Function of VvAIL12 gene in promoting grape hairy root regeneration and efficient hairy root transformation method

Abstract

The application discloses a function of VvAIL12 gene in promoting grape hairy root regeneration and a high-efficiency hairy root transformation method, and relates to the fields of plant biotechnology and molecular breeding. Through function gain and loss experiments, it is confirmed that VvAIL12 is a key positive regulation transcription factor for grape hairy root regeneration. Overexpression of VvAIL12 can significantly improve the transformation efficiency of transgenic hairy roots of a low-efficiency variety '5BB' from 9.92% to 27.78%. The application also establishes an optimized Agrobacterium rhizogenes-mediated hairy root transformation method. Through the best parameter combination of explants, bacterial liquid concentration and co-culture time, a transformation efficiency as high as 45.40% is realized on the 'Muscatlan' variety, and the method is successfully applied to 12 different genotypes. Based on the transgenic hairy roots obtained by the system, a subcellular localization method based on grape protocorm-like bodies is developed, and the nuclear localization of VvAIL12 is first confirmed in grape cells.

Description

Technical Field

[0001] This invention relates to the fields of plant biotechnology and molecular breeding, specifically to a novel function of the grape-specific gene VvAIL12 in regulating plant regeneration, and its application in improving the efficiency of grape genetic transformation. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Grape( Vitis vinifera L. Grapes are one of the most widely cultivated and economically important horticultural fruit crops globally. Their uses are diverse, including wine production, fresh consumption, juicing, and dried fruit processing. Genetic improvement of grapes using genetic engineering and gene editing technologies is of great value, and the key to achieving this goal lies in establishing an efficient genetic transformation system.

[0004] Currently, genetic transformation of grapes mainly relies on somatic embryogenesis and organogenesis pathways. However, these methods have significant limitations: low transformation efficiency (usually below 5%), long cycles (over a year), and strong genotype dependence, achieving success only in a very few varieties such as 'Seedless White' and 'Chardonnay'. Furthermore, the technical systems are highly dependent on precise control of plant growth regulators, resulting in poor experimental reproducibility and severely limiting the widespread application of transgenic grape technology.

[0005] In Agrobacterium-mediated transformation, Agrobacterium rhizogenes (Agrobacterium tumefaciens) Rhizobium rhizogenes ) and commonly used Agrobacterium tumefaciens ( A. tumefaciens Compared to other methods, it exhibits higher transformation efficiency in most species. The hairy root transformation system mediated by it has significant advantages: a short transformation cycle (transgenic hairy roots can typically be obtained within 4 weeks), the transformed roots can undergo hormone autotrophic growth and are genetically stable, making it highly suitable for rapid gene function studies. More importantly, this system has shown the potential to reduce genotype dependence in different species. For example, transformation systems based on Agrobacterium rhizogenes have been successfully established in various crops such as strawberry, poplar, apple, citrus, soybean, cabbage, and salvia miltiorrhiza, achieving efficient genetic transformation and gene editing. However, the application of this technology in grapes has progressed slowly, with limited reports of efficiency in only a few varieties such as 'Muscat' and 'Crimson Seedless'. Developing an efficient, robust hairy root transformation system applicable to multiple grape varieties is a pressing technical challenge in the field of grape biotechnology.

[0006] Overcoming bottlenecks in crop regeneration efficiency typically relies on two levels of strategies: optimizing external tissue culture systems and manipulating endogenous genetic regulatory networks. At the technical level, precise control of culture medium composition, carbon sources, plant growth regulator ratios, and light and temperature conditions can effectively improve callus induction and organ differentiation. For example, PureWheat technology significantly improved wheat conversion efficiency, and the N6 medium system improved maize regeneration. At the molecular level, nearly four decades of research have revealed that manipulating core regeneration regulatory genes can fundamentally enhance plant regeneration capabilities. Among them, the AINTEGUMENTA-LIKE (AIL) transcription factor family has been proven to be key to regulating plant cell totipotency, meristematic tissue maintenance, and organ regeneration. In numerous species such as Arabidopsis thaliana, maize, apple, sweet pepper, and Chinese cabbage, ectopic or overexpression of AIL family members (such as BBM and PLT5) has been shown to significantly improve somatic embryogenesis and organ regeneration efficiency, effectively shorten the transformation cycle, and successfully overcome the limitations of difficult-to-transform genotypes. This provides a clear theoretical basis and a feasible technical path for establishing an efficient and widely applicable genetic transformation system by mining and utilizing the key AIL gene in grapes themselves.

[0007] However, there are significant gaps in the existing technology: First, the systematic identification of members of the grape AIL gene family and their functional studies in regeneration, especially hairy root regeneration, are still lacking; second, there is a lack of a complete technical solution that effectively integrates key regeneration regulatory genes with efficient hairy root transformation processes and has been systematically optimized.

[0008] Therefore, there is an urgent need in this field for a systematic research project aimed at: 1) identifying key AIL genes in grapes that regulate hairy root regeneration; 2) establishing a highly efficient hairy root transformation system applicable to multiple varieties; and 3) applying key genes to this highly efficient system to explore its combined effects, thereby providing a complete technical system for grape functional genomics research and molecular breeding, from core target discovery to efficient genetic manipulation and precise functional verification. Summary of the Invention

[0009] To address the technical bottlenecks in existing technologies, such as low efficiency of grape genetic transformation, strong genotype dependence, and lack of reliable in situ functional verification tools, this invention provides a method for key gene discovery and efficient transformation.

[0010] The technical solution adopted in this invention is as follows:

[0011] In a first aspect of the invention, an isolated VvAIL12 gene or its encoded protein is provided for use in the preparation of a product for improving the efficiency of grape hairy root transformation.

[0012] Preferably, the product is an engineered Agrobacterium rhizogenes containing the VvAIL12 gene expression cassette, or an inoculum containing the engineered bacteria, or a screening medium required for transformation using the engineered bacteria.

[0013] Sixteen VvAILs were identified through genome-wide analysis, and VvAIL12 was found to be significantly upregulated during hairy root regeneration, with its expression level showing a significant positive correlation with transformation efficiency in different varieties. Key gain-and-loss experiments demonstrated that overexpression of VvAIL12 in the low-efficiency variety '5BB' increased its transformation efficiency from 9.92% to 27.78%, while knockdown of the gene in the high-efficiency variety 'Marselan' reduced efficiency from 45.40% to 20.72%. This definitively confirms that VvAIL12 is a key positive regulatory transcription factor regulating grape hairy root regeneration. This discovery not only reveals the key intrinsic mechanism of grape regeneration but also provides a direct molecular target and tool for significantly improving grape regeneration capacity by manipulating a single gene.

[0014] In a second aspect of the invention, a method for improving the transformation efficiency of grape hairy roots is provided, the method comprising increasing the expression level of the VvAIL12 gene or increasing the activity of its encoded protein in grape explants.

[0015] Preferably, the expression level or activity is increased by introducing and expressing the VvAIL12 gene into the grape explant.

[0016] Preferably, the introduction and expression are achieved by an expression vector containing the VvAIL12 gene; more preferably, the expression vector is transformed into grape explants by Agrobacterium rhizogenes-mediated transformation.

[0017] Preferably, the method is specifically used to improve the hairy root transformation efficiency in grape genetic transformation experiments.

[0018] Preferably, the method for improving the transformation efficiency of grape hairy roots specifically includes the following steps:

[0019] (a) Using leaves from grape tissue culture seedlings that have been subcultured for 40-50 days as explants;

[0020] (b) Infect the explants with a culture of *Agrobacterium rhizogenes* carrying the VvAIL12 gene expression cassette, wherein the OD of the culture is... 600 The value is 0.7-0.9;

[0021] (c) Co-culture the infected explants with the Agrobacterium rhizogenes for 1.5-2.5 days;

[0022] (d) The co-cultured explants were transferred to 1 / 2 MS selection medium containing antibiotics to induce the generation of transgenic hairy roots.

[0023] The explants were obtained from tissue culture seedlings that had been subcultured for 45 days; the OD of the bacterial solution... 600 The value was 0.8; the co-culture time was 2 days; the antibiotic was termethin, with a concentration of 250-350 mg / L.

[0024] Furthermore, the VvAIL12 gene expression cassette may contain a reporter gene or selection marker gene for identifying successful transformation, such as the green fluorescent protein (GFP) gene. By detecting the expression of the reporter gene (e.g., fluorescence), transgenic hairy roots can be distinguished and counted.

[0025] The establishment of this method first solved the fundamental operational challenges of grape genetic transformation. It involved systematically optimizing three key parameters: the physiological state of the explants (optimally determined to be leaves from seedlings at 45 days of subculture), the concentration of the inoculum (optimal OD...). 600 By adjusting the coefficient of variation (0.8) and co-cultivation time (optimal 2 days), this system achieved a hairy root transformation efficiency of up to 45.40% in the model variety 'Marselan', shortening the entire transformation cycle to 3-4 weeks. More importantly, the system was validated as effective in 12 grape varieties with different genotypes, including 'Beauty Finger', 'Chardonnay', and '5BB', significantly reducing the strong genotype dependence of traditional methods and providing a stable, efficient, and universally applicable basic operating platform for grape functional research.

[0026] In this invention, the nucleotide sequence of the VvAIL12 gene is shown in SEQ ID NO: 1, or the amino acid sequence encoded by it is shown in SEQ ID NO: 2.

[0027] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects:

[0028] (1) Core regulatory target discovered: The key positive regulatory role of VvAIL12 gene in grape hairy root regeneration was first clarified, providing a new tool gene for molecular breeding.

[0029] (2) Breakthrough in transformation technology bottleneck: Provides a set of efficient, stable and widely applicable hairy root transformation methods, improves the efficiency of grape genetic transformation to the practical level (up to 45.40%), and effectively alleviates genotype limitations.

[0030] (4) A systematic solution has been formed: the efficient transformation system and key regeneration genes are organically combined to form a complete innovation chain from genetic manipulation, functional regulation to mechanism verification, which systematically improves the efficiency and capability of grape functional genomics research and molecular breeding. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0032] Figure 1 A grape transformation system was constructed using Agrobacterium rhizogenes. (A) Workflow of the grape transformation system using Agrobacterium rhizogenes. Ti (+) indicates the addition of 300 mg / L termethin to the culture medium. Scale bar = 1 cm. (B) Grape tissue culture seedlings after 30, 45, and 60 days of growth. Scale bar = 1 cm. (C) Growth status of transgenic callus and transgenic hairy roots at the wound site after leaf infection. Red and yellow arrows indicate transgenic callus and transgenic roots, respectively. Scale bar = 1 cm. (D) Transformation efficiency of callus, hairy root, and transgenic callus and hairy root after leaf infection of tissue culture seedlings at different growth times. (E) Transformation efficiency of hairy roots and transgenic hairy roots under different bacterial concentrations and co-culture times.

[0033] Figure 2 Statistical analysis of the transformation efficiency of transgenic hairy roots and transgenic callus of twelve grape genotypes. (A) Leaves of 'Beauty Finger', 'Sunshine Rose', 'Seedless White', 'Red Globe', 'Marselan', 'Chardonnay', 'Cabernet Sauvignon', 'Left Youhong', 'SO4', '5BB', '1103P', and '101-14' bearing GFP-transgenic callus. Scale bar = 1 cm. (B) Leaves of 'Beauty Finger', 'Sunshine Rose', 'Seedless White', 'Red Globe', 'Marselan', 'Chardonnay', 'Cabernet Sauvignon', 'Left Youhong', 'SO4', '5BB', '1103P', and '101-14' bearing GFP-transgenic hairy roots. Scale bar = 1 cm. (C) Transformation efficiency of callus and transgenic callus after infection of leaves of different grape genotypes. (D) Transformation efficiency of hairy roots and transgenic hairy roots after infection of leaves with different grape genotypes. Callus transformation efficiency = (Number of leaves with callus / Total number of infected leaves) × 100%. Transgenic callus transformation efficiency (%) = (Number of leaves with transgenic callus / Total number of infected leaves) × 100%. Hairy root transformation efficiency = (Number of leaves with hairy roots / Total number of infected leaves) × 100%. Transgenic hairy root transformation efficiency = (Number of leaves with transgenic hairy roots / Total number of infected leaves) × 100%.

[0034] Figure 3Phylogenetic analysis, chromosome localization, interchromosomal homologous regions, and collinearity analysis of AILs. (A) Phylogenetic relationships among grape, apple, and Arabidopsis AILs. The phylogenetic tree shows the AIL genes in grape, apple, and Arabidopsis. Different colored branches represent different subfamilies. (B) From the innermost to the outermost, circles represent gene density heatmaps, followed by line graphs and chromosome names. (C) Analysis of AIL genes among grape, Arabidopsis, cultivated apple, tomato, rice, maize, and sorghum. Gray lines represent collinear relationships between grape and the other six species, and red lines represent collinear AIL gene pairs.

[0035] Figure 4 Conserved motifs and structures of VvAILs, and major cis-acting elements in VvAIL promoters. (A) Phylogenetic relationships of VvAILs. (B) Conserved motifs of VvAILs. Motifs 1, 2, and 3 are represented by blue, yellow, and red squares, respectively. (C) Gene structure of VvAILs. UTR and CDS are represented by yellow and blue squares, respectively, and introns are represented by black lines. (D) Locations of various cis-element types on chromosomes. Different types of cis-elements are represented by circles of different colors. (E) Heatmap of the number of different cis-element types. Red boxes indicate a larger number of each cis-element type, while blue boxes indicate a smaller number.

[0036] Figure 5 Tissue-specific expression profiles of 16 VvAILs are presented in heatmaps, plotted using computer analysis of VvAIL tissue-specific expression data obtained from the BAR database. Normalized log2 transformation values ​​from hierarchical clustering are used. Different VvAIL expression levels are represented by a color gradient from blue to red.

[0037] Figure 6qRT-PCR results of 16 VvAILs at 0, 1, 2, 3, 7, 14, and 21 days after Agrobacterium rhizogenes infection of leaves. (A) qRT-PCR results for gene AIL1. (B) qRT-PCR results for gene AIL2. (C) qRT-PCR results for gene AIL3. (D) qRT-PCR results for gene AIL4. (E) qRT-PCR results for gene AIL5. (F) qRT-PCR results for gene AIL6. (G) qRT-PCR results for gene AIL7. (H) qRT-PCR results for gene AIL8. (I) qRT-PCR results for gene AIL9. (J) qRT-PCR results for gene AIL10. (K) qRT-PCR results for gene AIL11. (L) qRT-PCR results for gene AIL12. (M) qRT-PCR results for gene AIL13. (N) qRT-PCR results for gene AIL14. (O) qRT-PCR results for gene AIL15. (P) qRT-PCR results for gene AIL16. Error bar = standard deviation. Identical lowercase letters indicate no significant difference at the P < 0.05 level, according to Duncan's multiple range test. VvAILs significantly upregulated after *Agrobacterium rhizogenes* infection of leaves are marked with red pentagrams.

[0038] Figure 7VvAIL12 positively regulates the regeneration of transgenic hairy roots in grapes. (A) Expression patterns of VvAIL12 during the regeneration of transgenic hairy roots in leaves of different grape varieties. Error bar = standard deviation. Identical lowercase letters indicate no significant difference at the P<0.05 level, Duncan multiple range test. (B) Schematic diagram of the constructs used for VvAIL12-OE and VvAIL12-RNAi. (C) Infection of grape variety '5BB' with Agrobacterium rhizogenes containing empty EV vector and VvAIL12-OE vector, and infection of grape variety 'Marselan' with Agrobacterium rhizogenes carrying empty EV vector and VvAIL12-RNAi vector. (D) PCR analysis of GFP inserted into the genome. (E) Quantitative analysis of VvAIL12 electrophoretic bands using ImageJ software. (F) Statistical analysis of the transformation efficiency of transgenic callus hairy roots after infecting grape variety '5BB' with Agrobacterium rhizogenes carrying both EV empty vector and VvAIL12-OE vector, and grape variety 'Marselan' with Agrobacterium rhizogenes carrying both EV empty vector and VvAIL12-RNAi vector. (G) Statistical analysis of the transformation efficiency of transgenic callus hairy roots in the two combinations. Transgenic callus hairy root transformation efficiency = (Number of leaves with transgenic hairy roots / Total number of leaves in all transgenic callus tissues) × 100%.

[0039] Figure 8 Conservative domain analysis of VvAILs.

[0040] Figure 9 Chromosomal location of VvAILs. The red line represents duplicated VvAIL gene pairs in the genome.

[0041] Figure 10 The sequence identifiers of three conserved motifs detected in the VvAIL protein; the overall height of each column represents the degree of conservation of that site, while the height of a single letter within the column represents the relative frequency of the corresponding amino acid. (A) represents the sequence identifier of motif 1. (B) represents the sequence identifier of motif 2. (C) represents the sequence identifier of motif 3.

[0042] Figure 11 Morphological changes during adventitious root regeneration of detached grape leaves after 1, 2, 3, 7, 14 and 21 days of culture on screening medium.

[0043] Figure 12 RT-PCR analysis of VvAIL12 in the roots of EV, VvAIL12-OE and VvAIL12-RNAi lines.

[0044] Figure 13Statistical analysis of transgenic callus transformation efficiency. Grape variety '5BB' was infected with *Agrobacterium rhizogenes* carrying the VvAIL12-OE vector, while grape variety 'Marselan' was simultaneously infected with *Agrobacterium rhizogenes* carrying the VvAIL12-RNAi vector. The transformation efficiency of transgenic callus from the two combinations was statistically analyzed. Transgenic callus transformation efficiency (%) = (Number of leaves producing transgenic callus / Total number of infected leaves) × 100%. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0047] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0048] The culture medium used in this invention includes:

[0049] 1 / 2 MS co-culture medium: formulation: 1 / 2 MS 2.17 g / L, sucrose 15 g / L, agar powder 7 g / L.

[0050] After preparation, adjust the pH of the culture medium to 5.2, and then autoclave it at 121°C for 20 min.

[0051] 1 / 2 MS screening (rooting) medium: Formula: 1 / 2 MS 2.17 g / L, sucrose 15 g / L, agar powder 6 g / L, timentin 0.3 g / L. After preparation, adjust the pH of the medium to 5.85, then autoclave at 121°C for 20 min. Add timentin (0.3 g / L) when pouring the plates at room temperature.

[0052] Example 1: Establishment and Material Preparation of the Hairy Root Transformation System

[0053] 1. Agrobacterium rhizogenes-mediated genetic transformation

[0054] Transgenic vectors were delivered using *Agrobacterium rhizogenes* strain K599. First, the transformation products were plated on agar plates containing specific antibiotics for initial screening using antibiotic markers, and morphologically sound, independent single colonies were selected. Subsequently, colony PCR was used for rapid identification of the insert fragment, followed by agarose gel electrophoresis analysis of band size to preliminarily screen for potential positive clones. Clones with positive PCR results were cultured overnight, and plasmid DNA was extracted. Further electrophoretic analysis verified the consistency of the vector with the expected restriction enzyme digestion pattern of the insert fragment. Finally, the plasmids with correct restriction enzyme digestion were sent for sequencing. Sequence alignment confirmed the absence of mutations, thus obtaining the final verified positive clones. Freshly prepared positive clones were cultured in LB medium containing 50 mg / L kanamycin and streptomycin at 28°C until OD (outcome limit). 600 The bacterial suspension was then transferred to 3 mL of fresh LB medium containing kanamycin and streptomycin and incubated overnight until the OD reached 0.6-1.0. 600 The concentration was adjusted to 0.6-1.0. The next day, 200 μL of this culture was inoculated into 20 mL of LB medium containing kanamycin and streptomycin and incubated at 28°C. Acetyleugenol was added 2 hours before infection to adjust the final concentration to 50 μM. The culture suspension was centrifuged at 5000 rpm for 5 minutes in an 80 mL centrifuge tube to precipitate the *Agrobacterium rhizogenes*. The collected *Agrobacterium rhizogenes* was resuspended in 1 / 2 MS infection medium, and the OD value was adjusted accordingly. 600 Adjust to 0.8. Immerse the prepared leaf explants in the infection solution for 1 minute, then transfer them to solid 1 / 2 MS co-culture medium (pH 5.2). After co-culturing in the dark for 2 days, transfer the leaves to solid 1 / 2 MS selection medium (pH 5.85). Timentin (300 mg / L) has been added to the 1 / 2 MS selection medium to inhibit the growth of Agrobacterium rhizogenes. Co-culture was carried out at 25°C with a photocycle of 16 hours light / 8 hours dark, and the medium was changed every 10 days (from infection until sufficient transgenic hairy roots were observed). The success of genetic transformation was assessed by detecting the fluorescence of hairy roots using a portable excitation lamp (Luyor-3415RG, Shanghai, China) approximately 3–4 weeks after Agrobacterium infiltration.

[0055] 2. Plant materials and sampling procedures

[0056] The tissue culture seedlings used for Agrobacterium rhizogenes-mediated transformation were derived from the following grape varieties: 'La Belle Finger', 'Sunshine Rose', 'Seedless Blanc', 'Red Globe', 'Marselan', 'Chardonnay', 'Cabernet Sauvignon', 'Le Rouge', 'SO4', '5BB', '1103P', and '101-14'. All tissue culture seedlings were stored under controlled environmental conditions at 25°C with a 16-hour light / 8-hour dark light cycle.

[0057] For gene expression analysis, leaf tissue was collected from 'Marselan' tissue culture seedlings 45 days after culture. Sampling was performed at 0, 1, 2, 3, 7, 14, and 21 days post-infection. At each time point, tissue was collected from the vicinity of the wound site (the area where the leaf was cut). Each time point included three biological replicates, each consisting of 18 independent leaves.

[0058] For cross-varietal analysis of the VvAIL gene, leaf tissue was collected from near the wound site in all grape varieties only 21 days post-infection. All collected tissues were immediately flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction.

[0059] 3. Implementation Results and Effect Data

[0060] 3.1. Establishment of an Agrobacterium rhizogenes-mediated genetic transformation system in grapes

[0061] The high transformation efficiency of hairy roots is significantly affected by the state of the experimental materials and the experimental transformation methods (including the Agrobacterium rhizogenes infection protocol and co-culture time). Therefore, their optimization and selection are key prerequisites for maximizing efficiency. In this study, we obtained transgenic hairy roots from grape leaves through Agrobacterium rhizogenes-mediated transformation. First, we selected well-grown 1–2 month old grape tissue culture seedlings as experimental materials. Leaves were cut from the grape tissue culture seedlings as explants, with one-third of each leaf removed. The cut leaves were transferred to small conical flasks and infected by adding Agrobacterium rhizogenes. After infection, they were transferred to 1 / 2 MS co-culture medium and co-cultured for 2 days. After the co-culture period, the explants were transferred to 1 / 2 MS selection medium to induce hairy root formation. Figure 1 A). To distinguish between transgenic and non-transgenic roots, a fluorescent protein excitation lamp was used. Successfully transgenic roots showed strong green fluorescence, while non-transgenic roots did not. To improve transformation efficiency, we optimized three key parameters: subculture time, bacterial concentration, and co-culture time. Infection experiments were conducted using leaf explants from 30-day, 45-day, and 60-day-old 'Marselan' tissue culture seedlings grown in vitro. Figure 1 B). The highest transformation efficiencies of transgenic callus and transgenic hairy roots were observed in the leaves of tissue-cultured seedlings after 45 days of subculture, reaching 45.19% and 32.85%, respectively. Figure 1 D). This indicates that overly tender or overripe leaves in tissue culture seedlings are detrimental to transformation and rooting. To further improve the transformation efficiency of transgenic hairy roots, we optimized the bacterial concentration and co-culture time. The results showed that a maximum transformation efficiency of 45.40% was obtained under the conditions of a co-culture time of 2 days and an OD600 value of 0.8 for the infection solution. Figure 1 E).

[0062] 3.2 Analysis of differences in transgenic hairy root regeneration efficiency among different grape genotypes

[0063] Genotypic variation is one of the important factors affecting the transformation efficiency of grape hairy roots. We further investigated the regeneration capacity of transgenic hairy roots after Agrobacterium rhizogenes infected leaves of 12 grape genotypes (including table grapes, wine grapes, and rootstocks). Figure 2 AB). Among the tested grape genotypes, the order of callus transformation efficiency from high to low was: 'Beauty Finger' > 'Seedless White' > 'Marselan' > 'Chardonnay' > '5BB' > 'Leo Red' > 'Sunshine Rose' > '101-14' > 'Cabernet Sauvignon' > '1103P' > 'SO4' > 'Red Globe' ( Figure 2 D). Among the tested grape genotypes, the order of hairy root transformation efficiency from high to low is: 'Marselan' > 'Beauty Finger' > 'Chardonnay' > 'Sunshine Rose' > '1103P' > 'Left Red' > '101-14' > '5BB' > 'Cabernet Sauvignon' > 'Seedless White' > 'Red Globe' > 'SO4' ( Figure 2 C). The results showed that the regeneration efficiency of transgenic hairy roots of 'Marselan' and 'Beauty Finger' was significantly higher than that of other grape genotypes.

[0064] The above results indicate that the Agrobacterium rhizogenes-mediated hairy root transformation system (HRTS) established in this invention has the following outstanding advantages: (1) The transformation cycle is short, requiring only 3-4 weeks from infection to obtaining analyzable transgenic hairy roots; (2) By optimizing key parameters (45-day seedling age of explants, OD600 of bacterial solution = 0.8, and co-culture for 2 days), a high transformation rate of 45.40% was achieved on the 'Marselan' variety; (3) The system successfully induced transgenic hairy roots in all 12 grape genotypes tested, confirming that it has low genotype dependence and wide applicability.

[0065] Example 2: Target gene family mining and bioinformatics analysis

[0066] 1. Identification and phylogenetic analysis of VvAILs

[0067] Eight Arabidopsis AIL protein sequences were retrieved from the Arabidopsis Information Resources (TAIR), namely At4g37750, At1g72570, At5g17430, At3g20840, At1g51190, At5g57390, At5g10510, and At5g65510. BLAST searches were performed on the Phytozome and Winebrige databases using the PN_T2T2 genome of grape as a reference. To ensure high confidence results, candidate sequences were selected based on an e-value threshold of <1e. -30Filtering was performed. The presence of AP2 or AP2 superfamily domains in the proposed VvAIL sequence was verified using NCBI-CDD and SMART tools. Furthermore, key physicochemical properties, including molecular weight (MW), size, and isoelectric point (pI), were analyzed using the Expasy ProtParam platform.

[0068] A rootless tree was constructed using the neighbor-joining (NJ) method in MEGA software to infer the phylogenetic relationship between Arabidopsis thaliana and grape AIL proteins. Sequence alignment was performed using ClustalW, and the robustness of the phylogenetic tree was assessed using a bootstrap analysis with 1000 replicates.

[0069] 2. Chromosomal localization, gene duplication, and collinearity analysis of VvAILs

[0070] Based on the annotation information of the grape genome, the positional information of chromosomes of VvAILs family members was obtained. To gain a deeper understanding of the evolution of VvAILs, we selected dicotyledonous representative species Arabidopsis thaliana, cultivated apple, and tomato, as well as monocotyledonous representative species rice, maize, and sorghum, and compared them with the grape genome sequence. The collinearity relationships between grape and these species were obtained, and collinearity maps between species were drawn using TBtools. Finally, non-synonymous substitutions (Ka) and synonymous substitutions (Ks) were calculated and analyzed using DNASP V5.0, and selection pressure was analyzed based on Ka / Ks values. Except for Arabidopsis thaliana, the genome and annotation information for all species were obtained from the Phytozome database.

[0071] 3. Analysis of gene structure, conserved motifs, and promoters of VvAILs

[0072] The gene structures of the 16 identified VvAILs were analyzed using GSDS, and structural features were visualized using TBtools. Conserved motifs in the VvAIL proteins were identified using the MEME suite; motif lengths ranged from 6 to 200 amino acids. The promoter region 2 kb upstream of the start codon for each VvAIL was extracted from the Winebrige database using TBtools. Cis-regulatory elements within these promoter regions were annotated using the PlantCARE database and subsequently visualized using TBtools for detailed interpretation.

[0073] 4. Tissue-specific expression pattern analysis of VvAILs

[0074] Expression profiles of the VvAIL gene in various grape tissues were retrieved from the BAR database, and FPKM values ​​were quality controlled to ensure data accuracy and reliability. Log2-normalized expression levels were calculated and visualized using heatmaps generated by TBtools to provide a comprehensive overview of tissue-specific expression patterns.

[0075] 5. Implementation Results and Effect Data

[0076] 5.1 VvAIL gene identification

[0077] Extensive genetic studies have shown that AIL proteins are key regulators of developmental processes such as meristem formation and organogenesis in plants. To identify potential AILs in the grape genome (PN_T2T2), a BLASTP search was performed using the Arabidopsis AIL protein sequence as a reference. The initial search yielded approximately 18 candidate proteins. Subsequently, SMART and NCBI-CDD tools were used to confirm that these candidate proteins contain two AP2 / ERF domains separated by a linker region. Figure 8 Ultimately, 16 VvAILs were identified and named VvAIL1–VvAIL16 based on their chromosomal locations. The molecular weights of the 16 VvAILs ranged from 37.99 kDa (VvAIL1) to 75.62 kDa (VvAIL5), and their amino acid lengths ranged from 336 amino acids (VvAIL1) to 681 amino acids (VvAIL5). Furthermore, the isoelectric points (pI) of these proteins ranged from 5.7 (VvAIL14) to 9.49 (VvAIL1) (Table 1).

[0078] Table 1. Characteristics of grape AILs

[0079]

[0080] 5.2 Phylogenetic analysis, chromosome localization, homologous regions and collinearity analysis of VvAILs

[0081] To investigate the evolutionary and phylogenetic relationships among 8 AtAIL, 27 MdAIL, and 16 VvAIL proteins, we constructed a rootless phylogenetic tree ( Figure 3 A). Based on the analysis results, AIL proteins were divided into four main groups (I–IV). Group I contained 6 proteins, Group II contained 1 protein, Group III contained 4 proteins, and Group IV contained 5 proteins.

[0082] The chromosomal location of VvAILs was determined using TBtools. Figure 9VvAILs are distributed across nine grape chromosomes, but the distribution is uneven. Specifically, there is one gene each on chromosomes 1, 14, and 18; two genes each on chromosomes 6, 8, 9, 11, and 13; and three genes on chromosome 7. Notably, there are more genes located alipids or distals than in the middle regions. To understand the evolutionary mechanism of VvAILs, replication events were analyzed. Sequence analysis did not reveal tandem duplications, while 62.5% of VvAILs (10 proteins) were involved in five fragment duplication pairs (…). Figure 3 B, Table 2).

[0083] Table 2. Repeat pairs of the VvAIL gene in grapes

[0084]

[0085] To further investigate the phylogenetic mechanisms of VvAILs, we analyzed the collinearity plots of grapes with six representative species (three dicotyledons: Arabidopsis thaliana, cultivated apple, and tomato; and three monocotyledons: rice, maize, and sorghum). Figure 3 C). Regarding homologous genes between VvAILs and other species, cultivated apple has the most homologous genes (15 pairs), followed by tomato (12 pairs), Arabidopsis (9 pairs), rice (7 pairs), sorghum (7 pairs), and maize (6 pairs). The difference in the number of homologous genes between grape and the other six species reflects their evolutionary distance. Overall, grape has more homologous gene pairs with dicotyledons than with monocotyledons. Furthermore, the number of homologous gene pairs between grape and dicotyledons (Arabidopsis, cultivated apple, and tomato) exceeds the number between grape and monocotyledons (rice, maize, and sorghum), which is consistent with the fact that grape is more closely related to dicotyledons (such as Arabidopsis, cultivated apple, and tomato) than to monocotyledons (such as rice, maize, and sorghum). To better understand the evolutionary process of VvAILs, the Ka, Ks, and Ka / Ks values ​​of repetitive gene pairs were calculated (Table 3). The results showed that 80% of the gene pairs had Ka / Ks ratios between 0.1 and 0.4, all of which were below 1, indicating that the evolution of VvAILs was influenced by strong purifying selection and that their gene functions tended to be conserved.

[0086] Table 3. Calculation of Ka / Ks ratio for VvAIL repeat gene pairs.

[0087]

[0088] Note: NaN indicates that it cannot be calculated.

[0089] Conserved motifs in proteins are a crucial aspect of evolutionary research. All VvAILs consist of two AP2 / ERF domains and a linker region, representing a characteristic sequence of the AP2 subfamily. Figure 8 The conserved motifs and gene structures of VvAILs were examined using GSDS 2.0 and MEME, respectively. The relationships between evolution, conserved motifs, and gene structures were analyzed by constructing a phylogenetic tree of NJ. Figure 4 A), and Figure 1 The findings are consistent with those in A. Conserved protein motifs must be identified to understand evolutionary processes. Three conserved motifs were identified in VvAILs, with lengths of 70, 78, and 100 amino acids, respectively. Figure 4 B and Figure 10 It is noteworthy that VvAILs within the same phylogenetic group exhibit consistent motif locations and distributions. Specifically, motifs 1 and 2 are conserved in groups I-IV, while motif 3 is unique to group I only.

[0090] Furthermore, the distribution of exons and introns is a key aspect of gene structure. Therefore, the structural features of 14 VvAILs were analyzed, including the location, number, and length of their exons and introns. Figure 4 C). Members within the same subfamily exhibit similar gene structures, reinforcing the results of grape AIL classification. Group I contains 7–9 exons, Group II contains 7–8 exons, Group III has the fewest exons (6), and Group IV contains 9–10 exons. Notably, all members of Group IV possess both 3'-UTR and 5'-UTR regions, while only a few members of Groups I and II lack both UTR regions. Variations in gene structure inevitably lead to functional differences between genes.

[0091] To predict the transcriptional characteristics and function of VvAILs, cis-regulatory elements in the promoter sequences of these genes were analyzed using PlantCARE. Five hormone-related elements were identified, including auxin, methyl jasmonate (MeJA), gibberellin, salicylic acid, and abscisic acid response elements. In addition, three putative cis-elements associated with stress response and eight cis-elements associated with plant growth and development were detected. Figure 4 D). The most common cis-regulatory elements in groups I–IV were anaerobic inducible elements, light-responsive elements, abscisic acid-responsive elements, and MeJA-responsive elements, respectively. Furthermore, the number of cis-regulatory elements associated with plant growth and development, stress response, and hormones differed between groups. Groups I and II had the most cis-regulatory elements associated with plant growth and development, while group III contained no cis-regulatory elements associated with stress response. Additionally, a large number of hormone-related cis-regulatory elements were identified in the promoter regions of genes in groups I and IV. Figure 4E). Notably, VvAIL2 and VvAIL12 showed the highest sequence homology with AtAIL3 / AtPLT1, AtAIL4 / AtPLT2, AtAIL2 / AtBBM, and AtAIL5. These findings suggest that VvAILs with different types and numbers of cis-regulatory elements may perform different biological functions.

[0092] 5.4 Tissue-specific expression profiles of 16 VvAILs and their expression patterns based on qRT-PCR data after Agrobacterium rhizogenes infection.

[0093] Tissue-specific expression patterns of genes are closely related to gene action sites and mechanisms. To gain a more comprehensive understanding of the potential functions of VvAILs, the expression levels of VvAILs in 21 organs or tissues at different developmental stages of grapes were examined using the BAR database. Figure 5 In general, VvAILs exhibited constitutive expression in almost all examined tissues. The VvAIL expression profile varied across different tissues and organs in different groups. VvAILs from group IV showed more extensive expression across various tissues and organs than those from groups I-III. Notably, VvAILs from group I showed relatively higher expression levels during bud development, suggesting that group I plays a crucial role in regulating bud development. Furthermore, even within the same group, expression profiles differed. For example, among the four genes in group IV (VvAIL3, VvAIL4, VvAIL6, and VvAIL13), VvAIL6 expression was significantly higher in different tissues and organs than the other three genes. In group III, VvAIL2 expression in pollen was significantly higher than the other three genes within the same group. In conclusion, our findings suggest that VvAILs are integrated regulators of grape growth and responses to environmental stress.

[0094] Based on these findings, we identified several putative cis-regulatory elements in the VvAIL promoter that may be involved in hormone response and plant growth and development. As a common wine grape variety, 'Marselan' exhibits high transgenic hairy root regeneration capacity and transformation efficiency, as revealed by our experimental results. Therefore, to explore the potential role of VvAILs in the hairy root regeneration process of detached grape leaves, we selected 'Marselan' as our experimental material and examined the gene expression levels in its leaves at 1, 2, 3, 7, 14, and 21 days after Agrobacterium rhizogenes infection. Figure 11The expression of *VvAIL4 / 5 / 6 / 12 / 16* increased significantly over time, with VvAIL5 and VvAIL12 showing a sustained upregulation. The relative expression level of VvAIL12 was higher than that of VvAIL5. The expression levels of VvAIL1 / 3 / 7 / 9 / 11 / 13 / 14 / 15 generally decreased over time, with VvAIL1 / 11 / 13 / 14 showing a significant downregulation, while the downregulation of VvAIL3 / 7 / 9 / 15 was not statistically significant. Interestingly, the relative expression levels of VvAIL2 and VvAIL10 initially decreased and then increased, while the relative expression level of VvAIL8 initially increased and then decreased. Figure 6 (AP). Notably, among these significantly upregulated genes, VvAIL4, VvAIL5, VvAIL12, and VvAIL16 all belong to Group I. This suggests that Group I genes may play an important role in regulating hairy root regeneration.

[0095] Example 3: Expression Validation and Functional Study of Key Genes

[0096] 1. qRT-PCR and semi-quantitative RT-PCR

[0097] Total RNA was extracted using the Quick RNA Isolation Kit (ZH120), and qRT-PCR was performed on a CFX96™ real-time system (Bio-Rad Laboratories) using TB Green Premix DimerEraser (TaKaRa) according to the manufacturer's protocol. VvUBI was used as an internal control gene for normalization. The primer sequences are as follows: forward primer: 5'-CCTCAACCCCAAGGCCAACAGA-3' (SEQ ID NO: 3); and reverse primer: 5'-ACCATCACCAGAATCCAGCACA-3' (SEQ ID NO: 4). The reaction mixture contained 10 μL of TB Green Premix Ex Taq II (Tli RNaseHPlus) (2X), 2 μL of DNA template, 0.8 μL each of the forward and reverse primers (10 μM), and 6.4 μL of sterile ddH2O, for a total of 20 μL. The cycling conditions were as follows: the reaction program was 95℃ pre-denaturation for 30 seconds, 1 cycle; 95℃ denaturation for 5 seconds, 60℃ annealing for 30 seconds, 40 cycles; melting curve analysis was performed at 95℃ for 5 seconds, 60℃ for 60 seconds, 95℃ for 1 cycle; and finally, cooling to 50℃ for 30 seconds. Fluorescence intensity was measured at the end of each cycle. All experiments included three biological replicates and three technical replicates. Relative quantification of miRNA expression was then performed. The relative gene expression level was calculated using the 2^−ΔΔCt method, with the formula: ΔΔCt = (ΔCt / ΔΔCt) / ΔΔCt.处理样品 -∆Ct 内参 )-(∆Ct 对照样品 -∆Ct 内参 ), Relative Expression Level (RQ) = 2 –∆∆Ct .

[0098] Semi-quantitative RT-PCR analysis was performed using gene-specific primers in a 20 μL reaction volume. The reaction mixture contained 1 μL of undiluted cDNA template, 10 μL of 2× Accurate Taq Master Mix, 0.5 μL each of forward and reverse primers (10 μM), and 8 μL of sterile ddH2O. Amplification was performed under the following cycling conditions: initial denaturation at 94°C for 5 min; followed by 25–35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The VvActin gene was used as an internal control for normalization. All PCR products were separated by electrophoresis on a 1% (w / v) agarose gel and observed and recorded using an ethidium bromide-stained DNA imaging analysis system. To ensure statistical reliability, three independent biological replicates were analyzed to obtain consistent results. Transgenic hairy roots were validated by PCR detection of GFP inserted into the genome. Gene-specific primer pairs (sequences listed in Table 4) were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0099] Table 4 Primer sequences used for expression analysis, where F represents the upstream primer and R represents the downstream primer.

[0100] ,

[0101]

[0102] 2. Carrier Construction

[0103] The construction of *pBWA(V)KS-VvAIL12-GFP* (i.e., 35S:VvAIL12-GFP) involved amplifying the full-length CDS sequence of VvAIL12 from grape 'Marselan' and cloning it into the pBWA(V)KS vector. For the *pROKII-VvAIL12-RNAi-GFP* vector, an interfering sequence of VvAIL12 was synthesized and ligated into the pROKII vector. The resulting recombinant vector was introduced into Agrobacterium rhizogenes strain K599. Primers used for cloning are listed in Table 5, and the VvAIL12 interfering sequence is shown in SEQ ID NO: 47.

[0104] Table 5 Primer sequences used for vector construction, where F represents the upstream primer and R represents the downstream primer.

[0105]

[0106] 3. Implementation Results and Effect Data

[0107] 3.1 VvAIL12 promotes the regeneration of transgenic hairy roots in grape leaves, thereby improving transformation efficiency.

[0108] As previously mentioned, *VvAIL4 / 5 / 6 / 12 / 16* were the most significantly upregulated genes in grape leaves at 1, 2, 3, 7, 14, and 21 days after Agrobacterium rhizogenes infection. Among them, the expression of VvAIL5 and VvAIL12 gradually increased over time, with VvAIL12 showing a higher relative expression level than VvAIL5. Furthermore, VvAIL12 showed high homology with BBM and AtAIL5, key regulators of early embryonic and root development in Arabidopsis thaliana. Figure 3 A). We also examined the expression of VvAIL12 in different grape genotypes after infection, revealing that its expression trend was consistent with the transformation efficiency of transgenic hairy roots. Figure 7 A). These results suggest that VvAIL12 may act as a key regulator in hairy root regeneration following Agrobacterium rhizogenes infection.

[0109] To investigate its role in this process, the interfering sequence of VvAIL12 was cloned into the pROKII vector containing green fluorescent protein (GFP), which is controlled by the 35S promoter. Simultaneously, an overexpression vector (*35S:VvAIL12-GFP*) was constructed, in which VvAIL12 and GFP were fused under the same promoter. Figure 7 B). To further explore the function of VvAIL12, we used '5BB' (low hairy root transformation efficiency) and 'Marselan' (high efficiency) as experimental materials. Agrobacterium rhizogenes containing empty vector (EV), VvAIL12-OE, or VvAIL12-RNAi constructs were used to infect 5BB and Marselan, respectively. Figure 7 C), and quantify the conversion efficiency ( Figure 7 F and Figure 7 G). PCR analysis confirmed that GFP was integrated into the transgenic hairy root genome ( Figure 7 D), and RT-PCR analysis confirmed that VvAIL12 was significantly upregulated in VvAIL12-OE hairy roots and downregulated in VvAIL12-RNAi hairy roots (D). Figure 7 E, Figure 12 ).

[0110] Infecting leaves of '5BB' tissue culture seedlings with Agrobacterium rhizogenes carrying the VvAIL12-overexpression vector significantly improved the hairy root transformation efficiency, increasing it from 9.92% to 27.78%. Figure 7F). We further evaluated the hairy root transformation efficiency of transgenic callus, which increased from 17.17% to 52.84% (F). Figure 7 G). Conversely, infection of 'Marselan' with Agrobacterium rhizogenes carrying the VvAIL12-RNAi vector significantly reduced the hairy root transformation efficiency, from 45.40% to 20.72% (G). Figure 7 F), and the hairy root transformation efficiency of transgenic callus tissue decreased from 65.24% to 32.34% (F). Figure 7 G). However, under VvAIL12-overexpression and VvAIL12-RNAi treatment, the total number of leaves carrying transgenic callus remained essentially unchanged (G). Figure 13 This indicates that VvAIL12 can promote the growth of transgenic hairy roots from leaves with transgenic callus, thereby improving the efficiency of transgenic hairy root transformation.

[0111] The above expression patterns and functional experiments together demonstrate that VvAIL12 is a key positive regulatory transcription factor in the regeneration process of grape hairy roots. Overexpression in low-efficiency varieties significantly improves transformation efficiency, while knockdown in high-efficiency varieties severely inhibits regeneration, indicating that its expression level is a key intrinsic factor determining grape regeneration capacity. Bioinformatics analysis suggests that VvAIL12 may exert its regulatory function by integrating hormone signaling pathways.

[0112] In summary, this invention establishes a highly efficient Agrobacterium rhizogenes-mediated hairy root transformation system (HRTS). Furthermore, we have, for the first time, systematically characterized the grape AIL gene family, elucidating its potential role in grape growth and development—particularly hairy root regeneration—and identified VvAIL12 as a key positive regulator of transgenic hairy root regeneration. These advances provide strong technical support and unique advantages for in-depth research into grape gene function, and have significant theoretical and practical value for advancing grape functional genomics research and molecular breeding work.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of an isolated VvAIL12 gene or its encoded protein in the preparation of a product for improving the transformation efficiency of grape hairy roots, characterized in that, The nucleotide sequence of the VvAIL12 gene is shown in SEQ ID NO: 1; The product is an engineered Agrobacterium rhizogenes containing the VvAIL12 gene expression cassette, or an inoculum containing the engineered bacteria, or a screening medium required for transformation using the engineered bacteria.

2. A method for improving the transformation efficiency of grape hairy roots, characterized in that, The method includes increasing the expression level of the VvAIL12 gene or increasing the activity of its encoded protein in grape explants; the nucleotide sequence of the VvAIL12 gene is shown in SEQ ID NO: 1; The expression level or activity is increased by introducing and expressing the VvAIL12 gene into the grape explants; the introduction and expression are achieved by an expression vector containing the VvAIL12 gene. The expression vector was transformed into grape explants via Agrobacterium rhizogenes-mediated transformation.

3. The method for improving the transformation efficiency of grape hairy roots as described in claim 2, characterized in that, The method specifically includes the following steps: (a) Using leaves from grape tissue culture seedlings that have been subcultured for 40-50 days as explants; (b) Infect the explants with a culture of *Agrobacterium rhizogenes* carrying the VvAIL12 gene expression cassette, wherein the OD of the culture is... 600 The value is 0.7-0.9; (c) Co-culture the infected explants with the Agrobacterium rhizogenes for 1.5-2.5 days; (d) The co-cultured explants were transferred to 1 / 2 MS selection medium containing antibiotics to induce the generation of transgenic hairy roots.

4. The method for improving the transformation efficiency of grape hairy roots as described in claim 3, characterized in that, The explants were obtained from tissue culture seedlings that had been subcultured for 45 days; the OD of the bacterial solution... 600 The value was 0.8; the co-culture time was 2 days; the antibiotic was termethin, with a concentration of 250-350 mg / L.

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