Vvsts3 gene and application thereof in low temperature stress
By cloning and characterizing the VvSTS3 gene, the accumulation of resveratrol and the activity of antioxidant enzymes in grapes were enhanced, solving the problem of insufficient cold resistance in grapes and improving the grapes' ability to withstand low-temperature stress and their quality.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO AGRI UNIV
- Filing Date
- 2026-03-16
- Publication Date
- 2026-06-05
AI Technical Summary
Grapes have poor cold resistance. Low temperature stress leads to damage to overwintering dormant tissues and leaves and new shoots during the growing season. Existing antifreeze measures are labor-intensive and material-intensive and affect quality. There is a lack of effective genetic resources to improve resveratrol accumulation and antioxidant capacity.
The grape VvSTS3 gene was cloned and characterized. By overexpressing or silencing this gene, resveratrol accumulation was significantly increased, antioxidant enzyme activity was enhanced, cell membrane stability was maintained, and the expression of cold-related genes was upregulated. Recombinant vectors and recombinant bacteria were constructed for the transformation of grapes and tobacco.
It significantly improved the low-temperature resistance of transgenic materials, enhanced the cold resistance of grapes, provided new genetic resources for grape cultivar improvement, and improved the stability of crop production and fruit quality.
Smart Images
Figure CN122146728A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, specifically to VvSTS3 Genes and their application in low temperature stress. Background Technology
[0002] Grapes are highly favored by consumers and are widely cultivated globally due to their significant nutritional and economic value. According to data analysis provided by the International Vine and Wine Organization (OIV), my country ranked first in the world in both fresh grape production and consumption in 2022. However, because the main cultivars cultivated in my country are Eurasian varieties, they have poor cold resistance. Low winter temperatures and late spring frosts often damage the overwintering dormant tissues of grapevines, as well as the leaves and new shoots during the growing season (Liu et al., 2022). Some grape-producing areas have been forced to adopt antifreeze measures such as smoke fumigation, irrigation, and ground burial, but these measures not only consume manpower, material resources, and financial resources but also damage the vines, affecting grape quality. In recent years, researchers have discovered that various plant growth regulators can effectively alleviate the damage to plants caused by low temperatures such as late spring frosts and early / late frosts, and these substances are widely used in production practices. Therefore, identifying growth regulators that enhance grape's resistance to low temperatures, analyzing their cold-resistance functions and synthetic regulatory mechanisms, and then utilizing related gene resources to improve the cold resistance of grape cultivars and select germplasm resources, and on this basis developing regulatory technologies to enhance the cold resistance of fruit trees, is of great significance for ensuring the sustainable development and economic benefits of the grape industry.
[0003] Plant abiotic stress refers to the phenomenon where the physiological or biochemical processes of plants are directly or indirectly affected by abiotic factors (temperature, salinity, heavy metals, etc.) or biotic factors (bacteria, viruses, etc.). Low temperature is one of the most severe stresses faced by plants. Within a suitable temperature range, plants can carry out normal life activities; exceeding this range leads to various functional disorders (Ding and Yang, 2022). The effects of low temperature stress on plants are a complex process. When low temperatures occur, the production and scavenging of reactive oxygen species (ROS) in plants become unbalanced, leading to a large accumulation of free radicals within cells. These free radicals then oxidize lipids in the plant membrane system, producing metastatic oxygen derivatives (MDA) (Anet et al., 2021). Lipid oxidation alters cell membrane permeability, leading to electrolyte extravasation and increased conductivity (Xu et al., 2023). To mitigate damage from free radicals, plants produce antioxidant enzymes such as SOD, POD, CAT, and ascorbate peroxidase (APX) (Keunen et al., 2013). Simultaneously, to reduce electrolyte leakage, plants accumulate osmotic regulators including proline, soluble proteins, and soluble sugars (Thomashow et al., 1999). Through long-term interaction with nature, plants have evolved complex mechanisms to sense and respond to low-temperature stress. Existing research indicates that plant resistance to low-temperature stress involves the expression of multiple genes and complex signal transduction networks.
[0004] Resveratrol (Res), chemically known as 3,4',5-trihydroxy-1,2-stilbene (trans-3,4',5-trihydroxystilbene), has the molecular formula C2. 14 H 12O3 is a non-flavonoid polyphenolic compound containing stilbene structures produced in plants (Tian and Liu, 2020). Resveratrol, as a natural stilbene compound, possesses diverse biological activities. It plays an important role not only in preventing or delaying cancer and various cardiovascular diseases, but also in protecting plants from pathogen infection and environmental degradation. Resveratrol has garnered widespread attention due to its unique biological activities and pharmacological effects (Spaleniak and Cuendet, 2023). Grapes are currently the main source of natural resveratrol worldwide (Weiskirchen and Weiskirchen, 2016). With the increasing demand for resveratrol, improving its accumulation in plants and identifying the gene regulatory networks controlling its synthesis have become hot topics in secondary metabolite research.
[0005] Studies have found that exogenous methyl jasmonate (MeJA) and Ca 2+ Treatment can enhance antioxidant enzyme activity and antioxidant capacity, reduce malondialdehyde and hydrogen peroxide content, significantly increase peanut sprout length and fresh weight, alleviate the inhibition of peanut sprout growth, and promote the accumulation of resveratrol in peanut sprouts (Yin et al., 2023). After ultraviolet light treatment, the MYB14-WRKY8-MYB30 molecular module promotes the accumulation of resveratrol, thereby alleviating stress damage to grapes, and can control its production when resveratrol accumulates to a certain level (Mu et al., 2023). However, there is currently no research on the relationship between resveratrol and low-temperature stress response. Summary of the Invention
[0006] The purpose of this invention is to provide VvSTS3 Genes and their application in low-temperature stress. This invention provides... VvSTS3 The gene can significantly increase the accumulation of resveratrol in plants, and achieve this by enhancing the antioxidant system, maintaining cell membrane stability, and upregulating the expression of cold-related genes, thus promoting the overexpression of resveratrol. VvSTS3 Genetically modified grape callus and tobacco tissues have been shown to have higher resistance to low temperatures.
[0007] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides VvSTS3 Genes, the ones mentioned VvSTS3 The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0008] The applicant cloned a low-temperature stress response gene from grapes. VvSTS3Bioinformatics analysis and expression characteristics studies were conducted on the gene. Real-time quantitative PCR analysis revealed high expression levels in older grape leaves, peels, and roots, with expression significantly induced by low-temperature stress. Further research was performed by constructing... VvSTS3 Gene overexpression and silencing vectors were used, and transient transformations were performed in grape leaves. Experiments confirmed that positive regulation of this gene significantly increased resveratrol content in leaves, while silencing the gene led to reduced resveratrol accumulation. Further research was conducted to obtain stable overexpression vectors. VvSTS3 Grape callus and tobacco plants were analyzed to demonstrate that the gene can effectively improve the tolerance of transgenic materials to low-temperature stress.
[0009] Secondly, the present invention provides a recombinant vector carrying the contents described above. VvSTS3 Gene.
[0010] Preferably, the recombinant vector is used for overexpressing pCAMBIA2301- VvSTS3 Or used for virus-induced gene silencing pTRV2- VvSTS3 Genetically modified materials.
[0011] Thirdly, the present invention provides a recombinant bacterium, comprising the recombinant vector described above.
[0012] Preferably, the recombinant bacteria is Agrobacterium GV3101.
[0013] Fourthly, the present invention provides, as described above VvSTS3 Application of genes, recombinant vectors as described above, or recombinant bacteria as described above in improving plant tolerance to low temperature stress.
[0014] As described above, under low-temperature stress, the application is at least one of the following (1) to (4): (1) VvSTS3 The gene was overexpressed in plant leaves, which increased the accumulation of resveratrol in plant leaves; (2) VvSTS3 The gene was transiently silenced in the plant leaves, reducing the accumulation of resveratrol in the plant leaves; (3) VvSTS3 The gene is overexpressed in plant leaves, and under low temperature stress, the overexpression of the gene... VvSTS3 Compared with leaves containing an empty vector, plant leaves with the gene showed reduced relative cell membrane permeability, MDA content, and superoxide anion accumulation. (4) VvSTS3 The gene is overexpressed in plant leaves, and under low temperature stress, the overexpression of the gene... VvSTS3Compared with leaves with empty vectors, plant leaves with the gene showed increased SOD, POD, and CAT activities, or increased chlorophyll content and Fv / Fm value.
[0015] As described above, the application will VvSTS3 The gene was overexpressed in plant leaves, and under low temperature stress, the expression of cold-related genes was increased. VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The expression level of at least one of them.
[0016] In the applications described above, the plant is either grape or tobacco.
[0017] Fifthly, the present invention provides, as described above VvSTS3 Application of genes, recombinant vectors as described above, or recombinant bacteria as described above in the genetic improvement of plant cold resistance.
[0018] Sixthly, the present invention provides a method for increasing the resveratrol content in plants by expressing the resveratrol as described above in the plants. VvSTS3 Gene.
[0019] In a seventh aspect, the present invention provides a method for improving the cold resistance of plants, the method comprising the following steps: (a) Provides containing as described above VvSTS3 Gene expression vectors; (b) Introduce the expression vector described in step (a) into plant cells or tissues; (c) Regenerate transgenic plants from the plant cells or tissues of step (b).
[0020] Compared with existing technologies, the effects and advantages of this invention are: 1. This invention is the first to clearly define grapes VvSTS3 The gene plays a crucial role in the response to low-temperature stress. Transient overexpression of this gene in grape leaves significantly increased resveratrol accumulation; stable overexpression in grape callus and tobacco effectively enhanced the low-temperature tolerance of transgenic materials, resulting in significant phenotypic improvement. This gene can provide new genetic resources for improving the cold tolerance of grape cultivars and for germplasm breeding, demonstrating clear application potential and market prospects.
[0021] 2. Overexpression in this invention VvSTS3 It not only directly promotes the synthesis of the stress-relief substance resveratrol, but also increases the activity of antioxidant enzymes such as SOD, POD, and CAT to scavenge reactive oxygen species, reduce the degree of membrane lipid peroxidation (MDA content), and upregulate... VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27The expression of key cold-related genes can synergistically enhance the plant's low-temperature adaptability from multiple levels, including metabolism, physiology, and signal transduction. This gene is applied to the genetic improvement of plant cold resistance and provides an important genetic resource for improving the stability of crop production in cold regions. Attached Figure Description
[0022] Figure 1 for VvSTS3 Electrophoresis images of gene clones, in which, Figure 1 A is VvSTS3 CDS sequence PCR amplification product, Figure 1 B is the recombinant plasmid pMD19-T- VvSTS3 Colony PCR; Figure 2 for STS3 Amino acid sequence alignment analysis; Figure 3 for VvSTS3 Bioinformatics analysis of genes, among which, Figure 3 A represents the predicted tertiary structure of the VvSTS3 protein. Figure 3 B represents the hydrophobicity / hydrophilicity analysis. Figure 3 C represents the predicted phosphorylation modification site. Figure 3 D represents the prediction of transmembrane structural domains; Figure 4 for VvSTS3 Gene expression characteristics analysis, among which, Figure 4 A is VvSTS3 Analysis of gene expression characteristics at low temperatures Figure 4 B is VvSTS3 Tissue expression characteristics analysis of genes; Figure 5 for VvSTS3 Subcellular localization of genes, among which, Figure 5 A is pMD19-T- VvSTS3 The plasmid was used as a template for amplification. VvSTS3 CDS fragments, Figure 5 B is the general VvSTS3 The CDS fragment was fused into the vector pSuper1300-GFP for colony PCR identification. Figure 5 C is pSuper1300- VvSTS3 -GFP recombinant plasmid was transformed into Agrobacterium GV3101 colonies for PCR identification. Figure 5 D is VvSTS3 Subcellular localization; Figure 6 pCAMBIA2301- VvSTS3 Construction of overexpression vectors, in which, Figure 6 A is pCAMBIA2301- VvSTS3 PCR amplification products, Figure 6 B is pCAMBIA2301- VvSTS3 Colony PCR, Figure 6 C is pCAMBIA2301- VvSTS3 Colony PCR identification of Agrobacterium GV3101; Figure 7 pTRV2- VvSTS3 Construction of VIGS vectors, in which, Figure 7 A is pTRV2- VvSTS3 PCR amplification products, Figure 7 B is pTRV2- VvSTS3 Colony PCR, Figure 7 C is pTRV2- VvSTS3 Colony PCR identification of Agrobacterium GV3101; Figure 8 for VvSTS3 Identification of transiently transformed grape leaves, including Figure 8 A represents the momentary silence in grape leaves. VvSTS3 Detection of expression levels Figure 8 B represents transient overexpression in grape leaves. VvSTS3 Detection of expression levels; Figure 9 for VvSTS3 The effect of transient conversion on resveratrol synthesis in grape leaves, among which, Figure 9 A represents momentary silence. VvSTS3 Resveratrol content in grape leaves Figure 9 B represents transient overexpression. VvSTS3 Resveratrol content in grape leaves; Figure 10 Transient silencing under low temperature stress VvSTS3 The effects of low temperature stress on the physiological parameters of grape leaves, including transient silencing. VvSTS3 In grape leaves, Figure 10 A represents the relative permeability of the cell membrane. Figure 10 B represents the MDA content. Figure 10 C is O2. - content, Figure 10 D represents SOD activity. Figure 10 E represents POD activity. Figure 10 F represents CAT activity. Figure 10 G represents chlorophyll content. Figure 10 H represents the Fv / Fm value; Figure 11 For the effect of low temperature stress on transient overexpression VvSTS3 The effects of low temperature stress on physiological indicators of grape leaves, including transient overexpression. VvSTS3 In grape leaves, Figure 11 A represents the relative permeability of the cell membrane. Figure 11B represents the MDA content. Figure 11 C is O2. - content, Figure 11 D represents SOD activity. Figure 11 E represents POD activity. Figure 11 F represents CAT activity. Figure 11 G represents chlorophyll content. Figure 11 H represents the Fv / Fm value; Figure 12 For the transient transformation of low temperature stress VvSTS3 The influence of cold-related genes in grape leaves, among which, Figure 12 A represents the effect of low-temperature stress on transient silencing. VvSTS3 The influence of cold-related genes in grape leaves Figure 12 B represents the transient overexpression effect of low-temperature stress. VvSTS3 The influence of cold-related genes in grape leaves; Figure 13 for VvSTS3 Screening and identification of grape callus overexpression, among which, Figure 13 A is VvSTS3 Screening for overexpression of grape callus Figure 13 B is VvSTS3 Identification of grape callus overexpressing; Figure 14 for VvSTS3 Screening and identification of heterologous overexpression tobacco, among which, Figure 14 A is VvSTS3 Screening for heterologous overexpression of tobacco Figure 14 B is VvSTS3 Identification of heterologous overexpression of tobacco; Figure 15 for VvSTS3 Effects on resveratrol content in grape callus and tobacco seedlings, among which, Figure 15 A is VvSTS3 Overexpression of resveratrol content in grape callus Figure 15 B is VvSTS3 Overexpression of resveratrol content in tobacco; Figure 16 For low temperature stress VvSTS3 The effect of overexpression on the cold resistance phenotype of materials, among which, Figure 16 A is VvSTS3 Overexpression of cold-resistant phenotype in grape callus tissue Figure 16 B is VvSTS3 Heterologous overexpression of cold-resistant phenotypes in tobacco seedlings; Figure 17 For low temperature stress VvSTS3 The effects of overexpression on physiological indicators of callus tissue, including, under low temperature stress VvSTS3 Overexpression in callus tissue, Figure 17 A represents the relative permeability of the cell membrane. Figure 17 B represents the MDA content. Figure 17 C is O2. - content, Figure 17 D represents SOD activity. Figure 17 E represents POD activity. Figure 17 F represents CAT activity; Figure 18 For low temperature stress VvSTS3 Overexpression of cold-related genes in grape callus VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The impact of expression; Figure 19 For low temperature stress VvSTS3 The effects of heterologous overexpression on physiological parameters of tobacco seedlings, including low temperature stress. VvSTS3 Heterologous overexpression in tobacco seedlings, Figure 19 A represents the relative permeability of the cell membrane. Figure 19 B represents the MDA content. Figure 19 C represents the DAB staining experiment. Figure 19 D represents the NBT staining experiment. Figure 19 E is O2. - content, Figure 19 F represents SOD activity. Figure 19 G represents POD activity. Figure 19 H represents CAT activity; Figure 20 The effects of low temperature stress on photosynthetic-related parameters of VvSTS3 heterologously overexpressing tobacco seedlings were investigated. Figure 20 A represents the chlorophyll content. Figure 20 B represents the Fv / Fm value. Detailed Implementation
[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.
[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0026] Example 1 VvSTS3 Cloning of genes Table 1. Gene amplification primers
[0027] Found from grape database VvSTS3 The CDS sequence is 708 bp in length. A pair of specific primers were designed using the biological software Primer Premier 5.0, as shown in Table 1, and their sequences are shown in SEQ ID NO. 3-4. PCR amplification was performed using cDNA from Chardonnay grape leaves as a template. The PCR products were detected by 1% agarose gel electrophoresis, as shown in Table 1. Figure 1 As shown in Figure A, a clear band was found around 750 bp, with a length similar to the target sequence, which was presumed to be the target band. After the PCR amplification product was detected by agarose gel electrophoresis, the target band was purified and recovered using the M5 Gel Extraction Kit (with column) (purchased from Beijing Polymer Biotechnology Co., Ltd.), and then ligated into the pMD19-T vector.
[0028] The ligation product was transformed into DH5α competent Escherichia coli cells (purchased from Sangon Biotech Co., Ltd.) using a heat shock method. The bacterial culture was then evenly spread on LB agar. Colonies that grew after incubation at 37°C for 12 h were identified by colony PCR. Figure 1 B shows a positive clone. The grown positive clone colonies were scraped off and dissolved in liquid culture medium, then shaken at 28°C and 200 rpm for 12 h. The bacterial cells were collected, plasmids were extracted, and sent to a biotechnology company for sequencing.
[0029] Sequencing results show that VvSTS3 The gene size is 708 bp. VvSTS3 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the sequence information is as follows: ATGTTGTATCATCAAGGCTGCTATGCAGGTGGGACTGTTCTCCGAACTGCTAAGGATCTTGCAGAGAATAATGCAGGAGCACGAGTTCTTGTGGTATGCTCTGAAATCACGGTTGTTACTTTTCGTGGGCCTTCTGAAACCCATTTAGACTCTTTAGTGGGTCAAGCCCTTTTTGGTGATGGGTCAGCAGCTGTAATTGTTGGATCAGATCCAGATACCTCCATTGAACGACCACTCTTCCAACTTGTTTCAGCGGCCCAAACATTCATTCCTAATACACAAGGTGCTATTGCTGGCAACTTACGTGAAGTGGGTCTAACCTTTCATTTGTGGCCTAATGTGCCTACTCTGATTTCCGAGAATATAGAGAAGTGCTTGACTCAAGCTTTTGGCCCACTTGGTATTAGTGATTGGAACTCCTTGTTTTGGATCGCTCATCCAGGTGGCCCGGCCATTCTGGATGCAGTCGAAGCAAAACTCAATTTGGAGAAGAAGAAACTTGAAGCAACAAGACACATCTTAAGTGAGTACGGTAATATGTCAAGTGCATGTGTGTTGTTTATTTTGGATGAGATGAGAAAGAAATCACTTAAGGAAGAAAGGACCACCACAGGTGAAGGATTGGACTGGGGTGTTTTATTTGGTTTTGGGCCAGGTTTGACCATTGAAACTGTTGTGCTGCATAGCGTTGTTGGGGCTACAAATTGA。
[0030] Encoding 235 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2, and having characteristic sites of STS and CHS families (GVLFGFGPGLT): MLYHQGCYAGGTVLRTAKDLAENNAGARVLVVCSEITVVTFRGPSETHLDSLVGQALFGDGSAAVIVGSDPDTSIERPLFQLVSAAQTFIPNTQGAIAGNLREVGLTFHLWPNVPTLI SENIEKCLTQAFGPLGISDWNSLFWIAHPGGPAILDAVEAKLNLEKKKLEATRHILSEYGNMSSACVLFILDEMRKKSLKEERTTTGEGLDWGVLFGFGPGLTIETVVLHSVVGATN.
[0031] Example 2 VvSTS3 Gene expression characteristics analysis one, VvSTS3 Gene bioinformatics analysis In order to analyze VvSTS3 The evolutionary relationships of genes among different species, through VvSTS3 The amino acid sequence was searched using BLAST in the NCBI database, and 14 highly similar STS3 amino acid sequences were selected from the sequence alignment results. These sequences are: [List of sequences would be inserted here]. CrSTS3 ( Cissus rhombifolia (AAM21772.1), hairy grape VqSTS3 ( Vitis quinquangularis , AFM56665.1), Mountain grape VaSTS3 ( Vitis amurensis (AFM22751.1), East China grapes VpSTS3 ( Vitis pseudoreticulata , AFN21530.1), Riverside grapes VrSTS3 ( Vitis riparia (XP_034698480.1), grapes VvSTS3 ( Vitis vinifera , RVW78731.1), Sang MaSTS3 ( Morus alba ,ARM20004.1), Sorghum SbSTS3 ( Sorghum bicolor , XP_002450870.1), Rheum palmatum RpSTS3 ( Rheum palmatum (AFX68803.1), Polygonum multiflorum FmSTS3 ( Fallopia multiflora (AFP97667.1), Polygonum cuspidatum PcSTS3 ( Polygonum cuspidatum , ABI78940.1), groundnut AhSTS3 ( Arachis hypogaea(BAA78617.1), European Spruce PaSTS3 ( Picea abies (AEN84236.1), European red pine PsSTS3 (Pinussylvestris, AAB24341.2), Pine-leaved Fern PnSTS3 ( Psilotum nudum (BAA87924.1). Phylogenetic analysis of the STS3 amino acid sequences in different species was performed using MEGA 5.1 software, and the results showed... VvSTS3 and VrSTS3 Closest kinship, such as Figure 2 As shown.
[0032] VvSTS3 Protein structure analysis revealed that the predicted tertiary structure of the protein showed... VvSTS3 Proteins contain a large number of α-helices, such as Figure 3 As shown in Figure A. The results for hydrophilicity and hydrophobicity are... VvSTS3 Proteins containing hydrophobic heads at both their C-terminus and N-terminus are hydrophobic proteins, such as... Figure 3 As shown in B. VvSTS3 Amino acid sequence phosphorylation modification prediction revealed the presence of multiple phosphorylation sites, such as Figure 3 As shown in C. For VvSTS3 Predictive analysis of transmembrane domains revealed that it does not possess transmembrane domains, such as... Figure 3 As shown in D.
[0033] II. Under Low Temperature Stress VvSTS3 Gene expression characteristics analysis 1. Experimental Method: Analysis of gene expression characteristics induced by low temperature: Grape leaves of the same leaf position from 2-month-old 'Cabernet Sauvignon' acclimatized seedlings were used as experimental materials. The leaves were treated at 4℃ for 2 h, then gradually decreased to -4℃ at a rate of 1℃ / h, followed by a warming to 4℃ for 2 h, and finally restored to room temperature for 3-4 h. Total RNA was extracted from the grape leaves at 3, 6, 9, 12, and 24 h after the low-temperature treatment and reverse transcribed into cDNA. Real-time quantitative PCR was used to detect the expression characteristics. VvSTS3 The relative expression levels were measured, and each treatment was repeated three times.
[0034] Gene expression characteristics analysis: Roots, stems, young leaves, mature leaves, old leaves, buds, tendrils and peels of 'Cabernet Sauvignon' grape seedlings were selected, and total RNA was extracted from the grapes and reverse transcribed into cDNA.
[0035] Total RNA was extracted from grapes using the CTAB method, and cDNA was synthesized using an MLV reverse transcription kit (purchased from Shanghai Shangbao Biotechnology Co., Ltd., catalog number T11238.50) as a template. Real-time quantitative PCR was used for detection. VvSTS3Gene expression levels. The TaKaRa PCR instrument was used for real-time quantitative PCR analysis. The reaction conditions were: 95℃ for 60 s, 95℃ for 10 s, 58℃ for 20 s, and 72℃ for 15 s, for 40 cycles. All experimental analyses were performed in triplicate. Relative gene expression levels were expressed as the target gene relative to an internal control gene. VvActin The change factor is expressed as 2. -△△CT Method calculations. Primers for real-time quantitative PCR are shown in Table 2. Results are as follows: Figure 4 As shown.
[0036] Table 2 qRT-PCR primers
[0037] 2. Experimental Results and Analysis: turn out, VvSTS3 Gene expression levels peaked after 12 hours of low-temperature stress, such as Figure 4 As shown in A; the expression level was significant in older leaves, pericarps, and roots, such as Figure 4 As shown in Figure B. The above results indicate... VvSTS3 Induced by low temperature stress, it may play a role in the cold resistance of grapes.
[0038] Example 3 VvSTS3 Subcellular localization (1) Agrobacterium transformation The successfully sequenced plasmid was transformed into GV3101 Agrobacterium competent cells (purchased from Sangon Biotech Co., Ltd.) using liquid nitrogen freezing. The transformed bacterial culture was stored at -80℃.
[0039] (2) Subcellular localization detection pMD19-T- VvSTS3 The plasmid was used as a template for amplification. VvSTS3 The CDS fragment was fused into the vector pSuper1300-GFP, and then the constructed pSuper1300- VvSTS3 -GFP and the positive control pSuper1300-GFP (purchased from Shanghai Beinuo Biotechnology Co., Ltd.) were transferred into tobacco leaves for infection. After 12 h of dark incubation, the leaves were placed in an artificial climate chamber for 3 days and then examined using a laser confocal microscope.
[0040] pSuper1300- VvSTS3 After the recombinant plasmid of -GFP was transformed into Agrobacterium GV3101, colony PCR was performed for identification, and positive bacteria were selected for subsequent experiments. Figure 5 A is pMD19-T- VvSTS3 The plasmid was used as a template for amplification. VvSTS3 CDS fragments, Figure 5B is the general VvSTS3 The CDS fragment was fused into the vector pSuper1300-GFP for colony PCR identification. Figure 5 C is pSuper1300- VvSTS3 -GFP recombinant plasmid was transformed into Agrobacterium GV3101 colonies for PCR identification. The constructed pSuper1300- VvSTS3 -GFP and the positive control pSuper1300-GFP were injected into tobacco leaves, and the expression was observed using a laser confocal microscope 3 days later. VvSTS3 Subcellular localization. The results showed that... VvSTS3 It is distributed on both the cell membrane and cytoplasm, such as Figure 5 As shown in Figure D, EV represents pSuper1300 unloaded.
[0041] Example 4 VvSTS3 Gene overexpression and VIGS silencing vector construction I. pCAMBIA2301- VvSTS3 Construction of overexpression vectors With the correct pMD19-T sequenced VvSTS3 The target fragment is amplified using a plasmid as a template, such as... Figure 6 As shown in A. The pCAMBIA2301 empty vector, after double enzyme digestion and ligation, was transformed into DH5α competent E. coli cells. Colony PCR identification was performed, as shown in... Figure 6 As shown in B, plasmids were then extracted from the positive bacterial culture and sequenced. The successfully sequenced pCAMBIA2301- VvSTS3 Plasmid was transformed into GV3101 Agrobacterium competent cells, and colony PCR was performed to identify the plasmid as follows: Figure 6 As shown in C, the bacterial culture is retained for the subsequent construction of overexpression materials.
[0042] II. pTRV2- VvSTS3 Construction of VIGS viral vector With the correct pMD19-T sequenced VvSTS3 The target fragment is amplified using a plasmid as a template, such as... Figure 7 As shown in A. The pTRV2 empty vector was ligated after double enzyme digestion and transformed into DH5α competent E. coli cells. Colony PCR identification was performed, as shown in... Figure 7 As shown in B, plasmids were then extracted from the positive bacterial culture and sequenced. The successfully sequenced pTRV2- VvSTS3 Plasmid was transformed into GV3101 Agrobacterium competent cells, and colony PCR was performed to identify the plasmid as follows: Figure 7 As described in C, the bacterial solution is retained for the subsequent construction of silenced materials.
[0043] Example 5 VvSTS3 Transient transformation and identification of genes in grape leaves 1. Experimental Method: Pour LB liquid medium containing 50 mg / L Kan and 50 mg / L Rif antibiotics into a sterile Erlenmeyer flask, add Agrobacterium tumefaciens bacterial suspension to the medium, and shake at 28℃ and 200 r / min for 12 h. Collect the bacterial cells, resuspend them in a liquid containing 2.13 g / L MES, 2 g / L MgCl2, and 20 g / L sucrose, and add 20 mg / L AS to adjust the OD600 to 1.0. Mix the target gene and empty vector at a 1:1 volume ratio and incubate at room temperature for 3 h. Transient transformation overexpression of grape leaves and VIGS technology were performed according to Wang Fusheng (2016) with slight modifications.
[0044] (1) Immerse the leaves in Agrobacterium resuspension, place them in a vacuum container, and evacuate for 20 min using a vacuum pump; (2) After rinsing the leaves with distilled water, they were transferred to a light incubator for cultivation. The petioles were wrapped with moistened degreased cotton and cultured in a culture room with a light / dark cycle of 16 / 8 h and a temperature of 25℃ for 2-3 days.
[0045] Low-temperature treatment method for grape leaves: Fresh grape leaves from the field were taken and treated at 4℃ for 2 h. Starting from 0℃, the temperature was gradually decreased to -4℃ at a rate of 1℃ / h, then increased to 4℃ for 2 h, and finally allowed to recover at room temperature for 3-4 h. Phenotypes were observed and relevant physiological indicators were detected.
[0046] To prove VvSTS3 The function of resveratrol in synthesis was investigated using 'Cabernet Sauvignon' leaves as experimental material. Agrobacterium-mediated transient transformation was employed, and vacuum filtration was used to convert leaves containing TRV2-... VvSTS3 plasmids and OE- VvSTS3 Agrobacterium tumefaciens solution containing plasmid (in this figure, EV represents the empty pCAMBIA2301 vector, and OE represents overexpression) was infiltrated into the leaves, and then the transformed grape leaves were subjected to... VvSTS3 Expression levels were detected. Results are as follows: Figure 8 As shown.
[0047] 2. Experimental Results and Analysis: The results showed that from Figure 8 It can be seen that TRV2- VvSTS3 grape leaves VvSTS3 The transcription level was lower in grape leaves transformed with the empty vector, such as Figure 8 A, and OE- VvSTS3 grape leaves VvSTS3 The transcription level was higher in grape leaves transformed with the empty vector than in those transformed with the empty vector. Figure 8 B indicates TRV2- VvSTS3 plasmids and OE- VvSTS3 The plasmid was successfully transferred into the grape leaf.
[0048] Example 6 Instantaneous Conversion VvSTS3 Resveratrol content in grape leaves 1. Experimental Method: The changes in resveratrol content in successfully transformed grape leaves and empty vector grape leaves were detected using HPLC.
[0049] Detection of resveratrol content: The method of resveratrol content was determined with slight modifications based on that of Feng Wenhua (2016).
[0050] (1) Extraction of resveratrol and preparation of resveratrol standard Weigh 2 g of grape leaves and grind them thoroughly under liquid nitrogen and in the dark. Extract with 10 mL of chromatographic grade methanol. Then, sonicate at 25℃ for 40 min (220V, 100W), extract at 4℃ in the dark for 24 h, centrifuge at 10000 rpm at 4℃ for 10 min, collect the supernatant, filter through a 0.22 μm organic filter, and store at -80℃ in the dark.
[0051] Weigh 200 mg of resveratrol standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., purity >99%) and dissolve it in 500 mL of methanol to prepare a 0.4 mg / mL stock solution, which was stored at -80℃ protected from light. Dilute the stock solution with methanol to prepare resveratrol standard solutions of 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, and 300 mg / L, which were then stored at -80℃ protected from light.
[0052] (2) Chromatographic conditions The Agilent 1290 system used ultrapure water and acetonitrile as the mobile phase for determining resveratrol content. The column temperature was 30℃, and the detection wavelength was 306 nm. Mobile phase A was ultrapure water, and mobile phase B was acetonitrile. The mobile phase elution program was as follows: the ratio of mobile phase A (ultrapure water) to mobile phase B (acetonitrile) was 20:80 (v / v), the flow rate was 0.3 mL / min, and the injection volume was 20 μL. The chromatographic column was SB-Aq C18, 4.6 mm × 250 mm, 5 μm, and the external standard method was used for quantification.
[0053] (3) Determination of linear relationship The peak elution time was approximately 4.9 min. A standard curve was plotted, with Y representing the peak area and X representing the standard concentration. Within the concentration range of 50-300 mg / L, the linear regression equation was: y = 97.588x - 1181.9, with a correlation coefficient R² = 0.9798. The resveratrol content in the sample solution was calculated based on the peak area.
[0054] To investigate the function of resveratrol under low-temperature stress, Cabernet Sauvignon leaves were used as experimental material. The leaves were treated at 4℃ for 2 hours, then gradually decreased to -4℃ at a rate of 1℃ / h, followed by a further increase to 4℃ for 2 hours, and finally allowed to recover at room temperature for 3-4 hours. The resveratrol content in the leaves of acclimatized grape seedlings was measured at different time points (0, 3, 6, 9, 12, and 24 hours) after the low-temperature stress treatment. Results are as follows: Figure 9 As shown.
[0055] 2. Experimental Results and Analysis: The results showed that, compared with grape leaves transformed with the empty vector, silencing... VvSTS3 The resveratrol content in grape leaves decreased, while overexpression... VvSTS3 The resveratrol content in grape leaves is elevated, such as Figure 9 A and Figure 9 As shown in Figure B, EV is pCAMBIA2301 under no-load conditions, which preliminarily proves... VvSTS3 It can synthesize resveratrol.
[0056] Example 7 Instantaneous Conversion VvSTS3 Analysis of cold resistance of grape leaves I. Effects of Low Temperature Stress on VvSTS3 Effects of transient silencing on physiological indicators of grape leaves 1. Experimental Method: Detection of physiological indicators under stress: SOD activity was determined by the nitroblue tetrazolium method, POD activity by the guaiacol method, MDA content by the thiobarbituric acid method, and CAT activity and O2 by colorimetric method. - The content was determined using an ultraviolet spectrophotometer (TU-1810). Cell membrane relative permeability was measured using a DDSJ-308A conductivity meter, Fv / Fm was measured using a chlorophyll fluorometer (FMS2-2521), and chlorophyll content was determined using a chlorophyll content analyzer.
[0057] In order to investigate VvSTS3 The function of genes in grape cold resistance was investigated using Agrobacterium-mediated transient transformation of grape leaves, and virus-induced gene silencing was employed to reduce the [function of genes]. VvSTS3 Gene expression in grape leaves. Successfully transformed TRV2- VvSTS3 Grape leaves and empty carrier grape leaves according to "Example 2 II. Low temperature stress" VvSTS3 The method of "analyzing gene expression characteristics" was used for low-temperature treatment. The results are as follows: Figure 10 As shown, Non-stress refers to room temperature treatment, Cold-stress refers to cold treatment, MDA is malondialdehyde, SOD is superoxide dismutase, POD is superoxide dismutase, and CAT is catalase.
[0058] 2. Experimental Results and Analysis: The results showed that, from Figure 10 As can be seen from the data, under low temperature stress, compared with grape leaves transformed with empty vectors, TRV2- VvSTS3 Relative permeability of leaf cell membranes, MDA, and O2. - Increased content, such as Figure 10 A, Figure 10 B and Figure 10 As shown in C; the activities of SOD, POD, and CAT decrease, such as Figure 10 D、 Figure 10 E and Figure 10 F indicates TRV2- VvSTS3 The leaf material exhibits lower resistance to low-temperature stress than the leaf material from an empty carrier. Meanwhile, TRV2- VvSTS3 The chlorophyll content and Fv / Fm value of the leaves were lower than those of grape leaves transformed with the empty vector, such as Figure 10 G and Figure 10 As shown in H.
[0059] II. Effects of Low Temperature Stress VvSTS3 Effects of transient overexpression on physiological parameters in grape leaves 1. Experimental Method: Detection of physiological indicators under stress: SOD activity was determined by the nitroblue tetrazolium method, POD activity by the guaiacol method, MDA content by the thiobarbituric acid method, and CAT activity and O2 by colorimetric method. - The content was determined using an ultraviolet spectrophotometer (TU-1810). Cell membrane relative permeability was measured using a DDSJ-308A conductivity meter, Fv / Fm was measured using a chlorophyll fluorometer (FMS2-2521), and chlorophyll content was determined using a chlorophyll content analyzer.
[0060] To further explore VvSTS3 The function of the gene in grape cold resistance was investigated using grape leaves as experimental material. Agrobacterium-mediated transient transformation of grape leaves was employed, injecting the gene containing pCAMBIA2301- VvSTS3 Agrobacterium and pCAMBIA2301 empty vector plasmid were used. Then, the successfully transformed grape leaves and the empty vector grape leaves were subjected to low-temperature treatment according to the method described in "Example 2, Section 2: Analysis of VvSTS3 gene expression characteristics under low-temperature stress". The results are as follows... Figure 11 As shown.
[0061] 2. Experimental Results and Analysis: The results showed that, from Figure 11 It can be seen that, compared with leaves without the empty vector, grape leaves transiently overexpressing the vector showed higher relative cell membrane permeability, MDA, and O2. - The content decreases, such as Figure 11 A, Figure 11 B and Figure 11As shown in C; the activities of antioxidant enzymes SOD, POD, and CAT are increased, such as Figure 11 D、 Figure 11 E and Figure 11 As shown in F; chlorophyll content and Fv / Fm value increase, such as Figure 11 G and Figure 11 As shown in Figure H, EV represents pCAMBIA2301 under no-load conditions. The above results indicate... VvSTS3 It improved the cold resistance of grape leaves.
[0062] III. Effects of Low Temperature Stress VvSTS3 Effects of transient transformation on the expression of cold-related genes in grape leaves 1. Experimental Method: Total RNA was extracted from grapes using the CTAB method and detected by real-time quantitative PCR. VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The relative expression levels were determined, and each treatment was repeated three times, using the same method as in Example 2. Real-time quantitative PCR primers are shown in Table 3. Results are as follows: Figure 12 As shown.
[0063] 2. Experimental Results and Analysis: By detecting changes in the expression of cold-related genes in transiently transformed grape leaves under low-temperature stress, it was found that compared with leaves without the vector, overexpression of these genes significantly increased the expression of cold-related genes. VvSTS3 Cold-related genes in grape leaves VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The expression is upregulated, such as Figure 12 As shown in B. And silence. VvSTS3 Cold-related genes in grape leaves VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The expression decreased, such as Figure 12 As shown in Figure A, EV represents pCAMBIA2301 under no-load conditions. Based on the above results, it can be inferred that... VvSTS3 It may participate in the grape's response to low-temperature stress through the CBF-COR pathway.
[0064] Table 3 qRT-PCR primers
[0065] Example 8 VvSTS3 Screening and identification of overexpression in grape callus and tobacco (1) Acquisition of callus tissue through overexpression: Pour LB liquid medium containing 50 mg / L Kan and 50 mg / L Lf antibiotics into a sterile Erlenmeyer flask. Add the Agrobacterium tumefaciens bacterial culture to the medium and shake at 28°C and 200 r / min for 12 h. Collect the bacterial cells and add MS liquid medium containing 20 mg / L AS (acetosyringone) to adjust the concentration to an OD600 value of 0.6-0.8.
[0066] Healthy grape callus tissue was placed in a petri dish containing filter paper. The prepared Agrobacterium tumefaciens bacterial suspension was added dropwise to the callus tissue using a pipette. After standing for 5 minutes, the callus was removed, and the residual Agrobacterium tumefaciens bacterial suspension was blotted dry with filter paper to complete the infection. The grape callus tissue, after removing the residual bacterial suspension, was then evenly distributed onto a co-culture medium containing 20 mg / LAS. It was then incubated in the dark at 25°C for 3 days.
[0067] Grape callus tissue cultured in the dark for 3 days was removed and transferred to callus selection medium containing antibiotics. One month later, RNA was extracted from the selected healthy, golden-yellow grape callus tissue for qPCR identification. Successfully identified overexpression materials were then propagated. These materials will be subsequently used for low-temperature phenotype observation and detection of stress resistance physiological indicators.
[0068] (2) Obtaining overexpression tobacco tissue culture seedlings: Pour LB liquid medium containing 50 mg / L Kan and 50 mg / L Rif antibiotics into a sterile Erlenmeyer flask, add Agrobacterium bacterial suspension to the medium, and shake at 28°C and 200 r / min for 12 h. Collect the bacterial cells and add MS liquid medium containing 20 mg / L AS.
[0069] Healthy tobacco K326 tissue culture seedlings were cut into appropriately sized cubes, placed in the bacterial solution, shaken for 5 minutes, and then removed. Residual Agrobacterium solution was blotted dry with filter paper to complete the infection. The leaves, after removing residual bacterial solution, were evenly placed on a co-culture medium containing 20 mg / L ALS. The plants were then incubated in the dark at 28°C for 2 days.
[0070] Tobacco leaves cultured in the dark were washed with sterile ddH2O and then placed in MS liquid medium containing 400 mg / L timetenidine (TM). After removal, residual moisture was blotted with filter paper. The leaves were then placed on selection medium containing antibiotics and cultured in the dark at 28°C for 3 days before being transferred to selection medium for screening. One month later, healthy tobacco buds were transferred to tissue culture flasks to grow into tissue culture seedlings, and DNA was extracted for semi-quantitative identification. This DNA will be used for subsequent observation of low-temperature phenotypes and detection of stress resistance physiological indicators.
[0071] First, use the prepared pCAMBIA2301- VvSTS3Agrobacterium plasmid was used to infect healthy seedless white grape callus. After 3 days of dark incubation at 25°C, the culture was transferred to selection medium for screening. Figure 13 As shown in Figure A. RNA was extracted from the selected healthy, golden-yellow callus tissue for qRT-PCR identification. Figure 13 As shown in B (WT is wild-type), overexpression VvSTS3 The expression level in callus tissue was significantly higher than that in wild type, achieving [the desired effect]. VvSTS3 Overexpression was performed on callus tissue, and successfully identified overexpression materials were propagated for subsequent experiments.
[0072] Next, the prepared bacterial solution was used to infect the pruned leaves of K326 tobacco tissue culture seedlings. After 3 days of dark incubation at 28℃, the seedlings were transferred to selection medium for screening. Positive tobacco buds were then transferred into tissue culture bottles to cultivate tissue culture seedlings. Figure 14 As shown in A. DNA was extracted for semi-quantitative analysis, ultimately yielding 16 positive tobacco seedlings. Two lines with high and low expression levels were selected (…). OE- VvSTS3-4 , OE-VvSTS3-16 Propagation will be carried out for subsequent purposes, such as... Figure 14 As shown in B.
[0073] The culture media used in this experiment are shown in Table 4.
[0074] Table 4 Culture medium formulation
[0075] Example 9 VvSTS3 Effects of resveratrol content on grape callus and tobacco seedlings 1. Experimental Method: To verify VvSTS3 The function of resveratrol in resveratrol synthesis was determined according to the HPLC detection method in "Example 6: Resveratrol Content in Grape Leaves After Instantaneous Conversion of VvSTS3" in wild-type grape callus and tobacco seedlings. VvSTS3 Changes in resveratrol content in grape callus overexpression and heterologously overexpressed tobacco seedlings. Results are as follows: Figure 15 As shown.
[0076] 2. Experimental Results and Analysis: The results showed that, compared with wild-type grape callus and tobacco seedlings, VvSTS3 The resveratrol content increased in grape callus tissue overexpressing resveratrol and in tobacco seedlings heterologously overexpressing resveratrol, such as Figure 15 A and Figure 15 As shown in B. The above results prove... VvSTS3 It is a key enzyme in the synthesis of resveratrol.
[0077] Example 10 VvSTS3Analysis of the cold resistance of overexpression materials 1. Experimental Method: Low-temperature treatment method for grape callus: Wild-type and overexpression 'seedless white' callus tissues with similar morphology and size were taken from 4-week-old grapes and treated at 4℃ for 12 h. Starting from 0℃, the temperature was gradually decreased to -8℃ at a rate of 1℃ / h, maintained for 2 h, and then treated at 4℃ for 12 h. Finally, the tissues were incubated in the dark at 25℃ for 3 days. Phenotypic characteristics were observed and relevant physiological indicators were detected.
[0078] Low-temperature treatment method for tobacco K326 seedlings: One-month-old wild-type and overexpression tobacco seedlings with similar morphology and size were taken and treated at 4℃ for 48 h, and then restored at room temperature for 3 h. Phenotypic characteristics were observed and relevant physiological indicators were detected.
[0079] Total RNA was extracted from grapes using the CTAB method and detected by real-time quantitative PCR. VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The relative expression levels were determined, and each treatment was repeated three times. The treatment method was the same as in "Example 2 II. Analysis of the expression characteristics of the VvSTS3 gene under low temperature stress". The real-time quantitative PCR primers are shown in Table 3.
[0080] SOD activity was determined using the nitroblue tetrazol method, POD activity using the guaiacol method, MDA content using the thiobarbituric acid method, and CAT activity and O2 content were determined using a colorimetric method. .- All instruments used were ultraviolet spectrophotometers (TU-1810). Cell membrane relative permeability was measured using a DDSJ-308A conductivity meter, Fv / Fm was measured using a chlorophyll fluorometer (FMS2-2521), and chlorophyll content was measured using a chlorophyll content measuring instrument.
[0081] 2. Experimental Results and Analysis: First, the obtained overexpression callus tissue was subjected to low-temperature treatment. The results showed that before low-temperature treatment, there were no significant phenotypic differences between wild-type and overexpression grape callus tissues. However, after low-temperature treatment, most of the wild-type grape callus tissue turned brown or black, and its growth was significantly inhibited. In contrast, the overexpression grape callus tissue remained golden yellow and showed less damage. Figure 16 A. Next, the heterologous overexpression tobacco seedlings were subjected to a 4℃ 48h low-temperature stress treatment. The results showed that before the low-temperature treatment, there were no significant phenotypic differences between wild-type and heterologous overexpression tobacco seedlings. However, after the low-temperature treatment, most wild-type tobacco seedlings showed wilting, while the heterologous overexpression tobacco seedlings only showed localized wilting, such as... Figure 16 B.
[0082] Under low-temperature stress, compared with wild-type grape callus, the relative permeability of the cell membrane in grape callus overexpressing the expression was reduced after low-temperature treatment, such as... Figure 17 As shown in Figure A, lower levels of MDA and O2 were accumulated. - Content, such as Figure 17 B and Figure 17 As shown in Figure C, this indicates that the overexpression grape callus suffered less damage. Simultaneously, the activities of antioxidant enzymes SOD, POD, and CAT in the overexpression grape callus were higher than those in wild-type seedless white grape callus. Figure 17 As shown in DF.
[0083] Furthermore, there was no significant difference in the relative expression levels of cold-related genes between wild-type grape callus and overexpressing grape callus before and after low-temperature treatment. However, after low-temperature treatment, the expression levels of cold-related genes in overexpressing grape callus increased compared to wild-type grape callus. VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The expression of was significantly upregulated, such as Figure 18 As shown.
[0084] After low-temperature treatment, heterologous overexpression VvSTS3 Tobacco seedlings have lower cell membrane permeability and lower MDA content than wild-type plants, such as... Figure 19 A and Figure 19 As shown in Figure B, since tobacco leaves cannot maintain their integrity after low-temperature treatment, small circular pieces, i.e., leaf discs, were removed from the tobacco leaves using a sterilized stainless steel perforator. DAB and NBT staining experiments were then performed to detect the accumulation level of ROS in tobacco seedlings under low-temperature stress. The results showed that under normal conditions, wild-type K326 tobacco seedlings and those with heterologous overexpression of ROS... VvSTS3 There was no significant difference in staining color among tobacco seedlings. Under low-temperature conditions, heterologous overexpression showed better results compared to wild-type K326 tobacco seedlings. VvSTS3 Tobacco seedlings are dyed a lighter color, such as... Figure 19 As shown in C and 19D. Simultaneous heterologous overexpression. VvSTS3 Tobacco seedlings O2. - The content is lower than that of the wild type, such as Figure 19 As shown in E, the activities of SOD, POD, and CAT were higher than those of the wild type. Figure 19 As shown in FH. Furthermore, heterologous overexpression VvSTS3 The chlorophyll content and Fv / Fm value of tobacco seedlings were higher than those of wild type, indicating overexpression. VvSTS3 It improves the efficiency of plants in utilizing light energy, such as Figure 20 A and Figure 20 As shown in B. In summary, observation of cold resistance phenotype and determination of physiological indicators indicate that overexpression of [the substance / method]... VvSTS3 Genes enable transgenic plants to have greater cold resistance.
[0085] It should be noted that the specific embodiments are merely representative examples of the present invention, and the technical solution of the present invention is obviously not limited to the above embodiments, and there can be many variations. Those skilled in the art who obtain the present invention based on its explicit disclosure or without objection from the written description should consider it to be within the scope of protection of this patent.
Claims
1. VvSTS3 Genes, characterized by, The VvSTS3 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. A recombinant vector, characterized in that, Carrying the as described in claim 1 VvSTS3 Gene.
3. A recombinant bacterium, characterized in that, Includes the recombinant vector as described in claim 2.
4. As described in claim 1 VvSTS3 The application of genes, the recombinant vector of claim 2, or the recombinant bacteria of claim 3 in improving the tolerance of plants to low temperature stress.
5. The application according to claim 4, characterized in that, During low-temperature stress, the application is at least one of the following (1) to (4): (1) VvSTS3 The gene was overexpressed in plant leaves, which increased the accumulation of resveratrol in plant leaves; (2) VvSTS3 The gene was transiently silenced in the plant leaves, reducing the accumulation of resveratrol in the plant leaves; (3) VvSTS3 The gene is overexpressed in plant leaves, and under low temperature stress, the overexpression of the gene... VvSTS3 Compared with leaves containing an empty vector, plant leaves with the gene showed reduced relative cell membrane permeability, MDA content, and superoxide anion accumulation. (4) VvSTS3 The gene is overexpressed in plant leaves, and under low temperature stress, the overexpression of the gene... VvSTS3 Compared with leaves with empty vectors, plant leaves with the gene showed increased SOD, POD, and CAT activities, or increased chlorophyll content and Fv / Fm value.
6. The application according to claim 4, characterized in that, Will VvSTS3 The gene was overexpressed in plant leaves, and under low temperature stress, the expression of cold-related genes was increased. VvCBF1 , VvCBF2 , VvCBF3 and VvCOR27 The expression level of at least one of them.
7. The application according to any one of claims 4 to 6, characterized in that, The plant in question is either grape or tobacco.
8. As described in claim 1 VvSTS3 The application of genes, the recombinant vector of claim 2, or the recombinant bacteria of claim 3 in the genetic improvement of plant cold resistance.
9. A method for increasing the resveratrol content in plants, characterized in that, Expression of the method described in claim 1 in plants VvSTS3 Gene.
10. A method for improving the cold resistance of plants, characterized in that, The method includes the following steps: (a) Providing a product comprising as described in claim 1 VvSTS3 Gene expression vectors; (b) Introduce the expression vector described in step (a) into plant cells or tissues; (c) Regenerate transgenic plants from the plant cells or tissues of step (b).