VvYUC10 gene and application thereof

Abstract

The invention provides a VvYUC10 gene, the nucleotide sequence of the VvYUC10 gene is as shown in SEQ ID No.1, or the nucleotide sequence which is obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence as shown in SEQ ID No.1 and expresses proteins with the same function; the invention also provides an application thereof. The grape VvYUC10 gene has the advantages that the function of the grape VvYUC10 gene is verified, it is found that the content of auxin in calluses of overexpressed VvYUC10 arabidopsis thaliana and grapes under salt stress is increased, the oxidation resistance is enhanced, and it is further found that the expression quantities of the VvRD22 gene, the VvDREB2A gene, the VvRD29A gene, the VvP5CS gene, the VvCAT1 gene and the VvSOD gene are increased; finally, it shows that the VvYUC10 gene positively regulates and controls the auxin signal channel to positively resist external adverse environmental factor salinization, and a theoretical reference basis is provided for grape salt-tolerant gene mining and breeding.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and specifically relates to the VvYUC10 gene and its application. Background Art

[0002] In grape (Vitis vinifera L.) cultivation, due to the increase in high-salinity groundwater, unreasonable irrigation methods, and the increase in soil evaporation caused by climate change, soil salinization occurs (Rengasamy et al., 2006). Excessive salt in the soil can cause water shortage and nutritional imbalance in grape plants, osmotic stress and ionic stress, oxidative stress and other physiological and biochemical stress responses (Zhu et al., 2002), and even affect the formation of fruit quality.

[0003] Flavin monooxygenase (YUC) is a rate-limiting enzyme in the auxin synthesis pathway in plants (Mashiguchi et al., 2002), participating in the catalysis of indole-3-pyruvate (IPA) in the tryptophan-dependent pathway of auxin synthesis (Naser et al., 2016), and IPA then generates the plant growth hormone (Indole-3-acetic acid, IAA) (Cao al., 2019). IAA exists in all land plants and is involved in regulating biological processes such as plant polarity, apical dominance, tropic movement, senescence, pathogen response, abiotic stress response, and fruit formation (Gao et al., 2024). Transgenic potatoes (Solanum tuberosum L.) overexpressing AtYUC6 showed high auxin accumulation, high water retention capacity, and low ROS levels, thus enhancing the drought tolerance of potatoes (Cheol et al., 2013; Kim et al., 2013). In poplar, overexpressing AtYUC6 increased the auxin level and the efficiency of photosystem II under drought stress, and reduced the ROS level in cells, thereby maintaining the cell membrane permeability (Ke et al., 2015). In addition, Chen et al. reported that the downregulated expression of LiYUC4 and LiYUC10 was observed while the content of endogenous IAA decreased in normally developing lily somatic embryos (Chen et al., 2020). Research reports show that members of the YUC gene family in plants such as Isatis indigotica, Vigna radiata, and lily all showed strong responses to abiotic stresses such as salt, drought, and high temperature, and also regulated the content change of IAA (Wu et al., 2023; Qin et al., 2020; Chen et al., 2020). In particular, Arabidopsis seedlings overexpressing the CsYUC11 gene showed salt tolerance in plant growth (Yan etal., 2016). Currently, there are few reports on the research of grape VvYUC10 in grape salt stress. Summary of the Invention

[0004] In view of the above-mentioned technical limitations, the present application provides the VvYUC10 gene and its application; it overcomes the deficiencies and defects mentioned in the background technology.

[0005] To achieve the above object, the present application adopts the following technical solutions:

[0006] The invention point of the present application is to provide the VvYUC10 gene, whose nucleotide sequence is shown in SEQ ID No.1, or a nucleotide sequence obtained by substituting, deleting, and / or adding one or more nucleotides to the nucleotide sequence shown in SEQ ID No.1 and expressing the same functional protein.

[0007] SEQ ID No.1:

[0008] > VvYUC10

[0009]

[0010] The second inventive point of the present application is to provide a protein, the coding gene of which is the above-mentioned VvYUC10 gene, and the amino acid sequence of the protein is as shown in SEQ ID No.2.

[0011] SEQ ID No.2:

[0012] MPEAAVIIVGAGPSGLAMAGCLCQLSIPYLILEREDCCASLWKKKAYDRLHLHLPKQYCTLPHMEMPADWPKYPSRQQFVQYLDDYADHFNIRPMYRRSVESGSFDESRGKWNVGVRNGESGELEEYSGLFLVVASGETSDAFVPDIDGLSTFIGKVIHSTQYKNGKEFADMKVLVVGSGNSGMEIALDLSNCGAKTSIVVRSPLHMLSREMVNLGLALLKYIPYNMVDSLMVILSKLVYGDLNKYGITRPEEGPFFLKVKYGKYPVVNTGTFGKIKSGEIQVLPKLIGIRGDEVVFEGGKSHPFDAIVFATGFKRSTSKWLKGDDYLLNEDGLPKPSFPNHWKGKNGLYCAGLARRGLYGSALDAQNIANDIKTQ.

[0013] The third object of the present application is to provide a biological material containing the above-mentioned VvYUC10 gene or the above-mentioned protein, and the biological material is selected from any one or more of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector and engineered bacteria.

[0014] The fourth object of the present application is to provide the application of the above-mentioned VvYUC10 gene, the above-mentioned protein or the above-mentioned biological material in enhancing the salt tolerance of plants.

[0015] The fifth object of the present application is to provide the application of the above-mentioned VvYUC10 gene, the above-mentioned protein or the above-mentioned biological material in increasing the content of plant auxin.

[0016] The sixth object of the present application is to provide the application of the above-mentioned VvYUC10 gene, the above-mentioned protein or the above-mentioned biological material in improving the reactive oxygen species scavenging ability of plants.

[0017] Optionally, for the above-mentioned application, the plant is selected from plants of the genus Vitis in the family Vitaceae, preferably grape.

[0018] The seventh objective of this application is to provide the amplification method of the above VvYUC10 gene. Using the cDNA of Pinot Noir grapes as a template, PCR amplification is carried out with primers to obtain the VvYUC10 gene.

[0019] Optionally, in the above amplification method, among the amplification primers, the upstream primer is as shown in SEQ ID No.3, and the downstream primer is as shown in SEQ ID No.4.

[0020] The objective of the present invention is to provide the grape VvYUC10 salt-tolerant gene and its use in solving the problem of grape salinization, to provide candidate genes for salt-tolerant grape breeding. This gene positively regulates grape salt tolerance, and up-regulating its expression in Arabidopsis thaliana and grape callus can improve the salt tolerance of grapes.

[0021] Compared with the prior art, this application has the following advantages:

[0022] This application provides the VvYUC10 gene and its application. By verifying the function of the grape VvYUC10 gene, it is found that under salt stress, the auxin content increases and the antioxidant capacity enhances in the overexpressed VvYUC10 Arabidopsis thaliana and grape callus. Furthermore, it is found that the expression levels of VvRD22, VvDREB2A, VvRD29A, VvP5CS, VvCAT1, and VvSOD genes all increase; finally, it is shown that the VvYUC10 gene positively regulates the auxin signaling pathway to actively resist the external adverse environmental factor of salinity, providing a theoretical reference basis for grape salt-tolerant gene mining and breeding. Description of the Drawings

[0023] Figure 1 Shown as the map of the overexpression vector.

[0024] Figure 2 Shown as the cloning of the VvYUC10 gene and the PCR amplification of the bacterial solution. Among them, Figure 2 A is the amplification result of the VvYUC10 target gene fragment, Figure 2 B is the PCR result of the Escherichia coli bacterial solution.

[0025] Figure 3 Shown as the subcellular localization analysis.

[0026] Figure 4 Shown as the identification of positive Arabidopsis thaliana plants; M, DL 2000 DNA Marker, P is the positive control, WT is the negative control, and 1-10 are transgenic positive plants.

[0027] Figure 5 Shown as the positive identification of grape callus; M, DL 2000 DNA Marker, P is the positive control, WT is the negative control, and 1-5 are positive transformed callus.

[0028] Figure 6 Show the phenotypes of transgenic Arabidopsis thaliana under salt stress, as well as the differences in auxin and genes. Among them, Figure 6 A shows the phenotypic differences under salt treatment, where WT represents wild-type Arabidopsis thaliana, and OE2, 3, and 6 represent Arabidopsis thaliana lines overexpressing the VvYUC10 gene; Figure 6 B shows the auxin content; Figure 6 C shows the relative expression level of the VvYUC10 gene.

[0029] Figure 7 Show the response of the antioxidant system of transgenic Arabidopsis thaliana under salt stress. Among them, Figure 7 A shows the superoxide dismutase activity, Figure 7 B shows the catalase activity, Figure 7 C shows the ascorbic acid content, Figure 7 D shows the glutathione content; each value (mean ± standard deviation) includes three replicates; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple range test (p < 0.05).

[0030] Figure 8 Show the changes in membrane lipid peroxidation products and osmoregulatory substances in transgenic Arabidopsis thaliana under salt stress. Among them, Figure 8 A shows the malondialdehyde content, Figure 8 B shows the proline content; each value (mean ± standard deviation) includes three replicates; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple range test (p < 0.05).

[0031] Figure 9 Show the expression levels of salt tolerance-related genes in Arabidopsis thaliana overexpressing VvYUC10 under salt stress, where each value (mean ± standard deviation) includes three replicates. Different lowercase letters at the same concentration indicate significant differences after Duncan's multiple range test (p < 0.05).

[0032] Figure 10 Show the analysis of the phenotypes of transgenic grape calli and key genes and metabolites under salt stress. Among them, Figure 10 A shows the phenotypes of different calli, Figure 10 B shows the relative growth of callus, and each point represents 1 replicate; Figure 10 C shows the auxin content; Figure 10 D shows the relative expression of VvYUC10; WT represents wild-type grape callus, and YUC-OE represents callus overexpressing VvYUC10; the values are expressed as mean ± standard deviation; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple range test (p < 0.05).

[0033] Figure 11Shown is the response of the antioxidant system in grape callus under salt stress, where Figure 11 A is the superoxide dismutase activity, Figure 11 B is the catalase activity, Figure 11 C is the ascorbic acid content, Figure 11 D is the glutathione content; each value (mean ± standard deviation) includes three replicates; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple test (p < 0.05).

[0034] Figure 12 Shown are the changes in membrane lipid peroxidation products and osmotic regulators in grape callus under salt stress, where Figure 12 A is the malondialdehyde content, Figure 12 B is the proline content; each value (mean ± standard deviation) includes three replicates; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple test (p < 0.05).

[0035] Figure 13 Shown is the expression level of salt tolerance-related genes in VvYUC10 transgenic callus under salt stress, where each value (mean ± standard deviation) includes three replicates; different lowercase letters at the same concentration indicate significant differences after Duncan's multiple test (p < 0.05). Detailed implementation manners

[0036] To make the objectives, technical solutions and advantages of this application clearer, the following further details this application. However, it should be understood that the description herein is only used to explain this application and not to limit the scope of this application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are all commercially available, and the characterization means involved can be referred to the relevant descriptions in the prior art and will not be elaborated herein.

[0038] To further understand this application, the following further details this application with reference to the best embodiments.

[0039] Example 1

[0040] The VvYUC10 gene has a nucleotide sequence as shown in SEQ ID No.1; the protein encoded by this gene has an amino acid sequence as shown in SEQ ID No.2.

[0041] This gene or protein can be incorporated into a biomaterial to play a role, and the biomaterial is selected from any one or more of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, and engineered bacteria.

[0042] The application scenarios of this gene, protein or biomaterial are as follows:

[0043] 1) Improve the salt tolerance of plants;

[0044] 2) Increase the auxin content in plants;

[0045] 3) Improve the ability of plants to scavenge reactive oxygen species.

[0046] The plant is selected from the plants of the genus Vitis in the Vitaceae family, preferably grape.

[0047] The amplification method of this gene is to use the cDNA of Pinot Noir grape as a template, and perform PCR amplification with primers to obtain the VvYUC10 gene; among the amplification primers, the upstream primer is as shown in SEQ ID No. 3, and the downstream primer is as shown in SEQ ID No. 4.

[0048] Example 2

[0049] 1. Experimental materials and methods:

[0050] 1.1 Experimental materials and treatments:

[0051] In this example, the materials used for grape gene cloning were the wine grape variety 'Pinot Noir'. Select the fully expanded leaves in the middle and upper parts, quickly freeze them in liquid nitrogen, and store them in a -80 °C refrigerator for later use.

[0052] The Arabidopsis thaliana Columbia wild type and Nicotiana benthamiana used in this example were both preserved and provided by the laboratory. They were planted in a mixed substrate (nutrient soil: vermiculite = 3:1) and grown in an artificial climate chamber at 25 °C, 10000 Lx, with a photoperiod of 16 h (light) / 8 h (dark), and a relative humidity of 70%.

[0053] The grape callus used in this example was induced from the wine grape variety 'Pinot Noir' and was preserved and provided by the laboratory. It was grown on a medium of B5 + 0.5 mg·L -1 NAA + 1 mg·L -1 of KT, and subcultured every 20 d for subsequent genetic transformation experiments.

[0054] Wild-type and transgenic Arabidopsis thaliana were sown in a cultivation substrate (nutrient soil: vermiculite = 3:1) and cultured in an artificial climate chamber until germination. The growth conditions were as follows: Arabidopsis thaliana was sown in the cultivation substrate (nutrient soil: vermiculite = 3:1), placed in an artificial climate chamber for cultivation, and Arabidopsis thaliana seedlings with consistent growth vigor were selected for transplanting and planting. Salt treatment was started after 20 days of growth. In this example, wild-type and transgenic Arabidopsis thaliana were treated with NaCl at concentrations of 0 (CK), 100 mmol·L -1 and 200 mmol·L -1 , and the control was watered with an equal amount of clear water. Seven days after salt treatment, the materials were harvested for subsequent experiments. Arabidopsis thaliana seedlings with consistent growth vigor were selected for transplanting and planting. Salt treatment was started after 20 days of growth. In this example, wild-type and transgenic Arabidopsis thaliana were treated with NaCl at concentrations of 0 (CK), 100 mmol·L -1 and 200 mmol·L -1 . The plants were watered thoroughly for the first time and supplemented with water on the 3rd and 5th days. The control was watered with an equal amount of clear water. Seven days after the end of salt solution irrigation, the materials were harvested for physiological index determination to analyze the differences in salt tolerance. The growth of the materials was recorded before and after treatment to compare phenotypic differences.

[0055] Wild-type and transgenic calli were respectively inoculated on B5 medium containing 0 (CK), 50 mmol·L -1 and 100 mmol·L - 1 NaCl (B5 + 0.5 mg·L -1 NAA + 1 mg L -1 KT + 20 g·L -1 sucrose + 7g L -1 agar + 1 g·L -1 inositol + 300 mg·L -1 PVP). After 20 days of treatment, the materials were harvested for physiological index determination. The growth of the materials was recorded before and after treatment to compare phenotypic differences.

[0056] 1.2 Vectors and competent cells:

[0057] The competent cells of Agrobacterium rhizogenes GV3101 were purchased from Shanghai Bioengineering Technology Co., Ltd. The overexpression vector pCAMBIA2300-EGFP was a gift from the College of Horticulture, Gansu Agricultural University. The vector map is as Figure 1 shown.

[0058] 1.3 Experimental methods:

[0059] 1.3.1 Primer design:

[0060] The cloning primers, qRT-PCR primers of the VvYUC10 (VIT_207s0104g01260) gene, and the primers for verifying positive plants were designed using CE Desgin 1.04. All primer sequences in the experiment are shown in Table 1.

[0061] Table 1

[0062]

[0063] 1.3.2 RNA extraction and reverse transcription:

[0064] 1.3.2.1 Extraction of grape RNA:

[0065] It was extracted according to the instructions of the RNAprep pure Plant Total RNA Extraction Kit (product number DP452, purchased from Tiangen Biochemical Technology Co., Ltd.).

[0066] 1.3.2.2 Reverse transcription of grape RNA (synthesis of the first strand of cDNA):

[0067] According to the Evo M-MLV RT Kit (product number AG11603, purchased from Aikerui Biotech)

[0068] The following steps were used to reverse transcribe the total RNA extracted above:

[0069] a. The RNA taken out from -80 °C was melted on ice; the reagents in the kit taken out from -20 °C and RNase-free ddH 2 O were melted at room temperature and then quickly placed on ice, and gently shaken and mixed before use.

[0070] b. Prepare the reaction system according to the following table (Table 2):

[0071] Table 2

[0072]

[0073] c. Carry out the first step of the reverse transcription reaction according to the following reaction program (Table 3):

[0074] Table 3

[0075]

[0076] d. Prepare the reaction system according to the following table (Table 4):

[0077] Table 4

[0078]

[0079] e. Carry out the first step of the reverse transcription reaction according to the following reaction program (Table 5):

[0080] Table 5

[0081]

[0082] f. After the reaction is completed, after detecting the concentration and purity of cDNA, store it at -20°C for later use.

[0083] 1.4 Amplification of target gene and construction of overexpression vector:

[0084] 1.4.1 PCR amplification of target gene:

[0085] Using grape leaf cDNA as a template, Green Taq Mix (product number P131, purchased from Novizan) was used to amplify the silencing fragment with primers added with the restriction enzyme BamH I site and protective bases. The system is as follows (Table 6):

[0086] Table 6

[0087]

[0088] After the operation is completed, gently shake and mix, then briefly centrifuge and react according to the following program (Table 7):

[0089] Table 7

[0090]

[0091] The VvYUC10 gene sequence was amplified from grape leaves by PCR method, and its nucleotide sequence is shown in SEQ ID No.1:

[0092] The nucleotide sequence of VvYUC10 is shown in SEQ ID No.1:

[0093] > VvYUC10

[0094]

[0095] 1.4.2 Ligation of the target gene with the cloning vector pCAMBIA2300-GFP

[0096] a. Take out the pCAMBIA2300-GFP vector from the -80°C refrigerator and melt it on ice.

[0097] b. The specific digestion system is shown in Table 8:

[0098] Table 8

[0099]

[0100] c. Calculate the volume of the added target fragment (the molar ratio of the vector to the target fragment = 3:1), and add the following components to a sterile 1.5 mL centrifuge tube (the whole operation is completed on ice), as shown in Table 9:

[0101] Table 9

[0102]

[0103] d. React at 37°C for 30 min; cool to 4°C or immediately place on ice.

[0104] 1.4.3 Transformation of DH5α Escherichia coli competent cells

[0105] Transform into DH5α Escherichia coli competent cells by heat shock method. Verify the bacterial liquid by PCR using the target gene sequence primers and sequence it (completed by Shanghai Sangon Biological Engineering Co., Ltd.). Transfer the plasmid with appropriate sequencing results into Agrobacterium tumefaciens GV3101 competent cells, add 50% glycerol at a ratio of 1:1 and store it in the -80°C refrigerator.

[0106] 1.5 Agrobacterium-mediated genetic transformation and identification of Arabidopsis thaliana

[0107] (1) Activate Agrobacterium tumefaciens carrying the VvYUC10 gene.

[0108] (2) Centrifuge the activated bacterial liquid to enrich the bacteria (6000 rpm, 10 min), and discard the supernatant.

[0109] (3) Resuspend the bacteria with infiltration buffer (1 / 2 MS, 5% sucrose, 0.03% Silwet-L77) and adjust the OD600 to 0.6 for later use.

[0110] (4) Use Arabidopsis thaliana at the flowering stage as the infection material. Cut off the existing pods, immerse the inflorescence completely in the infiltration solution for 30 s, take it out and blot dry the infiltration solution stained on other parts, then cover the Arabidopsis thaliana plants with a film and place them in the artificial climate chamber for dark culture for 24 h. Remove the film and adjust to the normal growth conditions of Arabidopsis thaliana.

[0111] (5) Repeat the above operations after 4 days to ensure the transformation efficiency. The materials after inflorescence infiltration were maintained under normal growth management until the seeds matured, and the seeds were the T0 generation transgenic Arabidopsis thaliana.

[0112] 1.5.1 Screening and identification of transgenic Arabidopsis thaliana:

[0113] (1) Disinfect the dried seeds of the T0 generation by the following process: soak them in 75% alcohol for 15 s, wash with sterile water 5 times, soak in 10% sodium hypochlorite for 5 min, and wash with sterile water 5 times. During this period, invert and mix well repeatedly.

[0114] (2) After surface disinfection, leave a little sterile water in the centrifuge tube, seal it and place it at 4 °C in the dark for vernalization for 2 days.

[0115] (3) After vernalization, sow the seeds on 1 / 2MS screening medium (+Kana 50 mg L -1 ) and culture them under the conditions of 22 °C, 16 h light / 8 h dark.

[0116] (4) Two weeks later, the positive transformed plants grew well, with two cotyledons being green, while the cotyledons of negative plants turned yellow. Select the Arabidopsis thaliana with normal growth on the plate and transplant it into the growth substrate. When the plants grow 7 - 8 true leaves, collect the leaves and extract DNA for PCR identification. The plants identified as positive were continued to be cultured in the artificial climate chamber until the seeds matured, and the T1 generation seeds were obtained.

[0117] (5) Continue to repeat the above screening process of steps 1 - 4 using the T1 generation as seeds until the homozygous transgenic Arabidopsis thaliana seeds of the T3 generation are harvested for subsequent salt tolerance function verification.

[0118] 1.6 Agrobacterium - mediated genetic transformation and identification of grape callus:

[0119] (1) Activate the Agrobacterium liquid containing the over - expressed VvYUC10 gene stored at ultra - low temperature. The specific method is as follows:

[0120] Prepare the grape callus required for infection;

[0121] Prepare the Agrobacterium infection solution: Pick the Agrobacterium (single colony of Agrobacterium carrying the recombinant plasmid) and inoculate it into the liquid LB medium containing kanamycin. Shake - culture it overnight on a constant - temperature shaker at 28 °C and 180 rpm until the OD 600 reaches 0.4 - 0.6; Centrifuge at 5000 rpm at room temperature for 10 min, discard the supernatant, and suspend the Agrobacterium cell pellet to be infected with sterile water containing 200 μmol·L -1 acetosyringone (ACE).

[0122] Infection and co-culture: Put the callus into the above-mentioned bacterial solution, shake it at 100 rpm on a shaker at 28 °C for 10 min, suck off the surface bacterial solution with sterile filter paper, and then use forceps to transfer the callus into the B5 co-culture medium without any antibiotics, and culture it in the dark at room temperature for 2 d;

[0123] Washing the bacteria: Wash the co-cultured callus with sterile water containing 300 mg / L cefalexin, dry it with sterile filter paper, and transfer it onto the MS screening medium for incubation at a constant temperature;

[0124] Screening: Spread the above-mentioned callus evenly on the callus screening medium (containing 300 mg / L cefamycin and 50 mg / L kanamycin); After culturing for about 30 d, transfer the newly grown callus to a new callus screening medium (containing 300 mg / L cefamycin and 50 mg / L kanamycin);

[0125] Detection: Subculture and screen the callus that grows normally on the screening medium to obtain resistant callus with stable growth for detection. Extract the DNA of the screened resistant callus and identify whether it is transgenic callus by PCR detection.

[0126] (2) After activation is completed, centrifuge at 6000 rpm for 10 min at room temperature to enrich the bacteria, and discard the supernatant.

[0127] (3) Suspend the bacteria with the resuspension solution (1 / 2 B5 + 20 g·L -1 sucrose + 200 μmol·L -1 acetosyringone) and adjust the OD 600 value of the bacterial solution to be between 0.5 - 0.6, and resuscitate at 28 °C and 180 rpm for 1 h for later use.

[0128] (4) In the ultra-clean workbench, select well-grown 'Pinot Noir' callus, dry the surface moisture with sterile filter paper, put it into a sterile centrifuge tube, and add the overexpression and RNA interference Agrobacterium bacterial solutions resuspended in the previous step respectively. The callus needs to be completely immersed in the bacterial solution.

[0129] (5) Use a portable vacuum pump to perform negative pressure infection for 10 min, and shake the centrifuge tube during this period to ensure sufficient infection of the tissue blocks.

[0130] (6) After the infection process is completed, suck off the excess bacterial solution on the surface of the callus with sterile filter paper and place it evenly on the callus medium (1 / 2 B5 + 20 g·L -1 sucrose + 7 g·L -1 agar) for co-culture for 48 h.

[0131] (7) After co - cultivation, the transformed materials were washed 3 times with an antibiotic (1 / 2 B5 + 20 g L -1 sucrose + 300 mg L -1 cef) aqueous mixture for 5 min each time, with shaking during the period, and finally rinsed 5 times with sterile water.

[0132] (8) The callus that had undergone the sterilization step was inoculated onto a callus screening medium containing antibiotics (B5 + 0.5 mg·L -1 NAA + 1 mg·L -1 KT + 20 g·L -1 sucrose + 7 g·L -1 agar + 1 g·L -1 inositol + 300 mg·L - 1 PVP + 300 mg·L -1 Cef + Kana 50 mg·L -1 ).

[0133] (9) After the callus grew for a period of time, new white callus grew out. It was transferred to the B5 screening medium for positive callus screening, and this was repeated three times. DNA was extracted and positive callus was identified by PCR. The tissues identified as positive were continuously transferred and proliferated for subsequent functional research.

[0134] 1.7 Fluorescent quantification of stress - related genes in over - expression lines of the target gene:

[0135] Total RNA of grape callus and Arabidopsis thaliana was extracted according to the RNAprep pure Plant Total RNA Extraction Kit (product number DP452, purchased from Tiangen Biochemical Technology Co., Ltd.), and reverse transcription was carried out through the FastKing One - Step Genomic DNA Removal and cDNA First - Strand Synthesis Premix Kit (product number KR118, purchased from Tiangen Biochemical Technology Co., Ltd.). Finally, the expression levels of stress - related genes such as VvRD22, VvDREB2A, VvRD29A, VvP5CS, VvCAT1, and VvSOD were detected by the SuperReal PreMix Plus (SYBR Green) Kit (product number FP205, purchased from Tiangen Biochemical Technology Co., Ltd.) ( Figure 13 ), and the specific method was referred to the instruction manual.

[0136] 2 Results and analysis:

[0137] 2.1 Amplification of the target gene fragment and construction of the over - expression vector:

[0138] Using grape leaf cDNA as a template, with primers as shown in Table 1, the full-length gene was amplified using the Green Taq Mix premix (Novizan), and the fragment size was detected by agarose gel electrophoresis, and the sequence was determined by sequencing ( Figure 2 ). The positive plasmid was ligated to the pCAMBIA2300-EGFP vector by double digestion, and amplified by colony PCR ( Figure 2 ), and the sequencing was successful.

[0139] 2.2 Subcellular localization prediction:

[0140] Analysis by transient injection in tobacco found that VvYUC10 is a cell membrane-localized protein that functions mainly in the cell membrane ( Figure 3 ).

[0141] 2.3 Identification of transgenic Arabidopsis thaliana and callus:

[0142] The 35s:VvYUC10 overexpression vector constructed in 2.1 was used to infect Arabidopsis thaliana and callus. After resistance screening and growth for a period of time, positive verification was carried out. All 10 transgenic Arabidopsis thaliana plants were positive (WT is wild-type Arabidopsis thaliana) ( Figure 4 ), and all 5 cell clusters were positive (WT is untransformed callus) ( Figure 5 ). Subsequently, the bands with brighter intensity were selected to detect the expression level of VvYUC10 in transgenic Arabidopsis thaliana and grape callus. The results showed that the expression level of VvYUC10 in overexpressing Arabidopsis thaliana and grape callus was significantly increased compared with the control ( Figure 6 ), and the auxin content increased.

[0143] 2.4 Determination of antioxidant enzyme activities in wild-type and transgenic Arabidopsis thaliana and callus under salt stress:

[0144] The antioxidant enzyme activities of the overexpressed 35S:VvYUC10 of the target gene and the control 35S:00 plants were detected. The results found that ( Figure 7 and Figure 11 ), after overexpression, the antioxidant enzyme (SOD, CAT) activities in Arabidopsis thaliana and grape callus under salt stress were significantly enhanced.

[0145] 2.5 Determination of membrane lipid peroxidation products and osmotic regulators in wild-type and transgenic Arabidopsis thaliana and callus under salt stress:

[0146] The membrane lipid peroxidation products and osmotic regulators of the overexpressed 35S: VvYUC10 of the target gene and the control 35S:00 plants were detected. The results found that ( Figure 8 and Figure 12), After overexpression in Arabidopsis thaliana and grape callus under salt stress, the antioxidant (AsA and GSH), Pro content in Arabidopsis thaliana and grape callus under salt stress was significantly enhanced, and the content of MDA was reduced.

[0147] 2.6 After salt stress treatment, the target gene will cause the expression levels of VvYUC10 and stress-related genes such as AtRD22, AtDREB2A, AtRD29A, AtP5CS1, AtCAT2, and AtSOD1 related to salt tolerance to increase in overexpressed Arabidopsis thaliana and grape callus ( Figure 9 and Figure 13 ).

[0148] In summary, after overexpressing VvYUC10 under salt stress, the auxin content, antioxidant enzyme activity, and osmotic adjustment substance content in transgenic Arabidopsis thaliana and callus increased, and the expression levels of antioxidant enzyme genes and salt tolerance-related stress genes were all up-regulated. Therefore, it is proved that the above genes positively regulate grape salt tolerance by regulating auxin content and scavenging excessive reactive oxygen species.

[0149] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. VvYUC10 A gene, whose nucleotide sequence is as shown in SEQ ID No.1, or a nucleotide sequence in which one or more nucleotides are replaced, deleted and / or added to the nucleotide sequence as shown in SEQ ID No.1 and the nucleotide sequence expresses a protein with the same function.

2. A protein, characterized in that The protein encoding gene is the one described in claim 1 VvYUC10 The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. A biomaterial, characterized in that: Containing the VvYUC10 The gene or protein according to claim 2, wherein the biological material is selected from any one or more of recombinant DNA, expression cassette, transposon, plasmid vector, viral vector and engineered bacteria.

4. The method according to claim 1 VvYUC10 Use of the gene, the protein described in claim 2 or the biological material described in claim 3 in improving plant salt tolerance.

5. The method according to claim 1 VvYUC10 Use of the gene, the protein according to claim 2 or the biological material according to claim 3 in increasing the content of plant auxin.

6. The method according to claim 1 VvYUC10 Use of the gene, the protein described in claim 2 or the biological material described in claim 3 in improving the active oxygen scavenging ability of plants.

7. The use according to any one of claims 4 to 6, characterized in that: The plant is selected from the genus Vitis of the Vitaceae family, preferably grapes.

8. The method according to claim 1 VvYUC10 A method for amplifying a gene, characterized in that Using the cDNA of Pinot Noir grapes as template, PCR amplification was performed using primers to obtain VvYUC10 Gene.

9. The amplification method according to claim 8, characterized in that Among the amplification primers, the upstream primer is shown as SEQ ID No.3, and the downstream primer is shown as SEQ ID No.4.

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