Application of autophagy-related gene VaATG6 in regulation of grape cold tolerance
By overexpressing or knocking out the VaATG6 gene in grapes and using genetic transformation, the gap in research on grape cold resistance was filled, autophagy activity and cold resistance of grapes were improved, and their tolerance to low temperature stress was enhanced.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- NINGXIA UNIVERSITY
- Filing Date
- 2024-10-21
- Publication Date
- 2026-04-24
AI Technical Summary
The lack of research on autophagy-related genes in grapes in terms of improving cold resistance makes grapes sensitive to low temperature stress, leading to vine tissue damage and reduced fruit quality.
By overexpressing or knocking out the autophagy-related gene VaATG6 in grapes, the cold resistance of grapes can be increased or decreased through genetic transformation mediated by immature zygotes.
It significantly improved the autophagy activity and cold resistance of grapes, enhanced their resistance to frost stress, provided a molecular tool for cold tolerance, and improved the performance of grapes in cold regions.
Smart Images
Figure CN119410651B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, and more particularly to autophagy-related genes. VaATG6 Application in regulating grape cold resistance. Background Technology
[0002] Autophagy is a highly conserved cellular degradation mechanism in eukaryotes, which transports damaged proteins and even intact organelles to vacuoles for degradation and recycling. In plants, autophagy is associated with various biotic and abiotic stresses, as well as developmental processes.
[0003] Studies show that autophagy plays a key role in improving the cold stress tolerance of plants. Overexpression of autophagy in alfalfa... MsATG13 It can improve cold resistance by promoting autophagy and activating the antioxidant defense system. Similar research results in tomatoes indicate that... SlATG8 , HY5, NBR1 Influencing autophagy activity plays a crucial role in mitigating cold-induced damage. Key autophagy-related genes have been identified in model plants such as Arabidopsis thaliana. ATG This has greatly advanced our understanding of the molecular mechanisms of autophagy.
[0004] However, despite these advances, autophagy-related genes remain largely undeveloped in grapes ( Vitis The specific role of grapes in perennial fruit crops such as grapes (spp.) remains unexplored, and there is a research gap in understanding and improving the cold resistance of these economically important crops. Grapes are a widely cultivated crop that is particularly sensitive to low-temperature stress, which can lead to damage to grapevine tissues and a decrease in fruit quality and quantity.
[0005] Therefore, discovering autophagy-related genes associated with grape cold resistance has become a pressing technical challenge in this field. Summary of the Invention
[0006] This invention aims to provide a grape autophagy-related gene. VaATG6 ( Vitis amurensis Rupr. autophagy-related gene and its application in improving plant cold resistance, aiming to solve problems in existing technologies. This is achieved through overexpression... VaATG6 Genes can significantly improve the cold resistance of plants.
[0007] This study found that, VaATG6 The expression level was significantly upregulated under low temperature stress, indicating that this gene plays a key role in plant cold resistance. Based on this, the present invention proposes the following technical solution:
[0008] 1. This invention provides a grape VaATG6The gene, whose nucleotide sequence is shown in SEQ ID No. 1.
[0009] 2. The present invention also provides a grape VaATG6 The protein has the amino acid sequence shown in SEQ ID No. 2.
[0010] 3. This invention provides an overexpression recombinant plasmid containing the above-mentioned grape... VaATG6 Gene.
[0011] 4. The present invention also provides a gene editing vector plasmid comprising the above-mentioned... VaATG6 Gene.
[0012] 5. After low-temperature treatment for different times VaATG6 Analysis of the relative expression levels of genes revealed that, VaATG6 Gene expression was significantly upregulated under low temperature stress.
[0013] 6. The present invention also relates to the above. VaATG6 Application of recombinant plasmids in improving plant cold resistance.
[0014] 7. The present invention also provides VaATG6 Application of gene overexpression in enhancing autophagy activity in plants under cold stress.
[0015] 8. This invention also relates to VaATG6 Application of gene overexpression in regulating malondialdehyde content and soluble sugar levels in plants under cold stress.
[0016] 9. This invention provides a method for improving the cold resistance of plants, specifically comprising: constructing a gene encoding the protein shown in SEQ ID No. 2 into an overexpression vector, and obtaining the overexpressed protein through immature zygote-mediated genetic transformation. VaATG6 This improves the cold resistance of grapevines.
[0017] 10. The present invention also provides a method for reducing plant cold resistance, specifically comprising: constructing the gene encoding the protein shown in SEQ ID No. 2 into a gene editing vector, and obtaining a knockout protein through immature zygotic embryo-mediated genetic transformation. VaATG6 The genes in grapevines can reduce the plant's cold resistance.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0019] This invention discloses for the first time VaATG6 The crucial role of genes in enhancing the cold stress tolerance of grapes. This was achieved through overexpression in grapes. VaATG6This gene significantly enhanced autophagy activity in plants, thereby strengthening the resistance of grapes to frost stress. The positive regulatory effect of this gene clearly indicates that... VaATG6 It is an important factor in improving the cold resistance of grapes. This invention not only provides an effective molecular tool for the breeding of new cold-resistant grape varieties, but also has broad agricultural application prospects, and can significantly improve the performance of grapes in cold regions. Attached Figure Description
[0020] Figure 1 yes VaATG6 Overexpression vector map.
[0021] Figure 2 yes VaATG6 Atlas of gene editing vectors.
[0022] Figure 3 Different treatment times under cold stress VaATG6 The expression level is represented by error bars, which indicate the standard deviation (SD).
[0023] Figure 4 It is under low temperature stress VaATG6 The growth status of genetically modified grapes; among them, Figure 4 A shows the phenotypic figures of different 8-week-old plants under low-temperature stress. Figure 4 B is a statistical graph showing the MDA content detection results of different 8-week-old plants under low temperature stress. Figure 4 C is a statistical graph showing the soluble sugar content of different 8-week-old plants under low-temperature stress; non-GMO grapes are represented by UT. VaATG6 Overexpression in grapevines is OE. VaATG6 The gene knockout grapevines are Cas9, and the error bars represent SD.
[0024] Figure 5 These are representative TEM images of autophagy structures in mesophyll cells of different plants; the arrows indicate autophagosomes; scale bar: 2 μm.
[0025] Figure 6 It is a statistical graph of the relative autophagy activity of different plants, and the structure is quantified using more than 10 cells.
[0026] Figure 7 Six hours after cold stress, different plants VvATGs Changes in expression (2 ℃); data are expressed as mean and SE of six replicates; based on one-way ANOVA and LSD multiple range test ( *P<0.05 , **P<0.01 ). Detailed Implementation
[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0028] Unless otherwise specified, all methods used in the examples were conventional or performed according to techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels.
[0029] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. Furthermore, depending on the experimental purpose, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals accompanying various commercial kits or entrust them to specialized companies, such as gene sequencing, plasmid sequencing, and molecular weight determination.
[0030] Example 1 VaATG6 Cloning of genes
[0031] Search for grapes in the NCBI database ATG6 The CDS sequence was obtained, and then primer sequences with adapters and no stop codons were designed (forward primer: SEQ ID NO.3, reverse primer: SEQ ID NO.4) for cloning. VaATG6 CDS sequences, using PrimeSTAR ® PCR amplification was performed using Max DNA Polymerase high-fidelity enzyme, followed by sequencing identification. The nucleic acid sequence is shown in SEQ ID NO.1, and the protein sequence is shown in SEQ ID NO.2.
[0032] Example 2 VaATG6 Construction of overexpression and knockout vectors and transformation of grapes
[0033] Construction of overexpression vectors:
[0034] Using the In-Fusion system, the PCR product from Example 1 was ligated to a PCR product that had undergone... BamH I and Sal I The enzyme-digested plant expression vector pCAMBIA2300-GFP was named pCAMBIA2300. -VaATG6 ( Figure 1The plasmid was transformed into E. coli DH5α, and positive clones were screened by PCR identification. The plasmid with the correct sequence was then transformed into Agrobacterium tumefaciens EHA105 to transform grape seeds.
[0035] Knockout vector construction:
[0036] Design on the Cas-Designer website (http: / / www.rgenome.net / cas-designer / ) VaATG6 The gene editing target site was identified, and primers were designed (forward primer: SEQ ID NO.5, reverse primer: SEQ ID NO.6) to construct a PCR product containing the target sequence into a pre-selected gene editing target site. EcoRI, XbaI Enzyme digestion Pp1C.4 On the carrier, named Pp1C.4-VaATG6 ( Figure 2 The correctly sequenced plasmid was transferred into Agrobacterium tumefaciens EHA105 to transform grape seeds.
[0037] pCAMBIA2300 -VaATG6 and Pp1C.4-VaATG6 The genetic transformation and screening of positive transgenic lines are carried out through the following steps:
[0038] (1) Seed pretreatment: Select plump and healthy Cabernet Sauvignon grape seeds and soak them in a conical container containing 2.5 g / L gibberellin. After a constant temperature water bath at 55°C for 15 min, place them in a shaker at 28°C and 180 rpm overnight for incubation. Rinse twice with sterile water in a laminar flow hood, add 70% alcohol and shake gently for 30 s, rinse 3-4 times with sterile water, add 15% NaClO and shake gently for 15 min for disinfection, and rinse 3-4 times with sterile water to complete the disinfection treatment. Finally, place the seeds in a petri dish with sterile absorbent paper to absorb excess moisture from the seed coat surface and perform skin cutting treatment. Then place them upright in a petri dish with sterile absorbent paper, add 3-5 mL of sterile water, and incubate in the dark at 25 ± 2°C until the seed coat cracks ≥0.1 mm for infection.
[0039] (2) Preparation of infection solution: Infect solution containing plasmid pCAMBIA2300 -VaATG6 or Pp1C.4-VaATG6 After activation, Agrobacterium was shaken, and the cells were collected by centrifugation at 5000 r / min. The cells were resuspended in 1 / 2 MS (2.215 g / L MS, 10 g / L sucrose, pH 5.85~5.9) suspension, and an infection solution with OD=0.6 was prepared. 100 μM AS (acetylsyleugenol) was added, and the cells were co-cultured in a shaker at 28 °C for 2-3 h.
[0040] (3) Infecting grape seeds: Place the cracked seeds in a sterile conical flask and pour in the infection solution; routinely infect (shake by hand) for 20 min, discard the infection solution, and place the explants into a culture dish containing sterile filter paper, and wait for the sterile filter paper to absorb the bacterial solution on the surface.
[0041] (4) Co-culture: Place sterile filter paper on co-culture medium (2.215 g / L MS + 10 g / L sucrose + 8 g / L agar, pH=5.85~5.9), moisten the filter paper with 1 / 2 MS liquid co-culture medium (about 3 mL), place the infected seeds on the sterile filter paper, and incubate in the dark at about 25 °C for 48 h.
[0042] (5) Sterilization: After co-culture, the explants were first washed with sterile water, and then rinsed with 500 mg / L cephalosporin and 500 mg / L carbenicillin solution for 5 min, and rinsed 3 times consecutively. Then rinsed 3-4 times with sterile distilled water. After inoculation into MEL-S1 medium (4.43 g / L MS + 2.0 mg / L 6-BA + 0.6 mg / L MEL + 30 g / L sucrose + 3 g / L plant gel + 0.5 g / L activated carbon + 200 mg / L CEF + 200 mg / L Carb, pH=5.85~5.9) for 3-4 weeks, they were transferred to rooting-S2 selection medium (MS + 300 μ / L IBA + 30 g / L sucrose + 7 g / L agar + 0.5 g / L activated carbon + 250 mg / L CEF + 250 mg / L Carb + 30 mg / L CEF + 200 mg / L Carb) for 3-4 weeks. (Kan, pH=5.85~5.9) until it germinates and grows.
[0043] Example 3: Real-time PCR
[0044] Potted seedlings of the wild grape variety "Zuoshan 1" were placed in a 2℃ incubator for 0, 3, 6, 12, 24, and 48 hours, and quantitative detection was performed using primers SEQ ID NO. 7 and SEQ ID NO. 8. VaATG6 Gene expression levels. The specific steps are as follows: Quantitative real-time PCR was performed using 2 × SYBR qPCR Mix (Aidlab, PC3301).
[0045] 1. System: 10 μL of 2 × SYBR qPCR Mix, 0.4 μL each of 10 μM forward and reverse primers, 1 μL of cDNA, and ddH2O to bring the total to 20 μL.
[0046] 2. qPCR program: pre-denaturation at 95℃ for 2 min, denaturation at 95℃ for 10 sec, annealing at 60℃ for 30 sec, amplification for 40 cycles.
[0047] 3. The ΔΔCt method was used for data analysis.
[0048] The results are as follows Figure 3 Compared to normal temperature conditions, under low-temperature stress treatment, starting after 3 hours, VaATG6 Gene expression levels were significantly increased, indicating that low temperature can induce... VaATG6 Gene expression.
[0049] Example 4 VaATG6 Cold resistance analysis of genetically modified grapes
[0050] 1. Low temperature treatment: We selected eight-week-old potted plants with similar growth and no pests or diseases, acclimatized them at 10°C for 2 days, and then treated them at -2°C for 22 hours to analyze their phenotypic changes.
[0051] 2. Physiological index detection: Three leaves from the same node below the tip of the new shoot were collected from WT (wild type) and transgenic grape varieties at 0 hours and 22 hours after treatment at -2°C, respectively, and the contents of soluble sugar and MDA were measured.
[0052] 3. Autophagy Activity Analysis: Leaves were collected at 0 h and 3 h after 2°C low-temperature treatment. A portion of the grape leaves were immediately cut into small pieces (∼1 mm × 4 mm) and fixed in 2.5% glutaraldehyde for 12 hours. Autophagy structures were examined using a transmission electron microscope (Hitachi, Tokyo, Japan). Another portion of the samples was rapidly frozen in liquid nitrogen and then stored in a -80°C freezer for the detection of autophagy-related gene expression levels.
[0053] like Figure 4 As shown, observations were made at room temperature. VaATG6 Overexpressing plants (OE) were significantly taller than UT lines (non-transgenic grapes), while VaATG6 The knockout line (Cas9) had a significantly lower number of cases than the UT line, indicating that... VaATG6 It may be involved in regulating plant growth and development. After -2℃ low-temperature stress, compared with the control UT, it overexpressed... VaATG6 The plants with the positive expression showed significantly less stress, while the knockout plants showed the opposite. This indicates that overexpression of the positive expression is effective under low-temperature stress. VaATG6 It can improve the tolerance of genetically modified grapes to low-temperature stress.
[0054] like Figures 5-7 As shown, after 2°C low-temperature stress, compared with the control UT, the overexpression of [a specific substance] was significantly reduced. VaATG6 Plants with positive expression accumulated more autophagosomes, while knockout plants showed the opposite. Furthermore, overexpression... VaATG6 Autophagy-related genes in plants ( ATG1 / 2 / 3 / 8a / 10 / 18f / 18h All three showed an upregulation trend, indicating that under low temperature stress conditions... VaATG6It can increase the autophagy activity and expression levels of autophagy-related genes in transgenic grapes, thereby improving their tolerance to low-temperature stress.
[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Overexpression of autophagy-related genes VaATG6 or VaATG6 Application of gene-encoded proteins in improving the cold resistance of grapes; the autophagy-related gene VaATG6 The nucleotide sequence of the protein is shown in SEQ ID No. 1; the amino acid sequence of the protein is shown in SEQ ID No.
2.
2. The application according to claim 1, characterized in that, VaATG6 The gene is overexpressed in grapes, which enhances the grapevines' low-temperature tolerance by increasing autophagy activity.
3. The application according to claim 1, characterized in that, get VaATG6 Overexpression vectors were obtained through genetic transformation mediated by immature grape zygotes. VaATG6 Cabernet Sauvignon grapes with genetic variations.
4. A method for enhancing the low-temperature tolerance of grapevines, characterized in that, By overexpressing in grapes VaATG6 The gene, under -2℃ stress conditions, reduces malondialdehyde content and increases soluble sugar content in plants; VaATG6 The nucleotide sequence of the gene is shown in SEQ ID No.
1.
5. A method for improving the cold resistance of grapes, characterized in that, By increasing the amount of SEQ ID No. 1 shown in the plant VaATG6 The expression of the gene or the protein shown in SEQ ID No. 2 enhances the cold resistance of grapes.
6. The application of the method according to claim 4 or 5 in cold-resistant grape breeding.