Mesotrione nitroreductase and its encoding gene and application
By providing high-energy activity nitroreductase PbMNR1 and its encoding gene, the problems of low degradation efficiency of nitroreductase and insufficient application of herbicide-resistant transgenic crops in the prior art are solved, and the effect of efficient degradation of nitroreductase and construction of herbicide-resistant transgenic crops is achieved.
Patent Information
- Application Number
- CN202410946945.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-16
- Publication Date
- 2025-03-28
- Estimated Expiration
- 2044-07-16
AI Technical Summary
In the prior art, the types and number of nitrosulone degraded strains are limited, the catalytic efficiency is low, and their application is not seen for herbicide-resistant transgenic crops.
It provides a highly enzyme-active nitroreductase PbMNR1 and its encoding gene, which is used to construct a transgenic crop that is resistant to nitrosine and to remove residual nitrosine in the environment.
The high-efficiency degradation of nitrosalone was achieved. The produced product AMBA could not inhibit the activity of rice HPPD, and solved the problem of residual contamination of nitrosalone and the cultivation of nitrosalone-resistant transgenic crops.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of environmental microorganisms and relates to a mesotrione nitroreductase and a coding gene and application thereof. Background Art
[0002] Mesotrione belongs to the class of HPPD inhibitor herbicides, which has control effects on annual broadleaf weeds and some grass weeds. Mesotrione can be used before and after seedlings, has systemic and contact effects, and has a long lasting effect. It is absorbed and utilized by the leaves, stems, and roots of plants, and is transmitted upward and downward through the xylem and phloem. The herbicidal principle of mesotrione is: by inhibiting the activity of p-hydroxyphenylpyruvate dioxidase (HPPD) in the tyrosine metabolic pathway, it prevents the conversion of p-hydroxyphenylpyruvate (4-HPP) into 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, and ultimately leads to plant death. At the same time, mesotrione has the advantages of broad spectrum, high efficiency, low toxicity, and slow development of resistance, and is considered to be an ideal herbicide.
[0003] Mesotrione has stable chemical properties and a half-life of up to 50 days in the soil. Microbial degradation is the main factor in eliminating mesotrione residues in the environment. Currently, the types and numbers of mesotrione-degrading strains that have been screened are still very small. Bacillus sp. 3B6, Bacillus megaterium Mes11, Pantoea ananatis CT 7673, Bacillus pumilus HZ-2 and Bradyrhizobium sp. SR1 These strains all degrade mesotrione to generate hydroxylamine intermediates through the nitroreduction pathway and finally convert it into 2-amino-4-methylsulfonylbenzoic acid (AMBA). The related genes cloned from these strains and the enzymes studied are all nitroreductases. However, these nitroreductases have low catalytic efficiency and there is no report on their use in herbicide-resistant transgenic crops. Summary of the invention
[0004] 1. Technical issues to be resolved
[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a mesotrione nitroreductase and a gene encoding the same and an application thereof. The reductase has a high specific enzyme activity and can be used to construct mesotrione-resistant transgenic crops, and can also be used to remove residual mesotrione in soil and water, thereby solving technical problems such as mesotrione residual pollution and the cultivation of mesotrione-resistant transgenic crops.
[0006] (II) Technical solution
[0007] In a first aspect, the present invention provides a mesotrione nitroreductase PbMNR1, whose amino acid sequence is shown in SEQ ID NO.2.
[0008] In a second aspect, the present invention provides a gene encoding mesotrione nitroreductase PbMNR1.
[0009] Preferably, the gene is the nucleotide sequence shown in SEQ ID NO:1.
[0010] Usually a specific protein corresponds to a specific gene sequence, but due to mechanisms such as genetic code degeneracy and gene splicing variation, different gene sequences may ultimately produce the same protein.
[0011] The degeneracy of the genetic code means that multiple different codons can encode the same amino acid. For example, three different codons (such as UUU, UUC) can all encode phenylalanine. Therefore, even if the nucleotide sequences of two genes differ in some parts, if these differences only involve degenerate codons, they can still be translated into the same amino acid sequence and ultimately produce the same protein. In addition, even if the gene sequences are different, if the splicing process selectively retains the same exon sequence, the same protein can theoretically be produced. Therefore, the nucleotide sequence shown in SEQ ID NO: 1 is only a specific embodiment of the gene that can encode mesotrione nitroreductase PbMNR1.
[0012] In a third aspect, the present invention provides an expression cassette comprising the gene under the control of an operably linked regulatory sequence (such as a promoter or terminator).
[0013] In a fourth aspect, the present invention provides a recombinant vector comprising the gene or expression cassette.
[0014] Preferably, the recombinant vector is obtained by inserting the gene or expression cassette between the Nde I and Xho I sites of pET-29a(+).
[0015] In a fifth aspect, the present invention provides a method for constructing a mesotrione-resistant transgenic crop, which comprises introducing the recombinant vector into the crop, so that the mesotrione nitroreductase PbMNR1 is expressed in the crop.
[0016] In a sixth aspect, the present invention provides a genetically engineered bacterium capable of degrading mesotrione, which comprises transferring the recombinant vector into a host strain, so that the mesotrione nitroreductase PbMNR1 is expressed in the host strain.
[0017] Preferably, the host strain is Escherichia coli BL21 (DE3).
[0018] In a seventh aspect, the present invention provides the use of the mesotrione nitroreductase PbMNR1 in degrading and removing residual mesotrione in the environment.
[0019] In an eighth aspect, the present invention provides the use of the gene in constructing mesotrione-resistant transgenic crops or mesotrione-degrading microorganisms.
[0020] (III) Beneficial effects
[0021] The mesotrione nitroreductase PbMNR1 provided by the present invention is composed of 247 amino acids, and can catalyze the conversion of mesotrione herbicide to produce product AMBA in the presence of reducing power NADH (reduced nicotinamide adenine dinucleotide), with a specific enzyme activity of 0.82U / mg. In addition, the product AMBA cannot inhibit the activity of rice HPPD, that is, AMBA has lost its herbicidal activity. Therefore, mesotrione nitroreductase PbMNR1 can be used to degrade and remove residual mesotrione in the environment (soil, water body).
[0022] The present invention also provides a nitroreductase encoding gene Pbmnr1, which has a full length (from the start codon to the stop codon) of 744 bp and can be used to construct transgenic crops resistant to mesotrione, and can also be used to construct mesotrione-degrading engineering strains to degrade mesotrione residual in soil and water bodies and reduce the harm of mesotrione to the environment. The gene has the advantages of good effect, low cost, no secondary pollution, etc. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the phylogenetic tree of strain P8 based on the 16S rRNA gene sequence.
[0024] Figure 2 HPLC detection of samples containing only mesotrione, AMBA standards and the products of mesotrione degradation by strain P8.
[0025] Figure 3 This is the SDS-PAGE detection result of exogenously expressed PbMNR1.
[0026] Figure 4 These are the HPLC detection spectra of PBS+NADH+mesotrione, PBS+PbMNR1+mesotrione, AMBA standard and PBS+PbMNR1+NADH+mesotrione.
[0027] Figure 5 This is the LC / MS detection spectrum for the analysis of mesotrione and the products of mesotrione catalyzed by PbMNR1 using liquid chromatography-mass spectrometry.
[0028] Figure 6 This is the chemical transformation pathway of mesotrione catalyzed by nitroreductase PbMNR1.
[0029] Figure 7 These are the results of the inhibition experiment of the degradation product AMBA on rice HPPD (OsHPPD). DETAILED DESCRIPTION
[0030] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.
[0031] Example 1
[0032] This example is a process for screening and identifying the mesotrione-degrading strain P8 from activated sludge of a pesticide factory, which specifically includes the following steps:
[0033] (1) Screening and purification of degradation strains
[0034] The enrichment matrix used to enrich the mesotrione herbicide degrading strain was taken from the activated sludge of a pesticide factory. 5.0 g of activated sludge was added to 100 ml of basal salt culture medium, and 100 µM mesotrione was added. The culture was cultured at 30°C and 180 r / min for 5 days, and then transferred to the same culture medium at a 5% (v / v) inoculation rate. The transfer was repeated 3 times. The residual amount of mesotrione in the enriched culture medium of the third transfer and whether new metabolites were generated were detected using an ultraviolet spectrophotometer and a high performance liquid chromatograph. The enriched solution that produced the degradation effect was gradient diluted and 10 -4 Up to 10 -7 0.1 ml of each gradient dilution enrichment solution was spread on R2A 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 basal salt liquid medium with 100 µM mesotrione, cultured at 30°C and 180 r / min for 5 days, and then each single colony was verified to have mesotrione degradation function.
[0035] The formula of basal salt medium (1L) is: 5.0g glucose, 1.0g NH4Cl, 1.0g NaCl, 1.5g K2HPO4, 0.5g KH2PO4, 0.2g MgSO4·7H2O, add water to 1L, pH 7.0. Add 15.0g agar to the solid medium.
[0036] A mesotrione-degrading bacterium was obtained through enrichment, acclimation, isolation and screening and was named P8. Figure 2 As shown, the HPLC detection spectrum of the sample containing only mesotrione ( Figure 2 A) and HPLC detection spectrum of AMBA standard ( Figure 2 B) was used as a control, and the sample of mesotrione treated with strain P8 for 36 hours was tested by HPLC, and the results were as follows Figure 2The HPLC spectrum is shown in C. After comparison, it can be found that the spectral peak of AMBA was produced in the sample of mesotrione treated with strain P8, and the peak intensity of mesotrione decreased. This shows that strain P8 can degrade mesotrione into AMBA, and the degradation rate of 100µM mesotrione by the bacterium is more than 90% within 36 hours. It was identified that strain P8 was Gram-positive. After 2 days of culture on R2A plate, the colonies were light yellow, moist on the surface, slightly rough on the edges, and convex in the middle. Using the genomic DNA of strain P8 as a template, PCR amplification was performed using universal primers for the bacterial 16SrRNA gene sequence to obtain the 16S rRNA gene sequence of strain P8 with a length of approximately 1515bp. The 16S rRNA gene was submitted to the online website Ezbiocloud for multiple sequence alignment. The results showed that the strain with the highest similarity to the 16SrRNA gene of strain P8 was Paenibacillus terreus D33T (98.0%), followed by Paenibacillus medicaginis CC-Alfalfa-19 T (96.1%) and Paenibacillus farraposensis UY79 T The phylogenetic tree of the 16S rRNA gene of strain P8 was constructed by the neighbor-joining method (e.g. Figure 1 The results showed that strain P8 and Paenibacillus The type strains of the genus are clustered together and are similar to the type strains Paenibacillus medicaginis CC-Alfalfa-19 T Therefore, strain P8 was preliminarily identified as Paenibacillus ( Paenibacillus sp.).
[0037] Example 2
[0038] This example is a process for cloning the mesotrione nitroreductase gene from strain P8, which includes the following steps:
[0039] (1) Sequencing analysis of bacterial genomes
[0040] After strain P8 was cultured in R2A liquid medium in large quantities, the genomic total DNA of P8 with high purity and large fragments was extracted by CTAB method, dissolved in TE buffer (pH 8.0), and stored at -20°C.
[0041] Bacterial genome sequencing requires the sample OD value to be between 1.8-2.0, the higher the concentration, the better, and the concentration should not be less than 30ng / μl. The prepared total genomic DNA of strain P8 was sent to Shanghai Lingen Biotechnology Co., Ltd. for sequencing under dry ice insulation. Sample testing includes: ① agarose gel electrophoresis to analyze the degree of DNA degradation and whether there is RNA contamination; ② NanoDrop to detect the purity of DNA (OD260 / 280 ratio); ③ Qubit to accurately quantify the DNA concentration. Among them, DNA samples with OD values between 1.8-2.0 and a content of more than 1.5μg were used to build the library.
[0042] (2) Analysis of draft genome sequencing results
[0043] The draft genome of strain P8 obtained by sequencing was uploaded to the Rast website (https: / / rast.nmpdr.org / ) for annotation. The annotated amino acid sequence was downloaded and a local protein database was constructed using the Bioedit software. The amino acid sequences of reported mesotrione nitroreductases NfrA1, NfrA2, NfsA, and NfsB were downloaded from the NCBI database. Using them as templates, the constructed protein database of strain P8 was searched for nitroreductase sequences with high homology to NfrA1, NfrA2, NfsA, and NfsB in the Bioedit software by homologous sequence alignment. After sequence alignment analysis, a nitroreductase was screened, which had the highest similarity with the reported mesotrione nitroreductase, at 39-47%. The nitroreductase was named PbMNR1.
[0044] (3) Construction of the mesotrione nitroreductase gene Pbmnr1 expression vector
[0045] The Pbmnr1 gene expression vector pET-Pbmnr1 was synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the requirements in the following table. The nucleotide sequence of the Pbmnr1 gene is shown in SEQ ID NO.1.
[0046] Target gene Pbmnr1 Target vector pET-29a(+) 5' end restriction site of target vector N i Target vector 3' end restriction site XOt
[0047] The above-synthesized expression vector pET-Pbmnr1 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 (Pbmnr1).
[0048] Example 3
[0049] This example is the expression, purification and functional identification of nitroreductase PbMNR1, and the specific process is as follows:
[0050] (1) Expression and purification of nitroreductase PbMNR1
[0051] The recombinant strain BL21 (Pbmnr1) was inoculated into LB medium and cultured at 37°C, 200 rpm shaking incubator until OD600nm was 0.6. IPTG was added to a concentration of 0.4 mM and induced at 16°C for 12 h. 100 ml of bacterial solution 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-650Bultrasonic processor, 30% intensity) for 10 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected. PbMNR1 was purified using a nickel ion affinity chromatography column, and the purification effect was detected by SDS-PAGE protein electrophoresis. The band size was consistent with the theoretically predicted size (27.3 kDa) (e.g. Figure 3 shown). Figure 3 In the figure, M is a protein marker, and lanes 1-4 are 30 mM, 50 mM, 100 mM and 150 mM imidazole eluents, respectively.
[0052] (2) Functional identification of nitroreductase PbMNR1
[0053] ① Determination of enzyme activity of PbMNR1 catalyzing the reduction of mesotrione
[0054] Enzymatic reaction system (1mL): In PBS buffer (pH 7.4), 100µM mesotrione, 1mM NADH (reduced nicotinamide adenine dinucleotide), 100μL purified PbMNR1 enzyme solution (purified in the previous step), 30℃ enzymatic reaction for 1h. Each reaction starts with the addition of enzyme, and an equal volume of acetonitrile is added after 1h to terminate the reaction. The activity of PbMNR1 was determined by high performance liquid chromatography (HPLC) to detect the reduction of mesotrione without adding enzyme as a control. One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the reduction of 1.0 μmol of substrate per minute under pH 7.4 and temperature 30℃. The enzyme activity of PbMNR1 for mesotrione was finally determined to be 0.82 U / mg.
[0055] ② Identification of products of mesotrione degradation catalyzed by PbMNR1
[0056] 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 (150mm×4.6mm, 5μm), column temperature 30℃, UV detector, detection wavelength 260nm and 330nm, injection volume 10μL. The external standard method was quantified by peak area. The HPLC analysis results of mesotrione degradation by nitroreductase PbMNR1 are shown in Figure 4 As shown in the figure, A is the HPLC detection spectrum of PBS+NADH+mesotrione; B is the HPLC detection spectrum of PBS+PbMNR1+mesotrione; C is the HPLC detection spectrum of AMBA standard; D is the HPLC detection spectrum of PBS+PbMNR1+NADH+mesotrione. The above-mentioned results indicate that reducing power or PbMNR1 enzyme alone cannot reduce and degrade mesotrione, but under the condition of the coexistence of reducing power and PbMNR1 enzyme, mesotrione (the signal peak of mesotrione is significantly weakened) can be reduced to AMBA (the signal peak of AMBA appears).
[0057] The degradation products were identified and analyzed by liquid chromatography-mass spectrometry (LC / MS). 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 mass scanning range (m / z): 100-550. The test results showed that PbMNR1 catalyzed mesotrione to generate hydroxylamine intermediates and finally converted them into 2-amino-4-methylsulfonylbenzoic acid (AMBA) ( Figure 5 ), the reaction pathway of PbMNR1 catalyzing the conversion of mesotrione to AMBA is speculated to be Figure 6 . Figure 5 In the figure, A is the LC-MS detection spectrum of mesotrione, B is the nitrososulfonate trichlorfon (C 14 H 13 NO6S) LC-MS detection spectrum, C is hydroxyaminosulfonate oxadiazine (C 14 H 15 NO6S) LC-MS detection spectrum, D is the LC-MS detection spectrum of AMBA; Figure 5 It was demonstrated that PbMNR1 catalyzes the 14 H 13 NO7S) is gradually reduced to nitrososulfonate oxadiazine (C 14 H 13 NO6S) and hydroxyaminosulfonate (C 14 H 15 NO6S), and finally transformed into the product AMBA.
[0058] Example 4
[0059] This example verifies whether AMBA, a product of mesotrione degradation catalyzed by PbMNR1, has an inhibitory effect on rice HPPD. The experimental method is as follows:
[0060] The recombinant expression strain BL21 (OsHPPD) of rice HPPD (OsHPPD) was obtained according to the method of Example 2. Experimental principle: Escherichia coli BL21 (DE3) has tyrosine transferase activity and 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 produces a reddish-brown substance after spontaneous oxidation and polymerization. Therefore, the activity of HPPD can be judged by the depth of color.
[0061] 200 µL of LB liquid medium supplemented with tyrosine was added to a 98-well plate, and different concentrations (0, 2, 4, 6, 8, 10, 12 µM) of mesotrione and AMBA were added thereto. The recombinant expression strain BL21 (OsHPPD) was then transferred to each well and placed in a constant temperature shaker at 37°C and 180 rpm for 12 hours. It was observed that 2 µM mesotrione could significantly inhibit the color formation of the recombinant strain BL21 (OsHPPD) ( Figure 7 ), indicating that OsHPPD is very sensitive to mesotrione and that low concentrations of mesotrione can inhibit its activity. However, within the same concentration range, AMBA did not inhibit the color production of the recombinant strain BL21 (OsHPPD) ( Figure 7 ), indicating that OsHPPD was insensitive to AMBA and AMBA lost its inhibitory effect on HPPD and its herbicidal activity.
[0062] 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 PbMNR1, characterized in that The amino acid sequence of mesotrione nitroreductase PbMNR1 is shown in SEQ ID NO.
2.
2. A gene, characterized in that It encodes the mesotrione nitroreductase PbMNR1 described in claim 1.
3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO:
1.
4. An expression cassette, characterized in that A gene according to claim 2 or 3 comprising the gene under the control of an operably linked regulatory sequence.
5. A recombinant vector comprising the gene according to claim 2 or 3 or comprising the expression cassette according to claim 4.
6. The recombinant vector according to claim 5, characterized in that The recombinant vector is obtained by inserting the gene described in claim 2 or 3 or the expression cassette described in claim 4 between the Nde I and Xho I sites of pET-29a(+).
7. A method for constructing a mesotrione-resistant transgenic crop, characterized in that: include: The recombinant vector according to claim 6 is introduced into crops to express the mesotrione nitroreductase PbMNR1 in the crops.
8. A genetically engineered bacterium capable of degrading mesotrione, characterized in that: include: The recombinant vector according to claim 6 is introduced into a host strain, so that the mesotrione nitroreductase PbMNR1 is expressed in the host strain.
9. Use of the mesotrione nitroreductase PbMNR1 according to claim 1 in degrading and removing residual mesotrione in the environment.
10. Use of the gene according to claim 2 or 3 in constructing mesotrione-resistant transgenic crops or mesotrione-degrading microorganisms.