Mesotrione nitroreductase Mtnr, and coding gene and application thereof

Abstract

The invention belongs to the field of agricultural biotechnology and environmental microbiological engineering, and discloses mesotrione nitroreductase Mtnr as well as a coding gene and application thereof. The nucleotide sequence of the gene is SEQ ID NO.1, the full length is 756bp, 251 amino acids are encoded, and the amino acid sequence is SEQ ID NO.2. The maximum homology of the Mstnr and reported nitroreductase is only 50.6%, so that the Mstnr is a new nitroreductase gene. The nitroreductase Mstnr 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 Mstnr gene has a huge application prospect in biodegradation detoxification and repair of the mesotrione.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and relates to a mesotrione nitroreductase Mtnr, a coding gene and uses thereof, and in particular to a nitroreductase capable of degrading and detoxifying mesotrione, a coding gene and uses 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] To achieve the above object, the present invention provides a protein, including: a mesotrione nitroreductase gene mtnr, whose nucleotide sequence is SEQ ID NO.1.

[0007] The protein Mtnr encoded by the mesotrione nitroreductase gene mtnr of the present invention has an amino acid sequence of SEQ ID NO.2.

[0008] A recombinant expression vector containing the mesotrione nitroreductase gene mtnr.

[0009] As a preferred embodiment of the present invention, the recombinant expression vector is obtained by homologous recombination of the mesotrione nitroreductase gene mtnr with a linearized pET-28a(+) plasmid.

[0010] A genetically engineered strain containing the mesotrione nitroreductase gene mtnr of the invention.

[0011] As a preferred embodiment of the present invention, the expression strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

[0012] The invention discloses an application of the mesotrione nitroreductase Mtnr in the biodegradation, detoxification and restoration of mesotrione.

[0013] The invention discloses an application of the mesotrione nitroreductase gene mtnr in the biodegradation, detoxification and restoration of mesotrione.

[0014] Application of the mesotrione nitroreductase Mtnr or the encoding gene mtnr of the present invention in the preparation of an economical mesotrione preparation.

[0015] Beneficial effects:

[0016] The invention provides a new mesotrione nitroreductase Mtnr, which can degrade mesotrione into a product 2-amino-4-methylsulfonylbenzoic acid (AMBA) and loses herbicidal activity. Mtnr 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 from the 16S rRNA gene of strain XHC-2

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

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

[0020] Figure 3 :Results of SDS-PAGE detection of exogenously expressed Mtnr

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

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

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

[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 farmland where mesotrione was used for a long time. 10g of soil sample was added to 100mL of basal salt medium containing 50mg / L mesotrione. After 1 week of shaking culture at 30℃ and 150r / min, the soil sample was transferred to the same medium at a 10% inoculation rate and then shaken for another week. This was repeated 4 times. The enriched solution with degradation effect was diluted in a gradient manner. 1mL of 10 -4 , 10-5 and 10 -6 , the dilution was coated on LB plates and cultured at 30°C for 3-4 days. Single colonies of different forms were picked on the plates, and further purified by streaking on the LB plates. The obtained pure bacteria were inoculated into a basic 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 20.0g agar per liter of solid culture medium.

[0029] LB medium formula: 5 g yeast extract; 10 g tryptone; 10 g sodium chloride; adjust the pH to 7.0 with 1 M NaOH solution.

[0030] A mesotrione-degrading bacterium was obtained by enrichment, acclimation, separation and screening, and named XHC-2. After growing on LB solid medium for 4 days, the colony of strain Md-42 was white, moist, smooth, with neat edges and a diameter of about 0.3-0.4 mm; the bacterial cells were rod-shaped (0.7-0.8×1.5-2.5μm), Gram-positive, with terminal spores and swollen spore capsules. Using the genomic DNA of strain XHC-2 as a template, PCR amplification with universal primers for bacterial 16S rRNA gene sequence was performed to obtain a 1447bp 16SrRNA gene sequence. Blast analysis was performed in NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and EZtaxon databases (http: / / eztaxon-e.ezbiocloud.net / ezt). The results showed that strain XHC-2 was most closely related to strains of the genus Paenibacillus. In the phylogenetic tree constructed using the 16S rRNA gene, strain XHC-2 was also clustered in the genus Paenibacillus. Figure 1 ), so strain XHC-2 was preliminarily identified as Paenibacillus.

[0031] 1.2 Degradation of mesotrione by strain XHC-2

[0032] Study on degradation characteristics: XHC-2 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 9cfu / 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 the 2-amino-4-methylsulfonylbenzoic acid (AMBA) standard had a characteristic absorption peak at 6.78 minutes ( Figure 2 A), while mesotrione has a characteristic absorption peak at 18.99 minutes ( Figure 2 B). After degradation, the characteristic absorption peak of mesotrione at 18.99 minutes ( Figure 2 C) almost completely disappeared, and a new peak appeared at 6.78 minutes, which was exactly the same as the peak time of the AMBA standard ( Figure 2 D), therefore strain XHC-2 converts mesotrione to the product AMBA by nitroreduction.

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

[0035] 2.1 Genome sequencing of strain XHC-2 and search for nitroreductase

[0036] The genome of strain XHC-2 was sequenced and annotated using RAST (Rapid Annotation with Subsystem Technology). The genome size of the bacterium is 6.9Mb, encoding 5820 ORFs. Through functional analysis of these ORFs, a suspected nitroreductase gene was found, named mtnr, with a nucleotide sequence of SEQ ID NO.1, a size of 678bp, encoding a 225 amino acid protein (Mtnr), 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 gene with the highest similarity to Mtnr was E. coli oxygen-insensitive NADPH nitroreductase, with a homology of only 49.2%.

[0037] 2.2 Construction of the mesotrione nitroreductase gene mtnr expression vector

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

[0039] Target gene mtnr Target vector pET-29a(+) 5' end restriction site of target vector N i Target vector 3' end restriction site XOt

[0040] The above-synthesized expression vector pET-mtnr 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 containing 50 mg / L kanamycin and cultured in a shaking table 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-mtnr).

[0041] Example 3. Functional verification of mesotrione nitroreductase Mtnr

[0042] 3.1 Expression and purification of Mtnr

[0043] BL21 (pet-mtnr) 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+ Mtnr was purified by ion affinity chromatography, and the purified enzyme was subjected to protein electrophoresis. Figure 3 .

[0044] 3.2 Mtnr enzyme activity detection

[0045] 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 Mtnr 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 Mtnr can reduce nitrotrione to AMBA and has nitroreductase activity. The specific enzyme activity for nitrotrione is 1.15U / mgprotein.

[0046] Example 4. Effect of Mtnr on the degradation and detoxification of mesotrione

[0047] 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.

[0048] 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 Mtnr 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.

[0049] 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 5 As 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 Mtnr were all red, with no significant difference from the control without mesotrione, indicating that the degradation products had no inhibitory effect on OsHPPD. Therefore, Mtnr can completely convert mesotrione into products that have no inhibitory effect on OsHPPD, achieving the degradation and detoxification of mesotrione.

[0050] 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 Mtnr, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene mtnr encoding the mesotrione nitroreductase Mtnr according to claim 1.

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

1.

4. A recombinant expression vector containing the mtnr encoding gene 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 mtnr according to claim 2 into the vector pET-29a(+).

6. A genetically engineered bacterium containing the mtnr 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 Mtnr according to claim 1 in the biodegradation, detoxification and restoration of mesotrione.

9. Use of the encoding gene mtnr according to claim 2 in the biodegradation, detoxification and restoration of mesotrione.

10. Use of the mesotrione nitroreductase Mtnr according to claim 1 or the encoding gene mtnr according to claim 2 in the preparation of an economical mesotrione preparation.

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