS / MS) was used to identify and analyze the degradation products. The mass spectrometry detection was performed using an AB Sciex high-resolution tandem mass spectrometer, Triple TOF 5600+LC / MS system, with ESI as the ion source, positive ion detection mode, and a mass scanning range (m / z): 100-550. The test results are as follows: Figure 5 As shown, Figure 5 This is the UPLC / MS / MS detection spectrum of mesotrione catalyzed by nitroreductase MnrA, wherein Figure A is metabolite A; Figure B is metabolite B, and Figure C is metabolite C, namely AMBA. Figure 5 The results showed that MnrA catalyzed mesotrione to generate hydroxylamine intermediate and finally converted it into 2-amino-4-methylsulfonylbenzoic acid (AMBA).

[0040] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A mesotrione nitroreductase MnrA, characterized in that: The amino acid sequence of the mesotrione nitroreductase MnrA is shown in SEQ ID NO.

2.

2. A gene encoding the mesotrione nitroreductase MnrA according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. An expression cassette, characterized in that The invention comprises the gene according to claim 2 under the control of an operably linked regulatory sequence.

4. A recombinant vector comprising the gene according to claim 2 or the expression cassette according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The recombinant vector is obtained by inserting the gene according to claim 2 or the expression cassette according to claim 3 between the NdeI and XhoI sites of pET-29a(+).

6. A genetically engineered bacterium capable of degrading mesotrione, characterized in that: The recombinant vector according to claim 5 is introduced into a host strain to express mesotrione nitroreductase MnrA in the host strain.

7. Use of the genetically engineered bacteria according to claim 6 in the degradation and transformation of mesotrione.

8. Use of the mesotrione nitroreductase MnrA according to claim 1 in the degradation and conversion of mesotrione.

9. Use of the gene according to claim 2 in constructing mesotrione-resistant transgenic crops.

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