Mesotrione nitroreductase gene Xhctnr, protein coded by mesotrione nitroreductase gene Xhctnr and application

Abstract

The invention discloses a mesotrione nitroreductase gene xhctnr, a protein coded by the mesotrione nitroreductase gene xhctnr and application of the mesotrione nitroreductase gene xhctnr The nucleotide sequence of the mesotrione nitroreductase gene xhctnr disclosed by the invention is as shown in SEQ ID NO.1, the encoded protein Xhctnr is mesotrione nitroreductase Xhctnr, and the amino acid sequence of the mesotrione nitroreductase gene xhctnr is as shown in SEQ ID NO.2. The mesotrione nitroreductase Xhctnr can be used for degrading mesotrione into a product AMBA, so that the herbicidal activity is lost. The mesotrione nitroreductase gene xhctnr and the protein coded by the mesotrione nitroreductase gene xhctnr have a huge application prospect in biodegradation detoxification and bioremediation of mesotrione.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and in particular to a mesotrione nitroreductase gene XHv , the proteins they encode and their uses. Background Art

[0002] The application of herbicide-resistant transgenic engineering can control farmland weeds efficiently and at low cost 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 worldwide, 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 resistance genes for 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. HGA 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 HGA, and thus failing to synthesize plastoquinone and tocopherol, causing the plant to produce whitening symptoms and die. 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 pyrazoles according to their chemical structures. Mesotrione, also known as methylsulfonamide, 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. Mesotrione has the advantages of high efficiency, low toxicity, high crop safety, and good relative environmental compatibility. At the same time, since weeds develop resistance to this type of herbicide slowly, it is considered to be an ideal herbicide-resistant transgenic target herbicide. Therefore, obtaining new mesotrione degradation and detoxification / resistance genes with excellent performance has very important theoretical and practical application value for the development of mesotrione pollution bioremediation technology and the construction of transgenic crops that can degrade and detoxify mesotrione. Summary of the invention

[0005] The purpose of the present invention is to provide a mesotrione nitroreductase gene XHv , the proteins encoded therein and their uses, so as to address the deficiencies of the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The first aspect of the present invention provides a mesotrione nitroreductase gene xhctn , whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention provides the mesotrione nitroreductase gene xhctn Mesotrione nitroreductase Xhctnr 。

[0008] The third aspect of the present invention provides a method comprising the above-mentioned mesotrione nitroreductase gene xhctn recombinant expression vector.

[0009] Furthermore, the mesotrione nitroreductase gene xhctn Obtained by recombination into the original vector, wherein the original vector includes pET-29a (+).

[0010] The fourth aspect of the present invention provides a method comprising the above-mentioned mesotrione nitroreductase gene xhctn of genetically engineered bacteria.

[0011] Furthermore, the expression strain of the genetically engineered bacteria includes Escherichia coli BL21 (DE3).

[0012] The fifth aspect of the present invention provides the mesotrione nitroreductase gene xhctnApplication in biodegradation, detoxification and bioremediation of mesotrione.

[0013] A sixth aspect of the present invention provides the use of the mesotrione nitroreductase Xhctnr in the biodegradation, detoxification and bioremediation of mesotrione.

[0014] Beneficial effects of the present invention: The present invention provides a mesotrione nitroreductase gene xhctn The nucleotide sequence is shown in SEQ ID NO.1. The encoded protein Xhctnr is mesotrione nitroreductase Xhctnr, and the amino acid sequence is shown in SEQ ID NO.2. Mesotrione nitroreductase Xhctnr can degrade mesotrione into product AMBA and lose its herbicidal activity. xhctn The protein encoded by it has great application prospects in mesotrione biodegradation, detoxification and bioremediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a colony photo of strain Xhctd-6.

[0016] Figure 2 This is a microscopic photograph of the strain Xhctd-6.

[0017] Figure 3 Phylogenetic tree of strain Xhctd-6 based on 16S rRNA gene sequence.

[0018] Figure 4 This is the UV scanning detection result of strain Xhctd-6 degrading mesotrione; the black arrow indicates the characteristic absorption peak of mesotrione (270 nm).

[0019] Figure 5 This is the SDS-PAGE detection result of exogenously expressed Xhctnr; lane M is protein Maker, and lane 1 is purified Xhctnr protein.

[0020] Figure 6 Figure 1 is the HPLC detection result of mesotrione degradation by Xhctnr-6; A is the mesotrione standard sample (characteristic peak 10.4 min), B is the AMBA standard sample (characteristic peak 7.7 min), C is the reaction time 0 min, and D is the reaction time 30 min.

[0021] Figure 7 This is a diagram showing the inhibitory effect of the products of mesotrione degradation by Xhctnr-6 on rice HPPD (OsHPPD). DETAILED DESCRIPTION

[0022] The present invention is further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0023] The culture medium formulas involved in the following examples are as follows: Basal salt liquid culture medium (1L system): 0.5 g KH2PO4, 1.5 g K2HPO4•3H2O, 1.0 g NH4NO3, 0.5 g NaCl, 0.2 g MgSO4•7H2O, pH 7.0.

[0024] R2A medium (1L system): 0.5 g yeast powder, 0.5 g peptone, 0.3 g sodium pyruvate, 0.5 g casein hydrolysate, 0.5 g soluble starch, 0.5 g glucose, 0.3 g KH2PO4, 0.024 g MgSO4, 15.0 g agar powder, pH 7.2. R2A liquid medium without agar powder.

[0025] LB liquid medium (1 L system): 5 g yeast extract, 10 g peptone, 10 g NaCl, pH 7.0.

[0026] Example 1 Isolation of mesotrione-degrading strains and their degradation of mesotrione 1.1 Enrichment, isolation and classification of mesotrione-degrading strains Sludge samples were collected from the sewer of a pesticide factory that produced mesotrione. 10 g of sludge sample was added to 90 mL of basal salt liquid culture medium containing 100 mg / L mesotrione. After shaking culture at 30°C and 180 rpm for about 15 days, 10 mL was transferred to 90 mL of basal salt liquid culture medium containing 100 mg / L mesotrione. Then, shaking culture was continued at 30°C and 180 rpm for about one week. This transfer culture was repeated 4 times, and the degradation of mesotrione was detected by ultraviolet scanning (mesotrione has a characteristic absorption peak at 270nm in the ultraviolet region). The enriched solution with obvious degradation effect was diluted in a gradient manner, and 1 mL of 10 -4 , 10 -5 and 10 -6 The dilution was coated on R2A medium plates and cultured at 30°C for 5-6 days. Single colonies of different morphologies on the plates were picked and further purified by streaking on R2A medium plates. The obtained pure strains were inoculated into a basal salt liquid medium containing 100 mg / L mesotrione and cultured at 30°C and 180 rpm for 5 days. UV scanning was used to detect whether each pure strain had the ability to degrade mesotrione.

[0027] A mesotrione-degrading bacterium was obtained by enrichment, acclimation, isolation and screening, and named Xhctd-6. After the strain Xhctd-6 was grown on R2A medium plates at 30°C for 5 days, the colonies were medium-sized (2-3 mm in diameter), creamy yellow, transparent, smooth and moist on the surface, without protrusions, irregular edges, and no soluble pigment was produced ( Figure 1 The bacterial cells are straight or slightly curved rods (0.9–1.0×3.0–4.5 µm) with flagella ( Figure 2 ). Gram staining is negative, oxidase and catalase are positive, it does not hydrolyze starch, does not liquefy gelatin, and can utilize glucose and fructose. Using the genomic DNA of strain Xhctd-6 as a template, PCR amplification was performed with universal primers for bacterial 16S rRNA gene sequences (27F (SEQ ID NO.4): 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R (SEQ ID NO.5): 5'-GGTTACCTTGTTACGACTT-3') to obtain a 1464 bp 16S rRNA gene sequence (its nucleotide sequence is shown in SEQ ID NO.3). Similarity analysis was performed with the 16S rRNA gene sequences of reported model strains in the EZtaxon database (http: / / eztaxon-e.ezbiocloud.net / ezt). The results showed that strain Xhctd-6 had a similarity to Delftia ( Delftia ) strains are most closely related to Delftia acidovorans 2176 T The highest homology is 99.1%. In the phylogenetic tree constructed based on the 16S rRNA gene sequence ( Figure 3 ), strain Xhctd-6 also clustered in Delftia genus, and thus strain Xhctd-6 was identified as Delftia Belong.

[0028] 1.2 Degradation of mesotrione by strain Xhctd-6 Study on degradation characteristics: Xhctd-6 was inoculated into R2A liquid medium, cultured at 30°C and 150 rpm until mid-logarithmic phase, centrifuged at 6000 g for 10 min, and the cells were collected. The cells were washed twice with fresh, sterile basal salt liquid medium, resuspended in basal salt liquid medium, and the cell concentration was adjusted to about 1.0×10 9 cfu / mL, inoculated into 20 mL of basal salt liquid medium containing 100 mg / L mesotrione at a 1% (v / v) inoculation rate, and cultured at 30°C and 150 rpm for 72 h. Samples were taken and UV scanning was used to determine the degradation of mesotrione by the strain.

[0029] The UV scanning results showed that mesotrione had a characteristic absorption peak at 270 nm. As the culture time increased, the characteristic absorption peak of mesotrione in the sample gradually decreased, indicating that strain Xhctd-6 was able to degrade mesotrione ( Figure 4 ). The results of sampling after 72 hours of cultivation showed that strain Xhct-6 could degrade 70% of mesotrione.

[0030] Example 2 Mesotrione Nitroreductase Gene xhctn clone 2.1 Genome sequencing and nitroreductase search of strain Xhctd-6 The initial step of microbial degradation of mesotrione is nitro reduction. This study intends to identify the suspected mesotrione nitroreductase gene by analyzing the genome of strain Xhctd-6. The genome of strain Xhctd-6 was sequenced using high-throughput sequencing technology (HiSeq 4000sequencer system, Illumina), and the genome was annotated using RAST (Rapid Annotation with Subsystem Technology). The bacterial genome size is 6.46 Mb, encoding 5013 ORFs. Through the possible functional analysis of these ORFs, a suspected nitroreductase gene was found, named xhctn , whose nucleotide sequence is shown in SEQ ID NO.1, is 627 bp in size, and encodes a protein (Xhctnr) of 208 amino acids, as shown in SEQ ID NO.2.

[0031] 2.2 Construction of mesotrione nitroreductase gene xhctn Recombinant expression vector xhctn Gene recombination expression vector pET-29a -xhctnr , synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the requirements of the following table. Among them, xhctn The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0032] Target gene Target vector (original vector) pET-29a (+) 5' end restriction site of target vector N i Target vector 3' end restriction site XOt The above synthesized xhctn Gene recombination expression vector pET-29a- xhctn The transformants were transferred into the E. coli expression strain E. coli BL21 (DE3). The transformants were selected and placed in LB liquid medium containing 50 mg / L kanamycin, cultured at 37°C and 180 rpm, and sequenced to verify whether they were correct. The recombinant expression strain with correct sequencing was named BL21 (pET-29a -xhctnr ).

[0033] Example 3 Functional verification of mesotrione nitroreductase Xhctnr 3.1 Expression and purification of Xhctnr BL21 (pET-29a- xhctn ) in 100 mL LB liquid medium, shake at 37°C and 150 rpm until OD 600 The concentration of isopropylthiogalactoside (IPTG) was 0.4-0.6, and then isopropylthiogalactoside (IPTG) was added to a concentration of 0.05 mM, and the culture was induced at 16°C and 150 rpm for 8 h. The bacterial solution obtained by the induced culture was centrifuged at 6000 g for 10 min, the bacteria were collected, and the bacteria were washed twice with PBS buffer (50 mM, pH7.4), resuspended with 10 mL of PBS buffer, ultrasonically disrupted (Auto Science, UH-650Bultrasonic processor, 30% intensity) for 5-10 min, and centrifuged at 12000 rpm for 30 min. The supernatant was collected and precipitated with Co 2+ Xhctnr was purified by ion affinity chromatography, and the purified Xhctnr was subjected to SDS-PAGE electrophoresis to detect its purity. The results of SDS-PAGE electrophoresis showed that the purified protein had a clear band at 22-25 kDa, which was consistent with the theoretical size of Xhctnr (22.8 kDa), and no other obvious miscellaneous bands were observed in the lane ( Figure 5 ), indicating that pure Xhctnr protein was obtained.

[0034] 3.2 Xhctnr enzyme activity detection 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 was 100 μL (containing 2 μg purified Xhctnr), and the reaction was carried out at 30°C for 30 min. Each reaction started with the addition of the enzyme, and was terminated by placing it in boiling water for 1 min after 30 min. The reaction solution was freeze-dried (-30°C, 0.2 mbar, 24 h), and 200 μl of methanol was added to dissolve the lyophilized material. HPLC was used to detect the amount of reduction in substrate (mesotrione). One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the reduction of 1 μM mesotrione per min under the conditions of pH 7.5 and 30°C. The HPLC analysis results of mesotrione degradation by Xhctnr are shown in the figure. Figure 6The results showed that mesotrione had a characteristic absorption peak at 10.4 min, while 2-amino-4-methylsulfonylbenzoic acid (AMBA) had a characteristic absorption peak at 7.7 min; after 30 min of enzyme reaction, the absorption peak of mesotrione decreased significantly, and at the same time, the characteristic absorption peak of AMBA appeared, indicating that Xhctnr can reduce mesotrione to AMBA and has mesotrione nitroreductase activity, and the specific enzyme activity for mesotrione is 1.24 U / μg protein.

[0035] Example 4 Effect of Xhctnr on Degradation and Detoxification of Mesotrione 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 can convert tyrosine into 4-HPP in LB liquid medium containing 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 expression strain BL21 (pET-29a-OsHPPD) was introduced with rice HPPD (OsHPPD) which is extremely sensitive to mesotrione (the construction of the recombinant expression strain BL21 (pET-29a-OsHPPD) refers to 2.2), so it was used in this experiment to detect the inhibitory activity of mesotrione and its degradation products on OsHPPD.

[0036] In a 100 mL enzyme reaction system, mesotrione was added to a final concentration of 4 μM, 8 μM, 12 μM and 16 μM, respectively, and then 60 μg of purified Xhctnr was added, and the reaction was carried out in a 30°C water bath for 5-6 h to completely degrade mesotrione, and then placed in boiling water for 1 min 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 deionized water to obtain the product of mesotrione degradation (4 μM, 8 μM, 12 μM or 16 μM) by Xhctnr.

[0037] The recombinant expression strain BL21 (pET-29a-OsHPPD) was cultured to the logarithmic growth phase (OD 6001.0), 20 μL was inoculated in a 96-well plate supplemented with 200 μL LB liquid medium containing 0.1 w / v% tyrosine, and the inducer IPTG was added at a final concentration of 10 mM, and then mesotrione with a final concentration of 0 μM, 4 μM, 8 μM, 12 μM and 16 μM or mesotrione degradation products of Xhctnr (4 μM, 8 μM, 12 μM or 16 μM) were added at a final concentration of 0 μM, 4 μM, 8 μM, 12 μM and 16 μM, respectively (the final concentration of the product of 4 μM mesotrione degradation by Xhctnr was 4 μM, the final concentration of the product of 8 μM mesotrione degradation by Xhctnr was 8 μM, the final concentration of the product of 12 μM mesotrione degradation by Xhctnr was 12 μM, the final concentration of the product of 16 μM mesotrione degradation by Xhctnr was 16 μM). μM), 30℃, 150 rpm shaking culture for 36 h, and then observe the color changes under different treatments. Figure 7 As shown in the figure, the control without mesotrione was red, indicating that OsHPPD was normally expressed and active; the treatment with 4 μM or higher concentration of mesotrione was light yellow, indicating that OsHPPD activity had been completely inhibited; and the products of mesotrione degradation by Xhctnr were all red in each treatment, and there was no significant difference from the control without mesotrione, indicating that the products of mesotrione degradation by Xhctnr had no inhibitory effect on OsHPPD. Therefore, Xhctnr can completely convert mesotrione into products that have no inhibitory effect on OsHPPD, thereby achieving the degradation and detoxification of mesotrione.

[0038] 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 gene xhctn , characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The mesotrione nitroreductase gene according to claim 1 xhctn The encoded protein Xhctnr is mesotrione nitroreductase Xhctnr.

3. A composition containing the mesotrione nitroreductase gene according to claim 1. xhctn recombinant expression vector.

4. The recombinant expression vector according to claim 3, characterized in that The mesotrione nitroreductase gene of claim 1 xhctn Obtained by recombination into the original vector, wherein the original vector includes pET-29a (+).

5. A composition containing the mesotrione nitroreductase gene according to claim 1 xhctn of genetically engineered bacteria.

6. The genetically engineered bacterium according to claim 5, characterized in that: The expression strain of the genetically engineered bacteria includes Escherichia coli BL21 (DE3).

7. The mesotrione nitroreductase gene according to claim 1 xhctn Application in biodegradation, detoxification and bioremediation of mesotrione.

8. Use of the mesotrione nitroreductase Xhctnr according to claim 2 in mesotrione biodegradation, detoxification and bioremediation.

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