CN109897860B - Wheat UDP-glucosyltransferase TaUGT6 and application thereof - Google Patents

Abstract

The invention discloses a wheat UDP-glucosyltransferase TaUGT6 and application thereof in degrading DON toxin and improving the tolerance of plants to the DON toxin; the nucleotide sequence of the enzyme is shown as SEQ ID NO.1, and the coded protein sequence is shown as SEQ ID NO. 2; in-vitro enzyme activity experiments prove that the protein can convert DON into D3G; the DON content can be obviously reduced by spraying the protein on high-pollution wheat grains and whole wheat flour; the gene is over-expressed in arabidopsis thaliana by utilizing an agrobacterium-mediated genetic transformation method, the tolerance capability of transgenic arabidopsis thaliana to DON is improved, and the new application of the gene in DON toxin degradation, DON toxin accumulation resistance and gibberellic disease resistance of plants is proved.

Description

Wheat UDP-glucosyltransferase TaUGT6 and application thereof

Technical Field

The invention relates to application of wheat UDP-glucosyltransferase gene, in particular to application of the gene in DON toxin degradation and improvement of plant DON tolerance, belonging to the technical field of plant genetic engineering.

Background

Wheat scab (FHB) is a major fungal disease that is widespread worldwide and caused by Fusarium graminearum as a major pathogenic bacterium, and is also one of the major diseases that endanger the development of the wheat industry in China. In recent years, due to the change of climate warming and farming systems, the incidence range of wheat scab in China is on a trend of continuously expanding, the current common area is expanded to the southern wheat area of Huang-Huai, and the disease incidence in the northern, southwest and northwest wheat areas of Huang-Huai is obviously increased. Scab causes a decrease in wheat yield, typically resulting in 10% -15% yield loss each year, and even failure in pandemics. Gibberellic disease not only causes the wheat yield to be seriously reduced, but also the quality and the food safety of wheat are influenced by mycotoxins such as Deoxynivalenol (DON) secreted by the gibberellic disease. DON has wide toxic effect on human and various animals, and can cause apoptosis of various cells, thereby generating toxic action on various systems of the organism, such as neurotoxicity, reproductive toxicity, immunotoxicity, teratogenicity and embryotoxicity, and possibly a cancer inducer. Therefore, the method has important significance for grain safety and food safety by actively and effectively preventing and controlling wheat scab and reducing the accumulation of DON toxin in grains.

The DON toxin is not only a great health hazard to humans and animals, but also inhibits the germination of wheat seeds, the growth of coleoptile, and root mitosis and differentiation. Research shows that the DON toxin is a pathogenic factor of fusarium graminearum, can promote the expansion of fusarium graminearum in wheat tissues, aggravate and aggravate the occurrence of gibberellic disease, reduce the loss or weakening of the DON toxicity, effectively prevent the expansion of pathogenic bacteria in plant tissues and improve the resistance to the gibberellic disease. Therefore, reducing DON accumulation is not only very important for food safety, but also a way to improve wheat scab resistance.

Plant UDP-glucosyltransferases (UGTs) are related detoxified proteins, and can reduce toxicity by glycosylation of toxins such as DON and the like, thereby improving the resistance of plants to gibberellic disease. Plant UDP-glucosyltransferase belongs to family 1 in the large family of glycosyltransferases, has a PSPG box (plant secondary product glycosylation transferase box) at the C-terminal, is a conserved sequence consisting of 44 amino acids, and generally takes uridine diphosphate glucose as glycosyl donor to participate in glycosylation reaction. The UGT members in plants are numerous, and more than 100 members are found in plants such as arabidopsis, maize and rice, but most of the functions of these UDP-glucosyltransferases are unknown.

The wheat UDP-glucosyltransferase TaUGT6 is a member of wheat glycosyltransferase family 1, the gene sequence is disclosed with wheat genome sequence, but the application of the gene, especially the application in DON toxin degradation and improvement of plant DON tolerance is not reported.

Disclosure of Invention

Aiming at the problems, the invention provides application of wheat UDP-glucosyltransferase TaUGT6 in DON toxin degradation and improvement of plant DON tolerance.

The invention is realized by the following technical scheme:

the invention firstly discloses a wheat UDP-glucosyltransferase TaUGT6, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 1.

Secondly, the invention provides a protein coded by the gene of the wheat UDP-glucosyltransferase TaUGT6, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

Thirdly, the invention provides the application of the wheat UDP-glucosyltransferase TaUGT6 in DON toxin degradation, which comprises the following steps: 1) the TaUGT6 sequence provided by the invention is introduced into a commercial expression plasmid pGEX-4T-1, the protein is expressed and purified by using escherichia coli exogenous sources, and DON-containing substances are used as substrates, so that DON toxin can be converted into deoxynivalenol-3-glucoside (D3G) and the like. 2) The TaUGT6 protein solution provided by the invention is sprayed in wheat polluted by DON toxin (the spraying amount is about 20 mu g/g) to reduce the content of the DON toxin; the wheat comprises wheat grains or whole wheat flour.

The invention also provides an application of the wheat UDP-glucosyltransferase TaUGT6 in improving DON tolerance of plants, which comprises the following steps: introducing a gene encoding UDP-glucosyltransferase TaUGT6 (the nucleotide sequence of which is shown in SEQ ID NO. 1) into a plant overexpression vector pHB, performing plant transgenic operation (Zhang X, Henriques R, Lin S, et al, Agrobacterium-mediated Transformation of Arabidopsis thaliana Using the floral dip method [ J ]. Nature Protocols,2006,1(2): 641.; Eva Medium Medveck, Harwood W A. wheat L.) Transformation Using genetic library antibodies [ J. Methods in Molecular Biology,2015,1223:199.) to obtain transgenic plants to improve the tolerance of plants to DON and improve gibberellic disease resistance; such plants include, but are not limited to, Arabidopsis, wheat, maize, to obtain transgenic material containing the gene for production.

The invention proves that UDP-glucosyltransferase TaUGT6 can reduce DON toxicity and improve the tolerance of plants to DON toxin by glycosylation of DON for the first time. And the content of DON in the polluted grains or whole wheat flour is reduced by spraying TaUGT6 protein for the first time, thereby providing a simple and feasible way for utilizing the high-pollution grains. In addition, the cloned gene is derived from wheat, so that the food safety is not influenced by the over-expression, and the gene can be widely used in disease resistance breeding of different plants.

Drawings

FIG. 1: the TaUGT6 sequence was amplified by RT-PCR technique and detected by agarose electrophoresis.

M is molecular weight standard DL 2000; lane 1 is a TaUGT6 fragment.

FIG. 2: and (3) carrying out double enzyme digestion identification on the recombinant plasmid pGEX-TaUGT 6.

M is a protein molecular weight marker; lane 1 is the plasmid pGEX-TaUGT6 not digested; lane 1 is pGEX-TaUGT6 plasmid double digested with BamHI and EcoR I.

FIG. 3: the purified fusion protein GST-TaUGT6 was detected by SDS-PAGE.

M is a protein molecular weight marker; lane 1 is a purified GST-TaUGT6 fusion protein.

FIG. 4: the catalytic product of TaUGT6 was identified by mass spectrometry (LC-MS).

A is a substrate in enzyme activity reaction, and the peak value accords with the characteristics of DON; b is a product of DON catalyzed by TaUGT6, and the peak value accords with the characteristics of D3G.

FIG. 5: the TaUGT6 protein reduces the content of DON in high-pollution grains or whole wheat flour.

A is grains polluted by gibberellic disease; b, measuring the DON content of grains sprayed with TaUGT6 protein for 4 hours; c is the whole wheat flour polluted by the gibberellic disease; d is DON content determination of whole wheat flour after being sprayed with TaUGT6 protein for 4 hours.

FIG. 6: and detecting the TaUGT6 gene in the transgenic plant.

In A, M is molecular weight standard DL2000, lanes 1-7 are PCR amplification products in transgenic plant leaves, lane 8 is a PCR product of a positive plasmid containing TaUGT6 gene, and lane 9 is a PCR product of wild type Arabidopsis thaliana leaf control; b is the quantitative detection of the gene qRT-PCR of TaUGT6 in the transgenic strains 1-7.

FIG. 7: tolerance of transgenic plants to DON.

A is water treatment; b is 100 mu M DON treatment; and C is root length statistics. The transgenic plant line grows faster than the WT plant root system under the DON treatment. WT was wild type, 2 transgenic line 2.

Detailed Description

Unless otherwise specified, the examples are all conventional methods and experimental reagents used in the art. The related wheat varieties Sumai No.3 and Arabidopsis are from the wheat research institute of food crop research institute of the academy of agricultural sciences of Jiangsu province.

Example 1 cloning of the wheat UDP-glucosyltransferase Gene TaUGT6

Ear RNA of wheat variety Sumai No.3 at heading stage was extracted using SV Total RNA Isolation System kit (Promega Co., Ltd., cat # Z3100), and cDNA was obtained using reverse transcription kit (TAKARA Co., cat # 6210A).

The cDNA sequence of the TaUGT6 gene was obtained by the public website http:// plants. ensemble. org/. A primer for amplifying the TaUGT6 gene is designed and synthesized, wherein the forward primer is 5'-CGGGATCCATGGCTGTCCACGACGAGCC-3' (the nucleotide sequence of the primer is shown as SEQ ID NO. 3), and the reverse primer is 5'-CGGAATTCGCTGGCCTGGATGTCTTGGC-3' (the nucleotide sequence of the primer is shown as SEQ ID NO. 4). Using this primer set and high fidelity Mix (Novozan, cat # P511), PCR amplification was performed using cDNA as a template.

The PCR reaction system is 2 in the design

Master Mix 25. mu.l, cDNA template 2. mu.l, SEQ ID NO.3 andprimers SEQ ID NO.4 were 1.5. mu.l each, and sterilized water was 20. mu.l. The reaction program is set to 95 ℃ for 1 min; at 95 ℃ for 10s, at 58 ℃ for 15s, at 72 ℃ for 50s, for 36 cycles; 5min at 72 ℃.

The PCR product was electrophoresed in 1% agarose gel, and the result of electrophoresis is shown in FIG. 1. The target fragment was recovered by gel (Novozan, cat # DC301), ligated into pEASY cloning vector (Takara, cat # CB111), and subjected to sequencing verification by Biotechnology, Shanghai, GmbH, and the plasmid with the correct sequencing was named T-TaUGT 6.

Example 2 application of wheat UDP-glucosyltransferase TaUGT6 in degradation of DON toxin

Construction of prokaryotic expression vector of TaUGT6

The T-TaUGT6 plasmid obtained in example 1 was digested with BamH I (NEB, cat R3136) and EcoR I (NEB, cat R3101) in two ways, and the desired gene fragment was recovered; meanwhile, a universal prokaryotic expression vector pGEX-4T-1 is disclosed by double enzyme digestion of BamH I and EcoR I, and a vector fragment is recovered. The target gene fragment is connected into the prokaryotic expression vector to obtain a prokaryotic expression vector pGEX-TaUGT6 of TaUGT6, and the pGEX-TaUGT6 vector is verified by double enzyme digestion of BamH I and EcoR I, as shown in figure 2.

2. Transformation of Escherichia coli, induction of protein expression and purification of protein

The plasmid pGEX-TaUGT6 was transformed into E.coli BL21(DE3) (Takara Shuzo Co., Ltd.; cat. No.: CD 601). The bacterial liquid of BL21 containing the positive clone was mixed with a mixture of 1: 100, adding into LB liquid culture medium containing Amp, and culturing for 2h at 37 deg.C with shaking table 200rpm until bacterial liquid OD600 is 0.6-0.8. IPTG was added to a final concentration of 1mM, and the mixture was induced overnight in a shaker at 25 ℃ to collect the cells for further use.

The bacterial suspension collected under overnight induction was added to 10mL of phosphate buffer (PBS, 20mM K2PO4, 0.15M NaCl, pH7.9) to suspend the cells, 100. mu.l of 1M phenylmethylsulfonyl fluoride (PMSF) was added thereto and mixed well, and then sonicated for 20 minutes (200W, 1s working, 2s batch), and the supernatant was centrifuged. The supernatant was filtered through a 0.45 μm syringe filter (Producer Co., Ltd.; cat # F513143), protein-purified using a ProteinIso GST Resin protein purification system (Takara Shuzo Co., Ltd.; cat # DP201) according to the protocol, subjected to column packing, equilibration, loading, washing, and elution, and then the eluate was collected in a 1.5ml centrifuge tube, and 30 μ l of a sample was subjected to SDS-PAGE to detect the concentration and purity of the recombinant protein, and the obtained recombinant protein was collected for use as shown in FIG. 3.

Enzymatic reaction of DON

Mu.g of recombinant protein, 50mM Tris-HCl pH 7.0, 5mM UDP-glucose, 14mM 2-mercaptoethanol and 1mM DON were included in 200. mu.l of the enzymatic reaction mixture, which was incubated at 30 ℃ for 3h, quenched with 20. mu.l 240mg/ml trichloroacetic acid, snap frozen in liquid nitrogen and stored at-20 ℃ in a refrigerator.

LC-MS \ MS detection of DON and production of D3G

Chromatographic conditions are as follows: agilent extended-C18 chromatography column (150 mm. times.3.0 mm, 3.5 μm); the column temperature was 40 ℃ and the sample temperature was 5 ℃. The injection volume was 5. mu.l. Mobile phase a was methanol and mobile phase B was 5mM ammonium acetate. The flow rate was 0.3 ml/min. Mobile phase elution gradient 0min 20% a, 1min 20% a, 5.5min 90% a, 6.5min 90% a, 7.0min 20% a.

Mass spectrum conditions: reaction monitoring mode (MRM) detection was selected. The temperature of a curve desolventizing tube (CDL) is 250 ℃, the atomizing gas and the drying gas are both nitrogen, and the flow rates are 3.0L/min and 15L/min respectively. The collision gas is high-purity argon, and the collision induced dissociation pressure is 230 kPa.

The enzymatic reaction solution was subjected to mass spectrometric detection under the above conditions, and subjected to peak pattern alignment analysis to find DON and D3G, as shown in FIG. 4. It follows that TaUGT6 can glycosylate the DON toxin to deoxynivalenol-3-glucoside (D3G), reducing DON toxicity.

Example 3 application of wheat UDP-glucosyltransferase TaUGT6 in degradation of toxin content in DON-contaminated grains or whole wheat flour

Preparation of a wheat UDP-glucosyltransferase TaUGT6 enzyme solution (2 ml): example 2. mu.g of the recombinant protein obtained in step 2, final content of 5mM UDP-glucose, final content of 14mM 2-mercaptoethanol, Tris-HCl (pH 7.0) to 2 ml.

In the enzyme solution, 2-mercaptoethanol can protect the activity of the enzyme; Tris-HCl can maintain a relatively stable enzymatic reaction environment.

Wheat UDP-glucosyltransferase TaUGT6 enzyme solution (protein concentration 10 μ g/ml) is sprayed on wheat grains (figure 5A) or whole wheat flour (figure 5C) polluted by gibberellic disease, the wheat grains or the whole wheat flour are placed for 4h at 30 ℃, a vomitoxin ELISA detection kit (Beijing Hua' an Mac Corp., product number: HEM0896/HEM0848) is used for quickly detecting the DON content according to the product instruction, as shown in figure 5, after 4h of reaction, the toxin content in the grains is reduced by 620 μ g/kg (figure 5B), the toxin content in the whole wheat flour is reduced by 660 μ g/kg (figure 5D), and water is used as a control group.

In the embodiment, 4ml of enzyme liquid is sprayed on a sample of 20g of wheat grains or whole wheat flour polluted by gibberellic disease, and the spraying amount of UGT protein is about 20 mu g/g of sample.

Example 4 application of wheat UDP-glucosyltransferase TaUGT6 in DON tolerance in plants

Plant overexpression vector construction of TaUGT6

Amplifying a target fragment by using a forward primer (the nucleotide sequence of which is shown as SEQ ID NO. 3) and a reverse primer 5'-GACTAGTGCTGGCCTGGATGTCTTGGC-3' (the nucleotide sequence of which is shown as SEQ ID NO. 5), carrying out double digestion by using BamH I and Spe I (NEB, cat # R3133S), and recovering to obtain a target gene fragment; and simultaneously carrying out double enzyme digestion on the plant expression vector pHB by using BamH I and Spe I, and recovering the vector fragment. And connecting the target gene segment into the expression vector to obtain a plant over-expression vector TaUGT6-OE of TaUGT6 driven by the maize ubiquitin promoter.

2. Agrobacterium-mediated genetic transformation

The plant overexpression vector TaUGT6-OE was transferred into Agrobacterium GV3101 (Shanghai Diego, cat. No.: AE1001) and verified by PCR. Agrobacterium GV3101 containing a plant expression vector was used to impregnate Arabidopsis flower buds using the general Arabidopsis transformation method "floral dip" (Zhang X, Henriques R, Lin S, et al, Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method [ J ]. Nature Protocols,2006,1(2): 641.). After the grown siliques are mature, collecting T1 generation seeds and screening on a screening culture medium, respectively harvesting T2 generation seeds, then carrying out next round of screening, transplanting green seedlings on the culture dish, and obtaining T3 generation seeds from a single plant, namely the homozygous transgenic plant.

3. Identification of overexpressed transgenic plant molecules

For the above transgenic plants, DNA was extracted from the leaves and PCR-detected to obtain 7 independent transgenic lines, which were designated 1-7, respectively, as shown in FIG. 6A. Respectively extracting RNA of the transgenic plant and the wild plant, carrying out qRT-PCR amplification by taking a forward primer with a nucleotide sequence shown as SEQ ID NO.6 and a reverse primer with a nucleotide sequence shown as SEQ ID NO.7 as primers after reverse transcription, and analyzing the gene expression difference of the over-expressed plant and the wild plant.

The expression level of TaUGT6 in the over-expression plants is obviously higher than that of the wild plants, as shown in FIG. 6B. The subsequent experiments were carried out using line 2 with a high expression level.

Validation of the role of TaUGT6 in DON tolerance

WT and transgenic line 2 were sown on 1/2MS medium, after 5 days, plants of similar size and growth phase were taken and transferred to a new 1/2MS medium, the surface of which was coated with a layer of 100. mu.M DON, and after 7 days of growth, as shown in FIG. 7, the root length of Arabidopsis plants over-expressed by TaUGT6 was significantly longer than that of wild type roots, indicating that TaUGT6 enhanced the tolerance of plants to DON toxins.

Sequence listing

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Claims (5)

1. Application of wheat UDP-glucosyltransferase TaUGT6 in degradation of DON toxin; the amino acid sequence of the wheat UDP-glucosyltransferase TaUGT6 is shown in SEQ ID NO. 2.

2. The use of claim 1, wherein said use is of wheat UDP-glucosyltransferase TaUGT6 sprayed onto wheat contaminated with DON toxin to reduce the level of DON toxin.

3. The use of claim 2, wherein the DON toxin-contaminated wheat comprises DON toxin-contaminated wheat grain or DON toxin-contaminated whole wheat flour.

4. Application of wheat UDP-glucosyltransferase TaUGT6 in improving tolerance of DON toxin in plants; the amino acid sequence of the wheat UDP-glucosyltransferase TaUGT6 is shown in SEQ ID NO. 2;

the plants are Arabidopsis thaliana, wheat and barley.

5. Application of wheat UDP-glucosyltransferase TaUGT6 in converting DON toxin into deoxynivalenol-3-glucoside; the amino acid sequence of the wheat UDP-glucosyltransferase TaUGT6 is shown in SEQ ID NO. 2.

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