Application of autophagy related gene VaATG6 in regulation and control of grape cold resistance

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of an autophagy related gene VaATG6 in regulation and control of grape cold resistance. The VaATG6 gene disclosed by the invention has a remarkable effect on regulating and controlling the low-temperature stress resistance of grapes. By overexpressing the VaATG6 gene in the grape, the autophagy activity is improved, and the anti-freezing capability of the grape is remarkably enhanced; and the autophagy activity is reduced by knocking out the gene, so that the grapes are more sensitive to low-temperature stress. The invention confirms that the VaATG6 gene is a positive regulatory factor for the cold resistance of grapes, provides an important molecular tool for cultivating a new variety of cold-resistant grapes, and has a wide agricultural application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to autophagy-related genes VaATG6 Application in regulating cold tolerance of grapes. Background Art

[0002] Autophagy is a highly conserved cellular degradation mechanism in eukaryotes that transports functionally damaged proteins and even intact organelles to the vacuole for degradation and recycling. In plants, autophagy is associated with various biotic and abiotic stresses, as well as developmental processes.

[0003] Studies have shown that autophagy plays a key role in improving plant tolerance to cold stress. MsATG13 It can improve cold tolerance by promoting autophagy and activating the antioxidant defense system. Similar research results in tomatoes showed that SlATG8 , HY5, NBR1 Influencing autophagy activity plays an important role in alleviating cold-induced damage. In model plants such as Arabidopsis, key autophagy-related genes were identified. ATG , which has greatly promoted our understanding of the molecular mechanism of autophagy.

[0004] However, despite these advances, autophagy-related genes remain largely unknown in grape ( Vitis spp.) remains unexplored, and there is a research gap in understanding and improving cold tolerance in these economically important crops. Grape is a widely cultivated crop that is particularly sensitive to low temperature stress, which can lead to damage to grapevine tissue and reduced fruit quality and quantity.

[0005] Therefore, discovering autophagy-related genes associated with grape cold tolerance has become a technical challenge that needs to be urgently solved in this field. Summary of the invention

[0006] The present invention aims to provide a grape autophagy-related gene VaATG6 ( Vitis amurensis Rupr. autophagy-related gene) and its application in improving plant cold resistance to solve the problems in the prior art. VaATG6 Genes can significantly improve the cold resistance of plants.

[0007] This study found that VaATG6 The expression level under low temperature stress is significantly upregulated, indicating that this gene plays a key role in the cold resistance of plants. Based on this, the present invention proposes the following technical solutions: 1. The present invention provides a grape VaATG6 The gene has a nucleotide sequence as shown in SEQ ID No.1.

[0008] 2. The present invention also provides a grape VaATG6 The protein has an amino acid sequence as shown in SEQ ID No.2.

[0009] 3. The present invention provides an overexpression recombinant plasmid comprising the above-mentioned grape VaATG6 Gene.

[0010] 4. The present invention also provides a gene editing vector plasmid, comprising the above VaATG6 Gene.

[0011] 5. After low temperature treatment for different time VaATG6 The relative expression analysis of genes revealed that VaATG6 The gene expression was significantly upregulated under low temperature stress.

[0012] 6. The present invention also relates to the above VaATG6 Application of gene recombination plasmids in improving plant cold resistance.

[0013] 7. The present invention also provides VaATG6 Application of gene overexpression in enhancing plant autophagy activity under cold stress conditions.

[0014] 8. The present invention also relates to VaATG6 Application of gene overexpression in regulating malondialdehyde content and soluble sugar levels in plants under cold stress.

[0015] 9. The present invention provides a method for improving the cold resistance of plants, which specifically comprises: constructing a gene encoding the protein shown in SEQ ID No. 2 into an overexpression vector, and obtaining an overexpression vector by genetic transformation mediated by immature zygotic embryos. VaATG6 of grape plants, thereby improving the plants' cold resistance.

[0016] 10. The present invention also provides a method for reducing the cold resistance of plants, which specifically comprises: constructing a gene encoding the protein shown in SEQ ID No. 2 into a gene editing vector, and obtaining a knockout gene by genetic transformation mediated by immature zygotic embryos. VaATG6 Genetic changes in grape plants, thereby reducing the plants' cold resistance.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses for the first time VaATG6 The key role of genes in enhancing the tolerance of grapes to low temperature stress. VaATG6 The gene significantly increased the autophagy activity of the plant and enhanced the resistance of grapes to frost stress. The positive regulation of this gene clearly shows that VaATG6It is an important factor in improving the cold resistance of grapes. The present invention not only provides an effective molecular tool for the cultivation of new cold-resistant grape varieties, but also has a wide range of agricultural application prospects and can significantly improve the planting performance of grapes in cold regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 yes VaATG6 Overexpression vector map.

[0019] Figure 2 yes VaATG6 Map of gene editing vectors.

[0020] Figure 3 Different treatment times under cold stress VaATG6 The error bars represent SD.

[0021] Figure 4 Under low temperature stress VaATG6 The growth of genetically modified grapes; Figure 4 A is the phenotype of different plants under low temperature stress at 8 weeks old. Figure 4 B is the statistical chart of MDA content test results of different plants under low temperature stress at 8 weeks old. Figure 4 C is a statistical chart of the soluble sugar content test results of different 8-week-old plants under low temperature stress; non-transgenic grapes are UT, VaATG6 Overexpressing grape plants are called OE. VaATG6 Gene knockout of grape plants is Cas9, and error bars represent SD.

[0022] Figure 5 Representative TEM images of autophagic structures in mesophyll cells of different plants; arrows indicate autophagosomes; scale bar: 2 μm.

[0023] Figure 6 It is a statistical graph of the relative autophagic activity of different plants, and the structures were quantified using more than 10 cells.

[0024] Figure 7 After 6 hours of cold stress, the VtG The data are presented as the mean and SE of six replicates; the data were analyzed by one-way ANOVA and LSD multiple range test ( *P<0.05 , **P<0.01 ). DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0027] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for technicians in the field of molecular biology when conducting experimental operations related to molecular biology. In addition, according to different experimental purposes, technicians in the field of molecular biology complete corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to conduct experiments, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0028] Example 1 VaATG6 Gene cloning

[0029] Search for grapes according to the NCBI database ATG6 CDS sequence, and then designed primer sequences with adapters and no stop codon (forward primer: SEQ ID NO.3, reverse primer: SEQ ID NO.4) to clone VaATG6 CDS sequences were generated using PrimeSTAR ® PCR amplification was performed using Max DNA Polymerase high-fidelity enzyme and sequencing was performed for identification. The nucleic acid sequence is shown in SEQ ID NO.1 and the protein sequence is shown in SEQ ID NO.2.

[0030] Example 2 VaATG6 Construction of overexpression and knockout vectors and transformation of grapes

[0031] Overexpression vector construction: Using the In-Fusion system, the PCR product of Example 1 was connected to BamH I and Sal I The plant expression vector pCAMBIA2300-GFP was cut and named pCAMBIA2300. -VaATG6 ( Figure 1), transformed into Escherichia coli DH5α, screened positive clones by PCR identification, and the correctly sequenced plasmids were transferred into EHA105 Agrobacterium to transform grape seeds.

[0032] Knockout vector construction: Designed on Cas-Designer (http: / / www.rgenome.net / cas-designer / ) VaATG6 The target site of gene editing was identified, and primers were designed (forward primer: SEQ ID NO.5, reverse primer: SEQ ID NO.6). The PCR product containing the target sequence was constructed into the EcoRI, XbaI Enzyme-digested Pp1C.4 On the carrier, named Pp1C.4-VaATG6 ( Figure 2 The correctly sequenced plasmid was transferred into EHA105 Agrobacterium to transform grape seeds.

[0033] pCAMBIA2300 -VaATG6 and Pp1C.4-VaATG6 The genetic transformation and screening of positive transgenic lines are as follows: (1) Seed pretreatment: Select plump and healthy Cabernet Sauvignon grape seeds and soak them in a cone containing 2.5 g / L gibberellin. After 15 min in a 55°C constant temperature water bath, place them in a 28°C shaker at 180 rpm overnight. Rinse them twice with sterile water in a clean bench, add 70% alcohol and shake gently for 30 s, rinse them 3-4 times with sterile water, continue to add 15% NaClO and shake gently for 15 min, then rinse them 3-4 times with sterile water to complete the disinfection. Finally, clamp the seeds into a culture dish with sterile absorbent paper to absorb excess water on the surface of the seed coat and perform skin cutting. Then, place them upright in a culture dish with sterile absorbent paper, add 3-5 mL of sterile water, and culture them in the dark at 25 ± 2°C until the seed coat cracks ≥ 0.1 mm for infection.

[0034] (2) Preparation of infection solution: -VaATG6 or Pp1C.4-VaATG6 After activation of Agrobacterium, shake the bacteria, collect the cells by centrifugation at 5000 r / min, resuspend them in 1 / 2MS (2.215 g / L MS, 10 g / L sucrose, pH = 5.85-5.9) suspension, prepare an infection solution with OD = 0.6, add 100 μM AS (acetosyringone), and culture them in a shaking incubator at 28 °C for 2-3 h.

[0035] (3) Infection of grape seeds: Place the cracked seeds in a sterile conical flask and pour in the infection solution; perform conventional infection (hand shaking) for 20 min, discard the infection solution, and place the explants in a culture dish filled with sterile filter paper, and wait for the sterile filter paper to absorb the bacterial solution on the surface.

[0036] (4) Co-cultivation: Place sterile filter paper in 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 culture in the dark at about 25 °C for 48 h.

[0037] (5) Degerming: After co-cultivation, 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. After rinsing for 3 times, they were rinsed with sterile distilled water for 3-4 times. After inoculation into MEL-S1 (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) medium, they were transferred to rooting-S2 screening 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 Kan, pH=5.85~5.9) and wait for it to germinate and grow.

[0038] Example 3 Fluorescence quantitative PCR

[0039] Potted seedlings of Vitis amurensis “Zuoshanyi” were placed in a 2°C incubator for 0, 3, 6, 12, 24 and 48 hours, and SEQ ID NO.7 and SEQ ID NO.8 primers were used for quantitative detection VaATG6 The specific steps are as follows: Fluorescence quantitative PCR was performed using 2 × SYBR qPCR Mix (Aidlab, PC3301).

[0040] 1. System: 2 × SYBR qPCR Mix 10 μL, 10 μM upstream and downstream primers 0.4 μL each, cDNA 1 μL, ddH2O to 20 μL.

[0041] 2. qPCR program: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 10 sec, annealing at 60°C for 30 sec, and amplification for 40 cycles.

[0042] 3. Data analysis used the ΔΔCt method.

[0043] The results are as follows Figure 3 Compared with the normal temperature state, under low temperature stress treatment, VaATG6The expression of the gene was significantly increased, indicating that low temperature can induce VaATG6 Gene expression.

[0044] Example 4 VaATG6 Analysis of cold tolerance of transgenic grapes

[0045] 1. Low temperature treatment: We selected eight-week-old potted plants with similar growth and no pests and diseases, acclimated them at 10°C for 2 days, and then treated them at -2°C for 22 h to analyze their phenotypic changes.

[0046] 2. Physiological index detection: Three leaves of WT (wild type) and transgenic grape lines were collected from the same node below the top of the new shoot at 0 h and 22 h after -2°C treatment, and the soluble sugar and MDA contents were measured.

[0047] 3. Analysis of autophagy activity: 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 h. Autophagy structures were detected using a transmission electron microscope (Hitachi, Tokyo, Japan). Another portion of the samples was quickly frozen in liquid nitrogen and then stored in a -80°C freezer for the detection of autophagy-related gene expression.

[0048] like Figure 4 As shown, it is observed at room temperature VaATG6 The plant height of the overexpression plant (OE) was significantly higher than that of the UT line (non-transgenic grape). VaATG6 The knockout strain (Cas9) is significantly lower than the UT strain, indicating VaATG6 It may be involved in regulating the growth and development of plants. After low temperature stress at -2℃, compared with the control UT, the overexpression VaATG6 The stress level of the plants with , was significantly lower, while the knockout plants were just the opposite. This indicates that under low temperature stress conditions, overexpression VaATG6 The tolerance of transgenic grapes to low temperature stress can be improved.

[0049] like Figure 5~Figure 7 As shown, after 2℃ low temperature stress, the overexpression VaATG6 The plants overexpressing α accumulated more autophagosomes, while the knockout plants showed the opposite. VaATG6 The autophagy-related genes in the plants ( ATG1 / 2 / 3 / 8a / 10 / 18f / 18h ) showed an up-regulated expression trend, which indicated that under low temperature stress VaATG6 The autophagy activity and expression of autophagy-related genes in transgenic grapes can be increased, thereby improving tolerance to low temperature stress.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An autophagy-related gene involved in cold resistance of grapes, characterized in that: The gene is VaATG6 Gene; its nucleotide sequence is shown in SEQ ID No.

1.

2. Coding VaATG6 A protein of a gene, characterized in that The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. The autophagy-related gene according to claim 1 VaATG6 Or use of the protein according to claim 2 in regulating the cold resistance of grapes.

4. The use according to claim 3, characterized in that: VaATG6 The gene is overexpressed in grapes, enhancing the low temperature tolerance of grape plants by increasing autophagy activity.

5. The use according to claim 3, characterized in that: VaATG6 Knocking out the gene in grapes makes the grape plants sensitive to low temperature by reducing autophagy activity.

6. The use according to claim 4 or 5, characterized in that: get VaATG6 Overexpression or gene editing vector, obtained through grape immature zygotic embryo-mediated genetic transformation method overexpression or gene editing transgenic Cabernet Sauvignon grapes.

7. The use according to claim 4 or 5, characterized in that: Transmission electron microscopy was used to observe the number of autophagic cells in grape plant leaves and determine the autophagic activity.

8. A method for enhancing low temperature tolerance of grape plants, characterized in that: By overexpression in grape VaATG6 The gene is used to reduce the malondialdehyde content in plants and increase the soluble sugar content under -2°C stress conditions; VaATG6 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

9. A method for reducing low temperature tolerance of grape plants, characterized in that: By knocking out VaATG6 The gene is used to increase the malondialdehyde content and reduce the soluble sugar content in plants under -2°C stress conditions; VaATG6 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

10. A method for improving the cold resistance of grapes, characterized in that: By increasing the plant body according to claim 1 VaATG6 The expression or activity of the gene or the protein according to claim 2, thereby improving the cold resistance of grapes.

11. A method for reducing the cold resistance of grapes, characterized in that: By inhibiting the plant body of claim 1 VaATG6 The expression or function of the gene or the protein described in claim 2 is reduced, the content of the protein is reduced, and a plant with reduced cold resistance is obtained.

12. Use of the method according to any one of claims 8 to 11 in plant breeding.

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