A mesotrione nitroreductase Xhnr, its encoding gene and uses

Abstract

The present invention discloses a mesotrione nitroreductase Xhnr, its encoding gene and uses. The mesotrione nitroreductase Xhnr of the present invention has an amino acid sequence as shown in SEQ ID NO.2, and its encoding gene xhnr has a nucleotide sequence as shown in SEQ ID NO.1. The homology of the mesotrione nitroreductase Xhnr with the reported nitroreductases is only 53.1% at most, so it is a novel nitroreductase. The mesotrione nitroreductase Xhnr can degrade mesotrione into the product AMBA and lose its herbicidal activity. The mesotrione nitroreductase Xhnr of the present invention and its encoding gene have potential 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 nicosulfuron nitroreductase Xhnr, its encoding gene 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 crop safety, creating a brand-new era for farmland weed control. Currently, the vast majority of transgenic crops being promoted and planted 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 serious weed resistance problems. So far, 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 for various herbicides to provide gene resources for constructing 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 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 methylsulfonyltrizone, 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 in 2016. Mesotrione has the advantages of high efficiency, low toxicity, high crop safety and good relative environmental compatibility. At the same time, because the resistance of weeds to this type of herbicide develops slowly, 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 Xhnr, its encoding gene and uses, so as 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 Xhnr is provided, and its amino acid sequence is as shown in SEQ ID NO.2.

[0008] In the second aspect of the present invention, the encoding gene of the above mesotrione nitroreductase Xhnr is provided xhnr。

[0009] Furthermore, its nucleotide sequence is as shown in SEQ ID NO.1.

[0010] In the third aspect of the present invention, a recombinant expression vector of the above encoding gene is provided xhnr of.

[0011] Furthermore, it is obtained by recombining the above encoding gene xhnr into the original vector, and the original vector includes pET-28a(+).

[0012] In the fourth aspect of the present invention, a genetically engineered bacterium of the above encoding gene is provided xhnr is provided.

[0013] Furthermore, the expression strain of the genetically engineered bacterium includes Escherichia coli BL21(DE3).

[0014] In the fifth aspect of the present invention, the above mesotrione nitroreductase Xhnr is provided for use in the biodegradation, detoxification and bioremediation of mesotrione.

[0015] The sixth aspect of the present invention provides the above-mentioned coding gene xhnr for use in the biodegradation detoxification and bioremediation of mesotrione.

[0016] Advantages of the present invention:

[0017] The present invention provides a mesotrione nitroreductase Xhnr, whose amino acid sequence is shown in SEQ ID NO.2, and its coding gene xhnr has a nucleotide sequence shown in SEQ ID NO.1. The homology of mesotrione nitroreductase Xhnr with the reported nitroreductases is only 53.1% at most, so it is a new nitroreductase. Mesotrione nitroreductase Xhnr can degrade mesotrione into the product AMBA and lose its herbicidal activity. The mesotrione nitroreductase Xhnr and its coding gene of the present invention have potential application value in the biodegradation detoxification and bioremediation of mesotrione. Description of the drawings

[0018] Figure 1 It is a colony photograph of strain EMD-1.

[0019] Figure 2 It is a phylogenetic tree constructed for strain EMD-1 based on the 16S rRNA gene.

[0020] Figure 3 It is a UV scanning detection result graph of the degradation of mesotrione by strain EMD-1. The characteristic absorption peak (270 nm) of mesotrione is indicated by the blue arrow.

[0021] Figure 4 It is a SDS-PAGE detection result graph of the exogenous expression of Xhnr. Lane 1, protein Maker; Lane 2, Xhnr protein eluted with 100 mM imidazole; Lane 3, Xhnr protein eluted with 200 mM imidazole.

[0022] Figure 5 It is an HPLC detection of the degradation of mesotrione by Xhnr. A, mesotrione standard; B, AMBA standard; C, reaction for 0 h; D, reaction for 1 h.

[0023] Figure 6Inhibitory effect diagram of the product of Xhnr-6 degrading mesotrione on rice HPPD (OsHPPD). A, negative control, adding BL21 (pET-29a-OsHPPD), without adding the substrate tyrosine, no HPPD activity; B, positive control, adding BL21 (pET-29a-OsHPPD) and the substrate tyrosine, with HPPD activity; C, adding BL21 (pET-29a-OsHPPD), the substrate tyrosine and mesotrione, the HPPD activity is completely inhibited by mesotrione; D, adding BL21 (pET-29a-OsHPPD), the substrate tyrosine and the product of Xhnr degrading mesotrione, with HPPD activity, showing no significant difference from the positive control, indicating that this product does not inhibit HPPD activity. Detailed implementation mode

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

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

[0026] Basic salt liquid 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.

[0027] LB liquid medium (1L system): 5 g yeast extract, 10 g peptone, 10 g sodium chloride, pH 7.0. 15 g agar powder is added to the LB medium on this basis.

[0028] Example 1 Isolation of mesotrione-degrading strains and search for their nitroreductase genes

[0029] 1.1 Enrichment isolation and taxonomic identification of mesotrione-degrading strains

[0030] Collect sludge samples from the sewer of a pesticide factory producing mesotrione. Add 5 g of sludge samples to 100 mL of basic salt liquid medium containing 50 mg / L mesotrione, and shake culture at 30°C and 160 rpm for about 10 d. Then transfer it to the basic salt liquid medium containing 100 mg / L mesotrione at an inoculation amount of 10 v / v%, and shake culture at 30°C and 160 rpm for about 10 d again. Continuously transfer and culture like this for 4 times, 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 10-6 The dilution solution was spread on an LB medium plate and cultured at 30 °C for 3 - 4 d. Different morphological single colonies on the plate were picked and further streaked and purified on the LB medium plate. The obtained pure strains were inoculated into a basal salt liquid medium supplemented with 100 mg / L mesotrione and cultured at 30 °C and 150 rpm for 5 d. UV scanning was used to detect whether each pure strain had the function of degrading mesotrione.

[0031] A mesotrione-degrading bacterium was isolated and screened through enrichment domestication, named EMD-1. After the strain EMD-1 grew on the LB medium plate at 30 °C for 4 d, as Figure 1 shown, the colonies were white, moist, smooth, with round edges, and about 3 mm in diameter; the bacterial cells were rod-shaped (0.6–0.8 × 2.0–3.5 µm); Gram-negative staining, non-spore-forming. Using the genomic DNA of strain EMD-1 as a template, universal primers for the bacterial 16S rRNA gene sequence (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 1450 bp (its nucleotide sequence is shown in SEQ ID NO.3). 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 strain EMD-1 had the closest genetic relationship with the strains of the genus Sphingobacterium ), and the highest homology with the type strain Sphingobacterium psychroaquaticum MOL-1 T was 98.9%. On the phylogenetic tree constructed based on the 16S rRNA gene sequence, strain EMD-1 was also clustered in the Sphingobacterium genus ( Figure 1 ), so strain EMD-1 was identified as the Sphingobacterium genus.

[0032] 1.2 Degradation of mesotrione by strain EMD-1

[0033] Study on degradation characteristics: EMD-1 was inoculated into an LB liquid medium and cultured at 30 °C and 150 rpm until the mid-logarithmic phase. Then it was centrifuged at 6000 g for 10 min to collect the bacterial cells. The bacterial cells were washed twice with fresh and sterile basal salt liquid medium and resuspended in the basal salt liquid medium. The cell concentration was adjusted to about 1.0×10 9cfu / mL, inoculated at an inoculation amount of 1% (v / v) into 20 mL of a basal salt liquid medium containing 100 mg / L of mesotrione, and cultured at 30 °C and 150 rpm for 72 h. Samples were taken and the degradation of mesotrione by the strain was determined by ultraviolet scanning.

[0034] The ultraviolet scanning results showed that mesotrione had a characteristic absorption peak at 270 nm. As the culture time extended, the characteristic absorption peak of mesotrione in the samples gradually decreased, indicating that the strain EMD-1 was able to degrade mesotrione ( Figure 3 ). The sampling and determination results after 72 h of culture showed that the strain EMD-1 was able to degrade 72.9% of mesotrione.

[0035] Example 2 Mesotrione nitroreductase gene xhnr Cloning

[0036] 2.1 Genome sequencing of strain EMD-1 and search for nitroreductase

[0037] The initial step of microbial degradation of mesotrione is nitroreduction. In this study, the suspected mesotrione nitroreductase gene was determined by analyzing the genome of strain EMD-1. The genome of strain EMD-1 was sequenced using high-throughput sequencing technology (HiSeq 4000 sequencer system, Illumina), and the genome was gene-annotated using RAST (Rapid Annotation with Subsystem Technology). The genome size of this bacterium was 6.5 Mb, encoding 5210 ORFs. Through the possible functional analysis of these ORFs, a suspected nitroreductase gene was found, named xhnr , whose nucleotide sequence is shown in SEQ ID NO.1, with a size of 675 bp, encoding a protein (Xhnr) of 224 amino acids, as shown in SEQ ID NO.2. The alignment results in the swissprot database of NCBI (https: / / blast.ncbi.nlm.nih.gov / ) showed that the one with the highest similarity to Xhnr was the nitroreductase Bacillus subtilis oxygen-insensitive NADPH nitroreductase YfkO, with a homology of only 53.1%.

[0038] 2.2 Construction of the mesotrione nitroreductase gene xhnr Recombinant expression vector

[0039] xhnr The gene recombinant expression vector pET-28a -xhnr, synthesized by Beijing Tsingke Biotechnology Co., Ltd. according to the requirements of the following table. Among them, xhnr The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0040] Target gene Target vector (original vector) pET-28a(+) Restriction enzyme cleavage site at the 5' end of the target vector NdeI Restriction enzyme cleavage site at the 3' end of the target vector XhoI

[0041] The above-synthesized xhnr gene recombinant expression vector pET-28a -xhnr 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-28a - xhnr ).

[0042] Example 3 Functional verification of nicosulfuron nitroreductase Xhnr

[0043] 3.1 Expression and purification of Xhnr

[0044] BL21(pet-28a- xhnr ) was cultured in 100 mL of LB liquid medium with shaking at 37 °C and 150 rpm 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 cells were induced to culture 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, and sonicated (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 - 10 min. After centrifugation at 12000 rpm for 30 min, the supernatant was collected, and Xhnr was purified using a Co 2+ ion affinity chromatography column. The purified Xhnr protein was subjected to SDS-PAGE electrophoresis (eluting proteins with 100 mM imidazole and 200 mM imidazole) to detect its purity. The results of SDS-PAGE electrophoresis are shown in Figure 4 , indicating that the purified protein had a clear band at 25 - 35 kDa, which was consistent with the theoretical size of Xhnr (the theoretical size of Xhnr plus 20 recombinant carrier amino acids and 6 histidine tags was 26.8 kDa), and no other obvious impurity bands were seen in the lane, indicating that pure Xhnr protein was obtained.

[0045] 3.2 Enzyme activity detection of Xhnr

[0046] Enzyme activity reaction system (3 mL): 20 mM Tris-HCl buffer (pH 7.5), 100 μM mesotrione, 1 mM NADH, 25 mM Mg 2+ , 100 μL of reaction enzyme amount (containing 1 μg of purified Xhnr), react at 30 °C for 1 h. Each reaction starts timing when the enzyme is added. After 1 h, place it in boiling water for 1 min to terminate the reaction. After the reaction solution is freeze-dried (-30 °C, 0.2 mbar, 24 h), add 200 μl of methanol to dissolve the freeze-dried product. The reduction amount of the substrate (mesotrione) is detected by HPLC. 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 Xhnr are as Figure 5 shown. The results show that mesotrione has a characteristic absorption peak at 10 min, while 2-amino-4-methylsulfonylbenzoic acid (AMBA) has a characteristic absorption peak at 4 min; after 1 h of the enzyme reaction, the absorption peak of mesotrione decreased significantly, and at the same time, the characteristic absorption peak of AMBA appeared, indicating that Xhnr can reduce mesotrione to AMBA, has the activity of mesotrione nitroreductase, and the specific enzyme activity for mesotrione is 1.02 U / μg protein.

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

[0048] 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), so it is used in this experiment to detect the inhibitory activity of mesotrione and its degradation products on OsHPPD.

[0049] In a 100 mL enzyme reaction system, mesotrione was added to a final concentration of 16 μM, and then 60 μg of purified Xhnr was added. The reaction was carried out 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 AMBA of Xhnr degrading mesotrione.

[0050] 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, and then mesotrione with a final concentration of 16 μM or the product of Xhnr degrading mesotrione with a final concentration of 16 μM was added. After culturing with shaking at 30 °C and 150 rpm for 36 h, the color change under different treatments was observed. At the same time, a negative control with BL21 (pET-29a-OsHPPD) added and no substrate tyrosine added, and a positive control with BL21 (pET-29a-OsHPPD) and substrate tyrosine added were set up. The results are as Figure 6 shown. The positive control without mesotrione added showed a red color, indicating that OsHPPD was normally expressed and had activity ( Figure 6 B); while the treatment with 16 μM mesotrione added was light yellow like the negative control ( Figure 6 A), indicating that the activity of OsHPPD had been completely inhibited ( Figure 6 C); while the treatment with the product of Xhnr degrading mesotrione added showed a red color ( Figure 6 D), and there was no significant difference from the positive control without mesotrione added ( Figure 6 B), indicating that the product of Xhnr degrading mesotrione had no inhibitory effect on OsHPPD. Therefore, Xhnr can completely convert mesotrione into a product that has no inhibitory effect on OsHPPD, achieving the degradation and detoxification of mesotrione.

[0051] 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 them; 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 Xhnr, characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding mesotrione nitroreductase Xhnr according to claim 1 xhnr.

3. The coding gene according to claim 2 xhnr, The feature of the invention is that its nucleotide sequence is shown as SEQ ID NO.

1.

4. Containing the coding gene according to claim 2 or 3 xh recombinant expression vector.

5. The recombinant expression vector according to claim 4, characterized in that: The coding gene according to claim 2 or 3 xh Obtained by recombination into the original vector, wherein the original vector includes pET-28a (+).

6. Containing the coding gene according to claim 2 or 3 xh of genetically engineered bacteria.

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

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