Vitis davidii jincheng 1 gene, protein encoded by the gene and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a Vitis davidii VdJUB1 gene, a protein coded by the gene and application. The nucleotide sequence of the Vitis davidii VdJUB1 gene is shown as SEQ ID No. 1. The protein coded by the gene has an amino acid sequence shown as SEQ ID No. 2. The application also relates to application of the Vitis davidii VdJUB1 gene in improving the resistance of grape to pathogenic bacteria after overexpression of the gene in grape callus. The Vitis davidii VdJUB1 gene provided by the application can improve the resistance of grape to anthracnose, and provides a theoretical basis for grape anthracnose resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a type of prickly grape. VdJUB1 Genes and their encoded proteins and their applications. Background Technology

[0002] Grape( Vitisvinifera L. Originally from West Asia, grapes are cultivated worldwide and are one of the most widely grown 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 fruit after ripening, but can also infect leaves, shoots, tendrils, and fruit stalks, although 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] NAC (NAP, ATAF1 / 2, and CUC2) transcription factors are among the largest transcription factors in plants. Their N-terminus possesses a highly conserved DNA-binding domain that precisely recognizes specific gene sequences, while their C-terminus produces diverse regulatory effects through combinations with different proteins. The conserved N-terminal DNA-binding domain can be further divided into five subdomains, A through E. Subdomains A and C primarily maintain protein stability, subdomains B and E are associated with functional differentiation, and subdomains C and D are rich in basic amino acids and are highly conserved. Studies have shown that NAC family members play important roles in plant disease resistance. For example, in bananas, MaNAC5 synergistically works with MaWRKY1 and MaWRKY2 to regulate the expression of a specific set of PR genes in disease responses and promote salicylic acid and methyl jasmonic acid-induced disease resistance. However, there is currently little functional validation of NAC transcription factors' involvement in anthracnose stress responses in grapes.

[0005] JUB1, a member of the NAC transcription factor family, plays a crucial role in delaying senescence and responding to abiotic stress in plants. However, the function of the JUB1 gene in grapes remains unclear. This invention elucidates the functional characteristics and mechanism of action of the transcription factor VdJUB1 in anthracnose stress response, providing a basis for enriching the theory of grape anthracnose resistance and for breeding grape varieties with anthracnose resistance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a prickly grape. VdJUB1 The gene and its encoded protein and its application: overexpression of this gene can improve the resistance of grapes to anthracnose.

[0007] This invention is implemented as follows: This invention first provides a prickly grape VdJUB1 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides the protein encoded by the VdJUB1 gene of the grape thorn, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] Finally, the present invention provides the aforementioned prickly grape. VdJUB1 The application of this gene in enhancing resistance to grape anthracnose after overexpression. This invention has been verified to enhance resistance under pathogen stress. VdJUB1 Gene expression was suppressed in disease-resistant varieties before inoculation with anthracnose, but upregulated 6 days after treatment; it was also overexpressed in callus tissue of seedless white grapes. VdJUB1 This enhanced the grape's resistance to anthracnose.

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

[0011] Furthermore, the grapes include seedless white grapes.

[0012] Furthermore, the overexpression vector pcambia2300-GFP was constructed for overexpression in grape callus tissue. VdJUB1 Gene.

[0013] Specifically, VdJUB1 was linked to the binary vector pcambia2300-GFP for overexpression of Grapevine. VdJUB1 Gene.

[0014] The present invention has the following advantages: This invention provides a prickly grape VdJUB1 The gene is 684 bp in length and encodes 227 amino acids. Its nucleotide sequence is shown in SEQ ID No. 1 and its amino acid sequence is shown in SEQ ID No. 2.

[0015] This invention analyzes the components of the pericarp of Grape argentea after infection with anthracnose. VdJUB1 Gene expression status. RT-qPCR results showed that its expression was suppressed in the early stage of infection, and by day 6, VdJUB1 Upregulation of expression levels indicates VdJUB1 It can respond to Staphylococcus aureus infection.

[0016] This invention utilizes cloning VdJUB1 The full-length CDS sequence was used to construct the vector VdJUB1-GFP carrying GFP overexpression. Transforming grape callus tissue into this vector resulted in milder disease symptoms compared to the control group after inoculation with *Staphylococcus aureus*. This verifies that VdJUB1 is a positive regulator in the grape response to *Staphylococcus aureus* infection. Therefore, the present invention provides… VdJUB1 Genes can enhance grapes' 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 thorny* and VdJUB1 from some species, where VdJUB1 represents *Vitis thorny*. Vitis davidii JUB1.

[0019] Figure 2 for VdJUB1 A schematic diagram of gene construction into the pCambia2300-GFP vector.

[0020] Figure 3 This is a diagram showing the domain structure analysis of the VdJUB1 protein from Grape thorn.

[0021] Figure 4 prickly grape VdJUB1 Chromosomal localization.

[0022] Figure 5 for VdJUB1 Response to anthracnose-induced expression levels in grapes (A) and expression levels in different tissues (B, where ML: mature leaves, MF: mature fruit, S: stem, T: tendrils, YL: young leaves).

[0023] Figure 6 for VdJUB1 Overexpression of the drug in grape callus tissue enhanced resistance to anthrax; A: RT-qPCR detection of expression levels in grapes; B: Overexpression. VdJUB1 Phenotypic characteristics of callus 5 days after inoculation with anthrax; VdJUB1-OE1 and VdJUB1-OE2 represent callus from different strains. 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 overexpression vector used in the experiment was pCambia2300-GFP; the Agrobacterium used in the overexpression experiment was strain 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 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.

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

[0030] Example 1 VdJUB1 CDS sequence cloning and vector construction Cloning of grape cDNA from the library was performed using the following primers. VdJUB1 The full length of the CDS.

[0031] SEQ ID No. 3: P2300- VdJUB1 -BamHI-F: tcggtacccggggatccATGGGAGATAAAGAGTGGTA; SEQ ID No. 4: P2300- VdJUB1 -SalI-R: gctcaccatggtgtcgacCTATCTACAGTCATAAACATGAGG; Note: Lowercase sequences are adapter primers (vector sequences), and uppercase sequences are gene sequences.

[0032] 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 (pCambia2300-GFP) 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℃ 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.

[0033] Example 2 VdJUB1 Bioinformatics Analysis Will VdJUB1 The amino acid sequences were compared using the BLAST database, and homologous sequences from different species were downloaded to construct a phylogenetic tree. The results are as follows: Figure 3 As shown, VdJUB1 It is 684 bp in length and encodes 227 amino acids. VdJUB1 The amino acid sequence similarity was analyzed using MEGA6 multiple comparisons. *Vitis thorn* VdJUB1 The gene is located on chromosome 12 of the grape thorn (Vitis thunbergii). Figure 4 The phylogenetic tree was constructed using the maximum likelihood (NJ) method, distributed in the region 9489581-9492752. VdJUB1 The amino acid sequence was compared with Pinot Noir (PN40024) and other amino acid sequences using the Clustw program, and it was found that... VdJUB1 and Pinot Noir JUB1 It has a high degree of homology. Figure 1 ).

[0034] Example 3 VdJUB1 Gene expression pattern analysis Acupuncture was used to treat thorny grapes ( Vitis davidii (Disease-resistant varieties) were inoculated with *Colletotrichum discoidus*, and grape skins infected with *Colletotrichum discoidus* were collected as experimental materials. RT-qPCR was performed and... VdJUB1 Gene expression patterns were analyzed. The results are as follows: Figure 5 As shown, on days 0, 1, 2, 3, and 6 after inoculation of Grapevine with anthracnose, the concentration of [unclear text - possibly related to fruit peel or fruit] in the pericarp [unclear text - possibly related to a specific culture or product]. VdJUB1 The expression level was suppressed in the early stage of pathogen infection as the infection progressed, but was induced to be upregulated on day 6 after inoculation. Figure 5 (A). Further findings revealed expression in different tissues. VdJUB1 The expression level is high in mature fruit. Figure 5 (B)

[0035] Example 4: Overexpression of grape callus tissue enhanced VdJUB1 Resistance to anthrax The loose callus tissue used in this invention was induced from the stems and leaves of 'Seedless White' grape tissue culture seedlings. The induction medium was: MS + 30 g / L sucrose + 0.3 mg / L 6-BA + 2.0 mg / L NAA + 7 g / L agar, and the pH was adjusted to 5.8. Agrobacterium-mediated transformation of the grape callus tissue was performed, and the specific genetic transformation method is shown below: Grape callus was cut into small pieces, not too large. The cut callus was placed in Agrobacterium-mediated bacterial suspension and gently agitated for 10 minutes. The suspension was discarded, and the remaining bacterial suspension on the surface of the callus was collected using sterile filter paper. The callus was then placed in co-culture medium and incubated in the dark for 48 hours. After dark incubation, the callus was placed in a sterilization solution (300-500 mg / L Cef, 200-300 mg / L Carb, 1 / 2 MS, and 20 g / L sucrose) and agitated for 10 minutes. The sterilization solution was discarded, and this process was repeated three times. The callus was rinsed three times with sterile water, and the remaining bacterial suspension on the surface of the callus was collected using sterile filter paper. The callus was then placed in selection medium (50 mg / L Kan (overexpression vector), 300 mg / L Cef, and 200 mg / L Carb). Finally, RT-qPCR was performed on the transgenic plants to determine their expression levels.

[0036] RT-qPCR results showed VdJUB1 Successfully transferred to grape callus tissue ( Figure 6 (A). Anthrax was inoculated into the transformed callus, and overexpression was observed after 5 days. VdJUB1 The grape callus showed smaller lesions, indicating that overexpression of [the substance]... VdJUB1 Grape callus tissue exhibited enhanced resistance to anthrax. Figure 6 (B)

[0037] 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 VdJUB1 Genes are characterized by: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The prickly grape as described in claim 1 VdJUB1 Gene-encoded proteins are characterized by: Its amino acid sequence is shown in SEQ ID NO.

2.

3. The prickly grape as described in claim 1 VdJUB1 Application of genes in enhancing grape resistance to pathogens through overexpression.

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 include seedless white grapes.

6. The application according to claim 3, characterized in that: The overexpression vector pcambia2300-GFP was constructed for overexpression in seedless white grape callus. VdJUB1 Gene.

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