Application of MdVQ22 gene in salt-tolerant genetic improvement of apple

Abstract

The invention discloses application of an MdVQ22 gene in salt-tolerant genetic improvement of apples, and relates to the technical field of plant genetic engineering. The key salt stress response gene MdVQ22 is identified, and the salt tolerance of apple plants can be improved by silencing the gene. The analysis of phenotypes and related physiological indexes of the MdVQ22 transgenic line before and after salt treatment shows that compared with a non-transgenic line, MdVQ22 overexpression transgenic apple callus has obvious salt sensitivity, and compared with a contrast, a silent line of a silent MdVQ22 gene has an obvious salt tolerance advantage; therefore, the MdVQ22 gene is a potential salt-tolerant breeding gene and can be used for salt-tolerant genetic improvement of plants. The invention provides a new gene resource and technical path for cultivating salt-tolerant apple varieties, can promote the development of the apple planting industry in saline-alkali soil, and breaks through the limitation of land salinization on the high-quality development of the apple industry.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to... MdVQ22 Application of genes in the genetic improvement of salt tolerance in apples. Background Technology

[0002] apple( Malus domestica Apple trees (Borkh.) are among the most economically valuable fruit trees. However, their growth and development are adversely affected by various abiotic stresses. Salt stress is one of the most widespread abiotic stressors globally, severely limiting the geographical distribution of apple trees and significantly reducing apple yield and fruit quality. Soil salinization is a major problem facing crop cultivation. Elucidating the molecular mechanisms of apple salt tolerance and developing new apple varieties or rootstocks with stronger salt tolerance is crucial for promoting the high-quality development of the apple industry.

[0003] Through long-term evolutionary adaptation, plants have established a complex regulatory network to coordinate responses to salt stress. This network relies on interconnected signaling pathways that can rapidly activate defense mechanisms. VQ proteins are a class of transcriptional regulatory cofactors containing VQ domains, characterized by 10 highly conserved amino acid residues in the sequence FxxhVQxhTG. Current research indicates that VQ proteins can interact with WRKY transcription factors to synergistically regulate specific biological processes and biotic stress responses, but studies on their involvement in plant responses to abiotic stresses are still limited. Summary of the Invention

[0004] The purpose of this invention is to provide MdVQ22 The application of genes in the genetic improvement of salt tolerance in apples aims to solve the problems existing in the above-mentioned prior art. This invention discovers the use of silencing genes. MdVQ22 Genes can improve the salt tolerance of apple plants, thus providing new genetic resources and technical pathways for breeding salt-tolerant apple varieties.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of a biomaterial that reduces the expression level of MdVQ22 protein in improving the salt tolerance of apple plants, wherein the amino acid sequence of the MdVQ22 protein is shown in SEQ ID NO.2.

[0006] Furthermore, the biomaterial inhibits MdVQ22 Gene expression was used to reduce the expression level of the MdVQ22 protein; The MdVQ22 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the biological material is a gene silencing vector.

[0008] Furthermore, the gene silencing vector is used to... MdVQ22 The target sequence of the gene is obtained by ligating it into the pTRV2 vector; The nucleotide sequence of the target sequence is shown in SEQ ID NO.9.

[0009] Furthermore, the biological material is a recombinant microbial strain containing a gene silencing vector.

[0010] Furthermore, the gene silencing vector is used to... MdVQ22 The target sequence of the gene is obtained by ligating it into the pTRV2 vector; The nucleotide sequence of the target sequence is shown in SEQ ID NO.9.

[0011] The present invention also provides a method for improving the salt tolerance of apple plants, including the step of reducing the expression level of MdVQ22 protein in apple plants; The amino acid sequence of the MdVQ22 protein is shown in SEQ ID NO.2.

[0012] Furthermore, by inhibiting MdVQ22 Gene expression was used to reduce the expression level of the MdVQ22 protein; The MdVQ22 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0013] This invention also provides a method for cultivating salt-tolerant apple varieties, including... MdVQ22 The steps involved in gene silencing to construct gene-silenced apple plants; The MdVQ22 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0014] Furthermore, the Agrobacterium transfection method was used to transfect the aforementioned... MdVQ22 Genes are silenced.

[0015] The present invention discloses the following technical effects: This invention identifies a key salt stress response gene. MdVQ22 Overexpression of this gene can increase the salt sensitivity of apple callus, while silencing the gene can increase the salt tolerance of apple plants. This invention analyzes the effects of salt treatment on callus tissue before and after salt treatment. MdVQ22 Phenotypic and related physiological indicators of transgenic lines indicate that, compared to non-transgenic lines, MdVQ22 Overexpression of transgenic apple callus tissue exhibited significant salt sensitivity, while silenced callus tissue... MdVQ22The pTRV2-MdVQ22 silenced lines showed a significant salt tolerance advantage compared to the control, indicating that... MdVQ22 The gene is a potential salt-tolerant breeding gene that can be used for the genetic improvement of salt tolerance in plants. This invention provides a new genetic resource and technical pathway for breeding salt-tolerant apple varieties, which can promote the development of the apple planting industry in saline-alkali land and overcome the limitations of soil salinization on the high-quality development of the apple industry. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The diagram shows the morphological characteristics (A), fresh weight (B), and relative electrical conductivity (C) of different strains subjected to salt stress treatment. Figure 2 TRV2-VQ22 silent plants and TRV2 empty control plants MdVQ22 Statistical graph of relative expression levels; Figure 3 Morphological characteristics of TRV2-VQ22 silent plants and TRV2 unloaded control plants subjected to salt stress for different durations. Figure 4 NBT and DAB staining images of TRV2-VQ22 silent plants and TRV2 unloaded control plants 2 days after salt stress treatment; Figure 5 A statistical graph showing the hydrogen peroxide content (A) and superoxide anion content (B) of TRV2-VQ22 silent plants and TRV2 unloaded control plants after 2 days of salt stress treatment. Figure 6 A statistical graph showing the superoxide dismutase (A), peroxidase (B), and catalase (C) activities of TRV2-VQ22 silent plants and TRV2 unloaded control plants after 2 days of salt stress treatment. Figure 7 The images show chlorophyll fluorescence imaging and quantitative fluorescence parameter analysis of TRV2-VQ22 silent plants and TRV2 unloaded control plants after 4 days of salt stress treatment; where A is the chlorophyll fluorescence imaging image; B and G are statistical graphs of F0, Fm, Fv / Fm, Y(II), Y(NO) and Y(NPQ) values, respectively. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The present invention relates to MdVQ22 The CDS sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0024] SEQ ID NO.1:ATGAGTAGTACTGGAACCATGTCCAGCAACCCTAATGATCACTGGCTCCAATTCTACAACCAAAACCTGAATTCTTCTACCCCTTCTTCACTGTTTTGCCATGACCAAGTCTCTGATGCCACCGTCGTCACCACCACCACTACAACTTCATCTTTACCAGGTCATGCCGGTTCAGGCCACCCGAGCTCCAGCACCAACAACAGTTTGAGTCCGGATCAAGGCCGCGTTTCGAAGCCGGCCCGGAAAAGGTCTAGAGCCTCAAGAAGAACTCCCACCACGCTGCTCAACACTGACACCACCAATTTTCGTGCCATGGTGCAGCAATTCACTGGGGGTCCTAGTACGGTGTCGTTTCCCACTGGT GCTTCGAATTTTACCTTTGGTC TCGGCCCCACTAGGCCACAGAGCTTCGTGGTGCCTCCCGGGGCGGCCGGGTGCCATCCTCTACAGCCTCAGCAGCAACAACTCTTACAACAACAACAACAACAACAACAACAACAACAACAGCAACAATTCCAACAACGCCATAACCAGCAATACTTGTTTTCGCTGAGGAACAATGACAACGTTAACGGTGATCATGGGTTTCTTGAGAGATTGTCATCAAGAGTGGGACCCACAAGCATGGTGATGGGACTGTCGTCCGATCATGATCAACATGAGTTTCTGAGGGAGGGTATTGCATCCCAATTGCCTTCTTCGGCTAGGCCGCCCAGCTCTAATAGTTCTG CCAATGAGAA CAGGAGTTACATG TTT。

[0025] SEQ ID NO.2: MSSTGTMSSNPNDHWLQFYNQNLNSSTPSSLFCHDQVSDATVVTTTTTTSSLPGHAGSGHPSSSTNNSLSPDQGRVSKPARKRSRASRRTPTTLLNTDTTNFRAMVQQFTGGPSTVSFPTGASN FTFGLGPTRPQSFVVPPGAAGCHPLQPQQQQLLQQQQQQQQQQQQFQQRHNQQYLFSLRNNDNVNGDHGFLELSSRVGPTSMVMGLSSDHDQHEFLREGIASQLPSSARPPSSNSSANENRSYMF.

[0026] The pCE2 TA / Blunt-Zero, pCAMBIA2300-GFP, pTRV1, and pTRV2 vectors used in the following examples were purchased from Nanjing Novizan Pharmaceutical Co., Ltd.

[0027] Example 1 Apple salt-tolerant genes — MdVQ22 Gene cloning: 1. RNA extraction: The RNAprep Pure plant total RNA extraction kit was used. For specific operating procedures, refer to the instructions for the RNA extraction kit from Tiangen Biotech Co., Ltd. The obtained RNA solution will be analyzed using Nanodrop and 1% agarose gel electrophoresis, respectively.

[0028] 2. Reverse transcription: Reverse transcription was performed using the All-In-One 5×RT Master Mix kit manufactured by ABM (Applied Biological Materials). Take 1 μg of total RNA, add 4 μL of 5×All-in-One RTMasterMix, and bring the volume to 20 μL with ddH2O. The reaction program was: 37℃ for 15 min, 65℃ for 10 min, and 95℃ for 3 min.

[0029] 3. MdVQ22 Gene cloning: according to MdVQ22 The CDS sequence of the gene was designed to obtain MdVQ22 Gene primer pairs: Upstream primer: ATGAGTAGTACTGGAACCATGTC (SEQ ID NO.3); Downstream primer: AAACATGTAACTCCTGTTCTCAT (SEQ ID NO.4); PCR amplification was performed using 2×Phanta Max Master Mix. The reaction mixture consisted of 25 μL Mix, 2 μL upstream primer, 2 μL downstream primer, 1 μL cDNA template, and ddH2O to a final volume of 50 μL.

[0030] The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 2 minutes, repeated for a total of 35 cycles; 72℃ extension for 5 minutes; and incubation at 4℃.

[0031] After the reaction, 1% agarose gel electrophoresis was performed at 220 V and 150 mA for 30 min, and images were taken using a gel imaging system. After electrophoresis, samples with molecular weights of similar sizes were cut off. MdVQ22 Consistent fragments were subjected to gel extraction. The gel extraction method was based on the Nanjing Novizan Agarose Gel DNA Recovery Kit.

[0032] 4. MdVQ22 Connect pCE2 TA / Blunt-Zero vector Will MdVQ22 The gel-recovered product was ligated into the pCE2 TA / Blunt-Zero vector for target gene amplification culture. The ligation system was: 1 μL of 5×pCE2 TA / Blunt-Zero Cloning Mix and 4 μL of PCR purified product. The reaction system was mixed and centrifuged, and the reaction was carried out at 20~37℃ for 5 min.

[0033] After the reaction, *E. coli* was transformed and plated on LB agar plates containing Amp for overnight growth. Single colonies were picked and placed in 1 mL of LB liquid medium containing antibiotics, and incubated at 37 °C for 200 rpm for 5 h for PCR identification. The bacterial culture with the correct PCR bands was added to 430 μL of 50% glycerol, and 200 μL was sequenced. The sequenced bacterial culture was stored in an ultra-low temperature freezer for subsequent experiments.

[0034] Example 2 MdVQ22 Gene overexpression reduces the salt tolerance of transgenic apple callus tissue 1. pCAMBIA2300-GFP- MdVQ22 Construction of overexpression vectors The target gene fragment obtained in Example 1 was ligated into the pCAMBIA2300-GFP vector to construct the pCAMBIA2300-GFP vector. MdVQ22 Overexpression vector: The empty vector pCAMBIA2300-GFP containing the 35S promoter was digested with Xba I and Xma I at 37°C for 1 h. Simultaneously, gene primers containing homologous arms, 2300-, were synthesized based on the restriction sites. MdVQ22 -Fhr: agaacacgggggactctagaATGAGTAGTACTGGAACCAT (SEQ ID NO.5); 2300- MdVQ22 -Rhr:cccttgctcaccatcccgggAAACATGTAACTCCTGTTCT (SEQ ID NO. 6).

[0035] PCR amplification was performed, followed by agarose gel electrophoresis. Correct bands were excised and recovered from the gel. Homologous recombination was used to ligate the recovered products. The reaction mixture consisted of: 7 μL linearized vector, 1 μL target gene fragment, 4 μL 5×CEII Buffer, 2 μL Exnase II, and 6 μL ddH2O. The reaction mixture was thoroughly mixed, centrifuged, and incubated at 37°C for 30 min.

[0036] After the reaction, *E. coli* transformation was performed, followed by sequencing of bacterial cultures with correct bands identified by PCR. *E. coli* strains containing the target vector that were correctly sequenced were then cultured, and plasmid DNA was extracted using the Nanjing Novizan plasmid extraction kit. Sequencing verification yielded the overexpression vector pCAMBIA2300-GFP-. MdVQ22 Agrobacterium EHA105 was transformed to obtain recombinant Agrobacterium, which is ready for subsequent experiments.

[0037] 2. Callus infection and tissue culture of 'Wang Lin' apple (1) Preparation of solutions and culture media: Y0 suspension formulation: 25 g / L sucrose and 4.4 g / L MS; sterilize at 121℃ for 20 min and then store in a refrigerator.

[0038] Y1 medium formulation: MS 4.4 g / L, sucrose 30 g / L, agar powder 7.5 g / L, 1 mg / L 2,4-D and 0.22 mg / L 6-BA, pH 5.8. After sterilization at 121℃ for 20 min, pour into sterile culture flasks and cool to solidify.

[0039] Y2 medium formulation: MS 4.4 g / L, sucrose 30 g / L, agar powder 7.5 g / L, 1 mg / L 2,4-D, 0.22 mg / L 6-BA, cephalosporin (Cef) 200 mg / L and kanamycin (Kan) 200 mg / L, pH 5.8. Sterilize at 121℃ for 20 min. Before it cools and solidifies, add the aseptically filtered cephalosporin and kanamycin in a laminar flow hood, then pour into a sterile culture flask as the screening medium.

[0040] (2) Transformation of callus tissue from 'Wang Lin' apples: 1) Activation of Agrobacterium: Take 20 μL of recombinant Agrobacterium culture into a 10 mL centrifuge tube, add 2 mL of LB liquid medium containing kanamycin, and incubate overnight at 28°C with shaking. The next day, take 1 mL of the culture and add it to a 100 mL sterile Erlenmeyer flask, add 30 mL of kanamycin liquid medium, and continue to incubate at 28°C with shaking until OD reaches 100%. 600nm =0.6-0.8.

[0041] 2) Transfer the liquid to a 50 mL centrifuge tube, centrifuge at 5000 rpm for 10 min, and discard the supernatant.

[0042] 3) Add 30 mL of Y0 suspension to suspend the bacterial cells and mix well by pipetting.

[0043] 4) Add 30 μL of acetylsalicylic acid and 'Wang Lin' callus tissue that is growing well (about 15 days).

[0044] 5) Infect at room temperature for 15 min.

[0045] 6) Filter the callus with sterile gauze, blot dry with filter paper, spread the callus evenly on Y1 medium without any antibiotics, and incubate in the dark at 24°C for 2 days.

[0046] 7) Spread the dark-cultured callus tissue evenly on Y2 medium until new callus tissue grows. Transfer the new callus tissue to Y2 medium for further identification and experiments.

[0047] 3. MdVQ22 Analysis of salt stress phenotype and physiological indicators in transgenic apple callus tissue overexpression To further explore MdVQ22 The invention achieves functionality under salt stress. MdVQ22 Transgenic apple callus tissues (OE-VQ22-1 and OE-VQ22-2) were overexpressed. Subsequently, wild-type (WT) apples and transgenic callus tissues OE-VQ22-1 and OE-VQ22-2, grown for 15 days, were placed on MS medium containing 150 mM NaCl and cultured for 15 days and 25 days, respectively. The results showed that the growth status and growth rate of the transgenic callus tissues were slower than those of the wild-type. Figure 1 (A). Under salt stress conditions, transgenic callus tissue exhibited higher electrical conductivity compared to wild-type. Figure 1 (C), and lower fresh weight ( Figure 1 (B)

[0048] In summary, the research results indicate that overexpression MdVQ22 It reduced the salt tolerance of callus tissue from genetically modified apples.

[0049] Example 3: Silence MdVQ22Genes can enhance the salt tolerance of apples. 1. Vector construction and transient transformation of apple tissue culture seedlings Construction of virus-induced gene silencing vectors: Using the target gene fragment obtained in Example 1 as a template, primer TRV2- with BmaHI and EcoRI restriction sites was used. MdVQ22 -Fhr: aggttaccgaattcGCTTCGAATTTTACCTTTGGTC (SEQ ID NO.7); TRV2-VQ22-Rhr: ctcggtaccggatccCATGTAACTCCTGTTCTCATTGG (SEQ ID NO.8), obtained by amplification MdVQ22 The non-conservative fragment (SEQ ID NO.9) was ligated into the pTRV2 vector to obtain the recombinant vector pTRV2- MdVQ22 pTRV2- MdVQ22 pTRV1 was introduced into Agrobacterium GV3101 to obtain gene-silenced recombinant Agrobacterium. Then, using a vacuum pump, the gene-silenced recombinant Agrobacterium was infiltrated into apple rootstock 'M9T337'. After the liquid on the plant surface was aspirated, the plant was inserted into the culture medium and cultured in a tissue culture room at a constant temperature of 25℃ for 2-3 days to obtain the TRV2-VQ22 transgenic line. Simultaneously, pTRV2- MdVQ22 The recombinant Agrobacterium was replaced with an empty vector pTRV2, and then infiltrated into apple rootstock 'M9T337' to construct the TRV2 transgenic line, which served as the control group.

[0050] The expression level of the target gene in transgenic plants was analyzed using qRT-PCR. Transgenic positive apple rootstocks were transferred to a medium containing 150 mM NaCl and cultured for 4 days. Phenotypic characteristics were observed, and relevant physiological and biochemical indicators were analyzed.

[0051] SEQ ID NO.9: GCTTCGAATTTTACCTTTGGTCTCGGCCCCACTAGGCCACAGAGCTTCGTGGTGCCTCCCGGGGCGGCCGGGTGCCATCCTCTACAGCCTCAGCAGCAACAACTCTTACAACAACAACAACAACAACAACAACAACAGCAACAATTCCAACAACGCCATAACCAGCAATACTTGTTTTCGCTGAGGA ACAATGACAACGTTAACGGTGATCATGGGTTTCTTGAGAGATTGTCATCAAGAGTGGGACCCACAAGCATGGTGATGGGACTGTCGTCCGATCATGATCAACATGAGTTTCTGAGGGAGGGTATTGCATCCCAATTGCCTTCTTCGGCTAGGCCGCCCAGCTTCTAATAGTTCTGCCAATGAGAACAGGAGTTACATG.

[0052] 2. MdVQ22- Analysis of salt stress phenotypes and physiological and biochemical indicators of TRV-silenced transgenic apple lines To further verify MdVQ22 To investigate the function of transgenic apples, transient transformation experiments were conducted using 'M9T337' apple rootstock to obtain TRV2-VQ22 transgenic lines and TRV2 control lines. The transformation was confirmed using reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR), and the function of transgenic apples in different transgenic lines was detected. MdVQ2 2. Downregulation of expression. After salt treatment, the TRV2-VQ22 transgenic lines and the TRV2 control lines showed... MdVQ22 Relative expression level, such as Figure 2 As shown.

[0053] After 2 and 4 days of salt stress treatment, TRV2-VQ22 plants showed less leaf wilting, curling, and wilt compared to the TRV2 control line. Figure 3 ).

[0054] Furthermore, staining with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) revealed that the ROS level in the leaves of the TRV2-VQ22 line was lower than that in the TRV2 control plant. Figure 4 ). For H2O2 and O2 - The quantitative determination results support the above staining results ( Figure 5Similarly, analysis of antioxidant enzyme activities (superoxide dismutase SOD, peroxidase POD, and catalase CAT) two days after salt treatment showed that the activities of these enzymes in TRV2-VQ22 plants were significantly higher than those in the TRV2 control plants. Figure 6 ).

[0055] Furthermore, chlorophyll fluorescence imaging revealed significant differences in fluorescence quenching characteristics among different plant lines after 4 days of sodium chloride (NaCl) stress treatment. In contrast, the quenching amplitude of TRV2-VQ22 leaves was far lower than that of the TRV2 control leaves. These results suggest that, compared to the control, the photosystem II (PSII) integrity of TRV2-VQ22 plants was largely maintained, while the PSII of TRV2 plants was severely damaged. Figure 7 (A)

[0056] Quantitative fluorescence parameter analysis ( Figure 7 The study (BG) showed that, compared with the TRV2 control, the TRV2-VQ22 line exhibited significantly higher F0 and Fm values, increased maximum quantum efficiency (Fv / Fm), and higher Y(II) values ​​compared to the TRV2 control. The Y(NO) value generated by non-photochemical pathways was decreased, while the Y(NPQ) value in the photoprotective system was increased. These findings indicate that the transgenic line silencing MdVQ22 enhances plant salt tolerance by maintaining the core responses of photosystem II (PSII) and optimizing energy allocation between photochemistry and photoprotection.

[0057] In conclusion, the research results confirm that silence... MdVQ22 The transgenic lines enhanced the salt tolerance of apples by scavenging reactive oxygen species (ROS) and optimizing the photosynthetic system.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of a biomaterial that reduces the expression level of MdVQ22 protein in improving the salt tolerance of apple plants, characterized in that, The amino acid sequence of the MdVQ22 protein is shown as SEQ ID NO.

2.

2. Use according to claim 1, characterized in that, The biomaterial reduces the expression level of the MdVQ22 protein by inhibiting MdVQ22 gene expression. The MdVQ22 The CDS sequence of the gene is shown as SEQ ID NO.

1.

3. Use according to claim 2, characterized in that, The biological material is a gene silencing vector.

4. Use according to claim 3, characterized in that, The gene silencing vector is obtained by ligating the target sequence of the gene into the pTRV2 vector. MdVQ22 The target sequence of the gene is ligated into the pTRV2 vector. The nucleotide sequence of the target sequence is shown as SEQ ID NO.

9.

5. Use according to claim 2, characterized in that, The biological material is a recombinant microbial strain comprising a gene silencing vector.

6. Use according to claim 5, characterized in that, The gene silencing vector is obtained by ligating MdVQ22 the target sequence of the gene into the pTRV2 vector. The nucleotide sequence of the target sequence is shown as SEQ ID NO.

9.

7. A method for improving salt tolerance in an apple plant, comprising, comprising the step of reducing the expression level of MdVQ22 protein in apple plants; The amino acid sequence of the MdVQ22 protein is shown as SEQ ID NO.

2.

8. The method of claim 7, wherein, by inhibiting MdVQ22 gene expression to reduce the expression level of said MdVQ22 protein; The MdVQ22 The CDS sequence of the gene is shown as SEQ ID NO.

1.

9. A method for breeding a salt tolerant apple variety, characterized by, comprising subjecting the gene to gene silencing, thereby obtaining a gene silenced apple plant; and MdVQ22 comprising subjecting the gene to gene silencing, thereby obtaining a gene silenced apple plant; and comprising subjecting the gene to gene silencing, thereby obtaining a gene silenced apple plant; The MdVQ22 The CDS sequence of the gene is shown as SEQ ID NO.

1.

10. The breeding method according to claim 9, characterized by, The genes were silenced by Agrobacterium-mediated transformation. MdVQ22 gene silencing.

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