A vdbhlh51 gene of ampelopsis brevifolia and the coded protein and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a Vitis davidii VdbHLH51 gene, a coding protein and application thereof. The Vitis davidii VdbHLH51 gene is located on chromosome 13 of Vitis davidii, is distributed in the region of 772522-773497, and the nucleotide sequence 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 of the Vitis davidii VdbHLH51 gene in improving the resistance of grape to pathogenic bacteria after overexpression of grape callus is provided. The VdbHLH51 gene provided by the present application can improve the resistance of grape to grape anthracnose, and provides a theoretical basis for grape anthracnose resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a VdbHLH51 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 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, 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 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, 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] bHLH (basichelix-loop-helix) is an important family of transcription factors in eukaryotes, playing a crucial role in plant growth and development, abiotic stress responses, and hormone signaling regulation. Its members recognize and bind to DNA through conserved bHLH domains, participating in gene expression regulation. A recent study (Gao Min. Expression of Grape Disease Resistance-Related Genes and Transcriptome Research on Resistance to Black Hole, 2016, Vol. 06) identified 126 bHLH transcription factors in the grape genome. The results showed that the expression patterns of grape bHLH transcription factors differed in different tissues; most bHLH genes were involved in responses to ABA and MeJA treatments; some bHLH genes responded to high salt (NaCl) or drought stress treatments; and a few bHLH genes were involved in responses to pathogen infection. However, there is currently no functional verification of bHLH genes' involvement in anthracnose stress responses.

[0005] This invention analyzes the functional characteristics and mechanism of action of transcription factor VdbHLH51 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 this invention is to provide a VdbHLH51 gene of grape thorn, its encoded protein and its application, which can improve the resistance of grapes to anthracnose.

[0007] This invention is implemented as follows:

[0008] The present invention first provides a VdbHLH51 gene of grape thorn, which is located on chromosome 13 of grape thorn and distributed in the region 772522-773497, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] Specifically, the protein encoded by the VdbHLH51 gene of the grape thorn has the amino acid sequence shown in SEQ ID NO.2.

[0010] Finally, this invention provides the application of the VdbHLH51 gene from *Grape argus* in enhancing resistance to grape pathogens through overexpression. Verification by this invention shows that under pathogen stress, the expression level of the VdbHLH51 gene is upregulated in resistant varieties and downregulated in susceptible varieties; overexpression of VdbHLH51 in callus tissue of seedless white grapes enhances the grape's resistance to anthracnose.

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

[0012] Furthermore, the grapes include seedless white grapes.

[0013] Furthermore, the overexpression vector pcambia2300-GFP was constructed to overexpress the VdbHLH51 gene in grape callus tissue.

[0014] Specifically, VdbHLH51 was linked to the binary vector pcambia2300-GFP for overexpression of the VdbHLH51 gene from Grape thorn.

[0015] The present invention has the following advantages:

[0016] This invention provides a VdbHLH51 gene from Grape thorn, which is 759 bp in length and encodes 252 amino acids. The amino acid sequence is shown in SEQ ID No. 2 and contains the bHLH_SF domain.

[0017] This invention analyzed the expression of VdbHLH51 in the peel of grape berries infected with anthracnose. Experiments showed that VdbHLH51 expression increased with the progression of infection, indicating that VdbHLH51 can respond to grape anthracnose infection.

[0018] This invention cloned the VdbHLH51 sequence and constructed the vector VdbHLH51-GFP carrying GFP overexpression. Transforming grape callus tissue into this vector resulted in milder disease symptoms compared to the control group after inoculation with *Actinidia chinensis*. This verifies that VdbHLH51 is a positive regulator in the grape response to anthracnose infection. Therefore, the VdbHLH51 gene provided by this invention can enhance grape resistance to anthracnose, providing a theoretical basis for anthracnose resistance breeding in grapes. 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 Vitis davidii and VdbHLH51 of some species, where the red box represents Vitis davidii VdbHLH51.

[0021] Figure 2 A schematic diagram of the VdERF054 gene constructed into the pCambia2300-GFP vector.

[0022] Figure 3 This is a diagram showing the domain structure analysis of the VdbHLH51 protein from Grape thorn.

[0023] Figure 4 Chromosome localization of VdbHLH51 from grape thorn.

[0024] Figure 5 Figure B shows the expression levels of VdbHLH51 in response to grape anthracnose induction (A) and in different tissues (where MF: mature fruit, ML: mature leaf, S: stem, T: tendril, YL: young leaf).

[0025] Figure 6 VdbHLH51 overexpression in grape callus improved resistance to anthrax; A: RT-qPCR detection of VdbHLH51 expression level in grapes; B: Phenotype of VdbHLH51 overexpressing callus 3 days after inoculation with anthrax (left: control WT, right: overexpressing VdbHLH51). Detailed Implementation

[0026] 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.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] The bacterial strains and plasmids used in the examples are as follows:

[0030] The overexpression vector used in the experiment was pCambia2300-GFP; the Agrobacterium strain used for the overexpression experiment was GV3101 (p19+pSoup); and the Escherichia coli Top10 competent cells used were purchased from TianGen.

[0031] 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.

[0032] The culture medium used in the induction experiment in this example was 1 / 2 MS liquid medium.

[0033] Example 1: Cloning and Vector Construction of VdbHLH51 CDS Sequence

[0034] 1. Use the following primers to clone the full-length, specific, and fragment-specific segments of VdbHLH51 from a grape cDNA library.

[0035] SEQ ID No. 3:

[0036] P2300-VdbHLH51-BamHI-F:

[0037] tcggtacccggggatccATGGAGAATTGCCTTTATTCTGGT;

[0038] SEQ ID No.4:

[0039] P2300-VdbHLH51-SalI-R:

[0040] gctcaccatggtgtcgacTCATTGTGGGAAATTAGAGGGC

[0041] Note: Lowercase sequences are adapter primers (vector sequences), and uppercase sequences are gene sequences.

[0042] 2. Constructing a carrier

[0043] Homologous recombination reactions were performed using seamless cloning kits from Bometech and Invitrogen (e.g.) Figure 2The 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°C 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.

[0044] Example 2: Bioinformatics Analysis of VdbHLH51

[0045] The amino acid sequence of VdbHLH51 was aligned 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, VdbHLH51 is 759 bp in length and encodes 252 amino acids. Multiple sequence similarity of VdbHLH51 was determined using MEGA6. The VdbHLH51 gene of *Vitis pisca* is located on chromosome 13 of *Vitis pisca*. Figure 4 The phylogenetic tree was constructed using the maximum likelihood (NJ) method, distributed in the region 772522-773497. The amino acid sequence of VdbHLH51 was compared with that of *Vitis simonii*, *Vitis rotundifolia*, and *Actinidia kiwifruit* using the Clustw program. The results showed that VdbHLH51 has high homology with bHLH51 of *Vitis simonii*. Figure 1 ).

[0046] Example 3: Analysis of the expression pattern of the VdbHLH51 gene

[0047] Anthracnose (discoidospora davidii) was inoculated into grapes (Vitis davidii, a resistant variety) and seedless white grapes (Thompsonseedless, a susceptible variety) using the needle prick method. Grape skins infected with anthracnose were collected as experimental materials. RT-qPCR was performed and analyzed.

[0048] The expression pattern of the VdbHLH51 gene was analyzed. The results are as follows: Figure 5 As shown, on days 0, 1, 2, 3, and 6 after inoculation with *A. sarcodactylis*, the expression level of VdbHLH51 in the pericarp of *Grape argentea* reached its highest value on day 6, while the expression level of VdbHLH51 in seedless white grapes continuously decreased. Figure 5 (A). Expression of VdbHLH51 was found to be higher in mature fruit (MF: mature fruit) in different tissues. Figure 5 (B) indicates that VdbHLH51 can be induced to express in response to grape anthracnose.

[0049] Example 4: Analysis of the effect of overexpression of grape callus on the resistance of VdbHLH51 to anthrax.

[0050] The loose grape callus used in this invention was induced from the stems and leaves of seedless white grape tissue culture seedlings. The induction medium consisted of MS medium + 30 g / L sucrose + 0.3 mg / L 6-BA + 2.0 mg / L NAA + 7 g / L agar, with the pH adjusted to 5.8. Agrobacterium-mediated transformation of the grape callus was performed, and the specific genetic transformation method is shown below:

[0051] 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, 1 / 2 MS, and 20 g / L sucrose). Finally, RT-qPCR was performed on the transgenic plants to determine their expression levels. RT-qPCR results showed that VdbHLH51 was successfully transferred into grape callus tissue. Figure 6 (A). Inoculation of transformed callus with anthracnose revealed smaller lesions in grape callus overexpressing VdbHLH51, indicating that grape callus overexpressing VdbHLH51 exhibited enhanced resistance to anthracnose. Figure 6 (B)

[0052] 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 overexpression of the VdbHLH51 gene from *Grape thorn* in enhancing grape resistance to pathogens, characterized in that: The VdbHLH51 gene of *Vitis piraceae* is located on chromosome 13 of *Vitis piraceae*, distributed in the region 772522-773497, and its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2; the pathogen is pathogenic anthracnose; and the grape is a seedless white grape.

2. The application according to claim 1, characterized in that: The overexpression vector pcambia2300-GFP was constructed to overexpress the VdbHLH51 gene in seedless white grape callus.

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