Novel mesotrione nitroreductase Bamnr as well as coding gene and application thereof

Abstract

The invention belongs to the field of agricultural biotechnology and environmental microbiological engineering, and discloses a novel mesotrione nitroreductase Bamnr, and a coding gene and application thereof. The nucleotide sequence of the gene is SEQ ID NO.1, the full length is 639bp, 212 amino acids are encoded, and the amino acid sequence of the gene is SEQ ID NO.2. The highest homology of Bamnr and reported nitroreductase is only 69.4%, so that Bamnr is a new nitroreductase gene. The nitroreductase Bamnr provided by the invention can catalyze nitro reduction of mesotrione, and the mesotrione is converted into a product 2-amino-4-methylsulfonyl benzoic acid (AMBA) without herbicidal activity, so that the Bamnr gene has a huge application prospect in biodegradation detoxification and repair of the mesotrione.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and relates to a new mesotrione nitroreductase Bamnr and a coding gene and application thereof. Background Art

[0002] The application of herbicide-resistant transgenic engineering can effectively and cheaply control farmland weeds while ensuring the safety of crops, creating a new era of farmland weed control. At present, most of the crops promoted for planting are glyphosate-resistant transgenic crops. In 2017, the global area of ​​glyphosate-resistant transgenic crops reached 150 million hectares, accounting for more than 80% of the global transgenic crop planting area. However, the long-term use of glyphosate has brought about serious weed resistance problems. So far, more than 40 major farmland weeds have been reported to be resistant to glyphosate, making it impossible to control weeds. One of the effective measures to solve this problem is to use herbicides with different weed-killing mechanisms at the same time and to construct corresponding herbicide-resistant transgenic crops in conjunction with them. Therefore, it is necessary to widely explore the resistance genes of various herbicides to provide genetic resources for the construction of transgenic crops resistant to various herbicides.

[0003] 4-Hydroxyphenylpyruvate dioxygenase (HPPDEC1.13.11.27) is a key enzyme involved in tyrosine metabolism in plants and some bacteria. Tyrosine generates 4-hydroxyphenylpyruvic acid (4-HPP) under the action of tyrosine aminotransferase (TAT), and then converts to homogentisic acid (HGA) under the catalysis of HPPD. Homodisic acid is a key precursor for the biosynthesis of plastoquinone and tocopherol in plants, while plastoquinone is a component of the photosynthetic electron transport chain, and tocopherol is an important antioxidant in plants. HPPD inhibitor herbicides competitively inhibit the activity of HPPD, hindering the synthesis of homogentisic acid (HGA), and thus failing to synthesize plastoquinone and tocopherol, resulting in albinism and death of plants. Therefore, this catalytic reaction also makes HPPD developed as an important herbicide target enzyme.

[0004] HPPD inhibitor herbicides are mainly divided into triketones, isoxazoles and phenylpyrazoles according to their chemical structures. Mesotrione, also known as mesotrione, is a triketone herbicide developed by Syngenta in 1984. It can control most broadleaf weeds and a few grass weeds. Since its launch in 2001, mesotrione has become the world's largest HPPD inhibitor herbicide, with global sales of US$650 million in 2016. Although mesotrione has the advantages of high efficiency, low toxicity, high crop safety and good environmental compatibility, weeds gradually develop resistance to this type of herbicide with long-term use. Therefore, obtaining new mesotrione degradation and detoxification / resistance genes with excellent performance and developing transgenic crops have very important theoretical and practical application value. Summary of the invention

[0005] The purpose of the present invention is to provide a new protein, a coding gene and use thereof, wherein the protein not only has mesotrione nitroreductase activity but also completely degrades mesotrione to lose its herbicidal activity.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A mesotrione nitroreductase Bamnr, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The gene bamnr encoding the mesotrione nitroreductase Bamnr of the present invention preferably has a nucleotide sequence as shown in SEQ ID NO.1.

[0009] A recombinant expression vector containing the mesotrione nitroreductase gene bamnr.

[0010] As a preferred embodiment of the present invention, the recombinant expression vector is obtained by homologous recombination of the mesotrione nitroreductase gene bamnr and a linearized pET-28a(+) plasmid.

[0011] A genetically engineered strain containing the mesotrione nitroreductase gene bamnr of the invention.

[0012] As a preferred embodiment of the present invention, the expression strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

[0013] Application of the mesotrione nitroreductase Bamnr of the present invention in the biodegradation, detoxification and restoration of mesotrione.

[0014] The invention discloses an application of the mesotrione nitroreductase Bamnr in the preparation of a mesotrione degradation preparation. The invention discloses an application of the mesotrione nitroreductase gene bamnr in the biodegradation, detoxification and repair of mesotrione.

[0015] Beneficial effects:

[0016] The invention provides a new mesotrione nitroreductase Bamnr, which can degrade mesotrione into a product 2-amino-4-methylsulfonylbenzoic acid (AMBA) and loses herbicidal activity. Bamnr and its encoding gene have potential application value in mesotrione biodegradation, detoxification and repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Phylogenetic tree constructed by 16S rRNA gene of strain P15 and its closest related model strain

[0018] Figure 2 :HPLC detection of mesotrione degradation by strain P15

[0019] A: AMBA standard sample; B: mesotrione standard sample; C: 100mg / L mesotrione degradation for 0h; D: degradation for 72h.

[0020] Figure 3 : SDS-PAGE detection results of exogenously expressed Bamnr

[0021] M: protein maker, 1: crude Bamnr enzyme, 2: purified Bamnr

[0022] Figure 4 :HPLC detection of mesotrione degradation by Bamnr

[0023] A: 0h reaction; B: 2h reaction

[0024] Figure 5 :Inhibitory effect of degradation products on rice HPPD (OsHPPD) DETAILED DESCRIPTION

[0025] Example 1. Isolation of mesotrione-degrading strain and search for its nitroreductase gene

[0026] 1.1 Enrichment, isolation and classification of mesotrione-degrading strains

[0027] Soil samples were collected from corn fields where mesotrione was used for a long time. 5 g of soil samples were added to 100 mL of basal salt medium containing 50 mg / L mesotrione. After 7 days of shaking culture at 30°C and 150 r / min, the soil samples were transferred to the same medium at a 5% inoculation rate. The transfer was repeated three times. The degradation of mesotrione by the enriched solution was determined by high performance liquid chromatography. The enriched solution with degradation effect was graded diluted and 1 mL of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The dilution was spread on LB plates and cultured at 30°C for 3 days. Single colonies on the plates were picked and further purified by streaking. The obtained pure bacteria were inoculated into a basal salt liquid medium with 100 mg / L mesotrione, and cultured at 30°C and 150 r / min for 5 days to verify whether each pure strain had the ability to degrade mesotrione.

[0028] The formula of basal salt medium (MSM): 1.5g K2HPO4·3H2O; 0.5g KH2PO4; 1.0g NH4NO3; 0.5g NaCl; 0.2g MgSO4·7H2O, add deionized water to make up to 1L, and add 15.0g agar per liter of solid culture medium.

[0029] LB medium formula: 5 g yeast extract; 10 g tryptone; 10 g sodium chloride; NaOH to adjust pH to 7.0.

[0030] A mesotrione-degrading bacterium was obtained by enrichment, acclimation, isolation and screening, and was named P15. After growing on solid culture medium for 3 days, the colony of strain P15 was milky white, moist, smooth, with regular edges and a diameter of about 0.3-0.5 mm; the bacteria were Gram-positive, facultatively anaerobic, rod-shaped (0.6-0.8×1.8-3.2μm), spores were formed at one end of the cell, and the spore capsule was swollen. Using the genomic DNA of strain P15 as a template, PCR amplification was performed with universal primers for bacterial 16S rRNA gene sequences, and a 16SrRNA gene sequence of about 1449bp in length was obtained (GenBank: PP526999). Blast was performed in the EZtaxon database (http: / / eztaxon-e.ezbiocloud.net / ezt), and the results showed that strain P15 was most closely related to Paenibacillus strains and Paenibacillus cavernae C4-5 T The highest homology was 93.3%. In the phylogenetic tree constructed by 16SrRNA gene, strain P15 was also clustered in the genus Paenibacillus ( Figure 1), so strain P15 was preliminarily identified as Paenibacillus.

[0031] 1.2 Degradation of mesotrione by strain P15

[0032] Study on degradation characteristics: P15 was inoculated into LB liquid medium and cultured at 30°C until mid-logarithmic phase. The cells were collected by low-speed centrifugation, washed twice with fresh, sterile basal salt medium, and resuspended in basal salt medium. The cell concentration was adjusted to about 1.0×10 9 cfu / ml, inoculated into 20 ml of basal salt medium containing 100 mg / L mesotrione at a 1% (v / v) inoculation amount, and cultured at 30°C for 72 hours. Samples were taken at regular intervals and the degradation of mesotrione by the strain was determined by HPLC.

[0033] The results showed that the bacteria could almost completely degrade 100 mg / L mesotrione within 72 hours. Liquid phase analysis results showed that mesotrione had a characteristic absorption peak at 18.99 minutes ( Figure 2 ). After degradation, the characteristic absorption peak of mesotrione at 18.99 minutes almost completely disappeared, and a new peak was generated at 6.78 minutes, which was exactly the same as the peak time of the 2-amino-4-methylsulfonylbenzoic acid (AMBA) standard. Therefore, strain P15 converted mesotrione into the product AMBA through nitroreduction.

[0034] Example 2. Cloning of the mesotrione nitroreductase gene bamnr

[0035] 2.1 Genome sequencing of strain P15 and search for nitroreductase

[0036] The genome of strain P15 was sequenced and annotated using RAST (Rapid Annotation with Subsystem Technology). The genome size of the bacterium is 7.0Mb, encoding 5940 ORFs. Through functional analysis of these ORFs, a suspected nitroreductase gene was found, named bamnr, with a nucleotide sequence of SEQ ID NO.1, a size of 639bp, encoding a 212 amino acid protein (Bamnr), as shown in SEQ ID NO.2 in the sequence table. The comparison results of the swissprot database of NCBI (https: / / blast.ncbi.nlm.nih.gov / ) showed that the nitroreductase YdgI (69.4%) had the highest similarity with Bamnr.

[0037] 2.2 Construction of mesotrione nitroreductase gene bamnr expression vector

[0038] The bamnr gene expression vector pET-bamnr was synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the requirements in the following table. The nucleotide sequence of the bamn gene is shown in SEQ ID NO.1.

[0039]

[0040]

[0041] The above-synthesized expression vector pET-bamnr was transferred into the E. coli expression strain E. coli BL21 (DE3). The transformants were picked and placed in a test tube containing LB liquid with 50 mg / L kanamycin and cultured in a shaking incubator at 37°C and 180 rpm. The sequencing was performed to verify whether the expression was correct. The recombinant expression strain with correct sequencing was named BL21 (pet-Bamnr).

[0042] Example 3. Functional verification of mesotrione nitroreductase Bamnr

[0043] 3.1 Expression and purification of Bamnr

[0044] BL21 (pet-Bamnr) was cultured in 100 mL LB liquid medium at 37°C and 150 rpm shaking until OD 600nm was between 0.6, and then IPTG was added to a concentration of 0.05 mM and induced at 16°C for 8 hours. 100 mL of bacterial liquid was centrifuged to collect the cells, washed twice with PBS (50 mM, pH 7.4), resuspended with 10 mL of PBS buffer, ultrasonically disrupted (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 10 minutes, centrifuged at 12000 rpm for 30 minutes, and the supernatant was collected and precipitated with Co 2+ Bamnr was purified by ion affinity chromatography, and the purified enzyme was subjected to protein electrophoresis. Figure 3 .

[0045] 3.2 Detection of Bamnr enzyme activity

[0046] Enzyme activity reaction system (3 mL): 20 mM Tris-HCl buffer (pH 7.5), 100 μM mesotrione, 1 mM NADH, 25 mM Mg 2+, the reaction enzyme volume is 100μL, and the reaction is carried out at 30℃ for 1h. Each reaction starts with the addition of the enzyme. After 1h, the reaction is terminated by placing it in boiling water for 1min. After the reaction solution is freeze-dried, 200μl of methanol is added to dissolve the lyophilized material, and the amount of substrate reduction is detected by HPLC. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the reduction of 1nM mesotrione per minute under the conditions of pH 7.5 and temperature 30℃. The HPLC analysis results of mesotrione degradation by nitroreductase Bamnr are shown in Figure 4 The graphical results show that after 2 hours of enzyme reaction, the absorption peak of nitrotrione decreased significantly. At the same time, the characteristic absorption peak of AMBA appeared, indicating that Bamnr can reduce nitrotrione to AMBA and has nitroreductase activity. The specific enzyme activity for nitrotrione is 0.95U / mgprotein.

[0047] Example 4. Effect of Bamnr on the degradation and detoxification of mesotrione

[0048] Experimental principle: E. coli BL21 has tyrosine transferase activity, which can convert tyrosine into 4-HPP. The recombinant expression strain introduced with exogenous HPPD (para-hydroxyphenylpyruvate dioxidase) can convert tyrosine into 4-HPP in LB liquid medium supplemented with tyrosine, and continue to convert 4-HPP into HGA. The generated HGA spontaneously oxidizes and polymerizes to produce reddish-brown substances. Therefore, the activity of HPPD can be judged by the depth of color. The recombinant strain E. coli (pet-29a-OsHPPD) was introduced with a rice HPPD (OsHPPD) that is sensitive to mesotrione and mesotrione, so it was used in this experiment to detect the inhibitory activity of mesotrione and its degradation products against OsHPPD.

[0049] In a 100 mL enzyme reaction system, mesotrione was added to a final concentration of 4 μM, 8 μM and 12 μM, and then an appropriate amount of purified Bamnr was added. The reaction was carried out at 30°C for 5-6 hours to completely degrade mesotrione, and then placed in boiling water for 1 minute to terminate the reaction. After the enzyme reaction solution was freeze-dried, 2 mL of methanol was added to dissolve the lyophilized material, and then the methanol solution was naturally evaporated, and the residue was dissolved in pure water to obtain the degradation product.

[0050] The recombinant strain E. coli BL21 (pET-29a-OsHPPD) was inoculated into a 96-well plate containing TLB medium (LB containing 0.1% Tyr), and the inducer IPTG was added at the same time. Then, 0μM, 4μM, 8μM and 12μM mesotrione and degradation products were added respectively. After culturing for 36 hours, the color changes under different concentration treatments were observed. The results are shown in Figure 2. Figure 5As shown in the figure, the control without mesotrione was red, indicating that OsHPPD was normally expressed and active; the treatment with 4μM or higher concentration of mesotrione was light yellow, indicating that OsHPPD activity had been completely inhibited; and the products of mesotrione degradation by Bamnr were all red, with no significant difference from the control without mesotrione, indicating that the degradation products had no inhibitory effect on OsHPPD. Therefore, Bamnr can completely convert mesotrione into products that have no inhibitory effect on OsHPPD, achieving the degradation and detoxification of mesotrione.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mesotrione nitroreductase Bamnr, characterized in that The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene bamnr encoding the mesotrione nitroreductase Bamnr according to claim 1.

3. The coding gene bamnr according to claim 2, characterized in that The nucleotide sequence is shown in SEQ ID NO.

1.

4. A recombinant expression vector containing the gene encoding bamnr according to claim 2 or 3.

5. The recombinant expression vector according to claim 4, characterized in that it is obtained by recombining the coding gene bamnr according to claim 2 into the vector pET-29a(+).

6. A genetically engineered bacterium containing the bamnr encoding gene according to claim 2 or 3.

7. The genetically engineered bacterium according to claim 6, characterized in that The expression strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

8. Use of the mesotrione nitroreductase Bamnr according to claim 1 in the biodegradation, detoxification and restoration of mesotrione.

9. Use of the mesotrione nitroreductase Bamnr according to claim 1 in the preparation of a mesotrione degradation preparation.

10. Use of the coding gene bamnr according to claim 2 in the biodegradation, detoxification and restoration of mesotrione.

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