A new mesotrione nitroreductase bamnr, its encoding gene and use

Abstract

The application belongs to the field of agricultural biotechnology and environmental microbial engineering, discloses a new mesotrione nitroreductase Bamnr and a coding gene and use thereof. The nucleotide sequence is SEQ ID NO. 1, the full length is 639 bp, and 212 amino acids are encoded, and the amino acid sequence is SEQ ID NO. 2. The highest homology of Bamnr with a reported nitroreductase is only 69.4%, and therefore it is a new nitroreductase gene. The nitroreductase Bamnr 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 Bamnr gene has great application prospect in mesotrione biodegradation detoxification and remediation.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural biotechnology, and relates to a new mesotrione nitroreductase Bamnr, a coding gene thereof and use. BACKGROUND

[0002] The application of herbicide-resistant transgenic engineering has created a new era of farmland weed control under the premise of ensuring crop safety. At present, most of the crops planted are glyphosate-resistant transgenic crops. In 2017, the area of glyphosate-resistant transgenic crops 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 serious weed resistance problems. So far, more than 40 major farmland weeds have been reported 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. Therefore, it is necessary to widely 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 to 4-hydroxyphenylpyruvic acid (4-HPP) under the action of tyrosine aminotransferase (TAT), and then converted to homogentisic acid (HGA) under the catalysis of HPPD. Homogentisic acid 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 homogentisic acid (HGA), and thus cannot synthesize plastoquinone and tocopherol, leading to 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 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 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, but also completely degrades mesotrione to lose herbicidal activity.

[0006] The purpose of the present application can be achieved by the following technical solutions:

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

[0008] The encoding gene bamnr of the mesotrione nitroreductase Bamnr of the present application, preferably the nucleotide sequence is 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 application, the recombinant expression vector is obtained by homologous recombination of the mesotrione nitroreductase gene bamnr and linearized pET-28a(+) plasmid.

[0011] A genetically engineered bacterial strain containing the mesotrione nitroreductase gene bamnr of the present application.

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

[0013] The mesotrione nitroreductase Bamnr of the present application is used in mesotrione biodegradation detoxification and repair.

[0014] The application of the mesotrione nitroreductase Bamnr in preparing mesotrione degradation preparation. The application of the mesotrione nitroreductase gene bamnr in mesotrione biodegradation detoxification and remediation.

[0015] Beneficial effects:

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

[0017] Figure 1 : 16S rRNA gene constructed system tree of the closest relative model strain of strain P15

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

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

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

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

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

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

[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 separation and classification identification of mesotrione degrading strain

[0027] Soil samples were collected from corn fields with long-term application of mesotrione, 5 g of soil sample was added into 100 mL of basal salt medium containing 50 mg / L mesotrione, and incubated at 30℃ with 150 r / min for 7 days, then 5% of the inoculum was transferred into the same medium for 3 times. The degradation of mesotrione by the enrichment liquid was determined by high performance liquid chromatography, and the enrichment liquid with degradation effect was gradiently diluted, 1 mL of 10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 The diluted liquid was spread on LB plates, and incubated at 30℃ for 3 days. Single colonies on the plates were picked and further purified by streaking, and the obtained pure bacteria were inoculated into basal salt liquid medium containing 100 mg / L mesotrione, and incubated at 30℃ with 150 r / min for 5 days to verify whether each pure strain had the function of degrading mesotrione.

[0028] The formula of basal salt medium (MSM) was 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 deionized water was added to 1 L, and 15.0 g of agar was added per liter of solid medium.

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

[0030] A mesotrione-degrading bacterium was obtained by enrichment domestication separation and screening, and was named as P15. After the strain P15 was grown on solid medium for 3 days, the colony was milky white, wet, smooth, and regular in edge, and the diameter was about 0.3-0.5 mm; the cell was gram-positive, facultative anaerobic, and bacillary (0.6-0.8 x 1.8-3.2 μm), and spores were formed at one end of the cell, and the spore cyst was swollen. The genomic DNA of the strain P15 was used as a template, and the universal primer of bacterial 16S rRNA gene sequence was used for PCR amplification, and a 16S rRNA gene sequence with a length of about 1449 bp was obtained (GenBank: PP526999). Blast was performed in the EZtaxon database (http: / / eztaxon-e.ezbiocloud.net / ezt), and the results showed that the strain P15 was closest to the Paenibacillus strain, and the homology with Paenibacillus cavernae C4-5 T was the highest (93.3%). In the phylogenetic tree constructed by 16S rRNA gene, the strain P15 also clustered in the Paenibacillus genus Figure 1), so strain P15 was initially identified as Paenibacillus.

[0031] 1.2 Degradation of bensultap by strain P15

[0032] Degradation characteristics: P15 was inoculated into LB liquid medium and cultured at 30°C to the mid-log phase, then the bacterial cells were collected by low-speed centrifugation, washed twice with fresh and sterile basal salt medium, resuspended in basal salt medium, and adjusted to a cell concentration of about 1.0 x 10 9 cfu / ml, inoculated into 20 ml of basal salt medium containing 100 mg / L bensultap at an inoculation amount of 1% (v / v), and cultured at 30°C for 72 h. Samples were taken at regular time intervals to determine the degradation of bensultap by the strain using HPLC.

[0033] The results showed that the strain almost completely degraded 100 mg / L bensultap within 72 h. Liquid chromatography analysis showed that bensultap had a characteristic absorption peak at 18.99 min Figure 2 ). After degradation, the characteristic absorption peak of bensultap at 18.99 min almost completely disappeared, and a new peak appeared at 6.78 min, which was exactly the same as the peak time of the 2-amino-4-methylsulfonylbenzoic acid (AMBA) standard, so strain P15 converted bensultap to product AMBA through nitro reduction.

[0034] Example 2. Cloning of bensultap nitroreductase gene bamnr

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

[0036] The genome of strain P15 was sequenced, and the genome was annotated using RAST (Rapid Annotation with Subsystem Technology). The genome of the strain was 7.0 Mb in size, 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 639 bp, and encoding a 212-amino-acid protein (Bamnr), 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 Bamnr was nitroreductase YdgI (69.4%).

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

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

[0039]

[0040]

[0041] The above-synthesized expression vector pET-bamnr was transformed into E. coli expression strain E. coli BL21(DE3). The transformants were picked into LB liquid test tubes containing 50 mg / L kanamycin and cultured at 37°C, 180 rpm on a shaking bed. Sequencing was performed to verify whether it was correct. The obtained recombinant expression strain with correct sequencing was named BL21(pet-Bamnr).

[0042] Example 3. Functional verification of nitrosulfuron 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, 150 rpm on a shaking bed until the OD 600nm was between 0.6, then IPTG was added to a concentration of 0.05 mM, and the culture was induced at 16°C for 8 hours. 100 mL of 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 of PBS buffer, ultrasonically broken (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 10 minutes, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and subjected to Co 2+ Bamnr was purified by ion affinity chromatography column. The purified enzyme was subjected to protein electrophoresis, as shown in Figure 3 .

[0045] 3.2 Detection of enzyme activity of Bamnr

[0046] Enzyme activity reaction system (3 mL): 20 mM Tris-HCl buffer (pH 7.5), 100 μM nitrosulfuron, 1 mM NADH, 25 mM Mg 2+, reaction enzyme amount 100 μL, 30 ℃ reaction 1 h. Each reaction was timed from the addition of enzyme, 1 h later placed in boiling water for 1 min to terminate the reaction. The reaction solution was freeze-dried, 200 μl of methanol was added to dissolve the freeze-dried material, and the reduction of the substrate was detected by HPLC. One enzyme activity unit (U) was defined as: the amount of enzyme required to catalyze the reduction of 1 nM of mesotrione per minute at pH 7.5, 30 ℃. The results of HPLC analysis of the degradation of mesotrione by nitroreductase Bamnr are shown in Figure 4 , the results show that after 2 h of enzyme reaction, the absorption peak of nitro-sulfosulfone is significantly reduced, and at the same time, the characteristic absorption peak of AMBA appears, indicating that Bamnr can reduce nitro-sulfosulfone to AMBA, has nitroreductase activity of mesotrione, and the specific enzyme activity of mesotrione is 0.95 U / mg protein.

[0047] Example 4. Detoxification effect of Bamnr on the degradation of mesotrione

[0048] Principle of the experiment: E. coli BL21 has tyrosine transferase activity itself, which can convert tyrosine to 4-HPP. The recombinant strain introduced with exogenous HPPD (hydroxyphenylpyruvate dioxygenase) can convert tyrosine to 4-HPP in LB liquid medium supplemented with tyrosine, and continue to convert 4-HPP to HGA. The red-brown substance is generated after the 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 to detect the inhibitory activity of mesotrione and its degradation products on OsHPPD in this experiment.

[0049] In a 100 mL enzyme reaction system, mesotrione was added to make the final concentration 4 μM, 8 μM and 12 μM, respectively, then a suitable amount of purified Bamnr was added, and the reaction was carried out at 30 ℃ for 5-6 h to completely degrade mesotrione. The reaction was terminated by placing it in boiling water for 1 min. The enzyme reaction solution was freeze-dried, 2 mL of methanol was added to dissolve the freeze-dried material, then the methanol solution was naturally volatilized, and the residue was dissolved in pure water to obtain the degradation products.

[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, 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. The results are shown in Figure 5As shown, the control without mesotrione is red, indicating that OsHPPD is normally expressed and active; the treatment with 4 μM or higher concentration of mesotrione is light yellow, indicating that the activity of OsHPPD has been completely inhibited; and the treatment with the product of Bamnr degrading mesotrione is red, which is not significantly different from the control without mesotrione, indicating that the degradation product has no inhibitory effect on OsHPPD. Therefore, Bamnr can completely convert mesotrione into a product that has no inhibitory effect on OsHPPD, achieving degradation detoxification of mesotrione.

[0051] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; 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 nitroreductase Bamnr, characterized in that, The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding nicosulfuron nitroreductase Bamnr as described in claim 1 bamnr.

3. The encoding gene according to claim 2 bamnr, Its features are, The nucleotide sequence is shown in SEQ ID NO.

1.

4. Containing the coding gene as described in claim 2 or 3 bamnr Recombinant expression vectors.

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

6. Containing the coding gene as described in claim 2 or 3 bamnr Genetically engineered bacteria.

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

8. The use of the nitroreductase Bamnr of claim 1 in the preparation of nitroreductase degradation formulations.

9. The encoding gene according to claim 2 bamnr Application in the preparation of nicosulfuron-methyl degradable formulations.

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