A mesotrione nitroreductase mtnr, its coding gene and use

Abstract

The application belongs to the field of agricultural biotechnology and environmental microbial engineering, and discloses a mesotrione nitroreductase Mtnr, a coding gene thereof and application. The nucleotide sequence is SEQ ID NO. 1, the full length is 756 bp, and 251 amino acids are encoded, and the amino acid sequence is SEQ ID NO. 2. The highest homology of Mtnr with a reported nitroreductase is only 50.6%, and therefore it is a new nitroreductase gene. The nitroreductase Mtnr provided by the application can catalyze the nitroreduction of mesotrione, and convert mesotrione into a product 2-amino-4-methylsulfonylbenzoic acid (AMBA) without herbicidal activity, indicating that the Mtnr gene has great application prospect in mesotrione biodegradation detoxification and remediation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, and relates to a mesotrione nitroreductase Mtnr, a coding gene thereof and use, in particular to a mesotrione nitroreductase capable of degrading and detoxifying mesotrione, a coding gene thereof and use. BACKGROUND

[0002] The application of herbicide-resistant transgenic engineering can control weeds in farmland efficiently and at low cost under the premise of ensuring crop safety, and has opened up a new era of weed control in farmland. At present, most of the crops planted are transgenic crops resistant to glyphosate, and in 2017, the area of transgenic crops resistant to glyphosate worldwide reached 150 million hectares, accounting for more than 80% of the global transgenic crop planting area. However, long-term use of glyphosate has brought about serious problems of weed resistance, and more than 40 major farmland weeds have been reported worldwide to have developed resistance to glyphosate, resulting in weeds that cannot be controlled. One of the effective measures to solve this problem is to use herbicides with different weed control mechanisms at the same time, and to construct corresponding herbicide-resistant transgenic crops in conjunction therewith. Therefore, it is necessary to extensively explore various herbicide resistance genes to provide gene resources for constructing transgenic crops resistant to various herbicides.

[0003] 4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) is a key enzyme involved in tyrosine metabolism in plants and some bacteria. Tyrosine is converted into 4-hydroxyphenylpyruvic acid (4-HPP) under the action of tyrosine aminotransferase (TAT), and then is converted into homogentisic acid (HGA) under the catalysis of HPPD. HGA is a key precursor for the biosynthesis of plastoquinone and tocopherol in plants. 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, block the synthesis of HGA, and thus cannot synthesize plastoquinone and tocopherol, resulting in the death of plants due to the occurrence of albinism. Therefore, this catalytic reaction has made HPPD an important target enzyme for herbicides.

[0004] HPPD inhibitors are mainly divided into three classes of herbicides according to chemical structure, namely triketones, isoxazoles and benzoheteroazoles. Mesotrione, also known as methyl sulfonyl, is a triketone herbicide developed by Syngenta in 1984, which 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 a global sales of 650 million US dollars in 2016. Although mesotrione has the advantages of high efficiency, low toxicity, high crop safety and good environmental compatibility, with long-term use, weeds gradually develop resistance to this type of herbicide. Therefore, it has very important theoretical and practical value to obtain new methyl sulfonyl degradation detoxification / resistance genes with excellent performance and develop transgenic crops. SUMMARY

[0005] The purpose of the present application is to provide a new protein, its encoding gene and use, which not only has mesotrione nitroreductase activity, and completely degrades mesotrione to lose herbicidal activity.

[0006] To achieve the above purpose, the present application provides a protein, comprising: a mesotrione nitroreductase gene mtnr, the nucleotide sequence of which is SEQ ID NO. 1.

[0007] The protein Mtnr encoded by the mesotrione nitroreductase gene mtnr of the present application 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 application, the recombinant expression vector is obtained by homologous recombination of the mesotrione nitroreductase gene mtnr and linearized pET-28a(+) plasmid.

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

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

[0012] The application of the mesotrione nitroreductase Mtnr of the present application in mesotrione biodegradation detoxification and repair.

[0013] The application of the mesotrione nitroreductase gene mtnr of the present application in mesotrione biodegradation detoxification and repair.

[0014] The application of the mesotrione nitroreductase Mtnr or the coding gene mtnr in preparing mesotrione economic preparation.

[0015] Advantages:

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

[0017] Figure 1 : The constructed phylogenetic tree of 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, 100 mg / L mesotrione degradation for 0 h; D, degradation for 72 h

[0020] Figure 3 : SDS-PAGE detection result of exogenous Mtnr

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

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

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

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

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

[0026] 1.1 Enrichment and separation of mesotrione degrading strain and classification and identification

[0027] Soil samples are collected from farmland where mesotrione has been applied for a long time, 10 g of soil sample is added into 100 mL of basic salt medium containing 50 mg / L of mesotrione, and then the mixture is cultured at 30 DEG C and 150 r / min for about 1 week, and then 10% of the inoculum is transferred into the same medium, and then the mixture is cultured again at 30 DEG C and 150 r / min for about 1 week. The above process is repeated for 4 times. The enrichment liquid with degradation effect is gradiently diluted, 1 mL of 10 -4 ,10-5 and 10 -6 , LB plate was coated with the diluent, and cultured at 30°C for 3-4 d. Different morphological single colonies on the plate were picked and further streaked on LB plates for purification. The obtained pure bacteria were inoculated into the basic salt liquid medium containing 100 mg / L mesotrione, and cultured at 30°C and 150 r / min for 5 d to verify whether each pure strain had mesotrione degradation function.

[0028] The formula of the basic salt medium (MSM) is as follows: 1.5 g of K2HPO4·3H2O, 0.5 g of KH2PO4, 1.0 g of NH4NO3, 0.5 g of NaCl, and 0.2 g of MgSO4·7H2O, and the volume is made up to 1 L with deionized water. For solid medium, 20.0 g of agar is added per liter.

[0029] The formula of the LB medium is as follows: 5 g of yeast extract, 10 g of tryptone, and 10 g of sodium chloride, and the pH is adjusted to 7.0 with 1 M NaOH solution.

[0030] A mesotrione-degrading bacterium was isolated and screened by enrichment domestication, and was named XHC-2. After the strain Md-42 was grown on LB solid medium for 4 d, the colony was white, wet, smooth, and had a neat edge with a diameter of about 0.3-0.4 mm. The cell was bacilliform (0.7-0.8 x 1.5-2.5 μm), gram-positive, and had a terminal spore with an inflated spore sac. The genomic DNA of the strain XHC-2 was used as a template for PCR amplification with universal primers for bacterial 16S rRNA gene sequences, and a 16S rRNA gene sequence with a length of 1447 bp was obtained. Blast was performed in the NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) and EZtaxon databases (http: / / eztaxon-e.ezbiocloud.net / ezt). The results showed that the strain XHC-2 was most closely related to the Paenibacillus strain, and in the phylogenetic tree constructed based on the 16S rRNA gene, the strain XHC-2 also clustered in the Paenibacillus genus (Paenibacillus sp.). Therefore, the strain XHC-2 was preliminarily identified as Paenibacillus. Figure 1

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

[0032] Study on degradation characteristics: XHC-2 was inoculated into LB liquid medium and cultured at 30°C to the mid-log phase. The bacterial cells were collected by low-speed centrifugation, washed with fresh and sterile basic salt medium for 2 times, resuspended in the basic salt medium, and the cell concentration was adjusted to about 1.0 x 10 9 ​The strain was inoculated into 20 mL of basal salt medium containing 100 mg / L of mesotrione at an inoculum of 1% (v / v) and incubated at 30°C for 72 h. The degradation of mesotrione by the strain was determined by HPLC at different time points.

[0033] The results showed that the strain almost completely degraded 100 mg / L of mesotrione within 72 h. Liquid chromatography analysis showed that 2-amino-4-methylsulfonylbenzoic acid (AMBA) standard had a characteristic absorption peak at 6.78 min (A), and mesotrione had a characteristic absorption peak at 18.99 min (B). After degradation, the characteristic absorption peak of mesotrione at 18.99 min (C) almost completely disappeared, and a new peak was generated at 6.78 min, which was exactly the same as the peak time of the AMBA standard (D), so strain XHC-2 converted mesotrione to product AMBA through nitro reduction. Figure 2 Figure 2 Figure 2 Figure 2

[0034] Example 2. Cloning of 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 the genome was annotated using RAST (Rapid Annotation with Subsystem Technology). The genome of the strain was 6.9 Mb in size, 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 678 bp, and encoding a 225-amino-acid protein (Mtnr), as shown in SEQ ID NO. 2 in the sequence listing. The swissprot database comparison results on NCBI (https: / / blast.ncbi.nlm.nih.gov / ) showed that the highest similarity to Mtnr was E. coli oxygen-insensitive NADPH nitroreductase, with a homology of only 49.2%.

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

[0038] The mtnr gene expression vector pET-mtnr was synthesized by Beijing Genesee 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] ​​​​ Gene of interest mtnr Target vector pET-29a(+) 5' end restriction site of target vector NdeI 3' end restriction site of target vector XhoI

[0040] The synthetic expression vector pET-mtnr was transformed into E. coli expression strain E. coli BL21(DE3). The transformants were picked into LB liquid tubes containing 50 mg / L kanamycin and cultured at 37°C, 180 rpm. Sequencing was performed to verify whether the correct recombinant expression strain was obtained. The recombinant expression strain obtained was named BL21(pet-mtnr).

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

[0042] 3.1 Expression and purification of Mtnr

[0043] BL21(pet-mtnr) was cultured in 100 mL LB liquid medium at 37°C, 150 rpm, to an OD 600nm of 0.6, then IPTG was added to a concentration of 0.05 mM, and the culture was induced at 16°C for 8 hours. The 100 mL bacterial solution was centrifuged to collect the bacterial cells, which were washed twice with PBS (50 mM, pH 7.4). The bacterial cells were resuspended in 10 mL PBS buffer, ultrasonically broken (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 10 minutes, and centrifuged at 12000 rpm for 30 minutes. The supernatant was collected and subjected to Co 2+ The purified Mtnr was subjected to protein electrophoresis, as shown in Figure 3 .

[0044] 3.2 Enzymatic activity detection of Mtnr

[0045] The enzyme activity reaction system (3 mL) was as follows: 20 mM Tris-HCl buffer (pH 7.5), 100 μM mesotrione, 1 mM NADH, 25 mM Mg 2+ , 100 μL of enzyme, and 30°C for 1 hour. Each reaction was timed from the addition of the enzyme, and terminated by placing in boiling water for 1 minute after 1 hour. The reaction solution was freeze-dried, 200 μL of methanol was added to dissolve the freeze-dried product, and the reduction of the substrate was detected by HPLC. One unit (U) of enzyme activity was defined as the amount of enzyme required to catalyze the reduction of 1 nM mesotrione per minute at pH 7.5 and 30°C. The HPLC analysis results of the degradation of mesotrione by the nitroreductase Mtnr are shown in Figure 4 , and the results show that the absorption peak of nitro-mesotrione decreased significantly after 2 hours of enzyme reaction, and a characteristic absorption peak of AMBA appeared, indicating that Mtnr can reduce nitro-mesotrione to AMBA, and has nitroreductase activity for mesotrione, with a specific enzyme activity of 1.15 U / mg protein.

[0046] Example 4. Detoxification effect of Mtnr on mesotrione degradation

[0047] Principle of experiment: E. coli BL21 has tyrosine transferase activity itself, which can convert tyrosine into 4-HPP. The recombinant strain with introduced exogenous HPPD (hydroxyphenylpyruvate dioxygenase) can convert tyrosine into 4-HPP in LB liquid medium with added tyrosine, and continue to convert 4-HPP into HGA. The red-brown substance is generated after spontaneous oxidation and polymerization of HGA, so the activity of HPPD can be judged by the color depth. The recombinant strain E. coli (pet-29a-OsHPPD) introduced a rice HPPD (OsHPPD) sensitive to mesotrione, so it was used in this experiment to detect the inhibitory activity of mesotrione and its degradation products on OsHPPD.

[0048] In a 100 mL enzyme reaction system, mesotrione was added to make the final concentration 4 μM, 8 μM and 12 μM, respectively, and then an appropriate amount of purified Mtnr was added. The reaction was carried out at 30°C for 5-6 h to completely degrade mesotrione, and the reaction was terminated by placing in boiling water for 1 min. The enzyme reaction solution was freeze-dried, then 2 mL of methanol was added to dissolve the freeze-dried material, and then the methanol solution was naturally volatilized. The residue was dissolved with pure water to obtain the degradation products.

[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, and then 0 μM, 4 μM, 8 μM and 12 μM of mesotrione and degradation products were added, respectively. After 36 h of culture, the color change under different concentrations was observed. As shown in Figure 5 , the control without added mesotrione was red in color, indicating that OsHPPD was normally expressed and had activity; the treatment with 4 μM or higher concentration of mesotrione was light yellow, indicating that the activity of OsHPPD had been completely inhibited; and the treatment with the product of Mtnr degrading mesotrione was red, which was not significantly different from the control without added mesotrione, indicating that the degradation product had no inhibitory effect on OsHPPD. Therefore, Mtnr can completely convert mesotrione into a product that has no inhibitory effect on OsHPPD, achieving the detoxification of mesotrione.

[0050] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

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

2.

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

1.

3. A recombinant expression vector comprising the coding gene of claim 2. mtnr 5.

4. The recombinant expression vector according to claim 3, characterized in that the coding gene according to claim 2 is mtnr recombined into the vector pET-29a (+).

5. A genetically engineered bacterium comprising the coding gene of claim 2. mtnr 5. A genetically engineered bacterium comprising the coding gene of claim 2.

6. The genetically engineered bacteria of claim 5, characterized in that mtnr The expression strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).

8. The coding gene of claim 2 7. The use of the mesotrione nitroreductase Mtnr in claim 1 in the biodegradation detoxification and remediation of mesotrione. Use in biodegradation detoxification and remediation of mesotrione.

9. The mesotrione nitroreductase Mtnr of claim 1 or the encoding gene of claim 2 mtnr mtnr use in the manufacture of a mesotrione degrading formulation.

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