CN116284286A - Wheat stripe rust fungus sugar transport protein, gene and application thereof - Google Patents

Abstract

The invention discloses a stripe rust glycotranslocator Pst25662, and the amino acid sequence is shown as SEQ ID NO. 2. The nucleotide sequence of the sugar transporter expression gene Pst25662 is shown in SEQ ID NO. 1. The sugar transport protein has the sugar and various hexose transport capacities, and the gene Pst25662 is up-regulated under the induction of the rust bacteria. The invention utilizes agrobacterium-mediated genetic transformation to obtain transgenic plants with gene Pst25662 silencing, and verifies that transgenic interference plants show resistance to stripe rust. The invention enriches the new cognition of the technicians in the field on the acquisition of the carbon source of the wheat stripe rust, provides ideas for the subsequent research of wheat stripe rust resistant varieties and sustainable regulation and control, and has substantial reference significance on the design of biological control drug targets.

Description

Sugar transporter, gene and application of wheat stripe rust

technical field

The invention belongs to the technical field of bioengineering, and relates to the selection and breeding of plant disease-resistant materials and biological control drug targets in agricultural biotechnology, in particular to a wheat stripe rust sugar transporter, a coding gene and an application thereof.

Background technique

Wheat is the food crop with the largest planting area and the widest distribution in the world. About 1/3 of the world's population uses wheat as the main food. Among all the biotic and abiotic factors that may lead to severe wheat yield reduction, wheat stripe rust is one of the most serious threats to wheat yield loss, which has the characteristics of wide occurrence range, fast epidemic speed, and large damage loss. In 2002, wheat stripe rust was included in the national list of first-class pests and diseases. Therefore, improving the prevention and control measures of wheat stripe rust to ensure food safety production has become an urgent need to be solved in agricultural production.

Chemical control is the main way to prevent and control wheat stripe rust. However, long-term and unreasonable use of a large number of drugs will not only increase environmental pollution and increase the burden on farmers, but also lead to many problems such as chemical drug residues and increased resistance of diseases and insect pests. The natural balance of agricultural ecosystems. Breeding disease-resistant varieties is one of the most cost-effective and environmentally friendly measures to control wheat stripe rust. However, due to the frequent variation of stripe rust races and the emergence of new toxic races, the main wheat disease-resistant varieties are usually Resistance is overcome within 3-5 years. Therefore, thoroughly understanding the pathogenic mechanism of stripe rust and creating broad-spectrum and durable disease-resistant materials are considered to be the most efficient and direct solutions.

Stripe rust is a biotrophic parasitic fungus whose survival and reproduction are highly dependent on nutrients absorbed from the host. Sugar is an important basic nutrient substance necessary for the growth and development of wheat stripe rust. Controlling the absorption of sugar by stripe rust can effectively limit its material and energy supply, thereby restricting the growth and reproduction of stripe rust and weakening its Hazards to wheat. However, there are relatively few studies on the sugar uptake mechanism of stripe rust during the interaction between wheat and stripe rust, and it is still unclear. The sugar transporter of wheat stripe rust is relatively conservative in gene evolution, and sugar is the main form of carbon source of pathogenic bacteria. Therefore, the key way to obtain sugar for pathogenic bacteria is clarified, so as to use the pathogenic factor of pathogenic bacteria to create wheat disease-resistant materials. Providing new ideas and breeding materials is of great significance for realizing broad-spectrum and long-lasting disease resistance in wheat breeding.

Contents of the invention

Carbohydrates are the main form of carbon source for pathogenic bacteria, and they are the main nutrients that are indispensable for the growth and development of stripe rust. The present invention aims to enrich the new knowledge of those skilled in the art on the carbon source acquisition of wheat stripe rust. Specifically, the present invention provides a sugar transporter gene Pst25662 of wheat stripe rust, and its encoded protein Pst25662.

The object of the present invention is to provide a sugar transporter Pst25662 of wheat stripe rust and the gene Pst25662 encoding the sugar transporter Pst25662. The invention also provides the application of the sugar transporter Pst25662 and its coding gene in the breeding of wheat varieties resistant to stripe rust.

In order to achieve the above object, the technical scheme adopted in the present invention is: a wheat stripe rust sugar transporter Pst25662, the sugar transporter Pst25662 plays a sugar transport function in the interaction process between stripe rust and wheat, and the sugar transporter The amino acid sequence of Pst25662 is shown in SEQ ID NO.2, and the nucleotide sequence of the gene Pst25662 encoding the sugar transporter Pst25662 is shown in SEQ ID NO.1.

Further, the sugar transporter gene Pst25662 of wheat stripe rust and the sugar transporter encoded by it perform sugar transport function during the interaction process between stripe rust and wheat. Silencing the specific fragment of the gene Pst25662 (the nucleotide sequence is shown in SEQ ID NO.3) can improve the resistance of wheat to the stripe rust pathogen.

The invention also provides the application of the sugar transporter Pst25662 and its coding gene in the breeding of wheat varieties resistant to stripe rust.

The present invention further provides a method for wheat disease-resistant breeding, the method comprising: constructing an expression vector comprising a silent fragment of the wheat stripe rust sugar transporter gene Pst25662; using Agrobacterium to mediate the transfer into wheat materials; obtaining the sugar transporter Transgenic wheat in which the protein gene Pst25662 is silenced; the expression vector contains the Pst25662 gene silence fragment.

In order to understand the technical solution of the present invention completely and unambiguously, it needs to be added that the sugar transporter gene of the wheat stripe rust in the present invention is represented by the italic font "Pst25662", and the sugar transporter of the wheat stripe rust is represented by the non-slanted font The font "Pst25662" indicates. Of course, those skilled in the art can clearly and completely understand the meaning and expression of related genes and their encoded proteins according to the description of the present invention.

Compared with the prior art, the present invention has the following beneficial effects or advantages:

(1) The sugar transporter Pst25662 of wheat stripe rust provided by the present invention is a protein with sugar transport function reported for the first time in wheat stripe rust, and can transport multiple monosaccharide substances at the same time. Silent plants of the sugar transporter Pst25662 of stripe rust were created with the help of host wheat, and the expression of the sugar transporter was inhibited when the stripe rust infected wheat, and inoculated with different physiological races of stripe rust, all of which would lead to a significant increase in the sporulation of the silent plants. decreased, and immune necrotic cells increased.

(2) Pst25662 transgenic plants show resistance to wheat stripe rust. Therefore, the sugar transporter Pst25662 can be used to create rust-resistant wheat lines, which provides excellent materials for the cultivation of wheat stripe rust-resistant varieties.

(3) The sugar transporter gene provided by the present invention is relatively conserved in different races, which lays the foundation for creating persistent and broad-spectrum disease-resistant wheat varieties. At the same time, it has important reference significance for the target research of new biological control drugs.

Description of drawings

Fig. 1 is the analysis chart of the expression level of the stripe rust sugar transporter gene Pst25662 provided by the embodiment of the present invention at different times after the stripe rust race CYR31 infects wheat, and the data in Fig. 1 are the mean ± SEM of three biological repetitions .

Figure 2 is a diagram of the fluorescent localization of the Pst25662 sugar transporter gene Pst25662 provided by the embodiment of the present invention. In Figure 2, Free GFP indicates the GFP protein, and Pst25662-GFP indicates the fusion protein of Pst25662 and GFP.

Figure 3 is a diagram of the colony growth of the sugar transport protein gene Pst25662 on different carbon sources for the identification test of sugar transport function; Among them, Control represents the pDR195 empty plasmid, Pst25662 represents the fusion plasmid of pDR195 and Pst25662, Maltose represents the carbon source is maltose, and Glucose represents carbon The source is glucose, Sucrose represents the carbon source is sucrose, Fructose represents the carbon source is fructose, Mannose represents the carbon source is mannose, Mannitol represents the carbon source is mannitol, Galactose represents the carbon source is galactose, Xylose represents the carbon source is xylose, Arabinose represents the carbon source as arabinose.

Figure 4 is a diagram of DNA detection results of transgenic plants containing Pst25662-specific silencing fragments; among them, M represents DNAMarker, H ₂ O represents water template, WT represents wild type, PC represents plasmid, and L19 and L43 represent two RNAi plants 19 and 43 strain.

Figure 5 is a phenotype diagram of transgenic silenced plants inoculated with different stripe rust races CYR32, CYR33, and CYR34; Fielder is a wild-type control, and L19 and L43 represent two transgenic lines of T3 generation 19 and 43.

Detailed ways

In the following, the technical solution of the present invention will be described in conjunction with examples, but the present invention is not limited to the following examples.

The experimental methods and detection methods in the following examples, if no special instructions, are conventional methods; the medicament and materials, if no special instructions, can be purchased in the market; the index data, if no special instructions , are conventional measurement methods.

Example 1

This example provides an analysis test of the expression level of the stripe rust fungus sugar transporter gene Pst25662 at different times after the stripe rust race CYR31 infects wheat.

1. Acquisition of real-time fluorescent quantitative PCR samples of stripe rust

Wheat seeds (water source 11) were planted in flower pots with a diameter of 10 cm, placed in a light/dark cycle of 16/8 h, and cultivated at a temperature of 16°C. Fresh uredia spores of wheat stripe rust were diluted, and 10 μL of the diluted spore solution was drawn up with a pipette gun and evenly spread and inoculated on wheat leaves. The inoculated wheat seedlings were kept moist for 36 hours in the dark at 15°C and then cultured under normal light conditions. Wheat leaves were sampled at 6h, 12h, 18h, 24h, 48h, 72h, and 120h after inoculation as real-time fluorescent quantitative PCR samples. For each group of samples, at least three leaves from different wheat seedlings were selected, and the middle part of the inoculated leaves was mainly collected. After sampling, immediately store at -80°C for later use.

2. Preparation of Stripe Template

The collected samples were prepared using the RNA extraction kit and vazyme reverse transcription kit (Beijing Huayueyang Biotechnology Co., Ltd.), according to the operating instructions, to prepare cDNA templates for real-time fluorescent quantitative detection of stripe rust.

3. Detection of real-time fluorescent quantitative PCR

The elongation factor gene PstEF of wheat stripe rust was used as an internal reference gene, and the relative quantitative method was used to analyze the gene expression.

The primer sequences of internal reference genes are:

PstEF-F: TTCGCCGTCCGTGATATGAACAA;

PstEF-R: ATGCGTATCATGGTGGTGGAGTGA.

Pst25662-specific primers were designed by Primer Premier 5.0 software, and the Pst25662-specific primer sequences were:

DL-25662-F: CGTCATTGGCTTAACCGTTCTG;

DL-25662-R: GTCGCCACCACGTTAACTACATT.

Use the UltraSYBR One Step RT-qPCRKit fluorescent quantitative PCR kit (Kangwei Century Biotechnology Co., Ltd.), use it according to the operating instructions, and use the Bio-Rad iQ5 real-time fluorescent quantitative PCR instrument to complete the relevant detection. The relative expression of different genes was calculated according to the 2 ^-ΔΔCt method with the expression of PstEF as a reference.

The results are shown in Figure 1. When wheat was infected by the stripe rust race CYR31 for 12 hours, a substomatal sac was formed in the cavity below the wheat stomata, and the stripe rust began to produce a large number of primary hyphae, and the sugar transporter gene Pst25662 was expressed in large quantities. , to make sufficient preparations for the absorption of sugar substances, and the expression level of the sugar transporter gene Pst25662 was the highest at 18h, when the stripe rust was transporting a large amount of sugar substances from the host body for itself. Since then, the protein has maintained high expression, indicating that it has been undertaking the task of obtaining carbohydrates from wheat tissues and has always played an important role.

Example 2

This example provides the localization test of sugar transporter Pst25662 in cells.

1. Amplification of the full-length sugar transporter gene Pst25662 of P.

Full-length amplification primers Pst25662-F and Pst25662-R were designed using Primer Premier 5.0 software. The primer sequences are:

Pst25662-F: ATGCCTGCCGTAGCCG;

Pst25662-R: TCAAATGTGCTCCAGCTTCTCT.

Using the cDNA of wheat stripe rust as a template, 2×TaqMasterMix PCR mixture (Kangwei Century Biotechnology Co., Ltd.), and the full-length primers of the gene were used for PCR amplification. The reaction system is:

The program of the PCR instrument was: 95°C for 5min pre-denaturation, 95°C for 15s denaturation, 58°C for 30s annealing, 72°C for 1min extension, after a total of 40 cycles, 72°C for 10min supplementary extension. After the PCR program is over, the PCR product is subjected to agarose gel recovery gel detection, and the band that meets the size of the target gene is recovered, and the omega gel recovery kit is used for gel recovery, and the operation is performed according to the instructions (Omega Bio-Tek Company).

The Pst25662 gene fragment recovered from the gel was connected to the vector pMD19-T Simple, transformed into Escherichia coli DH5α competent (Shanghai Weidi Biotechnology Co., Ltd.), and positive ones were screened. Afterwards, the positive samples were sent to Beijing Qingke Biotechnology Co., Ltd. for plasmid sequencing, and the plasmid with the correct sequencing result was kept for the next experiment. The plasmid was marked as: T-Pst25662.

2. Yeast subcellular localization plasmid construction

According to the sequence characteristics of the multiple cloning site of pDR195, GFP primers containing the BamHI homology arm sequence were designed. The primer sequences used are:

195-GFP-F: CGGCCGCGCGGATCCATGGTGAGCAAGGGCG;

195-GFP-R: TCCAAAGCTGGATCCTTACTTGTACAGCTCGTC.

Use the high-fidelity enzyme Primer STAR Max Premix (2×) produced by TAKARA, the primer pair 195-GFP-F, 195-GFP-R, and the plasmid containing the GFP sequence as a template to amplify the GFP containing the homology arm of the pDR195 vector BamHI site Sequence, and gel recovery of bands that meet the size. The pDR195 plasmid was digested with BamHI, and then the linearized pDR195 plasmid and the amplified GFP gel product were cloned in one step using the ClonExpressII One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct pDR195 -GFP plasmid, screen the positive ones, send the plasmid with the correct sequence and keep it. At this time, the pDR195 plasmid already contains the GFP sequence.

According to the sequence characteristics of the multiple cloning site of the pDR195 vector, the Pst25662 primer containing the XhoI homology arm sequence was designed. The primer sequences are:

195-25662GFP-F: ATATACCCCAGCCTCGAGATGCCTGCCGTAGCCG;

195-25662GFP-R:

CACCATGGATCCCTCGAGAATGTGCTCCAGCTTC.

Use the high-fidelity enzyme Primer STAR Max Premix (2×), primer pair 195-25662GFP-F, 195-25662GFP-R, T-Pst25662 plasmid as a template to amplify the Pst25662 sequence containing the homology arm of the pDR195 vector XhoI site, which will meet the Size bands for glue recovery. As above, use the ClonExpress II One Step Cloning Kit for one-step cloning. The following operations are the same as the construction of the pDR195-GFP plasmid to obtain the correct pDR195-25662GFP plasmid.

3. Yeast Subcellular Localization

Use YPD (with 2% maltose) liquid medium to inoculate a single colony of YSL2-1 yeast, culture at 30°C, 250rpm for 48 hours; transfer the yeast mother liquor to the above-mentioned YPD medium with 2% maltose at a ratio of 1:100 , shake the bacteria to a final concentration of OD ₆₀₀ of 0.6-0.8; collect 2 mL of bacterial liquid, centrifuge at 4500 rpm for 5 min at room temperature, discard the supernatant; add 2 mL of ddH ₂ O to resuspend, centrifuge at 4500 rpm for 5 min, discard the supernatant; add 100 μL of LiAc transformation solution ( (Shaanxi Point Biological Co., Ltd.) was gently resuspended and allowed to stand for 10 minutes; 50 μL of ssDNA, 3-5 μg of the target plasmid pDR195-GFP or pDR195-25662GFP, 400 μL of LiAc transformation solution were added in turn, mixed well, and placed in a shaker at 30 °C. Recover at 200rpm for 30min. Heat shock at 42°C for 15 minutes, gently invert and mix 7-8 times every 7.5 minutes, centrifuge at 4500 rpm for 5 minutes, and discard the supernatant. Resuspend the bacteria with 500 μL ddH ₂ O, centrifuge at 4500 rpm for 5 minutes, discard the supernatant; add 100 μL ddH ₂ O to resuspend, spread the transformed yeast strain on SD-Ura solid selection medium, and cultivate for 3 days to obtain positive clones .

Take the positive transformed strains transferred into pDR195-GFP and pDR195-25662GFP and inoculate them into the SD-Ura screening medium with 2% maltose as carbon source, culture with shaking at 220 rpm for 48 hours at 30°C, and transfer to Continue to cultivate in the new screening medium until the OD ₆₀₀ value of the bacterial solution is between 0.5-0.7, collect 2 mL of the bacterial liquid and centrifuge at 8000 rpm for 1 min at room temperature, discard the supernatant, add 1 mL of ddH ₂ O to resuspend, and take an appropriate amount of yeast liquid Make glass slides, use a super-resolution laser confocal microscope LSM880 for positioning observation and take pictures for recording. Among them, pDR195-GFP only expresses GFP protein, and pDR195-25662GFP expresses fusion protein of Pst25662 and GFP.

The results are shown in Figure 2. In the yeast expressing only GFP protein, green fluorescence was observed to be expressed in the whole cell. When the sugar transporter Pst25662 was fused with GFP protein, green fluorescence was only observed on the yeast cell membrane. The localization results showed that the sugar transporter Pst25662 was localized in the cell membrane, which was consistent with the membrane localization and transmembrane transport mode of sugar transporters.

Example 3

This example provides a validation test for the sugar transport function of the protein Pst25662.

According to the sequence characteristics of the pDR195 multiple cloning site, the Pst25662 primer containing the BamHI homology arm sequence was designed. Operate as in Example 2 to construct the pDR195-Pst25662 vector. The primer sequences are:

195-25662-F: CGGCCGCGCGGATCCATGCCTGCCGTAGCCG;

195-25662-R: TCCAAAGCTGGATCCTCAAATGTGCTCCAGC.

The constructed pDR195-Pst25662 and the empty vector pDR195 were transformed into yeast sugar transporter deletion mutant YSL2-1 strain by heat shock transformation method respectively. This strain has knocked out the sucrose and hexose transporters, and obtained positive transformants. The operation was the same. Example 2.

The positive transformed strains were inoculated into SD-Ura liquid selection medium with 2% maltose as the carbon source, and cultured with shaking at 30°C and 220 rpm until the OD ₆₀₀ value of the bacterial solution was between 0.5-0.7. Collect 2 mL of bacterial liquid, centrifuge at 8000 rpm for 1 min at room temperature, and discard the supernatant. The cells were resuspended with sterilized ddH ₂ O, centrifuged at 8000 rpm for 1 min, and the cells were washed twice repeatedly. Dilute the bacterial cell suspension to the same concentration with OD ₆₀₀ =0.6 with sterilized ddH ₂ O, continue shaking at 30° C. and 220 rpm, and starve for 5 hours. After the end, 10-fold gradient dilution was performed successively to obtain bacterial cell suspensions of different concentrations, and a total of 5 concentrations were diluted. Inoculate 5 μL of these bacterial suspensions into SD-Ura solid screening culture containing different sugars as carbon sources (maltose, sucrose, glucose, fructose, galactose, mannose, xylose, arabinose, mannitol) Basically, the sugar concentration was 2% (w/v), and the colony growth was recorded by photographing after inverting at 30° C. for 3 days.

The results are shown in Figure 3, control represents the pDR195 empty plasmid, which is the control group; Pst25662 represents the fusion plasmid of pDR195 and Pst25662, which is the experimental group. Different carbon sources were added to the SD-Ura solid screening medium to verify the transport function. Among them, maltose as the carbon source group showed that the yeast state was normal. Other experimental groups showed that the sugar transporter Pst25662 has sucrose, glucose, fructose, galactose, mannose, Mannitol transport capacity of various sugars.

Example 4

This example provides a verification test of the conservation of the Pst25662 gene-silenced transgenic plants against wheat stripe rust.

1. Construction of transgenic vector

Sequence alignment of the ORF region of the Pst25662 gene sequence of the stripe rust sugar transporter Pst25662 gene with wheat and wheat stripe rust homologous genes, and selection of the Pst25662 specific fragment sequence for the construction of the transgenic vector, the nucleotides of the Pst25662 gene silence fragment Sequence (shown as SEQ ID NO: 3). The selected target fragments were respectively inserted into two positions of the PC336 vector by homologous recombination (one fragment was inserted in the forward direction, and the other was inserted in the reverse direction, so that a hairpin structure could be formed), and the PC336-25662 plasmid was obtained.

The primer sequence containing the attB site is:

attB-25662-F:

GGGGACAAGTTTTGTACAAAAAAGCAGGCTTGGCTACATTGCGGGAGT;

attB-25662-R:

GGGGACCACTTTGTACAAGAAAGCTGGGTCCGATGGGATAAGC TAAAAGGGC.

2. Transformation of Agrobacterium

Thaw EHA105 competent cells stored at -80°C on ice, add the correctly sequenced PC336-25662 plasmid, ice bath for 30 minutes, liquid nitrogen for 2 minutes, 37°C water bath for 3 minutes, ice bath for 5 minutes, add an appropriate amount of liquid LB medium, and shake Cultivate for 3 hours; take 150 μL of the transformed bacteria solution and spread it on a solid plate (rifampicin and Kana antibiotics), and incubate in the dark upside down until positive transformants grow.

Using Agrobacterium-mediated wheat transformation technology, the recombinant silencing vector PC336-25662 was transformed into the wheat variety Fielder material to obtain RNAi silencing plants. DNA testing was performed on the obtained plants, and positive plants were retained for subsequent verification.

The results are shown in FIG. 4 , all the plants used in the identification of the transgene were positive plants, and all of them had been successfully transformed into the silencing vector.

3. Phenotype identification of transgenic plants

Two homozygous transgenic strains of the T3 generation were selected for disease resistance testing, and were inoculated with wheat stripe rust: CYR32, CYR33, and CYR34 respectively. The inoculation method was the same as in Example 1. Phenotype identification was carried out after sporulation of wheat leaves, and photographed and recorded.

The results are shown in Figure 5, the spore production of the stripe rust racee on the silenced plants inoculated with wheat stripe rust CYR32, CYR33, and CYR34 was significantly reduced. It shows that the sugar transporter Pst25662 proposed by the present invention is highly conserved among different races of stripe rust and plays a role as a key sugar transporter.

In summary, the sugar transporter protein Pst25662 of stripe rust proposed by the present invention plays a key role in the process of stripe rust infection of wheat, which is mainly reflected in its ability to transport a variety of sugars, and maintains its ability throughout the infection stage. High expression. By constructing transgenic RNAi plants to silence the expression of the gene, they can produce obvious disease resistance responses to a variety of wheat stripe rusts. This gene is highly conserved in stripe rusts, which has reference significance for the creation of broad-spectrum disease-resistant materials.

As mentioned above, the present invention can be better realized. The above-mentioned embodiment is only a description of the preferred implementation of the present invention, and does not limit the scope of the present invention. Various changes and improvements made by technicians to the technical solutions of the present invention shall fall within the scope of protection determined by the present invention.

Claims (10)

1. The rust protein Pst25662 is characterized in that the sugar transporter Pst25662 plays a sugar transporting function in the interaction process of rust bacteria and host plants, and the amino acid sequence of the sugar transporter Pst25662 is shown as SEQ ID NO. 2.

2. The sugar transporter Pst25662 of claim 1, wherein the sugar transporter Pst25662 is expressed at high levels in a infested host plant from which sugar material is transported for the puccinia striolata itself.

3. The sugar transporter Pst25662 of claim 1, wherein the sugar transporter Pst25662 is localized to a cell membrane and the sugar transport conforms to a transmembrane transport pattern.

4. A sugar transporter Pst25662 according to any of claims 1 to 3, characterized in that the sugar transporter Pst25662 is a transportable sugar comprising sucrose, glucose, fructose, galactose, mannose, mannitol.

5. A rust bacteria sugar transporter gene Pst25662 is characterized in that the nucleotide sequence is shown in SEQ ID NO.1, and the sugar transporter gene Pst25662 is encoded and expressed.

6. The sugar transporter gene Pst25662 of claim 5, wherein expression is upregulated in a rust infested host plant.

7. The sugar transporter gene Pst25662 of claim 5, having a silencing fragment with a nucleotide sequence as set forth in SEQ ID No. 3.

8. An expression vector, which is characterized by comprising a silencing fragment with a nucleotide sequence shown as SEQ ID NO. 3.

9. The application of the stripe rust sugar transporter gene Pst25662 and the coded protein thereof in the cultivation of stripe rust resistant materials.

10. The use according to claim 9, wherein silencing the expression of the stripe rust sugar transporter gene Pst25662 increases the resistance of the host plant to stripe rust.

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