Mesotrione nitroreductase Xhnr as well as coding gene and application thereof

Abstract

The invention discloses mesotrione nitroreductase Xhnr as well as a coding gene and application thereof. The amino acid sequence of the mesotrione nitroreductase Xhnr is shown as SEQ ID NO.2, and the nucleotide sequence of the coding gene xhnr of the mesotrione nitroreductase Xhnr is shown as SEQ ID NO.1. The invention further discloses a preparation method of the mesotrione nitroreductase Xhnr. The highest homology of the mesotrione nitroreductase Xhnr and reported nitroreductase is only 53.1%, so that the mesotrione nitroreductase Xhnr is a new nitroreductase. The mesotrione nitroreductase Xhnr can be used for degrading mesotrione into a product AMBA, so that the herbicidal activity is lost. The mesotrione nitroreductase Xhnr and the coding gene of the mesotrione nitroreductase Xhnr have potential application prospects 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 Xhnr, a coding gene thereof and uses thereof. 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 then 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 Xhnr, a coding gene and a use thereof, so as to solve the deficiencies of the prior art.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a mesotrione nitroreductase Xhnr, whose amino acid sequence is shown in SEQ ID NO.2.

[0007] The second aspect of the present invention provides a gene encoding the mesotrione nitroreductase Xhnr xhnr.

[0008] Furthermore, its nucleotide sequence is shown in SEQ ID NO.1.

[0009] The third aspect of the present invention provides the above-mentioned coding gene xh recombinant expression vector.

[0010] Furthermore, the above coding gene xh Obtained by recombination into the original vector, wherein the original vector includes pET-28a (+).

[0011] The fourth aspect of the present invention provides the above-mentioned coding gene xh of genetically engineered bacteria.

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

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

[0014] The sixth aspect of the present invention provides the above-mentioned coding gene xh Application in biodegradation, detoxification and bioremediation of mesotrione.

[0015] Beneficial effects of the present invention: The present invention provides a mesotrione nitroreductase Xhnr, the amino acid sequence of which is shown in SEQ ID NO.2, and the encoding gene xh The nucleotide sequence is shown in SEQ ID NO.1. The highest homology of mesotrione nitroreductase Xhnr with the reported nitroreductase is only 53.1%, so it is a new nitroreductase. Mesotrione nitroreductase Xhnr can degrade mesotrione into product AMBA and lose herbicidal activity. The mesotrione nitroreductase Xhnr of the present invention and its encoding gene have potential application value in mesotrione biodegradation, detoxification and bioremediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a colony photo of strain EMD-1.

[0017] Figure 2 Phylogenetic tree of strain EMD-1 based on 16S rRNA gene.

[0018] Figure 3 This is the UV scanning detection result of strain EMD-1 degrading mesotrione. The blue arrow indicates the characteristic absorption peak of mesotrione (270nm).

[0019] Figure 4 The results of SDS-PAGE detection of exogenously expressed Xhnr are shown in Figure 1. Lane 1, protein Maker; Lane 2, Xhnr protein eluted with 100 mM imidazole; Lane 3, Xhnr protein eluted with 200 mM imidazole.

[0020] Figure 5 HPLC detection of mesotrione degradation by Xhnr. A, mesotrione standard; B, AMBA standard; C, reaction time 0 h; D, reaction time 1 h.

[0021] Figure 6The inhibitory effect of the product of mesotrione degraded by Xhnr-6 on rice HPPD (OsHPPD). A, negative control, adding BL21 (pET-29a-OsHPPD), without adding substrate tyrosine, no HPPD activity; B, positive control, adding BL21 (pET-29a-OsHPPD) and substrate tyrosine, there is HPPD activity; C, adding BL21 (pET-29a-OsHPPD), substrate tyrosine and mesotrione, HPPD activity is completely inhibited by mesotrione; D, adding BL21 (pET-29a-OsHPPD), substrate tyrosine and the product of mesotrione degraded by Xhnr, there is HPPD activity, which is not significantly different from the positive control, indicating that the product does not inhibit HPPD activity. 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 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.

[0024] 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.

[0025] Example 1 Isolation of mesotrione-degrading strains and search for their nitroreductase genes 1.1 Enrichment, isolation and classification of mesotrione-degrading strains Sludge samples were collected from the sewer of a pesticide factory producing mesotrione. 5 g of sludge sample was added to 100 mL of basal salt liquid culture medium containing 50 mg / L mesotrione. After shaking culture at 30°C and 160 rpm for about 10 days, it was transferred to a basal salt liquid culture medium containing 100 mg / L mesotrione at an inoculum of 10 v / v%, and then shaken and cultured at 30°C and 160 rpm for about 10 days. This transfer culture was repeated 4 times, and the degradation of mesotrione was detected by ultraviolet scanning (mesotrione has a characteristic absorption peak at 270 nm 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 LB medium plates and cultured at 30°C for 3-4 days. Single colonies of different morphologies on the plates were picked and further purified by streaking on LB medium plates. The obtained pure strains were inoculated into basal salt liquid medium with 100 mg / L mesotrione, cultured at 30°C and 150 rpm for 5 days, and UV scanning was used to detect whether each pure strain had the ability to degrade mesotrione.

[0026] A mesotrione-degrading bacterium was obtained by enrichment, acclimation, isolation and screening, and named EMD-1. After strain EMD-1 was grown on LB medium plates at 30°C for 4 days, Figure 1 As shown, the colonies are white, moist, smooth, with rounded edges and a diameter of about 3 mm; the bacterial cells are rod-shaped (0.6–0.8×2.0–3.5 µm); Gram staining is negative and does not produce spores. Using the genomic DNA of strain EMD-1 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 1450 bp 16S rRNA gene sequence (its nucleotide sequence is shown in SEQ ID NO.3). Blast was performed in 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 was similar to Sphaerocystis spp. ( Sphingobacterium ) strains are most closely related to the type strain Sphingobacterium psychroaquaticum MOL-1 T The highest homology was 98.9%. In the phylogenetic tree constructed based on the 16S rRNA gene sequence, strain EMD-1 was also clustered in Sphingobacterium Genus ( Figure 1 ), so strain EMD-1 was identified as Sphingobacterium Belong to.

[0027] 1.2 Degradation of mesotrione by strain EMD-1 Study on degradation characteristics: EMD-1 was inoculated into LB 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.

[0028] The UV scanning results showed that mesotrione had a characteristic absorption peak at 270 nm. With the extension of the culture time, the characteristic absorption peak of mesotrione in the sample gradually decreased, indicating that strain EMD-1 was able to degrade mesotrione ( Figure 3 ). The results of sampling after 72 h of cultivation showed that strain EMD-1 could degrade 72.9% of mesotrione.

[0029] Example 2 Mesotrione Nitroreductase Gene xh clone 2.1 Genome sequencing of strain EMD-1 and search for nitroreductase 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 EMD-1. The genome of strain EMD-1 was sequenced using high-throughput sequencing technology (HiSeq 4000sequencer system, Illumina), and the genome was annotated using RAST (Rapid Annotationwith Subsystem Technology). The bacterial genome size is 6.5 Mb, encoding 5210 ORFs. Through the possible functional analysis of these ORFs, a suspected nitroreductase gene was found, named xh The 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 comparison results of the swissprot database of NCBI (https: / / blast.ncbi.nlm.nih.gov / ) showed that the most similar protein to Xhnr was nitroreductase Bacillus subtilis oxygen-insensitive NADPH nitroreductase YfkO, the homology is only 53.1%.

[0030] 2.2 Construction of mesotrione nitroreductase gene xh Recombinant expression vector xh Gene recombinant expression vector pET-28a -xhnr , synthesized by Beijing Qingke Biotechnology Co., Ltd. according to the requirements of the following table. Among them, xh The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0031] Target gene Target vector (original vector) pET-28a (+) 5' restriction site of target vector N i Target vector 3' end restriction site XOt The above synthesized xh Gene recombinant expression vector pET-28a -xhnr 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-28a - xh ).

[0032] Example 3 Functional verification of mesotrione nitroreductase Xhnr 3.1 Expression and purification of Xhnr BL21 (pet-28a- xh ) in 100 mL LB liquid medium and culture at 37°C and 150 rpm in a shaking incubator 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 hours. 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, pH 7.4), resuspended with 10 mL of PBS buffer, ultrasonically disrupted (Auto Science, UH-650Bultrasonic processor, 30% intensity) for 5-10 min, centrifuged at 12000 rpm for 30 min, and the supernatant was collected and precipitated with Co 2+ Xhnr was purified by ion affinity chromatography column, and the purified Xhnr protein was subjected to SDS-PAGE electrophoresis (100 mM imidazole, 200 mM imidazole elution protein) to detect its purity. The results of SDS-PAGE electrophoresis are shown in Figure 4, indicating that the purified protein has clear bands at 25-35 kDa, which is consistent with the theoretical size of Xhnr (the theoretical size of Xhnr plus 20 vector recombinant amino acids and 6 histidine tags is 26.8 kDa). No other obvious miscellaneous bands were seen in the lane, indicating that pure Xhnr protein was obtained.

[0033] 3.2 Xhnr 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 1 μg of purified Xhnr), and the reaction was carried out at 30℃ for 1 h. Each reaction started with the addition of the enzyme. After 1 h, the reaction was terminated by placing it in boiling water for 1 min. The reaction solution was freeze-dried (-30℃, 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 at pH 7.5 and 30℃. The HPLC analysis results of mesotrione degradation by Xhnr are shown in Figure 2. Figure 5 As shown, the results showed that mesotrione had a characteristic absorption peak at 10 min, while 2-amino-4-methylsulfonylbenzoic acid (AMBA) had a characteristic absorption peak at 4 min; after 1 h of 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 and has mesotrione nitroreductase activity, and the specific enzyme activity for mesotrione is 1.02 U / μg protein.

[0034] Example 4 Effect of Xhnr 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.

[0035] 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 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 AMBA of mesotrione degradation by Xhnr.

[0036] The recombinant expression strain BL21 (pET-29a-OsHPPD) was cultured to the logarithmic growth phase (OD 600 About 1.0), 20 μL was inoculated in a 96-well plate with 200 μL of 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 16 μM or the product of mesotrione degradation by Xhnr with a final concentration of 16 μM was added. After shaking culture at 30°C and 150 rpm for 36 h, the color changes under different treatments were observed. At the same time, a negative control with the addition of BL21 (pET-29a-OsHPPD) and no substrate tyrosine, and a positive control with the addition of BL21 (pET-29a-OsHPPD) and substrate tyrosine were set. The results are shown in Figure 6 As shown, the positive control without mesotrione was red, indicating that OsHPPD was normally expressed and active ( Figure 6 B); the treatment with 16 μM mesotrione was compared with the negative control ( Figure 6 A) is pale yellow, indicating that OsHPPD activity has been completely inhibited ( Figure 6 C); while the product of mesotrione degradation by Xhnr appeared red ( Figure 6 D), and the positive control without mesotrione ( Figure 6 B) There is no significant difference, indicating that the products of mesotrione degradation by Xhnr have no inhibitory effect on OsHPPD. Therefore, Xhnr can completely convert mesotrione into products that have no inhibitory effect on OsHPPD, achieving the degradation and detoxification of mesotrione.

[0037] 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 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).

8. Use of the mesotrione nitroreductase Xhnr according to claim 1 in mesotrione biodegradation, detoxification and bioremediation.

9. The coding gene according to claim 2 or 3 xh Application in biodegradation, detoxification and bioremediation of mesotrione.

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