A VdMYB4 gene from *Vitis pilosa*, its encoded protein, and its applications.

Abstract

This invention relates to the field of biotechnology, and more particularly to a VdMYB4 gene from *Grape spurs*, its encoded protein, and its applications. The VdMYB4 gene is located on chromosome 5 of *Grape spurs*, distributed in the region 8020439-8021562. The amino acid sequence of the protein encoded by the VdMYB4 gene is shown in SEQ ID NO. 2. The invention also relates to the application of the VdMYB4 gene in enhancing plant resistance to pathogens after silencing. The VdMYB4 gene provided by this invention, after silencing, can enhance the resistance of *Grape spurs* to anthracnose, providing a theoretical basis for breeding grapes with anthracnose resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a VdMYB4 gene of Grape thorn, its encoded protein, and its applications. Background Technology

[0002] Grapes (Vit. vinifera L.) are one of the world's oldest fruit tree species, originating in western Asia and cultivated worldwide. Approximately 95% of grapes globally are grown in the Northern Hemisphere, making it one of the most widely cultivated fruits in the world. Anthracnose, caused by fungi of the genus *Colletotrichum* in the Deuteromycetes, is a significant global disease affecting grapes, impacting both yield and quality. Grape anthracnose primarily affects the rachis of the grape bunch, but can also infect leaves, shoots, tendrils, and fruit stalks, although symptoms are less pronounced than on the fruit and rachis.

[0003] Currently, fungicides are mainly used to control anthracnose in grape cultivation. However, the use of fungicides not only harms the environment and health but also enhances the pathogen's resistance. Due to the adverse effects of using fungicides to control the spread of anthracnose, people are paying increasing attention to hybridization to breed anthracnose-resistant table grapes.

[0004] Previous studies have shown that only a few resistance genes have been reported in the defensive response mechanism of grapes to anthracnose infection, especially regarding the interaction network between grapes and anthracnose, and the isolation and functional analysis of key response genes. This invention elucidates the functional characteristics and mechanisms of transcription factors involved in the anthracnose stress response, providing a basis for enriching the theory of grape anthracnose stress and for breeding grapes with anthracnose resistance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a VdMYB4 gene of Grape thorn and its encoded protein, which can improve the plant's resistance to anthracnose after knockout.

[0006] This invention is implemented as follows:

[0007] This invention first provides a VdMYB4 gene from grapevine, located on chromosome 5 of grapevine, distributed in the region 8020439-8021562.

[0008] Specifically, the nucleotide sequence of the VdMYB4 gene of the grape thorn is shown in SEQ ID No. 1.

[0009] The present invention also provides a protein encoded by the VdMYB4 gene of the grape thorn, the amino acid sequence of which is shown in SEQ ID NO.2, contains a myb DNA-binding domain, and has the highest homology with European grapes.

[0010] Finally, this invention provides the application of the VdMYB4 gene from *Grape thorn* in enhancing plant resistance to pathogens after silencing. Under pathogen stress, the expression level of the VdMYB4 gene is first downregulated and then upregulated. Silencing VdMYB4 in *Grape 'Kyoho'* berries was found to enhance resistance to anthracnose infection.

[0011] Furthermore, the pathogen is pathogenic anthrax bacteria.

[0012] Furthermore, the plant includes grapes.

[0013] Furthermore, a silencing vector was constructed to silence the VdMYB4 gene of the grape thorn.

[0014] Furthermore, the silencing vector comprises the grapevine VdMYB4 gene, the pCambia2300-GFP vector operably linked thereto, and the virus-induced gene silencing vector pTRV-GATEWAY.

[0015] The present invention has the following advantages:

[0016] This invention provides a VdMYB4 gene from the grape thorn, the nucleic acid sequence of which is shown in SEQ ID No.1, is 762 bp in length, encodes 253 amino acids, and the amino acid sequence is shown in SEQ ID No.2, containing a myb DNA-binding domain and is highly homologous to that of European grapes. The encoded protein is located in the cell nucleus.

[0017] This invention analyzes the expression of VdMYB4 in the peel of grape berries after infection with anthracnose. The experiment shows that as the infection progresses, the expression level of VdMYB4 first decreases and then increases, indicating that VdMYB4 can respond to grape anthracnose infection.

[0018] This invention clones the VdMYB4 sequence, constructs the silencing vector TRV::VdMYB4, transforms it into Kyoho grapes, and after inoculation with anthracnose, the fruit showed milder disease symptoms compared to the TRV::00 (control). This verifies that VdMYB4 silencing enhances the resistance of Kyoho grapes to anthracnose. Therefore, the VdMYB4 gene provided by this invention, after silencing, can improve the resistance of Kyoho grapes to anthracnose, providing a theoretical basis for breeding grapes resistant to anthracnose. Attached Figure Description

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Figure 1 Cluster analysis of homologous protein sequences of VdMYB4 from *Vi tis davidii* and some other species, with yellow text representing VdMYB4 from *Vi tis davidii*.

[0021] Figure 2 A schematic diagram showing the construction of the VdMYB4 gene into the pCambia2300-GFP and pTRV2-GATEWAY vectors.

[0022] Figure 3 This is a diagram showing the domain structure analysis of the VdMYB4 protein from Grape thorn.

[0023] Figure 4 To locate the VdMYB4 chromosome of Grape thorn.

[0024] Figure 5 This is a graph showing the expression level of VdMYB4 in response to grape anthracnose.

[0025] Figure 6 Subcellular localization analysis map of VdMYB4 (scale bar is 25 μm) and yeast transcriptional activation activity analysis.

[0026] Figure 7 Figure 1 shows the results of RT-qPCR detection of Kyoho grapes after interference and the analysis of their resistance to anthracnose. A: RT-qPCR detection of interference efficiency in grapes; B: Phenotype of Kyoho grapes with the virus-induced gene silencing VdMYB4 5 days after inoculation with anthracnose (left is control, right is silenced VdMYB4), where the white part is the inoculated bacterial block and the part circled in black is the lesion. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention and is merely an example.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] The bacterial strains and plasmids used in the examples are as follows:

[0031] The overexpression vector pCambia2300-GFP and the interference vector pTRV2-GATEWAY were used in the experiment. The Agrobacterium tumefaciens strain used for tobacco epidermal cell localization analysis and virus-induced gene silencing experiment was GV3101 (p19+pSoup). The Escherichia coli Top10 competent cells used were purchased from TianGen.

[0032] The main reagents used in the examples include a reverse transcription kit, qRT-PCR supermix (Transgen), plasmid extraction kit, plant RNA extraction kit, gel extraction kit, LA high-fidelity enzyme, sucrose, MS powder, Tris, agar powder, CTAB, 75% ethanol, and anhydrous ethanol.

[0033] The culture medium used in the induction experiment in this example was 1 / 2 MS liquid medium.

[0034] Example 1: Cloning and Vector Construction of MYB4CDS Sequence

[0035] 1. Use the following primers to target *Vitis davidii* (Rom. Caill.). The full-length and specific fragments of MYB4 cloned from the cDNA library

[0036] SEQ ID No. 3:

[0037] p2300-VdMYB4-BamHI-F:

[0038] tcggtacccggggatccATGGTAAGAGCTCCTTGTTG

[0039] SEQ ID No.4:

[0040] p2300-VdMYB4-Sal IR:

[0041] gctcaccatggtgtcgacCTTACAGGAGCTTTGA

[0042] SEQ ID No. 5:

[0043] Attb1-VdMYB4:

[0044] ggggacaagtttgtacaaaaaagcaggcttcACGCGCTGTCCTCTGATAAT

[0045] SEQ ID No. 6:

[0046] Attb2-VdMYB4:

[0047] ggggaccactttgtacaagaaagctgggtcGCCGCCAGATCTAATGAAAA

[0048] Note: Lowercase sequences are adapter primers (vector sequences), and uppercase sequences are gene sequences.

[0049] 2. Constructing a carrier

[0050] Homologous recombination reactions were performed using seamless cloning kits from Bometech and Invitrogen (e.g.) Figure 2 The gel-recovered product obtained above was mixed with linearized vectors (pCambia2300-GFP and pTRV2 vectors) and reaction solution was added simultaneously. The reaction system was 10 μL / 1 μL. The mixture was reacted at 37℃ / 250℃ for 60 min, followed by an ice bath for 15 min for subsequent reactions. Only after PCR verification and sequencing confirmation of correct sequence could the product be used for subsequent experiments.

[0051] Example 2: Bioinformatics Analysis of VdMYB4

[0052] The amino acid sequence of VdMYB4 was compared with that of BLAST, and homologous sequences from different species were downloaded to construct a phylogenetic tree. Figure 1 The amino acid sequence similarity of VdMYB4 was analyzed using MEGA6 multiple comparisons. The VdMYB4 gene of *Vitis pisca* is located on chromosome 12, in the region 5138849-5140246. Figure 4 The phylogenetic tree was constructed using the maximum likelihood (NJ) method. The VdMYB4 amino acid sequence was compared with that of European grape, Arabidopsis thaliana, cultivated rice, tobacco, tomato, and soybean using the Clustw program. The results are as follows: Figure 3 As shown, VdMYB4 is 762 bp in length and encodes 253 amino acids.

[0053] Example 3: Analysis of the expression pattern of the VdMYB4 gene

[0054] Grapes arvensis were inoculated with anthracnose (Discocephalus discoidus) using the needle-pricking method, and grape skins infected with anthracnose were collected as experimental materials. RT-qPCR was performed to analyze the expression pattern of the VdMYB4 gene.

[0055] The results are as follows Figure 5 As shown, the expression level of VdMYB4 in the peel of Grapevine inoculated with Grape Anthracnose at 0, 1, 2, 4, and 6 days after infection first decreased and then increased, reaching its maximum on day 6. This indicates that VdMYB4 can be induced to express in response to Grapevine anthracnose.

[0056] Example 4: Subcellular localization and transcriptional autoactivation analysis of tobacco

[0057] The correctly sequenced recombinant plasmid was transferred into Agrobacterium GV3101 using electroporation. The correct bacterial culture was then expanded in 10 mL LB liquid medium containing the same vitamin. After centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the bacteria were resuspended in resuspension (MES 2.130 g / L + MgCl₂ 22.03 g / L + sucrose 20 g / L) and washed three times. The resuspension was diluted to OD₆₀ = 0.4, and then activated by adding AS (200 mmol·L⁻¹) to a light-protected strip and incubating at room temperature for 3 h. The resuspension was injected into the underside of healthy Tobacco Benzovia leaves using a needleless 1 mL syringe and cultured in a light incubator for 48 h. The distribution of the GFP fusion protein in the tobacco leaves was observed under a laser confocal microscope (Leica TCSSP8), and images were saved.

[0058] The results are as follows Figure 6 As shown, the VdMYB4-GFP protein is located in the cell nucleus. To verify whether VdMYB4 has transcriptional activation activity, this invention uses a yeast system to verify its transcriptional activation activity. The full-length VdMYB4 CDS was constructed into the pGBKT7 vector, transformed into the yeast strain Y2H Gold, and grown on tryptophan-deficient medium. The ability of the full-length VdMYB4 to activate the expression of reporter genes ADE2 and HIS3 was tested, with the empty pGBKT7 vector serving as a negative control (expressing only the GAL4 binding region). The results showed that the full-length VdMYB4 exhibits self-activation activity (CaWRKY41 was used as a positive control, which has been disclosed).

[0059] Example 5: Analysis of virus-induced gene silencing VdMYB4 in grapes and its resistance to anthracnose.

[0060] The gene silencing method used in this invention employs tobacco mosaic virus (TRV)-mediated VIGS, specifically by designing a gene-specific fragment and using the Gateway vector construction method to ultimately ligate a VdMYB4-specific fragment into the pTRV2 vector. After Agrobacterium transformation, single clones were selected for verification, and the culture was shaken. The concentration of the final resuspended Agrobacterium was adjusted to approximately OD600 = 1. Then, empty pTRV2 and pTRV2 carrying the target gene VdMYB4 were mixed with pTRV1 at a 1:1 ratio and incubated at 28°C and 80-100 rpm for 3-4 hours. Subsequently, Kyoho grape berries, 2-3 weeks after color change, were immersed in a separate mixture of pTRV1 and pTRV2-MYB4 under vacuum for 10 minutes. Co-immersion with empty pTRV2 and pTRV1 vectors served as a negative control. Wipe the surface of the grape berries with clean absorbent paper to remove any residual bacterial solution. Incubate for 24 hours in the dark at 26℃ and 60% relative humidity. Then, culture for 3 days at 26℃, 100 μmol m⁻² s⁻¹, 16 / 8h (light / dark) before proceeding with subsequent experiments. Inoculate the berries using the needle-pricking method, taking 5 mm diameter disc-shaped spores and applying them to the needle-pricking site. Incubate in a 25℃ constant temperature incubator while maintaining humidity. After 5 days, observe the disease symptoms and the size of lesions on the grape berries to assess the severity of anthracnose in the grapes.

[0061] To gain a deeper understanding of VdMYB4 resistance to pathogens in grapes, VdMYB4 was silenced in Kyoho grape berries after veraison by virus induction. RT-qPCR results showed that the expression level of VdMYB4 was significantly reduced in the silenced plants (TRV::VdMYB4). Figure 7 (A); Results from anthracnose inoculation of silent and control grape berries 5 days after inoculation showed that, compared to the control (TRV::00), the TRV::VdMYB4-silenced berries had less noticeable lesions. Figure 7 The result (B) indicates that silencing TRV::VdMYB4 can enhance the resistance of grape berries to anthracnose.

[0062] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A type of thorny grape VdMYB4 The application of gene silencing in enhancing plant resistance to pathogens is characterized by: The prickly grape VdMYB4 The gene is located on chromosome 5 of *Vitis piscifolium*, in the region 8020439-8021562, and its amino acid sequence is shown in SEQ ID NO. 2; the pathogen is *Bacillus anthracis*; the plant is *Vitis davidii*.

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