Vitis davidii susceptible gene VdTLP19 as well as encoding protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a vitis davidii susceptible gene VdTLP19 as well as an encoding protein and application of the vitis davidii susceptible gene VdTLP19. The vitis davidii susceptible gene VdTLP19 is located on a chromosome 2 of vitis davidii and is distributed in a region 3655997-3656642, and the nucleotide sequence of the vitis davidii susceptible gene VdTLP19 is shown as SEQ ID No.1. The amino acid sequence of the protein coded by the gene is as shown in SEQ ID No.2. And after the vitis davidii susceptible gene VdTLP19 is silenced, the resistance of grape fruits to pathogenic bacteria is enhanced. After the VdTLP19 gene provided by the invention is silenced, the resistance of grapes to anthracnose can be improved, and a theoretical basis is provided for grape anthracnose resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a disease-susceptibility gene VdTLP19 in grapes thorn, its encoded protein, and its applications. Background Technology

[0002] Grape( Vitis vinifera L. Grapes, native to western Asia, are one of the oldest and most widely planted fruit tree species in the world. Anthracnose, caused by fungi of the genus *Colletotrichum* in the subphylum 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] Thaumatin-like proteins (TLPs) are a class of proteins with acidic cleavage and typical natural structural features, playing a defensive role in plant responses to pathogen infection. At least 27 TLP genes exist in European grapes, encoding most TLP proteins that inhibit pathogen spore germination and germ tube elongation, collectively participating in the plant's antifungal response in a synergistic manner. VvTL-2 and VvTL-1 show high similarity and similar functions; their introduction into grapes enhances the plant's resistance to powdery mildew, gray mold, and black rot. Although TLP family genes have been found in many species, their functional involvement in anthracnose stress response has not yet been validated.

[0005] This invention analyzes the functional characteristics and mechanism of action of transcription factor VdTLP19 in anthracnose stress response, providing a basis for enriching the theory of grape anthracnose stress resistance and grape anthracnose resistance breeding. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a grape thorn disease susceptibility gene VdTLP19, its encoded protein and its application, which can improve the grape's resistance to anthracnose by silencing VdTLP19.

[0007] This invention is implemented as follows: The present invention first provides a disease susceptibility gene VdTLP19 for grape thorn, located on chromosome 2 of grape thorn and distributed in the region 3655997-3656642, and its nucleotide sequence is shown in SEQ ID NO.1.

[0008] Specifically, the protein encoded by the grape thorn disease gene VdTLP19 has the amino acid sequence shown in SEQ ID NO.2.

[0009] Finally, this invention provides the application of the grape thorn susceptibility gene VdTLP19 in enhancing grape fruit resistance to pathogens after silencing. Experiments show that under anthracnose stress, the expression level of the VdTLP19 gene first decreases and then increases, while silencing VdTLP19 enhances grape resistance to anthracnose.

[0010] Furthermore, the pathogen is pathogenic anthrax bacteria.

[0011] Furthermore, the grapes are either thorn grapes or Kyoho grapes.

[0012] Furthermore, the silencing vector TRV::VdTLP19 was constructed to silence the VdTLP19 gene.

[0013] Furthermore, the silencing carrier is pTRV2 and those carrying the target gene VdTLP19 pTRV2.

[0014] The present invention has the following advantages: This invention provides a VdTLP19 gene from Grape thorn, which is 1044 bp in length and encodes 347 amino acids. The amino acid sequence is shown in SEQ ID No. 1.

[0015] This invention analyzed the expression of VdTLP19 in the peel of grape berries infected with anthracnose. The results showed that VdTLP19 was highly expressed in mature berries, and its expression first decreased and then increased after inoculation with anthracnose, indicating that VdTLP19 can respond to grape anthracnose infection.

[0016] This invention clones the specific sequence of VdTLP19, constructs the silencing vector TRV::VdTLP19, and transiently transforms Kyoho grapes into their fruit. After inoculation with *Gnaphalium affine*, the resulting grapes showed milder disease symptoms compared to the TRV::00 (control). This verifies that VdTLP19 is a negative regulator in the grape's response to anthracnose infection. Therefore, silencing the VdTLP19 gene provided by this invention can enhance grape resistance to anthracnose, providing a theoretical basis for breeding grapes with anthracnose resistance. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Figure 1 Cluster analysis of homologous protein sequences of Vitis davidii and VdTLP19 from some species, with the red box representing VdTLP19 of Vitis davidii.

[0019] Figure 2 A schematic diagram of constructing the VdTLP19 gene-specific sequence into the pTRV2-GATEWAY vector.

[0020] Figure 3 This is a diagram showing the domain structure analysis of the VdTLP19 protein from Grape thorn.

[0021] Figure 4 To locate the VdTLP19 chromosome of Grape thorn.

[0022] Figure 5 Analysis of VdTLP19 expression in response to grape anthracnose. A: VdTLP19 expression level analysis over time; B: VdTLP19 expression level analysis in different tissues, where ML: mature leaves, YL: young leaves, MF: mature fruit, T: tendrils, S: stem.

[0023] Figure 6 To investigate the silencing of VdTLP19 in Kyoho grapes and their resistance to anthracnose. A: Interference efficiency of grapes detected by RT-qPCR; B: Phenotype of Kyoho grapes with the virus-induced gene silencing VdTLP19 5 days after inoculation with anthracnose, where the part circled in red is the lesion; C: Lesion area. Detailed Implementation

[0024] 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.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] The bacterial strains and plasmids used in the examples are as follows: 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 from TianGen.

[0028] 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.

[0029] The culture medium used in the induction experiment in the examples was 1 / 2 MS liquid medium.

[0030] Example 1: Cloning and Vector Construction of VdTLP19 CDS Sequence The following primers were used to clone the specific fragment of VdTLP19 from the grape cDNA library.

[0031] SEQ ID No. 3: Attb1-VdTLP19: ggggacaagtttgtacaaaaaagcaggcttcACATCCAAAACCGTTGCTCC; SEQ ID No.4: Attb2-VdTLP19: ggggaccactttgtacaagaaagctgggtcCATTCCCTGACGCATCGAAG.

[0032] Note: Lowercase sequences are adapter primers (vector sequences), and uppercase sequences are gene sequences.

[0033] 2. Constructing a carrier Homologous recombination reactions were performed using seamless cloning kits from Bometech and Invitrogen (e.g.) Figure 2 The gel recovery product obtained above was mixed with the linearized vector (pTRV2 vector) and the reaction solution was added at the same time. 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 it be used for subsequent experiments.

[0034] Example 2: Bioinformatics Analysis of VdTLP19 The amino acid sequence of VdTLP19 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, VdTLP19 is 1044 bp in length and encodes 347 amino acids. Multiple sequence similarity of VdTLP19 was determined using MEGA6. The VdTLP19 gene of *Vitis pisca* is located on chromosome 2 of *Vitis pisca*. Figure 4The phylogenetic tree was constructed using the maximum likelihood (NJ) method, distributed in the region 3655997-3656642. The VdTLP19 amino acid sequence was compared with that of Ripese grapes and wine grapes using the Clustw program, revealing a high degree of homology between VdTLP19 and Ripese grape TLP19. Figure 1 ).

[0035] Example 3: Analysis of VdTLP19 gene expression pattern Acupuncture was used to treat thorny grapes ( Vitis davidii Anthrax (Discocephalus discoidis) was inoculated, and grape skins infected with anthracnose were collected as experimental material. RT-qPCR was performed, and the expression pattern of the VdTLP19 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 VdTLP19 in the pericarp showed a trend of first decreasing and then increasing after treatment as infection progressed. Results from different tissue expression patterns revealed that the expression level of VdTLP19 was higher in mature fruit (MF: mature fruit). Figure 5 (B) indicates that VdTLP19 can be induced to express in response to grape anthracnose.

[0036] Example 4: Analysis of virus-induced gene silencing of VdTLP19 in grapes and its resistance to anthracnose. 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 VdTLP19 trans-3-UTR specific fragment into the pTRV2 vector. Agrobacterium transformation was performed, and single clones were selected for verification, followed by shaking. The concentration of the final resuspended Agrobacterium was adjusted to approximately OD600 = 1.0. Empty pTRV2 and pTRV2 carrying the target gene VdTLP19 were then mixed with pTRV1 at a 1:1 ratio and incubated at 28°C and 80-100 rpm for 3-4 hours. Subsequently, Kyoho grapes (VdTLP19 has the same gene sequence as the TLP19 gene in Kyoho grapes) that had undergone 2-3 weeks of color change were immersed in a separate mixture of pTRV1 and pTRV2-VdTLP19, vacuum-soaked for 10 minutes. Co-immersion of empty pTRV2 and pTRV1 vectors served as a negative control. TRV::00 Experiment. Wipe the surface of the grape berries with clean absorbent paper to remove any residual bacterial solution, incubate in the dark at 26°C and 60% relative humidity for 24 hours, and then at 26°C with 100 µmol / L... -2s-1 After culturing for 3 days under 16 / 8h (light / dark) conditions, subsequent experiments can be conducted.

[0037] First, the silent fruits were tested using quantitative real-time PCR (RT-qPCR). Figure 6 (A) The results showed that silencing the VdTLP19 gene expression in the fruit significantly reduced the expression level of TLP19 in the silenced fruit (TRV::VdTLP19). Subsequently, the needle inoculation method was used, and 5 mm diameter discoid spore blocks were inoculated at the needle puncture site and placed in a constant temperature incubator at 25℃ with humidity maintained. The results showed that the Kyoho grapes with the VdTLP19 gene silenced exhibited milder disease symptoms and smaller lesion area 5 days after inoculation with anthracnose, indicating that silencing VdTLP19 enhanced the grape's resistance to anthracnose. Figure 6 (B, C)

[0038] 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 susceptibility gene VdTLP19 for grapes with thorns, characterized in that: Located on chromosome 2 of *Vitis piscis*, in the region 3655997-3656642, its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the grape thorn disease susceptibility gene VdTLP19 as described in claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

2.

3. The application of the grape thorn susceptibility gene VdTLP19 as described in claim 1 in enhancing the resistance of grape fruits to pathogens after silencing.

4. The application according to claim 3, characterized in that: The pathogen is pathogenic anthrax bacteria.

5. The application according to claim 3, characterized in that: The grapes mentioned are either thorn grapes or Kyoho grapes.

6. The application according to claim 3, characterized in that: The silencing vector TRV::VdTLP19 was constructed to silence the VdTLP19 gene.

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