A vitis davidii yucca10 gene, its coded protein and application

Abstract

This invention relates to the field of biotechnology, and more particularly to a VdYUCCA10 gene from *Grape thorn*, its encoded protein, and its applications. The VdYUCCA10 gene is located on chromosome 7 of *Grape thorn*, distributed in the region 2271185-2274940, and its nucleotide sequence is shown in SEQ ID NO. 1. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2. The invention also relates to the application of silencing the VdYUCCA10 gene to enhance the resistance of grape fruits to pathogens. The VdYUCCA10 gene silencing provided by this invention can improve the resistance of grapes 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 VdYUCCA10 gene of Grape thorn, its encoded protein, and its applications. Background Technology

[0002] Grapes (Vitis vinifera L.) are one of the world's oldest fruit tree species, originating in western Asia and cultivated worldwide. Approximately 95% of grapes are grown in the Northern Hemisphere, making it one of the most widely cultivated fruits globally. Anthracnose, caused by fungi of the genus *Colletotrichum* in the Deuteromycetes, is a significant global disease affecting grapes, severely impacting yield and quality. Grape anthracnose primarily affects the rachis, but can also infect leaves, shoots, tendrils, and fruit stalks, though symptoms are less pronounced 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, there is increasing interest in breeding anthracnose-resistant table grapes through hybridization. Previous studies have shown that only the functions of a few resistance genes in grapes have been reported in response to anthracnose infection, especially regarding the interaction network between grapes and anthracnose, and the isolation and functional analysis of key response genes.

[0004] YUC (YUCCA) is the rate-limiting enzyme in the auxin indole-3-acetate tryptophan-dependent synthesis pathway, playing a crucial role in plant growth, development, and stress responses. As a multigene family, members of the YUCCA family have been identified in many plants, including Arabidopsis thaliana, rice (Oryza sativa L.), maize (Zeamays L.), alfalfa (Medicagosativa L.), and pear (Pyrus sorotina.). Existing research has shown that members of the YUCCA family are widely involved in various biological processes such as plant growth and development, and responses to abiotic stress. For example, 10 members of the YUCCA family have been identified in grape (Vitis vinifera L.), and YUCCA1A and YUCCA8 may be involved in the chlorosis of grape stalks during postharvest storage; 10 members of the YUCCA family have been identified in watermelon (Citrullus lanatus), and CIYUCCA4 and CIYUCCA10c can be involved in regulating the ripening of watermelon fruits; 11 members of the YUCCA family have been identified in peach (Prunus persica), and PpYUCCA6 / 12 may be involved in the formation of its drooping trait.

[0005] Although YUC family genes have been found in many species, there is very little research on the function of YUCCA genes in pathogen stress responses. Currently, there is no functional validation of the YUCCA gene's involvement in anthrax stress responses.

[0006] This invention analyzes the functional characteristics and mechanism of action of transcription factor VdYUCCA10 in response to anthracnose stress, providing a basis for enriching the theory of grape anthracnose resistance and grape anthracnose resistance breeding. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a VdYUCCA10 gene of grape, its encoded protein and its application, which can improve the resistance of grape to anthracnose by silencing VdYUCCA10.

[0008] This invention is implemented as follows:

[0009] The present invention first provides a VdYUCCA10 gene of grape thorn, which is located on chromosome 13 of grape thorn and distributed in the region 2271185-2274940, and its nucleotide sequence is shown in SEQ ID NO.1.

[0010] Specifically, the protein encoded by the VdYUCCA10 gene of the grape thorn has the amino acid sequence shown in SEQ ID NO.2.

[0011] Finally, this invention provides the application of the VdYUCCA10 gene from *Grape thorn* in enhancing the resistance of grape berries to pathogens after silencing. Under pathogen stress, the expression level of the VdYUCCA10 gene is downregulated, while grapes silenced with VdYUCCA10 show enhanced resistance to pathogens.

[0012] Furthermore, the pathogen is pathogenic anthrax bacteria.

[0013] Furthermore, the grapes mentioned are Kyoho grapes.

[0014] Furthermore, the silencing vector TRV::VdYUCCA10 was constructed to silence the VdYUCCA10 gene.

[0015] Furthermore, the silencing vector is pTRV2 and pTRV2 carrying the target gene VdYUCCA10.

[0016] The present invention has the following advantages:

[0017] This invention provides a VdYUCCA10 gene from Grape thorn, which is 1182 bp in length and encodes 393 amino acids. The amino acid sequence is shown in SEQ ID No. 2.

[0018] This invention analyzed the expression of VdYUCCA10 in the peel of grape berries infected with anthracnose. The results showed that the expression level of VdYUCCA10 decreased with the progression of infection, and was highly expressed in mature berries, indicating that VdYUCCA10 can respond to grape anthracnose infection.

[0019] This invention clones the VdYUCCA10 specific sequence to construct the silencing vector TRV::VdYUCCA10. When this vector was transformed into Kyoho grapes and inoculated with *Gnaphalium affine*, the resulting grapes showed milder disease symptoms compared to the TRV::00 (control) variety. This verifies that VdYUCCA10 is a negative regulator in the grape's response to anthracnose infection. Therefore, silencing the VdYUCCA10 gene provided by this invention can enhance grape resistance to anthracnose, providing a theoretical basis for breeding grapes with anthracnose resistance. Attached Figure Description

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Figure 1 Cluster analysis of homologous protein sequences of Vitis davidii and VdYUCCA10 from some species, where red text represents VdYUCCA10 of Vitis davidii.

[0022] Figure 2 A schematic diagram of constructing the VdERF054 gene-specific sequence into the pTRV2-GATEWAY vector.

[0023] Figure 3 This is a diagram showing the domain structure analysis of the VdYUCCA10 protein from Grape thorn.

[0024] Figure 4 To locate chromosome VdYUCCA10 of Grape thorn.

[0025] Figure 5 Figure B shows the expression levels of VdYUCCA10 in response to grape anthracnose induction (A) and in different tissue parts (YF: young fruit, ML: mature leaf, YL: young leaf, MF: mature fruit, T: tendril, S: stem).

[0026] Figure 6 To assess the resistance of Kyoho grapes to anthracnose after silencing VdYUCCA10. A: RT-qPCR detection of interference efficiency in grapes; B: Phenotype of Kyoho grapes with the virus-induced gene silencing VdYUCCA10 5 days after inoculation with anthracnose (left is control, right is silenced VdYUCCA10); C: Lesion area. 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 silencing vector used in the experiment was pTRV2-GATEWAY; the Agrobacterium strain used in the virus-induced gene silencing experiment was GV3101 (p19+pSoup); and the Escherichia coli Top10 competent cells used were purchased from TianGen.

[0032] The main reagents used in the examples include a reverse transcription kit, RT-qPCR supperMix (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 VdYUCCA10 CDS Sequence

[0035] 1. Use the following primers to clone the full length, specific fragment, and specific fragment of VdYUCCA10 from the grape cDNA library.

[0036] SEQ ID No. 3:

[0037] Attb1-VdYUCCA10:

[0038] ggggacaagtttgtacaaaaaagcaggcttcTGACCGCCTTCATCTTCACC;

[0039] SEQ ID No.4:

[0040] Attb2-VdYUCCA10:

[0041] ggggaccactttgtacaagaaagctgggtcAAACAGCCCCAGATGCATGA.

[0042] Note: Lowercase sequences are adapter primers (vector sequences), and uppercase sequences are gene sequences.

[0043] 2. Constructing a carrier

[0044] Homologous recombination reactions were performed using seamless cloning kits from Bometech and Invitrogen (e.g.) Figure 2 The obtained gel recovery product was mixed with the linearized vector (pTRV2 vector) and the reaction solution was added simultaneously. The reaction system was 10 μL / 1 μL. The reaction was carried out at 37℃ / 250℃ for 60 min, followed by an ice-water bath for 15 min for subsequent reactions. Only after PCR verification and sequencing to confirm the correct sequence could the product be used for subsequent experiments.

[0045] Example 2: Bioinformatics Analysis of VdYUCCA10

[0046] The amino acid sequence of VdYUCCA10 was compared with that of BLAST, and homologous sequences from different species were downloaded to construct a phylogenetic tree. The results are as follows: Figure 3 As shown, VdYUCCA10 is 1182 bp in length and encodes 393 amino acids. Multiple sequence similarity of VdYUCCA10 was determined using MEGA6. The VdYUCCA10 gene of *Vitis pisca* is located on chromosome 7 of *Vitis pisca*. Figure 4 The phylogenetic tree was constructed using the maximum likelihood (NJ) method, distributed in the region 2271185-2274940. The amino acid sequence of VdYUCCA10 was compared with that of Grape simonii and kiwifruit using the Clustw program, revealing a high degree of homology between VdYUCCA10 and YUCCA10 in Grape simonii. Figure 1 ).

[0047] Example 3: Analysis of the expression pattern of the VdYUCCA10 gene

[0048] The *Vitis davidii* grape variety was inoculated with *Colletotrichum discosum* using a needle prick method, and the grape skins after infection with *Colletotrichum discosum* were collected as experimental material. RT-qPCR was performed, and the expression pattern of the *VdYUCCA10* gene was analyzed. The results are as follows: Figure 5 As shown in Figure A, at 0, 1, 2, 3, and 6 days after inoculation of *Vitis thunbergii* with *A. thunbergii*, the expression level of VdYUCCA10 in the fruit peel decreased with the progression of infection. Results from different tissue expression patterns revealed that the expression level of VdYUCCA10 was higher in mature fruit (MF: mature fruit). Figure 5 (B) indicates

[0049] VdYUCCA10 can be induced to express in response to grape anthracnose.

[0050] Example 4: Analysis of virus-induced gene silencing VdYUCCA10 in grapes and its resistance to anthracnose.

[0051] The gene silencing 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 finally ligate the VdYUCCA10 trans-3-UTR specific fragment into the pTRV2 vector. Agrobacterium transformation was performed, and single clones were selected for verification and cultured. The concentration of the final resuspended Agrobacterium was adjusted to approximately OD600 = 1.0. Then, empty pTRV2 and pTRV2 carrying the target gene VdYUCCA10 were mixed with pTRV1 at a 1:1 ratio and incubated at 28°C and 80-100 rpm for 3-4 hours. Subsequently, Kyoho grapes (VdYUCCA10 has the same gene sequence as the YUCCA10 gene in Kyoho grapes) 2-3 weeks after color change were immersed in a separate mixture of pTRV1 and pTRV2-VdYUCCA10, and vacuum-soaked for 10 minutes. Co-infiltration of empty pTRV2 and pTRV1 vectors served as a negative control (TRV::00). The surface of the grape berries was wiped with clean absorbent paper to remove any residual bacterial culture. The berries were incubated in the dark at 26°C and 60% relative humidity for 24 hours, followed by culturing at 26°C, 100 μmol m⁻² s⁻¹, 16 / 8 h (light / dark) for 3 days before subsequent experiments.

[0052] First, the silent fruits were tested using quantitative real-time PCR (RT-qPCR). Figure 6 (A) The results showed that the expression of the VdYUCCA10 gene was silenced in the fruit. Subsequently, using the needle inoculation method, 5 mm diameter discoid spores were inoculated onto the needle-punched sites and cultured in a 25℃ constant temperature incubator with maintained humidity. The results indicated that the Kyoho grapes with the silenced VdYUCCA10 gene showed milder disease symptoms and smaller lesion areas 5 days after inoculation with anthracnose, suggesting that silencing VdYUCCA10 enhanced resistance to anthracnose. Figure 6 (B, C)

[0053] 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. An application of the VdYUCCA10 gene from *Grape thorn* in enhancing the resistance of grape fruits to pathogens after silencing, characterized in that: The nucleotide sequence of the VdYUCCA10 gene of the grape variety is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.

2. The pathogen is anthracnose. The grape variety is Kyoho grape.

2. The application according to claim 1, characterized in that: The silencing vector TRV::VdYUCCA10 was constructed to silence the VdYUCCA10 gene.

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