A mesotrione nitroreductase gene Xhctnr, the protein encoded thereby, and uses thereof

Abstract

The present invention discloses a mesotrione nitroreductase gene xhctnr , the protein encoded thereby and uses thereof. The mesotrione nitroreductase gene of the present invention xhctnr , its nucleotide sequence is as shown in SEQ ID NO.1, and the encoded protein Xhctnr, namely mesotrione nitroreductase Xhctnr, its amino acid sequence is as shown in SEQ ID NO.2. Mesotrione nitroreductase Xhctnr can degrade mesotrione into the product AMBA and lose its herbicidal activity. The mesotrione nitroreductase gene of the present invention xhctnr and the protein encoded thereby have great application prospects in the biodegradation detoxification and bioremediation of mesotrione.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to a mesotrione nitroreductase gene Xhctnr , the protein encoded thereby, and uses thereof. Background Art

[0002] The application of herbicide-resistant transgenic engineering has efficiently and low-costly controlled farmland weeds on the premise of ensuring the safety of crops, creating a brand-new era for farmland weed control. At present, the vast majority of transgenic crops being promoted and planted are glyphosate-resistant transgenic crops. In 2017, the global planting 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 serious weed resistance problems. More than 40 major farmland weeds have been reported worldwide to have developed resistance to glyphosate, resulting in the inability to control weeds. One of the effective measures to solve this problem is to simultaneously use herbicides with different herbicidal mechanisms and construct corresponding herbicide-resistant transgenic crops accordingly. Therefore, it is necessary to widely explore resistance genes of various herbicides to provide gene resources for constructing transgenic crops resistant to various herbicides.

[0003] 4-Hydroxyphenylpyruvate dioxygenase (HPPD EC1.13.11.27) 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, blocking the synthesis of HGA, and thus unable to synthesize plastoquinone and tocopherol, resulting in the death of plants due to albino symptoms. Therefore, this catalytic reaction has also enabled HPPD to be developed into an important herbicide target enzyme.

[0004] HPPD inhibitor herbicides are mainly divided into triketone, isoxazole and pyrazole types according to their chemical structures. Mesotrione, also known as methylsulfone herbicide, is a triketone herbicide developed by Syngenta in 1984, which can control most broadleaf weeds and a few gramineous weeds. Since its launch in 2001, mesotrione has now become the world's largest HPPD inhibitor herbicide, with a global sales volume of 650 million US dollars in 2016. Mesotrione has the advantages of high efficiency, low toxicity, high crop safety and good relative environmental compatibility. At the same time, due to the slow development of weed resistance to this type of herbicide, it is considered an ideal target herbicide for herbicide-resistant transgenic crops. Therefore, obtaining new and excellent mesotrione degradation / detoxification / resistance genes has very important theoretical and practical application values for the research and development of mesotrione pollution bioremediation technology and the construction of transgenic crops capable of degrading and detoxifying mesotrione. Summary of the Invention

[0005] The object of the present invention is to provide a mesotrione nitroreductase gene Xhctnr 、its encoded protein and uses to solve the deficiencies of the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a mesotrione nitroreductase gene is provided xhctnr whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] In the second aspect of the present invention, the above-mentioned mesotrione nitroreductase gene is provided xhctnr The encoded protein Xhctnr, namely mesotrione nitroreductase Xhctnr 。

[0009] In the third aspect of the present invention, a recombinant expression vector containing the above-mentioned mesotrione nitroreductase gene is provided xhctnr is obtained by recombining the above-mentioned mesotrione nitroreductase gene into an original vector, and the original vector includes pET-29a(+).

[0010] Furthermore, it is obtained by recombining the above-mentioned mesotrione nitroreductase gene xhctnr into the original vector, and the original vector includes pET-29a(+).

[0011] In the fourth aspect of the present invention, a genetically engineered bacterium containing the above-mentioned mesotrione nitroreductase gene is provided xhctnr is provided.

[0012] Furthermore, the expression strain of the genetically engineered bacterium includes Escherichia coli BL21(DE3).

[0013] In the fifth aspect of the present invention, the above-mentioned mesotrione nitroreductase gene is provided xhctnrApplication in biodegradation detoxification and bioremediation of mesotrione

[0014] The sixth aspect of the present invention provides the application of the above-mentioned mesotrione nitroreductase Xhctnr in biodegradation detoxification and bioremediation of mesotrione

[0015] Beneficial effects of the present invention

[0016] The present invention provides a mesotrione nitroreductase gene xhctnr The nucleotide sequence is shown in SEQ ID NO.1, and the encoded protein Xhctnr is the mesotrione nitroreductase Xhctnr, and its amino acid sequence is shown in SEQ ID NO.2. Mesotrione nitroreductase Xhctnr can degrade mesotrione into the product AMBA and lose its herbicidal activity. The mesotrione nitroreductase gene of the present invention xhctnr and its encoded protein have great application prospects in biodegradation detoxification and bioremediation of mesotrione Brief description of the drawings

[0017] Figure 1 It is a colony photograph of strain Xhctd-6

[0018] Figure 2 It is a microscopic photograph of the cells of strain Xhctd-6

[0019] Figure 3 It is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain Xhctd-6

[0020] Figure 4 It is a UV scanning detection result graph of the degradation of mesotrione by strain Xhctd-6; the characteristic absorption peak of mesotrione (270 nm) is indicated by the black arrow

[0021] Figure 5 It is a SDS-PAGE detection result graph of the heterologously expressed Xhctnr; Lane M is the protein Maker, and Lane 1 is the purified Xhctnr protein

[0022] Figure 6 It is an HPLC detection result graph of the degradation of mesotrione by Xhctnr-6; A is the mesotrione standard sample (characteristic peak at 10.4 min), B is the AMBA standard sample (characteristic peak at 7.7 min), C is the reaction at 0 min, and D is the reaction at 30 min

[0023] Figure 7 It is an inhibition effect graph of the product of the degradation of mesotrione by Xhctnr-6 on rice HPPD (OsHPPD) Detailed implementation manners

[0024] The present invention will be further explained below in conjunction with embodiments and the accompanying drawings. The following embodiments are only used to illustrate the present invention, but do not limit the scope of implementation of the present invention.

[0025] The culture medium formulations involved in the following examples are as follows:

[0026] Basal salt liquid medium (1L system): 0.5 g KH 2 PO 4 ,1.5 g K 2 HPO 4 •3H 2 O, 1.0 g NH 4 NO 3 ,0.5 g NaCl, 0.2 g MgSO 4 •7H 2 O, pH 7.0.

[0027] R2A medium (1L system): 0.5 g yeast extract, 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 KH 2 PO 4 ,0.024 g MgSO 4 ,15.0 g agar powder, pH 7.2. The agar powder is removed from the R2A liquid medium.

[0028] LB liquid medium (1L system): 5 g yeast extract, 10 g peptone, 10 g NaCl, pH 7.0.

[0029] Example 1 Isolation of mesotrione-degrading strains and their degradation of mesotrione

[0030] 1.1 Enrichment isolation and taxonomic identification of mesotrione-degrading strains

[0031] Collect sludge samples from the sewer of a pesticide factory producing mesotrione. Add 10 g of sludge samples to 90 mL of basal salt liquid medium containing 100 mg / L mesotrione. After shaking culture at 30°C and 180 rpm for about 15 d, take 10 mL and transfer it to 90 mL of basal salt liquid medium containing 100 mg / L mesotrione, and then shake culture at 30°C and 180 rpm for about one week. Continuously transfer and culture 4 times like this, and use ultraviolet scanning to detect the degradation of mesotrione (mesotrione has a characteristic absorption peak at 270 nm in the ultraviolet region). Gradient dilute the enrichment solution with obvious degradation effect, and pipette 1 mL of 10 -4 、10 -5 and 10 -6The dilution solution was spread on an R2A medium plate and cultured at 30 °C for 5 - 6 d. Different morphologically single colonies on the plate were picked and further streaked and purified on an R2A medium plate. 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 d. The ultraviolet scan was used to detect whether each pure strain had the function of degrading mesotrione.

[0032] A mesotrione-degrading bacterium was isolated and screened through enrichment and domestication, named Xhctd-6. After growing on an R2A medium plate at 30 °C for 5 d, the colonies of strain Xhctd-6 were of medium size (2 - 3 mm in diameter), milky yellow, transparent, with a smooth and moist surface, not convex, and an irregular edge, and did not produce soluble pigments ( Figure 1 ). The cells were straight or slightly curved bacilli (0.9–1.0×3.0–4.5 µm) and had flagella ( Figure 2 ). Gram-negative staining, positive for oxidase and catalase, did not hydrolyze starch, did not liquefy gelatin, and could utilize glucose and fructose. Using the genomic DNA of strain Xhctd-6 as a template, the 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’) were used for PCR amplification to obtain a 16S rRNA gene sequence with a length of 1464 bp (its nucleotide sequence is shown in SEQ ID NO.3). Similarity analysis was performed with the reported type strain 16S rRNA gene sequences in the EZtaxon database (http: / / eztaxon-e.ezbiocloud.net / ezt). The results showed that strain Xhctd-6 had the closest genetic relationship with the strains of the genus Delftia, and the highest homology with Delftia was 99.1%. On the phylogenetic tree ( Delftia acidovorans 2176 T constructed based on the 16S rRNA gene sequence, strain Xhctd-6 was also clustered in the Figure 3 genus. Therefore, strain Xhctd-6 was identified as the Delftia genus. Delftia genus.

[0033] 1.2 Degradation of mesotrione by strain Xhctd-6

[0034] Study on degradation characteristics: Inoculate Xhctd-6 into R2A liquid medium, culture at 30 °C and 150 rpm until the mid-logarithmic phase, centrifuge at 6000 g for 10 min, collect the cells, wash the cells twice with fresh and sterile basal salt liquid medium, resuspend them in the basal salt liquid medium, and adjust the cell concentration to about 1.0×10 9 cfu / mL. According to the inoculation amount of 1% (v / v), inoculate into 20 mL of basal salt liquid medium containing 100 mg / L of mesotrione, and culture at 30 °C and 150 rpm for 72 h. Sampling is used to determine the degradation of mesotrione by the strain by ultraviolet scanning.

[0035] The ultraviolet scanning results show that mesotrione has a characteristic absorption peak at 270 nm. As the culture time extends, the characteristic absorption peak of mesotrione in the sample gradually decreases, indicating that the strain Xhctd-6 can degrade mesotrione ( Figure 4 ). The sampling and determination results after 72 h of culture show that the strain Xhct-6 can degrade 70% of mesotrione.

[0036] Example 2 Mesotrione nitroreductase gene xhctnr Cloning

[0037] 2.1 Genome sequencing of strain Xhctd-6 and search for nitroreductase

[0038] The initial step of microbial degradation of mesotrione is nitroreduction. This study intends to determine the suspected mesotrione nitroreductase gene by analyzing the genome of strain Xhctd-6. The genome of strain Xhctd-6 is sequenced using high-throughput sequencing technology (HiSeq 4000 sequencer system, Illumina), and the genome is annotated using RAST (Rapid Annotation with Subsystem Technology). The genome size of this bacterium is 6.46 Mb, encoding 5013 ORFs. Through the possible functional analysis of these ORFs, a suspected nitroreductase gene is found and named xhctnr , whose nucleotide sequence is shown in SEQ ID NO.1, with a size of 627 bp, encoding a protein (Xhctnr) of 208 amino acids, as shown in SEQ ID NO.2.

[0039] 2.2 Construction of mesotrione nitroreductase gene xhctnr Recombinant expression vector

[0040] xhctnr Gene recombinant expression vector pET-29a -xhctnr, synthesized by Beijing Tsingke Biotechnology Co., Ltd. according to the requirements in the following table. Among them, xhctnr The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0041] Target gene Target vector (original vector) pET-29a(+) Restriction site at the 5' end of the target vector NdeI Restriction site at the 3' end of the target vector XhoI

[0042] The synthesized xhctnr gene recombinant expression vector pET-29a- xhctnr was transferred into the Escherichia coli expression strain Escherichia coli BL21 (DE3). The transformants were picked into an LB liquid medium containing 50 mg / L kanamycin and cultured with shaking at 37°C and 180 rpm. Sequencing was performed to verify whether it was correct, and the obtained recombinant expression strain with correct sequencing was named BL21 (pET-29a -xhctnr ).

[0043] Example 3 Functional verification of nicosulfuron nitroreductase Xhctnr

[0044] 3.1 Expression and purification of Xhctnr

[0045] BL21 (pET-29a- xhctnr ) was cultured with shaking at 37°C and 150 rpm in 100 mL of LB liquid medium until the OD 600 reached 0.4 - 0.6, then isopropyl β-D-thiogalactoside (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 induced culture broth was centrifuged at 6000 g for 10 min to collect the cells. The cells were washed twice with PBS buffer (50 mM, pH 7.4), resuspended in 10 mL of PBS buffer, sonicated (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 - 10 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected. Xhctnr was purified using a Co 2+ ion affinity chromatography column, and the purified Xhctnr was subjected to SDS-PAGE electrophoresis to detect its purity. The SDS-PAGE electrophoresis results showed that there was a clear band at 22 - 25 kDa for the purified protein, which was consistent with the theoretical size of Xhctnr (22.8 kDa), and no other obvious impurity bands were seen in the lane ( Figure 5 ), indicating that pure Xhctnr protein was obtained.

[0046] 3.2 Enzyme activity assay of Xhctnr

[0047] 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 amount of reaction enzyme is 100 μL (containing 2 μg purified Xhctnr), and the reaction is carried out at 30 °C for 30 min. Each reaction is timed starting from the addition of the enzyme. After 30 min, it is placed in boiling water for 1 min to terminate the reaction. After the reaction solution is freeze-dried (-30 °C, 0.2 mbar, 24 h), 200 μl of methanol is added to dissolve the freeze-dried product. HPLC is used to detect the reduction amount of the substrate (mesotrione). One enzyme activity unit (U) is defined as: the amount of enzyme required to catalyze the reduction of 1 μM mesotrione per minute under the conditions of pH 7.5 and 30 °C. The HPLC analysis results of the degradation of mesotrione by Xhctnr are as Figure 6 shown. The results show that mesotrione has a characteristic absorption peak at 10.4 min, while 2-amino-4-methylsulfonylbenzoic acid (AMBA) has a characteristic absorption peak at 7.7 min; after 30 min of the enzyme reaction, the absorption peak of mesotrione decreased significantly. At the same time, the characteristic absorption peak of AMBA appeared, indicating that Xhctnr can reduce mesotrione to AMBA, has the activity of mesotrione nitroreductase, and the specific enzyme activity against mesotrione is 1.24 U / μg protein.

[0048] Example 4 Detoxification effect of Xhctnr on the degradation of mesotrione

[0049] Experimental principle: Escherichia coli BL21 (DE3) itself has tyrosine transferase activity and can convert tyrosine into 4-HPP. The recombinant expression strain into which exogenous HPPD is introduced can convert tyrosine into 4-HPP in the LB liquid medium containing tyrosine and continue to convert 4-HPP into HGA. The generated HGA spontaneously oxidizes and polymerizes to produce a reddish-brown substance. Therefore, the activity of HPPD can be judged by the depth of the color. The recombinant expression strain BL21 (pET-29a-OsHPPD), which has introduced rice HPPD (OsHPPD) that is extremely sensitive to mesotrione (the construction of the recombinant expression strain BL21 (pET-29a-OsHPPD) refers to 2.2), is used in this experiment to detect the inhibitory activity of mesotrione and its degradation products on OsHPPD.

[0050] In a 100 mL enzyme reaction system, mesotrione was added to make the final concentrations 4 μM, 8 μM, 12 μM, and 16 μM respectively. Then, 60 μg of purified Xhctnr was added and reacted in a water bath at 30°C for 5 - 6 h to completely degrade mesotrione. It was placed in boiling water for 1 min to terminate the reaction. After freeze-drying the enzyme reaction solution, 2 mL of methanol was added to dissolve the lyophilized product. Then, the methanol solution was allowed to evaporate naturally, and the residue was dissolved in deionized water to obtain the product of Xhctnr degrading (4 μM, 8 μM, 12 μM, or 16 μM) mesotrione.

[0051] The recombinant expression strain BL21 (pET-29a-OsHPPD) was cultured to the logarithmic growth phase (OD 600 about 1.0). 20 μL was taken and inoculated into a 96-well plate containing 200 μL of LB liquid medium supplemented with 0.1 w / v% tyrosine. At the same time, an inducer IPTG with a final concentration of 10 mM was added. Then, mesotrione with final concentrations of 0 μM, 4 μM, 8 μM, 12 μM, and 16 μM or the product of Xhctnr degrading (4 μM, 8 μM, 12 μM, or 16 μM) mesotrione (the final concentration of the product of Xhctnr degrading 4 μM mesotrione added was 4 μM, the final concentration of the product of Xhctnr degrading 8 μM mesotrione added was 8 μM, the final concentration of the product of Xhctnr degrading 12 μM mesotrione added was 12 μM, and the final concentration of the product of Xhctnr degrading 16 μM mesotrione added was 16 μM) was added respectively. After culturing with shaking at 30°C and 150 rpm for 36 h, the color change under different treatments was observed. The results are as Figure 7 shown. The color of the control without mesotrione added was red, indicating that OsHPPD was normally expressed and had activity; while the treatments with 4 μM or higher concentration of mesotrione added were light yellow, indicating that the activity of OsHPPD had been completely inhibited; while the treatments with the product of Xhctnr degrading mesotrione all showed red, and there was no significant difference from the control without mesotrione added, indicating that the product of Xhctnr degrading mesotrione had no inhibitory effect on OsHPPD. Therefore, Xhctnr can completely convert mesotrione into a product that has no inhibitory effect on OsHPPD, achieving the degradation and detoxification of mesotrione.

[0052] 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 foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and 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).

Images